write a simple essay on saccharomyces cerevisiae

Saccharomyces Cerevisiae: Increasing the Fermentation Rate Essay

Saccharomyces Cerevisiae is discovered as one of the most rapid yeasts due to its quick fermentation speed. The primary goal of the paper is to determine the existence of the potential aspects to increase the fermentation rate of Saccharomyces Cerevisiae while disapproving the existent hypothesis (Shiroma et al. 1002). In this instance, suitable yeast strains were utilized as a sample size while using ethanol concentration. As for the methodology, various experiments and tests such as Western blotting and measurements of various parameters including weight, signal intensity, and cell area were selected to find support for the hypothesis (Shiroma et al. 1004). Nonetheless, the results were portrayed in the form of diagrams and graphs to be able to depict the potential fluctuations in the functioning of yeast in the changing environment.

As for the results, they contribute to the fact that the fermentation can be accelerated with the assistance of various factors. It could be said that the outcomes reveal that ethanol fermentation can be improved with the help of atg32 laboratory strain (Shiroma et al. 1008). It was also highlighted that ethanol fermentation can be infused by atg32 sake yeast mutant while using Ginjo sake’s environment and “minimal synthetic medium” (Shiroma et al. 1009). Finally, the outcomes of this study can have implications in the biofuel production due to the ability to enhance the speed of ethanol. In this instance, the hypothesis was approved as the fermentation speed can be easily accelerated with the assistance of influence on mitophagy.

Shiroma, Shodai, Lahiru Jayakody, Kenta Horie, Koji Okamoto and Hiroshi Kitagaki. “Enhancement of Ethanol Fermentation in Saccharomyces Cerevisiae Sake Yeast by Disrupting Mitophagy Function.” Applied and Environmental Microbiology 80.3 (2014): 1002-1010. Print.

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Bibliography

IvyPanda . "Saccharomyces Cerevisiae: Increasing the Fermentation Rate." April 19, 2022. https://ivypanda.com/essays/saccharomyces-cerevisiae-increasing-the-fermentation-rate/.

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SACCHAROMYCES CEREVISIAE

Saccharomyces cerevisiae is a unicellular yeast cell that is found in the Kingdom fungi (singular: fungus). S. cerevisiae is found in the genus Saccharomyces and family Saccharomycetaceae. Morphologically, the cells of S. cerevisiae are ellipsoidal or cylindrical; and they can be propagated in the laboratory on simple mycological media such as Sabouraud dextrose agar (SDA) and potato dextrose agar (PDA).

The cells of S. cerevisiae are usually round to ovoid measuring 5–10 micrometers in diameter. Nutritionally, all the strains of S. cerevisiae can grow aerobically on simple carbohydrate source such as glucose, maltose and trehalose – which serves as their sole source of carbon and energy. They do not grow on media containing lactose and cellobiose. S. cerevisiae can grow aerobically and anaerobically on different carbohydrate-based media. They use urea, amino acids, peptides and ammonia (NH 3 ) as their sole source of nitrogen.

Species of Saccharomyces also require some mineral elements like sulphur, phosphorus, iron, calcium and magnesium for optimum growth. S. cerevisiae reproduce asexually by the process of budding ( Figure 1 ). S. cerevisiae is very instrumental in many industrial productions; and this has made it one of the most useful yeast since ancient times. Several strains of Saccharomyces cerevisiae is used in the production of beer, bread, wine, feed yeasts, food yeasts, industrial alcohols and spirits.

• Benefits of Fermentation
• RIBONUCLEIC ACID (RNA)
• Overview of Genes
• PROTEIN STRUCTURE – types and function
• SOUTHERN BLOTTING TECHNIQUE

PROCESSES OF BEER PRODUCTION

• TOOLS OF BIOTECHNOLOGY

S. cerevisiae like Escherichia coli (a bacterium) is one of the most intensively studied eukaryotic model organisms in molecular and cell biology; and it has been applied in many biotechnological applications for the production of various economically important products ( Table 1 ). The use of S. cerevisiae as a model for industrial productions and for molecular biology techniques especially in studying the physiology and metabolic processes of eukaryotic cells is largely attributed to its unique characteristics. S. cerevisiae has a small size and it has a small generation (doubling) time which allows it to grow rapidly on simple media.

Table 1. Applications of various strains of Saccharomyces cerevisiae

It has the ability to adapt to changing substrates and S. cerevisiae is amenable to genetic manipulations. S. cerevisiae has a high yielding capacity, and it produces large amount of carbondioxide (CO 2 ) especially in flour dough. S. cerevisiae can be called several names depending on the type of industrial process it has been applied in.

Baker’s yeast, brewer’s yeast, top-fermenting yeast, ale yeast and budding yeast are some of the names that S. cerevisiae have been known for. For example, in bread production, S. cerevisiae is used in baking as a leavening agent – where it converts the fermentable sugars present in the dough into carbon dioxide gas. To the lay man, S. cerevisiae used in bread production is known as sodium bicarbonate (the leavening agent of bread).

The conversion of fermentable sugars in the dough by S. cerevisiae to CO 2 gas causes the dough to rise as gas forms pockets or bubbles in the dough or bread. And when the bread is finally baked, the yeast cells dies and the air pockets created in the dough sets; and this gives the baked product a soft and spongy texture after production.

Bader F.G (1992). Evolution in fermentation facility design from antibiotics to   recombinant proteins in Harnessing Biotechnology for the 21st century (eds. Ladisch, M.R. and Bose, A.) American Chemical Society, Washington DC. Pp. 228–231.

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Nester E.W, Anderson D.G, Roberts C.E and Nester M.T (2009). Microbiology: A Human Perspective. Sixth edition. McGraw-Hill Companies, Inc, New York, USA.

Steele D.B and Stowers M.D (1991). Techniques for the Selection of Industrially Important Microorganisms. Annual Review of Microbiology , 45:89-106.

Pelczar M.J Jr, Chan E.C.S, Krieg N.R (1993). Microbiology: Concepts and Applications. McGraw-Hill, USA.

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Summers W.C (2000). History of microbiology. In Encyclopedia of microbiology, vol. 2, J. Lederberg, editor, 677–97. San Diego: Academic Press.

Talaro, Kathleen P (2005). Foundations in Microbiology. 5 th edition. McGraw-Hill Companies Inc., New York, USA.

Thakur I.S (2010). Industrial Biotechnology: Problems and Remedies. First edition. I.K. International Pvt. Ltd. New Delhi, India.

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Chika Ejikeugwu (PhD, 2017, UNIZIK, Nigeria) is a Fellow of the Alexander von Humboldt (AvH) Stiftung in Germany. Dr. Chika Ejikeugwu is currently a Research Fellow at the Helmholtz-Zentrum für Umweltforschung GmbH-UFZ, Leipzig, Germany, where he is working on "the soilRESIST project to investigate the effects of antibiotic mixtures on soil microbiomes." He founded Africa's Number 1 Microbiology website, www.MicrobiologyClass.net. Dr. Chika Ejikeugwu was a DAAD postdoctoral fellow at Bundesanstalt für Materialforschung und -prüfung (BAM), Berlin, Germany (2021) and a MIF Postdoctoral Fellow at Kyoto University, Kyoto, Japan (2018). In 2021, he was awarded the Young Investigator Award on Antimicrobial Resistance (AMR) by Institute Mérieux in France. Dr. Chika Ejikeugwu is a member of the Global Young Academy in Germany, and a member of other professional (microbiology) societies including Applied Microbiology International (AMI), European Society of Clinical Microbiology and Infectious Diseases (ESCMID), Nigerian Society for Microbiology (NSM) and American Society for Microbiology (ASM). He holds a doctorate degree in Pharmaceutical Microbiology and Biotechnology. Dr. Chika Ejikeugwu is a Senior Lecturer & Researcher at Enugu State University of Science & Technology (ESUT), Nigeria where he mentors undergraduate and postgraduate students on microbiology & other aspects of life. He has a flair for teaching, research and community service.

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Saccharomyces cerevisiae

A Microbial Biorealm page on the genus Saccharomyces cerevisiae

• 1.1 Higher order taxa
• 1.2 Species
• 1.3 Major Strains of Saccharomyces cerevisiae
• 2 Description and significance
• 3 Genome structure
• 4 Cell structure and metabolism
• 6 Pathology
• 7 Application to Biotechnology
• 8.1 Lantana camara used as substrate for fuel ethanol production
• 8.2 Increased glycolytic flux due to whole-genome duplication
• 8.3 Effects of Aneuploidy on Cellular Physiology and Cell Division in Haploid Yeast
• 9 References

Classification

Higher order taxa.

Domain: Eukarya Kingdom: Fungi Subkingdom: Dikarya Phylum: Ascomycota Subphylum: Saccharomycotina Class: Saccharomycetes Order: Saccharomycetales Family: Saccharomycetaceae Genus: Saccharomyces Species: Cerevisiae

Major Strains of Saccharomyces cerevisiae

1. Wild-type Strains

Saccharomyces boulardii: Formerly used as a probiotic used to treat diarrhea caused by bacteria. Clinical tests have demonstrated that this and a species of S. cerevisiae were genetically identical. ( 1 )

Saccharomyces uvaruium: Used in fermenting lager-type beer. Due to a recent reclassification, this species is now considered to be another wild-type strain. ( 2 )

2. Laboratory Strains

While S. cerevisiae contains many different strains used in research, below are some of the strains most commonly used in laboratories. The choice of which strain to use depends on what part of the organism is being studied.

S288c: This strain was isolated in the 1950's by Robert K. Mortimer through genetic crosses. It was used as a parental strain when isolating mutants ( 3 ). S288c was the strain used when the genome of S. cerevisiae was fully sequenced in 1996. However, its low rate of sporulation and the lack of protein growth in the absence of nitrogen prompted scientists to pick alternative strains for their research. ( 4 )

A634A: Used in cell cycle studies. It is also closely related to S288c due to a cross with S288c and another unknown strain. ( 4 )

BY4716: Since this is nearly identical to S288c , it is often used as a reference or control stain. ( 4 )

CEN.PK: In Europe, this is used as a secondary reference strain alongside S288c when studying the yeast genome. Additionally, it can grow well on several different carbon sources as well as under anaerobic conditions. It is used when studying rates of growth and product formation.( 5 )

∑1278b: What distinguishes this strain is that it contains genes unique for nitrogen metabolism. ( 6 ). It is best studied when nitrogen is limited; cells become elongated and undergo a unique budding pattern where cells remain physically attached to each other. This is known as pseudohyphal growth. ( 4 )

SK1: Because this strain produces lots of spores, it is used in meiotic studies. ( 7 )

W303: Closely related to S288c due to a cross between S288c and an unknown strain, ( 4 ), it is used in genetic and biochemical analysis. ( 7 ).

Description and significance

Saccharomyces cerevisiae is an eukaryotic microbe. More specifically, it is a globular-shaped, yellow-green yeast belonging to the Fungi kingdom, which includes multicellular organisms such as mushrooms and molds. Natural strains of the yeast have been found on the surfaces of plants, the gastrointestinal tracts and body surfaces of insects and warm-blooded animals, soils from all regions of the world and even in aquatic environments ( 8 ). Most often it is found in areas where fermentation can occur, such as the on the surface of fruit, storage cellars and on the equipment used during the fermentation process ( 9 )

S. cerevisiae is famously known for its role in food production. It is the critical component in the fermentation process that converts sugar into alcohol, an ingredient shared in beer, wine and distilled beverages. It is also used in the baking process as a leavening agent; yeast releasing gas into their environment results in the spongy-like texture of breads and cakes. Because of its role in fermentation, humans have known about and used Saccharomyces cerevisiae for a long time. Archaeologists have found evidence of a fermented beverage in a pot in China as early as 7000BC ( 10 ), and molecular evidence of yeast being used in fermentation was found in a wine jar dating back to 3150BC ( 11 ).

Isolation of the species did not occur until 1938, when Emil Mrak isolated it from rotten figs found in Merced, California. ( 3 ). Taking advantage of its unique reproductive cycle, Robert Mortimer performed genetic crosses that used the isolated fig strain and other yeast strains obtained through other researchers. As a result, he created a new strain called S288c ( 3 ), which was then used as a parental strain in order to isolate most of the mutant strains currently used in research ( 3 ). Furthermore, this strain was then used to sequence the S. cerevisiae genome ( 9 ).

S. cerevisiae is also considered to be a "model organism" by scientists. Its big advantage is that it is both a unicellular and eukaryotic organism. As a eukaryote, a majority of the yeast genes and proteins have human homologs ( 12 ), and a greater understanding of the yeast genome would also help scientists understand the human genome. Another advantage is its fast growth grate. On a normal yeast medium, it takes 90 minutes for the yeast population to double. ( 13 ), and colonies are usually visible 2-3 days after placing them on fresh medium. Since the complete genome sequence is now available, mutants unique to eukaryotic organisms can now be expressed in an eukaryote as opposed to studying a similar gene in prokaryotes.

Genome structure

On April 24, 1996, the complete yeast genome sequence was available to the public. The genome contains 12,068 kilobases contained in sixteen linear chromosomes. ( 12 ). Unlike prokaryotes, DNA is concentrated in the nucleus, and are grouped into chromosomes during DNA replication. 70% the genome contains of open reading frames (ORF's), DNA sequences that would code for a protein, and the average ORF length is about 1450 bp long ( 14 ). Relative to more complex eukaryotes like nematodes (6kb) and humans (30kb), the yeast genome is more compact( 12 ). In the genome, 5,885 genes code for proteins, 275 code for tRNA, 40 code for snRNA's, and 140 genes on chromosome 12 code for ribosomal RNA. 4% of the genome is comprised of introns, which are pieces of mRNA cut by snRNA-protein complexes prior to translation( 12 ). Out of all the genes that code for proteins, 11% of the protenome is devoted to metabolism, 3% to energy production and storage, 3% to DNA replication, 7% to transcription and 6% to translation. Nearly 430 proteins are involved in intracellular trafficking, and 250 proteins have structural roles.

Protein-coding genes have been documented in the genome, but so far a few of those genes have been identified. Furthermore, the genome also shows signs of two or more copies of a gene in different locations. The genes that code for citrate synthase, an enzyme that converts acetyl CoA and oxaloacetate to citrate, is located in three different chromosomes. Chromosome 3 encodes the enzyme in the peroxidase, chromosome 12 encodes the enzyme in the mitochondria and another copy of the gene is located in chromosome 16. ( 12 ). One reason for the redundancy in the genome could be that multiple copies of a yeast gene are required in order for it to survive in its natural habitat ( 12 ).

Using the completed genome, scientists have reconstructed the metabolic network of S. cerevisiae . 708 ORF's were identified to take part in metabolism, with the possibility to conduct 1035 metabolic reactions. ( 15 ). More than 85% of these reactions involved transport in and out of the cytoplasmic or mitochondrial membrane, and the other reactions were mainly involved in the metabolism of amino acids, nucleotides and vitamins. ( 15 ). Additionally, ORF's involved in metabolism have been classified based on the pathway they are most involved with. Most ORF's take part in the electron transport chain and chemiosmosis, the final steps of aerobic respiration, followed by the breakdown of bigger carbohydrates. ( 15 ).

The mitochondrial DNA sequence has been attempted, but it is incomplete and contains many errors. The mitochondrial genome is about 85,000 base pairs long and contains seven hypothetical ORF's. Further experiments will determine if any of the seven ORF's are expressed in the mitochondria. In addition to the ORF's, the genome contains genes for three subunits of complex IV used in the electron transport chain, and three subunits of ATP synthase. ( 16 ).

S. cerevisiae strain A364A also contains a 2um circle plasmid. It is 6,318 base pairs long and constitutes 3% of the yeast genome. ( 17 ). Although the sequence contains coding regions for three proteins, the exact identity or function of the proteins is unknown. Like nuclear chromosomes, the plasmid is comprised of chromatin and histones, and can condense itself during mitosis. Unlike bacterial plasmids, which replicate independently of the bacterial chromosome, it replicates only once during the S phase of the cell cycle, and is regulated by the same genes that regulate nuclear DNA replication ( 17 ). While there is no evidence that it can integrate into chromosomal DNA, the yeast plasmid is capable of acting as vector in yeast transformation. ( 17 ). Foreign DNA extracted from eukaryotes could now be inserted directly into an eukaryote.

Cell structure and metabolism

Saccharomyces cerevisiae can exist in two different forms: haploid or diploid. It is usually found in the diploid form. ( 11 ). The diploid form is ellipsoid-shaped with a diameter of 5-6um, while the haploid form is more spherical with a diameter of 4um. ( 13 ). In exponential phase, haploid cells reproduce more than diploid cells. Haploid and diploid cells can reproduce asexually in a process called budding, where the daughter cell protrudes off a parent cell. The buds of haploid cells are adjacent to each other, while the buds of diploid cells are located in opposite poles. ( 13 ). Additionally, diploid cells can exhibit pseudohyphal growth if it is growing on a poor carbon source, exposed to heat or high osmolarity. Activated by cAMP, newly developed cells remain attached to the parent cell through a septum. ( 18 ).

In addition to budding, diploid cells can undergo a meiotic process called sporulation to produce four haploid spores. Haploid spores can be one of two mating type, a or α. These spores can also undergo budding to produce more haploid cells. a and α cells can also mate and fuse together, producing a diploid cell. S. cerevisiae strains are further distinguished by differences in the haploid stage. In heterothallic strains, the spores resulting from sporulation cannot undergo budding, and their mating type cannot be changed. . However, in homothallic strains, the presence of a HO gene allows the spores to change mating type as they grow ( 11 ). Sporulation can be induced if the yeast is exposed to either a poor carbon or nitrogen source or lack of a nitrogen source. Spores also have a higher tolerance to conditions such as high temperature. ( 11 ).

As a eukaryote, S. cerevisiae contains membrane-bound organelles. Its chromosomes are located in the nucleus, and it uses mitochondria to conduct cellular respiration. Like all other fungi, the cell's shape is based on its cell wall. The cell wall protects the cell from its environment as well as from any changes in osmotic pressure. The inner cell wall has a high concentration of β-glucans, while the outer cell wall has a high concentration of mannoprotein. Chitin is usually located in the septum. ( 19 ).

S cerevisiae can live in both aerobic as well as anaerobic conditions. In the presence of oxygen, yeast can undergo aerobic respiration, where glucose is broken to CO2 and ATP is produced by protons falling down their gradient to an ATPase. When oxygen is lacking, yeast only get their energy from glycolysis and the sugar is instead converted into ethanol, a less efficient process than aerobic respiration. The main source of carbon and energy is glucose, and when glucose concentrations are high enough, gene expression of enzumes used in respiration are repressed and fermentation takes over respiration ( 2 ). However, yeast can also use other sugars as a carbon source. Sucrose can be converted into glucose and fructose by using an enzyme called invertase, and maltose can be converted into two molecules of glucose by using the enzyme mannase ( 2 ).

It has been difficult to observe and collect Saccharomyces cerevisiae outside areas of human contact, so not much research has been done on its interactions in natural environments. Because it is rarely associated with any other environments other than areas that are close to sites of fermentation, people have wondered whether the yeast could ever be found in the wild. ( 19 ). So far, most interactions with its environment have been limited to fermentation. In 1871, Louis Pasteur discovered that grapes had to be crushed in order for fermentation to occur ( 9 ). The grape itself has been an ideal habitat for yeast due to its high sugar concentration and low pH, precluding the growth of rival species. ( 8 ). Despite this, not many intact grapes contain S. cerevisiae at any one time. In an estimate, only one intact grape berry has the yeast on its surface. ( 3 ).

While intact grapes have little to no yeast present on the skin, damaged grapes are more likely to contain the yeast as well as other organisms. Berries were damaged due to the weather, mold infections or birds feeding on the grapes. Additionally, insects may also appear more often if the berry is already damaged. ( 3 ) These insects would harbor the yeast in their bodies and deposit them unknowingly while feeding, and the yeast would divide upon exposure to the grape. While it is known that insects harbor microorganisms inside their bodies, it is unknown how yeast is introduced into the insect. ( 3 ).

Saccharoyces cerevisiae is not normally considered to be a pathogen. In healthy people, disease resulting from S. cerevisiae colonizing in a particular area are very rare, but have been reported. While yeast that normally colonize in the GI tract are not the direct cause of any disease, hypersensitivity to antibodies produced against could prove an irritant for people with Crohn's disease, an autoimmune disorder. ( 20 ). 1% of all vaginal yeast infections occur due to S. cerevisiae in the vagina, but symptoms associated with it are identical to the symptoms caused due to another organism more commonly associated with yeast infections, Candida albicans . ( 21 ). The only people susceptible to serious problems are immunosuppressed individuals, followed by those who have taken S. cerevisiae as an probiotic for diarrhea. For these individuals, the prevailing condition is fungemia ( 1 ). Caused by the presence of yeasts in the blood, its symptoms have been described as "flu-like".

Application to Biotechnology

1. Ethanol Production

One of the oldest applications of Saccharomyces cerevisiae in biotechnology is its role the creation of alcoholic beverages. In a process called fermentation, yeast feeds off sugars from their substrate and convert it to ethanol, giving these beverages their alcoholic content. Depending on the beverage, yeast is incorporated into the creation process in several ways.

Winemakers select their yeast based on several factors: type of grape, local climate, geographical area and the desired taste of the final product. ( 2 ). Yeast is then produced in the winery, then added to the crushed grapes when it is time for fermentation. Champagne is an exception where natural yeast strains are used, since yeast goes directly into the bottle instead of a huge vat. ( 2 ). More gas is trapped in the bottle, creating sparkling wine.

In brewing beer, two different types of yeasts are produced in the fermentation process, depending on the type of beer created. Top-fermenting yeasts, also known as ale yeasts, form foam on top of the wort, the liquid containing the sugars used to be converted into ethanol. The yeast stays at the top of the tank, and begins to ferment at warm temperatures. This process is used in the creation of ales, porters, stouts and wheat beers. ( 2 ). Bottom-fermenting yeasts, also known as lagers yeast, ferment at cooler temperatures, and the yeast settle at the bottom of the tank. ( 2 ). They are used in the production of most commercial beers sold in America.

Another process is used to create the beverages collectively known as spirits, such as vodka and tequilla. Yeast used in the fermentation of these beverages are isolated from beet or sugar cane. Selection criteria for these yeasts include high ethanol production, have high tolerance to ethanol concentration, and must be able to ferment various substrates specific to the beverage.( 2 ).

2. Food Production

S. cerevisiae also acts as a leavening agent. During preparation, dried yeast cells are added with the rest of the ingredients. While baking, yeast reacts with its environment and releases gas. This gas is trapped, forming holes as it bakes. This contributes to the spongy-like texture of breads and cakes seen after baking. While dried yeast cells include a leavening agent, unleavened yeast could also be used to add flavor to the bread. ( 2 ).

Yeast used to brew beer is still useful after the fermentation process. After fermentation is finished, the leftover yeast is dried and can be sold in liquid, tablet or powdered form. It is an excellent source of B vitamins, various minerals and proteins, and can be taken as a nutritional supplement ( 2 ). Yeast still contains these nutrients even after being broken down by its own enzymes, and the resulting yeast extract can be used as a flavor enhancer ( 2 ). One of the components of the famous food paste Vegemite contains yeast extract. Finally, S. cerevisiae has also been shown to survive living in the gastrointestinal tract while eliminating the potentially pathogenic bacteria residing. Since it does not colonize the GI tract permanently, it is used as a probiotic.

Current Research

Lantana camara used as substrate for fuel ethanol production.

Research is being conducted to find economically viable methods to produce ethanol, a possible alternate fuel source to petroleum since it can be made from renewable resources. Currently, starch-containing plants are used for ethanol production, but starch production is limited. Cellulose, another complex sugar, is preferred over starch because it is the more abundant sugar in plants, but cellulose is harder to break down than starch. Since the breakdown of cellulose would be more costly than the breakdown down starch, an abundant source of cellulose would be needed to offset the cost of its breakdown. Lantana camara , a hard-to-eradicate weed, would be used as the cellulose source. L. camara was first pre-treated with the enzyme cellulase as well as various strong acids and bases to break up the cellulose. A heat tolerant strain of Saccharomyces cerevisiae found from India would be used to conduct fermentation. 97.4 grams of sugar would be used to make 42 grams of ethanol, giving an ethanol yield of 0·431 per gram with a fermentation efficiency of 84.36% Despite the presence of fermentation inhibitors, the by-product of cellulose breakdown, the high carbohydrate content of the weed and the efficiency of the yeast proved that it could be viable to produce ethanol from a more abundant resource. ( 22 )

Increased glycolytic flux due to whole-genome duplication

Whole-genome duplication occurs when a cell replicates its DNA normally, but does not distribute its DNA equally during mitosis. While one copy is only needed for the organism to function, the excess genes are promptly deleted through mutations and gene loss. However, the genes coding for the enzymes involved in glycolysis have managed to survive in duplicate. This suggests that natural selection may have played a role in picking out yeast strains based on rapid growth on medium such as glucose. Surviving duplicate copies of the glycolytic enzymes would not only lead to increase glycolytic flux, but would eventually have the yeast prefer fermentation over aerobic respiration. This is possible since one unique aspect of S. cerevisiae is that even in oxygen, it would continue to convert sugar to ethanol despite being the more inefficient pathway in a glucose-rich environment ( 23 ).

Effects of Aneuploidy on Cellular Physiology and Cell Division in Haploid Yeast

Haploid cells of S. cerevisiae containing an extra copy of one or more chromosomes mated with another haploid cell with a normal amount of chromosomes, producing a diploid possessing three or more copies of the inherited chromosomes. To create yeast cells with additional chromosomes, researchers looked for haploid cells lacking the KAR1 gene, preventing nuclear fusion. Occasionally, chromosome transfer would still occur, and the union of this particular mating was then selected for. When compared with diploid yeast cells with the normal number of chromosomes, the aneuploid cells expressed unique traits. Doubling time was slightly increased in the aneuploids due to a delay in the G1 stage of the cell cycle. Aneuploid cells would also show increased glucose uptake mostly because as a result of extra chromosomes, certain genes located on the duplicated chromosome are overexpressed. In the presence of protein synthesis inhibitor chemicals such as cycloheximide, aneuploids were more likely to produce unfolded proteins. Since tumors in humans posses similar characteristics to yeast cells, studying phenotype expression in aneuploid yeast cells could provide a stepping stone to studying phenotypes in tumor cells ( 24 ).

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Industrially Important Fungi for Sustainable Development pp 393–427 Cite as

Saccharomyces and Their Potential Applications in Food and Food Processing Industries

• Vincent Vineeth Leo 7 ,
• Vinod Viswanath 8 ,
• Purbajyoti Deka 7 ,
• Zothanpuia 9 ,
• Dwivedi Rohini Ramji 7 ,
• Lallawmsangi Pachuau 7 ,
• William Carrie 7 ,
• Yogesh Malvi 7 ,
• Garima Singh 10 &
• Bhim Pratap Singh 11  
• First Online: 19 June 2021

1372 Accesses

Part of the book series: Fungal Biology ((FUNGBIO))

Genus Saccharomyces is one of the most explored yeast species, especially in the food processing and allied food industries. Among them S. cerevisiae , S. boulardii , and S. cerevisiae var. boulardii are the leading yeasts that find relatively major functional usages as natural fermenters in various food applications. As humanity as a whole is on the search for an easy cure into healthy living among their busy schedule, healthy gut, balanced diet, probiotics, and functional/fortified foods have gained immense importance. Yeast and Saccharomyces could play a substantial role in ensuring that such diet balances are maintained by utilizing their natural abilities to ferment food items according to their needs. In this context, this book chapter attempts to survey an overhaul of Saccharomyces in the current food market, food industry, and food technology and their role in food processing industries.

• Food applications
• Saccharomyces
• Wine yeasts

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The corresponding author is thankful to NIFTEM authorities for providing facilities which are used in writing this book review.

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Leo, V.V. et al. (2021). Saccharomyces and Their Potential Applications in Food and Food Processing Industries. In: Abdel-Azeem, A.M., Yadav, A.N., Yadav, N., Usmani, Z. (eds) Industrially Important Fungi for Sustainable Development. Fungal Biology. Springer, Cham. https://doi.org/10.1007/978-3-030-67561-5_12

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What is yeast?

What is Saccharomyces cerevisiae?

You may not know it, but you probably have used it already many times. We will give you a hint.

It is most commonly used for baking and brewing, but it can also be used for many other things.

Still not sure?

It is yeast! Or at least what most people refer to as “yeast.”

Saccharomyces cerevisiae : AKA “brewer’s yeast” or “baker’s yeast”

The scientific name “Saccharomyces” is derived from the Greek word meaning “sugar fungus” while “cerevisiae” comes from Ceres the Roman Goddess of the growth of food plants and crops. 

Saccharomyces cerevisiae ( S. cerevisiae ) is a species of yeast or single-celled fungus microorganism known since early times for its fermentation properties and used in baking, brewing, and winemaking. Today, it is also used as a unique probiotic to support gut health as well as being used for a variety of other applications. 

Despite its long history, S. cerevisiae ‘s name is relatively unknown even though it is one of the most common types of yeast used. In fact, without even knowing it, when most people speak of “yeast,” it is Saccharomyces cerevisiae that they are often speaking of. Today, however, most people are more familiar with it by the names “brewer’s yeast”, “baker’s yeast”, “budding yeast”, or as the main component of most nutritional yeasts and yeast extracts. 

Whether one is aware of it or not S. cerevisiae is everywhere and widely used and consumed across the globe.(1)

Saccharomyces cerevisiae is ALIVE! 

Saccharomyces cerevisiae is a unicellular fungus that reproduces by budding from a pre-existing cell, and which presents the main components of a typical eukaryotic cell. Its cell wall is a dynamic structure relatively rigid that provides cell protection, and osmotic support and determines cell shape.  S. cerevisiae cells are round to ovoid and are approximately 5–10 micrometers in diameter.(2)

All strains of S. cerevisiae can grow aerobically on glucose, maltose, and trehalose. However, they fail to grow on lactose and cellobiose. S. cerevisiae growth on other sugars varies. Galactose and fructose are shown to be two of the best fermenting sugars. The ability of S. cerevisiae to use different sugars can differ depending on whether it is grown aerobically or anaerobically. Some strains cannot grow anaerobically on sucrose and trehalose.(3)

In nature, S. cerevisiae is most commonly found on the skin of ripe fruits, such as grapes. S. cerevisiae can also be found in the bark of some tree species, as well as on some insects. 

S. cerevisiae is one of the most intensively studied eukaryotic model organisms in molecular and cellular biology.

Saccharomyces cerevisiae’s use in baking

If you have done any baking odds are the yeast that you used was Saccharomyces cerevisiae . 

During fermentation in bread-making, Saccharomyces cerevisiae as yeast produces carbon dioxide and modifies the physical properties of dough through the action of enzymes. 

First, the yeast ferments sugars which it directly assimilates and is naturally present in the flour. The second phase corresponds to the fermentation of sugar found in flour called maltose. Glucose is transformed by Saccharomyces cerevisiae into carbon dioxide (which gives volume to bread and the honeycomb shape of the crumb) and alcohol (evaporated when baked). Saccharomyces cerevisiae yeast also produces aromatic compounds that contribute to the aroma and taste of bread. Lastly, during baking, fermentation is activated by heat and ends when the temperature reaches 50°C. 

Making bread is easy!

Brewer and winemakers’ “secret ingredient” 

In the absence of air, Saccharomyces cerevisiae cells transform sugars into carbon dioxide and alcohol. This process is what changes simple barley or wheat into beer and grapes into wine.

In beer brewing, saccharomyces cerevisiae is sometimes referred to as a top-fermenting or top-cropping yeast. Yeast not only plays a major role in the fermentation process, in brewing but also the aromatic qualities of the end product. Different types of strains are often brewers’ “secret ingredient.” Depending on the temperatures used in fermentation, the same strain can produce different flavors.

Saccharomyces cerevisiae is also the main strain used in winemaking. Yeast in winemaking is used for the fermentation process just like in brewing. As the yeast makes alcohol through feeding, it also produces fermentation aromas (fruity, peach, rose, etc.) and secondary aromas, known as varietal aromas, associated with specific grape varieties. These secondary aromas can only be revealed by using specific types of yeast during the fermentation process (vanilla spice or toasty notes).

How Is Yeast Used in Fermented Drinks?

Nutritional yeast = saccharomyces cerevisiae  

In recent years, many people have started consuming a specific type of yeast called nutritional yeast or also known as NOOCH. In fact, most nutritional yeasts are made with the yeast strain Saccharomyces cerevisiae. It can be used in dietary supplements, seasonings, and functional foods. Nutritional yeast can also provide appealing nutritional contributions.

So, what exactly makes nutritional yeast made with Saccharomyces cerevisiae so exceptional?

Many things!

It is a valuable source of protein. These proteins contain all the essential amino acids that people should get in a healthy diet. Source of dietary fiber, Saccharomyces cerevisiae yeast also contains several vitamins like thiamine (B1), riboflavin (B2), niacin (B3), Pyridoxine (B6), and folic acid (B9) (4). This makes nutritional yeast a potentially interesting source of vitamins for all, including vegans and vegetarians. 

Nutritional Yeast, Your Health Ally

Now that you know what Saccharomyces cerevisiae is, you can tell your friends about all its wonderful uses and benefits the next time you are baking bread together or enjoying a tasty glass of wine or beer! 

Related posts

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Common Misconceptions About Yeast Infections

Everything You Need to Know about Yeast Allergies

An Introduction to Saccharomyces Cerevisia

JoVE Science Education Database. Model Organisms I: yeast, Drosophila and C. elegans. An Introduction to Saccharomyces cerevisiae. Journal of Visualized Experiments, Cambridge, MA, doi: 10.3791/5081 (2014).

Saccharomyces cerevisiae (commonly known as baker’s yeast) is a single-celled eukaryote that is frequently used in scientific research. S. cerevisiae is an attractive model organism due to the fact that its genome has been sequenced, its genetics are easily manipulated, and it is very easy to maintain in the lab. Because many yeast proteins are similar in sequence and function to those found in other organisms, studies performed in yeast can help us to determine how a particular gene or protein functions in higher eukaryotes (including humans).

This video provides an introduction to the biology of this model organism, how it was discovered, and why labs all over the world have selected it as their model of choice. Previous studies performed in S. cerevisiae that have contributed to our understanding of important cellular processes such as the cell cycle, aging, and cell death are also discussed. Finally, the video describes some of the many ways in which yeast cells are put to work in modern scientific research, including protein purification and the study of DNA repair mechanisms and other cellular processes related to Alzheimer’s and Parkinson’s diseases.

Watch the Video

Yeast Maintenance Research performed in the yeast Saccharomyces cerevisiae has significantly improved our understanding of important cellular phenomona such as regulation of the cell cycle, aging, and cell death. The many benefits of working with S. cerevisiae include the facts that they are inexpensive to grow in the lab and that many ready-to-use strains are now commercially available. Nevertheless, proper maintenance of this organism is critical for successful experiments.

This video will provide an overview of how to grow and maintain S. cerevisiae in the lab. Basic concepts required for monitoring the proliferation of a yeast population, such as how to generate a growth curve using a spectrophotometer, are explained. This video also demonstrates the hands-on techniques required to maintain S. cerevisiae in the lab, including preparation of media, how to start a new culture of yeast cells, and how to store those cultures. Finally, the video shows off some of the ways these handling and maintenance techniques are applied in scientific research.

Yeast Reproduction Saccharomyces cerevisiae is a species of yeast that is an extremely valuable model organism. Importantly, S. cerevisiae is a unicellular eukaryote that undergoes many of the same biological processes as humans. This video provides an introduction to the yeast cell cycle, and explains howS. cerevisiae reproduces both asexually and sexually Yeast reproduce asexually through a process known as budding. In contrast, yeast sometimes participate in sexual reproduction, which is important because it introduces genetic variation to a population. During environmentally stressful conditions, S. cerevisiae will undergo meiosis and form haploid spores that are released when environmental conditions improve. During sexual reproduction, these haploid spores fuse, ultimately forming a diploid zygote. In the lab, yeast can be genetically manipulated to further understand the genetic regulation of the cell cycle, reproduction, aging, and development. Therefore, scientists study the reproduction of yeast to gain insight into processes that are important in human biology.

Isolating Nucleic Acids from Yeast One of the many advantages to using yeast as a model system is that large quantities of biomacromolecules, including nucleic acids (DNA and RNA), can be purified from the cultured cells.

This video will address the steps required to carry out nucleic acid extraction. We will begin by briefly outlining the growth and harvest, and lysis of yeast cells, which are the initial steps common to the isolation of all biomacromolecules. Next, we will discuss two unique purification methods for the separation of nucleic acids: column binding and phase separation. Additionally, we will demonstrate several ways in which these methods are applied in the laboratory, including the preparation of nucleic acids for molecular biology techniques such as PCR and southern blotting, quantification of gene expression in response to environmental stimuli, and purification of large amounts of recombinant proteins.

Yeast Transformation and Cloning S. cerevisiae are unicellular eukaryotes that are a commonly-used model organism in biological research. In the course of their work, yeast researchers rely upon the fundamental technique of transformation (the uptake of foreign DNA by the cell) to control gene expression, induce genetic deletions, express recombinant proteins, and label subcellular structures.

This video provides an overview of how and why yeast transformation is carried out in the lab. The important features of yeast plasmids will be presented, along with the procedure required to prepare yeast cells to incorporate new plasmids. The presentation also includes a step-by-step protocol for the lithium acetate method of yeast transformation. Finally, examples of the many applications of this essential technique will be provided.

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1.8: Respiration and Fermentation

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• Brad Basehore, Michelle A. Bucks, & Christine M. Mummert
• Harrisburg Area Community College

Introduction

All organisms must break down organic molecules to release chemical energy to synthesize adenosine triphosphate (ATP). The energy stored in ATP can be released to perform cellular work. Organisms break down organic molecules, such as glucose, through the common processes of cellular respiration and fermentation (Figure 1). Cellular respiration is generally described as an aerobic process, requiring oxygen, which yields the most possible ATP generated from one molecule of glucose. But, technically, cellular respiration can occur in an anaerobic environment in some microorganisms. Anaerobic cellular respiration yields variable amounts of ATP, but much less than is generated in aerobic cellular respiration. In this laboratory, our discussion of cellular respiration will focus on aerobic cellular respiration .

Both aerobic cellular respiration and fermentation involve many chemical reactions that release high energy electrons from organic molecules and transfer the electrons to other molecules, often referred to as electron carriers (or coenzymes). These chemical reactions involving the transfer of electrons are called reduction-oxidation reactions, or redox reactions . In a redox reaction, one of the molecules gains electrons and becomes reduced (rig, r eduction i s g ain of electrons) and one of the molecules loses electrons and becomes oxidized (oil, o xidation i s l oss of electrons). In cellular respiration, electrons are often transferred to the electron carrier nicotinamide adenine dinucleotide (NAD+). When this redox reaction occurs, the organic molecule that loses the electrons has been oxidized. When NAD+ gains the electrons it forms NADH. NADH is the reduced form of NAD.

\[\ce{NAD^{+} + 2e^{-} + H^{+} ->[\text{Redox Reaction}] NADH}\]

Aerobic cellular respiration involves a series of three processes of enzymatic chemical reactions: glycolysis , the citric acid cycle (also known as the Kreb’s cycle), and the electron transport chain . Aerobic cellular respiration begins in the cytoplasm with glycolysis and ends in the mitochondria with the citric acid cycle and the electron transport chain. Aerobic cellular respiration results in fully oxidizing glucose, and can yield a maximum of 32 ATP per glucose molecule. At the culmination of the electron transport chain, the electrons are passed to oxygen, a highly electronegative element, to form water. Therefore, at the end of this process, the high energy electrons that were previously a part of glucose are now at a lower energy state, as they are held very closely by the electronegative oxygen.

Fermentation is an anaerobic process of breaking down organic molecules. It occurs in the absence of oxygen. Fermentation breaks down organic molecules, such as glucose, into smaller organic molecule end products. Fermentation begins with the process of glycolysis to produce pyruvic acid and 2 net ATP. Enzymes then carry out chemical reactions to convert pyruvic acid into various fermentation end products. Two common types of fermentation are named for their end products, alcoholic fermentation and lactic acid fermentation . Fermentation produces organic end products that still contain high-energy electrons. Fermentation does not fully oxidize glucose, and yields only 2 net molecules of ATP, along with organic end products.

PART 1: CELLULAR RESPIRATION

Exercise 1 : Investigating Cellular Respiration in Plants

This part of the lab investigates cellular respiration in pea seeds. Seeds of plants are stuffed full of sugars like starch. Cellular respiration involves breaking down sugars to generate ATP. Therefore, this process allows plants to harvest energy necessary to produce roots, shoots, and leaves. The process of cellular respiration also results in the release of carbon dioxide gas. Carbon dioxide will react with water to form carbonic acid. The formation of carbonic acid will affect the pH of an aqueous solution. Since carbon dioxide is colorless, odorless, and very hard to detect, we are going to use a pH indicator to detect the presence of carbonic acid and thus carbon dioxide. pH indicators, like red cabbage juice, bromothymol blue, and phenol red are chemicals that change color when pH is altered. In this experiment, we will observe cellular respiration in germinating pea seeds by detecting the production of carbon dioxide and monitoring the changes in the pH of the solution.

• Pea seeds (20 germinating/ lab group and 20 dormant / lab group)
• Large sealable bag
• Test tube rack (that can accommodate wide mouth test tubes)
• Wide mouth test tubes with rubber stoppers (3/ lab group)
• Distilled water or spring water (non-chlorinated water)
• Paper towels
• Nonabsorbent cotton plugs
• Glass beads (20 / group)
• Gloves and safety goggles
• Sharpie or red wax pencil
• Indicator reagent [Choose 1: red cabbage juice, or bromothymol blue (0.04% solution), or phenol red (0.04% solution)] (need 15 ml of indicator solution / lab group)

Overall Timeline:

Employing Steps in the Scientific Method:

• Record the Question that is being investigated in this experiment. ________________________________________________________________
• Record a Hypothesis for the question stated above. ________________________________________________________________
• Predict the results of the experiment based on your hypothesis (if/then). ________________________________________________________________
• Perform the experiment below and collect your data.
• Two days before beginning the experiment, pour half of the peas into a glass container and cover with several inches of non-chlorinated water to compensate for the expansion of the seeds as they swell. Allow the seeds to soak overnight.
• The next day, pour the water off of the seeds. Place the seeds onto a wet paper towel, place in a plastic sealable bag, seal the bag, and store the seeds overnight in the dark.
• On the day of the experiment carefully remove the rehydrated (germinating) seeds from the paper towel.
• Label 3 wide mouth tubes #1 - #3.
• Wear gloves when handling the indicator solution. Place 5 ml of the indicator solution into each test tube.
• Using the glass rod, push a plug of nonabsrobent cotton into each test tube until it sits right above the indicator solution.
• Tube 1: add 20 glass beads
• Tube 2: add 20 germinating peas
• Tube 3: add 20 dry dormant peas
• Tightly cap the tubes with rubber stoppers. ( If the rubber stoppers have a hole, cover the stoppers with cling wrap, and then place each stopper into a tube.)
• Observe the color of the indicator reagent at the beginning of the experiment and record your results in Table 1.
• Observe the color of the indicator reagent after the 2-hr incubation and record your results in Table 1.
• Observe the color of the indicator reagent after the 24-hr incubation and record your results in Table 1.
• Once the experiment has been completed, carefully pour the indicator reagent into the appropriate location as indicated by your instructor, being sure to collect the glass beads by pouring through a wire mesh filter.
• Rinse and wash the test tubes thoroughly.

Questions for Review

• What is the color of the indicator at at
• Neutral pH?
• What was the purpose of Tube #1 ?
• What specifically was produced as a result of cellular respiration that changed the color of the indicator?
• How is carbon dioxide an indicator that cellular respiration is taking place in these peas?
• Germination is the process by which a dormant seed begins to sprout and grow into a seedling. What are some possible metabolic processes that are required for seed germination?
• During respiration, a seed metabolizes sugars. What is the source of the sugar metabolized by the seed?
• What variables do you think may affect the respiration rate of the seeds?
• The equation for cellular respiration is:

\[\ce{C6H12O6 + 6O2 → 6CO2 + 6H2O + 32 ATP}\]

The energy released from the complete oxidation of glucose under standard conditions is 686 kcal/mol. The energy released from the hydrolysis of ATP to ADP and inorganic phosphate under standard conditions is 7.3 kcal/mol. Using the equation for cellular respiration above, calculate the efficiency of respiration (i.e. the percentage of chemical energy in glucose that is transferred to ATP). *For help with answering this question, refer to Concept 9.4 (Campbell Textbook).

• How might the process of photosynthesis affect pH? Form a hypothesis.

PART 2: AEROBIC RESPIRATION IN YEAST

Optional Activity or Demonstration

This part of the lab investigates aerobic cellular respiration by Saccharomyces cerevisiae , also referred to as “baker’s yeast” and “brewer’s yeast.” Yeast is a unicellular fungus that can convert glucose into carbon dioxide and ATP when oxygen is present. Methylene blue dye can be used as an indicator for aerobic respiration in yeast. Aerobic respiration releases hydrogen ions and electrons that are picked up by the methylene blue dye, gradually turning the dye colorless. This redox reaction can be observed when viewing a wet mount of yeast and methylene blue under the compound light microscope. The mitochondria of yeast cells undergoing aerobic respiration will appear as a clear area surrounded by a ring of light blue cytoplasm. If cellular respiration is not taking place, the mitochondria will absorb the blue dye and will not turn colorless.

• Yeast (not quick rise)
• Distilled water
• Transfer pipette
• Methylene blue dye (in dropper bottle)
• Compound light microscope
• Microscope slide and cover slip
• Electronic balance, spatula, and weigh paper
• Prepare yeast suspension: Add 7 grams yeast to 50 ml warm tap water. Stir to mix. Save the yeast suspension for Part 3.
• Place a drop of yeast suspension on a clean microscope slide with a transfer pipette.
• Add one drop of methylene blue dye and place a cover slip on the microscope slide over the yeast suspension.
• Observe the yeast using the scanning objective lens. Use the coarse adjustment knob to focus on the yeast cells. Switch to the low power objective lens and then to the high power objective lens.
• In the circle below, draw several yeast cells undergoing aerobic respiration and several yeast cells not undergoing aerobic respiration. Label the cytoplasm and nucleus if visible.

PART 3: ALCOHOLIC FERMENTATION IN YEAST

This part of the lab investigates alcoholic fermentation by Saccharomyces cerevisiae , also referred to as “baker’s yeast” and “brewer’s yeast.” Yeast converts pyruvate from glycolysis into acetaldehyde, releasing carbon dioxide gas. Acetaldehyde is then enzymatically converted by the enzyme alcohol dehydrogenase into ethanol (Figure 2). In this lab, we will measure the accumulation of carbon dioxide released in the first enzymatic reaction as an indicator of the progression of fermentation.

Exercise 1: Investigating Different Concentrations of Yeast

• 4 identical saccharometers (glass fermentation hydrometer with either a 10-cm or a 15-cm vertical tube, Figure 3) / lab group
• Wax pencil or Sharpie
• 10% glucose solution
• Transfer pipettes
• Test tube rack
• 4 large (20 ml) test tubes or small Erlenmeyer flasks for larger volumes
• Large plastic tray
• Masking tape or lab tape
• Large weigh boat (4/group)
• Metric ruler
• Electronic balance
• Weigh paper
• Red food coloring (optional)

*Double these amounts if using saccharometers that have a 15-cm vertical tube. See table below

• Prepare yeast suspension: Add 7 grams yeast to 50 ml warm tap water. Stir to mix. Alternatively, you can use the yeast suspension from Part 2. Optional: Add a few drops of red food coloring to the yeast to increase contrast, allowing easier measuring of the height of yeast in saccharometers.
• Label 4 test tubes and 4 saccharometers # 1- 4. Use a transfer pipette to add the appropriate amount of glucose and distilled water listed in Table 2 to the corresponding labeled test tubes.
• Use a transfer pipette to add the appropriate amount of yeast solution listed in Table 1 to the corresponding labeled test tubes. It is important to work carefully and quickly after adding the yeast solution to the glucose and water.
• Carefully pour the contents of the test tubes into the correspondingly labeled saccharometer, ensuring that the solutions are well mixed.
• Carefully tilt the saccharometers to allow any air bubbles that are trapped in the arms of the vertical tube to escape.
• Begin the timer for the experiment and measure the size of any bubbles (in mm) that are trapped in the vertical arms of the saccharometers. Record this measurement as the 0 time point.
• Position the saccharometers on the large plastic tray, positioning them around a plastic weigh boat to catch any fermentation overflow that may occur.
• Carefully tape the saccharometers to the large plastic tray to prevent them from falling and breaking.
• Every 2 minutes measure and record the total amount of bubbles that accumulate in the top of the vertical arm of the saccharometer. Record the mm of carbon dioxide (bubble) measurements in Table 3.
• Continue recording the total amount of carbon dioxide released every 2 minutes for 20 minutes.
• After completing the experiment carefully carry the saccharometers to a sink for washing. Carry only one saccharometer at a time . Spill the yeast mixture into the sink and wash the saccharometer carefully and thoroughly. Return the saccharometer to the plastic tray, laying it down on its side when not in use.

Extension Activity: (Optional)

The results of this experiment can be presented graphically. The presentation of your data in a graph will assist you in interpreting your results. Based on your results, you can complete the final step of scientific investigation, in which you must be able to propose a logical argument that either allows you to support or reject your initial hypothesis.

• Graph your results using the data from Table 3.
• What is the dependent variable? Which axis is used to graph this data? ___________________________________________________________________
• What is your independent variable? Which axis is used to graph this data? ___________________________________________________________________

Exercise 2: Investigating the fermentation of different carbohydrates

• 4 identical saccharometers (glass fermentation hydrometer with either a 10-cm or a 15-cm vertical tube) / lab group
• 1% starch solution
• 10% sucrose solution
• Masking or lab tape
• Large weigh boat
• Prepare yeast suspension: Add 7 grams yeast to 50 ml warm tap water. Stir to mix. Optional: Add a few drops of red food coloring to the yeast to increase contrast, allowing easier measuring of the height of yeast in saccharometers.
• Label 3 test tubes and 3 saccharometers # 1- 3. Use a transfer pipette to add the appropriate amounts of carbohydrates and distilled water listed in Table 4 to the corresponding labeled test tubes.
• Use a transfer pipette to add 6 ml yeast solution to each of the test tubes. It is important to work carefully and quickly after adding the yeast solution to the carbohydrate.
• Every 2 minutes measure and record the total amount of bubbles that accumulate in the top of the vertical arm of the saccharometer. Record the mm of carbon dioxide (bubble) measurements in Table 5.
• Graph your results using the data from Table 5.
• Fermentation involves redox reactions . Explain what happens to electrons during a redox reaction and how this changes a molecule’s potential energy.
• Why did we add the Saccharomyces cerevisiae (baker's yeast) to the fermentation tubes? Specifically, what did the yeast provide to the fermentation mixture?
• What is the purpose of Saccharomyces cerevisiae (“baker’s yeast) in the bread-making process?
• We measured the formation of what end product to determine the fermentation rate? Name the end product that we measured.
• List two specific factors (as they relate to the experiment performed in our lab) that affect the rate of fermentation.

Practical Challenge Questions:

• What other variables could be investigated that might affect the rate of alcoholic fermentation by yeast?

Saccharomyces cerevisiae and its industrial applications

Affiliations.

• 1 Molecular Biology Laboratory, Department of Biological applications and Technology, University of Ioannina, Ioannina, Greece.
• 2 Genetics Laboratory, Department of Biological Applications and Technology, University of Ioannina, Ioannina, Greece.
• PMID: 32226912
• PMCID: PMC7099199
• DOI: 10.3934/microbiol.2020001

Saccharomyces cerevisiae is the best studied eukaryote and a valuable tool for most aspects of basic research on eukaryotic organisms. This is due to its unicellular nature, which often simplifies matters, offering the combination of the facts that nearly all biological functions found in eukaryotes are also present and well conserved in S . cerevisiae . In addition, it is also easily amenable to genetic manipulation. Moreover, unlike other model organisms, S . cerevisiae is concomitantly of great importance for various biotechnological applications, some of which date back to several thousands of years. S . cerevisiae 's biotechnological usefulness resides in its unique biological characteristics, i.e., its fermentation capacity, accompanied by the production of alcohol and CO 2 and its resilience to adverse conditions of osmolarity and low pH. Among the most prominent applications involving the use of S . cerevisiae are the ones in food, beverage -especially wine- and biofuel production industries. This review focuses exactly on the function of S . cerevisiae in these applications, alone or in conjunction with other useful microorganisms involved in these processes. Furthermore, various aspects of the potential of the reservoir of wild, environmental, S . cerevisiae isolates are examined under the perspective of their use for such applications.

Keywords: Baker's yeast; Saccharomyces cerevisiae; bioethanol; cocoa fermentation; non-Saccharomyces yeast; wine yeast.

© 2020 the Author(s), licensee AIMS Press.

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Article Contents

Sequence and structure of telomeric regions, telomeric chromatin, the capping function, telomere replication, transcription at telomeres, telomeres and nuclear organization, acknowledgements, literature cited.

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Everything You Ever Wanted to Know About Saccharomyces cerevisiae Telomeres: Beginning to End

Present address: Department of Microbiology and Infectious Diseases, Faculty of Medicine, Université de Sherbrooke, Québec, J1E 4K8 Canada.

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Raymund J Wellinger, Virginia A Zakian, Everything You Ever Wanted to Know About Saccharomyces cerevisiae Telomeres: Beginning to End, Genetics , Volume 191, Issue 4, 1 August 2012, Pages 1073–1105, https://doi.org/10.1534/genetics.111.137851

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The mechanisms that maintain the stability of chromosome ends have broad impact on genome integrity in all eukaryotes. Budding yeast is a premier organism for telomere studies. Many fundamental concepts of telomere and telomerase function were first established in yeast and then extended to other organisms. We present a comprehensive review of yeast telomere biology that covers capping, replication, recombination, and transcription. We think of it as yeast telomeres—soup to nuts.

Eukaryotic chromosomes are linear DNA molecules with physical ends, called telomeres. It is estimated that as many as 10,000 DNA damaging events occur each day in every cell in the human body ( Loeb 2011 ). Perhaps the most hazardous of these events are double-stranded DNA breaks (DSBs), which create chromosome ends at internal sites on chromosomes. Thus, a central question is how cells distinguish natural ends or telomeres from DSBs. Telomeres on one hand are essential for the stable maintenance of chromosomes: they must be retained—they cannot be lost by degradation or fused with other ends. Exactly the opposite applies to DSBs: they must be repaired by either homologous or nonhomologous recombination, and this repair often involves regulated degradation of the DSB. In fact, unrepaired DSBs lead to cell cycle arrest to provide time for their repair. Capping is used to describe how telomeres prevent their degradation and recombinational fusion ( Muller 1938 ; McClintock 1939 ). Perhaps as a consequence of capping, the regions near telomeres are gene poor. In many organisms, telomere proximal genes are subjected to a special type of transcriptional regulation called telomere position effect (TPE), where transcription of genes near telomeres is metastably repressed. Another key role for telomeres is to provide the substrate for a special mechanism of replication. Telomere replication is carried out by telomerase, a specialized ribonucleoprotein complex that is mechanistically related to reverse transcriptases ( Greider and Blackburn 1987 ).

The biology of telomerase has broad ramifications for human health and aging. Therefore, the discovery of telomerase and studies on telomere capping by Elizabeth Blackburn, Carol Greider, and Jack Szostak, were honored with the 2009 Nobel Prize in Medicine. All three prize winners carried out research in single-cell organisms, including budding yeast. As described in this review, Saccharomyces cerevisiae continues to be a premier organism for telomere research.

Like most organisms whose telomeres are maintained by telomerase, the ends of S. cerevisiae chromosomes consist of nonprotein coding repeated DNA ( Figure 1A ). There are 300 ± 75 bp of simple repeats, typically abbreviated C 1-3 A/TG 1-3 . S. cerevisiae telomeric DNA is unusual, although not unique, in being heterogeneous. This sequence heterogeneity is due to a combination of effects: in a given extension cycle, only a portion of the RNA template is used and/or the RNA template and telomeric DNA align in different registers in different extension cycles ( Forstemann and Lingner 2001 ). The heterogeneity of yeast telomeric DNA is experimentally useful as it makes it possible to distinguish newly synthesized from preexisting telomeric DNA ( Wang and Zakian 1990 ; Teixeira et al. 2004 ). When many copies of the same telomere are sequenced from a given colony, the exact sequence of the internal half is the same from telomere to telomere while the terminal half turns over much more rapidly ( Wang and Zakian 1990 ). Thus, under most conditions, only the terminal half of the telomere is subject to degradation and/or telomerase lengthening. These repeats in conjunction with the proteins that bind them are necessary and sufficient for telomere function.

DNA structure and major protein components of telomeres. (A) DNA arrangement at telomeres indicating the subtelomeric X and Y′ elements as well as the terminal repeat sequences. Red strand, G-rich strand with 3′ overhanging end and blue strand, C-rich strand with 5′ end. Core X and STR ( s ub t elomeric r epeated elements; Louis et al. 1994 ) represent subareas in the X element. (B) Proteins are schematically positioned on the telomere drawing and the identity of the symbols explained on the bottom. Open circles represent nucleosomes (not to scale).

As in most eukaryotes, the very ends of S. cerevisiae chromosomes are not blunt ends. Rather the G-rich strand extends to form a 3′ single strand tail or G tail ( Figure 1A ). Throughout most of the cell cycle, G tails are short, only 12 to 15 nucleotides (nt) ( Larrivee et al. 2004 ). However, G tails are much longer, ≥30–100 nt in size, during a short period in late S/G2 phase when they can be detected readily by nondenaturing Southern hybridization ( Wellinger et al. 1993a , b ). Long G tails are not due solely to telomerase-mediated lengthening as they are seen in late S/G2 phase even in telomerase-deficient cells ( Wellinger et al. 1996 ; Dionne and Wellinger 1998 ). G tails are generated by cell-cycle–regulated C-strand degradation, which is dependent on the kinase activity of Cdk1p ( Cdc28p ; Frank et al. 2006 ; Vodenicharov and Wellinger 2006 ). This generation is obligatorily linked to semiconservative DNA replication, which occurs prior to C-strand degradation ( Wellinger et al. 1993a ; Dionne and Wellinger 1998 ).

Also similar to most organisms, yeast telomeric regions contain subtelomeric, middle, repetitive elements, often called TAS elements (telomere associated sequences; Figure 1A and http://www.nottingham.ac.uk/biology/people/louis/telomere-data-sets.aspx ). S. cerevisiae has two classes of TAS elements, X and Y′. Y′ is found in zero to four tandem copies immediately internal to the telomeric repeats ( Chan and Tye 1983a , b ). About half of the telomeres in a given strain lack Y′, and the identity of Y′-less telomeres differs from strain to strain ( Horowitz et al. 1984 ; Zakian et al. 1986 ). Y′ comes in two sizes, Y′ long (6.7 kb) and Y′ short (5.2 kb) ( Chan and Tye 1983a , b ), which differ from each other by multiple small insertions/deletions ( Louis and Haber 1992 ). X is present at virtually all telomeres and is much more heterogeneous in sequence and size. Although X is found on all telomeres, it is composed of a series of repeats, many of which are present on only a subset of telomeres. When telomeres contain both X and Y′, X is centromere proximal to Y′. Short tracts of telomeric DNA are sometimes found at the Y′–X and Y′–Y′ junctions ( Walmsley et al. 1984 ; Figure 1A ).

Subtelomeric regions are dynamic, undergoing frequent recombination ( Horowitz et al. 1984 ; Louis and Haber 1990 ). Moreover, subtelomeric repeats diverge rapidly even among related yeast strains ( Chan and Tye 1983a , b ). X and Y′ both contain potential replication origins or ARS elements ( a utonomously r eplicating s equences) whose presence probably contributes to the dynamic nature of subtelomeric regions. X and Y′ have binding sites for multiple transcription factors, whose identity differs from telomere to telomere ( Mak et al. 2009 ). Because the sequence of subtelomeric regions and the proteins that bind them are variable, their presence can confer distinct behaviors on individual telomeres.

Whereas complete loss of the C 1-3 A/TG 1-3 telomeric repeats from a chromosome end results in extremely high loss rates for the affected chromosome, chromosomes that lack X and Y′ at one ( Sandell and Zakian 1993 ) or even both ends have normal mitotic stability and go through meiosis with ease (S. S. Wang and V. A. Zakian, unpublished results). However, Y′ amplification by recombination can provide a telomere maintenance function to cells lacking telomerase ( Lundblad and Blackburn 1993 ). Y′ can also move by a transposition-like RNA-mediated process ( Maxwell et al. 2004 ).

Ty5 is a transposable element found only in heterochromatin, including subtelomeric DNA. The number of Ty5 elements varies from strain to strain. The S288C strain has eight Ty5 insertions: six near telomeres and two near the HMR silent mating type locus ( Zou et al. 1995 ). This chromosomal distribution is quite different from that of other classes of Ty elements, which are found close to tRNA genes. Movement of Ty5 to telomeres and HMR is regulated by the targeting domain of the Ty5-encoded integrase that interacts directly with Sir4p , one of the silencing proteins found in telomeric regions ( Xie et al. 2001 ).

Telomere binding proteins: direct binders and associated proteins

Table 1 presents a list of proteins that act at telomeres, divided into functional categories. Many of these proteins have multiple roles and could be listed in more than one category. The protein complexes associated with telomeres can be subdivided according to the three regions to which they bind: (A) subtelomeric areas containing Y′ and X, (B) double-stranded terminal repeat area, and (C) the 3′ G tail ( Figure 1 ).

Major genes affecting Saccharomyces cerevisiae telomeres

See text for details and references; although many genes are involved in more than one process, each is listed here under only one heading. Essential/nonessential refers to viability, not telomere maintenance. aa, number of amino acids; MW sizes are from the SGD website http:/www.yeastgenome.org/ .

(A) Subtelomeric regions are classified into XY′ and X-only ends. While most of the subtelomeric DNA is likely organized in nucleosomes ( Wright et al. 1992 ), the cores of the X elements have a low histone content, and nucleosomes near the Xs have histone modifications characteristic of silenced regions such as unacetylated lysine 16 on histone H4 (H4K16) ( Zhu and Gustafsson 2009 ). Consistent with these data, the NAD + -dependent histone deacetylase Sir2p , a H4K16 deacetylase, as well as Sir3p , are also enriched over X repeats ( Imai et al. 2000 ; Zhu and Gustafsson 2009 ; Takahashi et al. 2011 ), and the area around many X elements is transcriptionally silent ( Pryde and Louis 1999 ). The X elements on XY′ telomeres are organized similarly as on X-only telomeres ( Takahashi et al. 2011 ). However, on the distal Y′ elements, the overall density of nucleosomes as well as the occurrence of H4K16ac is similar to euchromatic areas. In addition, Sir2p and Sir3p are not detected in this region ( Zhu and Gustafsson 2009 ; Takahashi et al. 2011 ). Collectively, these data suggest that on X-only telomeres, the subtelomeric DNA elements are organized into silenced chromatin that demarcates the terminal area from more internal regions. On XY′ telomeres, the distal Y′ area is organized into chromatin that resembles that of expressed areas with the X element, again acting as a demarcation zone ( Fourel et al. 1999 ; Pryde and Louis 1999 ; Takahashi et al. 2011 ). Thus, emerging evidence points toward differences in behavior depending on subtelomeric repeat content and perhaps even individual chromosomal context.

γ-H2A, which is generated by Mec1p / Tel1p -dependent phosphorylation, is also enriched in subtelomeric chromatin ( Kim et al. 2007 ; Szilard et al. 2010 ). Since this modification normally marks damaged DNA, which activates checkpoints, it is unclear why it persists on telomeres and whether its occurrence has functional consequences. Finally, nucleosomes in certain areas within subtelomeric DNA contain the histone H2A variant H2A.Z. Nucleosomes containing H2A.Z often mark gene promoters for efficient activation and perhaps also function as heterochromatin–euchromatin boundary elements ( Guillemette et al. 2005 ; Albert et al. 2007 ).

Remarkably, there are a few precise matches to the vertebrate telomeric repeat sequence, (TTAGGG) n , within X and Y′ DNA, and the essential transcription factor Tbf1p ( Brigati et al. 1993 ) binds these repeats in vitro ( Liu and Tye 1991 ) and in vivo ( Koering et al. 2000 ; Preti et al. 2010 ; Figure 1A ). This Tbf1p binding is functionally significant as it participates in telomerase recruitment ( Arneric and Lingner 2007 ). Tbf1p can also provide Rap1p -independent capping on artificial telomeres consisting solely of vertebrate repeats ( Alexander and Zakian 2003 ; Berthiau et al. 2006 ; Bah et al. 2011 ; Ribaud et al. 2011 ; Fukunaga et al. 2012 ). The boundary between subtelomeric DNA and telomeric repeats appears special as it is preferentially accessible to DNases, restriction enzymes, and DNA modifying enzymes ( Conrad et al. 1990 ; Gottschling 1992 ; Wright et al. 1992 ; Wright and Zakian 1995 ). This behavior suggests a short stretch of DNA that is not strongly associated with proteins. Given this property, limited nuclease digestion can release the distalmost portion of chromosomes containing all telomeric repeat DNA in a soluble and protein bound form called the telosome ( Conrad et al. 1990 ; Wright et al. 1992 ). This telosome appears to be histone free and should contain all telomeric repeat binding proteins ( Wright and Zakian 1995 ).

(B) Double-stranded telomeric repeat DNA contains high-affinity Rap1p binding sites every ∼20 bp, which correlates well with the estimate that in vitro assembled Rap1 telomeric DNA contains 1 bound Rap1p molecule in 18 (±4) bp ( Gilson et al. 1993 ; Ray and Runge 1999a , b ; Figure 1B ). Therefore, given an average telomere length of 300 bp, individual telomeres are probably covered by 15–20 Rap1p molecules ( Wright and Zakian 1995 ). Rap1p is an abundant nuclear protein of 827 amino acids that was first discovered by its ability to repress or activate gene expression ( r epressor a ctivator p rotein 1) ( Shore and Nasmyth 1987 ). Indeed, given its abundance and the number of telomeric Rap1 binding sites, most (∼90%) Rap1p is not telomere associated. DNA consensus sites for Rap1p binding are quite heterogeneous, but those within telomeric DNA are among the highest affinity sites ( Buchman et al. 1988 ; Longtine et al. 1989 ; Lieb et al. 2001 ). Genetic evidence, chromatin immunoprecipitation (ChIP) and in vivo localization leave little doubt that Rap1p covers telomeric DNA in living cells ( Conrad et al. 1990 ; Lustig et al. 1990 ; Wright and Zakian 1995 ; Gotta et al. 1996 ; Bourns et al. 1998 ). Indeed, the amount of telomere bound Rap1p , along with its binding partners Rif1 /2 somehow establishes the actual telomere length ( Marcand et al. 1997 ; Levy and Blackburn 2004 ).

Although studied extensively, the functional domains for Rap1p are not completely defined ( Figure 2 ). Loss of up to 340 amino acids from the N-terminal region, which has a BRCT domain, is well tolerated ( Moretti et al. 1994 ; Graham et al. 1999 ). However, the double myb domain DNA binding module in the middle of Rap1p is essential for all functions of the protein, including those at telomeres ( Graham et al. 1999 ). For example, temperature-sensitive alleles of RAP1 can cause telomere shortening and telomere-bound Rap1p is required to prevent telomere fusions ( Conrad et al. 1990 ; Lustig et al. 1990 ; Marcand et al. 2008 ). The C terminus of Rap1p is key for its telomere functions as both the silencing proteins Sir3p / Sir4p and the length regulatory Rif1p / Rif2p bind this region ( Hardy et al. 1992a , b ; Moretti et al. 1994 ; Buck and Shore 1995 ; Wotton and Shore 1997 ; Figure 2 ).

Overall domain organizations and interaction areas for major telomeric proteins. Shown are Rap1p, members of the Cdc13 complex, and three protein subunits of the telomerase holoenzyme. Due to the paucity of information for Rif1p or Rif2p, they are omitted. For details on domain definitions, see text. Known interaction domains with other proteins, RNA, or DNA are indicated with a double arrow. Below the proteins, numbers define amino acid positions. Small up arrow indicates known amino acid modifications that affect functions and the red step on Est3p denotes a required +1 frameshift in protein translation.

Another key telomere binding protein is the yeast Ku complex (referred to as YKu), composed of Yku70p and Yku80p ( Boulton and Jackson 1996 ; Porter et al. 1996 ; Gravel et al. 1998 ). Given that YKu is essential for DNA repair via non-homologous end joining (NHEJ) and telomeres are protected from NHEJ, the association of YKu with telomeres is counterintuitive. Nevertheless, this association is critical for telomere function ( Gravel et al. 1998 ), not only in yeast but in many organisms ( Fisher and Zakian 2005 ). It is still uncertain where and how Yku associates with chromosomal termini, but there is evidence for two pools, one bound directly to telomeric DNA in a mode similar to that used for the nonspecific DNA end binding in NHEJ and another being associated with telomeric chromatin via a Yku80p – Sir4p interaction ( Martin et al. 1999 ; Roy et al. 2004 ). ChIP experiments suggest a Sir4p -independent association of YKu with some, but not all, core X sequences, and those bound areas also correlate with a high level of transcriptional and recombination repression ( Marvin et al. 2009a , b ). Furthermore, given the ability of YKu to associate with telomerase RNA, it has also been suggested that YKu functions to recruit telomerase to telomeres ( Peterson et al. 2001 ; Stellwagen et al. 2003 ; Fisher et al. 2004 ; Chan et al. 2008 ) and/or telomerase trafficking from the cytoplasm to the nucleus ( Gallardo et al. 2008 , 2011 ). Consistent with that proposal, YKu association with telomeres is independent of its association with TLC1 RNA and occurs throughout the cell cycle ( Fisher et al. 2004 ).

(C) The essential Cdc13p specifically and avidly binds single-stranded TG 1-3 DNA of at least 11 nt in vitro ( Lin and Zakian 1996 ; Nugent et al. 1996 ; Hughes et al. 2000 ) and is associated with telomeres in vivo ( Bourns et al. 1998 ; Tsukamoto et al. 2001 ). The DNA binding domain (DBD) of Cdc13p is confined to amino acids 497–694 of this 924-amino-acid protein ( Figure 2 ), and this domain reproduces the in vitro DNA binding characteristics of the full-length protein ( Hughes et al. 2000 ). Furthermore, structure determinations of this DBD bound to a telomeric G strand provide a model for the very high affinity and specificity of this association ( Mitton-Fry et al. 2002 , 2004 ). The relatively large N-terminal region (amino acids 1–455) may contain two OB fold domains plus a region defining an interaction with Est1p that is involved in telomerase recruitment (recruitment domain, RD) ( Nugent et al. 1996 ; Pennock et al. 2001 ; Figure 2 ). A direct Est1 –RD interaction is shown by in vitro experiments ( Wu and Zakian 2011 ). Finally, the N-terminal or first OB fold domain is important for an interaction with Pol1p and for Cdc13p dimerization ( Grandin et al. 2000 ; Qi and Zakian 2000 ; Gelinas et al. 2009 ; Sun et al. 2011 ).

Two other essential proteins with genetic and biochemical interactions with Cdc13p , namely Stn1p and Ten1p , also have a potential for direct interactions with the single-stranded 3′ overhangs ( Grandin et al. 1997 , 2001 ; Gao et al. 2007 ). The three-member protein complex composed of C dc13p/ S tn1p/ T en1p has been referred to both as the CST complex or telomeric RPA. Herein, we refer to it as the Cdc13 complex. There are several structural similarities between the three members of the Cdc13 complex and the three proteins making up replication protein A (RPA) ( Gao et al. 2007 ; Gelinas et al. 2009 ), and at least one essential OB fold domain can be swapped between Rpa2p and Stn1p ( Gao et al. 2007 ).

Stn1p and Ten1p may also act independently of Cdc13p . For example, a Stn1p / Ten1p complex when overexpressed can act as a chromosome cap in the absence of Cdc13p ( Petreaca et al. 2006 , 2007 ; Sun et al. 2009 ). Stn1p can be divided roughly into two parts, an N-terminal and a C-terminal domain ( Petreaca et al. 2006 , 2007 ; Puglisi et al. 2008 ; Figure 2 ). The N-terminal domain, which is necessary for its interaction with Ten1p , is required for its essential functions ( Petreaca et al. 2007 ; Puglisi et al. 2008 ). The C-terminal domain interacts with both Cdc13p and Pol12p , the latter protein a subunit of the DNA Polα complex that carries out lagging strand DNA replication ( Grossi et al. 2004 ).

Telomere dedicated proteins vs. proteins doing double duty

Remarkably, the majority of telomeric proteins have both telomeric and nontelomeric functions ( Table 1 ). For example, both Rap1p and Tbf1p are essential to regulate expression of a large number of genes, many of which are among the most highly transcribed genes in the genome ( Pina et al. 2003 ; Preti et al. 2010 ). The Rap1p -associated proteins Sir2p , Sir3p , and Sir4p promote transcriptional silencing not only at telomeres but also at the silent mating type or HM loci ( Rusche et al. 2003 ), and Rif1p has roles in establishing heterochromatin elsewhere than just at telomeres ( Hardy et al. 1992b ; Buck and Shore 1995 ; Buonomo 2010 ). The YKu complex is essential for NHEJ, in particular during G1 phase of the cell cycle (reviewed in Daley et al. 2005 ). The telomerase regulator Pif1p affects maintenance of mitochondrial DNA and replication of nontelomeric loci with the potential to form G-quadruplex structures ( Foury and Kolodynski 1983 ; Schulz and Zakian 1994 ; Ivessa et al. 2000 ; Ribeyre et al. 2009 ; Paeschke et al. 2011 ). Taken together, at least for budding yeast, it looks as if the proteins important for telomere function by and large are doing double duty.

How many more genes affect telomere biology?

It is not surprising that a large number of additional genes affect telomere length as genes with general roles in DNA replication, recombination, intra S checkpoint, protein and RNA synthesis pathways would be expected to affect them ( Dahlseid et al. 2003 ; Mozdy et al. 2008 ). Indeed, two systematic screens of the deletion collection of nonessential genes confirmed this idea ( Askree et al. 2004 ; Gatbonton et al. 2006 ). Of some concern, the gene sets from the two screens show little overlap, and it is not yet clear how many of the genes act directly.

Screens for suppressors of telomere-capping defects also yielded numerous new interactions ( Addinall et al. 2008 , 2011 ). For example, members of the KEOPS complex ( CGI121 , KAE1 , BUD32 , and GON7 ) were linked to telomere biology because they were identified by their ability to suppress the growth defect of cells harboring the cdc13 -1 allele incubated at slightly elevated temperatures ( Downey et al. 2006 ). KEOPS genes were also identified via an unrelated screen looking for suppressors of a splicing defect ( Kisseleva-Romanova et al. 2006 ), and one member of the KEOPS complex is linked to chromosome segregation ( Ben-Aroya et al. 2008 ). It appears now that the primary function of the KEOPS complex is to add a specific base modification to certain tRNAs (t6A addition; Srinivasan et al. 2011 ). Similarly, SUA5 , a gene first identified as a translational suppressor and then linked to telomere biology ( Na et al. 1992 ; Meng et al. 2009 ) is required for the same tRNA modification as the KEOPS complex ( Lin et al. 2010 ; Srinivasan et al. 2011 ). How this t6A tRNA modifying activity links with telomere biology is still a puzzle. In summary, with rare exceptions, we think it likely that all genes affecting yeast telomeres have been identified and would not be surprised if many of the genes identified by genome-wide approaches act indirectly.

Classical chromosome capping

Arguably the most important function of a telomere is that of providing protection to the end of the chromosome. This capping function is the property that prompted chromosome researchers in the 1930s to name the ends of chromosomes telomeres ( Muller 1938 ; McClintock 1939 ). Classically, the capping function prevents telomeres from being subject to DNA repair by homologous recombination or NHEJ. More recently, the capping function has expanded to include the concept of protecting telomeres from checkpoints as loss of a single telomere elicits a Rad9p -dependent cell cycle arrest ( Sandell and Zakian 1993 ). Loss of these capping functions can be determined by monitoring the integrity of both strands of telomeric DNA, presence of fused chromosome ends, and/or cell cycle arrest. The conservation among eukaryotes of the underlying structure of telomeres, duplex telomeric DNA with G-rich 3′ overhangs and corresponding sequence-specific duplex and single-strand DNA binding proteins, suggests that the mechanisms of capping are based on conserved principles.

The earliest demonstration that Cdc13p functions in chromosome capping was the discovery that in cells with a temperature-sensitive cdc13 -1 allele incubated at elevated temperatures, telomeres are degraded in a strand-specific manner such that their C strands are lost for many kilobases ( Garvik et al. 1995 ). In addition, at nonpermissive temperatures, cdc13 -1 cells arrest at the G2/M boundary of the cell cycle in a RAD9 -dependent fashion ( Weinert and Hartwell 1993 ). These phenotypes also occur in cdc13 Δ cells ( Vodenicharov and Wellinger 2006 ). Therefore, cells lacking Cdc13p display the two central hallmarks of telomere uncapping, unstable chromosome ends, and activation of a DNA damage checkpoint. Cdc13p undergoes cell cycle phase-specific post-translational modifications, including phosphorylation and SUMOylation that may affect capping ( Tseng et al. 2006 ; Li et al. 2009 ; Hang et al. 2011 ). Genetic and biochemical data indicate that these capping activities of Cdc13p involve Stn1p and Ten1p , both of which are also essential for capping ( Grandin et al. 1997 , 2001 ; Gao et al. 2007 ; Petreaca et al. 2007 ; Xu et al. 2009 ).

An inducible degron allele of Cdc13p combined with cell cycle synchrony experiments demonstrated that the Cdc13 complex is only required for capping during late S and G2/M phases, but not in G1 or early S ( Vodenicharov and Wellinger 2006 , 2007 , 2010 ). One might speculate that replication through the telomere would disrupt its capping function and therefore capping must be reassembled thereafter, creating a time-restricted situation of enhanced requirement for capping and hence Cdc13 complex function. This proposal is in line with the fact that during telomere replication, CDK-dependent end processing is at its peak ( Ira et al. 2004 ; Frank et al. 2006 ; Vodenicharov and Wellinger 2006 ). However, given that members of the Cdc13 complex interact with components of the lagging strand machinery, it is also possible that the capping functions of the Cdc13 complex are directly associated with the passage of the replication fork ( Nugent et al. 1996 ; Qi and Zakian 2000 ; Grossi et al. 2004 ; Vodenicharov and Wellinger 2010 ). In this context it is noteworthy that Cdc13p , although very sequence specific, does not require a physical 3′ end for its binding, as it can bind single-strand TG 1-3 DNA even if the telomeric DNA is on a circular plasmid ( Lin and Zakian 1996 ; Nugent et al. 1996 ). It thus remains unclear whether the C-strand–specific degradation of telomeres observed when Cdc13 complex-mediated capping is hampered is due to problems at the physical ends or problems associated with terminating replication of telomeric repeats ( Figure 3 ; Anbalagan et al. 2011 ).

Preventing DNA damage checkpoint signaling at telomeres. Schematic of hypotheses for how DNA damage checkpoint signaling is prevented (A) during the passage of the replication fork through the double-stranded telomeric repeat area and (B) after having passed the end. Symbols are as in Figure 1 .

Outside S phase, Rap1p is critical for capping. Rap1p with C terminus-associated Rif2p , and to a much lesser extent Rif1p , are important for preventing telomere fusions and limiting end resection ( Marcand et al. 2008 ; Bonetti et al. 2010 ; Vodenicharov and Wellinger 2010 ). Furthermore, Rif2p (but not Rif1p ) has a prominent role in preventing the association of Tel1p /MRX complex to telomeres ( Hirano et al. 2009 ; Bonetti et al. 2010 ). MRX is a heterotrimeric complex composed of Mre11p , Rad50p , and Xrs2p that serves important roles in both DSB recognition, telomere capping, and checkpoint activation ( Boulton and Jackson 1998 ; Nugent et al. 1998 ; Ritchie and Petes 2000 ; D’Amours and Jackson 2001 ; Grenon et al. 2001 ). Most likely there is a nucleolytic activity associated with the complex ( Llorente and Symington 2004 ), and it appears the complex also has the capacity to hold broken chromosome ends in proximity for eventual repair ( Kaye et al. 2004 ; Lobachev et al. 2004 ). On the other hand, Rif1p , and to a much lesser extent Rif2p , is important to maintain viability in cells where CDC13 capping is compromised ( Addinall et al. 2011 ; Anbalagan et al. 2011 ). Thus, Rap1p and the associated Rif1p and Rif2p proteins have important capping functions outside of S phase with Rif1p and Rif2p making specific and separable contributions to this capping.

Finally, Yku affects capping in G1 phase ( Vodenicharov and Wellinger 2007 , 2010 ; Bonetti et al. 2010 ) as telomeres in ykuΔ cells are resected at this time, even when bound by the Cdc13 complex. However, the G1 resection in ykuΔ cells is much more modest than, for example, the resection that occurs during late S phase in cdc13 -1 cells at elevated temperatures, and this limited resection does not activate a DNA damage checkpoint ( Bonetti et al. 2010 ; Vodenicharov and Wellinger 2010 ).

It is unclear whether telomerase has a capping function that is independent from its telomere elongation activity. Physical assays do not reveal increased end degradation in tlc1 Δ48 or yku80 -135i cells ( Vodenicharov and Wellinger 2010 ), mutations that result in reduced Est2p telomere binding ( Fisher et al. 2004 ). However, cdc13 -1 cells that also carry either the tlc1 Δ48 or yku80 -135i mutation are more temperature sensitive than cdc13 -1 cells, suggesting that capping is compromised further by reduced Est2p telomere binding in these backgrounds ( Vega et al. 2007 ). Moreover, cells lacking telomerase and the recombination protein Rad52p lose telomeric DNA more rapidly than if they lack telomerase alone ( Lundblad and Blackburn 1993 ). One explanation for these data are that telomerase protects ends from recombinational lengthening ( Lee et al. 2007 ).

Alternative ways of capping

While the Cdc13p -mediated capping of chromosome ends is essential, situations of telomere capping without Cdc13p have been described. In all such cases, chromosomes still end in canonical terminal TG 1-3 sequences and in some cases, the repeat sequences are still maintained by telomerase ( Larrivee and Wellinger 2006 ; Petreaca et al. 2006 ; Zubko and Lydall 2006 ; Dewar and Lydall 2010 ). In one particular case, capping requires the DNA polymerase α-associated Pol12p and overexpression of both an N-terminal part of Stn1p and Ten1p ( Petreaca et al. 2006 ). In another case, cdc13 Δ cells can be obtained by first deleting key genes involved in exonucleolytic degradation of DNA ends ( EXO1 , RAD24 , and SGS1 ) and DNA damage checkpoints ( RAD9 and PIF1 ) ( Zubko and Lydall 2006 ; Dewar and Lydall 2010 ; Ngo and Lydall 2010 ). In these cases, telomeres are still maintained by telomerase, if homologous recombination is impossible due to a deletion of RAD52 ( Zubko and Lydall 2006 ). Lastly, if telomere repeat maintenance is already accomplished by recombination, as in the survivors that arise in telomerase-deficient cells, then loss of Cdc13p can be tolerated in a small subset of cells. The fact that only a minor fraction of the culture survives suggests that additional events are required to maintain telomeres in such cells ( Larrivee and Wellinger 2006 ).

DNA structures can also provide an alternative mode of capping. For example, cells that lack both major pathways for telomeric repeat maintenance, i.e. , telomerase and homologous recombination, and that are also deficient in Exo1p , a 5′ to 3′ single-stranded exonuclease that processes DSBs, can divide and form colonies ( Maringele and Lydall 2004b ). Chromosomes in these survivor cells do not end in telomeric repeats but rather in DNA palindromes distal to the first essential gene on each chromosome arm.

Crosstalk between DNA damage checkpoint activation and DNA repair

Given that capping protects telomeres from repair and checkpoint activation, it seemed logical to think that proteins involved in DNA repair and checkpoints would not act at telomeres. Paradoxically, many checkpoint and DNA repair proteins associate with telomeres and contribute in important ways to telomeric functions, including capping. For example, the yeast YKu complex, which is critical for NHEJ, is telomere associated ( Gravel et al. 1998 ), and in its absence, telomeres are very short and have long G tails throughout the cell cycle ( Boulton and Jackson 1996 ; Porter et al. 1996 ; Gravel et al. 1998 ; Polotnianka et al. 1998 ). YKu contributes not only to capping but also protects telomeres from recombination, mediates nuclear import and/or retention of telomerase RNA, promotes TPE and telomere tethering ( Polotnianka et al. 1998 ; Peterson et al. 2001 ; Stellwagen et al. 2003 ; Hediger et al. 2006 ; Ribes-Zamora et al. 2007 ; Gallardo et al. 2008 ; Marvin et al. 2009a ) and is involved in telomere replication ( Cosgrove et al. 2002 ; Gravel and Wellinger 2002 ).

Mec1p , the most important checkpoint kinase in yeast, has a minor role in telomere length regulation ( Ritchie et al. 1999 ). Consistent with this, Mec1p binding is only detected at ultrashort telomeres that are probably already nonfunctional ( Abdallah et al. 2009 ; McGee et al. 2010 ; Hector et al. 2012 ). In fact, Cdc13p inhibits Mec1p binding to a DSB ( Hirano and Sugimoto 2007 ). Moreover, Mec1p prevents telomere formation at DSBs by phosphorylation of Cdc13p , which inhibits Cdc13p association with the DSB ( Zhang and Durocher 2010 ; Ribaud et al. 2011 ). In addition, Mec1p phosphorylation of Pif1p inhibits telomere addition to DSBs ( Makovets and Blackburn 2009 ). Normally, association of Mec1p to DSBs occurs after end processing and by binding to single-stranded DNA via the replication protein A heterotrimer (RPA) and Ddc2p ( Zou and Elledge 2003 ). An important issue is whether or not RPA binds the single-stranded G tails generated at the end of S phase ( Figure 3 ). RPA is detected transiently at telomeres at this time ( Schramke et al. 2003 ; McGee et al. 2010 ), but this binding could be explained by the RPA that associates with telomeres during semiconservative replication ( McGee et al. 2010 ). Mec1p binding is not detected at this time, suggesting that Cdc13p prevents RPA binding so that Mec1p -mediated DNA damage signaling is not elicited by the telomeric single-stranded G tails ( Figure 3 ; Gao et al. 2007 ; Gelinas et al. 2009 ; McGee et al. 2010 ).

Although Tel1p associates with DSBs ( Nakada et al. 2003 ; Shima et al. 2005 ), it has only minor functions in DNA repair. Rather, its major function is telomere length maintenance. Tel1p binds telomeres ( Bianchi and Shore 2007b ; Hector et al. 2007 ; Sabourin et al. 2007 ) via an interaction with the Xrs2p subunit of MRX. Indeed, Tel1p interacts preferentially with short telomeres and is thought to be involved in telomerase recruitment. However, in contrast to its binding at a DSB, its association with short telomeres does not elicit a checkpoint response, a difference that is not fully understood.

Other experiments involving the fate of DSBs made next to telomeric DNA emphasize the interconnections between telomeric DNA and checkpoints. For example, there is some evidence that a tract of telomeric DNA can affect cell cycle progression when it is adjacent to a DSB ( Michelson et al. 2005 ; but note conflicting data in Hirano and Sugimoto 2007 ). In these experiments, an inducible DSB is created such that one of the ends exposes telomeric repeats and the other does not. The exposure of telomeric DNA does not affect the initial checkpoint response, but it allows for an accelerated recovery from the checkpoint arrest and resumption of cell cycle progression ( Michelson et al. 2005 ). Intriguingly, this effect could be dependent on keeping the two ends created by the break in close proximity with Rif proteins at the DSB contributing to dampening of the checkpoint response ( Ribeyre and Shore 2012 ).

Regulated resection

Given that G tails are an essential feature of chromosome ends, they must be regenerated after DNA replication. This processing is particularly a problem for the end replicated by the leading strand polymerase, which is predicted to produce a blunt end ( Figure 4A ). This problem is solved by postreplication C-strand degradation ( Wellinger et al. 1996 ), which remarkably depends on the same genes that resect the ends of DSBs to generate the 3′ single-strand tails that initiate homologous recombination. This congruence is surprising as one of the key functions of telomeres is to prevent DNA repair at natural ends. Recent insights suggest a solution to this conundrum. C-strand resection at telomeres is strongly dependent on Sgs1p or Sae2p . Sgs1p is a 3′ to 5′ RecQ family DNA helicase, while Sae2p is an endonuclease whose phosphorylation by Cdk1 is critical for its activity ( Huertas et al. 2008 ; Bonetti et al. 2009 ). Indeed, Cdk1 activity is required for cell-cycle–dependent telomere resection ( Frank et al. 2006 ; Vodenicharov and Wellinger 2006 ). The MRX complex acts in the same pathway as Sae2p to generate G tails. Although G tails are shorter in mre11 Δ cells, they still increase in length in late S/G2 phase in this background ( Larrivee et al. 2004 ). However, the nuclease activity of Mre11 is not required to generate G tails ( Tsukamoto et al. 2001 ). Thus, MRX is not as critical as Sae2p for G-tail generation. Likewise, in sae2 Δ cells, C-strand degradation is not eliminated, as there is the second and partially overlapping degradation pathway that requires Sgs1p ( Bonetti et al. 2009 ). The fact that multiple nucleases are involved in telomeric end processing is also true at DSBs ( Zubko et al. 2004 ; Gravel et al. 2008 ; Mimitou and Symington 2008 ; Zhu et al. 2008 ). Indeed, on a DSB, a slow MRX-dependent and restrained resection soon gives way to fast and extensive resection carried out by Exo1p or Dna2p . At telomeres, the Cdc13 complex together with the YKu complex seems to inhibit this switch, as deep resection into telomere adjacent unique DNA rarely occurs. Consistent with this idea, there is rampant C-strand resection in cells expressing the temperature-sensitive cdc13 -1 allele and growing at high temperatures. The YKu complex also contributes to limiting C-strand resection as cells lacking YKu have constitutively long G tails, and this phenotype is suppressed by deletion of EXO1 ( Gravel et al. 1998 ; Polotnianka et al. 1998 ; Maringele and Lydall 2002 ). Furthermore, Rap1p and particularly the associated Rif2p act as inhibitors of MRX-dependent telomere resection ( Bonetti et al. 2010 ). Taken together, these data suggest that telomere processing in late S phase, which occurs right after conventional DNA replication, is triggered similarly at telomeres and DSBs: a Cdk1p -stimulated Sae2p /MRX-mediated activity generates a short G tail. However, at telomeres, further resection is inhibited by a combination of YKu, the Cdc13 complex, and the Rif proteins such that resection is limited to ∼30–100 nt, occurring only in the distal half of the telomere. Since no deep resection occurs, no unique sequence single-stranded DNA is uncovered, and no DNA damage checkpoint activity or cell cycle arrest is elicited. In this scenario, YKu association to telomeres is the primary inhibitor of initiation of resection, while the other factors limit deep resection once resection has begun ( Bonetti et al. 2010 ; Vodenicharov and Wellinger 2010 ). Telomeres on which resection generates G tails longer than the 10–15 nt must be processed prior to mitosis ( Wellinger et al. 1993a , b ). This processing probably involves C-strand resynthesis by conventional DNA replication, but there is also evidence for limited nucleolytic trimming of G tails ( Diede et al. 2010 ).

Molecular models for telomere replication. (A) DNA structures thought to be generated during telomere replication when the replication fork is still in the double-stranded telomeric repeats (left) and after having reached the physical end (right). Strand colors as in Figure 1 . Brown, subtelomeric sequences. (B) Proposed telomeric chromatin changes during a cell cycle. Note that telomerase elongation drawn for late S does not occur on all telomeres in every cell cycle. This step occurs preferentially on short telomeres. Bottom shows involved proteins and complexes as well as a sketch of the proposed secondary structure of the TLC1 RNA with associated proteins (telomerase). Short red line in RNA indicates templating area. Symbols for other proteins are the same as in Figure 1 .

Semiconservative replication of telomeric and subtelomeric DNA

Discussions of telomere replication usually focus on telomerase, a telomere-specific reverse transcriptase that replicates the very end of the chromosome. However, most of the telomeric repeats are replicated by standard semiconservative DNA replication. Conventional replication of telomeric DNA is one of the last events in S phase. Density transfer experiments reveal that Y′ repeats and the unique regions adjacent to telomeres replicate very late in S phase ( McCarroll and Fangman 1988 ; Raghuraman et al. 2001 ). This late replication is due primarily to late activation of origins near telomeres, such as the late firing ARS501 ( Ferguson and Fangman 1992 ). This late firing is independent of origin sequence as an origin that is normally activated in early S phase, such as ARS1 or the origin from the 2-μm plasmid, is activated late in S phase when placed near a telomere ( Ferguson and Fangman 1992 ; Wellinger et al. 1993a ). Likewise, ARS501 fires in early S phase when moved to a circular plasmid, while linearization of the ARS501 plasmid by telomere addition results again in its late activation ( Ferguson and Fangman 1992 ). One possibility is that late origin firing results from the topological freedom enjoyed by unrestrained ends. This model is ruled out by the finding that when a DSB is induced next to an early firing origin, that origin still activates in early S phase ( Raghuraman et al. 1994 ). Thus, telomeres exert a position effect on the timing of origin activation. Late activation of telomere adjacent origins is programmed in G1 phase. Thus, if a telomere proximal ARS is excised from the chromosome in late G1 phase, a circular plasmid containing it still replicates in late S phase ( Raghuraman et al. 1997 ). Late firing of telomere adjacent origins is affected by telomere length as origins next to short telomeres fire earlier in S phase than origins near wild-type (WT)–length telomeres ( Bianchi and Shore 2007a ).

It is tempting to speculate that late activation of telomeric origins is due to the same heterochromatic chromatin structure that causes TPE. However, depleting cells of Sir3p , which eliminates TPE, has little effect on replication timing of telomere adjacent DNA ( Stevenson and Gottschling 1999 ). In contrast, the YKu complex, whose absence causes telomere shortening, long G tails, and reduced TPE, is essential for late activation of telomeric origins yet it does not affect activation of more internal origins ( Cosgrove et al. 2002 ). Deletion of Rif1p , which causes telomere lengthening, also results in early replication of telomeric regions ( Lian et al. 2011 ).

Perhaps because of late replication, telomere length is particularly sensitive to mutations in conventional replication proteins. For example, telomeres lengthen in cells with temperature-sensitive alleles of several replication proteins, such as DNA polymerase α, DNA replication factor C, and Rad27p ( Carson and Hartwell 1985 ; Adams and Holm 1996 ; Parenteau and Wellinger 1999 , 2002 ; Adams Martin et al. 2000 ; Grossi et al. 2004 ). Since the telomere lengthening in these mutants is telomerase dependent ( Adams Martin et al. 2000 ), it likely reflects a competition between semiconservative DNA replication and telomerase extension, both of which occur in late S phase. The key player in this competition is probably the Cdc13 complex, as two of its subunits interact with subunits of the DNA polymerase α complex, Cdc13p with the catalytic subunit of DNA polymerase α ( Qi and Zakian 2000 ; Sun et al. 2011 ) and Stn1p with Pol12p ( Grossi et al. 2004 ). Cdc13p also interacts with Est1p , a telomerase subunit ( Qi and Zakian 2000 ; Pennock et al. 2001 ; Wu and Zakian 2011 ). Thus, when replication proteins are limiting, it may facilitate Cdc13p interaction with telomerase and promote telomere lengthening.

Semiconservative replication of telomeres is a prerequisite for the C-strand degradation that occurs in late S/G2 phase ( Wellinger et al. 1993a ; Dionne and Wellinger 1998 ). The two telomeres on each chromosome are synthesized differently, and these differences affect their need for C-strand degradation. At one end, the new strand is the product of leading strand synthesis while at the other end, it is the product of lagging strand synthesis ( Figure 4A ). Theoretically, the telomere replicated by leading strand synthesis can be replicated fully to generate a blunt end, while the other end will be left with a small gap at the 5′ end of the newly replicated strand after removal of the terminal RNA primer ( Figure 4A ). Although both ends of at least some DNA molecules are subject to C-strand degradation in a given cell cycle ( Wellinger et al. 1996 ), the leading strand and lagging strand telomeres are treated differently ( Parenteau and Wellinger 2002 ). While both bind Cdc13p , only the telomere replicated by the leading strand polymerase binds the MRX complex ( Faure et al. 2010 ).

When most people think about difficulties replicating chromosome ends, they think about telomerase and its role in solving the “end replication” problem. However, even semiconservative replication of telomeric DNA poses problems, as replication forks in yeast and other organisms move more slowly through telomeric DNA than through most other regions of the genome ( Ivessa et al. 2002 ; Miller et al. 2006 ; Sfeir et al. 2009 ). This difficulty is thought to arise from the GC-rich nature of telomeric DNA, which gives it a high thermal stability and also allows it to form stable secondary structures, such as G-quadruplex DNA, which can pose problems for DNA replication ( Lopes et al. 2011 ; Paeschke et al. 2011 ).

The first evidence that telomeric DNA, even at nontelomeric sites, slows replication forks came from two-dimensional gel analyses ( Ivessa et al. 2002 ). Additionally, there are multiple other sites in subtelomeric regions, such as inactive replication origins, that slow fork progression. Slow replication of telomeric regions is also seen in genome-wide studies that monitor DNA polymerase II occupancy ( Azvolinsky et al. 2009 ). The yeast replication fork also moves slowly through human telomeric DNA ( Bah et al. 2011 ).

Although fork slowing is detected in telomeric and subtelomeric DNA in wild-type cells, this slowing is 10-fold higher in the absence of Rrm3p , a 5′ to 3′ DNA helicase ( Ivessa et al. 2002 ; Azvolinsky et al. 2009 ). The effects of Rrm3p on fork progression are not limited to telomeres ( Ivessa et al. 2000 ; Ivessa et al. 2003 ) as it promotes fork progression at many nontelomeric loci, such as RNA polymerase III transcribed genes. All of the Rrm3p -sensitive sites are bound by stable protein–DNA complexes whose removal obviates the need for Rrm3p during DNA replication ( Ivessa et al. 2003 ; Torres et al. 2004 ). Eliminating any of the silencing proteins Sir2p , Sir3p , or Sir4p reduces replication pausing within telomeres in RRM3 cells. However, when both Sir proteins and Rrm3p are absent, telomeric pausing is still high ( Ivessa et al. 2003 ).Taken together, these data suggest that the sequence, as well as the chromatin structure, of telomeres contribute to their negative effects on fork progression.

Telomere maintenance via telomerase

End replication problems and the discovery of telomerase:.

All DNA polymerases synthesize DNA only in the 5′ to 3′ direction and are unable to start replication de novo . Thus, DNA polymerases require a primer, which for eukaryotic chromosomes is a short 8–12 nt stretch of RNA. A DNA polymerase can theoretically extend this primer on the so-called leading strand, until it reaches the end of the chromosome to produce a blunt end. In contrast, the lagging strand is made discontinuously, and each Okazaki fragment starts with an RNA primer. Removal of the most distal RNA primer leaves a gap of 8–12 nt at the 5′ ends of newly replicated strands that cannot be filled in by a conventional DNA polymerase. In the absence of a special end replication mechanism, the product is shorter than the starting template. This dilemma is the so-called end-replication problem, as classically defined ( Watson 1972 ).

Since eukaryotic chromosomes end with 3′ single-stranded G tails that are essential for chromosome stability, there is a second end-replication problem that affects leading strand replication ( Lingner et al. 1995 ). The leading strand DNA polymerase should generate a blunt ended DNA terminus, rather than a G tail ( Figure 4A ). Postreplication C-strand degradation at both ends of chromosomes can solve this problem ( Wellinger et al. 1996 ). In this scenario, the 5′ ends of the template for leading strand synthesis is degraded to generate long G tails. RNA primed C-strand resynthesis can fill in the C strand, but when the RNA that primes this synthesis is removed, a short G tail will be generated.

In the vast majority of eukaryotes, the continuous loss of DNA due to incomplete replication is solved by telomerase. This activity was first identified by a biochemical approach using extracts from the ciliate Tetrahymena ( Greider and Blackburn 1985 ). Telomerase consists of both protein and RNA subunits ( Greider and Blackburn 1987 ). During DNA extension, telomerase uses a short segment within its integral RNA subunit as the template to extend the 3′ end of the G-rich strand of the telomere ( Greider and Blackburn 1989 ). Thus, telomerase-generated telomeric repeats are templated not by the chromosome but by telomerase RNA. Once telomerase extends the 3′ strand, RNA primed DNA replication by a conventional DNA polymerase can fill in the complementary C strand.

C-strand degradation makes a de facto lagging strand-like terminus at the telomere that was lengthened by the leading strand polymerase. This degradation has the benefit of generating a G tail, but it will magnify the first end-replication problem as now, in the absence of telomerase, both the leading and the lagging strand telomeres lose ∼10 nt per S phase (assuming that the average RNA primer is 10 nt). However, the measured loss rate is only half this rate ( Lundblad and Szostak 1989 ; Singer and Gottschling 1994 ). A possible explanation for this discrepancy is that telomerase provides protection from a telomerase-independent lengthening activity, such as recombination. In this model, telomeres in telomerase-deficient cells would be lengthened by recombination that would partially compensate for sequence loss by incomplete replication. This proposal provides an explanation for why telomeric repeats are lost at a faster rate, ∼10 nt/generation, in strains that are both telomerase and recombination deficient compared to a strain deficient for telomerase alone ( Lundblad and Szostak 1989 ; Singer and Gottschling 1994 ; Lee et al. 2007 ).

Telomerase does not act on blunt-ended DNA molecules. Thus, C-strand degradation of the blunt end produced by leading strand replication generates not only a G tail for binding of the Cdc13 complex, it also creates a potential substrate for telomerase. With G tails at both ends of a chromosome, telomerase could theoretically act on telomeres replicated by either the leading or lagging strand polymerase. However, MRX, which recruits Tel1p and hence telomerase to telomeres, binds preferentially to telomeres replicated by the leading strand polymerase ( Faure et al. 2010 ), perhaps because MRX is needed to process blunt ends. MRX also binds preferentially to short telomeres ( McGee et al. 2010 ) and to DSBs next to short (81 bp) but not long (162 bps) tracts of telomeric DNA ( Negrini et al. 2007 ; Hirano et al. 2009 ). Since MRX is needed for efficient recruitment of telomerase, these data predict that telomerase acts preferentially at short telomeres replicated by the leading strand DNA polymerase.

Biochemical characterization of S. cerevisiae telomerase was slow in coming, perhaps because the enzyme is not abundant. In contrast, genetic analysis of telomerase was pioneered in S. cerevisiae . The first known telomerase subunit, EST1 ( e ver s horter t elomeres 1 ), was identified in a screen for genes with defective telomere function ( Lundblad and Szostak 1989 ). Although est1 Δ cells are viable, they slowly but progressively lose C 1-3 A/TG 1-3 telomeric DNA. Once telomeres become very short, chromosome loss and cell cycle length go up dramatically. After 50–100 generations, most est1 Δ cells die. The combination of progressive telomere loss and eventual chromosome instability and cell death is known collectively as the est phenotype ( Lundblad and Szostak 1989 ).

A similar screen identified an additional three genes whose deletion ( EST2 and EST3 ) or mutation ( EST4 ) also yields an est phenotype ( Lendvay et al. 1996 ). When the wild-type copy of est4 was cloned, it was found to be a separation-of-function allele of the previously identified essential CDC13 gene and renamed cdc13 -2 ( Nugent et al. 1996 ). Cells with the cdc13 -2 allele are telomerase deficient but viable because the end protection function of Cdc13p is intact. A separate screen to identify genes whose overexpression interfered with TPE, unexpectedly identified another est gene, called TLC1 ( t e l omerase c omponent 1 ) ( Singer and Gottschling 1994 ). TLC1 encodes a large RNA whose sequence has a 17-nt stretch complementary to the G strand of yeast telomeric DNA. Altering the putative template region in TLC1 produced mutant telomeric repeats in vivo , proving that TLC1 is indeed the templating RNA. Est2p was identified as the catalytic reverse transcriptase subunit of yeast telomerase when its sequence was found to be similar to that of the biochemically purified catalytic subunit of Euplotes aediculatus (a ciliated protozoan) telomerase ( Lingner et al. 1997 ).

Now that the entire yeast genome has been evaluated for telomeric roles, it is clear that TLC1 , EST1 , EST2 , EST3 , and CDC13 are the only genes whose mutation yields a telomerase null phenotype. However, certain double mutations also have an est phenotype. TEL1 encodes an ATM-like checkpoint kinase, but its major function is in telomere length maintenance. A tel1 Δ strain has very short but stable telomeres and does not senesce ( Lustig and Petes 1986 ; Greenwell et al. 1995 ; Morrow et al. 1995 ). The kinase activity of Tel1p is required for its role in telomere length maintenance as a kinase dead allele has the same phenotype as tel1 Δ ( Mallory and Petes 2000 ). Cells deficient for Mec1p , the yeast ATR equivalent and the major checkpoint kinase in yeast, have a very modest decrease in telomere length ( Ritchie et al. 1999 ). Although MEC1 is essential, both its checkpoint and telomere maintenance functions are dispensable for cell viability. Its essential function can be bypassed by deleting SML1 , an inhibitor of ribonucleotide reductase ( Zhao et al. 1998 ). Although neither tel1 Δ nor mec1 Δ sml1 Δ cells senesce, cells deficient in both kinases have an est phenotype ( Ritchie et al. 1999 ). Cells lacking any one (or all three) of the MRX subunits act in the same pathway as Tel1p to affect telomere length ( Nugent et al. 1998 ). Thus, like tel1 Δ cells, mrx mutants have short but stable telomeres and an est phenotype in combination with loss of Mec1p ( Ritchie and Petes 2000 ). Likewise, mrx ykuΔ cells have an est phenotype ( DuBois et al. 2002 ; Maringele and Lydall 2004a ).

Tel1p and the MRX complex are not part of the telomerase holoenzyme but have important roles in recruiting telomerase to telomeres. Consistent with this interpretation, fusion of Cdc13p to Est2p allows telomere maintenance in tel1 mec1 cells ( Tsukamoto et al. 2001 ). Moreover, tel1 mec1 cells have normal telomerase activity by in vitro assays and can maintain telomeres in a rif1 Δ rif2 Δ background ( Chan et al. 2001 ).

Characteristics of components of the telomerase holoenzyme:

The EST1 ORF predicts a 699-amino-acid protein with no strong structural motifs ( Figure 2 ; Lundblad and Szostak 1989 ). Est1 binds RNA and single-stranded TG 1-3 DNA in vitro ( Virta-Pearlman et al. 1996 ; DeZwaan and Freeman 2009 ). Unlike Cdc13p , Est1p binding to TG 1-3 DNA requires a 3′ OH end. Although Est1p is conserved through mammals, its sequence is divergent, even in fungi ( Beernink et al. 2003 ; Reichenbach et al. 2003 ; Snow et al. 2003 ). Unlike the other telomerase subunits, Est1p abundance is cell cycle regulated, low in G1 phase (∼20 molecules/cell) when telomerase is not active and higher in late S/G2 phase (∼110 molecules/cell) when it is ( Taggart et al. 2002 ; Wu and Zakian 2011 ). This cell cycle pattern is due primarily to proteasome-dependent cell-cycle–regulated proteolysis ( Osterhage et al. 2006 ), although Est1 mRNA degradation by Rnt1p also contributes to its cell-cycle–regulated abundance ( Spellman et al. 1998 ; Larose et al. 2006 ).

Although est1 Δ cells have a classic telomerase-deficient phenotype in vivo , standard primer extension assays for telomerase activity in vitro are not Est1p dependent ( Cohn and Blackburn 1995 ). Nonetheless, Est1p immunoprecipitates with both TLC1 RNA and telomerase activity, suggesting that it is an integral part of the telomerase holoenzyme ( Lin and Zakian 1995 ; Steiner et al. 1996 ). Est1p binds directly to a stem-bulge region in TLC1 , and disruption of this interaction confers an est phenotype in vivo ( Seto et al. 2002 ). The Est1p – TLC1 interaction is essential to bring both Est1p and Est2p to telomeres in late S/G2 phase ( Chan et al. 2008 ).

Genetic evidence using fusion proteins provided the first evidence that a Cdc13p – Est1p interaction recruits the telomerase holoenzyme to telomeres. Est1p is dispensable for telomere maintenance in cells expressing a fusion of the DNA binding domain of Cdc13p ( DBD Cdc13 ) and Est2p ( DBD Cdc13 – Est2 ) ( Evans and Lundblad 1999 ). These results suggest that the critical function of Est1p is to mediate the interaction between telomerase and the telomere. Two-hybrid and coimmunoprecipitation studies support this hypothesis by providing physical evidence of an interaction between the two proteins ( Qi and Zakian 2000 ). Moreover, this interaction is direct, as purified Cdc13p and Est1p interact in vitro to form a 1:1 complex ( Wu and Zakian 2011 ). The interaction is also specific, as Cdc13p does not interact with Est3p and is sufficient for recruiting Est1p to Cdc13p -coated TG 1-3 single-strand DNA in vitro .

The telomerase null phenotypes of certain mutations in CDC13 and EST1 , such as cdc13 -2 and est1 -60 , are proposed to be due to a disruption of the Cdc13p – Est1p interaction ( Pennock et al. 2001 ). These particular mutations are charge swap alleles: while each mutation alone confers an est phenotype in vivo , cdc13 -2 est1 -60 cells have short, stable telomeres and do not senesce. Because the charge interaction between the two proteins is restored in the double mutant, the telomerase proficiency of the double mutant can be explained by restoration of a physical interaction between Cdc13p and Est1p . Consistent with this interpretation, cdc13 -2 cells have low Est1p and Est2p binding to telomeres ( Chan et al. 2008 ) and DSBs ( Bianchi et al. 2004 ). However, the strengths of various combinations of interactions ( i.e. , Cdc13p – Est1p , Cdc13 –2p– Est1p , and Cdc13p – Est1 –60p) are indistinguishable in vitro ( Wu and Zakian 2011 ). The best model to fit all of the data is that these charge swap mutants support wild-type levels of Cdc13p – Est1p interaction, but the resulting complex is somehow defective in vivo such that it is unable to support wild-type levels of telomerase–telomere interaction or telomerase extension. Indeed, visualization of telomerase RNA in living cells suggests that it associates with telomeres in cdc13 -2 cells, but this association is transient ( Gallardo et al. 2011 ).

In addition to its role in telomerase recruitment, Est1p is thought to activate telomerase. The best evidence for this model also comes from studies with fusion proteins. Cells expressing a DBD Cdc13 – Est2 fusion protein have hyperelongated telomeres, presumably because telomerase is always telomere associated ( Evans and Lundblad 1999 ). However, telomeres are not hyperelongated in est1 Δ cells expressing the fusion. In line with an activating role for Est1p , biochemical studies show that Est1p interacts directly with Est3p , an interaction that is required for Est3p telomere binding ( Tuzon et al. 2011 ). The role of Est1p in recruiting Est3p might explain its activation function.

The EST2 ORF predicts an 884-amino-acid protein with motifs found in other reverse transcriptases including three invariant aspartate residues that are essential for catalysis ( Lingner et al. 1997 ). Mutation of any one of the conserved aspartates leads to an est phenotype equivalent to that seen in est2 Δ cells and also eliminates telomerase activity in vitro . Thus, Est2p is the catalytic reverse transcriptase subunit of S. cerevisiae telomerase.

Like other telomerase reverse transcriptases (TERTs), but unlike most other reverse transcriptases, Est2p contains a long basic N-terminal (TEN) domain that is essential for telomerase activity in vivo and in vitro ( Friedman and Cech 1999 ; Figure 2 ). The TEN domain supports multiple interactions within the holoenzyme, including interactions with TLC1 ( Friedman and Cech 1999 ) and Est3p ( Friedman et al. 2003 ; Talley et al. 2011 ). Est2p is a low abundance protein (<40 molecules/cell; Tuzon et al. 2011 ), and its levels are TLC1 dependent (reduced by ∼50% in tlc1 Δ cells; Taggart et al. 2002 ).

The EST3 ORF, which predicts an 181-amino-acid protein, has the unusual property of being generated by a programmed translation frameshift ( Figure 2 ) ( Morris and Lundblad 1997 ). Like Est1p , Est3p is essential for telomere maintenance in vivo but not for catalysis in vitro ( Lendvay et al. 1996 ; Lingner et al. 1997 ). Nonetheless, coimmunoprecipitation shows that Est3p is part of the telomerase holoenzyme ( Hughes et al. 2000 ). The association of Est3p with telomerase is Est1p dependent ( Osterhage et al. 2006 ), consistent with the direct interaction of purified Est1p and Est3p seen in vitro ( Tuzon et al. 2011 ). By genetic and biochemical criteria, Est3p also interacts with the TEN domain of Est2p ( Friedman et al. 2003 ; Talley et al. 2011 ), and Est3p association with telomeres is also Est2p dependent, especially in G1 phase ( Tuzon et al. 2011 ).

Although Est1p and Est3p have certain similarities, they do not have redundant functions. For example, a DBD Cdc13 – Est3 fusion protein can maintain telomeres in est3 Δ but not est1 Δ cells ( Hughes et al. 2000 ). Likewise, an Est1 – DBD Cdc13 fusion protein does not rescue the telomerase defect of est3 Δ cells, and a DBD Cdc13 – Est2 fusion bypasses the need for Est1p , but not Est3p ( Evans and Lundblad 1999 ).

So far Est3p is found only in budding yeasts. However, a possible key to its function comes from a predicted structural similarity between it and a mammalian telomere structural protein TPP1 ( Lee et al. 2008 ; Yu et al. 2008 ). Unlike Est3p , TPP1 is not a telomerase subunit but rather part of the multiprotein shelterin complex that protects telomeric DNA. However, TPP1 affects telomerase by cooperating with Pot1, a mammalian G-strand binding protein, to increase telomerase processivity ( Wang et al. 2007 ; Xin et al. 2007 ).

Like the Est proteins, the TLC1 RNA is not abundant, present in ∼30 molecules/cell ( Mozdy and Cech 2006 ). Transcription of TLC1 RNA by RNA polymerase II generates two populations, a slightly longer polyadenylated form (5–10% of total) and a polyA minus form (> 90%), the version in active telomerase ( Chapon et al. 1997 ; Bosoy et al. 2003 ). Akin to snRNAs and snoRNAs, the 5′ end of the TLC1 RNA has a trimethylguanosine cap ( Seto et al. 1999 ; Franke et al. 2008 ), while generation of the mature nonpolyadenylated 3′ end occurs via the Nrd1p -dependent noncoding RNA termination pathway ( Jamonnak et al. 2011 ; Noel et al. 2012 ). Similar to several fungal telomerase RNAs, TLC1 is >1000 nt in size, much larger than its ciliate (∼160 nt) or mammalian (∼450 nt) counterparts ( Singer and Gottschling 1994 ). However, a TLC1 RNA derivative that reduces the native RNA from 1157 to 384 nt is sufficient to maintain short, but stable yeast telomeres in vivo and to support catalysis in vitro ( Zappulla et al. 2005 ). Thus, much of TLC1 RNA is dispensable for enzyme activity.

Although the sequence and size of telomerase RNAs evolve rapidly, conserved secondary structures have been deduced. The structure predicted for the S. cerevisiae TLC1 RNA centers about a conserved pseudoknot domain that contains the templating region of the RNA and interacts with Est2p ( Livengood et al. 2002 ; Dandjinou et al. 2004 ; Lin et al. 2004 ; Zappulla and Cech 2004 ; Qiao and Cech 2008 ). The remainder of the RNA forms three largely duplex arms that are proposed to act as a flexible scaffold to organize TLC1 RNA interacting proteins ( Figure 4 ). One arm binds Est1p , and this binding is essential for telomerase activity in vivo ( Seto et al. 2002 ). One arm binds Yku80p , an interaction that is not essential for telomere maintenance but brings TLC1 to the nucleus and recruits Est2p to telomeres in G1 phase ( Stellwagen et al. 2003 ; Fisher et al. 2004 ; Vega et al. 2007 ; Gallardo et al. 2008 ). The third arm binds the seven-member Sm protein ring, an association that is dispensable for activity but important for TLC1 accumulation ( Seto et al. 1999 ).

Regulation of telomerase by the cell cycle:

Two experiments using quite different approaches show that telomerase-mediated lengthening is cell-cycle regulated. The first experiment followed telomerase action at a DSB induced next to a short stretch of telomeric repeats ( Diede and Gottschling 1999 ). When this break is made in G2/M arrested cells, it is lengthened by telomerase. However, the break is not lengthened in G1-arrested cells, suggesting that telomerase does not act at this time. However, in vitro assays show similar levels of telomerase activity in extracts prepared from cells arrested at these two points in the cell cycle.

The second assay studied the fate of a short telomere in cells with otherwise wild-type length telomeres by using site-specific recombination to generate a single short telomere ( Marcand et al. 2000 ). The resulting short telomere is preferentially lengthened by telomerase ( Marcand et al. 1999 ), but this lengthening does not occur in G1 or early S phase but rather only in late S/G2 phase ( Marcand et al. 2000 ).

One way to reconcile the finding that telomerase is active in vitro in extracts from G1-phase cells with its inability to lengthen telomeres in vivo in G1 phase is if the telomere is inaccessible to telomerase in G1 phase. An obvious way to test this model is to use chromatin immunoprecipitation (ChIP) to detect the presence of telomerase at telomeres as a function of position in the cell cycle. This type of experiment yields support both for and against regulated accessibility ( Taggart et al. 2002 ). Cdc13p is telomere associated throughout the cell cycle, but its binding increases dramatically in late S phase, as expected by the occurrence of long G tails at this time ( Wellinger et al. 1993b ). The telomere binding of Est1p ( Taggart et al. 2002 ) and Est3p ( Tuzon et al. 2011 ) is largely limited to late S/G2 phase, consistent with regulated accessibility. However, Est2p is telomere associated throughout most of the cell cycle, including in G1 and early S phase when telomerase does not act ( Taggart et al. 2002 ). Nonetheless, Est2p binding is not constitutive as there is a second peak of Est2p binding in late S/G2 phase.

The two peak pattern of Est2p telomere binding reflects two independent pathways of telomerase recruitment. Both pathways are TLC1 dependent as there is no telomere-associated Est2p in tlc1 Δ cells ( Taggart et al. 2002 ). However, Est2p telomere association in G1 phase requires a specific interaction between Yku80p and a 48-bp stem-loop structure in TLC1 RNA ( Fisher et al. 2004 ) while the late S/G2 phase binding requires Est1p binding to a stem-bulge region in TLC1 as well as its interaction with Cdc13p ( Chan et al. 2008 ). The Est2p that is telomere associated in G1 phase is likely not engaged with the very end of the chromosome as expected for active telomerase as much of it is bound >100 bp from the chromosome end ( Sabourin et al. 2007 ). Consistent with this view, the G1-phase association is not necessary for telomerase action as mutations that eliminate it ( tlc1 Δ48 ; yku80 -135i ) ( Fisher et al. 2004 ) result in only modest telomere shortening ( Peterson et al. 2001 ). Even this small reduction in telomere length may not be due to lack of G1-phase telomerase binding as nuclear levels of TLC1 are reduced in the absence of the TLC1 –Ku interaction ( Gallardo et al. 2008 ; Pfingsten et al. 2012 ). Thus, the short telomeres in tlc1 Δ48 and yku80 -135i cells could be a consequence of reduced amounts of holoenzyme being imported and/or retained in the nucleus. Recent data indicate that Yku binding to DNA and RNA are mutually exclusive ( Pfingsten et al. 2012 ). Since the binding of Est2p to telomeres in G1 phase requires a Yku80p – TLC1 interaction, it is likely that the Yku that is involved in this interaction associates with the telomere via protein–protein interactions, not by direct DNA binding.

Cell-cycle–limited telomerase activity at telomeres is also inferred from results in which TLC1 RNA is visualized in individual cells in real time ( Gallardo et al. 2011 ). Telomerase RNA marked with GFP is much more mobile than telomeres in G1 and G2 phases, whereas in late S phase, telomerase RNA movement slows. Thus, TLC1 association with telomeres is more transient in G1 and G2 phases than in late S phase. Genetic experiments argue that the more stably associated TLC1 reflects active telomerase, as these associations are less frequent in genetic backgrounds where telomerase recruitment is impaired. Thus, results with live cell imaging support previous findings that the association of telomerase with telomeres can occur throughout the cell cycle ( Taggart et al. 2002 ), but only the late S phase telomere-associated Est2p is important for telomere length regulation ( Fisher et al. 2004 ). This study also suggests that more than one telomerase complex is present on elongating telomeres as the TLC1 complexes, dubbed T-Recs ( t elomerase re cruitment c lusters), are brighter and larger in late S phase ( Gallardo et al. 2011 ).

Est1p is cell cycle regulated with peak abundance in late S/G2 phase ( Taggart et al. 2002 ; Osterhage et al. 2006 ). Moreover, Est3p telomere binding is Est1p dependent, so its telomere binding also occurs mainly in late S/G2 phase ( Tuzon et al. 2011 ). Thus, telomerase is cell cycle limited at least in part because the telomerase holoenzyme is assembled only during a narrow window in the cell cycle ( Osterhage et al. 2006 ). However, even when Est1p is expressed in G1 phase, which results in both Est1p and Est3p being Est2p – TLC1 associated, telomerase is still not active on telomeres in G1 phase ( Osterhage et al. 2006 ). Thus, Est1p abundance is not the whole answer to cell-cycle–regulated activity. Rif proteins also contribute to limiting telomerase action to late S phase as in the absence of either protein, short telomeres can be lengthened in G1 phase ( Gallardo et al. 2011 ). Cell-cycle–regulated changes in telomere structure, such as C-strand degradation, which is Cdk1 dependent, may also contribute to cell cycle limited telomerase action ( Frank et al. 2006 ; Vodenicharov and Wellinger 2006 ).

Regulation of telomerase by telomere length:

Two types of experiments indicate that short telomeres are preferentially lengthened by yeast telomerase. The first evidence comes from experiments where lengthening of a single short telomere is followed over time ( Marcand et al. 1999 ). It takes ∼50 generations to return a short telomere to a wild-type length. However, its rate of lengthening changes as it lengthens. When the telomere is at its shortest, it lengthens by ∼15 nt/generation. This rate progressively decreases until it is only ∼1 nt/generation when the once short telomere approaches wild-type length.

The preferential lengthening of short telomeres is best illustrated using the s ingle t elomere ex tension assay (STEX) that monitors lengthening of individual telomeres in a single S phase at nucleotide resolution ( Teixeira et al. 2004 ). STEX is particularly informative because it monitors events at individual telomeres rather than being a population average. In this assay, telomerase-deficient cells (recipient cells) are mated to telomerase proficient cells (donor cells). Telomere extension is monitored in the first generation after mating. The recipient cells contain marked telomere(s) that can be examined specifically by PCR because of differences in subtelomeric DNA from the same telomere in donor cells. Because the yeast telomeric sequence is heterogeneous, the starting telomeric DNA can be distinguished from newly added telomeric repeats simply by lining up telomeres and comparing their sequences.

In a given cell cycle, STEX finds that <10% of wild-type–length (∼300 bp) telomeres are lengthened by telomerase, while a 100-bp telomere is lengthened ∼50% of the time ( Teixeira et al. 2004 ). Thus, length-dependent extension is not an all or none event: many short telomeres are not lengthened while some long telomeres are. Although the frequency of telomerase action is dependent on length, the amount of telomeric DNA added is not until telomeres are very short (≤100 bp). On these very short telomeres, telomerase appears to be more processive. STEX is also useful to determine how different proteins affect telomerase. By STEX, Rif1p and Rif2p inhibit the frequency of telomere lengthening but not the amount of telomeric DNA added per S phase ( Teixeira et al. 2004 ). The preference for telomerase action at short telomeric tracts is also reflected during de novo telomere addition. A DSB induced next to an 81-bp stretch of telomeric DNA is more efficiently elongated than a break next to 162 bp of telomeric DNA ( Negrini et al. 2007 ; Hirano et al. 2009 ).

ChIP is useful to determine the protein content of short vs. wild-type–length telomeres. Using inducible short telomere assays ( Marcand et al. 1999 ), Est2p and Est1p have approximately fourfold higher binding at short telomeres specifically in late S/G2 phase, when telomerase is active ( Bianchi and Shore 2007b ; Sabourin et al. 2007 ). The similar level of increase for Est1p and Est2p argues against the idea that an elongation-incompetent Est2p binds all telomeres in G1 phase and then is activated in late S/G2 phase by Est1p binding.

Short telomere assays have also helped us understand how telomerase is targeted to short telomeres. Since Est2p recruitment to telomeres requires a Yku80p – TLC1 interaction in G1 phase and a Cdc13p – Est1p interaction in late S/G2 phase, if Yku80p and/or Cdc13p bound better to short telomeres, it could explain why short telomeres bind more telomerase. However, Yku80p and Cdc13p bind to similar extents at short and WT length telomeres ( Bianchi and Shore 2007b ; Sabourin et al. 2007 ). Another possibility is that Yku80p or Cdc13p is preferentially modified at short telomeres. For example, Cdc13p is phosphorylated by Cdk1p late in the cell cycle, and in the absence of this phosphorylation, Est1p telomere binding and telomere length are modestly reduced ( Li et al. 2009 ). Cdc13p is also sumoylated in early to mid S phase, a modification that limits telomerase action probably by increasing the Cdc13p – Stn1p interaction ( Hang et al. 2011 ).

In tel1 Δ cells, telomeres are very short ( Lustig and Petes 1986 ; Greenwell et al. 1995 ; Morrow et al. 1995 ) yet unlike other short telomeres, tel1 Δ telomeres bind very little Est1p or Est2p ( Goudsouzian et al. 2006 ). These data suggest that Tel1p might affect preferential lengthening of short telomeres. Indeed, while Tel1p binding to wild-type–length telomeres is low, transient, and limited to late S/G2 phase, Tel1p binding is about 10 times higher at short telomeres ( Sabourin et al. 2007 ). Tel1p binding to short telomeres is detectable even in early S phase, increases in magnitude as cells progress through the cell cycle, and persists for at least two cell cycles. In contrast, Mec1p telomere binding is extremely low, even in tel1 Δ cells where it is required for telomere elongation. Preferential binding of Tel1p to short telomeres is also seen when short telomeres are generated by deleting YKu or by deleting a telomerase subunit ( Hector et al. 2007 ).

Tel1p binding to telomeres is dependent on an interaction between Tel1p and the carboxyl terminus of Xrs2p ( Hector et al. 2007 ; Sabourin et al. 2007 ), just as it is at DSBs. Moreover, each of the three MRX subunits binds preferentially to short telomeres, and like high Tel1p binding, this high binding persists for at least two cell cycles ( McGee et al. 2010 ). MRX binding occurs mainly at telomeres that have been replicated by the leading strand DNA polymerase ( Faure et al. 2010 ). A unifying model for these data are that MRX binds preferentially to short telomeres replicated by the leading strand DNA polymerase; this binding recruits Tel1p , Tel1p phosphorylates one or more telomere proteins, and these changes in telomeric chromatin result in higher telomerase recruitment. Indeed, deleting TEL1 eliminates preferential binding of telomerase to short telomeres that lack subtelomeric repeats ( Arneric and Lingner 2007 ; Sabourin et al. 2007 ). However, when telomeres contain subtelomeric binding sites for Tbf1p , short telomeres are still preferentially lengthened in tel1 Δ cells, and tethering Tbf1p to a short telomere with no natural Tbf1p binding sites allows its preferential elongation in tel1 Δ cells ( Arneric and Lingner 2007 ). Thus, Tbf1p and Tel1p act in a partially redundant manner to distinguish short from wild-type–length telomeres. Like Rif2p , Tbf1p inhibits MRX, and hence telomerase, binding to short telomeres ( Fukunaga et al. 2012 ).

Although the kinase activity of Tel1p is required for its role in telomere length maintenance ( Mallory and Petes 2000 ), its critical targets at telomeres are not yet identified. Like other ATM-like kinases, Tel1p phosphorylates SQ/TQ motifs ( Mallory and Petes 2000 ; Tseng et al. 2006 ). Xrs2p and Mre11p are phosphorylated in a Tel1p -dependent process in response to DNA damage, but mutation of the phosphorylated residues in these proteins to nonphosphorylatable amino acids does not result in short telomeres ( Mallory et al. 2003 ). Cdc13p is another candidate for a Tel1p target as it contains 11 SQ/TQ motifs. Clusters of these motifs are in functionally important regions of Cdc13p , one in the DBD and one in the RD ( Figure 2 ; Tseng et al. 2006 ). In vitro , Tel1p phosphorylates Cdc13p on three RD serine residues (S225, 249, and and 255). Moreover, simultaneous mutation of two of these residues (S249 and S255) results in an est phenotype. In addition, if Est1p is targeted to the telomere using a DBD Cdc13 – Est1 fusion protein, phosphorylation of these residues is no longer required for telomerase action.

The phenotype of cdc13 -S249A , S255A cells makes a strong argument that their phosphorylation is critical for Cdc13p – Est1p interaction ( Tseng et al. 2006 ). However, cells expressing a cdc13 allele in which each of the S/TQ motifs is mutated to AQ supports near wild-type–length telomeres ( Gao et al. 2010 ). In addition, exhaustive mass spectrophotometric analysis of purified Cdc13p detects 21 sites of phosphorylation but no phosphorylation at S249 or S255, even in G2/M phase (W. Yun, P. A. DiMaggio, Jr., D. H. Perlman, V. A. Zakian, and B. A. Garcia, unpublished results). One possible way to reconcile these data are if cdc13 -S249A , S255A cells are telomerase defective because these mutations destabilize the RD domain (rather than preventing it from being phosphorylated). In any case, the key sites of Tel1p phosphorylation relevant to telomerase recruitment have probably not been identified.

Telomere length is proportional to the number of Rap1p binding sites at a given telomere ( Lustig et al. 1990 ). Rap1p recruits Rif1p , Rif2p , Sir3p , and Sir4p , each of which binds to the C terminus of Rap1p ( Figure 2 ). Sir proteins function mainly in TPE, not telomere length control, but both Rif proteins negatively regulate telomerase ( Teng et al. 2000 ). Deletion of either RIF1 or RIF2 results in telomere elongation ( Hardy et al. 1992b ; Wotton and Shore 1997 ). Since deletion of both proteins results in synergistic lengthening, Rif1p and Rif2p do not act redundantly ( Wotton and Shore 1997 ). Rif1p and Rif2p also act synergistically to inhibit telomere addition at DSBs induced near a 162-bp tract of telomeric DNA ( Negrini et al. 2007 ; Hirano et al. 2009 ).

By definition, telomeres progressively lose Rap1p binding sites concomitant with loss of telomeric DNA. Thus, an appealing model is that short telomeres are marked for MRX binding by their low content of Rif1p and Rif2p . Surprisingly, short telomeres have about the same amount of Rif1p binding as wild-type–length telomeres ( Sabourin et al. 2007 ; McGee et al. 2010 ). Thus, short telomeres are not distinguished from long telomeres by their Rif1p content, although Rif1p may be selectively modified at short telomeres. In contrast, Rif2p content is lower at short telomeres so its absence could mark short telomeres for elongation. Consistent with this possibility, Tel1p no longer binds preferentially to short telomeres in rif2 Δ cells ( McGee et al. 2010 ). The observation that shortening telomeres lose Rif2p before losing Rif1p suggests that the two proteins are distributed nonrandomly along the telomere with Rif2p being closer to the chromosome terminus than Rif1p ( Figure 1 ).

A mechanistic explanation for the effects of Rif2p on Tel1p binding comes from studies on DSBs made adjacent to telomeric DNA ( Hirano et al. 2009 ). Tel1p binding to these DSBs is increased in rif2 Δ cells, suggesting that Rif2p inhibits Tel1p association with the break. Moreover, tethering Rif2p to a nontelomeric DSB decreases Tel1p but not MRX binding to the break. Rif1 has similar but much smaller effects in these assays. Finally, coimmunoprecipitation shows that the N terminus of Rif2p , but not Rif1p , interacts with the C terminus of Xrs2p . Since Tel1p also binds this portion of Xrs2p , Rif2p , and Tel1p probably compete with each other for binding to MRX. If these results are applicable to telomeres, Rif2p could sequester Xrs2p in a manner that prevents its interaction with Tel1p .

Like Rif proteins, Pif1p , a 5′ to 3′ DNA helicase, is a negative regulator of telomerase. However, Rif proteins and Pif1p inhibit telomere elongation by different mechanisms as their absence has additive effects on telomere length ( Schulz and Zakian 1994 ). Genetic data suggest that Pif1p interacts with the finger domain of Est2p ( Eugster et al. 2006 ). Pif1p also reduces the number of gross chromosomal rearrangements, complex genetic changes of the type seen in cancers, by channelling DSBs toward recombination, rather than telomere addition ( Myung et al. 2001 ). Lengthening of existing telomeres by telomerase is also inhibited by Pif1p , and its effects at both DSBs and telomeres require its ATPase activity ( Schulz and Zakian 1994 ; Zhou et al. 2000 ). Although Pif1p inhibits telomerase at both telomeres and DSBs ( Schulz and Zakian 1994 ), it may help distinguish the two as Pif1p phosphorylation by Mec1p is required for its inhibition of telomerase at DSBs but not at telomeres ( Makovets and Blackburn 2009 ). Pif1p also appears to contribute to the preferential lengthening of short telomeres since in its absence, Est2p binds equally well to short and wild-type–length telomeres. This behavior can be explained by the finding that Pif1p itself binds preferentially to long telomeres. Like Est1p , Pif1p is cell cycle regulated by proteasome-dependent proteolysis such that nuclear Pif1p peaks in abundance in S/G2 phase ( Vega et al. 2007 ).

In vitro telomerase assays:

Although yeast telomerase is constitutively expressed, it is present at low levels, which probably explains why its detection by in vitro assays lagged behind other organisms. Even after in vitro assays were established, they were (and still are) inefficient, generating only short extension products ( Cohn and Blackburn 1995 ). Furthermore, although these assays require TLC1 and Est2p , they are not Cdc13p , Est1p , or Est3p dependent ( Cohn and Blackburn 1995 ; Lingner et al. 1997 ). However, as it is not clear whether the fractionated telomerase prepared from extracts from wild-type cells even contained Est1p , Est3p , or Cdc13p , their absence might explain why the in vitro reactions are not robust. Molecular chaperones such as Hsp82p affect telomerase activity in vitro as well as having modest effects on telomere length in vivo ( Toogun et al. 2008 ).

Definitive answers to the mechanistic contributions of Est proteins to telomerase activity will almost surely require purified components. Recombinant Cdc13p , Est1p , and Est3p (but not Est2p ) have recently been purified by multiple labs. All three proteins are reported to influence primer extension assays, although these analyses are in early stages and are sometimes contradictory. Cdc13p has been reported to inhibit ( Zappulla et al. 2009 ) and to stimulate in vitro telomerase activity ( DeZwaan and Freeman 2009 ). The reasons for this difference are not clear, but there are multiple experimental differences in the two studies. Another in vitro study found that, in the context of Stn1p and Ten1p , Hsp82p can modulate Cdc13p DNA binding and thereby its effects on telomerase activity ( DeZwaan et al. 2009 ). Purified Est1p is reported to increase the amount of product in a primer extension assay by up to 14-fold. Surprisingly, this stimulation does not require that Est1p be able to interact with the stem-bulge region in TLC1 RNA or to bind TG 1-3 single-strand DNA. An earlier report using a PCR rather than primer extension assay found that long extension products were Est1p dependent, perhaps providing additional evidence for an activating role for this subunit ( Lin and Zakian 1995 ). Finally, two groups report that purified Est3p from S. cerevisiae ( Talley et al. 2011 ) or the related S. castelli ( Lee et al. 2010 ) stimulates telomerase two- to threefold. This stimulation requires interaction of Est3p with the TEN domain of Est2p ( Talley et al. 2011 ). It is not clear whether the Est3p stimulation is Est1p dependent. Although more experiments are needed, the availability of in vitro assays should provide more detailed mechanistic information on the telomerase reaction.

S. cerevisiae telomerase is not very processive in vitro . The enzyme pauses after each nucleotide addition and rarely translocates on the DNA template as required for multiple rounds of synthesis. This lack of processivity is not due to enzyme falling off the DNA primer. Rather, after elongation, yeast telomerase remains tightly bound to its DNA substrate ( Prescott and Blackburn 1997 ). However, if the Pif1p DNA helicase is added to the in vitro reaction, Est2p is released into the supernatant ( Boule et al. 2005 ). As a result of this release, Pif1p reduces telomerase processivity in vitro . Likewise, in vivo , telomerase can dissociate and then reassociate with a given telomere in a single cell cycle ( Chang et al. 2007 ). The effects of Pif1p on telomerase require its enzymatic activity as Walker A box Pif1 mutant proteins bind single-stranded DNA as well as wild-type Pif1p but do not displace telomerase from DNA or reduce telomerase processivity ( Boule et al. 2005 ).

Telomere maintenance via recombination

Telomerase is not the only activity that can maintain telomeric DNA. Although discovered in S. cerevisiae ( Lundblad and Blackburn 1993 ), telomere maintenance by recombination is widespread occurring from yeasts to mammals. Recombinational maintenance of telomeres was detected by the finding that a small fraction of est1 Δ cells survive senescence and form viable colonies. The importance of recombination was inferred from the virtual absence of survivors in est1 Δ rad52 Δ strains. All est strains, except mec1 tel1 , produce survivors via recombination ( Figure 5 ).

Outline of the proposed sequence of events leading to telomere maintenance via recombination after telomerase loss. DNA strand coloring is as above. Tick marks on the brown sequence indicate a conserved Xho I restriction enzyme site. Most cells die after ∼50–100 generations of growth, but rare cells with the indicated two types of DNA arrangements can continue to divide. Virtually all events are dependent on RAD52 and POL32 . Bottom: Typical southern blot analysis using Xho I-digested DNA derived from indicated strains. The probe consisted of a 32 P labeled DNA fragment specific for telomeric repeat sequences. M, molecular size standards; yku , DNA derived from a strain lacking YKU80 and harboring short terminal repeat tracts. WT, DNA from a wild-type strain; type I, DNA from type I survivors; type II, DNA derived from type II survivors. Red square, location of terminal Xho I fragments. Blue square, signal for the amplified Y′ elements in type I survivors. Note that the fragment pattern for type II survivors is highly variable and unstable; thus the patterns shown in the last two lanes should be taken as an example for illustration purposes only.

Even in very early telomerase negative cultures, some cells stop growing when the average telomere length is expected to be near wild type ( Lundblad and Blackburn 1993 ; Enomoto et al. 2002 ; Khadaroo et al. 2009 ). Thus, the progressive shortening of the majority of telomeres is probably not the major determinant for growth arrest ( Abdallah et al. 2009 ; Khadaroo et al. 2009 ; Noel and Wellinger 2011 ). Most likely, this arrest is due to an occasional short telomere that arises during DNA replication and that cannot be relengthened by telomerase ( Hackett et al. 2001 ; Hackett and Greider 2003 ). Indeed, only one chromosome end that lacks a telomere is sufficient to trigger growth arrest, and this arrest occurs even in telomerase proficient cells ( Sandell and Zakian 1993 ; Abdallah et al. 2009 ; Khadaroo et al. 2009 ). The survivors that emerge from the arrested cultures continue to divide. However, individual survivor clones often grow considerably slower than wild-type cells, and some may even go through additional growth arrests.

Survivors have one of two different arrangements of telomeric DNA ( Lundblad and Blackburn 1993 ), now dubbed type I and type II survivors ( Teng and Zakian 1999 ; Figure 5 ). In addition to RAD52 , generation of both classes requires the replication protein Pol32p ( Lydeard et al. 2007 ), suggesting that the recombination that maintains telomeric DNA involves replication. Type I survivors are more common than type II survivors. For example, in one strain background, 90% of survivors have type I telomeres ( Teng et al. 2000 ). However, type I survivors are not stable and easily convert to type II cells, which owing to their faster growth rate, take over liquid cultures. This effect shows that the two major survivor pathways are not mutually exclusive.

Type I survivors:

These cells grow slowly with intermittent periods of growth arrest. The vast majority of telomeres in these cells contain multiple tandem Y′ repeats, but the very ends still have short (50–150 bp) tracts of duplex telomeric DNA and normal G tails ( Lundblad and Blackburn 1993 ; Larrivee and Wellinger 2006 ; Figure 5 ). The terminal arrays of Y′ repeats can be so substantial that individual cells can have up to 70-fold increase in Y′ elements ( Lundblad and Blackburn 1993 ). Type I survivors also contain extrachromosomal circular Y′ elements that are proposed to serve as substrates for Y′ recombination ( Larrivee and Wellinger 2006 ). Chromosomes of type I survivors do not enter agarose gels that are used to separate very large DNA molecules, probably because they contain a high fraction of highly structured recombination intermediates ( Liti and Louis 2003 ; E. Louis, personal communication).

About half of the Y′ repeats contain an ORF encoding a potential helicase called Y′-Help1 ( Louis and Haber 1992 ; Yamada et al. 1998 ). Expression of this ORF is greatly increased during growth arrest in telomerase lacking cells ( Yamada et al. 1998 ). Although amplification of Y′ usually occurs by recombination ( Lundblad and Blackburn 1993 ), in cells lacking telomerase, Y′ can also move by a transposition-like RNA-mediated process that relies on the Ty1 retrotransposon ( Maxwell et al. 2004 ). In addition to RAD52 and POL32 , the RAD51 , RAD54 , RAD57 , and presumably RAD55 genes are also required to generate type I survivors ( Le et al. 1999 ; Chen et al. 2001 ).

Type II survivors:

Telomeres in type II survivors show only minor amplifications of subtelomeric repeats but rather large increases in C 1-3 A/TG 1-3 telomeric repeats ( Figure 5 ). Telomeres in type II survivors are highly heterogeneous with some exceeding 12 kb in size and others being very short ( Teng and Zakian 1999 ; Teng et al. 2000 ; Figure 5 ). The long telomeres are not stable but progressively shrink during outgrowth and then are subject to stochastic and dramatic lengthening events, consistent with rolling circle replication as an initiating event ( Teng et al. 2000 ). In agreement with this hypothesis, circles of telomeric DNA are detected in type II survivors but not in wild-type cells ( Lin et al. 2005 ; Larrivee and Wellinger 2006 ). Unlike type I survivors, the generation of type II survivors requires the MRX complex, RAD59 and SGS1 , the yeast RecQ helicase, which is an ortholog of the Blm helicase ( Le et al. 1999 ; Teng et al. 2000 ; Chen et al. 2001 ; Huang et al. 2001 ; Johnson et al. 2001 ).

Telomeric length control by telomeric rapid deletions:

In wild type, telomerase positive cells, over-elongated telomeres can be shortened to approximately normal length via a single intrachromosomal recombination event between telomeric repeats, a mechanism dubbed t elomeric r apid d eletion (TRD) ( Li and Lustig 1996 ; Bucholc et al. 2001 ). This process contributes to keeping the average telomere length within a normal range ( Bucholc et al. 2001 ). As a side product of this reaction, extrachromosomal circular DNA molecules with telomeric repeats are generated. TRD could produce the circular telomeric DNA molecules necessary for rolling circle replication during generation of type II survivors ( Lustig 2003 ) as demonstrated in Kluyveromyces lactis ( Natarajan and McEachern 2002 ; McEachern and Haber 2006 ) and proposed for human cells ( Pickett et al. 2009 ; Cesare and Reddel 2010 ).

Telomere-associated RNA

Despite having hallmarks of heterochromatin, subtelomeric sequences are actually transcribed to yield a new class of noncoding RNAs called te lomeric r epeat-containing R N A (TERRA) ( Azzalin et al. 2007 ; Luke et al. 2008 ; Iglesias et al. 2011 ). TERRA occurs widely in eukaryotes as it has been detected in yeasts, plants, and vertebrates, including mammals, suggesting conserved functions ( Luke and Lingner 2009 ; Feuerhahn et al. 2010 ). For budding yeast, earlier investigations had already shown that an artificially constructed telomere where the C strand of the telomere is transcribed at high levels shortens by ∼25% of its overall initial length ( Sandell et al. 1994 ). However, transcription on a telomere per se did not induce signs of telomere dysfunction, such as high levels of chromosome loss, except that silencing of the adjacent gene was lost ( Sandell et al. 1994 ).

Naturally occurring TERRA is composed of composite RNAs containing both subtelomeric sequences, such as Y′ and X, and telomeric G-strand transcripts. The size of TERRAs range from 100 to 1200 nt, they are generated by RNA polymerase II, and are polyadenylated. In wild-type cells, TERRA is rapidly degraded by the essential RNA exonuclease Rat1p , which also functions in processing standard mRNAs ( Rosonina et al. 2006 ; Luke et al. 2008 ; Rondon et al. 2010 ). TERRA probably regulates telomere length and replication. For example, rat1 -1 cells grown at semipermissive temperatures and having increased levels of TERRA have shorter telomeres, and this telomere shortening is due to impairment of the telomerase pathway ( Luke et al. 2008 ). However, the telomere shortening due to reduced Rat1p levels can be reversed by overexpression of RNaseH. Since RNaseH removes RNA that is basepaired to DNA, this finding suggests that TERRA is associated with telomeric DNA when it inhibits telomerase. TERRAs transcribed from X telomeres vs. XY′ telomeres are subject to different regulation ( Iglesias et al. 2011 ). Both are repressed via a Rap1p -mediated pathway, but only the X TERRA is repressed by Sir proteins. X and Y′ TERRAs are repressed by Rif1p and to a lesser extent, Rif2p .

Telomere silencing or TPE

Telomeric silencing (or TPE) was discovered serendipitously in S. cerevisiae during attempts to generate a uniquely marked telomere that could be used for chromatin studies ( Gottschling et al. 1990 ). To mark the telomere, URA3 was inserted immediately adjacent to the left telomere of chromosome VII, in the process deleting the TAS sequences that are normally present at this telomere. Cells carrying URA-TEL, the URA3 marked telomere, are Ura + as expected but unexpectedly, many of them are also FOA resistant (FOA R ; FOA is a drug that kills cells expressing Ura3p ). The FOA R cells have not lost or mutated URA3 as the FOA R phenotype is reversible. These effects correlate with URA3 mRNA levels: cells growing on medium lacking uracil have ∼10 times more URA3 mRNA than FOA-grown cells ( Gottschling et al. 1990 ). Thus, TPE is due to repression of transcription, but this repression is reversible.

TPE is gene and telomere nonspecific. Expression of multiple RNA Pol II transcribed genes are repressed when they are near a telomere, and TPE is detected at other truncated telomeres ( Gottschling et al. 1990 ). The metastable nature of TPE is easily visualized when ADE2 is the telomeric marker, as Ade2 + cells produce white colonies while Ade2 − cells generate red colonies. A large fraction of ADE2 -TEL cells produce largely red colonies ( ADE2 expression repressed), while about an equal number produce largely white colonies ( ADE2 expressed). However, red colonies have many white sectors, and white colonies have many red sectors. These sectors reflect phenotypic switches in transcription state within individual cells during the ∼25 divisions it takes to generate a colony. This altered state is then inherited by their progeny to produce a sector of opposite color.

Over 50 genes affect TPE, although the effects of many are relatively minor, suggesting that some may act indirectly. Moreover, FOA medium can affect ribonucleotide reductase expression in such a way that assays using URA3 as a TPE reporter can misidentify genes, such as POL30 and DOT1 , as having roles in TPE when their effects are more likely due to metabolic changes ( Rossmann et al. 2011 ; Takahashi et al. 2011 ). In contrast, Sir2p , Sir3p , Sir4p ( Aparicio et al. 1991 ), and the YKu complex ( Boulton and Jackson 1998 ) are all essential for TPE, although ykuΔ cells are TPE proficient if they also lack RIF1 and RIF2 ( Mishra and Shore 1999 ). Since Rif1p , Rif2p , Sir3p , and Sir4p all interact with the C terminus of Rap1p , the absence of the two Rif proteins probably reduces their competition with Sir3p and Sir4p for the Rap1p interaction, which brings them to telomeres.

Sir2 , 3, 4, and YKu bind telomeres and thus act directly to promote TPE. The three Sir ( s ilence i nformation r egulator) proteins are part of the Sir silencing complex, which is also needed for transcriptional repression at the two silent mating type loci, HML and HMR . The carboxyl terminus of Rap1p , the major sequence-specific telomeric binding protein, interacts with both Sir3p and Sir4p , while Sir4p interacts with Sir2p ( Moretti et al. 1994 ; Moretti and Shore 2001 ). Thus, Sir3p / Sir4p – Rap1p and Sir2p / Sir4p interactions recruit these silencing proteins to telomeres. Sir4p also interacts with YKu ( Tsukamoto et al. 1997 ), which provides a Rap1p independent pathway to recruit silencing proteins to telomeres ( Martin et al. 1999 ; Luo et al. 2002 ). Both recruitment pathways are essential for TPE.

After TPE is initiated at telomeres, it spreads several kilobases from the Rap1p -bound telomeric repeats into subtelomeric nucleosomes. This spread is mediated by protein–protein interactions between Sir3p and Sir4p with the N-terminal tails of histones H3 and H4 ( Hecht et al. 1995 ; Strahl-Bolsinger et al. 1997 ). Thus, deleting the amino terminal tails of histones H3 and H4 abolishes TPE ( Aparicio et al. 1991 ; Mann and Grunstein 1992 ; Thompson et al. 1994 ). Spreading also requires the histone deacetylase activity of Sir2p , as acetylation, especially of histone H4 K16, decreases Sir3 /4p–histone interactions ( Hoppe et al. 2002 ). Many other genes that modify histones or that regulate these modifications also affect TPE.

Early studies suggested that TPE requires proximity to a telomere, not just telomeric sequence, as an 81-bp internal tract of telomeric DNA does not silence an adjacent gene ( Gottschling et al. 1990 ). However, long (≥300 bp) internal tracts of telomeric DNA can repress transcription, even if the affected gene and adjacent tract are on a circular chromosome ( Stavenhagen and Zakian 1994 ). This phenomenon is called C 1-3 A- b ased s ilencing, CBS. The fraction of cells exhibiting CBS increases with the length of telomeric sequence, but CBS is never as effective as TPE. However, CBS acts synergistically with TPE as the closer an internal tract is to a telomere, the more effectively it silences. This synergism suggests a higher order chromatin structure, such as looping, that brings internal telomeric tracts close to chromosome ends. Consistent with their ability to silence, internal tracts of telomeric DNA efficiently bind Rap1p and Sir proteins ( Bourns et al. 1998 ).

TPE was discovered using truncated telomeres that lack X and Y′, so it was not clear from early studies if this regulation affects genes that reside near native telomeres. This possibility was first tested by inserting a marker gene near a telomere without deleting its subtelomeric repeats. By this assay, only 6 of 17 telomeres (only half of the telomeres were tested) are subject to TPE ( Pryde and Louis 1999 ). X-only telomeres are more likely than XY′ telomeres to silence. However, there is enormous variation in the TPE phenotypes of different telomeres. These differences are largely due to telomere-to-telomere variation in the identity and precise sequence of X and Y′. Subtelomeric sequences, especially X, contain recognition sites for different transcription factors such as Reb1p , Tbf1p , and Abf1p . Indeed, computational analysis of genome-wide ChIP data for 203 transcription factors finds that >10% of these show preferential association with the 25-kb regions next to telomeres ( Mak et al. 2009 ). These enrichments are particularly high in stressed cells. Of these transcription factors, some activate and others repress TPE while others contribute to boundary activity, which limits the spread of silencing. The effects of these transcription factors on TPE may differ from their effects on transcription at nontelomeric loci. For example, Reb1p promotes transcription of ribosomal RNA ( Morrow et al. 1989 ), but in subtelomeric DNA, Reb1p has boundary activity ( Fourel et al. 1999 ). The binding of transcription factors to subtelomeric repeats explains why silencing is propagated differently at truncated vs. natural telomeres. At telomeres like URA-TEL, TPE extends inward continuously but dissipates quickly as the marker gene is moved further from the telomere ( Renauld et al. 1993 ). However, at natural telomeres, domains of silencing are discontinuous.

TPE in the context of native telomeres is also assessed by examining mRNA levels and the effects of Sir3p on these levels for genes that are naturally located near telomeres. Thus, transcription of a Ty5 transposon near the III-L telomere is low in wild-type cells but higher in sir3 cells ( Vega-Palas et al. 1997 ). Genome-wide studies also provide insights into the biological importance of TPE. For example, the 267 yeast genes that are within 20 kb of a telomere produce about five times fewer mRNA molecules (average of 0.5/cell) than nontelomeric genes, providing support for the repressive effects of telomere proximity ( Wyrick et al. 1999 ). However, transcription of only 20 of these genes is Sir3p inhibited, and almost all such genes are very close (≤8 kb) to a telomere. Thus, from the classical view of TPE as a Sir-dependent phenomenon, very few genes are regulated by TPE. However, if criteria other than Sir3p dependence are used, many more genes are affected by telomere proximity. For example, in hda1 Δ cells, which lack a histone deacetylase, genes that are 10–25 kb from telomeres are specifically derepressed ( Robyr et al. 2002 ). Thus, Hda1p -sensitive regions are near telomeres but are distinct from Sir3p sensitive regions, which are even closer to telomeres. Unfortunately, none of these studies include the classic test to establish a position effect, which requires moving the gene away from the telomere and showing that its expression pattern is telomere dependent.

Many of the genes located near telomeres are members of multigene families. The functions of these telomere-regulated genes include rapamycin resistance ( Ai et al. 2002 ), stress responsiveness, and ability to grow in nonstandard carbon sources ( Robyr et al. 2002 ). For example, four of five members of the FLO gene family are near telomeres and are usually repressed except under nutrient conditions that promote pseudohyphal growth ( Guo et al. 2000 ; Halme et al. 2004 ). Many of the transcription factors that bind X repeats ( e.g. , Rox1p , Gzf3p , and Oaf1p ) regulate TPE in response to either stress or metabolic change ( Smith et al. 2011 ). Thus, almost all genes that are naturally regulated by TPE, whether or not this regulation is Sir3p or Hda1p dependent, are genes that are not expressed under standard growth conditions. This pattern suggests a genomic logic where rarely or situationally expressed genes are located near telomeres where transcription is usually low.

Higher order chromatin structure and telomere folding

In some organisms, including mammals ( Griffith et al. 1999 ) and plants ( Cesare et al. 2003 ), telomeres end in t-loops. T-loops are formed by G tails looping back and invading the duplex region of the telomere. This invasion displaces the G-rich strand to form a single-stranded displacement (D)-loop. T-loops are thought to contribute to telomere capping by sequestering the 3′ end of the chromosome within the telomeric tract ( Griffith et al. 1999 ). Alternatively (or in addition), t-loops may be recombination intermediates ( Cesare and Griffith 2004 ).

Throughout most of the cell cycle, G tails on S. cerevisiae telomeres are probably too short to form t-loops. However, yeast telomeres do form a higher order fold-back structure or telomere loop ( Strahl-Bolsinger et al. 1997 ). Unlike t-loops, which are held together by DNA base pairing, the yeast telomere loop is maintained by protein–protein interactions. Telomere looping was proposed as an explanation for why Rap1p can be cross-linked in vivo not only to the telomeric repeats but also to subtelomeric DNA. As subtelomeric DNA is histone (not Rap1p ) associated, and Rap1p does not interact with histones, its detection in these regions is explained by a fold-back structure that puts the Rap1p -bound telomeric repeats in proximity to subtelomeric chromatin. Yku80p binds both C 1-3 A/TG 1-3 repeats and X elements, and it too is proposed to contribute to telomere folding ( Marvin et al. 2009a ). High rates of transcription through a telomere eliminate TPE in cis ( Sandell et al. 1994 ) as well as telomere looping, suggesting that TPE might require this fold-back structure ( de Bruin et al. 2000 ). Probably the best evidence for the importance of telomere looping comes from gene expression studies ( de Bruin et al. 2001 ). A yeast u pstream a ctivating s equence (UAS) is similar to enhancers in other organisms, except that it affects transcription only when it is upstream of a gene. However, a downstream UAS can activate transcription if the affected gene is next to a telomere. Telomere looping, which is expected to bring the downstream UAS close to the gene’s promoter, is a possible explanation for why the UAS works in the downstream context. In addition to a possible role in TPE, telomere folding is proposed to protect the telomere from ectopic recombination ( Marvin et al. 2009a , b ).

Telomere organization in mitotic cells

Telomeres are clustered and at the nuclear periphery in many organisms. However, in most cases, it is not clear whether this pattern is functionally important or is just a passive consequence of the way chromosomes segregate at mitosis with centromeres leading the way and telomeres lagging behind. Yeast chromosomes are small and thus hard to visualize. As a result, the first suggestion for their nonrandom localization came from the subnuclear distribution of telomere binding proteins. Rap1p concentrates in 7–8 spots (called Rap1p foci) on the nuclear periphery ( Palladino et al. 1993 ). These Rap1p foci also contain Sir2p , Sir3p , Sir4p , and the YKu complex and ∼70% of the Y′ repeats ( Palladino et al. 1993 ; Gotta et al. 1996 ; Laroche et al. 1998 ). As these studies were done in diploid cells where there are 68 telomeres, the much larger number of telomeres compared to the number of Rap1p foci suggests that individual Rap1p foci contain many telomeres.

Although yeast chromosomes are too small to localize by fluorescent in situ hybridization (FISH), they can be visualized if they are marked with multiple binding sites for a GFP–DNA binding protein expressed in the same cells ( Robinett et al. 1996 ). This system confirmed that the VII-L telomere is located at the nuclear periphery in ∼50% of cells ( Tham et al. 2001 ). This fraction changes throughout the cell cycle, being particularly low after DNA replication. Peripheral localization of the VII-L telomere does not require Sir3p or Yku70p and thus is independent of both Rap1p foci and TPE. Other telomeres are also at the periphery but the fraction localized and their requirements for localization vary from telomere to telomere ( Hediger et al. 2002 ). Thus, as with TPE, individual telomeres have different behaviors in terms of subnuclear localization. This variation is explained in part by differences in the TAS content of different telomeres ( Mondoux and Zakian 2007 ). The GFP–DNA binding protein visualization method also confirmed telomere clustering, but found that the clusters are quite transient and do not involve specific subsets of telomeres ( Therizols et al. 2010 ).

A priori , association with the nuclear periphery requires at least two proteins, one that is telomere associated and one located at the nuclear periphery. There are at least two nuclear envelope proteins that affect telomere tethering, Esc1p ( e stablishes s ilent c hromatin; Andrulis et al. 2002 ) and Mps3p ( m ono p olar s pindle; Bupp et al. 2007 ). Esc1p resides at the inner face of the nuclear envelope and interacts with the C-terminal portion of Sir4p (called the PAD4 domain; Andrulis et al. 2002 ). The Esc1p – Sir4p interaction can tether plasmid and chromosomal telomeres to the nuclear periphery ( Andrulis et al. 2002 ; Taddei et al. 2004 ). However, the PAD4 domain also interacts with Yku80p , and this interaction also affects telomere tethering ( Taddei et al. 2004 ). Although Mps3p was discovered as an essential subunit of the spindle pole body (yeast centrosome), a fraction of Mps3p is in the nuclear envelope ( Jaspersen et al. 2002 ). Mps3p spans the inner nuclear envelope with its nonessential N terminus extending into the nucleoplasm where it can interact with telomere bound Sir4p or Yku. These interactions are important for telomere positioning as cells expressing the N-terminally truncated mps3 Δ75-150 allele are viable but unable to tether telomeres ( Bupp et al. 2007 ).

Sumoylation is also important for telomere tethering. The two known telomere parts of the tether, Sir4p and Yku80p , are both sumoylated in vivo by the SUMO E3 ligase SIZ2 , and this modification affects their tethering functions ( Zhao and Blobel 2005 ; Ferreira et al. 2011 ; Hang et al. 2011 ). In siz2 Δ cells, tethering is lost, but TPE and Rap1p foci are unaffected. For Yku80p , loss of tethering is probably a direct result of loss of sumoylation, as an Yku80p –SUMO fusion increases tethering, and this tethering is now Siz2p independent ( Ferreira et al. 2011 ).

Telomerase has also been implicated in telomere tethering. By two-hybrid and coimmunoprecipitation, Mps3p interacts with Est1p ( Antoniacci et al. 2007 ). In early S phase, tethering requires a specific interaction between Yku80p and TLC1 ( Schober et al. 2009 ), the same interaction needed for Est2p telomere binding in G1 phase ( Fisher et al. 2004 ). The Mps3p – Est1p interaction raises the possibility that telomere tethering might regulate telomerase. However, while mps3 Δ75-150 cells are tethering deficient ( Bupp et al. 2007 ), they have wild-type–length telomeres (M. Paul and V. A. Zakian, unpublished results). Nonetheless, telomeres in siz2 Δ cells are modestly longer than wild-type telomeres, and this lengthening is telomerase dependent ( Ferreira et al. 2011 ; Hang et al. 2011 ). Moreover, epistasis analysis suggests that Siz2p and Pif1p act in the same pathway to affect telomere length as telomeres are no longer in pif1 siz2 cells than in the absence of Pif1 alone ( Ferreira et al. 2011 ). Because pif1 cells have higher levels of telomere-bound telomerase ( Boule et al. 2005 ), this result led to the hypothesis that siz2 Δ telomeres are longer because they bind more telomerase. Furthermore, pif1 Δ restores telomerase-dependent tethering in siz2 Δ cells, presumably by increasing the amount of telomere-bound Est2p / Est1p ( Ferreira et al. 2011 ). These data lead to the somewhat contradictory view that telomerase tethers telomeres to the periphery in a manner that is not permissive for telomere lengthening, while release of telomeres from the periphery promotes telomerase lengthening of the released telomere. Perhaps, when telomeres are bound at the periphery by an Mps3p – Est1p interaction, Est1p cannot interact with Cdc13p in a productive way. Consistent with this view, artificially tethering a telomere to the periphery results in telomere shortening without affecting the lengths of other telomeres in the cell ( Mondoux et al. 2007 ). However, given that many telomere proteins are modified by sumoylation ( Hang et al. 2011 ), it is probably wise to be cautious in attributing the modest telomere lengthening seen in siz2 Δ cells to telomeres being lengthened preferentially when released from the nuclear envelope.

Tethering is lost in siz2 Δ cells, yet TPE and Rap1p foci are normal. These results seem to rule out a critical role for tethering in TPE ( Ferreira et al. 2011 ). This conclusion is consistent with experiments indicating that TPE and tethering are separable phenotypes ( Tham et al. 2001 ; Mondoux and Zakian 2007 ). However, this conclusion is still surprising, given numerous examples in diverse organisms for a connection between the nuclear periphery, heterochromatin formation, and gene silencing. Perhaps the importance of concentrating silencing proteins at the periphery is not to support silencing but to sequester silencing proteins from the rest of the genome so that actively transcribed genes are not inadvertently repressed ( Taddei et al. 2009 ).

Finally, telomere tethering has been suggested to affect recombination and repair of telomeric regions. Deleting YKU80 , which reduces the association of some telomeres with the periphery, increases recombination between telomeres and nontelomeric sites in a pathway that acts through Yku80p -associated X elements ( Marvin et al. 2009a , b ). These data suggest that telomere tethering suppresses ectopic recombination within telomeric regions. In contrast, efficient repair of subtelomeric DSBs may require telomere localization at the periphery as such breaks within the XI-L telomere are less often repaired in genetic backgrounds where telomere tethering is lost ( Therizols et al. 2006 ).

Telomeres in meiosis

It has been known for many years that meiotic chromosomes in most organisms assume a characteristic conformation called the bouquet in early prophase of the first meiotic division with telomeres clustered at the nuclear periphery at a position near the spindle pole body. Progress has been made in S. cerevisiae in learning how the bouquet is set up, although its functional significance is still being established. The S. cerevisiae NDJ1 was discovered in a screen for genes whose overexpression causes mis-segregation of meiotic chromosomes ( Conrad et al. 1997 ). Ndj1p expression is limited to meiosis, and it localizes to meiotic telomeres in vivo . Cytological studies show that telomere clustering is Ndj1p dependent, making NDJ1 the first gene linked to bouquet formation in any organism. Meiotic chromosome segregation is also defective in ndj1 cells, suggesting that the bouquet configuration is important for normal meiotic chromosome behavior. Ndj1p interacts with nuclear envelope-localized Mps3p ( Conrad et al. 2007 , 2008 ). Mps3p is the yeast member of the conserved SUN family of inner nuclear membrane proteins. SUN proteins interact with chromosomal binding proteins in the nuclear interior and with outer nuclear membrane proteins in the space between the inner and outer nuclear membranes. Because the outer nuclear membrane protein can directly or indirectly bind to the cytoskeleton, the formation of a linker complex involving Mps3p and Ndj1p is able to move chromosome ends within the nucleus into a bouquet using energy derived from the cytoplasmic cytoskeleton. In contrast to Schizosaccharomyces pombe and multicellular eukaryotes, meiotic bouquet formation in S. cerevisiae is actin -, not tubulin dependent ( Scherthan et al. 2007 ; Koszul et al. 2008 ).

Time-lapse imaging reveals that meiotic chromosomes engage in rapid and sustained movements throughout prophase in virtually all eukaryotes. These rapid meiotic chromosome movements were first documented in fission yeast where they are particularly dramatic ( Chikashige et al. 1994 ). However, fission yeast meiosis is unusual in that it occurs in the absence of synaptonemal complexes. Therefore, the discovery of similar movements during S. cerevisiae meiosis was important because it made clear that these movements are not restricted to organisms with an atypical meiosis ( Trelles-Sticken et al. 2005 ; Scherthan et al. 2007 ; Conrad et al. 2008 ; Koszul et al. 2008 ). In S. cerevisiae , meiotic chromosome movements are often rapid, in excess of 1 μm/sec and are dependent on the nuclear envelope protein, Csm4p , whose expression is also limited to meiosis ( Conrad et al. 2008 ). The outcome of meiosis in csm4 Δ cells suggests that meiotic chromosome movements are important for meiotic progression, in part by preventing spindle checkpoint activation ( Zanders et al. 2011 ). However, since Csm4p is also needed for bouquet formation, it is not clear whether its effects on meiotic progression are due to its role in telomere clustering or chromosome movement ( Wanat et al. 2008 ). Current models suggest that meiotic chromosome movements test homology between chromosomes to facilitate pairing and synapsis of homologous chromosomes.

Classical genetic studies showed that meiotic recombination occurs at lower levels near telomeres than in the rest of the genome ( Barton et al. 2003 ). Genome-wide mapping of meiotic DSB positions confirms that DSBs are infrequent near telomeres with the best estimate being that they occur 3.5-fold less often in the 20 kb closest to telomeres than in most other genomic regions ( Pan et al. 2011 ). Although the mechanistic basis for the relative paucity of meiotic recombination in telomeric regions is not known, a plausible explanation for its significance is to prevent exchanges between nonhomologous chromosomes.

Given the multiple genome-wide approaches available in S. cerevisiae , it is likely that most genes affecting telomeres are identified. However, the functions of many of these genes have not been explored. Moreover, we lack mechanistic information even for well-studied telomere proteins. For example, although Rif1p has been known for years to act in cis to inhibit telomerase-mediated telomere lengthening, the mechanism(s) by which it does so is not understood. Likewise, the Tel1p kinase is critical for telomere length regulation yet there is no consensus on its phosphorylation targets nor information on how these targets differ at telomeres vs. DSBs. It is not known how the highly abundant RPA complex, which binds in a sequence nonspecific manner to single-stranded DNA, is excluded from TG 1-3 tails, a binding that is expected to trigger a checkpoint-mediated arrest. Even though Est1p was the first identified telomerase subunit, its exact role and that of Est3p are only beginning to be understood. The recently discovered TERRA opens up a whole new area of possibilities for telomerase regulation. There is a lot of information on telomere tethering, yet its functional role is not resolved. Of particular interest is to establish how telomere tethering can be telomerase dependent while at the same time telomere release from the periphery promotes telomerase action. Research over the past years demonstrates that telomeres have individual personalities: they differ by their subtelomeric repeats, TPE behavior, and nuclear localization, suggesting that we will only fully understand the impact of genes and conditions on telomere behavior if we study individual chromosome ends. The list of important unanswered questions goes on and on, making it clear that yeast telomere biologists still have a lot to do.

What do we think will be particularly important for future advances? Biochemical approaches for yeast telomerase have long been thwarted by difficulties isolating telomerase proteins and reconstituting telomerase. Given recent success with in vitro assays, this is an area that will likely yield new insights in the near future. The impact of in vitro studies will be increased enormously by the large number of mutants that have been generated and characterized in vivo , reagents that are not available to anywhere near the same extent in other organisms. So far, there is no successful mass spectrometry on yeast telomerase, yet this approach has been extremely fruitful for both mammalian and ciliate research. The small size of yeast chromosomes has limited cell biological approaches in telomere research. However, the ability to visualize specific telomere regions with GFP technology has largely solved this problem. We anticipate that soon these chromosome visualization techniques will be combined with new methods to visualize telomerase itself to yield important information on telomerase dynamics vis a vis nuclear organization at the single-cell level. TERRA is so newly discovered that it seems inescapable that its continued analysis will provide new and perhaps unanticipated findings.

Research in ciliates and to a lesser extent yeasts, pioneered the field of telomere biology. In the past decade or so, there has been a lamentable decline in the number of labs doing telomere research in ciliates. As a consequence, the importance of yeast as a model organism for telomere research has, if anything, become more apparent. Many important discoveries on mammalian telomerase were inspired by work in yeast. We expect this trend to continue. Given the clear links between telomere biology and human aging and cancer, there is little doubt that basic research in yeast telomere biology has an important place in biomedical research.

We are grateful to B. Brewer, I. Dionne, K. Friedman, S. Jasperson, J. Lingner, D. Lydall, C. Nugent, and Y. Wu for their reading of the manuscript and for courageously providing valuable input. The V.A.Z. lab is funded by the National Institutes of Health (RO1’s GM43265 and GM026938); the R.J.W. lab has funding from the Canadian Institutes for Health Research (grants MOP97874 and MOP110982). We thank the considerable physical distance separating our two universities, which prevented us from killing each other over minor details during the writing.

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Saccharomyces cerevisiae Essay Example

• Pages: 15 (4046 words)
• Published: August 29, 2018
• Type: Case Study

How do honey (strained honey), aloe vera, urine, and saliva affect the respiration rate of Saccharomyces cerevisiae? 

Background Information

China is one of the states with the longest period of history. Beginning with the Shang Dynasty till present, China has been creating and inventing numerous materials including paper, gunpowder, and the compass among others. The most intriguing development is the Chinese medicine which uses, natural herbs. The incorporation of Chinese remedies into today’s medical practice is both astonishing and fascinating, especially without any scientific background. Examples of these traditional medicines include honey, aloe vera, urine, and saliva to relieve burns. Knowledge of these remedies and their application prompts my investigation on their use and reliability.

Respiration is a vital process for the survival of a living organism. According to Aberts et al., both eukaryotic and prokaryotic organisms perform cellular respiration to yield energy. Ce

llular respiration occurs in two ways, which include anaerobic and aerobic respiration (476). Prokaryotes are single-celled organisms lacking specialized organelles. Based on the absence of complex organelles, prokaryotes perform anaerobic respiration. The process takes place either across the plasma membrane or the cytoplasm. Comparatively, eukaryotic organisms carry out aerobic respiration through the mitochondria to produce sufficient energy for the efficient performance of the complicated process of the specialized organelles (453).

Yeast cell, Saccharomyces cerevisiae, commonly known as baker’s yeast is one of the single-celled eukaryote frequently applied in scientific research globally. Genome sequencing provides that genes and proteins present in the yeast cells are human homologs allowing scientist to use it in place of the human’s cell (“Jove”). Therefore, I prefer to use Saccharomyces cerevisiae to replace human skin cell (Keratinocyte) in investigating the effectiveness of

the four traditional healing solutions. 

Saccharomyces Cerevisia: Hypothesis and Explanation

The optimum pH of Saccharomyces cerevisiae is about 4 to 4.5. Honey displays antibacterial properties based on the presence of royalism, a potent antibacterial protein. The activity of the protein in honey works under a pH range of 3.2 to 5, attributed to the production of hydrogen peroxide H2O2. Comparatively, aloe vera has a pH of about 4.4 to 5.5 allowing it to display anti-inflammatory effects superior to 1% hydrocortisone cream or a placebo gel. Additionally, researchers claim that aloe vera gel may be useful in treating inflammatory skin conditions (University of Maryland Medical). Both honey and aloe produce positive results in treating burns. However, Saccharomyces cerevisiae works better in aloe owing to the lower pH range of aloe. Therefore, aloe vera is the best solution to regenerate cells.

Measuring cylinder ( )

Gas syringe ( )

Beaker  

Balance ( 

Stop watch 

1. Prepare 10g of honey, aloe, saliva and urine.

2. Pour 50cm3 of deionized water into four conical flasks, label them with A, B, C, D.

3. Mix A with 10g of honey, B with 10g of aloe, C with 10g of saliva, D with 10g of urine. 

4. Stir A, B, C, and D with a glass rod separately.   

5. Attach one

end of the delivery tube to a rubber bung.

6. Attach the other end of delivery tube to a gas syringe.

7. Using a stand and clamp, secure the gas syringe horizontally onto the clamp. 

8. Prepare 1.00g of Saccharomyces cerevisiae in a weighing boat on an electronic balance. 

9. Pour 1.00g of Saccharomyces cerevisiae into the conical flask A.

10. Make sure the conical flask A may be quickly and securely attached to gas syringe connected rubber bung.

11. Make sure gas syringe is set to indicate 0 cm3.

12. Start the stopwatch and take the initial reading of gas syringe.

13. Take the reading of gas syringe every 15 minutes for 1 hour and 30 minutes.

14. Repeat the procedure 5-13 by using the conical flasks B, C, and D.

15. Record down the index.

16. Repeat the steps 5-13 five times and gather all the data.

Saccharomyces Cerevisiae Facts: Safety and Ethical Considerations

Based on the possible hazardous effects of the test solutions, it is necessary to handle them efficiently to minimize their effects on the individual performing the experiment and the environment. Human urine produces ammonia which odor is non-pleasant; therefore, to reduce the diffusion of the odor, air tight containers are most appropriate to handle urine. Additionally, body fluids pose a biological threat based on the numerous virus and bacteria present. Additionally it is necessary to wear appropriate Personal Protective Equipment (PPE), especially to the individual performing the experiment. 

Table1: Amount of gas volume produced in respect to time and type of solution

According to Aberts et al., cellular respiration occurs through a series of actions beginning with glycolysis, which is the splitting of sugar molecules for the production of Adenosine Triphosphate (ATP). Conversion of glucose into pyruvate involves two phases requiring the use of enzymes. The first phase requires the intake of energy allowing for the rearrangement of the sugar molecules and the attachment

of phosphate on either end of the molecule (469). The unstable molecule, usually fructose-1, 6- bisphosphate formed from glucose splits into two phosphates carrying three carbon sugars. The second phase involves the release of energy from the sugars formed in the previous steps. The reactions of the second phase lead to the production of two molecules of ATP and one of Nicotinamide Adenine Dinucleotide (NADH) illustrated by the equation below.

C6H12O6+ 2NAD + 2ADP+ 2P                     2 pyruvic acid, (CH3(C=O) COOH+ 2ATP + 2 NADH+ 2H

In regards to Lushchak et al., Krebs cycle follows glycolysis leading to a complete breakdown of the sugar and the release of carbon dioxide. The process releases energy through the electrons moving across the mitochondrial membrane (9). Additionally, it utilizes acetyl coenzyme A (CoA) a product of the oxidation process of the pyruvate. Acetyl-CoA associates with carbon accepting molecules forming citrate, which is a six-carbon molecule. Re-arrangement of the molecule releases two of its carbon particles producing carbon dioxide and NADH. The other four carbons undergo various chemical reactions leading to the formation of ATP, the reduction of the energy carrier from Flavin Adenine Dinucleotide (FAD) to Flavin Adenine Dinucleotide FADH2 and NADH. The final step in respiration is the movement of charged electrons through the mitochondrial membrane. Consequently, the movement results in attraction of oppositely charged molecules leading to the production of ATP, the primary source of energy.

Effects of Honey on the Respiration Rate of Saccharomyces Cerevisiae Infection

Saccharomyces cerevisiae belongs to the yeast family one of the eukaryotic organism. Yeast absorbs sugar breaking down the molecules into simple sugars such as the

monosaccharides and generating the energy-rich ATP. According to Kwakman, Paulus, and Sebastian, honey comprises of the sugars fructose, glucose, and sucrose. Sucrose is a disaccharide composed of the sugar glucose and fructose. The breaking down of sucrose into glucose involves the enzymes sucrase and isomerase. Sucrase facilitates the splitting of sucrose into fructose and glucose. Comparatively, enzyme isomerase converts fructose into glucose for respiration (49).

The mechanism of the enzyme sucrose involves a process referred to as hydrolysis requiring water to break the chemical bonds. Acidic conditions enhance the process of hydrolysis separating the hydrogen molecule from the water molecule. The equation below illustrates the process leading to glucose formation.

C12H22O11+ H2O                       C6H12O6+ C6H12O6

Respiration involving the use of honey produces more glucose resulting in significant energy levels. Additionally, there is an increased production of carbon dioxide caused by the breakdown of glucose to release energy. Although the process begins at a slow rate, it proceeds with a rapid production of carbon dioxide and later reduces to the minimum production of the gas as the products of fermentation increase. Table1 illustrate the performance of honey about the volume of gas with respect to time. An increase in osmotic concentrations of the sugar reduces the amount of water necessary for the growth of the yeast. Consequently, increased sugar levels shift the movement of water in the yeast cells.

In regards to Bento et al., the exponential growth rate of the yeast cells provides high and rapid metabolism. The characteristic of the process begins with an oxidative phase during the exponential period and increased fermentation towards the end of the phase

(3). Variations in carbon dioxide production with time provide the evidence for the different characteristic of the growth phases of the yeast cells. Additionally, decreased respiration across the exponential growth rate indicates changes in the metabolism process with an increase in fermentation. The data on honey and the amount of gas volume produce presents a typical example of changes in rate of respiration as metabolism of the sugar proceeds.

It is imperative to consider the pH of honey as a factor affecting cellular respiration rate. Saccharomyces cerevisiae thrives best in an acidic condition within the ranges of 4 to 4.5. Comparatively, honey contains both organic and amino acids influencing its pH range of 3.9 to 6.1. Organic acids include acetic, lactic, citric, formic and gluconic acid. Additionally, it is found to contain aromatic and aliphatic acids. The acidic properties of honey enhance its use in medicine, especially in inhibiting the growth of the microorganism. As respiration progresses, release of carbon dioxide lowers the pH enhancing the activity of the yeast cells. Comparatively, the increased acidity discourages the performance of enzymes significantly reducing the amount of glucose present in the cells and limiting the amount of energy produced. 

Effects of Aloe Vera on the Respiration Rate of Saccharomyces Cerevisiae Disease

The aloe vera plant provides numerous benefits as a result of its antibacterial properties. According to Marzieh et al., it promotes the growth of cells facilitating its use in the study (367). As for the chemical composition of the plant, it consists of vitamins, enzymes, sugars, amino acids, and minerals. The enzymes, with examples of the amylase, lipase, and acidic phosphatase contribute to digestion of particular biochemicals. The sugars comprise

of both the monosaccharides and the polysaccharides originating from tissue layer neighboring the parenchyma tissues.

The structure of the polysaccharides found present in aloe vera consists of long chains of mannose and glucose. Molecules of the mannose sugar occur in two forms of either the pyranose or the furanose structure. The metabolism process of the sugar involves the process of phosphorylation using the enzyme hexokinase yielding mannose-6-phosphate. Consequently, mannose-6-phosphate under the influence of the enzyme phospho-mannose isomerase leads to its conversion to the fructose-6-phosphate that undergoes glycolysis to produce glucose and the waste products of either anaerobic or aerobic respiration.  The breakdown of the polysaccharides in the Aloe Vera produces greater amounts of glucose resulting in increased energy levels in the cells. Results in Table 1 indicate that aloe vera produces the largest volume of the gas a result of metabolism of the polysaccharides present. According to Marzieh et al., in a study on the effects of Aloe Vera on fish skin cells, the plant increases the skin thickness. Additionally, the plant creates a hydrated and viscous environment around the mucosa cells and the epidermal cells creating a barrier to entry of pathogenic components to the body of the organism (369). The outcome of the research indicates that with increased doses of the plant concentration, the cell density increases enhancing the defense mechanism against the pathogen. Comparatively, Pranab, Ajit, Gogoi and Neeraj describe that both Aloe Vera and yeast are medicinal plants incorporated into the feed to enhance immunity (93). Study on the effects of the plant and yeast in the development of muscle indicates that yeast promotes the development of normal flora. It alters the

rate of metabolism through the action of the enzymes to improve the digestion process. Comparatively, Aloe Vera through the presence of the flavonoids provides an increased rate of metabolism increasing the levels of glycogen in both the liver and muscles (95).

Based on Tan, Benny, and Vanitha, the polysaccharide in Aloe Vera, acemannan, modulates the immunity in the host cells. The structure of the polysaccharide includes β (1, 4)-linked acetylated mannans, which act to increase phagocytosis (1425). The presence of polysaccharides, flavonoids, and proteoglycans in Aloe Vera influences their application in medicine to prevent the activity of microbes on the host cells (1427). About the pH level of Aloe Vera and its effects in influencing respiration rate in yeast, the plants’ pH range is 4.5 to 5.5. Its pH range enhances the activity of the yeast cells through providing an optimum pH for processes such as metabolism and respiration. The value corresponds with that of the skin providing a conducive and natural environment for the regeneration of cells. The acidic components of the plant promote the lower pH value limiting the activity of microorganism. 

Personal protective equipment (PPE) refer to equipment designed to reduce vulnerability of workers to serious injuries and illnesses which may result from contact with physical, chemical, electrical, or other hazards at workplace (Health and Safety Executive, 2013). Protective equipment includes gloves, earplugs, respirators, and protective glasses. PPE should only be used as a last resort when a hazard cannot be completely eliminated because it does not reduce hazard or give total protection (Health and Safety Executive, 2013). I t is the duty of employers to make the workplace safe for all employees, and

employee safety can be guaranteed through training and supervision, providing instructions, use of safety procedures, and PPE. Type of PPE used is determined by nature of tasks and hazard exposure.

History of Anti Saccharomyces Cerevisiae PPE

Use of PPE as safety a safety measure was inspired by equipment used in military due to the dangers associated with war. History of PPE can be traced to several years back when soldiers wore full body armor, which included protective headgear and face gear, when fighting their enemies to protect them from being killed (United States Department of Labor, 2017). Blacksmiths also wore hand gear and aprons or shields back in the Middle Ages to protect them from molten metal they were working on. 

Initially, use of PPE was not compulsory and there were no regulations on employee protection until the enactment of the Occupational Safety and Health Act (OSHA) of 1970 (United States Department of Labor, 2017). The Occupational Safety and Health Act advocated for a countrywide protection of workers from work-related injuries in the U.S. In April 1971, the U.S. Secretary of Labor James Hodgson incorporated OSHA into the Labor Department with the responsibility of investigating and preventing work-related accidents, and defining appropriate PPE for every job description in a company (United States Department of Labor, 2017). PPE has advanced as a result of OSHA’s initiatives, and industries or individuals who aspire to ensure safety of their workers.

Hazards and PPE Used

Use of PPE varies depending on the type of hazard an individual is exposed to, and part of the body at risk of harm. Below are description of type of hazard, part of the body affected, and the PPE

applicable in each case.

i. Whole body

Workers in different industries or companies can be exposed to hazards that can affect their entire body, such as excess heat, chemical splash, and leaks from spray guns (United States Department of Labor, 2017). In such a situation, boiler suits, chemical suits, aprons, and disposable aprons can be used to protect workers from injuries at workplace. Furthermore, materials used to make the PPE should be chemically impermeable, fire resistant, and clear for visibility. 

ii. Head and Neck

A worker’s head and neck are at risk of injuries from falling objects, extreme temperatures, hair getting tangled in machines, and head bumping. Hairnets, bump caps, industrial safety helmets, neck scarves, and firefighters’ helmet can be used to mitigate the injuries (United States Department of Labor, 2017). Tailor-made eye or ear protection can be fitted in the headgear, and damaged head protection should be replaced. 

Hazards that can harm eyes of workers and other individuals in industry environment include dust, gas, vapor, chemical splash, and projectiles. Employers and business owners can provide face screens, face shields, goggles, and safety spectacles to ensure no harm comes to individuals within their premises. The best eye protection should be effective and should fit the user perfectly.

In an industry environment, especially manufacturing industries, operation processes produce high sound that may affect ears individuals close to the manufacturing plant or machine. Impacts of noise can be reduced by use of earmuffs, earplugs, and canal caps. The type of ear protector should correspond to the type of work, and should be able to reduce to acceptable levels while making communication possible (United States Department of Labor, 2017). In addition, the

employer should ensure that employees know how to fit the ear protectors for effectiveness.

v. Hands and Arms

Most tasks in a company are performed using hands and arms, exposing them to risks such as to cuts, electric shocks, vibration, radiation, and scrapes. To reduce risk of injuries, employees can be provided with gauntlets, gloves, and long-sleeved wear that covers the arm. Safety measures to be observed by employees when using the PPE are; care in choice of glove used as not all gloves are fit for all conditions, avoid using gloves when operating machines because the gloves might tear, and avoid wearing gloves for a long period to avoid skin problems. 

vi. Feet and Legs

Employees’ feet and legs are exposed to workplace hazards such as slipping, cuts, falling objects, heavy loads, metal and chemical splash, and wet, hot and cold conditions. Safety boots and shoes with protective toecaps, foundry boots, and chainsaw boots can help in protecting feet and legs of individuals at the workplace. In addition, the type of boots worn should have sole pattern that can prevent slips in different conditions such as oil or chemicals.

Hazards that pose threat to lungs or respiratory system of individuals include gases, dust, vapors, and oxygen-deficient environments. Safety measure that can help in reducing risk of individuals developing lung problems caused by work environment the use of respiratory protective equipment such as face pieces, half masks, and full masks (United States Department of Labor, 2017).  Respirators used should have filters that are effective depending on the hazard an individual is exposed to, fit properly, and allow breathing. Breathing apparatus can also be used in areas with limited oxygen

to avoid cases of losing consciousness.

PPE can only serve its purpose when properly used, and used in the right situation. PPE should be designed in such a way that they are safe and comfortable to use and proper maintenance practice carried out to ensure that PPE are clean and in good condition. It is the duty of employers to provide PPE to their employees and ensure that the PPE are properly used by training employees on how to use and detect faults in PPE, when to use, and limitations to the type of hazard that a given PPE can protect them from. A company can implement a PPE program to look into company issues like hazards present, selection, maintenance, and use of PPE, training of employees, and constant checks on the program’s effectiveness. 

OSHA has set PPE standards for different industries such as construction, marine terminals, general industry, and shipyard employment, and requires PPE categories match standards established by American National Standards Institute (United States Department of Labor, 2017). Worker’s rights to PPE are protected by OSHA, and employees can complain to OSHA if they feel that serious hazards exist within their work environment and are not catered for by their employer.  To provide efficiency while using PPE, OSHA suggests that employers should ensure that PPE items that can be used together do not interfere with their individual performance, for example wearing goggles may interfere with respirator seal and cause air leaks.

When selecting PPE items for their employees, employers should have knowledge of the number of people exposed to a specific hazard, the duration in which they are exposed, and the volume or level

of hazard in question.  PPE items available in a company should be enough to cover all employees who are at risk of injury or infection should be able to cover the employees for the duration they are working in a hazardous environment, and be able to withstand the volume of hazard an employee is exposed to. For example, a respiratory mask should have enough breathable air for the duration an employee will be stuck in an area of short air supply.

PPE Regulation.

Regulation of implementation of PPE and protection of workers is done out by OSHA.  OSHA requires employers to pay for employees’ PPE, and the PPE used comply with the set standards, and workers who have their own PPE can only use the equipment if it can adequately protect them from hazards at workplace (United States Department of Labor, 2017). Examples of PPE that must be provided by the employers include face shields, metatarsal foot protection, rubber boots with steel toes, and firefighting PPE. However, employers are not obligated to cater for some PPE such as daily long-sleeve clothing, long pants, winter coats, sun creams, and normal work boots. 

Employers should ensure that they adhere to the standards and regulations set by OSHA because contrary to this, they may attract penalties. A breach of work health and safety arises when a person is put at risk of injury, illness or death occurs, no steps are taken to avoid risky situations, and failure to comply with regulatory requirements (Queensland Government, 2017). Examples of breaches of workers’ safety are; working at with no control over risk of falling, allowing unskilled workers to operate machines, and exposing workers excessive

Failure of companies to adhere to safety of their employees attracts penalties from OSHA depending on the magnitude of the offence (United States Department of Labor, 2017). An employer who intentionally and repeatedly violates workers’ safety may receive a civil penalty of $ 70,000 for each violation, an employer who has failed to correct safety violation attracts a penalty of $ 7,000, while a person who gives prior notice to a company about inspection to be conducted without permission from the secretary of OSHA is fined $ 1,000 or imprisonment for not more than six months. 

Employers and business owners should take measures to ensure that safety of employees and other individuals who visit company premises is guaranteed. Training employees on how to use PPE and constant monitoring and supervision of the implementation of PPE program is the best way to facilitate effectiveness of PPE items and promote employees’ safety. Employers should also avoid unnecessary charges on their companies by adhering to the regulations and standards of OSHA.

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Probiotic Yeast Saccharomyces : Back to Nature to Improve Human Health

Rameesha abid.

1 Department of Biotechnology, University of Sialkot, Sialkot 51310, Pakistan; moc.liamg@uaqilarafaj

2 National Agriculture Research Center, National Institute of Genomics and Agriculture Biotechnology (NIGAB), Islamabad 44100, Pakistan; moc.oohay@lamka_arikahs

Hassan Waseem

3 Department of Biological Sciences, Muslim Youth University, Islamabad 44100, Pakistan; [email protected]

Shakira Ghazanfar

Ghulam muhammad ali.

4 Pakistan Agricultural Research Council (PARC) 20, Ataturk Avenue, G-5/1, Islamabad 44000, Pakistan; moc.liamg@5ilamgrd

Abdelbaset Mohamed Elasbali

5 Department of Clinical Laboratory Science, College of Applied Sciences-Qurayyat, Jouf University, Al-Jouf P.O. Box 2014, Saudi Arabia

Salem Hussain Alharethi

6 Department of Biological Science, College of Arts and Science, Najran University, Najran 66262, Saudi Arabia; as.ude.un@ihtrahlahs

Associated Data

Not applicable.

Saccharomyces cerevisiae var. boulardii is best known for its treatment efficacy against different gastrointestinal diseases. This probiotic yeast can significantly protect the normal microbiota of the human gut and inhibit the pathogenicity of different diarrheal infections. Several clinical investigations have declared S. cerevisiae var. boulardii a biotherapeutic agent due to its antibacterial, antiviral, anti-carcinogenic, antioxidant, anti-inflammatory and immune-modulatory properties. Oral or intramuscular administration of S. cerevisiae var. boulardii can remarkably induce health-promoting effects in the host body. Different intrinsic and extrinsic factors are responsible for its efficacy against acute and chronic gut-associated diseases. This review will discuss the clinical and beneficial effects of S. cerevisiae var. boulardii in the treatment and prevention of different metabolic diseases and highlight some of its health-promising properties. This review article will provide fundamental insights for new avenues in the fields of biotherapeutics, antimicrobial resistance and one health.

1. Introduction

According to the latest definition of the World Health Organization, probiotics are active microbes that stimulate the growth of other probiotic bacteria in the gut and possess beneficial health effects to the host [ 1 ]. These microorganisms are able to produce anti-carcinogenic, antioxidant and anti-mutagenic agents and induce protection against different bacterial diseases including diarrhea and respiratory tract infections. Saccharomyces cerevisiae var. boulardii is the most significant probiotic yeast species. S. cerevisiae var. boulardii is a eukaryotic organism that has been used in scientific investigations since the time of its discovery [ 2 ]. This model organism has unique importance because of its alterable and flexible genome. The genome of S. cerevisiae var. boulardii was completely sequenced in 1950 and a genome size of approximately 11.3 Mb was reported. It has approximately 6000 genes and 275 additional tRNA genes. Almost 23% of the S. cerevisiae var. boulardii ’s genome is homologous to the hominid genome. This specific yeast is best known for its role in treating gastrointestinal diseases [ 3 , 4 ].

S. cerevisiae var. boulardii has gained the importance of the scientific community due to the production of different bioactive compounds [ 5 ]. This specie is an excellent protein source with high amino acid content, which is essential for the production of various foods and cosmetic supplements [ 6 ]. S. cerevisiae var. boulardii is also responsible for the formation of glutathione, an important antioxidant used in the food and drug industry [ 7 ]. The inactivated cells of S. cerevisiae var. boulardii are used as a rich protein source in probiotic feed supplements. Despite its high protein content and antioxidant nature, the thick and indigestible cell wall and high nucleic acid content limit the use of inactivated cells of S. cerevisiae var. boulardii in human food and nutrition. It can enhance its antioxidant properties by increasing the production of phytochemical constituents, such as isoflavones. S. cerevisiae var. boulardii is used preferably due to its unique digestible properties of starch and proteins. Reduction in trypsin-inhibitor activity and phytic acid content is responsible for its digestible behavior [ 8 ].

The oval to round cell shape of S. cerevisiae var. boulardii is composed of approx. 3 µm thickness and 2.5–10.5 µm length. This yeast is able to reproduce sexually and asexually by budding and unification [ 8 ]. The cell wall of S. cerevisiae var. boulardii is composed of a rigid inner polysaccharide layer with a 1,3-β-glucan branched structure while the outer layer is made up of mannoproteins. The total mass of S. cerevisiae var. boulardii in terms of dry weight is almost 30% and the estimated total polysaccharide and protein contents are 85% and 15%, respectively. Biochemical characterization of S. cerevisiae var. boulardii confirmed the presence of glucose, mannose and N -acetylglucosamine up to 90%, 20% and 2%, respectively. Glucose to glucose interaction is associated with β-1,3 and β-1,6 linkages. β-1,3 glucan is responsible for the elasticity and strength of the yeast cell wall. The lateral cell wall of S. cerevisiae var. boulardii is composed of straight chitin chains of 1–2% of total dry weight [ 9 ].

The nutritional value of S. cerevisiae var. boulardii is enhanced due to the presence of different minerals, vitamins and antioxidant compounds. Dietary yeast is composed of iron, manganese and copper, some trace minerals are also reported, i.e., ferric, manganic sulfate and cupric acetate [ 10 ]. Studies suggested that several toxic metals are easily accumulated by S. cerevisiae var. boulardii , which includes lead, cadmium, arsenic and mercury [ 11 ]. Nutritional yeast has the ability to enhance the energy level in an individual because of the presence of non-proteinaceous amino acids, proteinaceous amino acids and vitamin B, such as biotin, doxine, thiamin, vitamin B12 and riboflavin. It can also reduce antinutrient phytate levels and enhance the synthesis of folate. S. cerevisiae var. boulardii can also protect from bacterial infections along with increasing the glucose sensitivity to enhance the growth of skin, nails and hair [ 5 ].

Recently, it was observed that medical professionals are using nonpathogenic S. cerevisiae var. boulardii in the treatment of gut-related diseases. Clinical studies claimed that oral administration of S. cerevisiae var. boulardii can treat multiple gastrointestinal diseases including Traveler’s diarrhea [ 12 ], AIDS-associated diarrhea [ 13 ], antibiotic-associated diarrhea [ 14 ], Clostridium difficile- associated syndrome [ 15 ], Irritable Bowel Syndrome [ 16 ] and Crohn’s disease [ 17 ] ( Figure 1 ). This yeast can be used for the treatment alone or can be administered in combination with other probiotics resulting in enhanced treatment efficiency. One hundred grams per day consumption of S. cerevisiae var. boulardii can induce beneficial effects on human health. S. cerevisiae var. boulardii cells have the ability to stick on the gastric and intestinal linings of the mucosa and actively prevail in the gastrointestinal tract of animals and humans [ 18 ]. The antineoplasmic effects of S. cerevisiae var. boulardii were reported with major findings. Oral administration of S. cerevisiae var. boulardii can inactivate epidermal growth factor receptor (EFR), which can further suppress EGFR-Erk and EGFR-Akt pathways resulting in induced apoptosis in tumor cells and reducing the level of cell colony formation and cancer cell proliferation. In vitro study claimed that S. cerevisiae var. boulardii consumption can inhibit the expression of HER2, HER-3 and IGF-1R genes which leads to the prevention of intestinal neoplasia [ 19 ]. Diarrhea caused by the continuous use of antibiotics can also be treated by S. cerevisiae var. boulardii in adults and children ( Figure 1 ) [ 20 ]. A study claimed that this yeast can also effectively work against chronic permeability in patients with Crohn’s disease when administered orally for 3 months [ 21 ]. In HIV-linked diarrhea, exposure to a 3 g per day dose of S. cerevisiae var. boulardii can produce beneficial health effects [ 22 ]. The dose of S. cerevisiae var. boulardii in case of chronic diseases should be increased to meet the treatment criteria. Recently, an upsurge in multidrug-resistant organisms is reported due to the excessive consumption of antimicrobials [ 23 ]. Global healthcare authorities are trying to create awareness all over the globe via antibiotic stewardship programs, but the severity of antimicrobial resistance is continuously increasing [ 24 ]. To cope with this alarming situation, probiotics, especially S. cerevisiae and S. cerevisiae var. boulardii yeast, can be considered as an alternative method for the treatment of bacterial and fungal infections. A number of research and review articles describing the probiotic potentials of yeast and bacteria have been published in the last decade. A comprehensive review is needed to highlight the probiotic potential of S. cerevisiae var. boulardii in various aspects. Therefore, the aim of this review is to explore the diverse probiotic potential of S. cerevisiae var. boulardii through the combination of different meta-analyses. Utilization of S. cerevisiae var. boulardii as an alternate to antibiotics for the treatment and eradication of different metabolic diseases is investigated. Moreover, details of commercially available probiotic strains of S. cerevisiae var. boulardii and their clinical and beneficial detailed effects are provided. Finally, the significance of S. cerevisiae var. boulardii against cancer signaling cascades and safety attributes regarding its consumption among humans and livestock animals are thoroughly discussed.

Health-promoting effects of S. cerevisiae var. boulardii .

2. Enzymatic Potential of S. cerevisiae var. boulardii

S. cerevisiae var. boulardii can produce different enzymes which play a significant role in various industrial processes ( Figure 2 ). Some active enzymes, i.e., maltase and invertase, have the potential to enhance the flavor of fermented products specifically in the food industry. Maltase is responsible for the conversion of malt sugar into normal sugar while invertase converts granulated sugar into regular sugar. Another enzyme, zymase, transforms normal sugar into CO 2 and alcohol [ 25 ]. S. cerevisiae var. boulardii is able to produce intestinal enzymes including amylase, protease, cellulase and lipase and is unable to synthesize galactosidase, DNAase and gelatinase ( Figure 2 ) [ 26 ]. S. cerevisiae var. boulardii has antibacterial properties due to the presence of extracellular protease enzymes and cell surface hydrophobicity [ 27 ]. This yeast enhances the concentration of the enzymes by the production of polyamines that trigger the cells of the intestine. Cell surface hydrophobicity is responsible for the adherence of S. cerevisiae var. boulardii yeast to the cell wall lining of the human intestine. S. cerevisiae var. boulardii is critically responsible for the production of ethanol in anaerobic conditions. This species can also show tolerance against a high level of ethanol and gastric discharge, including bile salts and intestinal acids, hence, eliminating toxic bacterial strains from the host body in the form of fecal matter [ 28 ]. Interestingly, this yeast can work against both Gram-positive and -negative bacteria and boost the host immunity. Despite its antibacterial properties, S. cerevisiae var. boulardii also showed resistance against all broad and narrow-spectrum antibiotic drugs and does not disturb the normal microbiota of the gastrointestinal tract of the host [ 28 ].

Industrial significance of S. cerevisiae var. boulardii- based enzymes.

3. Factors Responsible for the Efficiency of S. cerevisiae var. boulardii as a Probiotic

Probiotics are being used to enhance treatment efficacy and to produce significant health benefits. S. cerevisiae var. boulardii is a unicellular, cost-effective active yeast species that has probiotic potential and is often used as a nutritional additive [ 29 ]. Different modes of actions were observed in favor of the host and against the antigenic microorganisms which include luminal action: (1) Antimicrobial activity: (a) Reduction in the intestinal bacterial growth [ 30 ], (b) lowering of gastrointestinal translocation of microbes [ 31 ], (c) nullifying the effect of bacterial pathogenicity [ 32 ], (d) reducing the binding affinity of the host cell with the bacterial population [ 33 ]. (2) Antitoxin effects: (a) obstructing the pathogenic receptor active sites [ 30 ], (b) enhancing the production of antibodies against Clostridium difficile toxin A [ 34 ], (c) mediating the synthesis of the phosphatases enzyme against Escherichia coli (E. coli ) [ 35 ], (d) cleavage of pathogenic enzymatic proteins [ 35 ]. (3) Trophic action associated with intestinal linings: (a) reducing the expression of tumor necrosis factor-alpha (TNFα) gene and inhibiting programmed cell death [ 36 ], (b) enhancing the synthesis of glycoprotein in the intestinal brush border [ 37 ], (c) inducing the production of intestinal polyamines [ 37 ], (d) repairing fluid transport pathways [ 37 ], (e) stimulating the production of membrane enzymes (28). (4) Mediation of immune system: (a) stimulating the production of regulatory T cells [ 32 ], (b) enhancing the level of IgG antibody against Clostridium difficile toxin A [ 34 ], (c) improving the adherence of WBCs (White Blood Cells) to the endothelial cells [ 38 , 39 ].

Probiotics have gained global beneficial additive status to use as a potential feed supplement [ 40 ]. Human probiotic administration is based on the development and viability of probiotics in the intestinal lumen of the host organisms. Probiotic yeast has more survival chances in the stomach due to the presence of digestive enzymes, bile and gastrointestinal juices in comparison to probiotic bacteria [ 41 ]. The Food and Drug Administration (FDA) has approved certain probiotic strains which are potentially used for the benefit of humans, but S. cerevisiae and S. cerevisiae var. boulardii are the only probiotic yeast species that are commercially used for human benefits ( Table 1 ) [ 42 ].

Commercially available probiotic yeast products.

S. cerevisiae var. boulardii has surpassed the affectivity of the commonest probiotic bacteria, i.e., lactobacillus due to its resistance against different antibiotics [ 43 ]. S. cerevisiae var. boulardii can be administered to patients as an alternative source of antibiotics due to its outrageous antibacterial properties. Probiotic consumption can also reduce the pathogenicity of harmful microbes present in the human gut [ 44 ]. The different strains of Saccharomyces sp. , including S. boulardii , S. cerevisiae , and S. unisporus , also showed antibacterial and antiviral properties. These strains were used to enhance the probiotic potential of different human food supplements. These probiotic strains are also effective against acute and chronic diarrhea. The combination of S. cerevisiae var. boulardii with other probiotics of the same or different genus can also enhance the efficacy of human feed supplements [ 45 ].

Several intrinsic and extrinsic factors are directly implicated in the efficacy of S. cerevisiae var. boulardii as a probiotic ( Figure 3 ).

Factors affecting the efficacy of S. cerevisiae var boulardii .

3.1. Temperature Fluctuations

S. cerevisiae var. boulardii strains can work effectively at a temperature range of 22–30 °C ( Table 2 ), while other S. cerevisiae var. boulardii strains are functional at 37 °C temperature and some can survive below 20 °C temperature. As a probiotic, S. cerevisiae var. boulardii is present in the form of capsules. The heat-dried S. cerevisiae var. boulardii capsules could not survive at 25 °C after opening due to their reduced potency. They retain their efficacy when stored at a 4 °C refrigerator. Lyophilized S. cerevisiae var. boulardii capsules can survive at room temperature and are viable for 1 year approximately. Studies suggested that S. cerevisiae var. boulardii can grow best at 37 °C. However, the death phase of this yeast usually appeared at 55–56 °C [ 46 , 47 ].

Parameters for the survival of S. cerevisiae var. boulardii .

3.2. Water Activity a w and Relative Humidity

Water activity a w and relative humidity can produce synergistic effects. Survival of S. cerevisiae var. boulardii can be influenced by water activity. A study reported that the cells of S. cerevisiae var. boulardii showed a 0.98% value of water activity when refrigerated at −20 °C, which ultimately increased the survival rate of the yeast S. cerevisiae var. boulardii ( Table 2 ). However, reduced water activity conditions can deteriorate the viability of S. cerevisiae var. boulardii ’s cells. Water activity can also be influenced via relative humidity, specifically in the case of opened or uncovered foods. The viability of S. cerevisiae var. boulardii is reduced when the rate of water activity and environmental humidity decreases [ 48 ].

3.3. pH and Acidity

S. cerevisiae var. boulardii showed resistance against less pH and more acidic conditions. However, some yeast species are too fragile to bear such a hard environment. The ideal pH range for S. cerevisiae var. boulardii development and maturation is 2–8 ( Table 2 ). Yeast species belonging to a genus other than Saccharomyces showed tolerance against extreme acidic and alkaline environments. Overall functionality of the yeast is better in the lyophilized form [ 29 , 47 , 51 ].

3.4. Antimicrobial Agents

S. cerevisiae var. boulardii showed antimicrobial properties due to these subsequent reasons: (i) synthesis of the extracellular enzyme, i.e., protease, it aids in the formation of colonic mucosa, (ii) excretion of toxins and SO 2 gas, it can halt the efficacy of toxins released by Clostridium difficile , (iii) secretion of enzyme-based proteins, (iv) cell surface hydrophobicity and autoagglutination, it is responsible for the attachment of S. cerevisiae var. boulardii to the patient’s intestinal lining. The viability of different Gram-positive and -negative bacteria can easily be reduced by the negative influence of S. cerevisiae var. boulardii on the host organism [ 26 ].

3.5. Nutrient Media for the Growth of S. cerevisiae var. boulardii

Yeast can grow on different nutrient media including Yeast extract peptone dextrose media, Sabouraud dextrose agar, but Oxytetracyclin yeast agar media (OGA) is considered as the best medium for its growth ( Table 2 ). Standard OGA media can be prepared by adding 8 g of yeast extract, 9 g of glucose, 12 g of nutrient agar and 0.1 uL of oxytetracycline in 500 mL of distilled H 2 O. Carbon (glucose, maltose, sucrose and fructose), nitrogen (Urea, peptone and powdered yeast extract) and a trace amount of minerals (zinc, copper, magnesium, sulfur) are required to enhance the growth of S. cerevisiae var. boulardii [ 49 , 50 ]. All these factors play a significant role in maintaining the viability of S. cerevisiae var. boulardii. The potential of this beneficial yeast may be disturbed when the optimum conditions of both intrinsic and extrinsic factors changes.

4. Clinical Significance of S. cerevisiae var. boulardii as a Probiotic in Acute and Chronic Diseases

4.1. acute diseases, 4.1.1. antibiotic-associated diarrhea.

Antibiotic-associated diarrhea (AAD) occurs due to the continuous consumption of antibiotics for a longer period. The use of probiotics, mainly S. cerevisiae var. boulardii, is the commonest method for the treatment against AAD. A total of 8 (80%) out of 10 (100%) controlled experiments confirmed the efficacy of S. cerevisiae var. boulardii for the prohibition of AAD specifically in adult patients ( Table 3 ). The beneficial impact of S. cerevisiae var. boulardii and the relative decline in AAD comparable to the control are categorized in the range of 7.4% and 25%, respectively [ 29 ]. The affectivity of S. cerevisiae var. boulardii against AAD in the pediatric population has also shown positive outcomes. Results of two meta-analyses confirmed the potential of S. cerevisiae var. boulardii against AAD with a pooled risk ratio of 0.47 and 0.43 and a 95% confidence interval [ 52 ].

Per capsule/tablet dose of S. cerevisiae var. boulardii for the treatment of different acute and chronic diseases.

4.1.2. Clostridium Difficile Infection (CDI)

Clostridium difficile (C. difficile) is a Gram-positive anaerobic rod-shaped bacteria that may cause antibiotic-associated Clostridium difficile diarrhea. It is responsible for the colon infection, it shows diarrhea (mild) to colon damage (severe) symptoms. Meta-analysis of six randomized control trials of different Saccharomyces strains including S. cerevisiae var. boulardii showed efficacy against CDI with a total risk ratio of 0.59 [ 53 ].

The beneficial impact of S. cerevisiae var. boulardii and the relative decline in CDI comparable to the control were categorized within the range of 19% and 33.3%, respectively ( Table 3 ). S. cerevisiae var. boulardii can prevent diarrhea caused by toxin A. It can also suppress colon inflammation and can block the intestinal toxin receptor sites via protease liberation. It can modulate the immune response by stimulating the production of IgA immunoglobulins. Moreover, probiotic use can block the activation of several kinases, Erk1/2 and interleukin 8 expression ( Figure 4 ) [ 53 ].

Pathways associated with S. cerevisiae var. boulardii .

4.1.3. Acute Diarrhea

S. cerevisiae var. boulardii administered to patients involved in two randomized control group trials showed clinical efficacy against acute diarrhea as compared to the control ( Table 3 ). S. cerevisiae var. boulardii consumption among 100 patients of an age less than 15 for 7 days resulted in reduced stool frequency and stabilized the normal stool condition [ 54 ]. A meta-analysis conducted among more than 600 patients that administered the S. cerevisiae var. boulardii probiotic strains for 60 days significantly reduce the rapid stool frequency [ 55 ]. Another meta-analysis of seven randomized controlled trials claimed to stabilize the childhood diarrhea consistency within 24 h as compared to the placebo treatment [ 56 ].

4.1.4. Persistent Diarrhea

Two randomized controlled trials suggested that S. cerevisiae var. boulardii significantly enhances treatment efficacy specifically in children with persistent diarrhea ( Table 3 ). The beneficial impact of S. cerevisiae var. boulardii and the relative decline in persistent diarrhea comparable to the control was 50%, respectively. However, a meta-analysis of S. cerevisiae var. boulardii against persistent diarrhea among pediatric and young populations has not been performed up till now [ 57 ].

4.1.5. Enteral Nutrition-Related Diarrhea

Diarrhea is the major complication associated with total enteral nutrition (TEN) and can also cause fluctuations in short-chain fatty acids (SCFA). Diabetes, gastrointestinal infection and malabsorption-related disorders are responsible for diarrhea-associated TEN ( Figure 5 ). Schneider et al. reported that patients who received S. cerevisiae var. boulardii can significantly enhance the levels of short-chain fatty acids in 10 TEN patients as compared to the normal controls. This treatment could increase the SCFA level in high stool frequency. The beneficial impact of S. cerevisiae var. boulardii and the relative decline in TEN-associated diarrhea comparable to control are categorized within the range of 5% and 8.2% in three randomized control trials, respectively [ 37 ].

Major causes of diarrhea.

4.1.6. Traveler’s Diarrhea

Traveler’s diarrhea is a common digestive illness that is responsible for frequent stool discharge. It occurs due to the intake of contaminated food or water ( Figure 5 ). Twelve randomized control trials of S. cerevisiae var. boulardii and other probiotic strains significantly reduced the severity of the infection caused by Traveler’s diarrhea in children ( Table 3 ). The beneficial impact of S. cerevisiae var. boulardii and the relative decline in Traveler’s diarrhea comparable to the control are categorized within the range of 5% and 11% in two randomized control trials, respectively [ 58 ].

4.2. Chronic Diseases

4.2.1. cancer.

S. cerevisiae var. boulardii is being potentially used to inhibit cancer cell development and progression ( Figure 4 ). It was observed that this probiotic yeast can reduce the tumorigenic effects of colorectal cells in humans. In vivo, high-throughput metagenomic analysis of 281 stool samples confirmed that S. cerevisiae var. boulardii has significantly inhibited colorectal cancer metastasis by stimulating cancer cell apoptosis and promoting gastrointestinal health via immune modulation. S. cerevisiae var. boulardii significantly downregulates the expression of various tumor-causing genes including TNFα, Interleukin-1β and Interleukin-17, the expression of NF- k B and mTOR signaling cascades was also inhibited ( Figure 4 ). However, the activity of different cytokines was not affected by S. cerevisiae var. boulardii treatment. HCT116 and DLD1 colorectal cell lines were used to analyze the apoptotic behavior of colorectal cells after the administration of S. cerevisiae var. boulardii . Results confirmed the presence of an enhanced percentage of apoptosis in probiotic yeast-treated cells [ 59 ].

4.2.2. Ulcerative Colitis

Broad-spectrum antibiotics are conventionally used to treat ulcerative colitis but due to antibiotic resistance, their efficacy has been reduced by a substantial level. Probiotics, especially S. cerevisiae var. boulardii and its derivatives, act as an alternative method for the maintenance of normal gut microbiota and help to treat chronic colitis diseased patients [ 60 ]. Studies suggested that the pathogenic strain of E. coli, known as adherent-invasive E. coli (AIEC), showed a strong binding affinity with the small intestinal lining of Crohn’s disease patients. This Gram-negative bacteria can easily invade the intestine of patients. Patients with Crohn’s disease showed strong adherence between AIEC bacteria due to its FimH adhesion potential and overexpressed mannose residues, which are present on the surface of intestinal glycoprotein CEACAM6 (carcinoembryonic antigen-related cell adhesion molecule). In vivo results reported that S. cerevisiae var. boulardii significantly blocked the adherence potential of LF82 to the intestinal brush border. Probiotic yeast also lowered the pro-inflammatory cytokine level and was confirmed to treat the pathogenesis of ulcerative colitis [ 61 ].

4.2.3. Crohn’s Disease (CD)

Typically, Crohn’s disease is a part of chronic inflammatory bowel disease, which can cause digestive inflammation, abdominal pain, weight loss, watery stool and malnutrition. Individuals with chronic Crohn’s disease may also come across inflammation of skin, liver, joints, anemia, kidney stones and maldevelopment. Bacteria associated with Crohn’s disease can damage the gastrointestinal tract (GI), especially the small intestine, colon and can cause erratic and multiwall GI inflammation. Dalmasso and colleagues reported that consumption of S. cerevisiae var. boulardii can significantly reduce the level of CD, can control chronic inflammation and reinforce epithelial reformation [ 62 ]. In a pilot study, 31 patients with Crohn’s disease were randomly treated with S. cerevisiae var. boulardii or an antimicrobial drug for 12 weeks. Patients treated with probiotics considerably reduced colonic permeability as compared to the antimicrobial drug-treated patients. Another pilot study of 20 patients with Crohn’s disease, who were administered S. cerevisiae var. boulardii for 49 days, showed remarkable improvement in the patient’s health ( Table 3 ). S. cerevisiae var. boulardii consumption after steroidal therapy does not produce health-promoting effects on Crohn’s disease patients [ 22 ].

4.2.4. Vaginal Candidiasis

Vaginal Candidiasis can be considered the most common fungus-associated vaginal infection globally. Candida albicans is the major causative agent of this disease. Vaginal Candidiasis is typically caused by large antibiotic consumption, which produces fluctuations in the normal composition of the vaginal microbiota. Studies have reported the efficacy of oral and intramuscular administration of S. cerevisiae var. boulardii- based probiotics [ 63 ] ( Table 3 ). Vaginal inoculation of S. cerevisiae var. boulardii live yeast or inactivated whole yeast can significantly lower the growth of Candida albicans in mice vaginas. Both these yeast types cause S. cerevisiae var. boulardii and fungus interaction which results in prohibiting the cohesion of Candida albicans to the vaginal epithelial cells. Probiotic administration can significantly reduce the pathogenicity of Candida albicans by lowering its ability to transform itself from yeast to mycelium and the capability of exhibiting aspartyl proteases. However, the efficacy of live yeast is greater as compared to the inactivated whole yeasts [ 63 ].

4.3. Health Benefits of S. cerevisiae var. boulardii as a Probiotic

4.3.1. antibacterial and antiviral properties.

The efficacy of S. cerevisiae var. boulardii on gastrointestinal microbiota has been critically investigated. S. cerevisiae var. boulardii can opt for different modes of action for antibacterial and antiviral activities in the human gut, which includes: (i) direct inhibition of pathogenic intestinal microbes and normalizing the pH of the gastrointestinal tract by reducing the pathogenicity of toxic microorganisms, (ii) producing an indirect impact on the gut microenvironment, (iii) producing an immunomodulatory effect on the host body [ 64 ]. The antibacterial effects of S. cerevisiae var. boulardii against different Gram-positive and -negative bacterial and viral pathogens including Bacillus anthracis , Shigella , E. coli , Vibrio cholera , Helicobacter pylori , C. difficile , Salmonella and Rotavirus have been previously reported [ 65 ]. S. cerevisiae var. boulardii can adhere to the toxin released by Vibrio cholera and inhibit its activity. The enhanced fluidity of sodium and chloride produced by Vibrio cholera can significantly be reduced by S. cerevisiae var. boulardii via inhibition of cyclic adenosine monophosphate-induced chloride secretion. Therefore, probiotic yeast can directly treat C. difficile disease by targeting its toxins and receptors. This infection can also be prevented by the action of the protease enzyme of S. cerevisiae var. boulardii against receptors and bacterial toxins. Moreover, this yeast can also block the pro-inflammatory pathways which are triggered by the toxins of C. difficile . It can also inhibit the expression of IL-8 and Erk1/2 genes and the activity of the NF- K B pathway ( Figure 4 ) [ 66 ]. Anthrax is a bacterial infection that is produced by virulence factors with a protective antigen, lethal factors and edematogenic factors, these peptides are responsible for causing morphological changes in the epithelial cells of the host [ 67 ]. The probiotic potential of S. cerevisiae var. boulardii against Salmonella enterica Typhimurium has been analyzed previously. Studies suggested that probiotic yeast can reduce the morbidity and mortality rate of the disease caused by pathogenic S. Typhimurium bacteria. It can limit the entry of bacteria into the host intestinal epithelial cells by inactivating the Rac pathway ( Figure 4 ). S. cerevisiae var. boulardii sticks to the surface of pathogenic bacteria and reduces its multiplication and growth by accelerating bacterial excretion via the stools [ 68 ]. S. cerevisiae var. boulardii can also produce antibacterial properties against peptic ulcer disease caused by Gram-negative Helicobacter pylori bacteria, which cause gastrointestinal tract infection and chronic gastric inflammation in the infected stomach. S. cerevisiae var. boulardii decreases the cytokine and chemokine levels into the stomach and significantly produces IgA antibodies against the toxic Helicobacter pylori [ 66 ]. This probiotic yeast is also effective against infection caused by viruses including rotavirus. It can suppress the level of oxidative stress in the host cells that are infected with rotavirus and also reduce the Cl - excretion caused by rotavirus [ 69 ].

4.3.2. Immune System Modulation

The probiotic effect of S. cerevisiae var. boulardii on the human immune system has been thoroughly investigated. The mechanisms that are mediated by the action of S. cerevisiae var. boulardii yeast are: (i) stimulation in the host immune activity, (ii) production of immunoglobulins, (iii) synthesis of cytokines and chemokines, (iv) assistance in the development of immune cells and (v) stimulate immune priming [ 70 ]. In a clinical study, S. cerevisiae var. boulardii administered to a child suffering from gastroenteritis showed a considerable rise in the IgA levels and a reduction in CRP (C-reactive protein) levels. After a 7-day treatment, a significant increase in the rate of CD8 lymphocytes in the S. cerevisiae var. boulardii -treated group as compared to the control group ( Figure 4 ) [ 71 ]. A combination of yeast and bacterial probiotic is capable of treating child-associated diarrhea. Results showed a significant increase in immune system modulation and CD3+, CD4+ and Th1/Th2 levels. Moreover, the clinical outcomes of the diarrheal disease were remarkably increased in the probiotic-treated group [ 72 ]. When the pathogenic bacteria enter into the gastrointestinal tract of the host, S. cerevisiae var. boulardii releases IgA antibodies which bind to the bacteria and excrete it from the host’s body via feces. S. cerevisiae var. boulardii consumption frequently increased the release of IgA antibodies when the host is exposed specifically to C. difficile toxin A [ 73 ]. A study on germ-free mice reported that yeast raised the level of IgM, cytokines and the total number of liver macrophages and cleared the infection caused by pathogenic bacteria from the host intestine in the treated group [ 74 ]. Briefly, S. cerevisiae var. boulardii can mediate different hormonal and molecular responses which are responsible to inhibit the activity of intestinal pathogens. Generally, the defensive mechanism of probiotic yeasts against several toxins is executed by stimulating the production of cytokines and interleukin (IL)-1β, IL-12, IL-6, TNFα, and IL-10 [ 75 ]. The in vitro and in vivo studies of S. cerevisiae var. boulardii showed significant modulation in the host early immune response, through this, the host body can show resistance against most microbial communities. It can also keep the equilibrium between pro and anti-inflammatory immune responses by the upregulation of several cytokines and inhibit the immune cell proliferation and maturation [ 8 ].

4.3.3. Antioxidant Properties

S. cerevisiae var. boulardii showed comprehensive antioxidant properties in the previous studies. S. cerevisiae var. boulardii extracted from the fermentation of guajillo pepper showed 66.1% alleviation in cholesterol level when placed in the incubator for 2 days. In a study, DPPH (1,1-diphenyl-2-picryl-hydrazyl free radical) assay calculated 63% of the total antioxidant potential of S. cerevisiae var. boulardii [ 34 ]. A DPPH scavenging assay of S. cerevisiae var. boulardii yeast also showed 2.3 mgTE/L antioxidant activity, which is beneficial for the manufacturing of beer with enhanced probiotic potential. Moreover, this assay also exhibited a 40% antioxidant level of S. cerevisiae var. boulardii yeast extracted from different Brazilian local fermented foods [ 76 , 77 ]. Studies suggested that S. cerevisiae var. boulardii whole cells possess superior antioxidant properties as compared to its extracts, it can be due to the presence of a high level of 1/3-b-D-glucan in the S. cerevisiae var. boulardii cell wall structure. Insoluble glucan and metabolites including phenyl ethyl alcohol, vitamin B6, cinnamic acid, vanillic acid and erythromycin are responsible for high antioxidant properties [ 78 ]. In a comparative study, raw and miscellaneous S. cerevisiae var. boulardii extracts were investigated for antioxidant level by DPPH test, superoxide radical scavenging assay by the total number of bioactive compounds. Results of this study confirmed the maximum antioxidant potential of S. cerevisiae var. boulardii raw extracts as compared to other extracts. S. cerevisiae var. boulardii antioxidant properties also showed beneficial effects on clinical therapeutics [ 79 ]. Another study demonstrated that S. cerevisiae var. boulardii can induce antioxidant activities of gastrointestinal-induced oxidative stress. In a human organ culture study, S. cerevisiae var. boulardii showed a reduction in the level of oxidative stress specifically in the rotavirus infected cells via human gastrointestinal examination [ 80 ].

4.3.4. Control of Antibiotic Resistance

S. cerevisiae var. boulardii showed resistance against both broad and narrow-spectrum antibiotic drugs. However, it cannot resist antifungal drugs and therapies. It is the most suitable and active therapeutic agent for the prevention and medication of all diarrheal-associated diseases which are specifically caused by the fluctuation in the normal gastrointestinal microbiota in patients with continuous antibiotics consumption for a longer run. Furthermore, bacterial probiotics are unable to exhibit such properties [ 34 ]. In order to increase the quality of functional food, different probiotic strains have been added to human foods and dietary supplements. The safety of human administered probiotics is significant due to their resistance against various antimicrobials [ 43 ]. Antibiotics are considered the fundamental tool to fight against pathogenic bacteria. These pathogenic microorganisms can acquire antibiotic resistance genes against advanced antibiotic drugs [ 81 ]. This resistance mechanism can negatively affect the treatment strategy against common bacterial infections [ 82 ]. Studies suggested that probiotic bacteria and yeast can act as the reservoir of antibiotic resistance genes. Antibiotic resistance in probiotic strains due to intrinsic or extrinsic mutations does not harm the host gastrointestinal tract. Moreover, they are useful to regain the lost gut microbiota of the host after continuous antibiotic intake. However, these probiotics can horizontally transpose resistance genes in harmful microbes. Tetracyclin and vancomycin resistance genes have been observed in various food and gut microbes [ 83 ].

5. Mechanisms of Action of S. cerevisiae var. boulardii Yeast

The responsibility of the host gut microbiota is not limited to just providing protection against pathogenic microbes [ 84 ]. It can also contribute to various mechanisms including cellular adhesion, reestablishment of lost gut microbiota, mediation of cancer signaling cascades, competition with pathogenic microbes, mucin production and regulation of nutritional and trophic effects. Adequate administration of S. cerevisiae var. boulardii can target and eliminate disease-causing microbes from the gastrointestinal tract of the host [ 85 ].

5.1. General Mode of Actions

Host gut dysbiosis due to the pathogenic microbial attack may reduce the overall probiotic bacterial load in the host gastrointestinal tract which may cause inflammation and secondary infections [ 86 ]. The adhesion potential of S. cerevisiae var. boulardii against pathogenic microbes may actively contribute to neutralizing the mechanism of antigen translocation from the gastrointestinal tract to other parts of the host body [ 87 ]. Continuous administration of S. cerevisiae var. boulardii for several weeks can stabilize the host gut microenvironment by reducing the severity of the disease and eventually eradicating the disease from the host body. Some probiotics are frequently eliminated from the host body, but before the elimination, they would have significantly modulated the host immune system. While other probiotics may recognize and bind to the active sites of the host intestinal mucosal layers [ 88 ]. Mucin production by intestinal epithelial cells of the host may also be influenced by the presence of probiotics in the host gut. Both pathogenic and beneficial microbes compete for binding to the gastrointestinal tract of the host. Cell wall proteins and mannose residues of S. cerevisiae var. boulardii are responsible for the direct binding of probiotic yeast to the intestinal receptors and reducing the probability of pathogenic microbes binding to the active sites [ 89 ]. However, if pathogens already adhere to the active sites, then probiotic administration may significantly induce the expression of exogenous sugars which can obstruct the binding of pathogenic microbes to the intestinal mucosal layers.

5.2. Mechanisms of Cancer Signaling Cascades

Cancer is considered as a major public health concern globally [ 90 ]. It is the leading cause of death not only in developing countries but also in developed countries. This deadly disease is a combination of more than 100 different diseases [ 91 ]. There are different types of cancer; all types have the same origin which is the abnormal growth of the cells. The growth rate of healthy and cancerous cells are different, healthy cells grow and proliferate in a controlled manner resulting to keep the body alive, while tumor cells grow in an abnormal fashion leading to cause anti-apoptotic effects [ 92 ]. To reduce the expression of oncogenes and protooncogenes, several clinical therapeutics including anticancer drugs, chemotherapy, radiotherapy and other strategies are conventionally used, however, the use of probiotics acts as an alternative treatment method for cancer prevention.

S. cerevisiae var. boulardii can significantly induce cancer signaling cascades by upregulating the expression of apoptotic proteins and downregulating the expression of protooncogenes and oncogenes. A recent study investigated the anti-tumorigenic activity of S. cerevisiae var. boulardii against gastric cancer cell lines and analyzed total cellular viability, apoptotic effects and activity of survivin gene after 3 days. Results of this study reported that targeted probiotic yeast significantly reduced the level of cellular viability, which stimulate apoptosis and lowered the activity of the survivin gene in gastric cancer cells ( Figure 4 ). This study strongly recommends the use of S. cerevisiae var. boulardii as a potential anti-gastric-cancer treatment therapy [ 93 ]. The probiotic potential of this yeast was also reported against human colorectal cancer cell lines (HT-29) and animal models. To evaluate the efficacy of S. cerevisiae var. boulardii on cell growth, development and apoptosis, this yeast was thoroughly spread over the HT-29 cells by using 4′,6-diamidino-2-phenylindole (DAPI) dye and 3-(4,5-dimethylthiazoyl-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The expression profiles of PTEN/caspase-3, Bclxl and RelA genes were evaluated by real-time PCR [ 94 ]. After 24 h, the activity of PTEN and caspase-3 gene was increased. However, the expression of Bclxl and RelA genes was significantly reduced ( Figure 4 ). After 2 days, the MTT assay showed inhibition in the growth of probiotic treated HT-29 cells. In another study, 1,3-beta-glucan part of the S. cerevisiae var. boulardii yeast showed anti-neoplastic effects on rat colon cancer cells when treated with dimethylhydrazine and S. cerevisiae var. boulardii orally [ 94 ]. Chen et al. reported that S. cerevisiae var. boulardii consumption can significantly block the activity of epidermal growth factor receptors when exposed to the targeted yeast and inhibit the Erk and Akt pathway ( Figure 4 ). According to the results, S. cerevisiae var. boulardii reduced the growth and proliferation of cancer cells and induces cancer cell apoptosis [ 19 ].

6. Discussion

Probiotics are non-digestible constituents of food, and when added in food or diet, confer useful and healthy effects to the host and stimulate the growth of a confined quantity of colon bacteria [ 95 ]. Natural strains of S. cerevisiae var. boulardii observed harsh environmental conditions as compared to the strains artificially cultured in the lab. This probiotic yeast has advanced conventional survival strategies which ensure its viability for the long run [ 96 ]. Mostly, the natural strains of this beneficial yeast are present in the nutrient-enriched soil environment. Some other environmental habitats of S. cerevisiae var. boulardii are the leaves and trunk surfaces of different medicinal and non-medicinal plants. It is also naturally present in intact grapes and other citrus fruits [ 27 ]. The natural transmission of this yeast to the human body is possible by the consumption of grapes, grape wine and different fruits. Studies suggested that this yeast is also insect-borne and is observed in wasps, Drosophila and other insects. These insects absorbed S. cerevisiae var. boulardii by feeding on the grapes and other fruits [ 97 ]. S. cerevisiae var. boulardii has shown direct and indirect effects on functional (fermented) food stuff. Direct effect indicates host-organism relationship, while indirect effects demonstrate the biogenic upshot (due to taking of microbial metabolites as a result of fermentation). This advances towards the efficient consequences of probiotics that seem to be applied in non-dairy food items as products related to chocolate, chewing gum, biscuit, honey, cereals, cakes, dressing, sweetness and tea [ 98 ]. In general, S. cerevisiae var. boulardii in the food industry somehow has difficulty in its multiplication and survival rate because of the distress conditions of the gastrointestinal tract [ 99 ].

To ensure the shelf-life of probiotics, novel probiotics are being designed through microencapsulation technology that opposes environmental conditions. Various factors can contribute to the beneficial aspects of probiotics but its proper mechanism of action is still vague. Studies suggested that lactation performance of the dairy animals was improved by S. cerevisiae var. boulardii yeast supplementation. It is found that the increased milk yield might be due to the stimulatory effect of probiotic yeast on the animal microbiota, which in turn increases cellulose digestion [ 100 ].

The presence of functional food in animal diets is responsible for increasing the productivity of livestock. Livestock can significantly improve human nutrition by providing essential nutrients in the form of milk, meat, and eggs [ 101 ]. An inadequate diet can drastically damage the health of livestock and reduced the overall yield. Due to poor feeding, animals are generally suffering from digestive and respiratory diseases leading to insufficient digestion and consequently retarded growth and productive performance. Dietary supplementation of S. cerevisiae var. boulardii is a viable and safe option for farmers to enhance the production of lactating dairy cattle and heifers [ 102 ]. This yeast has gained the Generally Recognized As Safe (GRAS) status from the Food and FDA, thus, can significantly be used to improve the animal feed supplements [ 103 ]. Moreover, the probiotic dose administered to animals is dependent on the (i) composition of feed, (ii) age of the animal, (iii) physical health of the animal and (iv) nature of the digestive system of the animal [ 29 ].

S. cerevisiae var. boulardii showed its applications in the wine industry for the benefit of humans. The natural grape was considered as a potential habitat of S. cerevisiae var. boulardii due to its high sugar content and acidic pH [ 104 ]. This yeast can significantly cope with all the fermentation stresses of the environment and has gained “the wine yeast” status and it is considered an important component of the wine industry globally. Moreover, the biosynthesis of primary and secondary alcohols is responsible due to their fermenting ability [ 105 ]. Theobroma cacao grains are significantly used for the manufacturing of chocolate. Fermentation of cacao grains can reduce the bitter and acrid effects of these grains. Direct exposure of cacao grains to probiotic yeast can induce fermentation reaction resulting in the production of ethanol and useful secondary metabolites [ 106 ]. The pectinolytic enzymes of S. cerevisiae var. boulardii can potentially metabolize citric acid produced by cacao grains. The increased growth of S. cerevisiae var. boulardii under high pH and stress conditions contributed to ethanol production [ 104 ]. S. cerevisiae and S. cerevisiae var. boulardii have remarkable applications in the bread and bakery industry. Sourdough, water and flour mixture are required for the manufacturing of bread. Various types of flours are commercially available which include spelt, barley, maize, einkorn, rye, khorasan, sorghum and many others [ 107 ]. Probiotic yeast and lactic acid bacteria are the main components of sourdough. A total of 2% of the fermenting yeast is added for the biosynthesis of bread. Atmospheric oxygen enters into the dough during dough mixing, which is adequately consumed by the yeast cells. Moreover, in an oxygen-limited environment, the rate of yeast cell reproduction was hindered and dough started to rise due to the fermentation process [ 108 ].

7. Conclusions

Continuous upsurge in multidrug-resistant organisms is responsible for causing millions of deaths annually. To control the spread of antimicrobial resistance, probiotic yeast S. cerevisiae var. boulardii can be considered as an alternative method for the treatment of bacterial and fungal infections. Several clinical and therapeutic studies confirmed the efficacy of S. cerevisiae var. boulardii against different pathogenic gastrointestinal diseases. The probiotic nature of this yeast has surpassed the effectiveness of different probiotic bacteria due to its gut microbiota protection potential. This probiotic yeast can actively participate in the manufacturing of bread, bakery products, wine, chocolate and large-scale bioethanol production. The consumption of S. cerevisiae var. boulardii in adequate amounts can also enhance the overall yield of milk and meat in poultry and livestock. The prescribed S. cerevisiae var. boulardii dose can also reduce the probability of co-morbidities that are caused by the continuous consumption of antibiotics for a long period. The combination of S. cerevisiae var. boulardii with other probiotics can enhance the treatment efficacy and reduce the pathogenicity of the disease. Despite its beneficial aspects, the use of this probiotic yeast should be according to the prescription of a physician. Moreover, this study will open up new insights for the development of novel probiotic strains, which will reduce the transmission of antimicrobial resistance genes among humans and farm animals.

Acknowledgments

A.M.E. extends his appreciation to the Deanship of Scientific Research at Jouf University for funding his work through Research Grant number DSR-2021-01-0363.

Author Contributions

R.A., H.W. and S.G. conceptualization, data analysis and wrote the first draft of the manuscript; G.M.A. revised the tabulated data and helped in graphical work; R.A. helped in data collection and drawing figures; J.A. critically revised the article; A.M.E. and S.H.A. provided funding and su-pervision. They also critically revised the article. All authors have read and agreed to the published version of the manuscript.

This article received external funding.

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