Chapter 7

work in progress

Leaven in a molecular era.

Not only does yeast now serve as one of the most important organisms throughout domestic history, in recent years it has also substantially contributed to biological research. The numerous molecular techniques that have evolved in yeast have allowed it to make an important contribution to a number of areas in science. Through studying various types of yeast and other microbes, scientists now know a great deal about the molecular processes involved in cell division, rapid evolution and disease. Large-scale experiments involving computers, robotics and new molecular techniques, such as polymerase chain reaction (PCR), to amplify genes, and DNA micro-arrays, that arrange hundreds of these amplified genes onto chips, have generated such a large amount of data that new scientific disciplines, eg., genomics, transcriptomics,  have evolved in order to process it all into meaningful results.

Fortunately, individuals with skin diseases are no longer thought of as unclean and are normally treated within the community. Scientists have greater understanding of disease management and although quarantine and hygiene are still practiced they are now carried out in order to reduce disease transmission. In the majority of cases, people are not ostracised when they are infected by disease, although fears and anxieties can still be generated through sensational media coverage. Nevertheless, even in this molecular age, some transmissible diseases are still associated with sins of the flesh and can lead to social ostracisation.

Yeast colonies in an array. Each spot contains thousands of yeast cells. Plate shows synthetic lethal interactions when the interaction of 2 or more genes cause cell death (shown by colonies with reduced/no growth colonies).
Image: Masur

There are still many diseases that generate fear because they are untreatable. Some of these have evolved through human activities, such as Bovine spongiform encepthalopathy (BSE) which gives rise to a human form of spongiform encepthalopathy called variant Creutzfeldt-Jakob Disease (CJD). The causative agent of BSE is a defective version of a protein called prion that is similar to one found in the brains of sheep with Scrapies. The prion protein is transmitted horizontically and causes disease through disrupting the normal function of the native protein. Studying the molecular mechanisms by which proteins change conformation to become prions in yeast has led to a greater understanding in the pathology of this disease. Many other human diseases, especially cancers, can be researched by studying molecular processes first in yeast.

Cancers arise when cells begin to divide abnormally due to mutations in DNA. Cancer research investigates the mechanisms that encourage these mutations to arise. The mechanism of cell division is often studied in fission yeast, Schizosaccharomyces pombe. Unlike Saccharomyces cerevisiae, which divides by budding, S. pombe divides symmetrically in a similar way to human cells. Fission yeast originates from Africa were it is found growing on banana skins and is used to ferment beer. Through research in this area scientists have reached many milestones in the mechanisms that have caused various cancers leading to greatly improved clinical treatments. Work yeast genetics has greatly contributed to our understanding of cell cycle research and has led to the award of a Nobel prize in 2001 to three scientists who led pioneering work in this area. The three scientists were: Paul Nurse, for his work in S. pombe and human model systems; Leland Hartwell, for his work in S. cerevisiae; and Tim Hunt who used sea urchins as a model system. Researchers later found similar cell division genes in human genomes.

Scanning electron micrograph of Fission yeast (Schizosaccharomyces pombe).
Image: David O Morgan.

In addition to investigating diseases, yeast is also used as a model system to research ageing. Saccharomyces cells can divide by budding a number of times but the new bud is always physiologically younger than the mother cell. Each cell produces about thirty buds depending on the environmental conditions and other factors and then it dies. About thirty genes in yeast have already been found to be involved in ageing. The main factors seem to be related to metabolic capacity, resistance to stress, gene dysregulation and genetic stability. Encountering certain environments that would overload any of these factors would also affect longevity. For instance, excessive oxidative damage or radioactivity would lead to a high level of mutations that will reduce the number of times that a cell can bud. Excessive oxidation is associated with the consumption of calories; so caloric restriction should result in increased longevity. This has been demonstrated in yeast, limiting the amount of nutrients and carbohydrates available in growth medium leads to a longer generation time and life span.

The development of yeast molecular biology can literally be used to assess the impact that the application of scientific research has had on 21st century society. Scientific researchers often describe yeast as the workhorse of eukaryotic molecular biology with many laboratories devoted to studying this single-celled organism, as much of the information derived from it can be equally applied to the study of human cells.

Most modern laboratory strains of yeast originate from one particular Saccharomyces cerevisiae strain, EM93, isolated from dried figs in Merced, California in the 1930’s byEmil Mrak. This strain turned out to be heterothallic, meaning that cells existed as two types of sterile haploids, with a single copy of each gene, that when fused together formed a fertile diploid that could perform meiosis in a similar way to that seen in human cells. Up until this point most strains studied were homothallic, this meant that all haploid cells were of the same mating type and capable of fusing together to form a fertile cell known as a zygote. The emergence of a heterothallic strain meant that the genetic stability of a culture could be placed under greater control, as it would remain haploid until the other haploid type was introduced and then through the production of mating phermones followed by cell fusion a diploid cell could be created.

Green Fluorescent report gene transformed into yeast cells.

Another reason why yeast is used as a molecular model system alongside other well-known microbes, such as Escherichia coli, is because it is a eukaryote. E. coli and other bacteria are prokaryotes, in contrast to eukaryotes they only have one chromosome housed in a cell without a nucleus. In yeast cells, DNA is packaged in chromosomes stored in a nucleus in a similar way as in human cells. Yeast has 16 individual chromosomes compared to 23 in humans. Surprisingly, there are only four chromosomes in the multi-celled fruit fly Drosophila another model organism used for biological research. Yeast also has the advantage of being able to grow just as happily with one set of chromosomes, in haploid cells, as with two or more sets of chromosomes, diploid and polyploid respectively. Additionally, as yeast is a single celled organism without the complexity of cellular differentiation it can be used to study the cell-cycle at a fundamental level. It can be used to study mitosis and meiosis. Many mutations that cause human disease are introduced during meiosis. Following cell fusion or mating, two haploid cells form diploids which can produce four individual haploid cells known collectively as an ascospore. After microscopic dissection of the ascospores, researchers can study recessive mutations and the complicated exchange of genetic material during meiosis by counting the numbers of surviving progeny. The information derived from yeast studies aids the study of genes involved in tissue development and cell differentiation in higher eukaryotes, such as Drosophila. Adding to all these factors many of the biochemical and cellular functions in yeast are conserved in human cells. Yeast therefore is a simple and practical system to study the mechanism of human cell division.So why has yeast become such a popular organism to study molecular biology and why is this microbe chosen in favour of others microorganisms? Firstly,Saccharomyces is non-pathogenic and does not present a threat to human safety. Therefore laboratory workers do not require expensive protective equipment to practice research. Saccharomyces is also easy to contain as is not usually airborne unless transported involuntarily by animals and insects. Another reason  is the ease by which it is cultured. Yeast can be grown easily and only requires a suitable carbon source, nutrients and appropriate physical conditions to continue multiplying. Additionally, these requirements can also be used to control the rate of cell division, for instance, by altering temperature or by creating metabolic mutants. Mutants are generated either through using a mutagen or by manipulating DNA through genetic engineering. Genes involved in yeast metabolism can be mutated and then used as molecular markers. For instance if the genes for the requirement of an essential amino acid are defective then the yeast will not grow without that amino acid added to its immediate environment. If the defective gene is artificially replaced by a functional one then the yeast cell will be able to continue growing without the need for that particular amino acid. Armed with this knowledge researchers are able to introduce fragments of DNA fused to these marker or reporter genes. If the yeast is able to grow without the selected amino acid this means that the DNA of interest to the researcher has been successfully introduced into the cell. This approach has led to the characterisation of countless genes and proteins in yeast and from other organisms.

That  establishes why yeast is used as a model organism to study molecular biology but how and what is it used for? The last century saw a molecular enlightenment, yeast was cemented as a key component of that movement. In the 1950’s, molecular biologists constructed a  Saccharomyces cerevisiae strain containing biochemical markers (antibiotic  resistance or amino acid selection) known as S288C from the fig strain mentioned in the previous post, EM93. It was soon discovered that self-replicating elements of DNA found in bacteria called plasmidscould also be made to function in yeast. Yeast cells multiply rapidly and the overall effect of a mutation in a certain gene can be measured biochemically or by observation under the microscope. If DNA fused to reporter genes is inserted into the self replicating DNA from bacteria and then introduced into the yeast cell, it can be propagated and then extracted. This technique is called cloning as it replicates an identical copy of a gene, has been used to mass-produce proteins and vaccines. In yeast, cloning was used as early as 1980 to produce Hepatitis B vaccine. Since then it has produced a multitude of proteins and vaccines including: insulin, growth hormone, haemoglobin, oestrogen receptor and interferons.

Cloning can also take place in bacteria such as Escherichia coli, these cells divide faster than mammalian cells but are a lot smaller so there is a limit in the size of the protein that can be cloned. As a consequence of this other cells types are now also used for cloning such as those derived from mammals, insects and viruses. Cloning provides an extremely economical way to reproduce human proteins. They replace the need for animal production and reduce the risk of transferring unwanted diseases, such as, CJD from growth factor. Although great advances have been made, the systems are still not perfect and have their limitations according to the type of protein that can be cloned, as some are toxic to the cell, and introduction of unwanted mutations occurs far more frequently when selection is not acting on the protein. Most organisms, including bacteria, have their own DNA repair systems that detect mutations. Foreign DNA has a higher chance of retaining mutations in a host cell as it is not detected through normal cell function, a problem to biotechnology that is successfully addressed in natural systems by selection. The use of these systems with limitations causes uncertainty and increases risk factors, subjects which are discussed later. It seems yeast occasionally retains its Biblical ability to behave in a corrupt way.

Protein interactions: Ribonuclease-inhibitor protein grabbing and surrounding the ribonuclease A enzyme. Image: Dcrjsr.

Following the heady days of protein engineering, yeast laboratories, through intra-science communication, successfully completed the enormous challenge to complete the first fully sequenced eukaryotic genome. This was achieved using strain S288C and relatively archaic apparatus compared to the robotic systems used to decode the human genome. Eventually, over 6,000 genes were unravelled from the yeast nucleus. The yeast genome is 200 times smaller than the human genome but almost four times larger than that of E. coli. This achievement marked a milestone in biological history. Yeast biologists did not stop at just sequencing the genome. In a striking example of inter-organisation collaboration, nominated laboratories began deleting single genes from individual yeast cells through advances in polymerase chain reaction (PCR), a technique that can amplify a single gene from the cells DNA. A marker/reporter gene flanked by target DNA is amplified by PCR technique and then inserted into the yeast cell. Non-homologous recombination replaces the genomic gene with the introduced marker/reporter gene. Biochemical tests were then carried out on the mutant yeast strains to uncover the functional analysis of hundreds of different gene products. This work elucidated many gene functions and undoubtedly contributed to the discovery of many analogous human genes. This information has been collated into several databases to provide a plethora of data available for bioinformatics across the internet. Genes placed on microarray slides and subjected to various environmental conditions and variations of DNA recombination techniques have increased the quantity of this information, enabling researchers to compose complicated hypotheses and uncover new cell processes without even entering a laboratory.

Many would expect yeast’s contribution to scientific research to stop at this point. Exhausted by constant, investigative probing. In contrast, the yeast story continues. It has also been used as a vehicle to investigate protein interactions first with native yeast proteins and then later with proteins from any other organism. Genes can be fused to protein tags, introduced into yeast cells and reporter genes within the cell can detect if the proteins produced from the introduced DNA interact. This procedure is known as the yeast 2-hybrid technique. Several variations to this technique exist, again modifying it to be used in other in cell cultures from other organisms. These techniques, in a rudimentary way, can also be used to evaluate post-translational modifications in proteins, to see how gene products are modified by the cell. Compared to the amount of gene sequencing data available the amount of protein interaction data is still fairly incomplete with the function of many gene products still unknown.

Yeast has also made a valuable impact in evolutionary biology as it has allowed the mechanisms of evolution to be scrutinised at
the molecular level and over short time-scales. In evolutionary terms, fungi, including yeasts, precede mammals and other bilatarians.  Bilaterians possess a left and right symmetry of body plan. The two predominate groups, deuterostomes and protostomes, differ from one another in skeletal development. They are believed to have separated in an early stage of evolution estimated to be 670 million years ago. Humans are likely to have diverged from apes only 4 to 5 million years ago. Plants and fungi are thought to have moved from water to land together, the earliest fossils of fungi are in Precambrian rocks dating back 900 million years. Comparing conserved DNA motifs between species of yeasts allows geneticists to estimate the evolution rate of proteins. Yeast can be compared with other yeasts and then with other model organisms such as nematodes or fruit flies. Comparative genomics evaluates the evolution of certain proteins and the processes and complicated pathways that they participate in.

Antibiotic resistance test: Antibiotic impregnated discs are placed on a lawn of Staphylococcus aureus. The width of the halo around each disc represents the efficiency of the antibiotics in clearing the bacterial cells.
Image: Don Stalons.

Fungal species are susceptible to disease and parasites that they control by producing antibiotics, such as, penicillin. In fact, the microbial world is full of toxins secreted by bacteria and fungi many being used as insecticides and other biological control agents. Yeast can also be used to study antibiotic resistance. Resistance to antibiotics and other stresses in yeast is often called rapid evolution. As yeast cells can evolve rapidly to overcome environmental challenge they provide a means to study the mechanisms of evolution. In addition the yeast cell susceptibility to mutagens make it an ideal organism to study the effects of mutagenesis and adaptation.

Yeast therefore provides a molecular tool to study cell biology and a model system that can add to our knowledge of evolution. In contrast to yeast in the biblical era, the molecular era now knows a great deal about this organism. In addition to great improvements in disease management, advances in genetics have led to new arguments surrounding the creation of living things, especially in respect to evolution and cloning. Yet, even though it exists as a simple single-celled organism that thousands of researchers have been studying intensely for centuries, a lot remains to be discovered.

Life on earth has evolved over millions of years through a complex network of processes that will take many years to unravel. Whether the molecular information we have derived from yeast is comparable to the corrupt leaven of the Pharisees or the leaven that the women kneaded into the dough to represent the kingdom of heaven  has yet to be established .