Yeast produces not only bread and wine

[The Leaven – exploring the relationship between science and religion (cont)]

The last post established 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 plasmids could 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  in future posts. 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 by 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.

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From figs trees to laboratories

[The Leaven – exploring the relationship between science and religion (cont)]

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 by Emil 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 reporter gene in yeast cells. Image: bio+ve

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.

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.

Leaven in a molecular era.

[The Leaven – exploring the relationship between science and religion (cont)]

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.

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. The 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 uploaded by 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: 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 micrographs of Fission yeast (Schizosaccharomyces pombe). Image by 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. 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.

…are women under represented in the history of yeast research because they don’t drink enough beer?

[The Leaven – exploring the relationship between science and religion (cont)]

Now, in the 21st cent, there are about 30 yeast factories in the European Union consuming about a million tons of cane molasses per annum. European yeast production alone generates an annual turnover of 800 million Euros. Until the turn of the 19th century yeast was supplied in a liquid form very similar to that found at the bottom of beer barrels. Perhaps, in a similar way to  how bread was made in early Egyptian civilisation from fermenting beer. Pliny the Elder noted in the first century BC that Gallic and Iberian bread was particularly light because it had been made with froth from the top of beer.

There are now several forms of yeast, compressed, crumbled and active/instant dried and genetically modified. The task of baking and brewing in earlier civilisations would have been difficult without the knowledge of sterilisation and pasteurisation. In ancient times, leaven or sourdough would have been left to rise in considerably unsterile conditions in a warm temperature. This environment would have been optimal not only for yeast but for all kinds of microbial growth including those that were pathogenic to humans. It is not surprising that leaven was associated with impurity and corruption. Excessive contamination would have certainly contributed to disease.

The desired characteristics of the yeast strain used in brewing and baking are different although they use the same species Saccharomyces cervisiae, which is also known as bakers or brewers yeast. Brewing yeast needs to have an agreeable flavour and an ability to flocculate so that the wort can settle quickly to achieve clear beer. In order to achieve these characteristics yeast are selected through generations, so that a specific yeast strain produces a desired flavour. In Darwinian terms this would be known  as directional selection.  So the variety of yeast varies with a particular industrial use. For instance, pizza dough is made with reduced power dry active yeast. Its slow fermentation allows the pizza to be shaped with reduced shrinkage after baking. Most commonly yeast for the baking industry is supplied as a compressed block because this form has a longer shelf life. Just 2.5 grams of this yeast in 100g of flour divides until it reaches a population size of 25 billion yeast cells.

Package of compressed yeast. Image by Hellahulla.

There is no question that yeast has transformed the structure of modern culture. In the food industry it provides baked goods, yeast extracts and alcoholic beverages. In scientific research it is a major model organism used mainly in molecular biology to discover information about the mechanisms of cellular processes. In fact, early in the 20th century, RNA was called yeast nucleic acid because it was first discovered in yeast.

Disappointingly, no women have been attributed to any of the early scientific discoveries associated with yeast. OK, they were less likely to encounter  Leeuwenhoek’s animalcule-containing sperm or beer during their daily routines but the reasons seem more likely to be associated with the status of women within religion.  As a consequence, they are largely excluded from early investigations were scientific endeavour was mainly to reveal the complexity of God’s creation. These investigations seem to be exclusively undertaken by men. Within the Bible it is clear that women were preferred to have a more subordinate role as revealed in a letter from Paul the Apostle to Timothy [Tim (1) 2, 11-15]:

Women should learn in silence and all humility. I do not allow them to teach or have authority over men; they must keep quiet. For Adam was created first, and then Eve. And it was not Adam who was deceived; it was the women who was deceived and broke God’s law.

In subsequent chapters, I will be addressing the portrayal of women in the progress of religion and science.

Knead me not into temptation

[The Leaven – exploring the relationship between science and religion (cont)]

Like other simple life forms, yeasts such as Saccharomyces cerevisiae are fully self-contained within one microscopic cell. S. cerevisiae cells are round and, providing they are well nourished with carbohydrates, spend most of their life-cycle reproducing vegetatively by growing buds. Buds separate from the parental cell when they reach a certain size in order to follow an individual pattern of growth. Upon maturity these too can start budding; each cell produces about thirty progeny. The loss of the bud leaves a scar on the parental cell that can be visualised with fluorescent dyes or electron microscopic techniques. The pattern and number of scars can reveal a lot about the condition and age of the yeast cell. Some yeasts do not reproduce by budding but by forming a cross-wall rather like the mitotic cell division observed in higher eukaryotes. Schizosaccharomyces pombe or fission yeast is an example of this. It divides in a similar way to human cells and therefore is used as a model system to study many human diseases, especially cancer.

Yeast cells stained with calcofluor white dye and observed under a fluorescent microscope. Newly budded cells take up less dye. Small rings on cell surfaces are budding scars. Image:bio+ve

The concept that living organisms produced leaven wasn’t seriously considered until Erxleben, in 1818, proposed that leaven and barm consisted of living vegetative organisms responsible for fermentation. Prior to this, in 1680, Leeuwenhoek, with his early microscope, observed yeast cells in fermenting beer. He referred to most of these single-celled creatures as animalcules because they were believed to be immature forms of larger animals. These first observations of microscopic cells were not further investigated for another century. Leeuwenhoek’s contemporaries were largely preoccupied with the argument centred on spontaneous generation, a belief that animals could materialise from other living or mineral things. Before groundbreaking experiments by Louis Pasteur in the mid 19th Century, which illustrated that excluding particles from sterile broth prevented contamination by microbes, many theorists believed in spontaneous generation.

Different theories and speculations concerning the creation of organic things occur in every religion, as most feel that the complexity of the natural world could not have arisen by chance. Many investigators began to challenge the image of creation as depicted in the Bible. Perhaps the most compelling of these arguments was the theory of natural selection presented by Charles Darwin in the mid 19th Century. His book entitled the Origin of Species created tensions between the Church and Science because it questioned a popular and largely excepted image of creation.

Religious devotees perhaps saw Science as being not only a threat to their faith but to their social acceptance and respect. Science innovation threatened to ridicule the basis of their fundamental beliefs and values. It is therefore understandable that there was a need to retain Biblical teachings in some form. In the 19th Century, the paradigm shift that was rapidly evolving Science was too extreme to evoke an equally rapid change in religious faith. In order to fully commit to a belief requires a great deal of conviction. This conviction can be impenetrable leading believers to imagine that an evil being is responsible for any deviancy from a steadfast commitment. Any element of uncertainty in religious belief seems to lead to the evolution of new religious theories to give meaning to situations that are too difficult to comprehend. In the New Testament an interesting method is used to quell sceptics and doubting critics. Individuals who questioned the ideals proposed by Jesus were thought to be influenced by the Devil:

Jesus is tempted by the Devil. Mosaic from Monreale Cathedral. Image by Sibeaster

After spending forty days and nights without food, Jesus was hungry. Then the devil came to him and said, “If you are God’s Son, order these stones to turn into bread.”
But Jesus answered, “The scripture, says, Man cannot live on bread alone, but needs every word that God speaks.”
[Matt. 4.1-11; MK. 1.12-13; Lk. 4.1-13]

This  not only discourages doubt from those with religious faith but also prevents others from persuading them away from their convictions. It is not surprising that scientific hypotheses that question religious beliefs are subject to contention.

…every living thing is a package of consumable energy

[The Leaven – exploring the relationship between science and religion (cont)]

Although it’s meaning still remains a mystery, Life, in itself, is hard work and requires a lot of energy. It’s now well established that the initial source of this energy is provided by the Sun in the form of light, which is absorbed by a photosensitive pigment called chlorophyll found in plants and other photosynthetic organisms. The energy is then trapped in molecules of glucose, a carbohydrate compound composed by a series of chemical reactions involving carbon dioxide and water. Plant consumers then transfer the energy stored within the glucose carbon source along the food chain. When the glucose is broken down it produces adenosine triphosphate (ATP), the compound required to release the energy that powers most cellular functions. The most efficient way for an organism to synthesise ATP, thereby releasing energy, is by an oxygen requiring process called cellular respiration. In humans oxygen is transferred into the body from the surrounding atmosphere by respiring, it is extracted from air in the lungs by haemoglobin, which is then circulated around the system in the blood.

Chloroplasts visible in the cells of Thyme-moss. Image by Kristian Peters.

As it contains the oxygen required for anaerobic energy production humans cannot survive without blood. Blood was therefore considered of extreme importance in the Biblical era, as it was the substance thought to contain an animal’s character and life force.

Every living thing is a package of consumable energy but not every organism can boast a sophisticated circulatory system that enables cellular respiration. Microbes and other lower life forms have to adopt fairly basic means to generate their energy. The energy generating processes of yeast produces by-products that have been exploited by human civilisations for centuries. One of the ways yeast requires its solar produced energy is by fermentation; a biochemical transformation that converts carbon sources such as glucose or sucrose into energy, producing alcohol, giving off carbon dioxide as a by-product.

Fermentation is not as efficient in producing ATP as aerobic respiration but enables yeast to convert glucose into energy without the aid of oxygen. Scientifically defined, fermentation is a catabolic process that makes a limited amount of ATP from glucose without an electron chain (supplied by oxygen) producing a characteristic end product, such as, ethyl alcohol or lactic acids. During fermentation, yeast not only generates energy from the carbon source but it also breaks it down into an industrially and socially important commodity, namely alcohol. Yeast also has the ability to perform aerobic respiration to give off carbon dioxide but this process does not produce alcohol. Being able to live with or without oxygen is undoubtedly ecologically advantageous to this microbe. Certainly explaining why it inhabited the Earth long before humans did and why it will still be here long after our fragile species has disappeared.

The mysteries surrounding fermentation were once, and to some extent still are, the subject of great scientific endeavour. It was mainly assumed that the reaction was chemically induced because investigators were unaware that miniscule creatures unseen by the human eye could exist. The yeast commercially responsible for transforming carbohydrate rich ingredients, like flour and fruit juice, into loaves of bread or alcoholic drinks is predominately Saccharomyces cerevisiae also known as baker’s, brewer’s or budding yeast. When sugar is plentiful the metabolic route that this type of yeast chooses is fermentation. During fermentation cells multiply rapidly by budding, when all carbon resources are depleted cells either enter a stationary phase of non-division or produce spores. Budding yeasts can also reproduce sexually. Adjacent cells of opposing mating types fuse together, in response to pheromones, by forming protruded structures called shmoos. The end product is a slightly larger round diploid cell that contains two sets of chromosomes; this is a way in which genetic variability is introduced into the cell. This diploid cell can either continue budding or enter meiosis to produce four ascospores.

Performing meiosis is a risky business to budding yeast as it has to temporarily stop increasing population size therefore it only faces this challenge when nutrients are low and its survival is threatened. In this state cells become resistant to stress and can remain dormant for several months, years, decades or even centuries. While dormant they lie at the bottom of the fermenting vessel to form a thick layer of pale brown sediment. Some yeast cells die but many retain the ability to begin dividing again when conditions improve, for instance when more sugar becomes available. This mode of survival allows them to remain viable in the face of adversity.  They are well suited to harsh industrial conditions and, also,  the arid  environment that forms the backdrop of the Biblical Testaments.