Download Biology response 5 yeast

Sample Response: Is curing cancer really as simple as Schizosaccharomyces pombe?
Yeast, or the most commonly used Saccharomyces cerevisiae, is a tiny micro-organism that
exists all around us - in soil, on plants, on fruit and even airborne. It has existed for so long, it is
referred to as ‘the oldest plant cultivated by man’. Wild yeasts are single-celled microbes that
are all around us. There are many different types of yeast in the environment, from those that
cause fungal infection such as Candida; to others that are used in the brewing industry and in
wine-making. There is also a strong traditional link between bread making and alcohol
production using Saccharomyces cerevisiae.
Yeast belongs to the fungi family. It is a very small single cell micro-organism. Like all other
fungi it doesn't have the power to produce food by photosynthesis, instead it ferments
carbohydrates (sugars) to produce carbon dioxide and alcohol.
The cell cycle is fundamental to the reproduction, growth and development of all living
organisms and defects in cell cycle controls can cause genomic instability. The generation of
form is a basic property of biological organisation, and changes in cell shape occur during cell
spreading and metastasis.
An understanding of these processes is therefore essential to understand the development of
cancer. Fission yeast Schizosacharomyces pombes is a good experimental model and has
been useful for providing insight into cellular and molecular problems of mammalian cells.
Uses of yeast in a research environment
S. cerevisiae, or budding yeast (and related interbreeding species) is the most common yeast
that is used commercially. The fission yeast Schizosaccharomyces pombe, which is only
distantly related to S. cerevisiae, has equally important features, but is not as well characterized.
a. Budding Yeast - Saccharomyces cerevisiae
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b. Fission Yeast - Schizosaccharomyces pombe
Although yeasts have greater genetic complexity than bacteria, containing 3.5 times more DNA
than Escherichia coli cells, they share many of the technical advantages that permitted rapid
progress in the molecular genetics of prokaryotes and their viruses. Some of the properties that
make yeast particularly suitable for biological studies include rapid growth, dispersed single
cells, the ease of mutant isolation, a well-defined genetic system, and most important, a highly
versatile naturally available DNA transformation system. Unlike many other microorganisms, S.
cerevisiae is viable with numerous genetic markers. Yeast is also nonpathogenic and therefore
can be handled without taking too many special precautions. As a commercial asset, large
quantities of normal bakers’ yeast are commercially available and can provide a cheap source
for biochemical studies.
The development of DNA transformation has made yeast particularly accessible to gene cloning
and genetic engineering techniques. Plasmids, containing changed or ‘mutated’ genes can be
introduced into yeast cells either as replicating molecules or by integration into the genome.
Also, researchers can use the yeasts’ high levels of gene conversion (crossover etc), for the
direct replacement of genetically engineered DNA sequences into their normal chromosome
locations. Thus, normal wild-type genes can be replaced with altered and disrupted alleles. The
phenotypes arising after disruption of yeast genes has contributed significantly toward
understanding of the function of certain proteins in vivo (at the site of function in the whole
organism). These techniques have been extensively exploited in the analysis of gene
regulation, structure-function relationships of proteins, chromosome structure, and other general
questions in cell biology. The overriding virtues of yeast are illustrated by the fact that
mammalian genes are being introduced into yeast for systematic analyses of the functions of the
corresponding gene products.
In addition, yeast has proved to be valuable for studies of other organisms, including the use of
the two-hybrid screening system for the general detection of protein-protein interactions and the
use of YACs for cloning large fragments of DNA.
During the last twenty years, an increasing number of molecular biologists have taken up yeast
as their primary research system because of its versatility and comparatively cheaper costs.
Most significantly, knowledge of the DNA sequence of the complete genome, which was
completed in 1996, has altered the way molecular and cell biologists approach and carry out
their studies.
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The Cell Cycle
The cell cycle is a process of division that allows the exact duplication of cells. During the cell
cycle the end products or daughter cells are an exact replica of the starting cell or mother cell.
All of the DNA in a cell must be copied correctly exactly once. This choreography must occur in
a timely fashion: where there is coordination of individual origins of replication to fire once and
only once. It is divided into a phasic cycle G1 - S - G2 - M phase, where interphase is
represented by G1/S/G2, S being the stage at which chromosome duplication occurs. The
mitotic stages are represented by prophase, metaphase, anaphase, and telophase.
Chromosome segregation occurs during the Mitotic phase. During prophase, DNA
chromosomes condense and become visible to allow more efficient movement. The cell then
assembles a mitotic spindle of microtubles. At metaphase, the chromosomes align upon the
spindle, attached via their kinetochores to the microtubules. Once all of the chromosomes are
attached and organised along the spindle they are segregated by releasing cohesion that
attaches the sisters together, the spindle is reeled in, which is anaphase. Chromosomes then
de-condense and form new nuclei, concluding one cycle at telophase.
For a cell cycle animation go to this page - http://www.cellsalive.com/cell_cycle.htm.
Checkpoints
These maintain the order of unrelated events by signalling if something goes wrong, for example
damage in G1 or G2, incomplete replication or incomplete establishment of mitotic apparatus.
A characteristic of checkpoints is that they are usually not essential. This is particularly true of
the checkpoints in yeast - as long as everything works all right, then no problem. However, if
anything is perturbed - damage occurs, S phase takes too long or the spindles fail to assemble,
then the checkpoint becomes essential to prevent continuation of the cell cycle. In checkpoint
mutants, mechanical apparatus of division is intact, and the problem is regulatory.
In multi-cellular organisms, some checkpoints are essential for viability of the organism.
Generally, even in cell types where some checkpoints are essential, we can think of them as
extrinsic to the normal engine of the cell cycle.
A checkpoint response consists of three components: something to generate the signal,
something to transduce it, and something to receive it. The transducers are typically considered
to be "checkpoint proteins".
How can one identify checkpoint proteins, if they aren't essential? Key is to make them
essential. For example, in response to irradiation, most yeast cells will arrest the cell cycle,
repair the damage, and then continue. A cell that cannot repair the damage will arrest
permanently. A cell that can repair the damage but can't arrest will go on to divide, with lethal
consequences – this can lead to cancer in multicellular organisms for example, humans.
S.Pombe vs S. cerevisiae
Genetic analysis in simple cells, like yeast, provides blueprint for biochemical studies that can be
extrapolated to the human genes for which there are yeast homologues; this is especially true in
cell cycle control studies.
Yeast cells, which are simple, single celled eukaryotes, undergo cell division cycles like human
cells. Two types of yeast are generally used Saccharomyces cerevisiae and
Schizosaccharomyces pombe.
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The key to the simplicity of using yeast in cell cycle and Cancer research is that yeast cells are
simple and haploid (one copy of each chromosome).
Saccharomyces cerevisiae, budding yeast; is a simple cell that divides by budding. This yeast
has a short G2 phase. It is usually found as Brewer's or Baker's yeast as it has been widely
used in baking and the brewing industry for many centuries; in fact, records date to the ancient
Egyptians. This yeast is a popular model organism for cell biology problems.
Schizosaccharomyces pombe: fission yeast has a more typical cell cycle with a long G2. This
model organism is therefore a good model for studies of growth control; therefore an excellent
model for the cell cycle and cancer initiation and progression.
For both yeasts to be used as model organisms the key insight was the idea that it might be
possible to identify genes required to regulate cell division by looking for mutant yeast cells. The
fact that the yeast cell is haploid is fundamental to the success of this type of analysis; you
would expect the genes for cell division to be key to the yeast cells survival. In order to execute
this type of research it is necessary to isolate cells with mutations, you can do this by altering the
temperature the cells are cultured in; cells will get "stuck" at a particular cell cycle stage.
Once mutants have been identified, how would one now determine the stage at which the cell
cycle blocked? This kind of analysis would be achieved by using landmarks within the cell cycle.
For example, did they replicate their DNA or not? For budding yeast: is there a bud and how
big? In fission yeast, cells elongate but don’t bud, for both cell types one can determine whether
there is a mitotic spindle? Or did the nuclei condense? Other landmarks often used are called
checkpoints, did the cell pass through all of the checkpoints during a cycle?
In yeast it is also possible to remove a key component of the yeast required for its successful
progression through the cell cycle, is it also possible to change regulation/timing? For example if
one slows down the fission yeast cell cycle, cells become elongated; if you speed up the cycle,
cells have less time to elongate and therefore remain short. If, during an experiment, one
isolated short or "wee" mutants, it would indicate a defect in timing or regulation of the cell cycle.
Using genetic methodology, it is also possible to determine how many genes are mutated and
which genetic pathways they fit into, this is an extremely powerful tool, especially in cancer cell
biology. For all mutants that arrest cells in G2, how can one determine how many different
genes they represent? One can also combine mutants together in order to establish the genetic
interactions and pathways between them, building a picture which can be directly extrapolated to
humans and their cell cycle.
Definitions
Yeast two hybrid system – this is a molecular biology technique used to discover protein-protein
interactions and protein-DNA interactions by testing for physical interactions, for example
binding between two proteins or a single protein and a DNA molecule respectively.
YAC – Yeast Artificial Chromosome. This is a vector that can be used to clone large DNA
fragments. It contains aspects of the actual sequence of the yeast chromosome – the telomeric
region, centromeric region and replication origin – therefore it can replicate and preserve the
yeast cell, it is also easy to manipulate for research purposes.
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