Introduction to Yeast Genetics
Molecular and Cellular Biology Graduate Program
at Dartmouth
February 16, 2007
Charles Brenner, Ph.D.
charles.brenner@dartmouth.edu
For the latest update, please reload http://www.dartmouth.edu/~brenner/102yeast.html
Please read these notes, explore the links, write your notes and questions in the margins, and then bring them to class.
There are excellent online resources for yeast. Start with Fred Sherman's introduction. Explore the Saccharomyces genome database and examine the literature on a protein by protein basis (site license and username required) at Proteome.
I. Culture: History of yeast and yeast in history
Most anthropologist consider humans to have established agrarian, wheat-growing civilization in 10,000 B.C.E. in the Fertile Crescent of Sumaria, present day Iraq. Beermaking began in the same area, at least six thousand years ago, i.e. before the birth of Abraham. In the upper, left hand corner of this ancient seal you can see a representation of Sumarians drinking beer through straws.
Wine vessels greater than 8000 years old have been identified in China.
Fermentation of fruits and grains was probably considered
a magical property of a properly cared-for vessel. We have been carrying
around these vessels ever since and thus the cultivation of yeast has always
been closely linked with human culture. It was not until Louis Pasteur's
time that yeast was colony-purified. Saccharomyces cerevisiae was purified from European beers. Schizosaccharomyces
pombe was purified from an African
millet beer. However, we know that cerevisiae was used in ancient times because S. cerevisiae ribosomal DNA was amplified and sequenced from 5000 year old Egyptian wine jars.
Today's lecture will be confined to Saccharomyces cerevisiae. It is also known and, of
course, used as baker's yeast. II. Yeast in relation to the tree of
life The five kingdom classification scheme (
plants, animals, fungi, protista and monera) was developed prior to existence
of nucleic acid comparisons between living things. Plants, animals and
fungi are all closely related eucarya. This tree of life is a figure we have revised from Norm Pace. III. The yeast life cycle and the
yeast cell cycle We will spend a significant amount of class
time on this figure. You should come away with a basic understanding of
the following key concepts: meiosis and sporulation, pseudohyphal
development, mitotic (or vegatative) division, mating pheromones, shmooing,
mating, heterothallism and homothallism. Both book chapters and Fred Sherman's online introduction are good sources for this material. The more time spent considering this figure,
the better. For example, you should consider what sorts of cellular processes
would be indicated by the following types of sterile mutants: A-mutants that are mating-proficient as a's but sterile as α's that can
respond to a-factor but can't make
α factor; IV. Yeast genetic nomenclature
Yeast genes are given 3 letter names with one
or two digits after them, such as CDC33. Classically, yeast gene names were given for the phenotype of
the mutant. Thus ste genes
such as ste2, ste3, etc., confer a sterile phenotype and his3 mutants require histidine. Genes can also be
named after the proteins or RNAs they encode, an example being CMD1, which encodes calmodulin. As you can see, we
use the italic (or underscore) to denote genes and upper case to denote
wild-type. Loss of function mutants are lower case. Known alleles
are given after a hyphen. A dominant mutant is usually upper case with an
allele designation, such as DAF1-1.
Corresponding proteins are capitalized as proper nouns, such as Kex2.
Phenotypes are usually indicated as follows: STE+ and TS+ mean not
sterile and not temperature sensitive. ts- and ste- mean temperature
sensitive and sterile. The plus means okay for that phenotype and the
minus means the particular strain has the phenotype. V. Alleles: wild-type, mutant,
dominant, recessive, loss of function, gain of function, etc. Anyone not clear on any of these concepts
will need to ask a question at this point in the lectures. VI. Tetrad analysis Put yourself in the position that you have a
mutant organism and have found an interesting phenotype. You would
obviously like to demonstrate that the mutation causes this phenotype.
There is probably no experimental organism in which this type of analysis is
faster or more definitive than in yeast. If you can separate the mutation
you are following from the phenotype, the mutation does not cause the
phenotype. If you cannot separate the mutation from the phenotype, you
can say they are linked and set
out to recover the allele and transfer it to another background to demonstrate
the genetic basis for the phenotype. Tetrad analysis is made possible by
doing crosses between haploid MATa
and MATα strains, putting the resulting diploid through
meiosis and sporulation, and physically dissecting the four ascospores from an
enzymatically digested tetrad. Spores are allowed to germinate on rich
media and the phenotypes of all segregants are determined. We will
provide examples of tetrad analysis in class. VI. Complementation and its
complications A.
Complementation groups as genes This is the simplest case. If you had
two leucine-requiring mutants and one was MATa and the other was MATα and you mated them you could test whether the
mutations were complemented by each other. If the resulting diploid were
LEU+, you would be tempted to say that each original strain has a mutation in a
different gene required for leucine biosynthesis. The diploid is
prototrophic because it is heterozygous for two different defects in leucine
metabolism. In the simplest case, the two defects define two different
enzymes encoded by two different genes, LEU1 and LEU2.
In the simplest case, the mutations complement each other by generating a
diheterozygous diploid of genotype leu1/LEU1 leu2/LEU2.
B. Intragenic
complementation as an indication of multiple domains or functions The careful reader has noted that we fell
short of allowing you to conclude
that the two mutations fall in two different genes. The degree of your
temptation to call the two complementable mutations two genes is directly
proportional to your subscription to the one gene, one enzyme hypothesis.
The more you consider exceptions to this rule, the less you will be certain
that mutations fall into the same gene. If the protein has two functional
domains that don't need to be part of the same polypeptide to work, the
mutations could be alleles that fall into different domains. Furthermore,
if the protein (or RNA) is multifunctional, particular alleles could be phenotypic
for different reasons even if they are close to each other in macromolecular
structure. Strictly, then, complementation allows one to conclude that
two mutations are either in different genes (like leu1 and leu2)
or are functionally different alleles of the same gene (such as cmd1-1 and cmd1-2).
We will show how one demonstrates intragenic
as opposed to extragenic complementation after we make the distinction between
forward and reverse genetics. C. Nonallelic
noncomplementation as an indication of assemblies In rare cases, two mutations in two different
genes fail to complement. If a gen1/GEN1 gen2/GEN2
mutant has the gen- phenotype, it is likely that Gen1 and Gen2 proteins are
required in a common assembly. As with intragenic complementation,
exceptions to rules are always informative. VII. Suppression A geneticist is a bit like a car mechanic
into whose garage an exotic machine is placed. The mechanic disturbs
parts and observes their effects alone and in combination. Genetic alterations
that diminish the phenotype of other alterations are suppressors. Before we continue, we need to make the
distinction between two types of mutant hunts: selections and screens.
In any mutant hunt, one decides on the mutant phenotypic criteria in advance.
In a screen, one examines every colony for the phenotype. In a selection,
one figures out a way to eliminate cells that retain the background phenotype
so that nonmutants do not form colonies. In general, selections are
considered more powerful, elegant and less time-consuming than screens but it depends
what the phenotype is. A microscopic screen (for example, looking for
bud-site alterations under the microscope) is time-consuming while a colony
color screen is not. Anything that confers resistance in a background of sensitivity is a selection. Anything that reduces viability suggests a screen unless a trick can be employed. Because there is much to be gained by looking
at suppressors of particular phenotypes, selections are often followed by screens
and vice versa. For example,
an investigator conducts a screen to find inositol-requiring mutants on
inositol-deficient media. In the background of the inositol-requiring
mutant, she now conducts a selection for inositol prototrophs. The
colonies that appear in the selection may be suppressors of the primary mutants
or they may be simple revertants.
If they are suppressors, they could either be extragenic suppressors or
intragenic suppressors. Intragenic suppressors restore function to a macromolecule at a site
different from the primary mutation. Extragenic suppressors reconstitute function in a system either by bending
one part to work with another bent part or by bypassing the requirement of the
first part. The most interesting examples of extragenic suppressors as
bent parts that fit other bent parts are cases of allele-specific
suppression in which only particular
alleles of one gene are suppressed by particular alleles of another gene.
These cases are usually taken to indicate specific protein-protein
contacts. Bypass suppression, on the other hand, usually involves one
mutation that is epistatic to
(masks the phenotype of) another mutation, rather than mutual suppression.
In class, we will use the Ras-cAMP system to
explore epistasis. Here are the three relevant observations: The genetic inference from these observations is that
Bcy1 protein acts downstream of Cyr1 protein. Before class, everyone should review a cartoon
of cAMP-dependent protein kinase in any biochemistry text book in order to
appreciate the power of epistatic inferences. SOME OF THE REMAINING NOTES ARE FOR A LONGER SERIES OF GRADUATE YEAST GENETICS LECTURES DEVELOPED AT JEFFERSON. We will not test on sections VIII, X or XI but they may be helpful for section IX, which we will try to cover.
Optional section VIII. Transformation of yeast and yeast
chromosome structure At last, some molecular genetics! We
taught this subject at Jefferson in Biochemistry 532
(references provided there). It should become clear that cloned chromosomal and
extrachromosomal elements give yeast molecular geneticists many options in DNA
transformations. The first functionally cloned yeast DNA
fragments encoded biosynthetic enzymes for amino acids and pyrimidine bases and
were inserted into pBR322. Transformation of these closed circular molecules
yielded few transformants. Linearization of the plasmids yielded more
transformants, the majority of which had stably integrated at the homologous
locus in the genome. Then came plasmid YRp7. YRp7 contains the TRP1 gene as an Eco RI fragment inserted into pBR322. YRp7 was found
to transform yeast with a high frequency and reduced mitotic stability.
The subfragment that conferred high frequency transformation was separable from
TRP1 and was named ARS1 for autonomously replicating sequence. As we
discuss in greater depth in the other lectures, ARS1 turned out to be the first cloned eukaryotic
replication origin. YRp plasmids can be made into more stable, high copy
YEp plasmids with addition of sequences from the endogenous yeast 2 micron
circle. YRp plasmids become highly stable, single copy YCp plasmids with
the addition of a cloned yeast centromere. All of these elements are
compact. Further, yeast plasmids are always constructed to be shuttle
plasmids compatible with replication and selection in E. coli as well as yeast. In vertebrate genetics laboratories you will encounter YACs
or yeast
artificial chromosomes. YACs have become favorite tools in mammalian
positional cloning projects because they allow large pieces of DNA to be
maintained in yeast. YACs are linear cloning vehicles with telomeres at
each end and yeast selectable markers, such as URA3 and LEU2,
to the right and left of a central insertion site. IX. Reverse genetics Classically, genetics starts with a mutant
phenotype and asks questions such as what sort of gene (defect) might be
responsible for color blindness or what sort of gene might be responsible for
arrest in response to radiation. Increasingly, many studies begin with a
gene (YFG1) and set out to
determine what the normal function of the gene is. This is reverse
genetics. Much of modern yeast genetics and mouse genetics are done in
reverse. Reverse genetics depends on the ability to introduce specific
mutations in genome. Yeast genes are usually knocked out by one-step
gene disruption. The
original scheme depended on cloning a selectable marker into a gene of interest
and transforming the linearized sequences into yeast, selecting for the
marker. Newer schemes utilize PCR to generate a selectable marker
surrounded by the 5' and 3' sequences of the gene to be disrupted. In
both schemes, homologous recombination transplaces the recombinant null
construct into the genome, generating an allele such as yfg1&Delta::URA3.
Frequently, it is not known whether a yeast
gene is essential for life as a haploid. In these cases, a
"wild-type" diploid with a genotype such as MATa/MAT&alpha ura3/ura3
will be transformed to heterozygosity for the yfg1&Delta::URA3allele.
(It should be noted here that laboratory strains are almost never fully
wild-type, typically containing mutations in several markers such as ura3, leu2, his3, trp1, lys2 and ade2.)
URA+ transformants are selected, restreaked, numbered, and assayed by PCR for
heterozygosity at the YFG1
locus. (Those transformants that remain homozygous for a nondisrupted YFG1 PCR product did not arise by homologous
recombination.) The heterozygous diploids are sporulated and
dissected. If YFG1 is
required for spore germination or for life as a haploid, then there will only
be two viable colonies per tetrad. Furthermore, when the surviving
colonies are scored on -ura medium, it will be found that the two expected URA+
colonies per tetrad are never recovered. If, on the other hand, YFG1 is not a haploessential gene, then there ought be be four viable colonies per
tetrad with the URA phenotype going 2:2 and URA+ marking yfg1&Delta. The next phase of research will focus on
uncovering the differences between the yfg1&Delta::URA3
mutants and their isogenic YFG1
brethren. Optional section X. Yeast genetic tricks A. URA3
URA3 is one of a small number of yeast genes that can be selected for or
against. As with any gene encoding a biosynthetic enzyme, ura3 auxotrophs provide a selection for URA3-transformation. However, because the Ura3
enzyme activity will convert 5-fluoroorotic acid into a toxin, one can select
for cells resistant to 5-FOA which have lost function in URA3. There are many important applications of 5-FOA
selection. Here is one application. Let's say that you have found that the yfg1&Delta::URA3
mutants fail to grow on galactose medium. In a recent sequence alignment,
you found that Yfg1 is related to a family of enzymes that utilize a conserved histidine
as the nucleophile and a conserved cysteine for metal coordination.
Having found the gal- phenotype for the complete deletion mutant, you now wish
to see whether His168 or Cys195 are required for function. You generate
His168Asn, His168Ala, Cys195Ser and Cys195Ala alleles of YFG1, transform them
into your yfg1&Delta::URA3
strain, and select for 5-FOA resistant colonies. If you can verify that
you have faithfully transplaced the point-mutant alleles in place of yfg1&Delta::URA3, you should be able to do the perfect experiment: evaluating each allele
in an isogenic background, with every allele under the control of the wild-type
YFG1 promoter in its normal
chromosomal context. Many times, one would like to identify
mutants (mut1) that are inviable
in a specific condition. The specific condition might be a background
that lacks your favorite gene (yfg1).
If the hypothetical mut1 yfg1
double mutant is dead, how are you going to recover this mutant if you can get
it? C. One-step site-directed
mutagenesis Homologous recombination can be used not only
to direct integration of an in vitro constructed allele into the genome, it can
also be used to direct recombination between a PCR product and plasmid
sequences. Thus, it has been possible to introduce site-directed
mutations into plasmids in yeast without bacterial transformation. Optional section XI. Yeast genomics A.
Global gene expression monitoring Array hybridization has changed the way in
which people can look at gene expression changes. Chips containing
immobilized probes of every yeast open reading frame have been
constructed. A two-color experiment can be done by preparing fluorescent
total cDNA from two different conditions (or genotypes) with two different
fluorescently labeled PCR reactions. The PCR reactions are mixed, denatured,
and hybridized to the chips. In this manner the differences in mRNA
accumulation for all 6220 ORFs can simulatneously be assessed for any pair of
conditions. Recently, such analyses have been performed for the mitotic cell cycle as well as for sporulation. B. Global genetic
screens The yeast barcoded
disruption project, genetic
footprinting, and allelic
variation scanning are three techniques that the interested student is
encouraged to explore further. Back to the Brenner Group
Home Page. 
B-mutants that are mating-proficient as a's but sterile as α's that can
make α-factor but can't respond to a-factor;
C-mutants that are mating-proficient as α's but sterile as a's that can respond to α-factor
but can't make a-factor;
D-mutants that are mating-proficient as α's but sterile as a's that can make a-factor but can't respond to α-factor;
E-mutants that are sterile as a's and α's that have
diploid patterns of gene expression; and
F-mutants that are sterile as a's and α's that have
haploid patterns of gene expression but are nonetheless resistant to mating
pheromones.