Download Genetic Techniques for Biological Research Chapter4

Genetic Techniques for Biological Research
Corinne A. Michels
Copyright q 2002 John Wiley & Sons, Ltd
ISBNs: 0-471-89921-6 (Hardback); 0-470-84662-3 (Electronic)
4 Mutant Hunts - To Select or to Screen
(Perhaps Even by Brute Force)
The first step in a genetic analysis of a process it to isolate mutant individuals that
areunable to carry outthat process orcarry it out in anaberrant way. The
researcher must hypothesize what characteristics, changes in growth capabilities,
morphology, etc. will be exhibited by this individual and to develop a means of
identifying such individuals from among a larger group of normal individuals.
A mutation is a heritable change in the genetic material, and mutagenesis is the
process of producing such changes. Spontaneousmutations are the by-product of
natural processes, particularly errors resulting from DNA replication and repair and
the movement of transposable elements. The spontaneous rate of mutation varies
from species to species and the rate of spontaneous production of different classes
of mutation(pointmutation,insertion,
deletion) varies. The geneticist alsohas
available a variety of methods for enhancing the rate
of mutagenesis above the
spontaneous, orbackground, rate including treatment with various chemical agents,
ultraviolet light, X-rays, and transposon-mediated events. Different mutagens
produce different types of genetic change. The best choice of mutagenic agent and
the best method of delivering thatmutagen
will depend on one’s particular
experimental needs and the organism under study.
Regardless of whether the mutation is spontaneous or induced and regardless of
which type of mutagen is used, the researcher is unableto direct mutations to
specific sequences or particular genes. To a first approximation, mutagenesis is a
random process, and any particular gene has an equal chance of being damaged by
a mutagen or a spontaneous error in a cellular process such as DNA replication or
repair. So how doesone find those individuals carrying mutations in a geneof
interest, i.e. a gene involved in the process under study? In other words, how does
one distinguish the interesting mutant individuals from all the other individuals in
the pool of potential mutant individuals? One searches for them or carries out what
geneticists sometimes call a mutant hunt. To do this, one must develop a method of
identifying mutants. This is the first job of the geneticist and, in many respects, it is
the most difficult job.
An individual carrying a heritable genetic change is referred to as a mutant and
the process of identifying mutants of interest is called either a selection or a screen,
depending upon how it is carried out. Note thatone identifies mutant individuals by
observing their phenotype.Phenotype is an indirect expression of genotype and is the
product of the biochemical and physiological interplay among all the genes of an
individual’s genome. For this reason, geneticists prefer to work with isogenic strains,
i.e. strains that are genetically identical at all but perhaps one or a limited number
of known genes. In this way the effectof a newly induced mutationonthe
phenotype of the individual can clearly be associated with that specific single genetic
alteration. If isogenic strains are not available, then efforts should be made to work
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with very closely related strains such as a congenic strain. A congenic strain is one
obtained by repeated backcrosses to a common parent. The products of the tenth
backcross should be better than 99.9% identical.
In the earlier yeast literature, prior to the early 1970s, researchers did not seem to
pay a great deal of attention to working with genetically related strains. The influx
of bacterial and phage geneticists into the yeast research community brought an
awareness of the importance of working with isogenic strains where possible. In the
prokaryotic systems the technology forconstructing
isogenic strains is rather
straightforward.In yeast the ability toconstruct isogenic strains came with the
development of Saccharomyces DNA technology. One can now change the mating
type of a strain by the simple use of a plasmid expressing the H 0 gene.
To identify mutant individualscarryingalterations
in a gene of interest, the
geneticist will hypothesize the probable phenotype of these mutant individuals and
develop a method of identifying them based on their phenotypic difference from the
starting strain. The best method is to select for the mutant individuals. A selection
method is one in which only individuals with the desired phenotype are able to grow,
or move toward a light source, or carry out a function. For example, one is interested in studying the mechanism of action of an antibacterial drug, and the drugresistant mutant bacteria capable of growing in the presence of the drug is isolated.
This is a selection because millions of bacteria will be spread on thesurface of a petri
dish containing nutrient medium
with the drug. Almost all the bacteria will die
because of the action of the drug, but afew will grow to form acolony because these
individuals carry a mutation that makes them resistant to the drug.
A screen is where the individuals of a population are observed one by one following growth in a particular condition and individualswith a particular phenotype are
chosen. For example, one is interested in tryptophan synthesis, and needs to isolate
mutant strains unable to synthesize tryptophan. Such mutants would be unable to
grow in the absence of added tryptophan in the medium because tryptophan is an
essential amino acid. Colonies (clones of cells) are grown on the surface of a solid
(agar-containing medium in a petri dish or petri plate) medium containing tryptophan. Clones are genetically identical individuals. Each clone is tested by a simple test
called replica plating to see if it grows on the same medium only lacking tryptophan.
(Replica plating is a method of making duplicate copies of the pattern of colonies
growing on the surface of solid media in a petri dish. Colonies are grown on the
media in a petridish; these colonies are transferred to a sterile velvet cloth by pressing
the cloth to the surface of the plate. Then the colonies are transferred to other plates
by pressing the cloth to the surface of new plates.) Colonies grown on a medium plus
tryptophan arereplica plated to plates containing thesame medium with and without
tryptophan. Those colonies that do notgrow on the plate lacking tryptophan but do
grow on the plate containing tryptophan have a tryptophan minus phenotype.
Only when all else fails will the geneticist resort to a brute force screen in which
individuals are screened for a complex phenotypic trait by a difficult procedure.
These are few and far between, and the geneticist would have to be desperate.
Before undertaking a brute force screen, the geneticist will attempt to enrich for
mutants of the desired type. Penicillin enrichment for E. coli mutants is an example.
Penicillin kills only growing cells because it blocks cellwall growth causing the
expanding cytoplasm to burst the cell. Mutagenized cells asked to grow in penicillin-
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containing medium lacking histidine should be enriched for histidine minus mutants
because these would have been unable to grow in the absence of histidine and
therefore would have survived penicillin treatment.
Geneticists refer to the original strain isolated fromnatureandpriortoany
mutagenesis as the wild-type strain and the allele of each gene in this strain as the
wild-type allele. Any alteration in a gene, whether associated with an altered
phenotype or not, is a mutant allele. Most often, the strain used in a selection or
screen will carry one or more known mutations that are forthe use of the geneticist.
These mutant genes are often referred to as genetic markers or marker genes because
they mark the existence and position of the gene in the genome and are used to
expedite the genetic analysis. Marker genes with easily identifiable phenotypes are
generally chosen, such asnutritionalmutationsor
drug-resistant alleles. Thus,
because the original strain used to isolate new mutation is not truly wild-type, it is
usually referred to as the parental strain.
A mutant allele may be dominant or recessive to the wild-type, and this canonly be
determined by makingthe heterozygous diploid individual and determiningits
phenotype. If the mutant phenotype is exhibited by the heterozygous diploid, then
the mutation is dominant. If the wild-type phenotype is exhibited by the heterozygous diploid, then the mutation is recessive, i.e. the wild-type allele dominates the
phenotype. A recessive mutation is a loss of function mutation;that is, the gene either
no longer produces a product or the product produced
is so abnormal that it is
unable to carry out thefunction.Adominantmutation
is a changeoffunction
mutation; that is, the product of the gene has retained all or part of its original
functional characteristics but also has acquired
new functions or carries out its usual
functions differently.
The more rare a particular
type of mutation, the more
aggressively one must
look. Selection is more aggressive than screening because more individuals can be
tested. This statement does not contradict the fact that mutagenesis is a random
process. What it does say is that the genetic alteration needed to produce a certain
phenotype can be more or less difficult to achieve. For example, to knock out all
function of a gene one can delete it, insert a DNA fragment into it, or change its
sequence in any of a variety of ways. One does not carebecause no function need be
retained. But if one wants a mutant with a slightly changed phenotype, such as
resistance to a drug, then the product of the altered gene must be able to carry out
its normalfunctionbut
with different characteristics.Alterationscapable
of
achieving this are few and far between and only certain select changes will do the
trick. In other words, it takes a scalpel to make a dominant mutation but you can
use a grenade to make a recessive mutation. A priori, the geneticist does not know
how rare the searched-for defects will be so, ideally speaking, one tries to devise a
selection. This is not always feasible, and so a screen may have to be used. The
geneticist will often repeat a mutant search using subtle variations in the selection/
screening method.Theparticularmethod
used undoubtedly biases the types of
mutation isolated. By varying the isolation method, it is hoped that the additional
genes involved in the process will be identified or that different alleles of an already
identified gene with subtly different phenotypes will be obtained.
If several genes are expected to be involved in a particular process or pathway,
large numbers of mutant individuals must be obtained so asto ensurethat
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mutations in all of these genes are isolated. This is referred to as saturating the
system. Mutations in different genes involved in the same process may have the
same or slightly different phenotypes. Sometimes different mutations in the same
gene may have different phenotypes. A great deal may be learned about the normal
function of a gene by carrying out a careful phenotypic analysis of the different
mutant alleles obtained.
A forwardmutation is a genetic alteration of a wild-type gene. Itproducesa
mutant strain with a mutant phenotype and mutant genotype. A reverse mutation,
also called a reversion, is an alteration that produces a revertant strain with a wildtype or pseudo wild-type phenotype. If the reverse mutation exactly reproduces the
original wild-type genetic sequence, then the revertant strain is a true revertant and
has the wild-type phenotype and genotype. As we will see in Chapter 8, it is possible
for a mutation at a different site, even in a different gene, to produce a revertant
strain with what appears to be a wild-type phenotype even though the genotype is
not wild-type but is actually a double mutant. In this situation the second mutation
interacts with theoriginalmutationtosuppressits
mutant phenotype, and the
secondmutation is called a suppressormutation. Suppression is animportant,
interesting, and very valuable tool for themolecular geneticist, and we will explore it
in great detail in future chapters.
A null mutation is one in which the alteration completely eliminates gene function.
Null mutations result from deletions of the gene that include large sections of the
ORF or the 5' end of the gene, nonsense mutations near the 5' end of the ORF,
transposon insertions, or other changes that block gene expression or production of
the gene product. Null mutations are always recessive.
A strain carrying a conditional mutationis one that exhibits the mutant phenotype
under some conditions and the wild-type phenotype under other conditions. The
strain is a mutant strain but whether the strain exhibits the mutant phenotype or the
wild-type phenotype depends on the growth conditions. The condition that is most
often varied is the growth temperature. Most organisms prefer to grow at a particular temperature. For example, the ideal growth temperature of Saccharomyces is
30"C, but wild-type strains are able to grow at temperatures from 14°C to 42°C.
Mutantstrainsthat
exhibitthe mutant phenotype at temperaturesabovethe
preferred growth temperature are called temperature sensitive, and the mutant gene
is referred to as a temperature-sensitive or ts allele. Mutant strains that exhibit the
mutant phenotype at temperatures below the preferred growth temperature are said
to be cold sensitive, and the mutant gene is referred to as a cold-sensitive or CS allele.
Since there is a condition where the mutant gene functions somewhat normally,
conditional alleles are not null alleles and most often are changes of a single amino
acid residue in theencodedprotein.Thegrowthcondition
at which the mutant
phenotype is exhibited is called the nonpermissive or restrictive condition.The
permissive or nonrestrictive condition is the one in which the wild-type phenotype is
expressed.
Researchers interested in essential cellular processes like cell division or transcriptionmust
isolate conditionalmutations.
An essentialgene encodes aproduct
required for normal growth and cell division and its function cannot be by-passed
by nutrient supplementation of the medium. A cell carrying a mutation in a gene
encodinga necessary component of RNA polymerase I1 cannot survive if the
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mutant exhibits the mutant phenotype under
all conditions. There is no way of
adding a nutrient or drug to the medium or altering the growth conditions that will
enable this cell to grow. Such a mutation in an essential gene is a lethal mutation. A
conditional mutation in an essential gene allows the researcher to culture the mutant
strain under the permissive condition and to observe the mutant phenotype under
the nonpermissive condition. This type of mutation is referred to as a conditional
lethalmutation. The useof conditional mutations is a very powerful tool of the
geneticist and its many uses will become obvious as we read the literature.