Download Project 1 Mutagenesis

Project 1 Mutagenesis
Goals and Road Map
After isolating mutants, the goal is to characterize the genetics of the mutation. There is some
gene that has been altered in the mutant. We can work towards understanding what the function
of that gene is. The best test will be molecular mapping, which we will do in the mapping project
for one mutation, but that requires determining the basic properties of the mutation. Do not
despair if your mutants do not go through all these tests. There are genetic dead-ends that require
methods we are not prepared to do. Please share mutants and collaborate with other students in
the class. A mutation discussion board is set up for this. You can use another student’s mutant if
your mutants fall into the dead-end category. However, the tests require multiple attempts.
Persistence and thinking about alternate approaches is valuable.
Self-crosses:
For Arabidopis and C. elegans, this is easiest genetic test since they will do it with no
intervention.
Do you see the mutant phenotype in the F1?
-was the phenotype from environmental variation or genetic variation?
-are there are environmental or developmental factors which limit the appearance of the
phenotype? Temperature, food, stress, crowding, stage of development?
What proportion of progeny display the phenotype?
-if the mutant was recessive, could it have <100% penetrance?
-if the mutation was dominant, was the original mutant heterozygous?
-could there be contamination from other worms when picking (darn eggs and F1)?
-could mutations in multiple genes cause the phenotype and they are segregating?
~separation of progeny to separate plates and looking at selfed progeny in each
will distinguish the above possibilities
- variation in the degree of phenotype is expressivity - could not be 100%
-is there environmental dependence causing phenotypic variation?
Troubleshooting: Are there no selfed progeny?
-could the phenotype be interfering with self fertilization?
~try growing at 16oc to reduce temperature-dependent alleles, or 25oC to speed
lifecycle of slow-growing mutants.
~try crossing with a male in case the mutant lost male function. In Arabidopsis,
you can cross to a female (imp1) if mutant lost female function. However,
progeny are heterozygous- look at next generation.
~try to characterize where lethality or infertility is occurring with compound
microscope (last ditch)- are there no eggs?
Characterization of mutant:
The self crosses above provide near clones of the original mutant to characterize how it is
altered. This will be important in placing your mutant into the context of the literature.
-What stage does the phenotype appear?
-Observe under the compound microscope to see the cells and organs affected. C. elegans
can be immobilized with sodium azide or levamisole.
-crossing into GFP-containing strains in C. elegans allows examination of specific
tissues. Muscle-GFP, nerve GFP, egg GFP, and subset of nerves & vulva GFP
strains are available. The latter two throw males because of a him-8 background.
Outcrossing:
A: Crosses with “wild-type”
In Arabidopsis, imp-1 plants lack male function and supply and easy “wt” female parent. In C.
elegans, “wt”males are available from him-8 and him-5 strains that have 30% males. Also, any
successful cross will produce 50% males (XX x XO gives 50% XX and 50% XO) which will
give more males to cross. Alternately, one can find males in the mutant strain (:mutant males”)by
inducing males development. This is available by i) heat shock of L4, ii) ethanol treatment with
male enhancer solution, iii) RNAi with him-5 gene. These enhance male appearance in the next
generation.
-did the cross work or is it just selfed progeny? (C. elegans- look for presence of males)
-is your mutant allele recessive or dominant?
-are the ratios skewed or sex dependent? Note behavior of genes on the X chromosome if
males are different than hermaphrodites.
B: Crosses with other mutants
These crosses require a mutant male. This is easy in arabidospis. In C. elegans, it will need
crosses above to create a few mutant males.
-cross with another mutant with a similar phenotype for a complementation test. This
can be from another student or a known stock mutant. Is your mutant altered in
the same gene or a different gene than the other mutant?
-cross with another mutant that displays a different phenotype. What phenotype is
displayed in progeny?
Heterozygous progeny- complementation for different alleles in same gene?
Self the heterozygotes- are the phenotypes additive or do they display epistasis?
-Cross with GFP-containing worms to show GFP in specific tissues. Does your mutation
alter expression or the tissues? GFP is dominant but you may need to self to get
homozygous recessive mutant phenotypes.
Troubleshooting: Outcrosses do not work?
-always suspect selfing in crosses. In C. elegans, count males as evidence of outcrossing. A
perfect outcross gives 50% males.
-some mutants do not outcross easily. Use temperature-dependence or <100% penetrance or
expressivity to your advantage. Try to use WT phenotype worms that carry mutant
genotype.
-avoid using immobile males. Hermaphrodites show absolutely no inherent interest in the males!