BIO 621 Advanced Genetic Analysis
Spring 2007
Exam I
Name_____KEY__________
1. Imagine that you are carrying out a screen to isolate ethylnitrosourea-induced mutations in
zebrafish that result in an eye-less phenotype. Using the following diagram describing your
screen protocol, respond to the following questions (5 pts. each).
random pair matings
a. If a new recessive mutation arises, what proportion of animals among the F2 do you expect to
exhibit the defect?
zero
b. Randomly selected pairs of F2 animals are mated, each pair producing a clutch of F3 progeny.
If an F2 family contains a new recessive eye-less mutation, what is the likelihood that any single
clutch of F3 siblings will exhibit the defect?
Likelihood that any random pair is heterozygous is 1/4
c. If an F3 clutch exhibits the defect, what proportion of the animals within that clutch do you
expect to exhibit the defect?
If parents are heterozygous, likelihood of a homozygote is 1/4
d. What type of mutation is usually created by NMU mutagenesis?
Nucleotide substitution (one type of “point” mutation – other is frameshift)
2. Transgenic mouse strains are commonly created by two general methods: a) injection of
DNA into zygotic nuclei, b) gene targeted replacement. Briefly describe and compare these to
approaches, being sure to include the following topics in your comparison
 genome site selection by the transgene insertion process
 usual number of copies and arrangement of inserted DNA
 mendelian segregation pattern of the transgene insertion in subsequent generations.
(20 pts.)
Injection into zygotic nuclei: random insertion sites (but usually only one site per
genome); multiple/many copies (often concatameric repeats); initial transgenic is heterozygous
for the insertion and insertion segregates as a simple mendelian factor.
Gene targeted replacement: site-specific insertion through homologous exchange;
typically a single copy (replacement of homologous sequence); initial transgenic cell is
heterozygous for the replacement and the insertion segregates as a simple mendelian factor in the
eventual transgenic animals.
3. Describe and distinguish the uses of the following types of yeast transgenic vectors: YEp,
YIp, YCp (10 pts.).
Yep: episomal plasmid (usually mu replicon); used to create high-copy-number cells
YIp: no yeast replicon, therefore requires integration into yeast genome to be maintained;
used for gene replacement/disruption (homology-dependent gene targeting)
YCp: yeast centromere replicon; used to create low-copy-number cells
4. Provide brief definitions for each of the following terms (3 pts each):
a. Missense nucleotide substitution
Nucleotide substitution resulting in amino acid change in protein
b. Frameshift mutation
Insertion/deletion of one or more nucleotides that changes translational reading frame of
(results in downstream missense and nonsense codons)
c. Haplo-insufficient gene
In a diploid cell, one normal gene copy is insufficient for survival or normal phenotype
(i.e., a null mutation is dominant)
5. In the Simon et al paper we covered in class (Cell 67:701, 1991), screens were carried out for
dominant enhancers of the rough-eye phenotype produced by sev mutant flies that receive the
sevB4 loss-of-function mutation in a transgene. The authors hypothesized that such enhancer
mutations should include mutations of other components of the signal transduction pathway in
which the sevenless protein normally works (5 pts. each).
a. Why did the authors select the sev gene for this screen, rather than some other PTK that
initiates a Ras cascade?
Eye morphology provides a range of phenotypes, that are easy to score, and does not kill
the animal; mutations of the Ras signaling pathway (the target of this work) would affect the
viability of the animal, if expressed in most other tissues.
b. Did they expect to obtain gain-of-function or loss-of-function mutations, and why – describe
the rationale.
Loss-of-function. Because sevB4 is a partial loss-of-function (hypomorph), the rationale
was that loss-of-function mutations in other pathway steps (further reducing the signaling
pathway) would exacerbate/enhance the rough eye phenotype of sevB4 flies.
c. Could they expect to recover mutations in genes that are not rate-limiting in the overall
pathway? Explain.
Possibly. The rationale (above) is that null mutations in another pathway step should
reduce that step to 50% of wild-type activity. Certainly, they should be able to recover any step
that is rate-limiting at 100% of wild-type activity. If 50% reduction makes that step ratelimiting, overall pathway flow would be reduced, resulting in enhancement of the sevB4
phenotype.
d. The authors tested the sevB4 enhancers for their effects on Elp, a dominant gain-of-function
mutation of the torpedo gene (an EGF receptor homolog). What result did they get and how did
they interpret it?
At least some of the mutations that enhanced sevB4 acted as suppressors of Elp. This
result supports two hypotheses/assumptions of the screen: enhancer mutations are likely loss-offunction mutations; enhancer mutations represent genes in the Ras signaling pathway (same
pathway).
6. Because the entire yeast genome is sequenced and all genes have been presumably identified,
it has been possible to use a single strategy to create uniquely “tagged” knock-outs of each gene.
This has been accomplished using specially designed primers to do PCR, then introducing the
PCR product into the genome in a targeted manner. Draw a schematic of the features that are
included in each primer set (pair for each gene), explain why each feature is there, and briefly
explain how it is targeted to the correct site in the genome.
Two slightly different tag systems using the same basic strategy were discussed in lecture. A
schematic for one is shown below.
The blue sequence in the primers matches 5’ and 3’ sequence of the target gene and is used to
direct homologous recombination into the genome. A unique 20bp tag is used on each primer to
identify the locus that is knocked-out when mixed populations of KO strains are grown and
selected. The 3’ end of each primer is homologous to the KanR cassette. The primer pair for
each gene to be knocked-out is used to amplify the KanR cassette. The amplified cassette is
introduced into the yeast and homologous recombination is directed by the gene-specific
sequences at the ends of the cassette DNA. Recombinants are identified by the presence of the
selectable marker (resistant to Kanamycin).