1. The cut site for HindIII is A^AGCTT (^ = position of
cut). The RNA codons for lysine are AAA and AAG. The codons for
asparagine are AAC and AAU. CUU codes for leucine. Mouse enzyme
E has an internal amino acid sequence arginine-lysine-X-proline
that must be preserved to maintain function (X = any amino acid).
A cDNA probe for enzyme E (prepared from a full length mRNA) detects
two bands whose sizes are 3.0 and 2.0 kb on a Southern blot of
genomic DNA digested to completion with Hin dIII.
a. A naturally-occurring missense mutation in mice causes loss
of enzymatic activity and the appearance of a single band of 5.0
kb in a Southern blot with HindIII. Which amino acid in
the essential site has been replaced by the missense mutation?
b. When you sequence the mutant DNA, you find a sense strand sequence
of ...AATCTT... What has the amino acid that was replaced been
changed to?
c. You would like to clone the gene for enzyme E in a vector with
a HindIII cut site without having to worry about the problem
of it being cut into two pieces by HindIII. You decide
to start with the 5.0 kb mutant restriction fragment and restore
the enzymatic activity of its gene product through use of site-directed
mutagenesis. How could you do this without also restoring the
HindIII cut site?
d. Sequence analysis of the 5.0 kb cloned gene (from c) reveals
that the beginning of the coding sequence is 315 bp from the 5'
end of the clone and the polyadenylation signal is 247 bp from
the 3' end. However, the full-length cDNA is only 1897 bp in length.
How do you account for the discrepancy in lengths? Sketch a reasonable
hypothetical diagram of the 5.0 kb genomic clone.
e. You decide to do RFLP analysis and enzymatic studies on a number
of unrelated mice to determine how frequently loss of function
of enzyme E is associated with a mutation that affects the HindIII
site. Sketch the RFLP patterns you would expect to detect for
a mouse that was heterozygous for the mutation described in part
b above. Also sketch patterns expected from mice that are homozygous
for the mutation and homozygous wild type.
f. Which of the mice in part e would you expect to lack the enzymatic
activity and why?
g. You find that about 10% of cases where the HindIII cut
site is lost still possess enzymatic activity. Describe TWO
different ways in which this could occur.
h. You use the RFLP pattern as a genetic marker to study linkage
of gene E (coding for enzyme E) to markers that are known to be
on certain chromosomes. E23 refers to bands of 2 and 3
kb, E5 refers to a single band at 5kb, and E235
refers to bands at 2,3, and 5 kb. A/a, B/b,
and C/c are autosomal genes with the recessive form
indicated by a lower case letter. You cross true breeding A
E5 mice with true breeding a E23 mice.
What will be the phenotype and genotype of the F1 progeny?
i. You cross the F1 progeny from part h with true breeding a
E23 mice. What ratios of phenotypes do you expect in the progeny
if the two genes are not linked?
j. In 100 progeny of the cross described in i, you observe the
following phenotypes. A E235, 44; A
E23, 6; a E235, 4; a
E23, 46. What is the map distance between the A/a
locus and the E locus?
k. Why do you never observe an E5 phenotype in the cross
described in part j?
l. In one part of your study on the polymorphic Hin dIII
site in gene E, you examine a group of mice that have been maintained
in your departmental animal room for many generations. You find
an allelic frequency for E5 of 0.2 and an allelic frequency
for E23 of 0.8. What percentage of the mice would you expect
to exhibit the phenotype E235?
m. Your data show that the actual frequency of phenotype E235
in the population in part l is 0.02. What conclusions would you
draw about that population?
n. In a separate study of wild mice living in a field that has
been repeatedly sprayed with pesticides, you find the following
phenotypic distribution: E23, 0.16; E235, 0.48;
E5 0.36. Further tests confirm that most of the E5 individuals
lack enzyme E activity. Other studies have shown that wild mice
living in an undisturbed environment have a phenotypic distribution
that is over 99% E23. What conclusions would you draw about
the reasons for the altered distribution in the mice living in
the field.
o. Gene E is imprinted in the gametes produced by female mice.
A true-breeding E5 male is mated to a true-breeding E23
female. What would you expect the RFLP phenotype of the offspring
to be and why? Be careful, this is a trick question designed to
entrap you.
p. Based on the information provided in part d, would you expect
the cloned gene E to be expressed if it were successfully transfected
into a cultured mouse cell? Explain your answer.
q. You have the sense strand sequence for the first 350 base pairs
of the cloned gene in part d. What sequences would you look for
to determine whether your answer to part p is correct?
r. Enzyme E is part of a metabolic pathway involving enzymes D,E,F,
and G in that order. You cross a mutant mouse lacking enzymes
D and F with your strain that lacks enzyme E. Would you expect
the enzymatic pathway to be functional in the offspring? Explain
your answer.
s. You use primers based on the exact ends of the coding sequence for
enzyme E to perform PCR on DNA from a mouse that exhibits an E235 phenotype.
What is the length of the PCR product that is recovered.
t. You digest the PCR product from part s to completion with Hin
dIII, and do a southern blot on the digestion products, using the
cDNA as a probe. How many digestion fragments will you obtain and what
will their lengths be? Assume that the original genomic restriction fragments
were exactly 3000 and 2000 bp in length for this calculation.
u. You hybridize the PCR produce from part S with the cDNA that has
been removed from its vector. You then examine the resultant hybrid
molecules with an electron microscope. Describe what you expect to see
in as much detail as you can.
v. Based on the results observed in part n, what phenotype would you expect to
see in knockout mice that totally lacked functional genes coding for
enzyme E?
w. When transfected into a cultured human cell, the modified 5.0 kb gene
that you prepared in part c is transcribed at a low rate, far below the
level of expression observed for the intact mouse gene
in a comparable type of mouse cell. Describe the porocedures that you would
use to look for an ehnancer located upstream from the mouse gene.
x. Describe procedures that could be used to identify the essential sequences
in the promoter for the gene coding for enzyme E.
y. What is a reporter gene and why might you want to use one for the
studies described in parts w and x?
z. Eco RI cuts the 3.0 kb dIII fragment into pieces
that are 1.7 and 1.3 kb in length.It does not cut the 2.0 kb fragment at
all. When mouse DNA is cut with Eco RI only, fragments of 7.0 kb and
4.0 are detected with the cDNA, When the 2.0 kb fragment is used as a probe,
only the 4.0 kb fragment is detected. Construct a restriction map showing
cuts by both enzymes within the genomic region defined by the two Eco
RI fragments.
2. A short deletion completely eliminates the TATA box for a mouse
gene with no damage to the coding sequence.
a. In very general terms, what phenotypic effect would you expect this
mutation to have if the protein it codes for is not essential for survival
of the mouse? What pattern of inheritance would you expect in this case?
b. Under what conditions might such a mutation exhibit a
dominant pattern of inheritance?
c. If the gene product is
both essential for survial and haplo-insufficient, what phenotypic
ratio would you expect among the progeny of two parents that were both
heterozygous for the mutation?
d. You have cloned the defective gene including its promoter region.
How would you demonstrate that the coding sequence is undamaged? (You
should be able to think of at least two answers).
e. The entire genomic clone minus its defective promoter is cloned into a
lambda phage expression vector that has its own promoter and transfected into
E. coli . Would you expect the protein to be expressed? Explain
your answer.
f. Do you think your answer in part e would have been different if you
had cloned a cDNA for the gene into the expression vector? Explain
the reasoning behind your answer.
g. You isolate a complete wild type genomic clone for the same gene, including an intact promoter that extends 300 base pairs upstream from the
transcription start site. When you transfect that clone (carried in an
appropriate vector that does not supply its own promoter) into a cultured
mouse cell line, you obtain only very weak expression of the gene. Explain
what else might be needed for more vigorus expresssion.
h. You transfer the genomic sequence form part e into a retroviral vector
with a strong constituitive promoter and use that vector to generate a
strain of transgenic mice. Would you expect the mice to be healthy and normal? Explain your answer. (You will have to do a bit of
speculation, but it should not be too difficult).
i. You create a transgenic mouse strain with an easily-assayed reporter
gene attached to the normal promoter for the gene you have been studying,
together with several thousand basepairs of upstream DNA. What would you
expect to be able to learn from studying the patterns of expression of the
reporter gene in such mice?
j. How would you use the molecular constructs from part i to identify
essential components of the minimal promoter for the gene.
k. How would you expand on the studies in part j to identify sequences that
increase or decrease transcription relative to the basal level