Download Midterm Exam Due: Wednesday 10/31 before 5 PM at Weiner Lab

Midterm Exam
Due: Wednesday 10/31 before 5 PM at Weiner Lab (CVRI, Room 384)






The midterm examination is to be worked on individually
TAs will not be allowed to review class material during the exam
Be explicit and detailed in your answers in order to receive full credit
Type out answers (you can draw in any diagrams)
Include your name, question #, and page number on each page
(example: John Smith, Question #1, Page 1 of 2)
Start each question on a new page and staple each question together separately
.
1. λ lysogens are immune to superinfection by λ, but not with other lambdoid phages
such as 434. λimm434 (a lambdoid hybrid phage that has mostly λ genes except for cI
and the surrounding “immunity” region of 434 phage) also can grow on E. coli(λ).
a. If you take a 1ml culture of E. coli(λ) (this is wild-type E. coli) and superinfect with
λimm434 at an MOI of 1, the tube will clear after about an hour. Describe how you
would determine whether the released phages from this infection are λimm434 or λ
(induced from the prophage). Do you expect there will be more λimm434, more λ, or
equal numbers of both phages in the lysate?
b. You perform infection experiments using wild-type E. coli and E. coli(λ), infecting
them at an MOI = 1 with either λ, 434, λimm434, or λimm434 Ram (an amber mutant in
the R gene) and get the following result:
λ
E. coli
+
E. coli(λ)
+ = lysis, - = no lysis
Infecting Phage
434
λimm434
+
+
+
+
λimm434 Ram
+
Explain, in detail, why λimm434 Ram is able to lyse the λ lysogen but not wild-type E.
coli.
c. Do you expect there are viable phages in the lysate from the infection of E. coli(λ) with
λimm434 Ram and, if so, what phage genotype will be present?
2. For all questions, please include enough details so that we know exactly what you are
doing. E.g. Transform a wildtype strain with a plasmid containing X and plate at 23C.
Replica that to a plate containing Y at 36C. Mate this to a strain with a deletion of gene
Z. If you start with a strain, say how you would get it (I’d mate my mutant to a strain
with a deletion of gene X and I would select the segregants that had these characteristics),
etc
i. You want to identify bypass suppressors of the essential function of the gene YFG1. That
is, you want mutations that allow a yfg1∆ to live. You have no temperature sensitive
allele of YFG1 and don’t want to make one.
a) Describe the screen that you would do to find these suppressors of YFG1
(the SOY genes).
b) How would you clone (find the gene for) a recessive SOY mutant? No
mapping.
c) How would you clone a dominant SOY gene? No mapping.
d) In class we discussed the use of a collection of all the deletions of all the
nonessential genes. How could you use the deletion collection to find
more SOY genes in a comprehensive way?
e) Why might you not isolate all the genes in part d that you had isolated as
recessive suppressors in part a/b?
ii.
You also want to do a screen for suppressors of YFG1 that are not bypass suppressors.
That is, these are things that allow a ts allele of yfg1 to grow at the normally nonpermissive temperature of 36° C.
a) First, you would like to generate a ts allele of YFG1. Exactly how can this
be done?
b) After isolating your temperature sensitive allele, you sequence it (to make
sure there is really a mutation in the YFG1 gene) and do a suppressor
screen. You isolate four extragenic suppressors. These suppressors allow
an allele of YFG1, yfg1-1, to grow at 36°C. There are two
complementation groups, each with two alleles (SOY10-1, SOY10-2,
SOY11-1, SOY11-2). How exactly did you determine this? Describe how
you would generate all strains used.
c) Neither of these extragenic suppressors are bypass suppressors and both
are allele specific (you know this because you had isolated two different ts
alleles of YFG1 originally, and neither of these suppressors suppressed
your other allele, yfg1-2) and dominant. Such a screen could identify both
interesting and uninteresting suppressors. What is an example of an
uninteresting class of suppressor that you might get. How could you set
up the screen to avoid this uninteresting class of suppressors.
iii.
Your advisor wants you to do a project in which you need to delete 50 genes in
one strain. You hate your advisor. You decide to do this serially, deleting each
gene in turn. The problem is, there aren’t 50 markers available. Design a system
in which you can delete the first gene without “using up” the marker
3. Sookie, a student in Genetics 200A, is a little too obsessed with vampire TV shows.
She keeps a mirror on her bench that she uses to see if any of her mutants fail to show a
reflection (a telltale sign of yeast vampirism). Sookie recently obtained a number of wild
S. pombe strains that were found growing outside the nuclear reactor sites around the
world. Interesting in the question of how heterochromatin is kept within its normal
boundaries, she inserted into each these strains a ura4 reporter gene just outside the
boundaries of the mat2/3 silent mating type locus in each of her wild S. pombe isolates.
After crossing two strains found outside of different reactors, she was astonished to see
that an occasional progeny that did not produce a reflection. She also replica-plates the
colonies to 5-FOA. The plates below show the patterns observed.
a) What types of tetrads are observed and in what ratios?
b) What does this tell you about the location of the mutations that are segregating in
this genetic cross?
c) What does this tell you about how genotype is connected to phenotype?
d) Assuming that silencing of a ura4+ gene that is placed outside of heterochromatin
means that there has been a lateral spread of heterochromatin outside of its normal
boundaries, suggest a hypothesis (or two) for what might be the molecular cause
of fission yeast vampirism.