Introduction

1. If base pairs are arranged at random in a double stranded DNA molecule 106 base pairs long,
and if the base composition of this DNA is 10% A, how many times should the restriction
endonuclease site 5'-GAATTC-3' occur in this molecule?

2. When the DNA of a virus is subjected to Cot analysis, it renatures with a Cot1/2 of 3 X 10-1.
Represent the expected results on a graph.
Evaluate the size of the genome.
(Use E. coli as reference genome)

3. You are given two test tubes containing 10 mg of cow DNA and 50 mg of DNA from a
bacterium that is closely related to E. coli. You want to compare the complexity of the DNA
from these two organisms. Explain how you would compare the complexities of these DNAs and
draw the Cot curves you might expect.

DNA replication

1. The genome of Aspergillus niger is about 8 X 107 base pairs long and each replicon is about
34 micrometers long.
a) How many replicons are there in the A. niger genome.
b) If the A. niger genome is replicated in 25 minutes how many base pairs will be replicated:
i) every minute per replicon?
ii) every minute per replication fork?

2. The size of the Saccharomyces cerevisiae (yeast) genome is about 1.5 X 107 base pairs. Yeast,
which uses 400 replication origins to replicate its 17 chromosomes, takes 30 minutes to complete
S phase (replicate its genome). Assume all replicons are the same size, initiate replication at the
same time during the cell cycle and are replicated at the same rate.
a) How long does it take to replicate one replicon?
b) How fast does a yeast replication fork move (give your answer in micrometers per minute)?
c) Telomeres are special and necessary for the proper replication of eukaryotic chromosomes.
Telomeres are not required for the replication of bacterial chromosomes. What important role do
the telomeres serve and what replaces the function of the telomere in bacteria?

3. Explain why a significant proportion of newly synthesized DNA is found as very short DNA
fragments which are later incorporated into high molecular weight DNA. Use diagrams as part of
your answer.

4. The duration of the S phase in yeast is about 5 minutes. If yeast cells replicate their DNA at a
rate of about 50 base pairs per second per fork, how many bidirectional replication origins are
required to replicate the 3.6 x 107 base pair yeast genome during the S phase.

5. a) The linear chromosome of phage lambda is 17 µm long. Since its chromosome is double
stranded, its genome is about 50% GC and the weight of the average nucleotide pair weighs 660
daltons, what is the molecular weight of the lambda molecule?
b) What are topoisomerases and what role do they serve?

6. a) An E. coli replication fork moves forward at about 600 base pairs per second, how often
must the DNA ahead of the fork rotate every minute to accommodate this rate of replication?
b) List the steps that must be completed to attach a newly formed Okazaki fragment to a pre-
existing DNA strand. What enzymes participate in each of these reactions?

Gene regulation 1

1. The following mutations (mutant bases are shown in small letters) occur in the leader
polypeptide gene region of the tryptophan operon. Also shown is the wild type sequence of the
coding strand of the leader peptide gene.

Note: (i) Assume that there is only one methionine codon (ATG) between the 5' end of the
tryptophan operon encoded mRNA and the start codon for the trpE gene.

Note: (ii) The codon GGG codes for glycine (Gly).

fMet Lys Ala Ile Phe Val Leu Lys Gly Trp Trp Arg Thr Ser Stop

wild type- ATG AAA GCA ATT TTC GTA CTG AAA GGT TGG TGG CGC ACT TCC TGA

mutant #1- ATG AAA GCA ATT TTC GTA CTG AAA GGT TGa TGa CGC ACT TCC TGA

mutant #2- ATG AAA GCA ATT TTC GTA CTG AAA GGT gGG gGG CGC ACT TCC TGA

mutant #3- cTG AAA GCA ATT TTC GTA CTG AAA GGT TGG TGG CGC ACT TCC TGA

mutant #4- ATG AAA GCA ATT TTC GTA CTG AAA GGT TGG gGG CGC ACT TCC TGA

Question: What effect will these four mutant leader peptide genes have on the regulation of the
tryptophan operon? Justify your answer.

2. Describe the fate of the following mutant lambda phages after they infect E. coli. Explain your
rational.

mutant #1- inactive CI gene.

mutant #2- inactive cro gene.

mutant #3- inactive Prm promoter.

mutant #4- mutant causing a defective att site.

b) Are Oc mutations dominant or recessive, and do they act in cis or trans? Please explain.

c) You are given a strain of E. coli that constitutively expresses the lactose genes lacZ, lacY and
lacA. How would you determine what type of mutation caused these genes to be constitutively
expressed?

4. How does the lac repressor prevent transcription of the lactose operon?

5. What is the advantage to E. coli of the lactose operon being under both positive and negative
control?

6. Explain how it is that is is dominant over i+, and why i+ is dominant over i-. Your explanation
should explain the relative dominance of these mutations in terms of how the lac repressor
functions, the type of mutations present and how these mutations alter the activity of the lac
repressor.

7. Regulation of the tryptophan operon involves a repressor protein and tryptophan as a co-
repressor, whereas although regulation of the lactose operon also involves a repressor protein it
does not utilize a co-repressor but rather lactose (actually allolactose) acts as an inducer. Please
outline the roles that tryptophan and lactose play in regulating expression of the tryptophan and
lactose operons respectively.

8. The transcription activator protein GAL4 has three functionally distinct regions. What are
these regions and what are their roles in galactose expression. What would you expect the
phenotype of the following three yeast strains to be with respect to galactose gene regulation and
their ability to grow on galactose.
i)Strain one has a mutation that inactivates the GAL80 binding region of GAL4 so that it cannot
bind the GAL80 protein.
ii)Strain two has a mutation that inactivates the DNA binding region of the GAL4 protein.
iii)Strain three has a mutation that deletes the acidic blob region of the GAL4 protein.

9. You isolated two classes of mutants (A and B). Both classes cause the SUC2 gene (SUC is
short for sucrose) to be expressed at high levels even when sucrose is not added to the growth
medium (Note: SUC2 is normally not expressed unless sucrose is present). SUC2 encodes an
enzyme activity (invertase) that is essential for the utilization of sucrose as a carbon source. A
detailed analysis of these mutants shows that Class A mutants map adjacent the SUC2 gene and
can only affect the expression of invertase genes that are on the same molecule. Class B mutants
are not linked to SUC2. What can you tell about the role of the sequences affected by Class A
and Class B mutants in the expression of SUC2?
10. You discover that yeast is able to utilize the carbon source sugarose. You also discover that
induction of the genes coding for the enzymes required for sugarose breakdown (the SUG1, 2, 3
& 4 genes) by sugarose is dependent upon the SUG8 gene. You suspect that the SUG8 product
codes for a trans-acting transcription factor that binds upstream of the enzyme encoding SUG
genes. Your experiments tell you that the SUG8 protein is always bound to the DNA upstream of
the four enzyme encoding genes regardless of whether or not sugarose is present.
a)Explain how this factor (the SUG8 product) might operate to regulate expression of the
sugarose genes.
b)Design an experiment to test the model you proposed in (a).

11. What will happen to the expression of the tryptophan operon of E. coli when tryptophan is
removed from the following cultures of mutant strains that are growing in the presence of
tryptophan? Note: Assume that attenuation accounts for a ten fold regulation and that the trp
repressor protein accounts for about a 70 fold regulation.
a)The tryptophan repressor protein is mutant so that it cannot bind tryptophan.
b)The tryptophan repressor is mutant so that it cannot bind the trp operator.
c)The start codon for the leader peptide is mutant.
d)The series of T=A base pairs at the end of the attenuator region is deleted.

12. What will happen to the expression of the tryptophan operon of E. coli when tryptophan is
added to the following cultures of mutant strains that are starved for tryptophan. Note:Assume
that attenuation accounts for a ten fold regulation and that the trp repressor protein accounts for
about a 70 fold regulation. a)The tryptophan repressor protein is mutant so that it cannot bind
tryptophan.
b)The tryptophan repressor is mutant so that it cannot bind the trp operator.
c)The start codon for the leader peptide is mutant.
d)The series of T=A base pairs at the end of the attenuator region is deleted.

13)What are the fundamental differences between the mechanisms employed by phages SP01,
T7 and lambda to switch transcription from one class of genes to another?
14) (i)If you infect E. coli with a lambda phage carrying a mutation in the CI gene, can it
lysogenize (form a stable association with the bacterial chromosome)? Why?

(ii)If you infect a bacterium simultaneously with a wild-type CI- and a CI+ mutant of lambda,
can you obtain stable lysogeny? Why?

Gene regulation 2

1) Compare and contrast the transcription process including its regulation, initiation and
termination in eukaryotes with the transcription process in prokaryotes.

2) In the sapce below draw a diagram of the DNA sequence coding for a eukaryotic gene. Please
accurately indicate the location of the information that must be found in this DNA sequence if it
harbours a gene that expresses a 200 amino acid protein.
3) Describe the two differences that you think are the most important, between DNA replication
and transcription.

4) A gene has three introns and four exons. You do R-loop hybridization between this gene's
RNA and a genomic DNA clone. What product would you expect to generate from this
hybridization? Draw the hybrid molecule.

5) A gene has three introns and four exons. You do DNA RNA hybridization between this RNA
and a cDNA clone of this gene. What product would you expect to generate from this
hybridization. Draw the hybrid molecule.

6) a) If you could radioactively label the phosphate between the first exon and the adenosine at
the 5'-end of the adjoining intron the tetrahymena rRNA precursor ie. exon-P-intron Where
would the radioactivity end up after splicing was completed (show a diagram).

b)What is the role of the sigma factor polypeptide in the bacterial RNA polymerase?

c)Why are purified eukaryotic RNA polymerases incapable of transcribing genes accurately in
vitro?

7) Eukaryotic mRNA molecules are modified significantly between their initial transcription and
their export from the nucleus for translation. Describe four modifications events that alter the
primary transcript during the production of mature mRNA.

Recombinant DNA technology

1. Your friend cloned the yeast gene immediately adjacent a gene (YFG) that you are interested
in. The BamH1 fragment he cloned contains some but not all of YFG. Using your friend's clone
and a yeast clone bank in pBR322 (fragments generated by partial digestion with the restriction
endonuclease Sau3A) how would you isolate a clone containing YFG?

2. A liner DNA molecule is labelled at both ends with P32 and digested with restriction enzymes.
HindIII produces fragments of sizes 2.9, 4.5, 6.2, 7.4 and 8.0 kb. An autoradiogram developed
from a Southern blot of this digest shows radioactivity associated with the 6.2 and 8.0 kb
fragments. When Sst1 cleaves the same fragment it generates three bands of 6.0, 10.1 and 12.9
kb with the 6.0 and 10.1 being radioactive. When the same molecule is digested with both
HindIII and Sst1, fragments of sizes 1.0, 2.0, 2.9, 3.5, 6.0, 6.2 and 7.4 kb are produced. Draw a
restriction enzyme recognition site map showing the Sst1 and HindIII sites.

3. The following restriction fragments were obtained by a graduate student when she performed
agarose gel electrophoresis analysis of a bacterial plasmid.

BamH1 (4.3 kbp and 6.0 kbp).

Draw a restriction endonuclease recognition site map that shows the location of the BamH1 and
EcoR1 sites present in this plasmid.

4. Draw the banding pattern you would expect to see on a DNA-sequencing gel if you annealed
the primer 5'-T-C-G-A-A-3' to the following single-stranded DNA fragment and carried out a
DNA sequencing experiment using the dideoxy chain termination method. Assume:

a) all four DNA precursors were labelled.

5'-A-G-C-T-T-G-G-A-C-T-C-A-G-T-A-G-C-T-T-C-G-A-3'

5. Saccharomyces cerevisiae has a haploid genome that is about 1.4 X107 base pairs. How many
40 kbp pieces would you have to clone into a library if you wanted to be 90% certain of
including a particular sequence?