BIO4320 Mid-Term Examination Oct.

24, 2000
Name: Student ID:
Part II (Dr. Lam’s Portion; total 32 marks) Answer all questions.

Question 1

A special lambda phage vector (λYES) was constructed as shown in the following diagram:

λ Arm λ Arm

Promoter Promoter
GAL1 lac

lox ori ARS1 Apr URA3 lox

sequence Single sequence
Cloning Site:
XhoI
ori: plasmid replication origin (ColE1 type) that functions in E. coli

ARS1: replication origin that works in yeast

Apr: ampicillin (an antibiotic) resistance gene

Promoter GAL1: yeast promoter inducible by galactose

Promoter lac: E. coli promoter inducible by IPTG

URA3: yeast URA3 gene required for uracil biosynthesis

lox sequence: Cre-lox system is a site specific recombination mechanism found in P1 phage
(P1 phage is not a filamentous phage); two lox sequences will recombine in
the presence of the Cre protein

lox lox

+ Cre Protein

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BIO4320 Mid-Term Examination Oct. 24, 2000
Name: Student ID:

A modified λYES vector containing an amber mutation in a coat protein gene was used to
generate a cDNA library of mammalian cells.

(a) What are the two major properties of the E. coli strain used to amplify the library. (2
mark)

It should be a suppressor strain (1) and free of Cre protein activities (1).

(b) A putative mammalian cell cycle gene was identified in the above cDNA library. You are
provided with a clone of the Cre gene. What strategies will you use to convert the λ
phage clone into a plasmid clone AND propagate it in E. coli? (5 marks)

Choose a non-suppressor strain (1) and transform it with a clone containing the Cre gene (1).
Infect the resulting strain with the λ phage clone (1). The Cre protein will release the circular
(0.5) plasmid DNA between two lox sites (0.5) on the λ phage DNA (1). The λ phage cannot
lyse the cell because the amber mutation (0.5) it carries will prevent protein coat synthesis
(0.5) in a non-suppressor strain. Selection of the bacteria containing the desire plasmid clone
can be done using the ampillicin resistant marker (1).

(c) Draw a diagram representing the plasmid clone resulting from (b). (2 marks)

Putative
Promoter mammalian Marking Note
GAL1 cell cycle gene
Reduce 0.25 mark for each
Promoter
Ap r missing item; reduce 0.5 mark
lac for 2 lox sites

XhoI Wrong arrangement: no mark;

XhoI
Only lox site on a circular
ARS1 plasmid: 0.5 mark

Ura3

ori

lox

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BIO4320 Mid-Term Examination Oct. 24, 2000
Name: Student ID:
The mammalian cell cycle gene in the plasmid clone obtained in (c) was sequenced and
found to locate at the right orientation relative to the lac promoter (i.e. sense RNA will be
generated using the lac promoter). With only the information given above, describe how you
will reconstruct the clone so that the mammalian cell cycle gene is now located at the right
orientation relative to the yeast GAL1 promoter on the plasmid. (4 marks)

Cut out the mammalian cell cycle gene from the plasmid clone using XhoI (1). Purified the
vector and cDNA by gel electrophoresis (0.5). Remove the 5’ phosphate group from the cut
vector by phosphatases (0.5) to prevent self-ligation of vector (0.5). Ligate the cDNA
fragment with the cut vector (1). Since it is a non-directional cloning (1), the cDNA fragment
can be inserted in both orientation (1) and some will at the right orientation as that of the
promoter GAL1 (1).

OR

Cut out the mammalian cell cycle gene from the plasmid clone using XhoI (1). Generate
probes using the target genes (1) and used the probes to re-screen the λ phage library
mentioned above (1). λ phage clones with positive signals will be purified (0.5) and plasmid
clones will be obtained as described in (b) (1). Clones with desired the orientation will be
identified by DNA sequencing (0.5) or restriction mapping (0.5).

(d) Since the function of the mammalian cell cycle gene cannot be tested in E. coli, the new
plasmid clone constructed in (d) was transformed into yeast for further experiments.
Describe how you can select for the successful transformation event. (3 marks)

Transform the plasmid into a URA3- mutant (1). Since the plasmid carries a normal URA3
gene (1), successful transformants can be selected as survivors (0.5) on uracil free medium (1;
0.5 for minimal medium).

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BIO4320 Mid-Term Examination Oct. 24, 2000
Name: Student ID:
Question 2

A cDNA fragment encoding a glutelin protein from wheat was cloned. No EcoRI site was
found in this cDNA fragment. The wheat genomic DNA was isolated and cut with EcoRI
before separated on an agarose gel. Southern blot experiments were performed using high
stringency conditions that do not allow mismatch.

(a) If no DNA sequence information is available, which method you will use to generate
probes based on this cDNA fragment. (1 mark)

Random primed labeling (1) or nick translation (1).

(b) Two (instead of one) bands were found to hybridize to the probes generated in (a).
Explain this observation. (1 mark)

Presence of an intron (0.5) with a EcoRI site (0.5) on the genomic region spanned by the
cDNA fragment (0.5).

OR

More than one copy of the gene existed in the genome (1).

(c) With only the information mentioned above, describe the key experimental conditions
that you will use to check if similar glutelin genes exist in rice. (3 marks)

Extract genomic DNA from rice (0.5) and perform Southern blot (0.5; accept description of
procedure without stating the term Southern blot). Using the wheat glutelin gene as probe
(0.5), low stringency hybridization and washes (1) will be performed. Low stringency
conditions can be achieved by lowering the hybridization temperature (0.5), increase the
NaCl concentration (0.5), or reducing the amount of formamide (0.5).

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BIO4320 Mid-Term Examination Oct. 24, 2000
Name: Student ID:
Question 3

RNA samples were prepared from normal cells and cancer cells respectively. After reverse
transcription, the cDNAs were subject to real time PCR measurement. Two pairs of primers,
using for the amplification of an unknown gene “X” and a housekeeping gene “Y”
respectively, were tested. The following results were obtained. Standard Curve

Gene Sample Number of Cycle 20

Number of PCR Cycle

X Normal 16 18
X Cancer 12
16
Y Normal 18
Y Cancer 16 14

12

10 1.0 0.1 0.01 0.001

Relative
Relative Amount
Amount of
ofPCR
PCRProducts
Templates

(a) Describe the relative expression of Gene “X” in normal cells and cancer cells. (4 mark)

In normal cells, expression of Gene X relative to Gene Y = 0.1/0.01 = 10 (1).

In cancer cells, expression of Gene X relative to Gene Y = 10/0.1 = 100 (1).
Using Gene Y as the reference (0.5), expression of Gene X in normal cells relative to cancer
cells = 10/100 = 0.1 (1).
Therefore, Gene X is induced in cancer cells as compared to normal cells (1).

(b) Explain why it is important to measure the expression of Gene “Y”. (2 marks)

Gene Y is a housekeeping gene that will express at relatively constant level (1) in different
cell types. Gene Y thus provides a reference point for comparison (0.5) and will eliminate
experimental artifacts during tissue collection (0.5), RNA preparation (0.5), and reverse
transcription (0.5).

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BIO4320 Mid-Term Examination Oct. 24, 2000
Name: Student ID:
Question 4

An AFLP (amplified fragment length polymorphism) experiment was performed using the
enzyme EcoRI and MseI.

EcoRI MseI BamHI

5’GAATTC3’ 5’TTAA3’ 5’GGATTC3’

3’CTTAAG5’ 3’AATT5’ 3’CCTAAG5’

(a) If BamHI was used instead of MseI in the first step of the experiment, what problems will
occur? Explain. (2 marks)

BamHI is a 6 bp cutter and MseI is a 4 bp cutter (0.5) and hence restriction by MseI will be
more random (0.5) and more fragments will be generated (0.5) compared to the restriction by
BamHI. Therefore, the number of bands in the final gel electrophoresis would be decreased
if BamHI was used (1).

(b) What subsequent step can be altered to minimize the problem caused in (a)? Explain. (3
marks)

One way to increase the number of bands in the final gel electrophoresis is to use a less
selective primer (1) during the PCR steps involved. For example, the Primer (+2) can be
used instead of Primer (+3) (1) during the selective amplification step (1).

- End of Part II -

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