0001 Mid

BIO4320 Mid-Term Examination Name:

Oct. 24, 2000 Student ID:

Part I (Dr. Fung’s portion; 16 marks total) Answer ALL questions. 1. A unique protein produced by a cancer cell was purified and sequenced. The amino acid sequence of the N-terminal of this protein was Met-Lys-Thr-Trp-Tyr-Cys-Glu-Asn-GlySer. A cDNA library was constructed from the mRNA of this cancer cell. (i)

Write down the sense and anti-sense sequences of the 5’ end of the corresponding mRNA. (4 marks)

(ii)

Design an oligonucleotide with 17 bases for screening this cDNA library. (4 marks)

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BIO4320 Mid-Term Examination Name:

Oct. 24, 2000 Student ID:

2. E. coli was first cultured in a medium with glucose and then changed to a medium with lactose as its only carbon source. Explain in terms of gene regulation, how the E. coli coped with the new environment. (8 marks)

- END of Part I -2-

BIO4320 Mid-Term Examination Name:

Oct. 24, 2000 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 GAL1 lox sequence

ori

ARS1

Promoter lac

Apr

lox sequence

URA3

Single 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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lox

BIO4320 Mid-Term Examination Name:

Oct. 24, 2000 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)

(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)

(c)

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

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BIO4320 Mid-Term Examination Name:

Oct. 24, 2000 Student ID:

(d) 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)

(e) 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)

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BIO4320 Mid-Term Examination Name:

Oct. 24, 2000 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)

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

(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)

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BIO4320 Mid-Term Examination Name:

Oct. 24, 2000 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 Sample Normal Cancer Normal Cancer

Number of Cycle 16 12 18 16

Number of PCR Cycle

Gene X X Y Y

20 18 16 14 12 10

0.01 0.001 1.0 0.1 RelativeAmount Amountof ofPCR PCRTemplates Products Relative

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

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

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BIO4320 Mid-Term Examination Name:

Oct. 24, 2000 Student ID:

Question 4 An AFLP (amplified fragment length polymorphism) experiment was performed using the enzyme EcoRI and MseI. EcoRI

MseI

BamHI

5’GAATTC3’ 3’CTTAAG5’

5’TTAA3’ 3’AATT5’

5’GGATTC3’ 3’CCTAAG5’

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

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

- End of Part II -

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