0203 Fin Mark

ANSWER ALL QUESTIONS (1)

The efficiency of transforming genes into rice via Agrobacterium is found to be variety dependent. It is much easier to transform japonica rice than indica rice. (a) Based on your current knowledge on plant tissue culture, plant regeneration and Agrobacterium-plant interaction, what parameters you will test in order to improve the Agrobacterium-mediated transformation of indica rice? Explain your answer. (6 marks)

The success of plant transformation mainly depends on (i) the interaction between plants and Agrobacterium (1) via signal exchanges (1); and (ii) the ability of the tissue explants to become callus (0.5) and regenerate to an intact plant (0.5) after transformation. Therefore, the following parameters should be tested: (i) different Agrobacterium strains (1) (ii) different indica rice lines (1) (iii) addition of putative signal molecules (phenolic compounds) (1) (iv) try different tissue explants (1) (v) try different phytohormone combination during regeneration (1) (b) Will there be variety differences if we use gene gun bombardment instead? is the major disadvantage of the gene gun approach? (2 marks)

What

The variety differences should be reduced (1). However, gene gun may lead to multiple insertions (1). (2)

A team of scientists has discovered several kinds of well preserved ancient insects inside a cave in an ice mountain. Based on their morphology, they do not belong to any of the recent insect taxa. (a) Will you recommend them to use SSR or ISSR experiment to generate a molecular phylogeny for these ancient insects? Explain your answer. (2 marks)

ISSR should be used (1). For ancient insect, no DNA sequence information is available(1) and hence SSR cannot be used. Moreover, ISSR is multilocus and will examine DNA at a genome-wide scope (1) and hence is better for phylogenic studies. (b) Can you use the same method to study phylogeny of distinctly related organisms? Explain your answer. (2 marks) No (1). For distinctly related organisms, the number of common bands in ISSR will be too few (1).

(3) Gene therapy and animal cloning are controversial topics in our society. (a)

What are the fundamental differences between somatic gene therapy and germ line gene therapy? (3 mark)

Somatic gene therapy only changes genetic composition of somatic cells (0.5) and the changes will not be passed to subsequent generations (1). Germ line gene therapy changes genetic composition of germ cells/reproductive cells (0.5) and the changes will be passed to subsequent generations (1). (b)

The success of animal cloning also brought new insight in the regulation of cell cycle. How will this affect somatic gene therapy? (3 marks)

In the past, stem cells are absolutely needed for somatic gene therapy because these cells can divide and differentiate (1) but not the somatic cells (0.5). Using starvation techniques, somatic cells can be returned to their resting stage (1). These cells can restart the cell cycle and regain ability to divide and differentiate (1). In theory, we can now use somatic cells in place of ES cells (1).

C

G

H

I

A

B

D

E

J

F

L

M

N

O

P

(4) The relationship of individuals in the above pedigree is as follows: Square: male; Circle: female A/B, C/D, E/F are couples D and E are monozygotic twins, sons of A and B G, H, I, and J are sons and daughters of C and D L, M, N, and O are sons and daughters of E and F Only C, F, G, H, I, J, and P are still alive (a)

What proportion of genes is in common between J and L? (2 marks)

Explain your answer

D and E have the same genetic component (0.5). J and L will be similar to the case of half siblings (1) and the proportion of genes in common is ¼ (1). (b)

z z z z z

A child of E and F was kidnapped when he was a baby. Many years later, P came and claimed that he is a son of E and F. Since E was died, no DNA sample can be obtained. Based on the information stated above, can you suggest a way to determine of P is really a son of E and F.? (5 marks)

Perform DNA fingerprinting on C, F, G, H, I, J. and P (0.5) The bands from G, H, K and J should come from either C or D (0.5). Combine the DNA fingerprints of G, H, K, J (1) and subtract those bands common to C (1) will regenerate the DNA fingerprint for D (1). D is identical to E (0.5). Compare DNA fingerprints of P to D and F (0.5). If ½ bands of P are identical to F and ½ bands of P are idential to D, P is the son of E and F (2).

(5)

A group of scientists tried to investigate seed development related genes in rice. After constructing a cDNA library from development seeds, they find that most cDNA clones are those encode seed storage proteins (mainly glutelins, prolamins, and albumins). Suggest an effective approach to handle this situation. Explain your experimental strategy in details. (6 marks)

Use subtractive library techniques (1). Total mRNA from development seeds as tester (1) and RNAs of glutelins, prolamins, and albumins (1) as drivers. Explanation of how to construct subtractive library using the above materials (3). Accept alternative answers like pre-screening to get rid of redundant clones. (6)

A group of scientists tried to perform proteomic studies on developing seedlings. However, they found that there too many proteins on the 2-D protein gel so that spots overlapped to each other. Suggest some ways to circumvent this problem. (4 marks)

Use zoom gel (1) to enhance the separation of a particular pH range (1). Separate the proteins in different subgroups (1), using techniques such as multi-dimension liquid chromatography (1), SELDI (1), etc. (7) You have learnt the yeast two-hybrid technique in class. A more sophisticated technique called yeast three-hybrid technique was invented to study RNA-protein interaction. The principle is that if one RNA molecule contains two protein binding sites, it will act as a linker to bring the two proteins to close proximity (analogous to the yeast two hybrid system that requires physical interaction of two proteins). Suppose that you have obtained the following constructs: (i)

A yeast vector expressing a fusion protein shown below: LexA DNA binding domain

The RNA binding MS2 coat protein

(ii) A yeast vector expressing a hybrid RNA shown below: Binding site for MS2 coat protein

Binding site for a RNA binding protein X

(iii) A cDNA library constructed in a yeast vector expressing fusion proteins shown below: Protein portion encodes by cDNAs inserted in the vector

LexA DNA activation domain

Describe the strategy you will used to clone protein X.

(10 marks)

Construct a reporter strain with two reporter genes (GUS and URA3, etc) (1). These reporters were driven by a promoter having UAS (1) binding to the LexA transcription factor (1). Transform both constructs (i) and (ii) into the reporter strain (1) and use as bait (1). Then, transform the cDNA library (iii) into the bait strain (1) and select positive transformants by the reporter genes (1). Using diagrams or text to describe how the two fusion proteins and one RNA molecule interaction and drive the expression the reporter genes (5 marks max.) Description of confirmation tests (2 marks max.) - END -