Biology 4, Exam#2, 2004S

1. Explain why individuals in a population that are the most efficient at utilizing available resources will be the most likely to survive. (5 points)

When resources such as food get to be very scarce, those individuals that can live on the least amount of resources will survive, while those that are not as efficient at utilizing these resources will die.

 

2. Explain why the cell needs to regulate the Lac operon both positive and negative means - what is the biological purpose of this. (5 points)

The Lac operon contains genes that are involved in the utilization (metabolsim) of lactose. The negative control featured in the Lac operon allows the operon to make its products when lactose is present but will turn off the operon when there is no lactose around. This ensures that the cell will not waste energy making products that cannot be used. The positive control aspect of the Lac operon has to do with regulation of the Lac operon in response to glucose levels. The cell is most efficient in using glucose as an energy source so if both lactose and glucose are present, the cell would be better off (more efficient) in utilizing glucose. The positive control system will a small level of tanscription of the Lac operon in the presence of glucose, but if there is not glucose around, the positive control system will dramatically boost the level of Lac operon transcription.

 

3. Explain how T4 phage infection is regulated into three stages: early, middle, and late. (6 points)

Early genes are transcribed using a promoter that is recognized by the bacterial holoenzyme (RNA polymerase core + host sigma factor). One of the products of the early genes is a "middle" specific sigma factor, while another product of the early genes is a protein that interferes with the host sigma factor. This serves to stop initiation at the early promoter (no host sigma is available) and start initiation at the middle promoter (core + middle sigma). Switching from middle to late works in a similar manner. One product of the middle genes is a "late" specific sigma; another product blocks the middle sigma. This causes a switch to late genes.

 

4. Normally in the Lac operon, the promoter and the operator regions overlap slightly, with the promoter being upstream of the operator region. What would be the effects of the following changes in the DNA on the ability of the cell to regulate transcription from the Lac operon according to the level of lactose (2 points each) :

a) a separation between the promoter and the operator of 20 extra nucleotides

This will not have any effect on Lac operon regulation (a repressor bound to the operator will still block transcription)

 

b) reversing the order of the promoter and operator so that the operator is upstream (5'-wards) of the promoter.

This will eliminate repression of the operon since binding of repressor to this operator will not interfere with the ability of the RNA polymerase to start transcription of the genes. The operon will always be ON. It will not matter whether lactose is present or not.

 

c) moving the operator from its normal upstream location to between the first and second genes of the Lac operon

Since the operator will not interfere with the ability of RNA polymerase to initiate transcription, the first gene will always be on (± lactose). However, when the repressor is bound to the operator, it will now block transcription of genes 2 and 3 of the operon. Thus, in the presence of lactose, all three genes will be transcribed (and protein products made), but in the absence of lactose, only gene 1 products will be made.

 

5. Which kind (class) of RNAs do the three different eukaryotic RNA polymerases transcribe. (3 points)

RNA plolymerase I - rRNAs

RNA polymerase II - mRNA (protein coding genes)

RNA polymerase III - tRNAs

 

6. What are the three main processes in maturation of a mRNA in the nucleus? (3 points)

capping, splicing, polyadenylation

 

7. Why is it thought that regulation of eukaryotic genes in general does not utilize repressors that are so prevalent in prokaryotic cells? (4 points)

In contrast to prokaryotes, only a very small percentage of genes are expressed in any one cell at any one time. The general biological requirement for most genes, therefore, is to be off. It is much more efficient to activate the 1-5% of the genes that are needed than to repress (inhibit) the 95-99% of the genes that are not needed. In other words, the default condition is OFF.

 

8. Explain how transcription factors and multiple promoter motifs in eukaryotes can lead to the very subtle and fine level of control necessary in multicellular organisms. (5 points)

Each promoter sequence element can be the target for one or more transcription factors, and each transcription factor can recognize and bind to one or more promoter element. The level of transcription from a promoter is determined by the number and nature of the transcription factors that are bound to that promoter. Further, different transcription factors can have different binding affinities to any given promoter element. So, in any given tissue at any given time, it is the population of transcription factors and their ratios that determine the level of transcription from any given promoter. Regulation of expression of the gene is therefore determined by the transcription factors that are available to interact with the promoter.

 

9. Explain how tryptophan levels serve to regulate the Trp operon (a labelled diagram is OK). (4 points)

The Trp operon is responsible for synthesis of Trp. In the absence of Trp the operon is fully turned on (to make more Trp). In the presence of Trp, the Trp repressor (which by itself is not functional) binds to Trp and becomes functional. This Trp repressor-Trp complex then represses the Trp operon to stop further Trp synthesis.

 

10. One of the goals of pharmacogenomics is to customize drug treatments to individual patients. In choosing an appropriate chemotherapy for cancer patients, this could mean the difference between life and death. Briefly discuss the economic implications of this testing and the role of the insurance company. (You may use the back of this page if necessary) (12 points)

some topics that might be included: cost of testing, consequences of NOT testing (balancing money vs. human costs), does this make economic sense for the insurance company, doing this procedure for serious conditions (chemotherapy) vs less serious (Prozac treatment). Many other points are valid.

 

11. Describe the nature of the globin gene clusters in mammals and describe how it is thought they evolved to this current arrangement. (5 points)

There are two globin gene clusters in mammals - alpha and beta. Each consists of several copies of a globin gene, with each gene copy being slightly different from the others. This arrangement is thought to have arisen from by gene duplication of an original gene and then divergence of the sequence(s) after duplication. If the divergence of one copy of the gene benefited the organism, then the change would have been kept and passed on to offspring.

 

12. What is meant by bacterial transformation? (3 points)

The incorporation of foreign DNA into the host bacterial cell by picking up that DNA from the environment and bringing it inside the cell.

 

13. What is a transducing phage? (3 points)

A transducing phage is a phage that can package host DNA into its progeny, insted of only the phage DNA.

 

14. Explain why plasmids are well suited to be used as vectors in genetic engineering experiments. (4 points)

Plasmids are well suited because they can carry genes that provide antibiotic resistance and therefore provide a means for cells containing plasmids to be selected from a mixed population of cells. Plasmids also replicate naturally in the cell as separate entities and do not have to be integrated into the host genome. Finally, unlike phage vectors, they do not kill the cell. That they can exist and not kill the cell means they can be used to express products in the host cell.

 

15. Explain how the restriction-modification acts to protect bacteria against foreign DNA without attacking its own DNA. (4 points)

Each strain of bacterial cell has a pair of enzymes that recognize a specific sequence on DNA. The restriction enzyme will cut any DNA containing that sequence, while the modification enzyme will modify that sequence to prevent the restriction enzyme from cutting the site. All the cell's own DNA is modified and thus protected from degradation by the restriction enzyme. Foreign DNA, such as a phage DNA injected into the cell, is not modified and will therefore be attacked by the restriction enzyme and rendered useless.

16. Describe the steps needed to create a genomic library in the plasmid pBR322 (the one we discussed in class). Include the manipulations of the DNA and the selection of host bacteria by appropriate conditions. (8 points)

1. mix genomic DNA fragments with plasmid DNA cut in the Tet gene (tetracycline resistance)

2. ligate DNAs

3. transform bacteria with this mixture of DNAS

4. grow bacteria in ampicillin - only cells that have picked up plasmids (either with or without genomic DNA) will grow

5. now grow cells in presence of tetracycline and D-cycloserine. The D-cycloserine will kill all growing cells. Since the recombinant DNA plasmids will not have a functioning Tet gene, they will not grow in tetracycline. Recircularized plasmids (without a genomic DNA insert) will grow in tetracycline and therefore they will be killed. All that will remain is the bacteria containing recombinant plasmids.

 

17. a) What is cDNA and how is it generated? (4 points)

cDNA is DNA that is complementary to a mRNA. It can be double stranded or single stranded. It is made by incubating mRNA(s) with oligo(dT) which will basepair to the poly(A) tail on the mRNAs. This annealed oligo(dT) can serve as a primer for DNA synthesis by an enzyme called reverse transcriptase, which will read the mRNA sequence and synthesize a complementary DNA strand (a cDNA).

 

b) Describe one use of cDNA as contrasted to genomic DNA when carried in a vector. (2 points)

Since the sequence is a direct copy of the mRNA sequence, cDNAs can be used in expression vectors to create a protein product in bacterial cell hosts. This will not work with genomic DNA because of the presence of introns that can not be removed because bacteria do not carry out splicing.

 

18. What are RFLPs and why are they useful? (5 points)

A R estriction F ragment L ength P olymorphism is a site on genomic DNA that will produce different restriction enzyme digest patterns in different individuals. These differences can be thought of as being due to different "alleles" of a piece of DNA, each of which have a slightly different sequence resulting in different restriction site locations. It is useful in identifying individuals for forensics, paternity, or genetic screening reasons.

 

19. You perform a genomic blot using DNA extracted from human, rat, and chicken and then probe the blot with a human insulin cDNA probe? You observe a single band in the chicken DNA but you see two bands in the rat and human DNAs. What is a likely explanation for this? (3 points)

This suggests that there was a change in the DNA sequence during evolution that introduced a new restriction enzyme site sometime after chickens and rats diverged.

 

20. The goal of gene therapy is to replace a damaged gene with a "good" copy of that gene. Assume that you have developed a method of delivering a "good" gene into a targeted cell. Describe two possible obstacles that you would still need to overcome in order for this approach to work reliably in the target cells and create a cure. (6 points)

a number of possibilities including:

· "bad" product is still being made and may still cause harmful effects

· the introduced gene may be inserted into the host cell at an inappropriate location and could disrupt the functioning of a normal gene

· the introduced gene may be inserted into a heterochromatic region and therefore not be transcribed

· the introduced gene might be transcribed and translaged at too high a level and make too much product - ie it is not regulated to a specific level

· the introduced gene might never get integrated into the host cell DNA and therefore might only provide a transient expression

 

Biology 4: Genes and Society