1. Genes are regulated in prokaryotic organisms in clusters called operons.
a. Explain why it is necessary to regulate expression of the Lac operon genes according to both the level of lactose and the level of glucose in the cell. 4 points

The lactose operon synthesizes products that metabolize lactose, thus allowing lactose to serve as the cell's energy source. When lactose is present, the operon is turned on to metabolize the lactose. However, when lactose is not present, the operon is off to help the cell conserve energy. The prefered energy source of the cell is glucose, though, so when glucose is present, the cell will use glucose instead of any other energy source (including lactose). In order to not waste energy synthesizing the lac operon products when glucose is present in addition to lactose, the cell needs an additional control system to prevent lac operon expression in the presence of glucose.

b. Describe how this dual level of control is accomplished (a detailed labeled diagram is acceptable for this answer). 6 points

An operator region exists on the DNA which stands between the promoter and the genes of the operon. In the absence of lactose, a lac repressor molecule binds to the operator and prevents any RNA polymerases which bind to the promoter from actually accessing and transcribing the operon genes. In the presence of lactose, the repressor interacts with lactose and undergoes a change in shape that prevents it from binding to the operator. This removes the repression and RNA polymerases that bind to the promoter can transcribe the operon genes. This allows the operon to be turned on in the presence of lactose and off in the absense of lactose.

In addition to the repressor region, there is an additional binding site on the DNA that overlaps the promoter on the "upstream" side. This site binds a cAMP-CRP protein complex that actually enhances the ability of RNA polymerases to bind to the promoter. When it is present there is at least a 10x increase in initiation of transcription. In the presence of glucose, however, the cAMP concentration in the cell drops dramatically and therefore causes a decrease in the c-AMP-CRP protein complex concentration. This causes a decrease in initiation of transcription from the lac promoter, and therefore a decrease in lac promoter transcription -- even if lactose is present.

2. Explain how the shift from early to late operons is made in a T7 phage infection? 5 points

The promoter for the early operon is recognized by the E. coli RNA polymerase, which transcribes the early operon and leads to the production of all the early proteins from this one polycistronic mRNA. The late promoter is not recognized the the E. coli RNA polymerase so thte late operon is off. One of the products of the early genes is a T7 specific RNA polymerase. This RNA polymerase will recognize and bind to the late promoter, causing transcription of the late operon genes and leading to late operon products.

3. The products of the Trp operon are responsible for the synthesis of the amino acid tryptophan.
a. What would be the effect on regulation of tryptophan levels if there were a mutation in the operator so that it could no longer be recognized by repressor? Explain your answer. 3 points

The trp operon is normally on and is synthesizing tryptophan for the cell. As trp levels increase, a complex forms between trp and a trp operon repressor, which is in an inactive state. The trp-repressor complex becomes active and binds to the operator to turn off trp operon synthesis. If there were a mutation in the trp operator so that repressor could no longer bind, the operon would always be on and synthesizing trp irregardless of trp levels.

b. What would be the effect on regulation of tryptophan levels if in this same cell (part a) a new and entirely functional trp operon was introduced on a plasmid? Explain your answer. 3 points

The new operon would be regulated in response to trp levels as it would be in a normal cell. The "genomic" original trp operon would still have a defective operator, however, and would be non-responsive to changing levels of trp in the cell. Thus, the cell would have a constant level of trp operon synthesis from the constantly expressed genomic trp operon. The high levels of trp this would produce in the cell would cause a trp-repressor complex to form that would totally turn off the "new" trp operon. Thus, the cell would always be synthesizing trp at the level of one operon's activity, while the second operon would always be off due to the high intracellular levels of trp. This cell would also be non-responvie to changes in trp levels.

4. Prokaryotic promoters are often comprised of a single sequence to which a control molecule can bind (e.g. a repressor). Eukaryotic promoters, on the other hand, are much more complex, reflecting the complexity of eukaryotic multicellular organisms.
a. Explain the basic structure/organization of a typical eukaryotic promoter (you may use a diagram) 3 points

A typical eukaryotic promoter consists of a number of sequence elements. Each of these sequence elements is actually a binding site for a transcription factor. The promoter elements could each be different, but there may be more than one copy of a given element. Each different promoter element can serve as a binding site for a different transcription factor.

b. How does this structure facilitate the precise regulation of transcription of specific genes by transcription factors in different tissues or in the same tissue under different conditions? 7 points

5. Explain why it is believed that the a-globin cluster genes, the b-globin cluster genes, and the myoglobin gene all were derived from a common ancestral gene. 3 points

Each of these genes show a similar sequence and a similar organization of introns and exons. The proteins they code for are all very similar in their behaviors. Because of this marked similarity (and dissimilarity to other genes) and because gene duplication has been observed, it is believed that the original globin gene was duplicated and that one of the copies underwent a few changes (making for a slightly different gene product). These two genes each subsequently underwent a gene duplication and mutation process to generate even more descendents from the original gene.

6. Lambda phage can infect E. coli in two different ways. Name the two mechanisms (4 points) and describe them (6 points).

Lambda can infect either by a lytic or lysogenic pathway. The lytic pathway leads to the immediate replication of phage DNA and synthesis of phage proteins which are assembled into new phage. The E. coli are then killed by a late gene product release the new progeny phage. The lysogenic pathway occurs when the lambda DNA integrates into the E. coli genome (it is then called a prophage) where it remains dormant until a later time. At some time in the future, the prophage might be activated and start synthesizing all its products to create a lytic infection -- ultimately causing the release of many progeny phage and the bursting of the E. coli host.

7. Explain how new gene expression can be turned on by regulating transcription termination. 5 points

This occurs in lambda phage, as well as some other organisms. The organization of genes is p-A-B-t-C, where p is a promotor, t is a rho dependent termination site, and A, B, and C are genes. Initially, the RNA polymerase binds to the promoter, transcribes genes A and B, and stops transcription at the termination site, t. However, anti-termination can occur if the product of gene A or gene B can interfere with the normal action of rho at the termination site. In this case, the RNA polymerase would initiatie transcription at the promoter, read throuhg genes A and B, and since rho is not able to cause termination, the polymerase would continue to transcribe throught the termination signal, t, and transcribe gene C.

8. In eukaryotes, methylation of DNA near or in a promoter often prevents that gene from being activated, yet we know that during development methylated regions of DNA can be activated (and transcribed) in cells descended from one containing methylated sites near the gene of interest. No “demethylases” have ever been found. How is the activation of such genes brought about? 5 points

Since there are no demethylases, the only way to generate non-methylated DNA from DNA that is methylated is to go through several cell generations and NOT methylate newly synthesized DNA in the new generations. After a few rounds of replication, the new DNA will not be methylated and can be activated.

9. You have cloned a piece of eukaryotic DNA (DNA-Z) that, although it is not transcribed, has some unusual properties you wish to explore. To do this, you transfect various kinds of eukaryotic cells with this DNA and observe how the newly inserted DNA-Z alters the recipient cell’s behavior. In each different cell you observe that a different gene is activated after DNA-Z insertion. In each case the activated gene had been expressed at very low levels before transfection, but after DNA-Z is inserted nearby, the gene gets transcribed at a high level. What does this behavior say about DNA-Z (how is it functioning)? Explain your answer. 6 points

It suggests that DNA-Z contains an enhancer. Enhancers are sequences in DNa that can serve to activate transcription from any nearby promoter. What appears to be happening in this experiment is that DNA-Z is being inserted into a different place in the genome in each of the cells being examined. Since it has a different location in each transfected cell, it is activating different nearby promoters.

10. Explain how gene duplication and the ability to perform RNA splicing is thought to be one of the factors that allowed eukaryotes to evolve from primitive cells. 6 points

The primitive genes coded for small proteins, each of which performed a specific job, such as binding DNA. When these primitive cells developed the ability to splice RNA, large transcripts that spanned more than one of these genes could be spliced to produce a mRNA that contained more than one function and could be of benefit to the cell. Thus, if a gene were duplicated and translocated to another region of the DNA, it might be able to combine with a neighboring gene to produce a novel product. This rearrangement could be approximate because the splicing would remove the intervening RNA. On the other hand, if rearrangement at the DNA level was required to produce the "new" gene, then the DNA translocation would have to be exact (same reading frame). If it was not beneficial, the cell would have lost the ability to use that translocated sequence. By "experimenting" at the RNA level with splicing, the genome is not being permanently altered/damaged.

11. Explain how bacterial conjugation can lead to the exchange of genetic information between two individuals. 4 points

In conjugation, two bacterial cells connect through a bridge or tube. One of the pair will start to duplicate its DNA which is transfered through the tube into the other cell. The recipient cell therefore will have two copies of the genes that are transferred across the bridge before the bridge breaks. The two DNAs in the recipient cell can exchange genetic nformation through DNA cross-over.

12. Explain how a reporter gene might be used to study the developmental regulation of a gene. 5 points

A reporter gene is one whose activity can be readily assayed. In this case, lets say that the gene product is GFP, green fluorescent protein. The GFP gene is placed downstream of a developomentally regulated promoter and this construct is placed into a fertilizerd egg. As the egg matures and begins to develop, each of the cells will contain a copy of GFP under the control of the promoter to which it is attached. At any time, the cells in which GFP is being expressed (e.g. - in which the promoter is active) can be observed by placing the developing embryo in ultraviolet light. Any cells with GFP will glow green.

13. You have determined that there is a protein that binds very strongly to red dye#17 (a suspected carcinogen) and want to study the gene that codes for this protein. You do not have a sequence for this protein (or for the coding DNA), yet you want to isolate a cloned DNA that codes for the protein to learn more about it. Explain what steps are necessary to isolate such a clone starting from a collection of cells and ending with bacteria containing cloned DNA containing the coding information of the gene of interest. Please be careful to state your answer accurately. Use the available space as a guide to the level of detail needed (e.g. it is OK to state that you “screen a library by hybridizing with a cDNA probe” without going into the details of hybridization and autoradiography. Note that this comment is not necessarily part of the answer.). 10 points

We need to take advantage of the fact that RD#17 binds to the protein we are interested in as a screen in selecting our clone. Here are the steps that need to be taken. Starting with the cells, extract the mRNA population and make a cDNA library from that mRNA population. The library should consist of cDNAs inserted into an expression vector. This will allow the host cell to synthesize a product from any gene it contains on the expression vector. To screen the libarary, the bacteria are plated out on a petri dish and then exposed to RD#17. Those cells that are synthesizing the gene of interest will pick up the dye and will "stain" red. Cells not synthesizing the protein will not be very red. Picking the cells off the plate that are red will result in a population of cells, each of which contains the gene coding for a RD#17 binding protein.

14. What are RFLPs and how can they be used to identify individuals? 6 points

RFLPs are Restriction Fragment Length Polymorphisms. They result when different alleles of a gene are cut with a restriction enzyme. Each allele has a slightly different sequence from the other alleles of a gene. This slightly different sequence might result in the addition or removal of a restriction enzyme recognition site -- so that the sizes of DNA fragments resulting from such a digest will be different. In fact, the RFLP site does not have to be within an allele; it can be any location on the DNA that has a different restriction enzyme pattern in different individuals. Since different individuals will have different sets of alleles (or RFLP sites), they will each show a unique set of RFLPs. This is a DNA fingerprint of the individual.

15. Restriction enzymes are thought of today as molecular biologist’s tools, yet they evolved naturally for a different purpose. What natural role does a restriction enzyme play for the cell in which it resides? Explain your answer. 4 points

Restriction enzymnes serve as a kind of defense system for bacterial cells. Since they have the ability to recognize specific sequences on DNA and then cut the DNA at those locations, they will cut up any invading phage DNA and prevent the phage DNA from starting a successful infection.

16. You have identified a specific RFLP location on a chromosome that always seems to be present in individuals with a specific genetic disorder (e.g. cystic fibrosis). How would you go about finding the actual gene that is defective if the actual RFLP location did not reside in that gene? 4 points

Since the RFLP and the trait are passed on together, they must be near each other along the chromosome. Chromosome walking, starting with the clone of DNA containing the RFLP should allow identification of nearby genes.

17. Gene therapy holds the promise of treating or even curing individuals suffering from a genetic disorder. Yet just delivering the correct gene into the individual is not sufficient - there are other obstacles that need to be overcome for such a treatment to become effective. Describe one such obstacle and how it might prevent successful treatment. 5 points

There are a number of obstacles including (but not limited to) the following. Note that only one has to be discussed in the answer.

the gene must be delivered into the correct cells in order for its protein product to function correctly.

if the gene is delivered into the correct cell, it must still be regulated appropriately. For example, it is unlikely that a liver gene will be appropriately regulated in the brain.

the gene has to be inserted into the host cell's DNA in such a way that it does not disrupt a critical gene just by its insertion.