Bio 4 Exam#2, Feb 23, 2000

1. Molecular biologists learned about the ways in which operons work by creating situations in which cells have two copies of an operon. This can be accomplished by putting into the cell a plasmid that contains a copy of the operon being studied. In each of the following cases, explain how the Trp operon will be regulated (how is it turned on, off, and regulated). Unless stated otherwise, “operon” refers to the entire operon - repressor, promoter, operator, and coding sequences.

a. a wildtype (normal) Trp operon on the genomic DNA and a Trp operon on the plasmid that synthesizes a repressor that does not recognize Trp. 2 points
Since Trp-repressor complex is needed to turn off transcription and the genomic operon can provide that, the operons both will be regulated normally.

b. a wildtype Trp operon on the genomic DNA and a Trp operon on the plasmid that is missing an operator region. 2 points
Since the plasmid operon is missing an operator region, it can never be turned off (repressor cannot bind). So the genomic operon will be regulated normally but the plasmid operon will be always on - no matter how much Trp is around.

c. a wildtype Trp operon on the genomic DNA and a Trp operon on the plasmid with a mutation in the repressor causing it to bind to Phe instead of Trp. 2 points
Since the repressor is diffusible within the cell, and both normal Trp repressor and the mutant Trp repressor are being synthesized, whenever levels of Phe OR levels of Trp increse in the cell, there will be more functioning repressor formed and the operons will be turned off. So... the operon will be regulated by the levels of Phe as well as the levels of Trp.

d. a wildtype Trp operon on the genomic DNA and a Trp operon on the plasmid that synthesizes a repressor that binds to but does not release from an operator. 2 points
The mutant repressor will bind to operators on both the plasmid and genomic operons and keep transcription of both operons off - irregardless of Trp levels.

e. a Trp operon on the genomic DNA that is missing a repressor gene and a Trp operon on the plasmid that synthesizes a repressor that binds to Trp but does not release the Trp. 2 points
The only repressor present is that from the plasmid operon. This repressor will bind Trp to become an active repressor but will not release the Trp, so both operons will be turned off as soon as any Trp is present and they will remain off since the repressor cannot release the Trp.

2. Explain why it is necessary for phage to regulate the expression of their genes? 4 points

The phage genes are responsible for making more phage. This is accomplished in an orderly fashion by having the phage DNA replicate and then associate with newly synthesized phage proteins, and finally having the cell lyse to release all the progeny phage. If phage products are synthesized in the wrong order (e.g. lysing cell before phage particles are assembled) the process will be inefficient at best and will produce few if any progeny phage.

3. Explain how the shift from middle to late genes is made in T4 phage infection. 4 points

One of the middle gene products is a new “sigma” factor that can interact with the E. coli core RNA polymerase to form a new holoenzyme that recognizes the late promoter. A different middle gene product inhibits the middle “sigma” factor to turn off transcription from the middle promoter.

4. Eukaryotes have three different RNA polymerases. Explain what kinds of genes each of the RNA polymerases transcribe. 6 points

RNA polymerase I - synthesizes ribosomal RNAs
RNA polymerase II - synthesizes pre-mRNAs/mRNAs
RNA polymerase III - synthesizes tRNAs and 5S rRNAs

5. Transcription factors are involved in defining the startpoint of transcription in eukaryotes while sigma plays a similar role in prokaryotes. Describe the basic difference in the mechanisms through which these two processes work. 4 points

The basic difference is that in prokaryotes, sigma factor binds to the RNA polymerase to allow the complex to bind to the DNA at the promoter while in eukaryotes, transcription factors bind to the DNA of the promoter and then the RNA polymerase can bind.

6. Describe how enhancers work. 5 points

Enhancers serve as binding sites for transcription factors. The transcription factors bound to enhancers can then interact with transcription factors which are bound to promoters in the area. The larger complex of transcription factors is a stronger signal to start transcription than just having the transcription factors bound to the promoter.

7. Describe how alternative splicing can be used to produce different protein products from the same gene in different tissues. 5 points

Alternative splicing uses different splicing pathways to remove introns in different tissues. For example, if a pre-mRNA contains four exons A-B-C-D, it might be spliced in one tissue to give a mRNA of ABD, while splicing in a different tissue could give ACD. Since the two mRNAs contain different coding information they will produce different proteins.

8. What is a phage plaque and how is it produced? 4 points

A phage plaque is produced when a single phage is placed onto a lawn of bacteria. That phage will infect a bacterium, which will then lyse and release phage to infect adjacent bacteria. The process continues and the area of lysed cells appears as a clear circular area in the hazy lawn of bacteria - called a plaque.

9. Explain how phage transduction can result in the transfer of genomic DNA from one bacterial cell to another. 5 points

Lysogenic phage can insert their DNA into the E. coli genomic DNA at specific locations. At a later time, the phage DNA can be removed to initiate a lytic infection. When the phage is removed from the genomic DNA errors are sometimes made and a bit of the neighboring genomic DNA is also excised. The excised DNA is then packaged into phage particles which can infect other bacterial cells. If the phage particle contains a bit of the genomic DNA, that DNA will be introduced into the new target cell when the phage injects its DNA, thus transferring the genomic DNA from one bacterial cell to another.

10. Explain what advantage there appears to be for us to have embryonic (d, z) and adult (a, b) forms of globin genes. 5 points

The embryonic forms of globin have a higher affinity for oxygen than the adult forms. This enables the mother to bring oxygen in her red blood cells to the placenta and then have the embryonic form of hemoglobin “pull” the oxygen from the mother’s hemoglobin to the embryo’s hemoglobin. This is only possible becuase of the higher oxygen affinity of the embryonic globin.

11. Restriction enzymes have been utilized extensively by molecular biologists, yet these enzymes are naturally occurring enzymes that play a role in their own organisms.

a. How do restriction enzymes provide bacteria with a primitive kind of immune response? 3 points
The restriction enzymes in the host cell will degrade any foreign DNA that enters the cell, such as phage DNA.

b. Explain why bacteria containing restriction enzymes do not kill themselves. 4 points
Bacteria contain a modification enzyme in addition to the restriction enzyme. The modification enzyme recognizes the same site on the DNA as does the restrictoin enzyme and modies the DNA chemically so that it is not cut by the restriction enzyme. All of the bacterial DNA is modified by the modification enzyme so that it is not attacked by the restriction enzyme.

12. We have discussed two kinds of libraries that can be constructed using molecular biology cloning technologies.

a. What is the difference between a genomic library and a cDNA library? 3 points
A genomic library contains genomic DNA sequences and represents the entire genome of the organism. A cDNA library contains sequences found in a mRNA population from a particular tissue and represents only those genes expressed in that tissue.

b. Give an example of what each kind of library might be used for. 6 points
A genomic library might be used to study promoters, the intron/exon structure of a gene, or to do chromosome walking. A cDNA library might be used to synthesize a protein product of a gene or to study transcription levels for that gene. [other answers are possible]

13. The gel at the right shows the result of some digests you have just performed on a gene you isolated called pBio4. Lane 1 is a digest using ApaI, lane 2 is a digest using MseI, and lane 3 shows a double-digest using both enzymes. The size of each fragment is indicated just above the band. Using this information, determine the restriction map for this 3000 nucleotide long DNA. (you can draw a diagram) 10 points

                        

                                                   -- OR --

                        

 

 14. You have isolated a complete genomic DNA clone for a cancer causing gene you are studying (upstream sequences, all introns and exons, and downstream sequences). You want to evaluate a number of drugs designed to inhibit the expression of this gene. How would you utilize this clone in conjunction with a GFP reporter gene to determine the levels of expression of this gene in a tissue after treatment with these various drugs? 8 points

I would use restriction enzymes to isolate the promoter from this gene and join it to the GFP reporter gene. I would then introduce this construct into the tissue being studied. The tissue then can be treated with various drugs and the level of expression of this gene can be measured by measuring the level of green fluorescence in the tissue after drug treatment and comparing it to the level of expression in untreated tissue. Since the level of GFP synthesized is related to the level of activity of the promoter, measuring GFP levels is an indication of the level of expression that would be occurring from the intact gene containing the same promoter.

15. Explain what an RFLP is? 5 points

A restriction fragment length polymorphism is the result of small differences in the DNA sequences of different individuals in a population. Single base changes from one allele to another might cause the appearance of or the elimination of a restriction enzyme site. This would lead to different sized DNA restriction fragments correspoonding to a particular location on the DNA from different individuals. These different patters can be used to identify individuals.

16. PCR technology has been used for many different processes from forensics to making dinosaurs (Jurassic Park). Explain how this process works to amplify minute quantities of DNA. 5 points

See figure 7.10 in your text book.

17. You have isolated a complete wildtype cDNA clone for the gene product that is defective in cystic fibrosis and want to use it for gene therapy for patients with cystic fibrosis. Your goal is to treat the symptoms present in the lungs of CF patients in your initial gene therapy trials. Discuss some of the difficulties you might encounter, assuming that your method of deliver into lung cells works flawlessly. 8 points

Briefly... the gene that is inserted might insert itself into a dormant region on the chromatin and might not get expressed; the original defective CF gene product will continue to be synthesized and might interfere with the ability of the “correct” product to function; the level of expression of the CF gene product might be too high or too low; the regulation of the level of synthesis of the correct CF mRNA might be inappropriate so that it cannot respond to changing physiological needs. Other possibilities might exist -- only two are needed for your test answer.