Advanced Molecular Biology-Dr. Schleif

October 6, 2009

90 minute exam

Each question is worth 10 points.

 1.  Why is there a tendency in eukaryotic chromosomes for the dinucleotide AA/TT to occur at 10 base intervals?

 Such spacing of these nucleotides bends the DNA which facilitates positioning a nucleosome and winding DNA around the nucleosome.

 2.  Suppose bacterial cells are grown many generations in the presence of ³²PO₄, then the radioactivity is replaced by nonradioactive phosphate.  At intervals of about a generation, samples are removed from the culture, small numbers of cells are immobilized on slides, lysed in place and extensively digested with proteases and RNases.  Then the slides are exposed to photographic film.  Finally, the relative amounts of radioactivity present in each cell from each of the slides is determined.  By analysis of the data, what might you be able to learn about the cells or DNA of the cells?

 With increasing growth in the unlabeled medium, the labeled strands will be distributed to daughter cells.  Finally, after sufficient cycles of replication and cell division, if a cell contains any labeled DNA at all, that DNA will be at most one strand.  Hence, from this time onwards, the distribution of radioactivity in the cells that contain any radioactivity, will be constant.  The time required to reach this plateau region can give a rough idea of the number of chromosome copies present in cells, and the maximum amount of radioactivity in cells will indicate the size of the chromosome.

 3.  If, as seems to be the case, Drosophila DNA is not methylated, what might be something interesting to investigate?

 Whether the cells repair mispaired bases shortly after passage of the replication fork, and if they do, how they identify newly replicated DNA strands.

I expect they would not have supercoiling densities in this region, but a well reasoned answer reaching the opposite conclusion was also satisfactory.  My reasoning is that if DNA gyrases in the cell are set to add supercoiling to a density the forms the plectonemic supercoils, they very likely would continue to add and add and add supercoiling, which would at some point be removed by topo I isomerase.  Hence all that is being accomplished is ATP is being consumed to no useful end.  Also, operating in such a regime removes the possibility of using supercoiling as a global means of adjusting transcription levels.

5.  What can be learned about mRNA by measuring protein synthesis following the addition of rifamycin to growing cells?

Rifamycin will block further RNA initiation.  After about a minute, there will be no further completions of mRNA molecules, and their levels will decay to nothing.  As the levels of mRNA decay, the rate of protein synthesis will drop to zero.  Measuring this rate of turning off will give the average half-life of mRNA in the cells.

6.  Which category(ies) of amino acid type(s) would you expect to find on the surfaces of cytoplasmic proteins?

Charged and hydrophilic.

7.  Why is life near absolute zero impossible (even if water didn’t freeze)?

It’s the universal answer this semester!  Brownian motors would come to a halt

8.  If silencing existed in E. coli, what is the minimum size of RNA in a silencing complex that would be required to have a good chance of being appropriately sufficiently selective?

9.  What kind of evidence, other than X-ray crystallography, could show that a protein binds to DNA making use of contacts within the minor groove of DNA rather than the major groove?

 Once the binding position of the protein is determined using the same methods as were described for locating histone binding sites, the sequence of the binding site could be changed. A/T cannot be distinguished from T/A nor G/C from C/G in the minor grove.  If the protein still binds normally despite such changes, it likely is binding in the minor grove.

 10.  Which step(s) in the initiation of RNA synthesis, K_D or k₂, do repressors affect? (It would be best to provide a short explanation for your answer.)

 Either or both.  Blocking access would change K_D and blocking movement to form the open complex would change k₂.  Additionally, repressors could block elongation.

Advanced Molecular Biology-Dr. Schleif

November 10, 2009

90 minute exam

Each question except number one is worth 10 points.

 1.  One morning when class was to begin, only 12 students were present.  At that time I announced a magic word and stated that in the next exam I would assign 120 points total to this question, and would divide them equally amongst those correctly providing the magic word. What was that magic word? Maximum of 10 points per correct answer.

             A few took this too seriously.  It was a light-hearted attempt to poke fun at the difficulty of getting to class on time.

 2. When seeking substrates for a particular tRNA synthetase, not only was an 85 nucleotide RNA, presumed to be a tRNA found, but also an RNA on the order of 300 nucleotides.  What is a likely possibility for the function of this second RNA?

             This question comes from one of the assigned readings.

             It could be the RNA that functions both as a tRNA and a messenger that for a peptide tail specifying destruction of the protein by a protease, and contains a normal translation termination signal.

 3.  In about five short statements briefly mention the steps one could use to determine in cells what chromosomal sequences lie in close proximity to each other.

             This was described in class.  Because so many people were thinking in one dimension rather than three, I regraded and gave full credit for answering genetic recombination.  What I really wanted was the following.

 Crosslink DNA segments that lie near one another with formaldehyde.

Cut with a restriction enzyme.

Fill in sticky ends with a biotin-labeled nucleotide.

Dilute and ligate.

(Lightly digest with an exonuclease to remove biotin from ends that have not ligated. This step was not mentioned in class.)

Isolate biotin-labeled DNA

Sequence and analyze to see who lies near whom.

 4.  Explain how primers of the following type, where Q is a fluorescent quencher that quenches the fluorescence of F when Q is within 15 Å of F, can be used for real-time monitoring of the  progress of a PCR amplification temperature cycle by temperature cycle.

 5.  Estimate the chances that a random 50 residue amino acid sequence possesses biologically significant (has a good chance of being a homolog) sequence similarity to a sequence found in nature? (“High” or “Low” won’t be worth as much as numbers or numerical limits.)

             Your results with the Namesake problem should have stimulated consideration of this issue.

            If the human genome codes for ~25,000 proteins, and lots of proteins, from yeast to humans are related, perhaps in nature there are 100,000 different proteins. For for each of these there may be as many as 100,000 variants, coming to a total of 10¹⁰ different sequences.  On the other hand, from 50 residues, one can form (20)⁵⁰= 10⁵⁰ x 2⁵⁰~= 10⁶⁵ different sequences.  Hence, the chances are exceedingly low, 1 in 10⁵⁵.

 6.  Suppose for a molecular biology experiment you needed to produce a set of chromosomal fragments with one primer ligated onto one end, and a different primer on the other end. How would you accomplish this?

             This was a homework problem.  Many people gave the answer of using two different restriction enzymes.  Only one addressed the issue of how to keep multiple linkers from being added to an end.  Too many did not know that only double stranded DNA is a substrate for DNA ligase.

            Ligate a mixture of the two primers to the chromosomal fragments and then select just those fragments containing each of the two kinds of linker.  One way to do this is have one linker type labelled with biotin.  Both linkers should be double-stranded at one end only so the other end cannot be ligated. Use streptavidin-coated beads to select fragments containing the biotin linkers. What will be bound contains either the biotin linker at one end, or at both ends. Melt without destroying the biotin-streptavid binding.  The released strand therefore had the biotin linker at one end only. Can use immobilized DNA complementary to the correct strand of the other linker to select out of the released linkers, those with sequence complementary to the linker.

 7.  It was largely of academic interest that RNA molecules could be isolated that bind a small molecule like ATP.  Now that we know how to make them, what is a “practical” use of the ability to make RNA aptamers?

             The construction and use of aptamers to study transcription was described in one of the assigned readings.

            You can make aptamers that bind to a protein and use them to block its activity and study what and how activity of the protein is blocked by your different aptamers. You could make aptamers against some molecule you wish to detect, e.g. doxin in the environment.

 8.  How would you select for a clone carrying the methylase partner of a restriction enzyme you have purified?

            Another homework problem.

            From cells that contain the methylase gene you could ligate DNA fragments into a cloning plasmid carrying the ampicillin resistance gene.  Transform into cells, selecting for ampicillin resistance. Then prepare plasmid DNA from the transformed cells and digest with the purified restriction enzyme. Plasmids surviving the digestion must have been appropriately methylated by the methylase which they, themselves carry, and hence, are resistant to the restriction enzyme.

 9.  Outline how you would identify candidate genes or alleles that significantly contribute to high-level tennis play.

             Do snp mapping of the world’s top two hundred tennis players to identify chromosome regions containing genes that apparently affect tennis playing abilities.

 10.  Devise an efficient selection for operator negative mutations in the lac operon.  Note that you really want to minimize the number of  i minus mutations you get as unwanted background.

             Use cells containing an extra lacI gene, but only one promoter-operator-lacZ.  Use phenol-galactoside in the medium as the source of carbon and energy.