Download GENETICS 603 EXAM 1 Part 1: Closed book October 3, 2014 NAME

GENETICS 603 EXAM 1 Part 1: Closed book October 3, 2014 NAME 1. Autoradiographs of replicating DNA revealed that the E. coli chromosome is circular. a) What molecule was labeled for these experiments and what isotope was used for the label? Thymine or thymidine for DNA specificity, H3 for locating on film b) What extra problem(s) did this revelation add to the models that previously assumed replicating DNA molecules were linear, and how is/are the problem(s) resolved? Lack of free ends meant unwinding had to begin within the circle which leads to extreme supercoiling and ‘interlocking’ products. Resolution: defined origins, topoisomerases for relieving the supercoiling and topoisomerase and gyrase to separate the two circles. 2. A number of cases where RNA molecules other than mRNA or tRNA are critical for
cell function and viability were described in class. List several and describe their
function. rRNA for ribosomes: large subunit is part of the petptidyl synthase SRPs signal receptor particles for docking with mRNAs that are to be extruded into the ER Telomerase includes an RNA component for maintaining ends of chromosomes snRNPS function n splicing pre-­‐mRNAs guides function in RNA editing RNA primers are made and removed in DNA replication of the lagging strand snoRNAs are important in editing rRNA (micro RNAs and siRNA etc OK, but were not covered in the test material) 3. In searching for proline auxotrophs in Neurospora, mutations of 3 different genes were identified. Further, a mutant called arg-­‐4 which had been isolated based on a requirement for arginine was also found to require proline. Double mutants of arg-­‐4 and other arginine auxotrophs did not accumulate ornithine, citrulline or AS, which had been shown to be intermediates in the arg pathway, even if grown on limiting arginine. The table below shows the results of growth tests of the mutants on minimal medium, supplemented as indicated. Supplement(s) added to minimal medium Mutant Proline G5C GSA Pro + Arg Orn pro-­1 + -­‐ -­‐ + -­‐ pro-­3 + + + + -­‐ pro-­4 + + -­‐ + -­‐ arg-­4 -­‐ -­‐ -­‐ + + a. Suggest a screening procedure that might have been used to originally identify arg-­‐4 as an arginine auxotroph. It likely was identified first as growing on complete but not minimal medium. However to consider it as an “arg” auxotroph based on response to arginine, proline (or a precursor) must have also been present in the medium b. What is the significance of the fact that arg-­‐4 containing double mutants did not accumulate intermediates on limiting arginine? On limiting arginine, the pathway should be ‘on”. This means that arg-­‐4 acts early in the pathway so no other compounds can be made in the double mutants. c. Show a pathway and identify mutational blocks compatible with these data. Arg4 pro3 pro4 pro1 Orn GSA G5C Pro Arg d. pro-­2 mutants had the same responses on the test media as pro-­1 mutants. List some alternative reasons this might occur. 1. there is another intermediate “X” in the pathway 2. pro-­1 and pro-­2 encodes soubunits of the same enzyme 3. both mutations are defects in the same gene 4. Some his-­‐ auxotrophs in Salmonella that Ames analyzed in selecting the strains later used to test for chemical mutagenicity proved to be less accurate in specifying the precise base changes induced than those included in the test. This was true even though they were single base-­‐pair point mutations, including nonsense mutations that fully eliminated enzyme activity. Suggest an explanation for these observations. The Ames tests uses strains with different type of his-­‐ mutations (transition, transversion and frameshift) so that in addition to detecting mutagens based on increased rates of reverse mutation, the type of change can also be predicted. Base changes in some sites may create changes that can be readily corrected by second site mutations or suppressors, while others cannot. For example, if only TRP at a specific position will allow the enzyme to function, a change from UGG to UGA could only be corrected by true reversion or a nonsense suppressor that inserted TRP for UGA, while other nonsense mutations may be functional if any of several tRNA anticodons change to recognize the nonsense mutation. 5. Shown below are a series of strains of E. coli with two copies of the lac operon per cell. Tell whether the synthesis of the enzymes beta-­‐galactosidase, permease and transacetylase would be regulated (R), constitutive (C) or absent (A) in each case. FS is a frameshift mutation with extreme polarity. Strain β−gal’ase permease TAase -
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R R R Y A
C C A -
C C A -
A A A 1. P I P O Z Y A/ P I P O Z Y A
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FS
2. P I P O Z Y A / P I P O Z
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3. P IS P O Z Y A/ P I P O Z Y A
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4. P IS P O Z Y A/ P I P O Z Y A
Which strains, if any, if any would give different results by using lactose rather than IPTG to test for induced expression? NONE 6. Linkage among the genes genes tal (resistance/susceptibility), nic1 (nicotinamide biosynthesis) and xyl1 (ability to use xylose) was tested in B. subtilis using transformation with DNA from a talR, nic+, xyl+ donor into a recipient with opposite markers. When selected for only one donor marker first and then using replica plates to test for the others, most of the transformants had all 3 donor alleles. a) What is the significance of this observation? Show potential gene orders that could be correct thus far. The 3 genes are very close together Orders: talR, nic+, xyl+ or talR, xyl+, nic+ or nic+, talR, xyl+ )(ie any of the 3 genes could be in the middle b) What media were used in the first round of screening where only each single donor marker was selected? For talR: glucose minimal (not xylose) with β-­‐thienylalanine + nicotinamide For nic+: glucose minimal (no xylose or β-­‐thienylalanine For Xylose utilization: xylose medium + nicotinamide c) When talR was first selected, replica plating showed that almost no colonies that were also nic1+ were xyl1-­‐, whereas xyl1+, nic1-­‐ transformants were much more common. How can this be explained and how does it affect prospects for map order? Since all 3 markers are very close together the best (not only, perhaps) is if the markers are in the order talR, xyl+, nic+ since then xyl-­ would require 4 rather than 2 exchanges (crossovers) d) What human gene test needs to be aware of the fact that there are such things as β-­‐
thienylalanine resistance in bacteria? PKU phenylketonuria 7. B. lactis, a gram positive anaerobic bacterium that is used in making fermented foods such as yogurt can digest sucrose, which is a 12 carbon sugar and raffinose, an 18 C sugar. Sucrose is made of glucose + fructose, and raffinose is sucrose with a galactose added. Genetic analysis has shown 3 key genes for sucrose digestion, ScrP, ScrT and ScrR are found in a cluster. ScrP is a phosphorylase involved in splitting sucrose, ScrT has 12 membrane spanning regions and ScrR has motifs for binding sucrose and DNA. The table below shows relative mRNA levels for the 3 genes when grown on media with specific carbon sources: Relative mRNA levels Carbon source ScrP ScrT ScrR glucose 12 0 0 glucose & sucrose 48 0 0 sucrose 80 30 16 raffinose 80 15 16 a) Given that mutants lacking ScrR showed no change in mRNA levels when grown on sucrose from those grown on glucose, propose a model that could explain regulation of this system. Information provided suggests, ScrP is a digestive enzyme that is increased by the presence of sucrose and seems also to function with other compounds since the gene is also transcribed in the absence of sucrose; ScrT is a sucrose transporter and ScrR encodes a regulator that can bind both sucrose and DNA. Based on the observation that ScrR-­‐ mutants aren’t constitutive, positive regulation seems likely. On Glucose On sucrose b) When the promoter regions of the ScrP was analyzed, it did not have sequences typical of the most common E. coli promoters at -­‐10 or -­‐35, but yet when the gene was transferred to E. coli intact with its own promoter, it was transcribed under some conditions. i. What is a typical E. coli promoter “-­‐10” sequence? TATA box ii. How can the ability to transcribe the mRNAs be explained? Likely involves a different sigma factor than the common one and it’s also present in E. coli. c) The mRNA made did have the sequence AGGAGG located 6-­‐11 bases before the ATG start. Would you expect the mRNA to be translated? Explain AGGAGG is is a typical leader sequence for binding the mRNA on ribosomes to position the start codon (generally called ATG these days, sorry if that was confusing) so translation would be expected. d) The ScrR gene promoter was similar to that for ScrP, but also had a symmetrical site analogous to the CAP binding site found in the lac operon. What does this suggest about the regulation of the sucrose degradation pathway. It is affected by levels of ATP 8. Which codons could be converted to a nonsense codon by a transition in the a) first, b) second or c) third codon positions? a: Arg CGA to UGA and Gln AAA and CAG to UAA and UAG respectively b: Trp UGG to UAG (UGA and UAA switch also OK) c) Trp UGG to UGA Take home portion-­ Open Notes Questions: No Internet Searches!!
1) 4′-Ethynyl-2-fluoro-2′-deoxyadenosine triphosphate, the phosphorylated version of a
nucleoside analog that was being tested as a flavor additive to soy sauce was found to be
rather effective in blocking reverse transcriptase, an enzyme found in retroviruses that
cn make a DNA complement of an RNA strand. After being added to the growing DNA
strand by reverse transcriptase with release of a pyrophosphate, the remaining
phosphate is out of position for use in further extension.
a) Suppose the nucleoside was included in food as was being considered. Would you
have concerns if it readily phosphorylated in cells? Explain why or why not. What
cellular events might be affected?
(lots of possibilities here!) I would be especially concerned if the nucleoside is converted
to a nucleotide triphosphate that also might interfere with any DNA polymerase, in
which case it could interfere with replication or repair or perhaps lead to high mutation
rate as in the case of 5-bromouracil. Even if it does not, it could potentially interfere
with telomere maintenance since that does involve a reverse transcriptase component.
b) Suppose it is not phosphorylated. How does this affect your concerns, if any?
Less concern with any effects it may have as far as acting as a mutagen or interfere with
DNA replication or reverse transcriptase. However, base analogs like caffeine may
mimic natural signaling molecules with undesired effects.
c) Do you see any prospects for the phosphorylated version? Anti-AIDS?
2. Before DNA sequencing methods were developed, amino acid sequences of fragments of well-­‐studied proteins were easier to determine than base sequences, so were used to assess the effects of mutation. Part of a normal catalase enzyme from E. coli had the sequence his•cys•met•asp•gly. No activity was found in an acridine (ICR-­‐170) induced mutation, but in a revertant found after a second treatment with ICR-­‐170, the equivalent sequence of amino acids was found to be his•ala•trp•thr•gly. a. Would the same kind of changes be expected if EMS had been used as the mutagen? Explain: No: acridines cause frameshifts and EMS causes mostly transitions and some transversions which won’t account for the changes seen without multiple rare events in a very short region of DNA. b. Propose base sequences for the normal, mutant and revertant strains that account for the changes seen. ORIGINAL CAU/C•UGU/C•AUG•GAU/C•GGX !CAU/C•GCA•UGG•ACG• MUTANT ? Frameshift If the red U is deleted and the C rather than U options for the third base is used, then adding any base before the GGX codon will restore the reading frame REVERTANT: CAU/C•GCX•UGG•ACX•GGX ! CAU/C•GCA•UGG•ACX•GGX (X added) 3. A single base change in one of the 5 arginine tRNA genes for AGA codons in a particular mouse strain leads to a neurodegenerative disorder. a) What is/are any potential anticodon(s)? UCU or UCI (someone told me mouse does not use I which may be true but would not have been anticipated from class material) b) The base change is not in the anticodon, but seems to destabilize the tRNA. Provide an explanation of how/why such a mutation might affect tRNA function. The folding of the tRNA is critical to its ability to be activated and to bind to the ribosome A and P sites, and possibly also to having correct base modification, so a change that disrupts one the regions of foldback could eliminate normal function. c) Based on just this information and assuming AGA codons are fairly common, why isn’t the mutation lethal? With 4 other copies of the gene competing for the AGA sites, there may be enough cases where normal protein can be made. It may also be that the unstable form is less efficient in competing. Why it should be made only in the brain is another question! d) It turns out that in mice, this particular tRNA is made only in the brain and that it does not lead to neurodegeneration in mice that have a functional mechanism to release stalled ribosomes. How does this observation tie in with the previous information? This suggests that ribosome binding does occur but the amino acid may not be in position to add to the growing polypeptide, thus stalling translation. If so, accumulation of stalled ribosomes will prevent normal levels of all proteins from being made. When the release works, the normal proteins can still be made and generally. Mal-­‐folded proteins are rapidly destroyed.