Answer8 - MCB 161 Study Problems Lecture 9 Transposition...

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Unformatted text preview: MCB 161 Study Problems Lecture 9: Transposition; Overview of gene expression 4-96 The Hox gene clusters are packed with complex and extensive regulatory sequences that ensure the proper expression of individual Hox genes at the correct time and place during development. Insertions of transposable elements into the Hox clusters are eliminated by purifying selection, presumably because they disrupt proper regulation of the Hox genes. Comparison of the Hox cluster sequences in mouse, rat, and baboon reveals a high density of conserved noncoding segments, supporting the idea of a high density of regulatory elements. Reference: Lander ES et al. (2001) Initial sequencing and analysis of the human genome. Nature 409, 860–921. 5-107 False. Transposable elements integrate nearly randomly and genes often are destroyed or altered by the integration event. While it is true that some of these events are lethal to the cell and to the transposable element, most events are not. Spreading throughout the genome, even at the cost of a few cells (and transposons), ensures that the transposable element will survive with the species. 5-108 Cre ­mediated recombination between oppositely oriented LoxP sites inverts the sequences between the sites, whereas recombination between loxP sites in the same orientation deletes the sequences (Figure 5–67). This result should remind you of the similar outcome obtained for homologous recombination between direct repeats and inverted repeats in Problem 5–94 (see Figure 5–64, substrates 3 and 4). As in that problem, the easiest way to work out the products is to align the LoxP sites and then follow the crossover between them. 5-111 A. Transposition of the Ty element depends on reverse transcription of an RNA intermediate. Normally, reverse transcriptase is expressed at a very low level. Your modified plasmid, however, places the gene under control of the galactose control elements. In the presence of glucose (absence of galactose) the galactose control elements turn the gene off and, as a result, the expression of reverse transcriptase is very low. In the presence of galactose the reverse transcriptase gene is expressed at very high levels. Thus, the frequency of transposition increases substantially. (5 111 cont.) B. The frequency of Ty ­induced His+ colonies is low because a very specific kind of transposition event is required to activate the defective histidine gene: the Ty element must transpose to a site near the 5’ end of the gene. Thus, even though nearly all cells show evidence for transposition, insertion near the defective histidine gene is still relatively rare. C. The data in Figure 5–47 indicate that nearly every cell harboring the Ty ­bearing plasmid suffers one or more transposition events when grown on galactose. Each Ty transposition has the potential for altering the function or expression of genes near the site of integration. If the element integrates into the coding portion of a gene, it can eliminate the encoded function; if it integrates in the noncoding region near a gene, it may alter the gene’s expression. In organisms such as yeasts, which have been finely tuned to their environmental niche by evolutionary pressure, it is unlikely that random insertion of a Ty element will improve growth characteristics. Thus it is not unreasonable that a high rate of transposition should cause cells to grow poorly. These data do not prove that the cells grow more slowly because of the high rate of transposition, even though that explanation is very likely to be correct. As the authors point out, the high level of expression of reverse transcriptase might interfere directly with RNA metabolism. For example, mRNA molecules could be inactivated by reverse transcription. Alternatively, the reverse transcripts of the cellular mRNAs could be mutagenic to the nuclear genes. Errors introduced during reverse transcription into DNA could be incorporated into the nuclear genes by recombination. Reference: Boeke JD, Garfinkel DJ, Styles CA & Fink GR (1985) Ty elements transpose through an RNA intermediate. Cell 40, 491–500. 6-12 True. Errors in DNA replication have the potential to affect future generations of cells, while errors in transcription have no genetic consequence. Errors in transcription lead to mistakes in a small fraction of RNAs, whose functions are further monitored by downstream quality ­control mechanisms. The essential feature is that errors in DNA replication change the gene and, thereby, affect all the copies of RNA (and protein) made in the original cell and all its progeny cells. By contrast, errors in transcription are limited to a small number of defective RNAs (and proteins), and are not passed on to progeny cells. These considerations are reflected in the intrinsic error rates for RNA and DNA polymerases: RNA polymerases typically make 1 mistake in copying 104 nucleotides, while DNA polymerases make about 1 error per 107 nucleotides. Such significant differences in error rates suggest that natural selection is stronger against errors in replication than against errors in transcription. 6-13 False. The s subunit associates with the bacterial RNA polymerase core enzyme to form the RNA polymerase holoenzyme only during the initiation phase of RNA synthesis. The σ subunit helps the core enzyme bind to the promoter and stays associated with the core enzyme until RNA synthesis has been properly initiated, and then it dissociates. 6-17 The answer is best given by Francis Crick himself, who coined the terms ‘the sequence hypothesis,’ which proposes that genetic information is encoded in the sequence of the DNA bases, and ‘the central dogma,’ which states that DNA makes RNA makes protein, in 1957. “I called this idea the central dogma, for two reasons, I suspect. I had already used the obvious word hypothesis in the sequence hypothesis, and in addition I wanted to suggest that this new assumption was more central and more powerful. I did remark that their speculative nature was emphasized by their names. As it turned out, the use of the word dogma caused almost more trouble than it was worth. Many years later Jacques Monod pointed out to me that I did not appear to understand the correct use of the word dogma, which is a belief that cannot be doubted. I did apprehend this in a vague sort of way but since I thought that all religious beliefs were without serious foundation, I used the word in the way I myself thought about it, not as most of the rest of the world does, and simply applied it to a grand hypothesis that, however plausible, had little direct experimental support.” Reference: Crick F (1988) What Mad Pursuit: A Personal View of Scientific Discovery, p 109. New York: Basic Books, Inc. 6-37 The consensus sequence for this set of promoters is shown in Figure 6–53. In this set of 13 promoters there are clear examples of common nucleotides outside the – 35 and –10 regions. Also, one of the accepted consensus nucleotides (the terminal A in the –35 sequence) doesn’t even show up as common. When 300 promoters recognized by σ70 are compared, the consensus sequence is TTGACA (–35) and TATAAT (–10). It’s always better to compare more sequences! ...
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