Hartl, Jones - Genetics. Principlers and analysis - 1998 (522927), страница 69
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The keyword site summarizes the development of this key method. If assigned to do so, write aparagraph describing two innovations that were instrumental in the maturation of the method, and explain why eachwas important.3. In DNA sequencing, there is sometimes uncertainty in the identification of a particular base because the gelpattern for that base is not completely unambiguous. An ambiguous base is denoted in a sequence by any one of astandard set of abbreviations. For example, the letter M stands for the possibilities ''either A or C," V stands for"either A or C or G," and N (or X) stands for "unknown base." The keyword will access the complete set ofabbreviations.
If assigned to do so, organize the abbreviations in a systematic way of your choosing.MUTABLE SITE EXERCISESThe Mutable Site Exercise changes frequently. Each new update includes a different exercise that makes use ofgenetics resources available on the World Wide Web. Select the Mutable Site for Chapter 5, and you will belinked to the current exercise that relates to the material presented in this chapter.PIC SITEThe Pic Site showcases some of the most visually appealing genetics sites on the World Wide Web.
To visit theshowcase genetics site, select the Pic Site for Chapter 5.Analysis and Applications5.1 What is the base sequence of a DNA strand that is complementary to the hexanucleotide 3'-AGGCTC-5'? Labelthe termini as to 5' or 3'.5.2 What is a nuclease enzyme, and how do endonucleases and exonucleases differ?3' or 3'5') does a DNA polymerase move along the template strand? How do5.3 In what direction (5'organisms solve the problem that all DNA polymerases move in the same direction along a template strand, yetdouble-stranded DNA is antiparallel?5.4 What is the chemical difference between the groups joined by DNA polymerase and DNA ligase?5.5 What are three enzymatic activities of DNA polymerase I?Page 2185.6 Why can RNA polymerases and primases initiate DNA replication, whereas DNA polymerases cannot?5.7 In gel electrophoresis, do smaller double-stranded molecules move more slowly or more rapidly than largermolecules?5.8 Consider a hypothetical phage whose DNA replicates exclusively by rolling-circle replication.
A phage withradioactive DNA in both strands infects a bacterium and is allowed to replicate in a nonradioactive medium.Assume that only daughter DNA from the elongating branch ever gets packaged into progeny phage particles.(a) What fraction of the parental radioactivity will appear in progeny phage?(b) How many progeny phage will contain radioactive DNA, and how will the number be affected byrecombination?5.9 What is the fundamental difference between the initiation of θ replication and that of rolling-circle replication?5.10 When the base composition of DNA from the bacterium Mycobacterium tuberculosis was determined, 18percent of the bases were found to be adenine.(a) What is the percentage of cytosine?(b) What is the entire base composition of the DNA and the [G] + [C] content?5.11 The double-stranded DNA molecule of a newly discovered virus was found by electron microscopy to have alength of 34 µm.(a) How many nucleotide pairs are present in one of these molecules?(b) How many complete turns of the two polynucleotide chains are present in such a double helix?5.12 An elegant combined chemical and enzymatic technique enables one to identify "nearest neighbors" of bases(adjacent bases in a DNA strand).
For example, if the single-stranded tetranucleotide 5'-AGTC-3' were treated inthis way, the nearest neighbors would be AG, GT, and TC (they are always written with the 5'terminus at the left).Before techniques were available for determining the complete base sequence of DNA, nearest-neighbor analysiswas used to determine sequence relations. Nearest-neighbor analysis also indicated that complementary DNAstrands are antiparallel, a phenomenon that you are asked to examine in this problem by predicting some nearestneighbor frequencies.
Assume that you have determined the frequencies of the following nearest neighbors: AG,0.15; GT, 0.03; GA, 0.08; TT, 0.10.(a) What are the nearest-neighbor frequencies of CT, AC, TC, and AA?(b) If DNA had a parallel (rather than an antiparallel) structure, what nearest-neighbor frequencies could youdeduce from the observed values?5.13 What is the chemical group (3'-P, 5'-P, 3'-OH, or 5'-OH) at the sites indicated at the positions labeled a, b, andc in the accompanying figure?5.14 Identify the chemical group that is at the indicated terminus of the daughter strand of the extended branch ofthe rolling circle in the accompanying diagram.Challenge Problems5.15 The following sequence of bases is present along one chain of a DNA duplex that has opened up at areplication fork, and synthesis of an RNA primer on this template begins by copying the base in red.3'-. .
. TCTGATATCAGTACG. . . -5'(a) If the RNA primer consists of eight nucleotides, what is its base sequence?(b) In the intact RNA primer, which nucleotide has a free hydroxyl (-OH) terminus and what is the chemical groupon the nucleotide at the other end of the primer?(c) If replication of the other strand of the original DNA duplex proceeds continuously (with few or no interveningRNA primers), is the replication fork more likely to move in a left-to-right or a right-to-left direction?Page 2195.16 A DNA fragment produced by the restriction enzyme SalI is inserted into a unique SalI cloning site in a vectormolecule. Digestion with restriction enzymes produces the following fragment sizes that originate from the insertedDNA:(a) SalI:20 kb(b) SalI + EcoRI:7 kb, 13 kb(c) SalI + HindIII:4 kb, 5 kb, 11 kb(d) SalI + EcoRI + HindIII:3 kb, 4 kb, 5 kb, 8 kbWhat restriction map of the insert is consistent with these fragment sizes?5.17 The illustrated DNA sequencing gel was obtained by the dideoxy sequencing method.
What is the nucleotidesequence of the strand synthesized in the sequencing reaction? What is the nucleotide sequence of thecomplementary template strand? Label each end as 3' or 5'.5.18 For the following restriction endonucleases, calculate the average distance between restriction sites in anorganism whose DNA has a random sequence and equal proportions of all four nucleotides. The symbol R meansany purine (A or G), and Y means any pyrimidine (T or C), but an R–Y pair must be either A–T or G–C.TaqI5'-TCGA-3'3'-AGCT-5'BamHI5'-GGATCC-3'3'-CCTAGG-5'HaeII5'RGCGCY-3'3'YCGCGR-5'Further ReadingBauer, W. R., F.
H. C. Crick, and J. H. White. 1980.Supercoiled DNA. Scientific American, July.Cairns, J. 1966. The bacterial chromosome. Scientific American, January.Danna, K., and D. Nathans. 1971. Specific cleavage of Simian Virus 40 DNA by restriction endonuclease ofHemophilus influenzae. Proceedings of the National Academy of Sciences, USA 68: 2913.Davies, J. 1995. Vicious circles: Looking back on resistance plasmids. Genetics 139: 1465.DePamphilis, M. L., ed. 1996. DNA Replication in Eukaryotic Cells. Cold Spring Harbor, NY: Cold Spring HarborPress.Donovan, S., and J. F.
X. Diffley. 1996. Replication origins in eukaroytes. Current Opinion in Genetics &Development 6: 203.Grimaldi, D. A. 1996. Captured in amber. Scientific American, April.Grunstein, M. 1992. Histones as regulators of genes. Scientific American, October.Hubscher, U., and J. M. Sogo. 1997. The eukaryotic DNA replication fork. News in Physiological Sciences 12:125.Kelley, T., ed. 1988. Eukaryotic DNA Replication. Cold Spring Harbor Laboratory.Kornberg, A. 1995. DNA Replication. 2d ed. New York: Freeman.Kornberg, R.
D., and A. Klug. 1981. The nucleosome. Scientific American, February.Mullis, K. B. 1990. The unusual origin of the polymerase chain reaction. Scientific American, April.Neidhardt, F. C., R. Curtiss III, J. L. Ingraham, E. C. C. Lin, K. B. Low, B. Magasanik, W. S. Reznikoff, M. Riley,M. Schaechter, and H. E. Umbarger, eds. 1996. Escherichia coli and Salmonella typhimurium: Cellular andMolecular Biology (2 volumes).
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