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Name: ______KEY_________________________ Student ID #: _______________________ 1/5 MCB 110. Midterm 1. 2007 Please put your name and student ID # on the first page. 1. (20 pts.) Find the one letter below that best matches the following statements. You may use each letter only once. A. major groove K. stimulates primase B. DNA glycosylase L. minor groove C. dnaA M. dnaC D. DNA ligase N. replicative helicase E. DNA pol III O. Dpn1 F. 5’-->3’ P. uracil G. DNA ligase Q. 3’-->5’ H. adenine R. primer I. plasmid S. junk DNA J. ribonuclease H T. LINE i) ___C__ recognizes bacterial origin of DNA replication ii) ___R__ location of the mutation in site-directed mutagenesis iii) ___E__ binds sliding clamp iv) ___K__ SSB v) ___J__ hydrolyzes RNA in a RNA:DNA heteroduplex vi) ___D/G__ performs last step in base-excision, nucleotide-excision and mismatch repair pathways vii) ___T__ the most common repeated motif in the human genome viii) ___P__ the RNA base without an exocyclic amino group ix) ___F__ direction of DNA synthesis in a DNA sequencing reaction x) ___A__ displays H-bonding groups specific for each base pair 2. (10 pts.) Your graduate advisor has given you three tubes containing solutions of either ssDNA, dsDNA or ssRNA of the same length. The labels have washed off the tubes. Briefly describe two experiments you would do to determine which solution is in which tube. How would these two experiments distinguish the samples? 1. Distinguish ss from ds by thermal denaturation (ds melts most) 2. Distinguish RNA from DNA by base hydrolysis (RNA gets hydrolyzed) Other combinations will workName: ______KEY_________________________ Student ID #: _______________________ 2/5 3. A BLAST search of the bacterial genome database using the sequence of the 76-amino-acid, human ubiquitin (Ub) gave the following results: E Sequences producing significant alignments: Value ref|YP_973019.1| hypothetical protein [Acidovorax avenae] 5e-28 ref|YP_213464.1| hypothetical protein [Bacteroides fragilis] 3e-22 ref|ZP_01741221.1| branched-chain alpha-keto acid dehydrogena... 5.3 ref|ZP_01514519.1| 3-isopropylmalate dehydratase, small subun... 7.0 3A. (8 pts.) Which of these bacterial proteins is most closely related to human ubiquitin? Explain your answer. YP_973019. The E-value, which denotes the number of random hits of this quality in a data base of this size, is the lowest for this sequence. 3B. (8 pts.) The top hit produced the following alignment: Length=287 Identities = 60/73 (82%), Positives = 71/73 (97%), Gaps = 0/73 (0%) Query 1 MQIFVKTLTGKTITLEVEPSDTIENVKAKIQDKEGIPPDQQRLIFAGKQLEDGRTLSDYN 60 MQ+FVK L+G+T+TL+VEPSD+IENVKAKIQD++ IPP++QRLIFAGKQLEDGRTLSDYN Sbjct 40 MQVFVKMLSGETLTLDVEPSDSIENVKAKIQDQKDIPPERQRLIFAGKQLEDGRTLSDYN 99 Query 61 IQKESTLHLVLRL 73 IQK+STLHLVLRL Sbjct 100 IQKDSTLHLVLRL 112 Because Ub is thought to occur only in eukaryotes and a Ub homolog was found in only 2/867 bacterial genomes, Dr. Alber suggested that this sequence might be an error in the database due to contaminating DNA from another species. Based on the BLAST results, do you think this “bacterial” sequence could come from contaminating human DNA? Why or why not? It’s not human DNA. There are too many changes between the bacterial and human sequences. 3C. (8 pts.) Suggest one computational experiment and one laboratory experiment you would do to test the idea that this ubiquitin gene does not occur in the sequence of the bacterial genome, but rather comes from contaminating eukaryotic DNA. Briefly describe what results you expect from these specific experiments if Dr. Alber was wrong (i.e. if the bacterial DNA really does contain this gene and the Ub sequence is not a contaminant). Computational experiment: BLAST the nr data base with the bacterial sequence and see if there is any eukaryotic ubiquitin with th4e identical sequence. All the eukaryotic sequences would be different if Alber was wrong. Laboratory experiment: 1. Isolate genomic DNA from Acidovorax and Bacteroides and do PCR with Ub primers or a Southern blot with a Ub probe. If Alber was wrong, PCR products would be produced and the expected band would hybridize to the probe in the Southern. 2.A microarray of cDNA made from the bacterial RNA preps could also show if the Ub genes are expressed.Name: ______KEY_________________________ Student ID #: _______________________ 3/5 4A. (8 pts.) John Watson says that to use RFLP analysis to distinguish DNA from different people (for example to identify a person from a sample collected at a crime scene), it’s best to use a unique-sequence probe. On the other hand, Frances Crick says that it’s easier to use a minisatellite probe. Who’s right and why? Frances was right. The minisatellite probe gives more shots on goal (potential differences) with avery restriction enzyme used to digest the genomic DNA. 4B. (8 pts.) In searching for the golden mutation in zebra fish, scientists found a RFLP that separated from the light pigmentation trait once in 1100 fish. If 1 centimorgan represents 740,000 base pairs in the zebra fish genome, what was the approximate distance from the RFLP to the mutation? 1 recombinant/1100 fish x 1 centimorgan x 740,000 bp = 67,272 base pairs 1 recombinant/100 fish centimorgan 4C. (8 pts.) Given this distance, do you think the RFLP could be in the golden gene? Why or why not? Yes, this could easily be in the same gene, because some genes are big and the recombination frequency could easily be overestimated with only one recombinant. Or No, the RFLP is likely in a different gene, because most genes are smaller than 67,000 base pairs and the recombination frequency could easily be underestimated with only one recombinant. 4D. (8 pts.) Inactivating mutations in the ras oncogene are found in many tumors. Surprisingly, much milder, partially inactivating ras mutations were recently found in a small number of patients with Noonan’s syndrome, an inherited disease characterized by abnormal skull features and cardiac problems. In broad terms, how would you identify candidate genes that might


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