Bacterial Genetics Micro 475 Problem Set 1 Name: 1. In genetics, any observable characteristic of an organism constitutes a Phenotype . 2. Different versions of the same gene are referred to as alleles of that gene. 3. You are synthesizing DNA in vitro using the template 5'-CCGTTCCAACGTCTAAGCTAATGC-3' and the primer 5'-GCATTAGCTT-3'. a. In the first reaction you use polymerase plus all four dNTPs (dATP, dCTP, dGTP, and dTTP). Show the sequence of the DNA strand you make (including the primer sequence). b. In the second reaction you use the same template and primer with a combination of dATP, dCTP, dGTP, and ddTTP in place of dTTP (ddTTP is dideoxythymidine triphosphate which lacks both the 2'- and 3'-OH groups of the ribose sugar moiety). Show the sequence of the DNA strand you make. Explain the difference in the two sequences. 4. Relaxed chromosomal DNA forms a helix where the two strands wrap around each other every 10.5 bp. Supercoiling, however, changes this number. In a strain of E. coli that has the gene encoding gyrase inactivated (a gyrase mutant), would the strands wrap around each other more or less than in the wild-type organism? Do you think the DNA would be positively or negatively supercoiled in the mutant? The strands would wrap around each other less than in the wild-type organism, because gyrase relaxes positive supercoils/induces negative supercoils. The DNA would be positively supercoiled in the mutant since gyrase is inactivated. 5. When a nick occurs in the chromosomal DNA of an organism, most of the chromosome remains supercoiled. Why doesn’t the entire chromosome become relaxed DNA? The nucleoid is segregated into domains. 6. Briefly describe how you would isolate a cold-sensitive mutant. You would grow a bunch of the organism, 7. If extra copies of OriC were introduced to E. coli on a plasmid, would initiation of replication of the chromosome occur more, less or at the same frequency as cells without the plasmid? (A plasmid is a small piece of DNA that replicates independently of the chromosome). 8. If the gene encoding SeqA were inactivated, would the resulting mutant E. coli strain initiate replication of the chromosome more, less, or at the same frequency as the wildtype strain? 9. Why is an RNA polymerase, such as DnaG, necessary for DNA replication? 10. What is the importance of methylation in: A. mismatch repair? B. restriction systems?