1) A meteorite landed on earth carrying with it a sample of extraterrestrial bacteria. Homer Simpson was assigned the task of determining the mechanism of DNA replication in this organism.

Homer grew the cells in unlabeled medium and then transferred them to 15N containing medium for exactly one generation. He extracted the DNA and subjected a portion of it to CsCl density gradient centrifugation. The banding pattern is shown below with reference to control DNA isolated from cells grown in 14N or 15N media.

 

(a) Homer claims to have demonstrated that extraterrestrial DNA replicates in a semiconservative manner. Why is his claim premature?

 

 

 

(b) Unfortunately, Homer used all of the bacteria to extract the DNAs and the organism is now extinct on earth--D'oh! Devise an experiment using the leftover DNA samples and CsCl centrifugation, that would distinguish between semiconservative replication and the remaining alternative mode(s) of replication.

 

 

2)Temperature sensitive mutations are useful for analyzing essential biochemical pathways. A variety of ts mutations affecting DNA synthesis have been isolated in E. coli. Assume that the proteins affected by the following mutations function normally at 30˚ but are inactive at 42˚C.

 

dnaBts (helicase)

dnaEts (pol III polymerase function)

dnaGts (primase)

dnaQts (pol III 3'-5' exonuclease function)

ligts (DNA ligase)

polAts (DNA polymerase I: Polymerase function, 3'-5' exonuclease, 5'-3' exonuclease)

 

(a) Which of the mutants would be expected to accumulate Okazaki fragments after shift from 30˚ to 42˚ C?

 

 

(b) DNA mutants can be categorized by whether they lead to a rapid (within seconds) or a slow (20 min - 1hr) cessation of DNA synthesis after shift to non-permissive conditions. (DNA synthesis is measured by incorporation of radioactive thymidine.) Which of the mutants would produce a fast-stop phenotype and which a slow-stop phenotype? If you think some of your answers may be ambiguous state why.

 

 

(c) A mutation called mutD (for "mutator gene D") is allelic to one of the mutations listed above. The mutD phenotype is manifested as an ~ 105-fold increase in the rate of spontaneous mutation. Which of the genes listed above is most likely to be altered by the mutD allele? Why?

 

 

 

3) When DNA from bacterial virus T4 grown in 14N medium, is denatured and mixed with denatured DNA from T4 that was grown in 15N medium and allowed to renature three bands are observed in a equilibrium density gradient centrifugation experiment. When denatured 14N T4 DNA is mixed with denatured 15N DNA from bacterial virus T5 and allowed to renature only two bands are observed. Explain these results.