17. Mutation, Repair, and Recombination

Induced Mutations

Open Question
A fragment of a wild-type polypeptide is sequenced for seven amino acids. The same polypeptide region is sequenced in four mutants.Wild-type polypeptide N . . . Thr–His–Ser–Gly–Leu–Lys–Ala . . . CMutant 1 N . . . Thr–His–Ser–Val–Leu–Lys–Ala . . . CMutant 2 N . . . Thr–His–Ser–CMutant 3 N . . . Thr–Thr–Leu–Asp–CMutant 4 N . . . Thr–Gln–Leu–Trp–Ile–Glu–Gly . . .Use the available information to characterize each mutant.
Open Question
Experiments by Charles Yanofsky in the 1950s and 1960s helped characterize the nature of tryptophan synthesis in E. coli. In one of Yanofsky's experiments, he identified glycine (Gly) as the wild-type amino acid in position 211 of tryptophan synthetase, the product of the trpA gene. He identified two independent missense mutants with defective tryptophan synthetase at these positions that resulted from base-pair substitutions. One mutant encoded arginine (Arg) and another encoded glutamic acid (Glu). At position 235, wild-type tryptophan synthetase contains serine (Ser) but a base-pair substitution mutant encodes leucine (Leu). At position 243, the wild-type polypeptide contains glutamine and a base-pair substitution mutant encodes a stop codon. Identify the most likely wild-type codons for positions 211, 235, and 243. Justify your answer in each case.
Open Question
Alkaptonuria is a human autosomal recessive disorder caused by mutation of the HAO gene that encodes the enzyme homogentisic acid oxidase. A map of the HAO gene region reveals four BamHI restriction sites (B1 to B4) in the wild-type allele and three BamHI restriction sites in the mutant allele. BamHI utilizes the restriction sequence 5′-GGATCC-3′. The BamHI restriction sequence identified as B3 is altered to 5′-GGAACC-3′ in the mutant allele. The mutation results in a Ser-to-Thr missense mutation. Restriction maps of the two alleles are shown below, and the binding sites of two molecular probes (probe A and probe B) are identified.DNA samples taken from a mother (M), father (F), and two children (C1 and C2) are analyzed by Southern blotting of BamHI-digested DNA. The gel electrophoresis results are illustrated.Using A to represent the wild-type allele and a for the mutant allele, identify the genotype of each family member. Identify any family member who is alkaptonuric.
Open Question
Using your knowledge of DNA repair pathways, choose the pathway that would be used to repair the following types of DNA damage. Explain your reasoning.

a cytosine that has been deaminated to uracil
Open Question
Using your knowledge of DNA repair pathways, choose the pathway that would be used to repair the following types of DNA damage. Explain your reasoning.

a double-stranded break that occurs during G₁ and prevents completion of DNA replication
Open Question
Using your knowledge of DNA repair pathways, choose the pathway that would be used to repair the following types of DNA damage. Explain your reasoning.

a double-strand break that occurs just after replication in an actively dividing cell
Open Question
Using your knowledge of DNA repair pathways, choose the pathway that would be used to repair the following types of DNA damage. Explain your reasoning.

a thymine dimer induced as a result of UV exposure
Open Question
Common baker's yeast (Saccharomyces cerevisiae) is normally grown at 37°C, but it will grow actively at temperatures down to approximately 25°C. A haploid culture of wild-type yeast is mutagenized with EMS. Cells from the mutagenized culture are spread on a complete-medium plate and grown at 25°C. Six colonies (1 to 6) are selected from the original complete-medium plate and transferred to two fresh complete-medium plates. The new complete plates (shown) are grown at 25°C and 37°C. Four replica plates are made onto minimal medium or minimal plus adenine from the 25°C complete-medium plate. The new plates are grown at either 25°C or 37°C and the growth results are shown.Classify the nature of the mutations in colonies 1, 2, and 5.
Open Question
Common baker's yeast (Saccharomyces cerevisiae) is normally grown at 37°C, but it will grow actively at temperatures down to approximately 25°C. A haploid culture of wild-type yeast is mutagenized with EMS. Cells from the mutagenized culture are spread on a complete-medium plate and grown at 25°C. Six colonies (1 to 6) are selected from the original complete-medium plate and transferred to two fresh complete-medium plates. The new complete plates (shown) are grown at 25°C and 37°C. Four replica plates are made onto minimal medium or minimal plus adenine from the 25°C complete-medium plate. The new plates are grown at either 25°C or 37°C and the growth results are shown.

Which colonies are prototrophic and which are auxotrophic? What growth information is used to make these determinations? <>
Open Question
Common baker's yeast (Saccharomyces cerevisiae) is normally grown at 37°C, but it will grow actively at temperatures down to approximately 25°C. A haploid culture of wild-type yeast is mutagenized with EMS. Cells from the mutagenized culture are spread on a complete-medium plate and grown at 25°C. Six colonies (1 to 6) are selected from the original complete-medium plate and transferred to two fresh complete-medium plates. The new complete plates (shown) are grown at 25°C and 37°C. Four replica plates are made onto minimal medium or minimal plus adenine from the 25°C complete-medium plate. The new plates are grown at either 25°C or 37°C and the growth results are shown.What can you say about colony 4?