Lecture 11 Gene Mutations Lets say that we are investigating the lacz gene, which encodes the lactose hydrolyzing enzyme B-galactosidase. There is a special compound known as X-gal that can be hydrolyzed by B-galactosidase to release a dark blue pigment. When X-gal is added to the growth medium in petri plates, Lac E coli colonies turn blue whereas Lac- colonies with mutations in the Lacz gene are white. By screening many colonies on such plates it is possible to isolate a collection of E coli mutants with alterations in the lacz gene. PCR amplification of the lacz gene from each mutant followed by dNa sequencing allows the base changes that cause the Lacz- phenotype to be determined. A very large number of different Lacz mutations can be found but they can be categorized into three general Mutation Type Description Missense a base change that converts one codon into another many missense mutations are silent because the encoded amino acid remains the same or the amino acid substitution is sufficiently subtle so as not to compromise activity of the enzyme. Missense mutations that have a marked effect often lie in the active site or grossly disrupt protein folding Nonsense a base change that converts a codon within the coding sequence into a stop codon. Note that there is only a limited set of sense codons that can be converted to a stop codon by a single base change Nonsense mutations lead to a truncated protein product. Nonsense the gene. Sometimes nonsense mutations that lie late in the gene Late mutations that lie early in the gene sequence will completely inactivate sequence will not disrupt gene function Frameshift The addition or deletion of a base or bases such that the coding sequence is shifted out of register. Note that addition or deletion of a multiple of three bases does not cause a frameshift. After the frameshift mutation is encountered, missense codons will be read up to the first stop codon. Like nonsense mutations, frameshif mutations usually lead to complete inactivation of the geneLecture 11 Gene Mutations Let’s say that we are investigating the LacZ gene, which encodes the lactose hydrolyzing enzyme ß-galactosidase. There is a special compound known as X-gal that can be hydrolyzed by ß-galactosidase to release a dark blue pigment. When X-gal is added to the growth medium in petri plates, Lac+ E. coli colonies turn blue whereas Lac– colonies with mutations in the LacZ gene are white. By screening many colonies on such plates it is possible to isolate a collection of E. coli mutants with alterations in the LacZ gene. PCR amplification of the LacZ gene from each mutant followed by DNA sequencing allows the base changes that cause the LacZ– phenotype to be determined. A very large number of different LacZ mutations can be found but they can be categorized into three general types. Mutation Type Description Missense A base change that converts one codon into another. Many missense mutations are silent because the encoded amino acid remains the same or the amino acid substitution is sufficiently subtle so as not to compromise activity of the enzyme. Missense mutations that have a marked effect often lie in the active site or grossly disrupt protein folding. Nonsense A base change that converts a codon within the coding sequence into a stop codon. Note that there is only a limited set of sense codons that can be converted to a stop codon by a single base change. Nonsense mutations lead to a truncated protein product. Nonsense mutations that lie early in the gene sequence will completely inactivate the gene. Sometimes nonsense mutations that lie late in the gene sequence will not disrupt gene function. Frameshift The addition or deletion of a base or bases such that the coding sequence is shifted out of register. Note that addition or deletion of a multiple of three bases does not cause a frameshift. After the frameshift mutation is encountered, missense codons will be read up to the first stop codon. Like nonsense mutations, frameshift mutations usually lead to complete inactivation of the gene