2. In yeast the gene hisA codes for an enzyme necessary for histidine biosynthes

Question

2. In yeast the gene hisA codes for an enzyme necessary for histidine biosynthesis.

A certain spontaneous mutation is mapped in hisA, rendering that gene inactive.

a) Suppose the mutation reverts to wild type spontaneously at a low frequency, and can be

reversed by frame-shift mutagens. Explain the molecular basis of the original mutation and

its effect on hisA. What kind(s) of fairly common mutations might yield this phenotype?

b) Now, suppose that the mutation is not reversible by either frame-shift or base- analog

mutagens. Spontaneous reversion occurs quite frequently, but the rate of spontaneous

reversion is not enhanced by any mutagen. Discuss the possible nature of the original

mutagenic agent.

Explanation / Answer

(a) The first comprehensive review of histidine biosynthesis was written by Brenner and B. N. Ames in 1971. This extraordinary article consolidated information about control of the histidine biosynthetic pathway and posed many of the questions about histidine biosynthesis and his operon control that were the subject of investigation in subsequent years. Later reviews, including the first two versions of this review in 1987 and 1996, and reviews by Blasi and Bruni and Artz and Holzschu , covered the developments of the molecular structure of the his operon, the mechanisms of his operon metabolic regulation and attenuation control, and the function of the his regulatory loci, including identification of hisW mutations.

The flow of intermediates through the histidine biosynthetic pathway can be adjusted by varying the enzymatic activity of the HisG enzyme, which catalyzes the first reaction in the pathway. Modulation of HisG enzyme activity is brought about by four interrelated forms of inhibition: (i) classical, noncompetitive feedback inhibition by histidine; (ii) inhibition by ppGpp in the presence of partially inhibiting concentrations of histidine; (iii) competitive inhibition by ADP and AMP in response to the overall energy status in the cell; and (iv) competitive product inhibition by phosphoribosyl-ATP (PR-ATP). In wild-type bacteria growing in minimal medium, the rate of histidine biosynthesis seems to be controlled primarily by regulation of HisG enzymatic activity. Several feedback-resistant and feedback-hypersensitive mutations were mapped in a region that encodes the carboxyl-terminal portion of the HisG protein. Feedback-resistant mutants selected for their growth in the presence of the analog 2-thiazolealanine excrete histidine into the culture medium. This important observation indicates that feedback inhibition holds histidine biosynthesis far below its full capacity, even when histidine is not supplied as a supplement. Some feedback-hypersensitive mutants also have a distinct phenotype; they are growth restricted at 20°C because of severe inhibition of the mutant HisG enzyme by histidine at lower temperatures.

(b) In genetics, a mutagen is a physical or chemical agent that changes the genetic material, usually DNA, of an organism and thus increases the frequency of mutations above the natural background level. Mutagens can cause changes to the DNA and are therefore genotoxic. They can affect the transcription and replication of the DNA, which in severe cases can lead to cell death. The mutagen produces mutations in the DNA, and deleterious mutation can result in aberrant, impaired or loss of function for a particular gene, and accumulation of mutations may lead to cancer. Mutagens may therefore be also carcinogens. However, some mutagens exert their mutagenic effect through their metabolites, and therefore whether such mutagens actually become carcinogenic may be dependent on the metabolic processes of an organism, and a compound shown to be mutagenic in one organism may not necessarily be carcinogenic in another.

A spontaneous mutation is one that occurs as a result of natural processes in cells. We can distinguish these from induced mutations; those that occur as a result of interaction of DNA with an outside agent or mutagen. Since some of the same mechanisms are involved in producing spontaneous and induced mutations, we will consider them together. Some so-called "spontaneous mutations" probably are the result of naturally occurring mutagens in the environment; nevertheless there are others that definitely arise spontaneously, for example, DNA replication errors.

DNA replication error

Mistakes in DNA replication where an incorrect nucleotide is added will lead to a mutation in the next round of DNA replication of the strand with the incorrect nucleotide.The frequency at which a DNA polymerase makes mistakes (inserts an incorrect base) will influence the spontaneous mutation frequency and it has been observed that different polymerases vary in their accuracy.

Base alterations and base damage

The bases of DNA are subject to spontaneous structural alterations called tautomerization: they are capable of existing in two forms between which they interconvert. For example, guanine can exist in keto or enol forms. The keto form is favored but the enol form can occur by shifting a proton and some electrons; these forms are called tautomers or structural isomers. The various tautomer forms of the bases have different pairing properties. Thymine can also have an enol form; adenine and cytosine exist in amino or imino forms. If during DNA replication, G is in the enol form, the polymerase will add a T across from it instead of the normal C because the base pairing rules are changed (not a polymerase error). The result is a G:C to A:T transition; tautomerization causes transition mutations only. Another mutatgenic process occurring in cells is spontaneous base degradation. The deamination of cytosine to uracil happens at a significant rate in cells.

Spontaneous frameshift mutations

Streisinger observed in the 1960's that frameshift mutations in bacteriophages tended to occur in areas with "runs" of repeats of one nucleotide.

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