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Here are some full-credit student answers that are solid enough to receive full credit on an exam.

1. To obtain "o^c" mutants, Jacob et al started with a strain with the genotype i⁺z^-/i⁺z⁺.

a. In your own words, why did Jacob et al start with a strain that was diploid for the gene encoding the lac repressor?

Jacob et al suspected that another DNA factor besides the already characterized lac repressor was involved in lac operon expression control (the operator region). They hoped to mutagenize cells to obtain mutations in this other factor, but in a haploid cell it would be difficult to determine if the mutation was in this other element or in the lac repressor gene. By having a cell diploid for the repressor, the chances of both alleles of the repressor mutating were greatly reduced. Thus, mutated cells exhibiting constitutive expression of the lac operon could be expected to have a mutation in the operator region.

b. Given the information about lac z mutants described above (i.e. that they encode protein that is stable, nonfunctional, and distinguishable from the wild-type protein), what was the usefulness of beginning with a strain that was heterozygous for lac z? What information was obtained in this way that would not have been obtained from a lac z homozygote?

Using a strain heterozygous for lac z, they could distinguish between cis and trans effects of the operator mutations. The products synthesized by each chromosome of the heterozygote were different (beta-galactosidase and protein Cz); they could detect which chromosome controlled constitutive expression according to which gene was constitutively expressed. Because beta-galactosidase or protein Cz was only constitutively produced when cis to the o^c, Jacob et al could conclude that the operator was only cis-acting and did not affect the trans allele.

2. Study the data in the table.

a. Is the i^-₃ mutation dominant or recessive? How can you tell?

The i^-₃ mutation is recessive because its phenotype (unregulated expression of the lac-related genes) is not seen in heterozygotes.

b. Explain this dominance or recessiveness physiologically, given what you know about the product of the lac i gene.

The product of the lac i gene is a repressor that binds to the operator of the lac operon, blocking transcription. The repressor protein is produced and diffuses throughout the cytosol of the cell, binding to DNA at the lac operator. The protein is able to bind to any lac operator within the genome, thus only one functional copy of the gene is necessary to repress both copies of the lac operon in this experiment.

3. The "o^c" mutations described in this paper are classic examples of "cis dominant" mutations.

a. Given the phenotype of these mutants as described in the paper, explain what might be meant by the term "cis dominant" (this term is not used in the paper).

A mutation in the operator is dominant and cannot be complemented by a wild-type operator in a heterozygote, but it is cis-acting because it does not affect the operon trans to the mutation. When there is a mutation of the operator (o^c), there is constitutive expression of the genes in the cis operon.

b. Explain the physiological reason why these mutations would be cis dominant, and why this characteristic would be expected from mutations in a DNA element that regulates transcription.

The physiological basis of cis-dominance arises due to the location of the operator within the lac operon. The operator is located directly upstream of the coding regions. This operator binds the repressor and thus prevents the polymerase from transcribing the gene. When a mutation occurs in the operator, or the binding sequence specific to the repressor, the repressor no longer can bind and therefore the genes are consitutively expressed. This trait is considered dominant because only one mutated allele is required to give rise to the new phenotype of constitutive beta-galactosidase expression.

4. Jacob et al also describe the isolation of "o^o" mutants.

a. Jacob et al mapped these o^o mutations to a region between the lac z and the lac i genes, which is where the operator is located. Their interpretation was that these are operator mutations. Explain this interpretation--in order for an operator mutation to have this phenotype, what effect would you expect it to have on the binding of the lac repressor?

o^c mutants result in the loss of the ability to synthesize permease and beta-gal. These are deduced to be operator mutations because they affect expression of both genes. [If these were indeed operator mutations, ] I would expect that the lac repressor irreversibly binds to the mutated operator.

b. These mutations do not turn out to be in the operator itself. Given your more current understanding of the regulation of the lac operon, explain what you think these mutations might represent. Where might they be located, and what effect might they have on protein binding?

It makes sense to think that the o^o muatation is in fact a mutation in the promoter region. Were this true the lac operator could be wild-type or mutated, the i gene could be expressing repressor or not, and there would still be no transcription. RNA polymerase needs to bind to the promoter in order to transcribe the following genes. If, for example, there was a mutation in the critical -10 or -35 regions of the promoter it may be too weak to successfully bind the sigma 70 subunit of the RNA polymerase.

5. Summarize the main point of this paper in your own words. Describe how the data presented address this point. Why was this important, in the historical context of this paper?

The main point of the paper is that Jacob et al had articulated a bipartite mechanism of gene expression control. One unit of the mechanism involves a structural unit on the DNA itself (o region) that acts in a "cis" fashion and the other unit involves a regulator (lac repressor), which is a gene product and acts in a "trans" fashion. Jacob et al's method of using cells diploid for the lac operon region allowed them to show the presence of two discrete types of mutations in the operon region, occuring at different loci and causing different phenotypes of lac expression. At the time that this paper was written the idea of a cis-acting DNA element was especially improtant, because DNA sequences were thought to serve solely as instructions for proteins, with no other kinds of sequences included. This study also brought to light the fact that gene expression may be quite complicated, involving two or more discrete elements.

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