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Jacob et al, "The Operon," 1960
(see also Lodish CH10, especially p. 343-344)
Due at the beginning of class (8:00) on Monday, Oct 30
"Diploid" bacteria
Jacob et al used a bacterial genetic technique in this paper that we
haven't discussed in class. Specifically, they exploited the fact
that bacteria can "mate" (at least in a sense). There is a factor
called "F" that is a conjugation factor in bacteria--it promotes the
transfer of DNA from one bacterium to the next. Researchers have
created and maintained various altered versions of this "sex factor"
in order to create "diploid" bacteria. These bacteria are only
diploid at particular regions of the genome (lac, in this case) and
haploid at all other loci. Still, this "diploid" state is stable and
heritable, so that a cell that is diploid in this sense for the lac
region of the genome gives rise to a colony (or a culture) of cells
that are all also diploid in this region. This provides a convenient
system in which to ask genetic questions about dominance. It also
facilitates genetic complementation analysis.
Historical context
Remember that this paper was published in 1960. It was known that
DNA was the genetic material, and that it carried instructions for
making proteins. mRNA had not yet been discovered and characterized,
although it was becoming apparent that some "cytoplasmic replica" of
the DNA instructions must be involved. Needless to say, the
mechanisms of transcription were not known. The existing model was
that genes were "structural," carrying the instructions for building
proteins. No other function of DNA sequences had been discovered. The
proposition that a DNA sequence (an operator) could be involved in
gene expression without itself coding for protein was thus a novel
and unorthodox idea. Promoters had not been discovered yet.
The tools that were on hand
It was known that there were at least three genes specifically
involved in lactose metabolism, and mutations had been isolated in
each of these genes. LacZ encodes beta-galactosidase, the enzyme that
actually breaks down lactose into glucose and galactose. LacY encodes
lac permease, the transmembrane protein that allows lactose to enter
the cell in the first place. Lac I encodes the lac repressor, the
mechanism of action of which was unknown.
It was known that these three genes were located close to each other in the bacterial genome, in the order I..Z..Y. It was also possible to "map" any new mutations obtained relative to these known mutations, using genetic techniques.
There were existing mutants that did not make functional lac permease, and their mutations mapped to lacY (lac y-R). There were also existing mutants that did not make functional beta-galactosidase, and their mutations mapped to lac z. Some of these mutations (e.g. z-1 and z-4) permit the synthesis of beta-galactosidase protein which can be detected using antibody-based methods, is distinguishable from wild-type beta-gal in these methods, and does not catalyze the metabolism of lactose. Beta-gal protein encoded by these mutant lac z genes is referred to as "protein Cz" in the data table.
Jacob et al weren't right about everything!
This work was done very early in the exploration of gene
expression. Not every conclusion that Jacob et al draw turned out to
be correct. For example, "oo" mutants don't turn out to
carry mutations in the operator (see below). Also, the model of gene
expression being controlled in units of related genes (operons), each
of which would be regulated by a repressor, did not turn out to be as
general as Jacob et al propose in their discussion. This provides a
good example of how science works. Specific data are obtained about a
model system and interpreted in the context of that system, and these
results are extrapolated to explain more general phenomena. This
extrapolation provides hypotheses that guide further research. The
fact that all of the hypotheses thus generated do not turn out to be
true is not important--in fact, it's what makes the process
interesting. It's the ability of a model to generate testable
predictions about larger questions that matters most.
and now for some questions... (2 points per question, for 10 pts total)
1. To obtain "oc" 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?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?
2. Study the data in the table.
a. Is the i-3 mutation dominant or recessive? How can you tell?b. Explain this dominance or recessiveness physiologically, given what you know about the product of the lac i gene.
3. The "oc" 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).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.
4. Jacob et al also describe the isolation of "oo" mutants.
An aside (not directly related to the question): These recessive mutations revert at a frequency (10-7 to 10-8) that suggests that they are single nucleotide substitutions. This is about the frequency with which any given nucleotide change will occur at random in E. coli, due to spontaneous events such as DNA damage or polymerase errors. A reversion frequency in this range suggests that reversion--i.e. a return to a wild-type genotype--requires only a single nucleotide change rather than a more complex mutational event.
a. Jacob et al mapped these oo 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?
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?
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?
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Updated: 25 Oct 00