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Why Do Genetics
More Basic Concepts
Mutagenesis in vitro
Effects of Mutations
Two Factor Crosses
Other Mapping Methods
Problem Set 1
Problem Set 2
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©2002 written by Gary Roberts, edited by Timothy Paustian, University of Wisconins-Madison
In the course of analyzing a regulated gene or genes, it is often useful to monitor the regulatory system by putting a foreign gene under the control of that system. The foreign gene typically encodes an easily assayed product, (a reporter molecule), like the enzyme -galactosidase, encoded by lacZ. For the activity of the reporter to reflect the regulatory scheme of interest, the foreign gene needs to be separated from its own regulatory signals and placed transcriptionally downstream from the regulatory sites under study. Such a merger of the two different regions is termed a fusion, and is used when the regulation of the system is the important topic. The inverse case, where a gene of interest is placed downstream of a strong or tunable promoter, is typically called an expression vector and is employed when the function of the gene product is the critical issue.
Fusions fall into two classes that require separate definitions. In transcript fusions, the reporter gene lacks a promoter, but possesses a functional ribosome binding site, so the reporter gene product is made whenever the "target gene" is being transcribed. This allows you to monitor transcriptional regulation of the target gene and ignores any possible posttranscriptional effects. In translational fusions, the reporter gene lacks both a promoter and a functional ribosome binding site, so the reporter gene product is made whenever the "target gene" is being transcribed and translated. In this case, you test for the presence and degree of both transcriptional and translational regulation, though such a fusion by itself does not allow you to tell which is present; the comparison of results with transcriptional and translational fusions identifies how much regulation is being done at each level.
The nature of reporter gene ("gene X" in the figures) product is obviously crucial, you want an easily assayable product (lacZ, -galactosidase, for which there are easy color assays; galK, galactokinase, also with color assays; lux, luciferase, with photometric assays) or a selectable gene product (for example, most any gene causing drug resistance by degradation or modification of the drug). Further, the reporter protein must be functional even when synthesized as a fusion protein.
X B. UTILITY
Easy assay for transcriptional/translational regulation: Many genes are expressed at relatively low levels so that direct measurements of RNA synthesis is technically difficult and therefore the regulation of transcription of the gene cannot be so determined. If the reporter gene encodes an easily assayable product, transcriptional regulation of the affected transcript becomes possible (but see section X D below).
Selection/screening of mutants: In a wild-type situation, there may be no easy selection or screen for mutants that abnormally regulate expression of a given gene. However, introduction into that transcript of a gene with an easily selectable or screenable gene product can allow the production of desired mutants because the regulatory system is now regulating a more technically addressable gene and gene product. The strain desired for biochemical/physiological analyses should typically contain the regulatory mutation, but not the fusion used to select that mutation. That is, you want to see the effect of your new regulatory mutation in an otherwise wild-type background and determine how it affects the norally controlled genes, not merely the reporter. The mode of construction of such a strain would depend on which alleles are selectable, as well as the linkage of the regulatory mutation to the fusion (see the section on strain construction).
Protein isolation/localization: When fused or hybrid proteins are produced, antibodies recognizing either portion of the hybrid will typically precipitate the entire hybrid. Thus, antibody directed toward the product of the introduced gene will allow the isolation of a protein with some portion of gene B product which in turn can be used to raise B-specific antibodies. Alternatively, the hybrid protein often behaves like the wild-type B protein and can be used to monitor that behavior. For example, if the hybrid protein is detected in the cell membrane, it is likely that the B product is normally there (assuming, of course, that the reporter product was already known not to be associated with the membrane by itself).
X C. GENERATION OF FUSIONS
As noted in sections IV A and IV D, fusions can be generated by deletions and duplications. Such modes of generation have a number of disadvantages if the fusion is to be "useful" as described above. Most notably, the frequency of any given fusion will be low, and there will be severe limitations on which genes can be so fused. More useful systems involve use of specially constructed transposons for the generation of fusions in vivo and cloning methodology for in vitro fusion generation.
Transposons: A small variety of transposable elements have been constructed to carry a gene (encoding an assayable product) that is automatically fused to the transcript (or gene) into which the transposon inserts. Different versions of these transposons generate either transcript or protein fusions as described above. Examples of such fusions are shown and described below. Desired fusions can be sought either by the phenotype that the fusion causes (after mutagenesis with a fusion-forming transposon, a newly generated His- strain will probably have a fusion in the his region, but perhaps not in the "proper" orientation) or the regulatory phenotype itself (strains that express the reporter gene in response to a growth condition or other stimulus). This latter point is particularly interesting since it allows regions to be mutationally identified on the basis of their regulation, rather than on the phenotype caused by their loss.
in vitro construction: It should be evident that having access to the DNA of the two genes of interest allows one to fuse them "chemically" through the use of appropriate restriction enzymes. This approach has the advantage that one can precisely build the fusion desired, but requires extensive knowledge of the genes of interest such as their restriction map, the location of the coding regions, etc.
X D. CONTROLS/CONCERNS
More than one fusion per genome: If a fusion is generated by an in vivo transposition event, the possibility exists that one will end up with a strain containing fusions at two different sites due to two transposition events (this is more likely with Mu-generated events than with other transposons). Such a strain would be inappropriate for a study of regulation since the assayable product would be produced by two differently regulated promotors. Selections for altered regulation would be flawed for similar reasons. Unfortunately, it is sometimes not easy to verify that the strain carries only one fusion. If the desired fusion confers a deleterious phenotype (His-, for example) then transduction or reversion to His+ should select for restoration of the wild-type genotype with concomitant loss of the fusion. If such a selection results in His+ strains that still retain a fusion (scored by the assayable gene product or the drug-resistance gene typically carried), one may assume the original fusion strain actually contained two fusions and a different strain should be chosen. Alternatively, Southern analysis might indicate that the strain contains two copies of the fusion system (this would be tested with a probe for the reporter genes themselves).
Perturbation of the regulatory system by the fusion: The fusion is itself a mutation with respect to the wild-type genotype, and a polar one at that. As such, it can perturb the very regulatory scheme under analysis in the following ways: (a) the products of either the mutated gene or one transcriptionally downstream from the insertion could be autoregulatory or (b) the absence of the product of the mutated gene or of those downstream might perturb the physiology of the cell so as to alter the regulation detected by the fusion. The latter perturbation is particularly common, especially in anabolic pathways where regulation is typically based on the level of the product of the pathway. Arguably, most gene products are autoregulatory in some way.
If the fusions are being used to monitor regulation, an obvious control is the introduction of a wild-type version of the affected region, typically on a low-copy number plasmid, on an integrated specialized phage, or in the normal chromosomal location. This copy supplies the mutated gene products and their metabolic products, making the merodiploid much more like the wild-type situation. It is, however, still not precisely the same as wild type, since there are two copies of the region in the cell and one is not in the "normal" position on the chromosome. For most systems, it will be close enough, but for some systems of extremely delicate regulation, the appropriate control strains cannot be constructed.
If the fusions are being used to generate regulatory mutations, the analysis of these mutations in the absence of the fusion will eliminate perturbation by the fusion. On the other hand, certain regulatory mutations may not be selectable because of the perturbation by the fusion.
Finally, if the system under analysis employs post-transcriptional regulation, then the fusion will possibly perturb that regulation by its effect on the mRNA produced.
Use of appropriate strains: Without belaboring the obvious, if one wishes to monitor the regulation of an operon with a lacZ fusion and the compound X-gal (which -galactosidase enzymatically cleaves, producing color), one needs to start with a strain which is itself lacZ-(preferably by deletion to avoid complications due to recombination with the fusion system), and which is capable of transporting X-gal into the cell. If the fusion system uses a transposon, it should be capable of transposing in that strain. Lastly, if fusions are selected using a drug-resistance marker in the vector, the starting strain must be sensitive to that drug and the resistance gene must be expressed in that strain.
Functionality of reporters from translational fusions: If you have translational fusions to different positions within the target gene, the spacific activity of the different reporter proteins might be different because of the "extraneous" protein attached to the amino terminus of each one. The hybrid proteins might also possess different degrees of stability and therefore show different accumulated activity, which could be misinterpreted as a difference in regualtion of the target gene.
[See sample problem 25]
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