Suppression Analysis

OVERVIEW

Suppression analysis is an elegant and highly favored molecular genetic tool used to identify genes that are functionally related to the gene of interest. It dates back to the very earliest days of genetics and the work of Sturtevant (1920) and Beadle & Ephrussi (1936) but it was not until the 1960s that the variety of suppression mechanisms and the capabilities of suppressor analysis were fully appreciated. Increased use of suppressor analysis, particularly in model genetic organisms like Saccharomyces, has refined the method making it one of the two accepted genetic methods for identification of functionally related genes or gene products. The second method, enhancer analysis, will be described in Chapter 9. Functionally related genes encode products capable of carrying out the same or similar functions, are different components of the same metabolic or regulatory pathway, or function together in a structural or enzyme complex. Mutations in these genes might not have been revealed by the original mutant hunt for a variety of reasons. The mutant alleles might have a different and unpredicted phenotype or might not produce a detectable phenotype unless in conjunction with another mutation. Thus, a search for suppressor mutations has the potential for revealing an entirely new spectrum of genes.

A suppressor mutation is a mutation that counteracts the effects of the original mutation such that the double mutant individual containing both the original mutation and the suppressor mutation has a phenotype similar to that of the wildtype. Suppressors are isolated when a mutant strain is 'reverted' to restore the wildtype or wild-type-like phenotype. As with the isolation of the original mutation, one must devise a selection/screen that allows the identification of revertants having the 'wild-type' phenotype. One determines whether a revertant is a true revertant, that is restores the original gene sequence, or if it carries a suppressor mutation by crossing the revertant to a wild-type strain and seeing whether the original mutation can be recovered in the progeny. Recovery of the original mutation can only occur if there has been a recombination event separating the original mutation from the suppressor mutation. The suppressor mutation alone will frequently have a mutant phenotype, but this phenotype could be novel or could be similar to the phenotype of the original mutation.

INTRAGENIC SUPPRESSORS

If the suppressor mutation and the original mutation are in the same gene, this is referred to as intragenic suppression. Recombination between the original mutation and the intragenic suppressor is expected to be rare because the two alterations are very tightly linked. Intragenic suppression can occur by a variety of mechanisms. A classic example is found in Crick et al. (1961). The original mutation used in this analysis was a +1 frameshift mapping to the 5' end of the rllB ORF of the T2 phage of E. coli. Intragenic suppressors of this mutation were produced by a nearby -1 frameshift thereby restoring the correct reading frame. One can also get intragenic suppression of a missense mutation. A missense mutation might functionally inactivate a protein by altering its shape or by increasing its rate of degradation to such an extent that inadequate amounts of the protein are produced. An intragenic suppressor could restore function if it enables the doubly mutant protein to fold into a more active shape or reduces the rate of degradation sufficiently to provide adequate levels of the protein. Changes in the promoter could increase the level of expression of a partially active mutant protein to a level adequate to restore a wild-type-like phenotype.

INTERGENIC SUPPRESSION

If the suppressor mutation and the original mutation are in different genes, this is referred to as intergenic suppression. Recombination between the original mutation and the suppressor mutation is likely to occur at a high rate since with rare exceptions the suppressor mutation will be unlinked to the original mutation. Intergenic suppressors fall into two broad categories: information suppressors and what we will call function suppressors.

Information suppression

Information suppressors are mutations in genes involved in the transmission of information from DNA to protein. As such, they act by improving the expression of the mutant gene. Moreover, an information suppressor will suppress any other gene, even those that are functionally unrelated to the original mutant, so long as the mutation in that gene has the same effect on the information transfer process as the mutation in the original gene. The reader is already familiar with nonsense, missense and frameshift suppressors that are mutations in tRNA genes. These were among the first types of information suppressor identified. Mutations affecting ribosome components also can be information suppressors. This type of information suppression acts at the level of the translation process and alters the reading of the encoded information. Information suppression can also act by increasing the amount of the transcript, such as by altering components of the transcription machinery or complexes involved in post-transcriptional processing. In summary, information suppressors suppress particular types of mutational alteration affecting the information transfer process and can do this in any gene that has that type of alteration. In genetic terms, information suppressors are allele-specific but not gene-specific.

Researchers interested in the processes of information transfer can use suppressor analysis to obtain mutations in genes involved in these processes but it is important to choose the original mutant strain carefully. For example, if one is interested in translation start-site selection one should work with a mutation in the leader sequence of a gene that exhibits reduced protein production but not reduced transcript levels. Intergenic suppressors that increase translation should be obtained, for example, in translation initiation factors or the small ribosomal subunit. Starting with a promoter mutation or an alteration in the coding region of the gene would not give the desired types of suppressor. As part of the initial characterization of the suppressors, one should test the ability of the suppressor to suppress a comparable mutation in the leader sequence of another unrelated gene. This should sort the suppressor mutations into the desired class of information suppressor from uninteresting mutations affecting some other aspect of the expression of the original mutant gene.

Function suppression

Function suppressors act to restore or replace the altered gene function. This is accomplished by several means. The activity of a gene product can be appropriately modulated by mutations that affect its post-translational modification, subcellular localization, degradation, or interaction with activators, inhibitors, or other regulatory factors. Mutational changes can substitute another gene product for the mutant gene product either by changing the specificity or abundance of that alternate protein. Or, if the mutant gene encodes a component of a switch regulatory pathway, suppression can occur by the constitutive activation of a downstream component.

The isolation of function suppressors provides a means of exploring the role of a gene product in a cellular process and identifies other functionally related gene products or other components of a pathway. Again, if one is interested in isolating function suppressors of a mutant gene, it is important to start the analysis with an appropriate mutation in that gene. The original mutation should affect the gene function, not expression, and thus should be confined to the coding region. Depending on the type of function suppressor desired, one could start with a null mutation, such as a deletion or an insertion, or with a missense mutation. Temperature-sensitive and cold-sensitive mutant alleles are frequently used for function suppression analysis because these types of mutation are almost always missense mutations.

There are three mechanisms of function suppression: by-pass suppression, allele-specific suppression, and suppression by epistasis.

BY-PASS SUPPRESSION

A by-pass suppressor by-passes the need for the mutated gene product by providing the function of that gene product by alternate means. Two mechanisms of by-pass suppression of mutations in hypothetical GEN1 are shown in Figure 8.1. In Model 1, GEN1 and GEN2 carry out related but distinct processes that do not functionally overlap in the wild-type strain. The mutation of GEN1 blocks the reaction thereby creating a mutant. An alteration in GEN2 allows the GEN2 gene product to acquire a new function that enables it to substitute for the GEN1 product thereby bypassing the GEN1 mutation. The short arrow indicates that the by-pass may be less effective than the wild-type function. In Model 2, expression of another gene is increased so that its product can substitute for the GEN1 product. Gen3 protein is capable of carrying out the same or a similar function as the Genl protein but not at rates adequate for a wild-type phenotype. Perhaps the specific activity of Gen3p

GENETIC TECHNIQUES FOR BIOLOGICAL RESEARCH Model 1

Wild-type

GEN2

Mutant

gen I

GEN2

geni

Suppressed

GEN2-sup

Model 2

GENI

Wild-type

Mutant

I GEN3 X

Suppressed

GEN3-SUP

Figure 8.1 Models of by-pass suppression for the Genlp substrate R is very low. An increase in the expression levels of GEN3 by the suppressor mutation provides levels of Gen3 protein sufficient to allow it to substitute for the loss of GEN1.

It is important to note that by-pass suppression is not dependent on the original gene product and thus by-pass suppressors are able to suppress either missense mutations or null mutations of the original gene. In addition, the suppressor allele is dominant in both Model 1 and Model 2.

ALLELE-SPECIFIC SUPPRESSION

In allele-specific suppression, a suppressor mutation in GEN2 is able to suppress only a particular non-null allele or select a group of non-null alleles in GEN1. Experience has demonstrated that allele specificity of function suppressors implies that the products of the two genes interact directly, i.e. they make physical contact with each other. Both by-pass suppression and suppression by epistasis (see below) can occur with null alleles, i.e. alleles that produce no protein product. Therefore, if a suppressor is not capable of suppressing a null mutant allele but is only capable of suppressing missense mutations of a gene, then the mechanism of suppression is allele specific. Allele-specific function suppression is considered strong evidence that the gene products of the original mutant gene and the suppressor gene physically interact. This interaction could indicate an enzyme-substrate type of interaction (as between a protein kinase and its target protein), an interaction between components of a heterodimer or heteromultimeric complex, or an interaction between the catalytic subunit of an enzyme complex and its regulatory subunit(s).

To conceptualize allele-specific suppression, consider the fact that two proteins interact with each other only over a small portion of their surfaces, and that within

Figure 8.2 Model of allele-specific suppression

these defined regions of interaction specific residues on the surfaces of the proteins make the most significant contributions to the binding strength of the interaction. This is illustrated in Figure 8.2. Mutations that alter these residues affect the binding strength of the interaction either by weakening or strengthening it. If the original mutation in GEN1 decreases the binding of the Genl protein to the Gen2 protein, a suppressor mutation in GEN2 might act to restore strong binding of Genlp to Gen2p. Suppression will occur if, and only if, the suppressor mutation in GEN2 alters a residue mapping within the surface region of Gen2p that physically makes contact with Genlp.

In the context of this explanation, the gen2 suppressor mutation can only be expected to suppress genl mutations that alter residues in the region of Genlp involved in the Genlp-Gen2p interaction; that is, they would be specific to alleles altering residues in this region. Suppressor mutations in GEN2 would not be expected to suppress null mutations of GEN1 (large deletions, 5' nonsense or frameshift mutations, insertions, or other rearrangements). Allele-specific suppressors also would not be expected to suppress mutations elsewhere in Genlp outside of the surface region of Genlp that interacts with Gen2p, for example in the Gen3p binding site. The suppressor mutation of gen2 alone, that is when not in combination with the original genl mutation, is likely to have a mutant phenotype similar to the genl mutant phenotype because both affect the Gen 1 p Gen2p interaction.

Allele-specific suppression is not limited to protein-protein interactions. It can be used to characterize DNA-protein or RNA- protein interactions. When two proteins (or a DNA sequence and a protein) are known to interact directly, allele-specific suppression can be used to explore the details of the binding, i.e. which specific residues and/or basepairs are involved. Alternately, one can identify novel proteins that interact with the product of a known gene by identifying allele-specific suppressors of a mutant allele of that gene.

SUPPRESSION BY EPISTASIS

Suppression by epistasis occurs between genes whose products are components of a switch regulatory pathway as described in Chapter 6 on epistasis. The component proteins of switch regulatory pathway alternate between the 'on' state and the 'off'

state. Mutations in these proteins can permanently shift them to either state. Such mutations act to separate the switch regulatory pathway from the upstream signal. The pathway can be constitutive (that is, unregulated) by activating the pathway in the absence of the stimulatory signal or despite the presence of inhibitory signal. Alternately, mutations can block the pathway despite the presence of the stimulatory signal or in the absence of an inhibitory signal. The ultimate effect of a mutation in a component protein depends on whether the component is a positive regulator or a negative regulator.

For example, in the switch regulatory pathway shown below, the signal activates protein 1, protein 1 activates protein 2, activated protein 2 inhibits protein 3, and protein 3 is an inhibitor of GEN4 expression.

Signal-*■ protein 1 -»- protein 2-1 protein 3-' |

Recessive mutations in GEN1 or GEN2 lead to a lack of GEN4 expression. Recessive mutations in GEN3 lead to the constitutive expression of GEN4. A strain doubly mutant for recessive mutations in GEN1 and GEN3 or GEN2 and GEN3 would be constitutive for GEN4 expression. Thus, a loss of function mutation in GEN3 will suppress loss of function mutations in GEN1 and GEN2, and restore GEN4 expression to genl and gen2 mutant strains. This suppression of gen 1 and gen2 mutations by a mutation in gen3 is suppression by epistasis. Of course, in this case the suppression does not restore normal regulation but it does restore expression of GEN4, albeit constitutively.

Suppression by epistasis can occur with null or non-null alleles of either the original mutant gene or the suppressor gene. For example, in the above pathway, a deletion of GEN3 will suppress either a missense mutation or a deletion mutation of GENl. A dominant constitutive mutation in GEN1 would activate this pathway in the absence of a signal. This constitutive activation would be suppressed by recessive mutations in GEN2.

OVEREXPRESSION SUPPRESSION

Because of the variety of plasmid vectors available for Saccharomyces it is possible to modulate the expression levels of a desired gene in a number of ways and not simply by in situ alterations in the promoter region of genes. This type of suppression is called multicopy suppression or suppression by overexpression or overproduction. Multicopy plasmids are frequently used to obtain overproduction of a particular protein. A gene carried by a YEp vector will be present at up to 50 copies/cell and thus one might expect a 50-fold overproduction of the protein. Vectors are available that allow one to fuse the coding region of a gene to any one of several different Saccharomyces promoters that have different expression levels and some of these are easily regulated by environmental signals such as nutrient availability. These are both high and low copy vectors and, as a result, a wide range of expression levels of the inserted gene can be achieved (see Chapter 1 ; Mumberg et al., 1995; Labbé & Thiele, 1999). Libraries can be made with these vectors and screened for suppression of mutant strains. The major strength of this type of suppressor hunt is that one can very simply identify the suppressing gene by recovering the suppression plasmid and sequencing its insert fragment. All three types of suppression can be obtained by multicopy or overexpression suppression.

BY-PASS SUPPRESSION BY OVEREXPRESSION

Overproduction of a gene product can lead to by-pass suppression. This is similar to Model 2 of by-pass suppression (above) except that the overproduction is from the plasmid-borne copy of the suppressing gene.

ALLELE-SPECIFIC SUPPRESSION BY OVEREXPRESSION

One might compensate for the reduced binding constant between mutant Protein 1 and wild-type Protein 2 by overproduction of wild-type Protein 2. Abundant amounts of Protein 2 could kinetically drive complex formation despite the lower binding constant by a mass action effect. This is a form of allele-specific suppression because it could not occur with null alleles of the gene encoding Protein 1. Mutant Protein 1 is necessary for the suppression. As is discussed above, overexpression allele-specific suppression of genl mutant alleles by GEN2 implies that the product of GEN2 binds to the product of GEN1 at the site of the mutant residue. Similar overproduction of Protein 3 should not suppress the same genl mutations as overproduction of Protein 2.

OVEREXPRESSION SUPPRESSION BY EPISTASIS

In a switch regulatory pathway, overproduction of an activator or repressor could, respectively, activate or block a pathway regardless of the upstream signal. For example, overproduction of an activator (positive regulator) might activate a blocked pathway by overcoming an inhibitory regulator (perhaps by binding to it and titrating out its effects). Or, if the activator has a very low constitutive activity, its overproduction might be sufficient to restore progress through the pathway. Comparable situations can be proposed for repressors (negative regulator). The suppressing gene product necessarily would be downstream of the original mutant gene product. Moreover, the effect of overexpression of a particular component will depend on the role of the component in the pathway.

REFERENCES AND FURTHER READING

Beadle, G.W. & B. Ephrussi (1936) Development of eye colors in Drosophila: transplantation experiments with suppressor of vermillion. Proc. Natl Acad. Sei. USA 22: 536. Crick, F., L. Barnett, S. Brenner, & RJ. Watts-Tobin (1961) General nature of the genetic code for proteins. Nature 192: 1227-1232. Gorini, L. (1970) Informational suppression. Ann. Rev. Genet. 4: 107-134. Gorini, L. & J. Beckwith (1966) Suppression. Ann. Rev. Microbiol. 20: 401-422. Hartman, P.E. & J.R. Roth (1973) Mechanisms of suppression. Adv. Genet. 17: 1-105.

Labbe, S. & D.J. Thiele (1999) Copper ion inducible and repressible promoter systems in yeast. Meth. Enzymol. 306: 145-153. Mount, S. & P. Anderson (2000) Expanding the definition of informational suppression. Trends Genet. 16: 157.

Mumberg, D., R. Muller, & M. Funk (1995) Yeast vectors for the controlled expression of heterologous proteins in different genetic backgrounds. Gene 156: 119-122. Prelich, G. (1999) Suppression mechanisms: themes from variations. Trends Genet. 15: 261266.

Rine, J. (1991) Gene overexpression in studies of Saccharomyces cerevisiae. Meth. Enzymol. 194: 239-251.

Sturtevant, A.H. (1920) The vermillion gene and gynandromorphism. Proc. Soc. Exp. Biol. Med. 17: 70-71.

Genetic Techniques for Biological Research Corinne A. Michels Copyright © 2002 John Wiley & Sons, Ltd ISBNs: 0-471-89921-6 (Hardback); 0-470-84662-3 (Electronic)

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