Genetic Techniques for Biological Research Corinne A. Michels Copyright q 2002 John Wiley & Sons, Ltd ISBNs: 0-471-89921-6 (Hardback); 0-470-84662-3 (Electronic) 8 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 thatthe variety ofsuppression mechanisms andthe capabilities of suppressor analysis werefully 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 methodsfor identification of functionally related genes or gene products.The second method,enhancer analysis, willbe described in Chapter 9. Functionally related genes encode products capableof 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 avariety 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 forrevealing an entirely new spectrum of genes. A suppressor mutation is a mutation that counteracts the effects of the original mutation such that thedouble mutant individual containingboth the original mutation and the suppressor mutation has a phenotype similar to that of the wild- type. Suppressors are isolated when a mutant strain is ‘reverted’ to restore the wild- type 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 arecombination event separatingthe original mutation from the sup- pressor 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 . 92 GENETIC TECHNIQUES FOR BIOLOGICAL RESEARCH analysis was a +l frameshift mapping to the 5' end of the rIIB 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 mutationmight 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 suppressormutation islikely to occur ata high rate sincewith rare exceptions the suppressormutation willbe unlinkedto the original mutation. Intergenic suppressors fall into two broad categories: information suppressors and what we will call function suppressors. Information suppression Information suppressors aremutations ingenes 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 suppressorwill 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 encodedinformation. 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 translationstart-site selection oneshould work with a mutation in the leader sequence of a gene that exhibits reduced protein productionbut not reduced transcript levels. Intergenic suppressors that increase translation shouldbe obtained, for example, in translation initiation factors or the small ribosomal subunit. Starting SUPPRESSION ANALYSIS 93 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 com- parable mutation in the leader sequence of another unrelated gene. This should sort the suppressormutations into the desiredclass of information suppressor from uninteresting mutations affecting some other aspect of the expression of the original mutant gene. Function suppression Functionsuppressors 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 regu- latory factors. Mutationalchanges can substitute another gene productfor the mutant gene product either by changing the specificity or abundance of that alter- nate protein. Or, if the mutant gene encodes a component of a switch regulatory pathway, suppression canoccur by the constitutive activation of adownstream 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 shouldbe confined to the coding region. Depend- ing on the type of function suppressor desired, one could start with a null mutation, such as a deletion or aninsertion, 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 alwaysmissense mutations. There are three mechanisms of function suppression: by-pass suppression, allele- specific suppression, and suppression by epistasis. BY-PASS SUPPRESSION A by-pass suppressorby-passes the need for the mutated gene product by providing the function of that gene product by alternate means. Two mechanismsof by-pass suppression of mutations in hypothetical GENI are shown in Figure 8.1. In Model 1, GENl and GEN2 carry out related but distinct processes that do not functionally overlap in the wild-type strain. The mutation of GENl 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 GENl product thereby by- passing the GENl mutation. The short arrow indicates that the by-pass may be less effective than the wild-type function. In Model 2, expressionof another geneis increased so that its product can substitute for the GENl 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 94 GENETIC TECHNIQUES FOR BIOLOGICAL RESEARCH Model 1 Wild-type Mutant Suppressed genl GEN2-sup R GENl GENZ genl