Fundamentals of Molecular Biology. Part 2: Molecular Genetic Mechanisms - A. N. Ogurtsov 2011

Alleles and Mutations
Analysis of Double Mutants

Based on a thorough Analysis of the mutant phenotype associated with a given cellular process, researchers can determine the order in which genes—and consequently, the Proteins they express—function. In principle, such a sequence can be determined in Two Types of processes:

1) in metabolic pathways, where an initial substance is converted into a product of biochemical synthesis through several intermediate steps,

2) in signaling systems that regulate cellular processes and utilize informational signals rather than chemical intermediates.

8.6.1. Sequence of Metabolic Transformations.

Let us consider Tryptophan Biosynthesis in Bacteria as an example. Each of the Enzymes required for tryptophan synthesis catalyzes the chemical conversion of an intermediate compound, passing the results of its work along to the next enzyme. In E. coli, the genes encoding these enzymes are organized into the trp Operon (see Figure 12).

The order of action of the various genes encoding these enzymes, and consequently The sequence of biochemical reactions in this pathway, was initially determined based on the intermediate compounds that accumulated in each mutant.

However, in the case of complex synthetic pathways, phenotypic analysis of mutants with a defect in a single step of a metabolic chain can yield contradictory and ambiguous results, making it impossible to determine the order of reactions in the pathway. In such cases, determining the sequence of steps requires The Use of double Mutations that create defects in two steps of the pathway (Figure 80).

Suppose, for example, that a mutation in enzymes A and B leads to the accumulation of certain intermediate compounds 1 and 2 in The Cell, respectively. Then, if a double mutation results in the accumulation of intermediate compound 1 in the system, this indicates that enzymes A and B belong to the same metabolic pathway, with the action of enzyme A preceding that of enzyme B.

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Figure 80 - Analysis of the metabolic sequence

8.6.2. Sequence of Events in a Signaling Pathway. The expression of Many eukaryotic genes is regulated by cellular signaling systems initiated by extracellular Hormones, growth factors, and other signals. Such signaling pathways can involve numerous components, and analysis using double mutations can clarify the Functions and interactions of these components.

A necessary condition in this case is that the two mutations must have opposite effects on the operation of the same signaling pathway (Figure 81).

Generally speaking, one mutation should repress the expression of the Gene responsible for responding to an external signal, even in the presence of that signal. Conversely, another mutation should stimulate the expression of this gene even in the absence of an external signal (i.e., ensuring constitutive expression). As shown in Figure 81, if two simple regulatory mechanisms satisfy these requirements (a mutation in protein A represses, while a mutation in protein B stimulates the expression of the reporter gene), the double mutant phenotype can determine the order in which the proteins act and whether they function as negative or positive regulators.

In the first case (Figure 81, top), the double mutation in proteins A and B represses the expression of the indicator (reporter) gene. This unambiguously indicates that protein A positively regulates (activates) the expression of the reporter gene while, at the same time, protein A itself is negatively regulated (inhibited) by protein B.

Figure 81 - Analysis of the signaling sequence

In the second case (Figure 81, bottom), the double mutation in proteins A and B causes constant (constitutive) expression of the reporter gene. This clearly demonstrates that protein B negatively regulates the expression of the reporter gene, and in turn, protein B is negatively regulated by protein A.

Two other Genetic Methods can provide information on how proteins participating in the same cellular process interact with one another. Both methods rely on double mutations, where the Phenotypic effect of the first mutation is modified by the presence of the second mutation.

8.6.3. Suppressor Mutations. The first technique is based on genetic suppression (Figure 82).

Figure 82 - Suppressor and lethal double mutations

Suppose a point mutation leads to structural changes in one protein (A) that result in the loss of its ability to associate with another protein (B) involved in the same cellular process. Similarly, mutations in protein B lead to structural alterations that inhibit its ability to interact with protein A.

Let us further assume that the normal functioning of proteins A and B depends on their interaction. Theoretically, specific structural changes in protein A caused by the first mutation can be compensated for (suppressed) by alterations in protein B caused by the second mutation, thereby restoring the ability of proteins A and B to interact.

In those rare cases where such suppressor mutations actually occur, strains carrying both mutant alleles will appear normal, whereas strains carrying only one of the mutant alleles will exhibit a mutant phenotype (Figure 82(a)).

Thus, if single mutations in proteins produce a mutant phenotype while double mutations yield the original wild-type phenotype, there is reason to suspect that the functions of these proteins depend on their mutual interaction. Such genetic suppression has been observed in Yeast strains carrying a mutant Actin allele (act1-1) and another mutation (sac6) in a different gene, suggesting that the proteins encoded by these genes interact within the living cell. Indeed, subsequent biochemical studies demonstrated that the two proteins, Act1 and Sac6, interact during The formation of actin structures in the cell.

8.6.4. Synthetic Lethal Mutations. Another phenomenon, known as synthetic lethality, produces a phenotypic effect opposite to that of suppression. In this case, the deleterious effect of the first mutation is significantly enhanced (rather than suppressed) by a second mutation in the same gene (or in a closely related gene), resulting in a cumulative effect.

One such synthetic lethal mutation is illustrated in Figure 82(6). Here, a heterodimeric protein is partially (rather than completely) inactivated by mutations in either of the non-identical subunits. However, when a double mutation affects both genes encoding these subunits, the interaction between the subunits is reduced

to such an extent that protein function ceases entirely, resulting in a distinct phenotypic manifestation of the defect.

Synthetic lethal mutations with a cumulative effect can also reveal auxiliary genes that encode proteins functioning in backup (redundant) pathways responsible for synthesizing essential cellular components. As shown in Figure 82(b), if either of the two pathways is inactivated by a mutation, the remaining pathway can single-handedly supply the cell with the necessary product. However, if both pathways are inactivated simultaneously, the essential product will not be synthesized, rendering such a double mutant unviable.

Conclusions

Diploid organisms possess two copies (alleles) of each gene, whereas haploid organisms contain only a single copy.

Recessive mutations result in a loss of function, but they are not expressed if a normal allele of the gene is present. For a recessive mutation to manifest in the phenotype, both alleles must be recessive.

Dominant mutations are expressed in the phenotype even in the presence of normal alleles. The phenotype corresponding to a dominant mutation typically reflects The Emergence of a new function, although in some cases it also results from a loss of function.

During Meiosis, a diploid cell undergoes one round of METABOLISM/36.html">DNA Replication followed by two cell divisions, yielding four haploid Cells in which maternal and paternal alleles are distributed randomly (Figure 17.2).

Dominant and recessive mutations exhibit characteristic distribution patterns upon crossing (Figure 17.3).

In haploid yeast, Temperature-sensitive mutations are practically employed to identify and investigate genes essential for survival.

The number of functionally related genes involved in a given cellular process can be determined through complementation analysis.

The operational order of genes within a biosynthetic or signaling pathway can be deduced by analyzing the phenotype of double mutants carrying defects in two steps of the cellular process under consideration.

Functionally important Protein-Structure/156.html">Protein Interactions can be uncovered by analyzing the phenotypic expression of allele-specific suppressor mutations or synthetic lethal mutations.

Review Questions

1. What is an allele?

2. What substance is referred to as a mutagen?

3. WHAT IS A genotype, and how does it differ from a phenotype?

4. What is a phenotype, and how does it differ from a genotype?

5. Which genotype is termed the wild-type genotype?

6. What are haploid organisms, and how do they differ from diploid organisms?

7. What are diploid organisms, and how do they differ from haploid organisms?

8. What are heterozygous organisms, and how do they differ from homozygous organisms?

9. What are homozygous organisms, and how do they differ from heterozygous organisms?

10. What is a dominant mutant allele, and how does it differ from a recessive allele?

11. What is a recessive mutant allele, and how does it differ from a dominant allele?

12. What is the proportion of individuals exhibiting mutant traits in the second generation when crossing a homozygous mutant strain with a dominant mutant allele to a homozygous wild-type strain?

13. What is the proportion of individuals exhibiting mutant traits in the second generation when crossing a homozygous mutant strain with a recessive mutant allele to a homozygous wild-type strain?

14. In what form (haploid or diploid?) can yeast cells of Saccharomyces cerevisiae exist?

15. What are "conditional" mutations? Provide Examples.

16. What temperature is referred to as the restrictive (non-permissive) temperature?

17. What temperature is referred to as the permissive temperature?

18. What are cdc mutations?

19. What are complementary mutations?

20. What is The phenomenon of genetic complementation?

21. In which processes is it possible to determine the order of gene function using the double-mutant method?

22. What phenomenon is known as genetic suppression?

23. What are suppressor mutations?

24. What phenomenon is referred to as synthetic lethality?

25. What are synthetic lethal mutations?