3.2: Pathways and Complementation Analysis

Pathway Analysis: The "One Gene, One Enzyme" Hypothesis

Once you've performed a screen and recovered mutants, you can begin to use the tools of genetics, cell biology and biochemistry to understand how the mutated genes are involved in the biological process you're interested in. How can you begin to use those mutants to understand the genetic basis of the biological process?

There are two key insights for the practical use of forward genetics. The first, as we've already seen, is epistasis -- the fact that multiple genes are likely involved in any process, and the effect of the alleles of one locus often depend on the alleles at other loci. For example, a flower may get its color from a compound produced by two enzymes in a biosynthetic pathway (see the figure below.) Mutating either of the genes will result in the pigment not being produced.

In This Simplified Biochemical Pathway, Two Enzymes Encoded by Two Different Genes Modify Chemical Compounds in Two Sequential Reactions to Produce a Purple Pigment. Loss of either of the enzymes disrupts the pathway and no pigment is produced. [Long description]

Synthesis of even a relatively simple molecule requires many steps, each with a different enzyme. Each enzyme works sequentially on a different intermediate in the pathway. For example, the genes involved in synthesizing the amino acid arginine were identified in Neurospora crassa using a screen where the progeny of a mutagenized spore could grow on minimal medium only when it was supplemented with arginine (Arg). (Such strains that can only grow with supplements like this are called auxotrophs.)Thus, the mutant strain must bear a mutation in the Arg biosynthetic pathway and was called an “arginineless” strain (arg-).

A Simplified Version of the Arg Biosynthetic Pathway, Showing Citrulline (Cit) and Ornithine (Orn) as Intermediates in Arg Metabolism. These chemical reactions depend on enzymes represented here as the products of three different genes.

Our screen has told us that genes A, B and C are necessary to synthesize arginine -- but what order are they in in the biosynthetic pathway? To answer this question, we can use a method called pathway analysis (or sometimes genetic analysis). For arginine (Arg), two biochemical intermediates are ornithine (Orn) and citrulline (Cit). Thus, mutation of any one of the enzymes in this pathway could turn Neurospora into an Arg auxotroph (arg-). Srb and Horowitz extended their analysis of Arg auxotrophs by testing the intermediates of amino acid biosynthesis for the ability to restore growth of the mutants (Figure 5.4.3).

  

Testing Different Arg Auxotrophs for Their Ability to Grow on Media Supplemented with Intermediates in the Arg Biosynthetic Pathway 

They found that only Arg could rescue all the Arg auxotrophs, while either Arg or Cit could rescue some (Table 5.4.1). Based on these results, they deduced the location of each mutation in the Arg biochemical pathway, (i.e., which gene was responsible for the metabolism of which intermediate).

Table 5.4.1 Ability of Auxotrophic Mutants of Each of the Three Enzymes of the Arg Biosynthetic Pathways to Grow on Minimal Medium (MM) Supplemented with Arg or Either of its Precursors, Orn and Cit. Gene names refer to the labels used in the figure above.

Mutants in:	MM + Orn	MM + Cit	MM + Arg
Gene A	Yes	Yes	Yes
Gene B	No	Yes	Yes
Gene C	No	No	Yes

 

Complementation Analysis

If epistasis is the first key to using forward genetics, the second is this: you may have isolated more mutants than there are genes in the pathway. Geneticists often observe two independently derived mutants with similar phenotypes, through a mutant screen or in natural populations. Is mutant phenotype is due to a loss of function in the same gene, or are they mutant in different genes that both cause the same phenotype (e.g., in the same pathway)? In other words, are they allelic mutations or non-allelic mutations, respectively? This question can be resolved using complementation tests, which bring together the two mutations into the same organism to assess the combined phenotype.

The easiest way to understand a complementation test is by example in the figure below. Let's assume, as above, that the purple flower pigment is due to a ciochemical pathway involving two genes. A diploid plant that lacks the function of gene A (genotype aa) would produce mutant white flowers that phenotypically looked just like the white flowers of a plant that lacked the function of gene B (genotype bb). Both A and B are enzymes in the same pathway that leads from a colourless compound #1, through colourless compound #2, to the purple pigment. Blocks at either step will result in a mutant white flower instead of the wild type purple flower.

If these two strains are crossed together the resulting progeny will all be AaBb. They will have both a wild type, functional A gene and B gene and will thus have a pigmented, purple flower, a wild type phenotype. This is an example of complementation. Together, each strain provides what the other is lacking (AaBb). The mutations are in different genes and are thus called non-allelic mutations.

Now, if we are presented with a third pure-breeding, independently derived, white-flower, mutant strain, we won’t initially know if it is mutant in gene A, gene B, or some other gene altogether. We can use complementation testing to determine which gene is mutated. To perform a complementation test, two homozygous individuals with similar mutant phenotypes are crossed.

If the F₁ progeny all have the same mutant phenotype (Case 1), then we infer that the same gene is mutated in each parent. These mutations would then be called allelic mutations — mutant in the same gene locus. These two mutations FAIL to COMPLEMENT one another (still mutant). These could either be exactly the same mutant alleles (same base pair changes), or different mutations (different base pair changes, but in the same gene — allelic).

Conversely, if the F₁ progeny all appear to be wild type (Case 2), then each of the parents most likely carries a mutation in a different gene. These mutations would then be called non-allelic mutations — mutant in a different gene locus. These mutations DO COMPLEMENT one another.

Figure 5.2.2 Observation: In a Typical Complementation Test, the Genotypes of Two Parents Are Unknown. (Although, they must be pure breeding, homozygous mutants.) If the F1 progeny all have a mutant phenotype (Case 1), there is no complementation. If the F1 progeny are all wild-type, the mutations have successfully complemented each other. [Long description]

Figure 5.2.3 Interpretation: The Pure Breeding, Homozygous Mutant Parents had Unknown Genotypes Before the Complementation Test. It could be assumed that they had either mutations in the same genes (Case 1) or in different genes (Case 2). In Case 1, all of the progeny would have the mutant phenotype, because they would all have the same, homozygous genotype as the parents. In Case 2, each parent has a mutation in a different gene, therefore none of the F1 progeny would be homozygous mutant at either locus. Note that the genotype in Case 1 could be written as either aa or aaBB. [Long description]

Note: For mutations to be used in complementation tests they are (1) usually true-breeding (homozygous at the mutant locus), and (2) must be recessive mutations. Dominant and semi-dominant mutations CANNOT be used in complementation tests, since these mutations won’t show complementation effects of two non-allelic genes.

Extending the Complementation Test

So, with the third mutant strain above, we could assign it to be allelic with either gene A or gene B, or some other locus, should it complement both gene A and gene B mutations. If they came from different natural populations or from independently mutagenized individuals, we could have a fourth, fifth, sixth, etc. white flower strain, then we could begin to organize the allelic mutations into groups, which are called complementation groups. These are groups of mutations that FAIL TO COMPLEMENT one another (a group of NON-complementing mutations) and are assumed to have mutations in the SAME gene; hence they are grouped as complementation group. A group can consist of as few as one mutation and as many as all the mutants under study. Each group represents a set of mutations in the same gene (allelic). The number of complementation groups represents the number of genes that are represented in the total collection of mutations.

It all depends on how many mutations you have in a gene. For example, the white gene in Drosophila has >300 different mutations within the white gene described in the literature. If you were to obtain and cross all these mutations to themselves, you would find they all belonged to the same complementation group or same white gene. Each complementation group represents a gene.

If, however, you obtained a different mutation, vestigial for example, which affects wing growth, and crossed it to a white eye-colour mutation, the double heterozygote would result in red eyes and normal wings (wild type for both characters) so the two would complement and represent two different complementation groups: (1) white, (2) vestigial. The same would be true for the other eye-colour mutations mentioned elsewhere in this text. For example, if you crossed a scarlet eye-colour mutant to a white eye-colour mutant, the double heterozygote would have wild type red eyes. Each mutant has the wild type allele of the other. Again, remember that all the other genes in the diploid genome are assumed to be wild type.

To drive home the concept of complementation groups, we will look at two hypothetical examples.

Example 1: Multiple Mutant Complementation Test

The first example shows the results of a series of crosses as a complementation test table with six mutants labelled a to f. The mutants fall into three complementation groups in total: (1) a (2) b, c, f, and (3) d, e. Notice that a complementation group can consist of only one mutant, or more than one.

Complementation Test Table Showing Which Flower Mutant Strains Complement Each Other and Vice Versa. “w” stands for the white flowers, which is mutant (no complementation) and “p” stands for purple which represents wild type (complementation). Blanks are for crosses not completed. [Long description]

Example 2: Double Hit Strain

The second example is similar, but has a twist. It has five mutants labelled 1-5, with 1-4 being mutations in only a single gene each, while mutant #5 has mutations in two different genes, and thus is unable to complement the mutations in two, different genes. A double-hit strain like strain #5 is normally a very rare event, but is included here to make a point. A double-hit strain may appear to belong in two different groups. In this case, mutants #3 and #4 complement (different genes) but #5 fails to complement both #3 and #4, indicating it has mutations in both the mutant genes in #3 (gene B) and #4 (gene C).

Complementation Test Table with Pink as Mutant and Green as Wild Type. (Black is for crosses not completed.) Note: mutant #5 has two mutations. [Long description]

Chromosomes of the Organisms Used in Complementation Tests to Decide if Genes are Allelic or Non-Allelic. [Long description]