Genetics

BIOC/MCB 568 -- Fall 2009
John W. Little--University of Arizona

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Dominance test

The concept of dominance was discussed in class. This is a genetic test that can give considerable insight into the mechanisms by which mutations change the function of a protein.

To do a dominance test, one uses genetic methods to make a cell containing one copy of the wild-type allele of a gene, and one copy of a mutant allele, symbolized  +/m. One then examines the behavior or phenotype of this cell and compares it with the phenotype of the wild-type and mutant cells.

If the +/m cell has the same phenotype as the wild-type, the mutation is said to be recessive to wild-type. Note that almost all mutations that abolish function ("knockout mutations") are recessive to wild-type. Hence, a dominance test can help you identify whether a mutation is a knockout mutation.

If the +/m cell has the same phenotype as the mutant, the mutation is said to be dominant to wild-type. This behavior often means that the mutant protein can continue to function under a condition in which the wild-type protein cannot.  Geneticists are usually delighted to find dominant mutants.

An example that shows how this test relates to protein function is provided by Lac repressor. This protein represses the lac operon in the absence of an inducer (a commonly used inducer is IPTG); in the presence of inducer, inducer binds to Lac repressor and weakens its affinity for DNA. Accordingly, a knockout mutation in lacI (the gene for Lac repressor) would be constitutive (lac operon always on), and would be recessive. A mutation abolishing binding of IPTG to the repressor would be non-inducible (lac operon always off). This mutation would be dominant; it would continue to act as a represor in the presence of IPTG, even though the WT repressor could not.

Epistasis test 

Epistasis tests are used to order events in a regulatory pathway.

To do an epistasis test, one constructs a strain containing mutations in two different genes. Individually, these mutations must confer different phenotypes on the cell for the test to be interpretable. One then determines the phenotype of the double mutant and compares it to that of the single mutant. If the phenotype of the double mutant is like that of one of the single mutants, we say that the latter mutation is epistatic to the other one.

Example: Suppose mutation A makes a gene regulatory circuit non-inducible, while mutation B makes it constitutive. If the A - B double mutant is constitutive, B is epistatic to A. In this case, we can infer that B lies downstream in the regulatory pathway from A.

Note that the interpretation of an epistasis test is different from this in a metabolic pathway. In this case, the epistatic gene lies upstream of the other one.

Note also that, if B is epistatic to A, then one can't tell from the phenotype whether A is mutant or wild-type.

Nomenclature

In bacterial genetics, the name of the gene is given in italics, using a code with three lower-case letters followed (usually) by an upper-case letter, such as recA or lexA. The name of the protein is given as RecA or LexA. In some cases the protein (for historical reasons) doesn't follow this convention; e.g., lacZ codes for b-galactosidase (often called beta-gal). The wild-type allele of the gene is given by a superscript "+", as recA⁺, and mutant alleles are given with a number, such as recA142.

The convention for budding yeast (Saccharomyces cerevisiae) is different. Here the wild-type allele is given by capital letters, such as GAL4, the mutant allele with small letters, such as gal4-1. In this case the protein is termed Gal4p.

Genotype vs phenotype: In bacterial genetics, the genotype is given as lac⁺; the phenotype is given by "Lac⁺ ". The latter means "able to grow on lactose". "Lac^- " means "not able to grow on lactose", without specifying the genetic defect.

Screens and selections

Screens and selections are used by geneticists to isolate mutants with altered phenotypes. They differ in that, in a selection, only cells with the desired phenotype can grow.

In a screen, all cells can grow (and, for bacteria or yeast, form a colony on an agar plate), but the desired mutants have a different phenotype. For instance, a mutant that can make beta-galactosidase might form a blue colony (on a plate with X-gal), while one that can't make beta-gal will form a white colony. The most useful screens are those in which the desired phenotype (blue colony in this example) is easy to recognize. As a practical matter, it is much easier for the eye to pick out one blue colony in a large number of white ones than to find a white colony in a sea of blue ones.

In a selection, only the cells with the desired phenotype are able to grow. For instance, cells that can make beta-galactosidase (i.e., Lac⁺, see above for nomenclature) would be able to utilize lactose (a disaccharide) as their only source of carbon, since the enzyme breaks lactose down to glucose + galactose; cells not making beta-gal cannot grow under this condition. Hence the selection would be to grow the cells on a defined growth medium (that is, one in which specific compounds neede for growth are provided, such as phosphate, ammonium chloride, magnesium sulfate, a bit of calcium, and a source of carbon) containing lactose as sole carbon source. Only Lac⁺ cells can grow.

Conditional mutants

Geneticists frequently want to analyze the effects of mutations on cell behavior and function. A common way to do this is to inactivate the gene product completely, but this can't be done when the gene product is an essential function, because there is no way to propagate the cells. To get around this limitation, we use "conditional mutants", that is, ones that can grow under one condition but not under another. There are two common approaches to this, though the second is mostly used in work with viruses. The first and most common is to use temperature-sensitive (ts) mutants. These affect the gene product so that it can function at the "permissive" temperature, but not at the "restrictive" temperature. For E. coli, these temperatures might be 25º C and 42º C, respectively, and somewhat lower for budding yeast. Then the cells can be propagated at the permissive temperature, and the experiment to test the effect of losing function can be done at the restrictive temperature. Generally, ts mutants affect the protein so that it doesn't function well at the high temperature. Cold-sensitive (cs) mutants also exist but are not so widely used. The second approach is to use nonsense mutants, in which the normal codon in a gene is replaced with a chain-terminating codon (usually UAG, the amber codon, less commonly UAA, the ochre codon). This approach is commonly used with viruses (such as E. coli phages lambda and T4), which are then allowed to infect either a host carrying a "nonsense suppressor"--a special tRNA that inserts an amino acid at the nonsense codon--or a host lacking a nonsense suppressor. These are the permissive and restrictive conditions, respectively. Again, the mutant can be propagated in the host with the suppressor, then analyzed in the restrictive host.

Genetic mapping, and linkage

This process is used to order genes on the chromosome. A genetic map is determined by genetic crosses, but gives the same order of genes as a physical map made by DNA sequencing. The basic idea is that organisms with different genotypes are crossed. In diploid organisms, this occurs when the two homologs carry different arrangements of genetic markers. Recombination occurs during meiosis, giving crossovers between the two homologs. The closer two genes (or genetic markers) are, the more infrequently recombination will separate them. Hence, a genetic map is a measure of the frequency of recombination between two markers, and a lot of information must be combined to generate a detailed map. See a genetics text for more detail. The idea of "linkage" is that two genes are closely linked if they lie close together on the genetic map; they are termed "unlinked" if recombination between them is very frequent or, if (as in eukaryotes) they lie on different chromosomes.

Complementation test

The purpose of this test is to determine whether two mutations that confer the same phenotype lie in the same gene or in two different genes. It only works for recessive mutations (can you see why?). Again it's easiest to visualize with a diploid organism. If one homolog carries one of the mutations, and the other homolog the other mutation, then there are two possibilities: Either the mutations are in the same gene, and there is not a good copy of that gene, so the phenotype is still mutant; or the mutations are in two different genes, there are good copies of each of the genes, and the phenotype is wild-type. In this case the two mutations are said to "complement" each other.

As an example, consider two mutations in yeast that individually make the cells unable to make histidine (and hence are phenotypically His^-). Two his genes are HIS3 and HIS4. If there are two his mutations, and both are his3 alleles, then the diploid cell will not have a good copy of HIS3, and will phenotypically be His^-. If one his mutation is a his3 allele, and one a his4 allele, then in the diploid there will be one good copy each of HIS3 and HIS4, and the cell will be able to make histidine, so it will be phenotypically His⁺.