Part F: A Closer Look at....

Adenine-Requiring Mutants

To understand the mutants that require adenine, we need to understand how normal cells produce their own adenine. Yeast cells don't actually make adenine as a separate compound. Instead, they make it as adenosine monophosphate -- adenine combined with the sugar ribose and phosphate -- abbreviated AMP (Jones & Fink 1982). AMP is usually made from a sugar-phosphate compound called phosphoribosylpyrophosphate (PRPP) through a series of 12 sequential enzymatic steps, in which the adenine molecule is built up attached to the sugar-phosphate compound. The steps in this biosynthetic pathway are illustrated in Figure 1. However, if adenine is in the medium, the cell can convert it to AMP by a single step, using a single enzyme to add the adenine molecule to the sugar-phosphate molecule. There are many enzymes required specifically for the biosynthesis of AMP, and there is a different gene to code for each enzyme. Therefore, mutations in any one of several different genes will cause an adenine requirement. Figure 1 illustrates the AMP pathway with each of the genes and enzymes involved.

The information in Figure 1 is very useful. It helps us understand many thing about the behavior of these mutants. Let's see how the information came to be known in the first place. To begin with, this was a genetics problem, not a biochemical one. Scientists knew very little about the biosynthesis of adenine when they discoveried these mutants, so understanding the biochemistry resulted from understanding the genetics. When a mutation occurs in a gene, the mutant phenotype results from the loss of some enzyme function. With this information, biochemists can identify the enzymes and the reactions they catalyze. With enough mutants, they can piece together the entire pathway.

The first step is to isolate a large number of adenine-requireing mutants in both mating types. The next step is to determine which mutations affect the same genes. When enough mutants are studied, all of the genes involved should be represented.

The genetic test for determining whether two mutations are in the same gene or in different genes is simple. Haploid strains of opposite mating type containing the two mutations are crossed to form a diploid. If the mutations are in the same gene, then neither parent strain will be able to provide a working copy of that gene, so the diploid will have the mutant phenotype. It will be homozygous for the mutant allele. On the other hand, if the mutations are in different genes then one parent will contribute a functional copy of one gene and the other parent will contribute a functional copy of the other one. The diploid will be heterozygous for each of the genes. In other words, if the mutations are in different genes then the functional allele in each parent will complement the defect in the other. This is called a complementation test.

As expected, when a large number of adenine-requiring mutant strains are tested for complementation in all possible combinations, two at a time, they define a number of different genes. All of the mutant strains that do not complement each other are placed in a separate complementation group. Through this process, the adenine-requiring mutants have been divided into twelve complementation groups. All the mutants in two of these groups form red colonies, while those in the other groups form the normal cream-colored colonies. Each group of mutants defines an enzyme. The enzymes act sequentially in the biosynthetic pathway that converts precursor compounds, step by step, into the compound adenosine monophosphate (AMP). Mutants that are defective in any of the steps in this pathway can grow when provided with adenine, which they can convert into AMP in a single step.

Red Adenine-requiring Mutants:

One intermediate compound in this pathway is of special interest because it is responsible for the red color of some of the mutants. For obvious reasons, we shall refer to this compound by the abbreviation AIR, rather than its official chemical name (P-ribosylaminoimidazole, which used to be called aminoimidazoleribotide). The mutants that turn red are those blocked in either of the two steps just beyond AIR in the pathway. A mutation in either step causes AIR to accumulate in the cell. AIR itself is not red, but cells that are growing aerobically oxidize it to a red pigment.

The mutant forms of these "red adenine" genes are called ade1 and ade2. These red mutants are most obviously useful because they are visible, but their utility goes far beyond this, because they provide a set of phenotypic characteristics that make it possible to demonstrate a variety of genetic and physiological principles with a few simple methods. For practical reasons we start with a set of representative mutant (ade1 ADE2, ADE1 ade2, ade1 ade2) and non-mutant (ADE1 ADE2) strains of each of the mating types. In principle, students could isolate these themselves, but there are advantages in starting with mutants we have already characterized.

Several conditions, in addition to an ade1 or ade2 mutation, are necessary for the formation of the red pigment. The cell must form AIR and oxidize it to the red pigment. Anything, genetic or physiological, that impairs one of these steps results in a "white" phenotype. The "white" phenotype includes a group of mutants that range from the normal cream-colored to pure white. Genetically, any mutation that blocks the formation of AIR, or any mutation that impairs oxidative metabolism, will block formation of the red pigment. Consequently, this provides a simple way to isolate and study a large number of additional "white" adenine-requiring mutants.

"White" Adenine-requiring Mutants:

We can use this approach to isolate mutations in several genes that control earlier steps in the adenine pathway (ade4 through ade8). These mutants appear as cream-colored spots in old red colonies. The spots are clones of mutant cells, the progeny of individual mutant cells that arise during the growth of the colony. They are particularly prominent in old colonies because the cream-colored mutants grow better than red ones. We can characterize these new mutants by complementation tests.

In addition to cream-colored mutants, you may also find pure white ones. These are especially common in cultures of red mutants that have been exposed to ultraviolet radiation. These mutants also form smaller colonies and are, therefore, called "petite colony" mutants, or just "petites" (Dujon 1981). Rather than being blocked in the adenine pathway like the cream-colored mutants, they fail to make the red pigment because they lack oxidative metabolism -- respiration. Most of them have some kind of defect in their mitochondria. Since mitochondria are complicated organelles necessary for respiration, there are many mutations that produce this phenotype, so petite mutants are very common. These mutations are not lethal because the cells can grow (more slowly) by fermentation, deriving energy from just the initial steps of sugar metabolism. The initial steps do not involve respiration. Normal yeast grow this way when they are anaerobic, converting six-carbon sugars -- hexoses -- to three-carbon products, such as glycerol. Consequently, these mutants require a six-carbon sugar, such as glucose or fructose, as their carbon source, and cannot use the smaller, non-fermentable carbon sources such as acetate, ethanol, or glycerol. In contrast, normal yeast cells can use acetate, ethanol, glycerol in place of glucose.

Physiology of Red Pigment Formation:

Any environmental condition that blocks the formation of AIR or impairs respiration will prevent formation of the red pigment, including, obviously, anaerobic growth conditions. Another, particularly instructive way to prevent pigment formation is to use feedback inhibition, an important physiological control mechanism that cells use to conserve energy. If the environment -- growth medium -- contains an excess of adenine, then there is no need for the cell to make AMP the hard way, from PRPP. Consequently, the cell responds to high levels of adenine in the medium as a signal to stop producing AMP as well as all the intermediates in the pathway, including AIR, from PRPP; it simply makes it directly from adenine. Therefore, a large excess of adenine added to the medium inhibits the formation of the pigment. Since there is a preferential selection for white mutants in red cultures, we suggest storing working stocks of adenine mutants on high-adenine medium to prevent accumulation of unwanted mutants.

Another environmental condition that inhibits the formation of the red pigment is anaerobic growth. When red mutants are grown in the absence of oxygen, they do not turn pink or red. But when the anaerobically grown colonies are exposed to air, they turn red within a hour or two.

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Last updated Saturday August 14 1999