The two fundamental aspects of genetics are gene action and
heredity. The genes, of course, are the elemental units of genetic information;
their actions, or expression, result in all the physiological processes
that make up an organism. Although the genes can also be considered as
the elemental units of heredity, this is not precisely true. In a sense,
the individual base pairs of the DNA are the elemental units of genetic
transmission, and by sophisticated techniques one can demonstrate the inheritance
of portions of genes. But for the basic concepts of heredity that everyone
should understand, the gene is the fundamental element. It is the identity
of the unit of function and a unit of heredity that is important.
Genetics is unique among the sciences because in no other case is the scientific method so integral to the way science is taught. Usually, we teach only the results or findings of science, because we cannot teach how those results were achieved. In other scientific disciplines the experimental methods are so complex, the data are so extensive, and reducing and analyzing them is so tedious, that teaching them at the junior high and secondary school levels is neither practical nor desirable. Genetics is different. The study of genetics is the study of how scientists -- geneticists -- know what they know. In teaching genetics, we can really teach scientific method. In genetics, the results and the way the results are achieved need not -- indeed, cannot -- be separated.
The key is to realize that basic genetic concepts are operational definitions, meaning that genetic facts are defined by the operations we use to observe and demonstrate them. The experiments described in this book are designed to allow students to observe these operations for themselves and thereby to develop genetic concepts first-hand.
So, how do we define "the gene" operationally? In fact, we don't. We define specific, individual genes, operationally, by their genotypes (heredity) and phenotypic traits (physiological expression). The genotype is the information coded in the DNA, while phenotype refers to the describable, heritable characteristics of an organism, such as the color of some part of it. We could use virtually any describable characteristic, but some are more useful than others. We define the gene or genes that determine a phenotypic trait through controlled experiments.
The mainstay of genetics is the controlled experiment, where we change only one variable at a time, so that we can identify the change in the outcome with a change in a variable. But in genetics one can often conduct several controlled experiments simultaneously, in parallel, which is much more fun than doing them one at a time. We use experimental organisms that differ by a small number of well-defined, single-gene traits. By using mutants, we generate strains that have one or the other of two alternative forms, or alleles of a gene. For example, we will use two mutant genes (ade1 and ade2) that result in the cells turning red under some conditions (Roman 1956). The alternative alleles of these genes are the non-mutant forms (ADE1 and ADE2), which result in cream-colored cells. The appearance of the red color defines the presence of the mutant allele in a particular strain, and the absence of red color usually defines the absence of the mutant allele, and therefore, the presence of the non-mutant allele.
When we have succeeded, through controlled experiments, in identifying a unit of heredity with a phenotype, we have operationally defined the gene for that trait. The genotype seems more abstract, and therefore more vague, than the phenotype. Experimentally, observing the genotype directly has always been more difficult. However, since molecular biologists can now isolate genes and determine their exact nucleotide sequences, the genotype has become more concrete. These methods are too complicated to allow students to observe the genotype first hand, so at this point we are forced to revert to teaching the results, or using models and simulations. From experiments on inheritance, in which we observe patterns of transmission of traits from one generation to another, we can develop operational definitions of organizational units of genetic information, which we call genes and chromosomes. These, too, can then be identified with physical structures.
We mark individual genes of interest with mutations to distinguish
them from the thousands of genes that make up the genome of an organism.
Although hundreds of genes have been defined by mutations in yeast, we
need only a few of them to teach the fundamentals of genetics, so we have
chosen some that are particularly instructive and easy to work with.
(or
just ) -- determine the two opposite mating
types (Herskowitz & Oshima 1981). They are defined, operationally,
by their ability to mate with each other. Therefore, we can determine the
mating type of a haploid strain by a mating-type test -- determining if
it can mate with one or the other of two strains whose mating types we
know. Strains used to define particular alleles of genes in this manner
are called "tester" strains.
When haploids mate they produce a diploid that is heterozygous
for mating type. This a/ diploid cannot mate
with either mating type, but it can sporulate, which is something that
haploids cannot do. When a diploid does sporulate it produces two spores
of each mating type, which demonstrates that a and
are indeed determined by two alternative alleles of a single genetic locus.
The mating-type alleles are unique in that neither is simply
a mutant form of the other. Each is a functional gene, but normally each
haploid cell expresses only one of the two. In this way they behave as
alleles. The phenotype of the heterozygous diploid is different from either
of the two haploids. From extensive genetic, physiological, and biochemical
studies we know that the mating type alleles are control genes that determine
whether or not other genes are expressed. Another advantage of yeast is
the ease with which these regulatory genes can be studied.
Many of these small molecules are essential for growth, but they need not be made by the cell itself. If they are present in the growth medium the cell will take them in and use them instead of making its own. If a mutation occurs in one of the genes that codes for an enzyme involved in the biosynthesis of a particular small molecule, then that enzyme may be inactive, and the cell will be unable to make that product. That mutant cell, and all of its progeny, will be able to grow only on medium that contains the required product as a nutrient. A mutant strain that requires a new nutrient for growth is called an auxotroph; the strain that does not require that nutrient is a prototroph. In practice, we call a particular strain auxotrophic or prototrophic for a particular compound, as a shorthand way of saying it has, or doesn't have, a particular growth requirement. Once an auxotrophic mutant has been isolated and characterized, the presence or absence of the mutant gene can be defined by whether or not the cell requires that nutrient in the growth medium. However, a requirement for a particular nutrient does not necessarily define a particular gene because many genes are involved in the biosynthesis of each compound. Clearly, mutations in any of the genes in the same pathway will result in the same requirement. Using additional tests to distinguish mutations in one gene from another is necessary in this case. Furthermore, some genes are involved in the biosynthesis of many different compounds, while others are specific for only one. The latter type are obviously easiest to work with because each mutation will result in a single new growth requirement.
In our experiments we will use two types of auxotrophic mutants: mutants that require adenine and mutants that require tryptophan.
Some alleles are neither dominant nor recessive. If the phenotype
of a heterozygous diploid -- one that carries different alleles of a gene
--is intermediate between the respective homozygous phenotypes, we call
them codominant. In some cases, however, such as with mating type, the
phenotype of the heterozygous diploid is entirely different from either
of the homozygous phenotypes. The concept of dominance does not apply at
all here. We call this type of interaction complementary, to denote that
each gene is producing a different functional product, and the phenotype
results from the interaction of the two products. This is analogous to
the case we have already discussed, in which a strain that has a mutation
in one of the red adenine genes (for example, ADE1 ade2) was crossed
with one having a mutation in the other red adenine gene (ade1 ADE2).
The diploid has the normal cream-colored, adenine-independent phenotype
because the two mutants have complementary genotypes. In this case, the
complementation is between different genes. Many forms of complementary
gene expression occur in cells. The term complementation denotes a type
of interaction that is operationally defined, rather than a particular
molecular mechanism.
,
but they are usually abbreviated to simply a and .
In printing or with word processors it is conventional to put gene symbols
in italics to distinguish them from phenotype descriptions. In typing,
gene symbols are underlined.
One of the most fundamental properties of living organisms is their ability to reproduce. Since the phenotype is determined by the genotype, reproduction of the organism requires reproduction of all the genes that determine the phenotype. This total set of genes is called the genome. The genome of each cell is duplicated prior to each cell division and the two copies are then divided between the progeny cells. This happens both in mitotic and meiotic cell divisions, but in the two cases the outcome is different because the function of the cell division is different.
Whereas mitosis produces nearly identical progeny cells, meiosis is guaranteed to yield progeny that are different from the parent cell and usually different from each other. While mitosis is the mode of asexual division in both haploid and diploid cells, meiosis occurs only in diploids and yields haploid progeny. Both processes begin with a round of chromosome replication, in which the DNA is replicated and distributed between two replicate copies of each of the chromosomes. Since these replicate copies do not separate from each other immediately, it is useful to give them a name. They are called chromatids, or simply strands. They are separate structures, but remain joined together at one short region, called the centromere. (Note that each chromatid -- or strand -- consists of a DNA double helix, which, in turn, consists of two polynucleotide strands. In other words, what we call a strand at the cytological level consists of two strands at the molecular level.) So, before replication, in the single-strand stage, each chromosome consists of a single chromatid. After replication, but before the chromatids disjoin (separate at the centromere), they are at the double-strand (two chromatid) stage. Both mitosis and meiosis are extremely complicated in their detailed events, but simple in terms of the information flow -- the way various genes are transmitted from parent to progeny. We will concentrate on this informational level of the process.
Figure 4: Mitosis Without Crossing Over
Information Flow at Mitosis:
The usual mitotic division guarantees that each progeny cell receives a set of chromosomes -- genome -- that is identical to the parent cell (not counting occasional mutations, which normally occur at the time of DNA replication). Figure 4 shows how a diploid heterozygous for ADE2/ade2 produces two identical heterozygous progeny. At the onset of cell division each cell becomes polarized by a structure called the spindle. The chromosomes, at the two-strand stage, congregate at the center of this structure, separate into two independent single-stranded chromatids, and move along the fibers of the spindle to its poles, becoming organized within the cell so that when it divides the set of chromosomes at one pole ends up in one cell and those at the other pole end up in the other cell. This separation of the chromatids, called disjunction, occurs independently for each chromosome, so that each cell receives one copy of each of the chromosomes of its parent. If the parent is haploid then each progeny cell is haploid; if the parent is diploid, the progeny are diploid.
Information Flow at Meiosis:
Meiotic division (Figure 5) is a little more complicated. Since the net effect is to go from a diploid state to a haploid state, there are two divisions. In the first meiotic division, the centromeres do not disjoin. Instead, they pair: the two copies of each chromosome in the diploid nucleus come together, side-by-side, with their centromeres together.
Figure 5: Meiosis Without Crossing Over
The two copies are not necessarily identical, because they may be heterozygous at any number of genes; instead, they are said to be homologous and are sometimes referred to as homologs. Since each of the paired homologous chromosomes has two-strands, we call this the four-strand stage. The paired chromosomes separate and move along the spindle with each double-stranded member going to an opposite pole. This step may be described as centromere separation. The second meiotic division is analogous to mitosis in a haploid cell: the chromosomes, still in the two-strand stage, become organized at the center of a new spindle, and then the centromeres disjoin and the chromatids migrate to the spindle poles, yielding a total of four haploid sets of chromosomes at the single-strand stage. These become incorporated into the four nuclei of the meiotic products, in the case of yeast, the ascospores. Figure 6 shows how a diploid heterozygous for ADE2/ade2 (represented as +/-) produces four haploid spores, two ADE2 (+), and two ade2 (-).
Obviously, if the homologous chromosomes are not identical -- that is, some of the genes are heterozygous -- then the four product genomes will not be identical. Since the segregation of the chromatids at the first meiotic division is independent, from one chromosome to another, the segregation of genes on different chromosomes will be independent. Indeed, this is the pattern of assortment Mendel made famous. For each heterozygous pair of alleles, there will be two products -- ascospores -- of one type, and two of the other. If there are two heterozygous pairs on different chromosomes, they will segregate independently, so that all possible combinations will be represented among a sample of the progeny spores. The combinations that are the same as the parent-cell combinations are termed parental types, while those that are not like the parent-cell combinations are termed non-parental or recombinant, which is why the process is called recombination.
But chromosome segregation is not the only way that recombination can occur. According to this model, all the genes on the same chromosome would segregate together, and would never recombine, but we do not observe this. In fact, nearly all genes segregate from each other to some extent, although not always at the frequency predicted by random assortment. Recombination of genes on the same chromosome occurs when the homologous chromosomes exchange portions of their chromatids while paired at the four-strand stage in preparation for the first meiotic division (Figure 6). These exchanges -- crossovers -- occur at random along the chromatids, so the chance of two heterozygous genes on the same chromosome recombining depends on how far apart they are. The closer together they are, the less often they will recombine. The tendency of alleles of genes on the same chromosome to segregate together more often than expected by chance alone, is called linkage. The less frequently they recombine, the more tightly they are linked.
In yeast, crossing over occurs quite frequently, so even genes on the same chromosome appear to segregate independently unless they are very close together. The three specific genes that we are using in these experiments, mating-type and the two red adenine genes, are on three different chromosomes, so we will not observe any cases of linkage.
Figure 7: Meiosis With Crossing Over
Mitotic Recombination:
We usually think of mitosis as a process in which there is no segregation of heterozygous characteristics. Each mitotic product is considered identical to the parent. We can easily demonstrate that mitotic segregation, or recombination, does occur at a low frequency in yeast (Figure 7) (Mortimer & Hawthorne 1969). It also occurs in the somatic cells of higher organisms, but is more difficult to demonstrate. Mitotic segregation, as we shall demonstrate, makes genes that are heterozygous become homozygous, resulting in the expression of recessive alleles that otherwise would not be expressed. Since most deleterious mutations are recessive, and are carried unexpressed in the heterozygous condition, this has important consequences. In higher organisms, including people, it may be a mechanism for development of some diseases, including cancer. In experimental organisms, especially in yeast, it provides an alternative to meiotic segregation for studying the genotype of diploid strains.
Figure 8: Mitosis With Crossing Over
We can understand mitotic recombination if we assume that at mitosis the homologous chromosomes actually do pair, as they do in meiosis. Since we cannot observe this under the microscope, it probably only happens occasionally. If we then assume that crossing over occurs between the centromere and the position of a pair of heterozygous alleles at this four-strand stage, then, when the chromatids disjoin, segregation could indeed occur. In fact, if the disjoined chromatids are randomly distributed to the poles, then in half of the divisions in which such a crossover occurs, the heterozygous alleles would become homozygous in the progeny cells, resulting in two clones of homozygous cells growing side-by-side. When it occurs in a developing colony, the colony becomes sectored. Since cells that are heterozygous for one of the red adenine mutations (such as ADE1/ade1 or ADE2/ade2) are white and those that are homozygous for one of the recessive alleles (ade1/ade1 or ade2/ade2) are red, mitotic segregation results in the appearance of red sectors in the normally white colonies (Figure 8).
Figure 9: Formation of Sectored Colonies
Homozygous ade2/ade2 clone produces red sector in otherwise cream-colored colony. Segregation at first division produces half-red and half-cream-colored colony (right), while segregation at later division produces smaller red sector (left).
Gene Conversion:
Mitotic recombination can be induced by radiation, such as ultraviolet or x-rays. If we irradiate single cells that are heterozygous for ADE2/ade2 with a dose that kills about half of them, then several percent of the survivors will form colonies with red sectors. When radiation induces this effect, the segregation occurs early in the development of the colony, so the sectors are large, sometimes half of the colony. If the segregation results from a reciprocal crossover, then the cream-colored half of the colony should be homozygous for the dominant ADE2 allele. However, the cream-colored sector often turns out to still be heterozygous ADE2/ade2. This is easy to detect, because if it is heterozygous it will segregate red sectors, but if homozygous, it won't. This type of sectored colonies appears to arise from some sort of nonreciprocal event. We frequently observe nonreciprocal segregation or recombination events in yeast, and other fungi. They usually occur less frequently than the reciprocal events, but not always. We now consider them a distinct type of genetic event called gene conversion, and this process has gained more general interest recently as an attractive model for explaining the events occuring at the genetic sequences associated with the production of antibodies in higher animals (Fogel, Mortimer & Lusnak 1981).
Return to contents
Last updated Wednesday, 04-Dec-2002 14:34:24 CST