Genetics - A. V. Sivolob 2008

Genetics of Bacteria, Viruses, and Unicellular Eukaryotes
Unicellular Eukaryotes
Yeast

Among the wide variety of Unicellular Eukaryotes, which can be viewed as an evolutionary stepping stone Cell/4.html">From Prokaryotes to Multicellular Organisms, we will focus on two Examples. The first concerns an experimental model Organism of fundamental importance to genetics, while the second illustrates a species that undergoes unusual rearrangements of its hereditary apparatus during development.

Yeasts are, on the one hand, eukaryotes with all their characteristic features (see Chapter 6). On the other hand, they are among the simplest eukaryotes, which makes them (particularly Saccharomyces cerevisiae) a popular model system in genetics, molecular biology, cell biology, and biotechnology. Much like Bacteria, yeasts multiply rapidly and are quite easily cultured and transformed (i.e., modified by the Introduction of foreign DNA, see Chapter 9).

The S. cerevisiae genome contains ~12 Mb of Base Pairs (16 Chromosomes in the haploid set; In addition to chromosomal DNA, yeasts often harbor autonomous Plasmids, much like bacteria). Just over 1% of The Genome consists of repetitive sequences located in the centromeric and telomeric regions of chromosomes, and slightly more than 2% comprises mobile elements (the so-called Ty elements, which belong to the LTR-retrotransposon Class — see Chapter 6). Overall, repetitive sequences (including rRNA and tRNA genes) account for ~10% of the genome. Coding sequences for the ~6.7 thousand protein-encoding genes make up about 70% of the genome. The average coding sequence length is 480 codons (ranging from 40 to 5,000). Only about 3.5% of genes contain introns. Thus, the S. cerevisiae genome represents a clear transitional link between the genomes of prokaryotes and higher eukaryotes (see Table 1.1; Chapter 6).

The developmental cycle of S. cerevisiae is illustrated in Fig. 5.10. A diploid cell is capable of multiplying by budding: this involves mitosis, nuclear division, Cell wall formation, and cell Separation. Under conditions of nutrient deprivation, sporulation takes place, during which the diploid cell undergoes Meiosis. Four haploid progeny form ascospors encapsulated together within a Structure known as an ascus. Sporulation is only possible for diploid Cells heterozygous at the MAT locus—that is, those carrying two alleles of this locus, designated as MATa and MATα. Consequently, haploid ascospores of two types are formed: a and α.

The spore type constitutes the so-called mating type, or a peculiar "sex" of haploid cells. When an ascus is transferred to a nutrient medium, the spores multiply vegetatively, and mating occurs between "opposite-sex" spores to yield a diploid cell. Using asci from different strains, one can perform crosses between them and analyze their progeny using genetic analysis Methods. To obtain "hybrid" strains, complementary genetic markers are typically employed: for example, if cells of one strain cannot grow on a medium lacking Tryptophan, while another requires Histidine, hybrid diploid colonies can be readily selected on the appropriate medium.

Fig. 5.10. Life Cycle of the ascomycete Saccharomyces cerevisiae

If an individual ascospore is isolated, its progeny derived through budding should theoretically consist of cells of a single type. However, such a strain will not truly be monosporic if it carries the HO Gene, which enables cell-type switching: an a-type cell produces an α-type bud (or vice versa). Naturally, in this case, the accumulating cells of different types will mate to form diploid heterozygotes, which ultimately predominate in the culture.

Cell-type switching in S. cerevisiae is a prime example of programmed genomic rearrangement. The MAT locus is located on the right arm of the third chromosome: the Y segment sequence element (either Ya or Yα), which determines the allelic form of the locus, is flanked by several other elements on both sides (Fig. 5.11, showing the configuration corresponding to the MATa allele). Near the left and right telomeres lie two so-called cassettes, which represent the a and α alleles, respectively. However, these cassettes (designated as HMLa and HMRα) reside in sub-telomeric heterochromatic regions and are therefore transcriptionally inactive—they are inaccessible to the METABOLISM/31.html">Transcription machinery due to additional Chromatin compaction (see Chapter 6).

During cell-type switching, the HO gene is activated, encoding a specific endonuclease. The HO endonuclease cuts the MAT locus within the Y element zone (either Ya or Yα). Subsequent events follow the Homologous Recombination pathway (cf. Figs. 5.11 and 1.25): the double-stranded break is resected to generate two 3'-single-stranded tails, a loop forms in the chromosome, and the homologous HMRα cassette is brought to the break site, where repair DNA Synthesis proceeds using the cassette DNA strands as a template. The result is Gene Conversion: replacement of the MATa locus with the MATα locus. Both cassettes remain intact in the process, meaning that in future generations, the opposite cassette can be used to switch the MAT locus back.

Fig. 5.11. Schematic of mating-type switching in S. cerevisiae.

When the MATa locus is in the center, the HMLa cassette is used for conversion

The DNA synthesis depicted in the inset of Fig. 5.11 leads to The formation of two Holliday junctions (cf. Fig. 1.26). As explained in Chapter 1, There are two equally probable ways to resolve these junctions: with or without Crossing Over between the two duplexes (see also Fig. 1.28). In the scenario shown in Fig. 5.11, crossing over between two duplexes belonging to the same chromosome would result in a deletion—excising the region between the MAT locus and the HMLa cassette. Thus, in this case, resolution pathways that do not lead to Crossing over and, consequently, Chromosomal aberrations are specifically favored.

It should be noted that even during interchromatid homologous recombination, crossing over and non-crossover conversion are not always equally probable events. For instance, during mitotic recombination in diploid yeast cells, crossing over occurs in only about 10% of recombination events. Thus, at least in some cases, homologous recombination processes may be specifically executed to convert a genomic region without being accompanied by crossing over.

The cassette mechanism of gene activity switching is quite widespread: similar processes have been described in other ascomycete species, trypanosomes, and certain bacteria. Furthermore, such a mechanism undoubtedly operates during intrachromosomal homologous recombination at tandemly repeated genes (following the principle illustrated in Fig. 5.11, without crossing over) to maintain the identity of tandem copies. A mutation in one such copy is highly likely to be eliminated by using one of the many normal homologous regions as a template.

Returning to cell-type switching in S. cerevisiae, it also serves as a well-studied example of gene interaction at the level of Transcriptional Regulation. Approximately in the center of the MATa locus lies an operator region that activates two promoters driving transcription in opposite directions. The products of these genes are two transcription factors, Proteins a1 and α2. The former activates transcription of a group of genes that determine the specific traits of a-type cells, whereas the latter acts as a repressor for a set of α-specific genes (Fig. 5.12). Meanwhile, haploid cells of both types express a group of genes specific to haploid cells in general, driven by another transcription factor. In a-type cells, the MATa locus expresses protein a1, which by itself does not affect the Transcription of the aforementioned gene groups, but the absence of proteins a1 and α2 leads to the activation of a-specific genes (in the absence of a repressor) and the shutdown of α-specific genes (in the absence of an activator) (Fig. 5.12). In diploid cells heterozygous for both MAT loci, protein a1 forms a complex with α2, which effectively blocks transcription of a1 (thereby turning off a-specific genes once more) as well as the transcription of genes specific to haploid cells (Fig. 5.12).

Additionally, the a1–α2 complex acts as a repressor of the HO gene, preventing it from initiating the recombination process shown in Fig. 5.11 in diploid cells. Activation of this gene in haploid cells requires a set of specific transcription activators whose appearance is coordinated with Cell Cycle regulation: they appear only during the late G1 phase, which is when cell-type switching can take place.

Fig. 5.12. Schematic of Transcriptional Regulation in diploid and haploid Cells of the two S. cerevisiae mating types. Red arrows indicate mRNA, and ovals represent the corresponding protein products acting as transcription factors for gene groups specifically expressed in haploid cells in general (hsg), a-cells (asg), and α-cells (αsg)

However, The regulation of HO gene activity is even more complex and not yet fully understood. Specifically, following mitosis, a haploid spore produces Two Types of cells that differ slightly in their properties—the so-called mother and daughter cells. Cell-type switching occurs exclusively in the mother cell; the daughter cell is incapable of it because the HO gene is not activated. Consequently, the mother cell changes its mating type—for instance, from a to α—and divides to yield two α-cells, one of which is now a mother cell that will switch its type back to a, while the other is a daughter cell that remains of the α type.

Such Asymmetry in mitotic products is also characteristic of Cell Differentiation in multicellular organisms, where a stem cell gives rise to another stem cell and a more specialized cell that has lost some of its developmental potential. The examples discussed here reflect only a small fraction of the genetic mechanisms studied in S. cerevisiae, which help elucidate the General Principles governing the functioning of the eukaryotic hereditary apparatus.