Читать книгу Handbook of Enology: Volume 1 - Pascal Ribéreau-Gayon - Страница 26

The yeast nucleus is spherical. It has a diameter of 1–2 μm and is barely visible using a phase‐contrast optical microscope. It is located near the principal vacuole in non‐proliferating cells. The nuclear envelope is made up of a double membrane attached to the ER. It contains many ephemeral pores, whose locations are continually changing. These pores enable the exchange of small proteins between the nucleus and the cytoplasm. Contrary to what happens in higher eukaryotes, the yeast nuclear envelope is not dispersed during mitosis. In the basophilic part of the nucleus, the crescent‐shaped nucleolus can be seen by using a nuclear‐specific staining method. As in other eukaryotes, it is responsible for the synthesis of ribosomal RNA. During cell division, the yeast nucleus (Williamson, 1991) also contains rudimentary spindle threads composed of microtubules of tubulin, some discontinuous and others continuous (Figure 1.10). The continuous microtubules are stretched between the two spindle pole bodies (SPB). These corpuscles are permanently included in the nuclear membrane and correspond to the centrioles of higher organisms. The cytoplasmic microtubules depart from the SPB toward the cytoplasm.

The size of the yeast genome, about 12,800 kb, is low compared to that of higher eukaryotes (about 10 times lower than that of Arabidopsis). It has a genome almost three times larger than in Escherichia coli, but its genetic material is organized into true chromosomes. Each one contains a single molecule of linear double‐stranded DNA associated with basic proteins known as histones, to form chromatin that contains repetitive units called nucleosomes. Because of their small size and their weak condensation, yeast chromosomes cannot be observed under the microscope.

Pulsed‐field electrophoresis (Carle and Olson, 1984; Schwartz and Cantor, 1984) enables the separation of the 16 chromosomes in S. cerevisiae, whose sizes range from 200 to 2,000 kb. This species has a very large degree of chromosomic polymorphism. This characteristic has made karyotype analysis one of the main criteria for the identification of S. cerevisiae strains (Section 1.9.3).

The full chromosomal DNA sequence of S. cerevisiae (S288C) was established in 1996. It has 6,275 genes, including 23% in common with humans (Goffeau et al., 1996). In 2009, the EC1118 diploid genome of a yeast strain in wine was fully sequenced. It reveals the gene transfer mechanisms between Saccharomyces and non‐Saccharomyces. These works show that the genome of winemaking yeast may be constantly remodeled by the addition of exogenous genes (Novo et al., 2009). This detailed knowledge of the yeast genome will constitute a powerful tool, both for the molecular understanding of its physiology and for the selection and improvement of winemaking strains. Current research done in the field of synthetic biology, as well as in enology, aims to create and assemble a full artificial genome of S. cerevisiae yeast with a number of potential medical and industrial applications, including in the field of enology (Richardson et al., 2017).

The yeast chromosomes contain relatively few repeated sequences. Most genes are only present in a single copy in the haploid genome, but the ribosomal RNA genes are highly repeated (about 100 copies).

FIGURE 1.10 The yeast nucleus (Williamson, 1991).

The genome of S. cerevisiae also contains transposable elements or transposons—specifically, transposon yeast (Ty) elements. These comprise a central ε region (5.6 kb) framed by a direct repeated sequence called the δ sequence (0.25 kb). The δ sequences have a tendency to recombine, resulting in the loss of the central region and a δ sequence. As a result, there are about 100 copies of the δ sequence in the yeast genome. The Ty elements code for noninfectious retrovirus particles. This retrovirus contains Ty messenger RNA as well as a reverse transcriptase capable of copying the RNA into complementary DNA. The latter can reinsert itself into any site of the chromosome. The random excision and insertion of Ty elements in the yeast genome can modify the genes and play an important role in strain evolution.

A single plasmid, called the 2 μm plasmid, has been identified in the yeast nucleus. It is a circular molecule of DNA, containing 6 kb, and there are 50–100 copies per cell. Its biological function is not known. However, it is a very useful tool, used by molecular biologists to construct artificial plasmids and genetically transform yeast strains.