THE YEAST SACCHAROMYCES CEREVISIAE, A MODEL EUKARYOTIC ORGANISMS

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Genome evolution

Saccharomyces cerevisiae still provides a unique opportunity to explain molecular mechanisms that underlie yeast evolutionary genomics, for instance, gene duplications and mutations (Lynch M. et al., 2001), as well as acquisition of novel genetic material (reviewed by Dujon B., 2010). The evolutionary period, which starts with the ancestor, corresponds to a time during which the Saccharomyces cerevisiae lineage became increasingly adapted to rapid fermentative growth (Thomson J. M. et al., 2005). This period faced the extensive rearrangement of the genome, including the deletion of thousands of redundant copies of duplicated genes (Scannell D. R. et al., 2007) that mainly appeared at the time of the WGD event (see below).
It was nevertheless published that in experimental cultures of Saccharomyces cerevisiae, duplications of large chromosomal segments (tens to hundreds of kilobases) containing many genes appeared spontaneously at high frequency in both haploid and diploid (Koszul R. et al., 2004; 19 Schacherer J. et al., 2007). Four types of chromosomal structures are formed with different degrees of stability during subsequent generations (Koszul R. et al., 2006). This process of spontaneous segmental duplications in Saccharomyces cerevisiae was recently reviewed (shown in Figure 2. 2) (Dujon B., 2010). Whatever their origin, the existence of sets of two or more genes encoding proteins with identical or very similar sequences (redundancy) provided the raw material for the evolution of novel functions (Ohno S., 1970). Duplication is indeed in charge of the formation of paralogous gene families, and functional diversification may occur within this process (Lynch M. et al., 2001) through subtle alteration of structures and catalytic motifs to achieve new functions. Studies of proteins have demonstrated that aspects of catalysis are often conserved between evolutionarily related proteins that catalyze different reactions. Understanding the evolution of enzymes has implications for many areas of biology, including genome annotation, function prediction and protein engineering (Glasner M.E et al., 2007).

The whole genome duplication (WGD)

Gene duplication is recognized as a crucial mechanism in evolution. Gene duplication allows asymmetric evolution of preexisting promiscuous function in a protein, such that these prior functions can be further optimized (subfunctionalization) (Ohno S., 1970). Mewes et al. have systematically observed the genome for clusters of genes that have been produced by local duplication events (Mewes H.W. et al., 1997). The large number of fungal genome sequences has provided firm support for the idea that the S. cerevisiae genome resulted from a whole-genome duplication (Wolfe K.H and D. C. Shields, 1997; reviewed in Scannell et al., 2007). After the genome duplication, one gene copy is apt to preserve the initial function while the other copy is free to diverge. Most studies indeed assume that enzymes change function from one specialized catalytic role to another specialized role, after being freed to change function, usually through an extra copy of the gene for the enzyme being created through gene duplication (Zhang J.Z., 2003). Duplicated genes can supply genetic raw material for the emergence of new functions through the forces of mutation and natural selection (Kellis B. et al., 2004). In principle, coordinate duplication of an entire genome may allow for large-scale adaptation to new environments. Kellis and coworkers published the model of WGD followed by massive gene loss of Kluyveromyces and Saccharomyces, see Figure 2. 3 (Kellis B. et al, 2004). To counterbalance the uncontrolled genome expansion, massive gene loss and gene inactivation was researched following the WGD (Liti G. and E. J. Louis, 2005).

Multigene families

Multigene families are a group of genes that have descended from a common ancestral gene and therefore have comparable functions and similar DNA sequences (Li W.H, 1997). Most of these multigene families in S. cerevisiae are found either in subtelomeric regions (Viswanathan M. et al., 1994) or organized as tandem repeats (Leh-Louis V. et al., 2004). The concentration of multigene families in the telomere-adjacent regions has been proposed to reflect a recombination-mediated dispersal mechanism (Zakian, V. A. 1996). As compared to non-subtelomeric genes, their subtelomeric location, which is characterized by an extraordinary instability, mainly explains their fast expansion through gene recombination, and evolution through functional divergence of the alleles (Brown C.A. et al., 2010; Sickmann A. et al., 2003)). Besides, a recent study by Liao et al. showed that duplication is an important contributor to phenotypic evolution and a large number of morphological and physiological traits are controlled by multigene families (Liao B.Y. et al., 2010), promoting adaptation to novel niches.

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Yeast Sugar metabolism

Sugars are preferential carbon sources for yeasts, and vast amount of information is available on the components of the pathways for sugar utilization in the yeast Saccharomyces cerevisiae. The glycolysis is the general pathway for conversion of glucose to pyruvate and energy, while an alternative mode of glucose oxidation is the hexose phosphate pathway also known as the pentose phosphate cycle. Two principal modes of pyruvate utilization for energy production can be distinguished: respiration and fermentation (Figure 2. 4). Under aerobic condition, the yeast is capable of degrading glucose (or similar monosaccharides such as fructose and mannose) by an oxidative metabolism, leading to the respiration of glucose into CO2 and H2O. This is an energetic metabolism and allows an increase in the cellular biomass with a high yield. While under anaerobic or aerobic condition with high glucose concentration, it is converts the sugar into ethanol and CO2 by the process of fermentation. These sugars are called fermentable sugars and the transformation is energetically favorable, although less energy than oxidative metabolism. The relationship between respiration and the utilization of sugar is reviewed by Fukuhara (Fukuhara Hiroshi, 2003).The use of other sugars such as sucrose, galactose, maltose or trehalose in S. cerevisiae depends on both the genetic variability of strains and the regulatory mechanisms.

Table of contents :

1 INTRODUTION
2 BIBLIOGRAPHY
2.1 THE YEAST SACCHAROMYCES CEREVISIAE, A MODEL EUKARYOTIC ORGANISMS
2.1.1 Generality
2.1.2 General aspects of genome and evolution in Saccharomyces cerevisiae
2.2 YEAST SUGAR METABOLISM
2.2.1 The monosaccharides
2.2.2 The di- and tri-saccharides
2.2.3 The polysaccharides
2.3 ENZYMES IN CARBOHYDRATE METABOLISM
2.3.1 Carbohydrate metabolic process genes
2.3.2 Classification of Glycoside hydrolases
2.3.3 GH13 family
2.3.4 Alpha-glucosidases
2.3.5 Isomaltases
2.4 ISOMALTASES FROM THE YEAST SACCHAROMYCES CEREVISIAE
2.4.1 Characterization of the IMA multigene family
2.4.2 Biochemical evidence for isomaltase activity
2.4.3 Amino acid sequence and structural elements of isomaltase
2.4.4 Enzyme evolution
3 RESULTS AND DISCUSSIONS
3.1 PURITY AND STABILITY OF IMA PROTEINS
3.1.1 Expression and purification of IMA proteins
3.1.2 Evaluation of conditions for stabilization and perservation
3.1.3 Effects of chemical reagents and metal ions
3.2 BIOCHEMICAL CHARACTERIZATION OF IMA PROTEINS
3.2.1 Optimal pH
3.2.2 Optimal Temperature
3.2.3 Half-Life
3.2.4 Melting Temperature
3.2.5 Thermostabilization by single proline substitution
3.2.6 Other possible issues for thermostabilization of Ima proteins
3.2.7 Conclusion
3.3 ENZYMOLOGICAL CHARACTERIZATION OF IMA PROTEINS
3.3.1 Substrates Specificities
3.3.2 Impact of point mutations
3.3.3 Kinetics parameters of purified isomaltases
3.3.4 Inhibition of isomaltase
3.3.5 Transglycosylation acitivity
3.3.6 Conclusion
4 CONCLUSIONS AND PERSPECTIVES
4.1 GENERAL CONCLUSION
4.2 PERSPECTIVES
5 EXPERIMENTAL PROCEDURES
5.1 STRAINS AND CULTURE CONDITIONS
5.1.1 Bacteria strains
5.1.2 Yeast strains
5.2 MOLECULAR BIOLOGY METHODS
5.2.1 Primers
5.2.2 PCR amplification
5.2.3 Purification of DNA fragment
5.2.4 Cloning in pYES2.1/V5-His-TOPO® (TOPO® Cloning)
5.2.5 Mutation construction
5.3 YEAST TRANSFORMATION BY LITHIUM ACETATE
5.4 EXPRESSION AND PURIFICATION
5.4.1 Preparation of crude extract
5.4.2 Purification of proteins by affinity chromatography
5.4.3 SDS-PAGE/ Coomassie Blue staining
5.4.4 Measurement of proteins concentration ( Bradford assay)
5.4.5 Removal imidazole (The ZebaTM Desalt Spin Columns)
5.5 ENZYMATIC ASSAYS
5.5.1 Colorimetric reaction with pNPG
5.5.2 Coupled enzymatic reactions for other subtrates
5.6 BIOCHEMICAL PARAMETERS
5.6.1 Optimal temperature
5.6.2 Optimal pH
5.6.3 Half Life
5.6.4 Melting Temperature
5.6.5 Influence of chemical compounds and metal ions
5.6.6 Kinetic parameters
5.6.7 Measurement of transglycosylation and analysis by HPAEC-PAD
6 APPENDIX
6.1 ENZYME BIOCHEMISTRY AND KINETICS
6.1.1 Michaelis-Menten mechanism
6.1.2 Enzyme inhibition
7 REFERENCE

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