The human genome holds an extraordinary trove of information about human development, physiology, medicine and evolution. Here we report the results of an international collaboration to produce and make freely available a draft sequence of the human genome. We also present an initial analysis of the data, describing some of the insights that can be gleaned from the sequence.

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Initial sequencing and analysis of the human genome.

Fundamental features of microbial cellulose utilization are examined at successively higher levels of aggregation encompassing the structure and composition of cellulosic biomass, taxonomic diversity, cellulase enzyme systems, molecular biology of cellulase enzymes, physiology of cellulolytic microorganisms, ecological aspects of cellulase-degrading communities, and rate-limiting factors in nature. The methodological basis for studying microbial cellulose utilization is considered relative to quantification of cells and enzymes in the presence of solid substrates as well as apparatus and analysis for cellulose-grown continuous cultures. Quantitative description of cellulose hydrolysis is addressed with respect to adsorption of cellulase enzymes, rates of enzymatic hydrolysis, bioenergetics of microbial cellulose utilization, kinetics of microbial cellulose utilization, and contrasting features compared to soluble substrate kinetics. A biological perspective on processing cellulosic biomass is presented, including features of pretreated substrates and alternative process configurations. Organism development is considered for "consolidated bioprocessing" (CBP), in which the production of cellulolytic enzymes, hydrolysis of biomass, and fermentation of resulting sugars to desired products occur in one step. Two organism development strategies for CBP are examined: (i) improve product yield and tolerance in microorganisms able to utilize cellulose, or (ii) express a heterologous system for cellulose hydrolysis and utilization in microorganisms that exhibit high product yield and tolerance. A concluding discussion identifies unresolved issues pertaining to microbial cellulose utilization, suggests approaches by which such issues might be resolved, and contrasts a microbially oriented cellulose hydrolysis paradigm to the more conventional enzymatically oriented paradigm in both fundamental and applied contexts.

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Microbial cellulose utilization: fundamentals and biotechnology.

The genome of the yeast Saccharomyces cerevisiae has been completely sequenced through a worldwide collaboration. The sequence of 12,068 kilobases defines 5885 potential protein-encoding genes, approximately 140 genes specifying ribosomal RNA, 40 genes for small nuclear RNA molecules, and 275 transfer RNA genes. In addition, the complete sequence provides information about the higher order organization of yeast's 16 chromosomes and allows some insight into their evolutionary history. The genome shows a considerable amount of apparent genetic redundancy, and one of the major problems to be tackled during the next stage of the yeast genome project is to elucidate the biological functions of all of these genes.

Life with 6000 Genes

Publisher Summary The yeast Saccharomyces cerevisiae is now recognized as a model system representing a simple eukaryote whose genome can be easily manipulated. Yeast has only a slightly greater genetic complexity than bacteria and shares many of the technical advantages that permitted rapid progress in the molecular genetics of prokaryotes and their viruses. Some of the properties that make yeast particularly suitable for biological studies include rapid growth, dispersed cells, the ease of replica plating and mutant isolation, a well-defined genetic system, and most important, a highly versatile DNA transformation system. Being nonpathogenic, yeast can be handled with little precautions. Large quantities of normal baker's yeast are commercially available and can provide a cheap source for biochemical studies. The development of DNA transformation has made yeast particularly accessible to gene cloning and genetic engineering techniques. Structural genes corresponding to virtually any genetic trait can be identified by complementation from plasmid libraries. Plasmids can be introduced into yeast cells either as replicating molecules or by integration into the genome. In contrast to most other organisms, integrative recombination of transforming DNA in yeast proceeds exclusively via homologous recombination. Cloned yeast sequences, accompanied by foreign sequences on plasmids, can therefore be directed at will to specific locations in the genome.

Getting started with yeast.

A set of yeast strains based on Saccharomyces cerevisiae S288C in which commonly used selectable marker genes are deleted by design based on the yeast genome sequence has been constructed and analysed. These strains minimize or eliminate the homology to the corresponding marker genes in commonly used vectors without significantly affecting adjacent gene expression. Because the homology between commonly used auxotrophic marker gene segments and genomic sequences has been largely or completely abolished, these strains will also reduce plasmid integration events which can interfere with a wide variety of molecular genetic applications. We also report the construction of new members of the pRS400 series of vectors, containing the kanMX, ADE2 and MET15 genes.

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Designer deletion strains derived from Saccharomyces cerevisiae S288C: a useful set of strains and plasmids for PCR-mediated gene disruption and other applications.

Heterogeneity among the 2μm plasmids in Saccharomyces cerevisiae : a new sequence for the REP1 gene

We report here the isolation of temperature-sensitive mutants of the yeast Saccharomyces cerevisiae which exhibit cdc phenotypes. The recessive mutations defined four complementation groups, named ore1, ore2, ore3 and ore4. At the non-permissive temperature, strains bearing these mutations arrested in the G1 phase of the cell cycle. The wild-type allele of the gene altered in ore2 mutants was cloned. The nucleotide sequence of a fragment which can complement the mutation showed the presence of an open reading frame capable of encoding a protein with 286 amino acid residues. The deduced amino acid sequence showed 25% identity with that of the Escherichia coli Δ1-pyrroline-5-carboxylate reductase, an enzyme of the pathway for the biosynthesis of proline. The ore2 mutants, correspondingly, were found to be capable of growing at the non-permissive temperature on a synthetic medium supplemented with proline. In addition, the chromosomal location of the gene and its restriction map were compatible with those previously reported for the PRO3 gene which encodes the S. cerevisiae Δ1-pyrroline-5-carboxylate reductase.

ore2, a mutation affecting proline biosynthesis in the yeast Saccharomyces cerevisiae, leads to a cdc phenotype

Malolactic fermentation, a crucial step in winemaking, results mostly in degradation by lactic acid bacteria of L-malic acid into l-lactic acid. This direct decarboxylation is catalysed by the malolactic enzyme. Recently we, and others, have cloned the mleS gene of Lactococcus lactis encoding malolactic enzyme. Heterologous expression of mleS in Saccha-romyces cerevisiae was tested to perform simultaneously alcoholic and malolactic fermentations by yeast. mleS gene was cloned in a yeast multicopy vector under a strong promoter. Malolactic activity was present in crude extracts of recombinant yeasts. Malic acid degradation was tested during alcoholic fermentation in synthetic media and must. Yeasts expressing the mleS gene actually produced l-lactate from l-malate; nevertheless malate degradation was far from complete.

Research letterFunctional expression in Saccharomyces cerevisiae of the Lactococcus lactis mleS gene encoding the malolactic enzyme

Several yeast strains of the species S. cerevisiae, S. bayanus and S. paradoxus, first identified by hybridization experiments and DNA/DNA hybridization were characterized by using Polymerase Chain Reaction/Restriction Fragment Length Polymorphism (PCR/RFLP) of the MET2 gene. The concordance between this tool and classical genetic analyses did not reveal any exception for all the strains analysed, so PCR/RFLP of the MET2 gene proves to be a reliable and fast tool for delimiting S. cerevisiae and S. bayanus. OEnological strains race bayanus, chevalieri, capensis gave S. cerevisiae restriction patterns, whereas most of strains race S. uvarum belong to S. bayanus and displayed a specific chromosomal band patterns different from band patterns of S. cerevisiae strains. To avoid confusion in oenological terminology, oenologists should no longer use the name of bayanus to designate industrial or wild S. cerevisiae Gal- strains, and should consider S. bayanus as a distinct species.

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Genetic determination of Saccharomyces cerevisiae et Saccharomyces bayanus species by PCR/RFLP analysis of the MET2 gene

The invention relates to a nucleic acid containing: (1) either the DNA sequence itself contained in the nucleic acid represented in figure 5 and stretching from position 210 to position 1829; (2) or a portion of this sequence, this sequence being however sufficient to express in the appropriate host, after having been included beforehand in a vector allowing the transformation of this host, a polypeptide having a malolactic enzymatic activity; (3) or a sequence derived from either of the sequences defined in (1) or (2) by substitution of one or more of their nucleotides, or even addition or deletion of certain codons, as long as this sequence continues to code for a polypeptide having the abovementioned enzymatic activity

Michel Aigle

Papers

Heterogeneity among the 2μm plasmids in Saccharomyces cerevisiae : a new sequence for the REP1 gene

ore2, a mutation affecting proline biosynthesis in the yeast Saccharomyces cerevisiae, leads to a cdc phenotype

Research letterFunctional expression in Saccharomyces cerevisiae of the Lactococcus lactis mleS gene encoding the malolactic enzyme

Genetic determination of Saccharomyces cerevisiae et Saccharomyces bayanus species by PCR/RFLP analysis of the MET2 gene

Identification, cloning and expression of the malolactic enzyme