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.

In Saccharomyces cerevisiae, the actin cytoskeleton is depolarized by NaCl stress. In this study, the response was maximal after 30 min, and then actin patches repolarized. Rvs161p was required for actin repolarization because the rvs161Δ mutant did not repolarize actin patches after growth in a salt medium. Mutations suppressing the rvs161Δ-related salt sensitivity all occurred in genes required for sphingolipid biosynthesis: FEN1, SUR4, SUR2, SUR1, and IPT1. These suppressors also suppressed act1-1-related salt sensitivity and the defect in actin repolarization of the rvs161Δ mutant, providing a link between sphingolipids and actin polarization. Indeed, deletion of the suppressor genes suppressed the rvs161Δ defect in actin repolarization in two ways: either actin was not depolarized at the wild-type level in a set of suppressor mutants, or actin was repolarized in the absence of Rvs161p in the other suppressor mutants. Rvs161p was localized as cortical patches that concentrated at polarization sites, i.e., bud emergence and septa, and was found to be associated with lipid rafts. An important link between sphingolipids and actin polarization is that Rvs161p was required for actin repolarization and was found to be located in lipid rafts.

Rvs161p and sphingolipids are required for actin repolarization following salt stress.

Direct detection of yeast mutants with reduced viability on plates by erythrosine B staining

The Rvs161p and Rvs167p proteins of Saccharomyces cerevisiae, homologues of higher eukaryotes' amphiphysins, associate with actin and appear to be involved in several functions related to the actin cytoskeleton. In order to identify partners of the Rvsp proteins, yeast libraries constructed in two-hybrid vectors were screened using either Rvs167p or Rvs161p as a bait. The selected candidates, representing 34 ORFs, were then tested against both Rvsp proteins, as well as domains of Rvs167p or Rvs161p. Among the most significant ones, 24 ORFs were specific preys of Rvs167p only and two gave interactions with Rvs161p only. Interestingly, five ORFs were preys of both Rvs161p and Rvs167p (RVS167, LAS17, YNL094w, YMR192w and YPL249c). Analysis of putative functions of the candidates confirm involvement of the Rvsp in endocytosis/vesicle traffic, but also opens possible new fields, such as nuclear functions. Copyright © 2000 John Wiley & Sons, Ltd.

A network of proteins around Rvs167p and Rvs161p, two proteins related to the yeast actin cytoskeleton

A plasmid containing the gene coding for the Saccharomyces cerevisiae F0Fl ATPase subunit 4 was isolated from a yeast genomic DNA library using the oligonucleotide probe procedure. The gene and the surrounding regions were cloned into M13 tg 130 and M13 tg 131 phage vectors. A 732-base-pair open reading frame encoding a 244-amino-acid polypeptide is described. The nucleotide sequence predicts that subunit 4 is probably derived from a precursor protein with a hydrophilic and basic 35-amino-acid leader sequence. Mature subunit 4 contains 209 amino acid residues and the predicted molecular mass is 23250 Da. This subunit presents amphiphilic behaviour with two distinct domains. A high α-helix content of 77% was predicted from the sequence. Subunit 4 shows homology with the b subunit of Escherichia coli ATP synthase.

https://febs.onlinelibrary.wiley.com/doi/pdf/10.1111/j.1432-1033.1988.tb13745.x

ATP4, the structural gene for yeast F0F1 ATPase subunit 4.

https://febs.onlinelibrary.wiley.com/doi/pdf/10.1016/S0014-5793%2800%2902276-6

Michel Aigle

Papers

Rvs161p and sphingolipids are required for actin repolarization following salt stress.

Direct detection of yeast mutants with reduced viability on plates by erythrosine B staining

A network of proteins around Rvs167p and Rvs161p, two proteins related to the yeast actin cytoskeleton

ATP4, the structural gene for yeast F0F1 ATPase subunit 4.

Genomic Exploration of the Hemiascomycetous Yeasts: 5. Saccharomyces bayanus var. uvarum