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.

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

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 the endoplasmic reticulum (ER), secretory and transmembrane proteins fold into their native conformation and undergo posttranslational modifications important for their activity and structure. When protein folding in the ER is inhibited, signal transduction pathways, which increase the biosynthetic capacity and decrease the biosynthetic burden of the ER to maintain the homeostasis of this organelle, are activated. These pathways are called the unfolded protein response (UPR). In this review, we briefly summarize principles of protein folding and molecular chaperone function important for a mechanistic understanding of UPR-signaling events. We then discuss mechanisms of signal transduction employed by the UPR in mammals and our current understanding of the remodeling of cellular processes by the UPR. Finally, we summarize data that demonstrate that UPR signaling feeds into decision making in other processes previously thought to be unrelated to ER function, e.g., eukaryotic starvation responses and differentiation programs.

The mammalian unfolded protein response

We have constructed and tested a dominant resistance module, for selection of S. cerevisiae transformants, which entirely consists of heterologous DNA. This kanMX module contains the known kanr open reading-frame of the E. coli transposon Tn903 fused to transcriptional and translational control sequences of the TEF gene of the filamentous fungus Ashbya gossypii. This hybrid module permits efficient selection of transformants resistant against geneticin (G418). We also constructed a lacZMT reporter module in which the open reading-frame of the E. coli lacZ gene (lacking the first 9 codons) is fused at its 3' end to the S. cerevisiae ADH1 terminator. KanMX and the lacZMT module, or both modules together, were cloned in the center of a new multiple cloning sequence comprising 18 unique restriction sites flanked by Not I sites. Using the double module for constructions of in-frame substitutions of genes, only one transformation experiment is necessary to test the activity of the promotor and to search for phenotypes due to inactivation of this gene. To allow for repeated use of the G418 selection some kanMX modules are flanked by 470 bp direct repeats, promoting in vivo excision with frequencies of 10(-3)-10(-4). The 1.4 kb kanMX module was also shown to be very useful for PCR based gene disruptions. In an experiment in which a gene disruption was done with DNA molecules carrying PCR-added terminal sequences of only 35 bases homology to each target site, all twelve tested geneticin-resistant colonies carried the correctly integrated kanMX module.

New heterologous modules for classical or PCR‐based gene disruptions in Saccharomyces cerevisiae

The first step in executing the genetic program of a cell is production of mRNA In yeast, almost every gene is transcribed as multiple distinct isoforms, differing at their 5' and/or 3' termini However, the implications and functional significance of the transcriptome-wide diversity of mRNA termini remains largely unexplored In this paper, we show that the GAT1 gene, encoding a transcriptional activator of nitrogen-responsive catabolic genes, produces a variety of mRNAs differing in their 5' and 3' termini Alternative transcription initiation leads to the constitutive, low level production of 2 full length proteins differing in their N-termini, whereas premature transcriptional termination generates a short, highly nitrogen catabolite repression- (NCR-) sensitive transcript that, as far as we can determine, is not translated under the growth conditions we used, but rather likely protects the cell from excess Gat1

https://www.tandfonline.com/doi/pdf/10.1080/15476286.2015.1058476

Premature termination of GAT1 transcription explains paradoxical negative correlation between nitrogen-responsive mRNA, but constitutive low-level protein production.

In S. cerevisiae, the synthesis of ureaamidolyase is subject to at least two different forms of regulation: nitrogen catabolite repression and induction by allophanate. Two positive regulatory genes DURM and DURL are involved in the induction process. We have measured the levels of mRNA homologous to the DUR2,1 gene in conditions of ureaamidolyase induction and in regulatory mutants. The amounts of DUR2,1 enzyme and messengers are well coordinated; moreover, the half life of DUR2,1 messengers is identical in the presence or absence of inducer. These data suggest that the ureaamidolyase production is probably controlled at the level of transcription. From a pool of hybrid plasmids carrying Sau3A fragments representing the entire yeast genome, a 13 kb DNA fragment containing the regulatory gene DURM was cloned by complementation of a durM mutation which prevents the growth on allantoin as sole nitrogen source. Cells containing the cloned DNA recover the inducibility of ureaamidolyase by allophanate. Four RNA transcripts have homology to this 13 kb DNA fragment but the study of subcloned restriction endonuclease fragments allowed us to map the DURM regulatory gene within a 4 kilobase pair region. This fragment encodes a 1 kb transcript. The level of this RNA is the same in induced and non-induced cells.

Regulation of ureaamidolyase synthesis in Saccharomyces cerevisiae, RNA analysis, and cloning of the positive regulatory gene DURM.

The TorC1 protein kinase complex is a central component in a eukaryotic cell’s response to varying nitrogen availability, with kinase activity being stimulated in nitrogen excess by increased intracellular leucine. This leucine-dependent TorC1 activation requires functional Gtr1/2 and Ego1/3 complexes. Rapamycin inhibition of TorC1 elicits nuclear localization of Gln3, a GATA-family transcription activator responsible for the expression of genes encoding proteins required to transport and degrade poor nitrogen sources, e.g., proline. In nitrogen-replete conditions, Gln3 is cytoplasmic and Gln3-mediated transcription minimal, whereas in nitrogen limiting or starvation conditions, or after rapamycin treatment, Gln3 is nuclear and transcription greatly increased. Increasing evidence supports the idea that TorC1 activation may not be as central to nitrogen-responsive intracellular Gln3 localization as envisioned previously. To test this idea directly, we determined whether Gtr1/2- and Ego1/3-dependent TorC1 activation also was required for cytoplasmic Gln3 sequestration and repressed GATA factor-mediated transcription by abolishing the Gtr-Ego complex proteins. We show that Gln3 is sequestered in the cytoplasm of gtr1Δ, gtr2Δ, ego1Δ, and ego3Δ strains either long term in logarithmically glutamine-grown cells or short term after refeeding glutamine to nitrogen-limited or -starved cells; GATA factor−dependent transcription also was minimal. However, in all but a gtr1Δ, nuclear Gln3 localization in response to nitrogen limitation or starvation was adversely affected. Our data demonstrate: (i) Gtr-Ego-dependent TorC1 activation is not required for cytoplasmic Gln3 sequestration in nitrogen-rich conditions; (ii) a novel Gtr-Ego-TorC1 activation-independent mechanism sequesters Gln3 in the cytoplasm; (iii) Gtr and Ego complex proteins participate in nuclear Gln3-Myc13 localization, heretofore unrecognized functions for these proteins; and (iv) the importance of searching for new mechanisms associated with TorC1 activation and/or the regulation of Gln3 localization/function in response to changes in the cells’ nitrogen environment.

GATA Factor Regulation in Excess Nitrogen Occurs Independently of Gtr-Ego Complex-Dependent TorC1 Activation.

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Nitrogen catabolite repression-sensitive transcription as a readout of tor pathway regulation: The genetic background, reporter gene and GATA factor assayed determine the outcomes (Genetics 181, (861-874))

https://www.jbc.org/content/289/5/2918.full.pdf

Evelyne Dubois

Papers

Premature termination of GAT1 transcription explains paradoxical negative correlation between nitrogen-responsive mRNA, but constitutive low-level protein production.

Regulation of ureaamidolyase synthesis in Saccharomyces cerevisiae, RNA analysis, and cloning of the positive regulatory gene DURM.

GATA Factor Regulation in Excess Nitrogen Occurs Independently of Gtr-Ego Complex-Dependent TorC1 Activation.

Nitrogen catabolite repression-sensitive transcription as a readout of tor pathway regulation: The genetic background, reporter gene and GATA factor assayed determine the outcomes (Genetics 181, (861-874))

Constitutive and Nitrogen Catabolite Repression-Sensitive Production of Gat1 Isoforms