Genome-wide studies have recently revealed the unexpected complexity of the genetic response to apparently simple physiological changes. Here, we show that when yeast cells are exposed to Cd(2+), most of the sulfur assimilated by the cells is converted into glutathione, a thiol-metabolite essential for detoxification. Cells adapt to this vital metabolite requirement by modifying globally their proteome to reduce the production of abundant sulfur-rich proteins. In particular, some abundant glycolytic enzymes are replaced by sulfur-depleted isozymes. This global change in protein expression allows an overall sulfur amino acid saving of 30%. This proteomic adaptation is essentially regulated at the mRNA level. The main transcriptional activator of the sulfate assimilation pathway, Met4p, plays an essential role in this sulfur-sparing response.

A class of alleles at the VNTR (variable number of tandem repeat) locus in the 5' region of the insulin gene (INS) on chromosome 11p is associated with increased risk of insulin-dependent diabetes mellitus (IDDM), but family studies have failed to demonstrate linkage. INS is thought to contribute to IDDM susceptibility but this view has been difficult to reconcile with the lack of linkage evidence. We thus investigated polymorphisms of INS and neighbouring loci in random diabetics, IDDM multiplex families and controls. HLA-DR4-positive diabetics showed an increased risk associated with common variants at polymorphic sites in a 19-kilobase segment spanned by the 5' INS VNTR and the third intron of the gene for insulin-like growth factor II (IGF2). As INS is the major candidate gene from this region, diabetic and control sequence were compared to identify all INS polymorphisms that could contribute to disease susceptibility. In multiplex families the IDDM-associated alleles were transmitted preferentially to HLA-DR4-positive diabetic offspring from heterozygous parents. The effect was strongest in paternal meioses, suggesting a possible role for maternal imprinting. Our results strongly support the existence of a gene or genes affecting HLA-DR4 IDDM susceptibility which is located in a 19-kilobase region of INS-IGF2. Our results also suggest new ways to map susceptibility loci in other common diseases.

A continuous array of overlapping clones covering the entire human chromosome 21q was constructed from human yeast artificial chromosome libraries using sequence-tagged sites as landmarks specifically detected by polymerase chain reaction. The yeast artificial chromosome contiguous unit starts with pericentromeric and ends with subtelomeric loci of 21q. The resulting order of sequence-tagged sites is consistent with other physical and genetic mapping data. This set of overlapping clones will promote our knowledge of the structure of this chromosome and the function of its genes.

RSC is an essential, multisubunit chromatin remodeling complex. We show here that the Rsc4 subunit of RSC interacted via its C terminus with Rpb5, a conserved subunit shared by all three nuclear RNA polymerases (Pol). Furthermore, the RSC complex coimmunoprecipitated with all three RNA polymerases. Mutations in the C terminus of Rsc4 conferred a thermosensitive phenotype and the loss of interaction with Rpb5. Certain thermosensitive rpb5 mutations were lethal in combination with an rsc4 mutation, supporting the physiological significance of the interaction. Pol II transcription of ca. 12% of the yeast genome was increased or decreased twofold or more in a rsc4 C-terminal mutant. The transcription of the Pol III-transcribed genes SNR6 and RPR1 was also reduced, in agreement with the observed localization of RSC near many class III genes. Rsc4 C-terminal mutations did not alter the stability or assembly of the RSC complex, suggesting an impact on Rsc4 function. Strikingly, a C-terminal mutation of Rsc4 did not impair RSC recruitment to the RSC-responsive genes DUT1 and SMX3 but rather changed the chromatin accessibility of DNases to their promoter regions, suggesting that the altered transcription of DUT1 and SMX3 was the consequence of altered chromatin remodeling.Transcription occurs in the crowded context of the nucleus in which genes are wrapped in chromatin. The first step in gene expression involves the modification and/or the remodeling of repressive chromatin by specialized complexes. For polymerase II (Pol II)-transcribed genes, these steps are followed by the recruitment of Mediator, the general transcription factors (GTFs) and the Pol II itself, although in an order that can vary from one promoter to another (9, 34). The pathway leading from silent chromatin to transcription by Pol I and Pol III has not been studied as thoroughly but is globally similar, with an additional contribution of cognate GTFs. In yeast and human cells, the Pol III-specific transcription factor TFIIIC has been found to be required for the proper nucleosomal organization of Pol III genes (4, 23, 32). In the case of Pol I transcription, the mammalian termination factor TTF-I is able to activate transcription by promoting chromatin remodeling in synergy with ATP-dependent cofactors in vitro (24). Transcription initiation is not the only step at which chromatin might interfere with transcription. Nucleosomes residing in the transcribed region can inhibit the movement of RNA polymerases during elongation. To contend with this, the FACT complex helps human Pol II transcribe through nucleosome-induced blocks (28, 38). These observations suggest that factors that relieve the repressive effect of nucleosomes might act in conjunction with the transcription machinery at the successive stages of the transcription cycle.The repressive effect of nucleosomes is overcome by two cooperative mechanisms. The first involves the covalent modification of the histones, including the acetylation of specific histone tail lysines by acetyl transferases (...

A set of 1091 human skeletal muscle cDNA clone inserts representing more than 800 human gene transcripts were spotted as PCR products at high density on nylon membranes. Replicas of the filters were hybridized in stringent conditions with 33P-radiolabeled cDNA probes transcribed from skeletal muscle poly(A)+ RNA. Hybridization signals were collected on phosphor screens and processed using a software specifically adapted for this application to identify and quantitate each spot. Parameters likely to influence the hybridization signal intensity were assessed to eliminate artifacts. Each clone was assigned to one of four intensity classes reflecting the steady-state level of transcription of the corresponding gene in skeletal muscle. Differential expression of specific gene transcripts was detected using complex cDNA probes derived from nine different tissues, allowing assessment of their tissue specificity. This made it possible to identify 48 novel gene transcripts (including 7 homologous or related to known sequences) with a muscle-restricted pattern of expression. These results were validated through the analysis of known muscle-specific transcripts and by Northern analysis of a subset of the novel gene transcripts. All these genes have been registered in the Genexpress Index, such that sequence, map, and expression data can be used to decipher their role in the physiology and pathology of human muscles.