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Background: Parent-of-origin-dependent expression of alleles, imprinting, has been suggested to impact a substantial proportion of mammalian genes. Its discovery requires allele-specific detection of expressed transcripts, but in some cases detected allelic expression bias has been interpreted as imprinting without demonstrating compatible transmission patterns and excluding heritable variation. Therefore, we utilized a genome-wide tool exploiting high density genotyping arrays in parallel measurements of genotypes in RNA and DNA to determine allelic expression across the transcriptome in lymphoblastoid cell lines (LCLs) and skin fibroblasts derived from families., Results: We were able to validate 43% of imprinted genes with previous demonstration of compatible transmission patterns in LCLs and fibroblasts. In contrast, we only validated 8% of genes suggested to be imprinted in the literature, but without clear evidence of parent-of-origin-determined expression. We also detected five novel imprinted genes and delineated regions of imprinted expression surrounding annotated imprinted genes. More subtle parent-of-origin-dependent expression, or partial imprinting, could be verified in four genes. Despite higher prevalence of monoallelic expression, immortalized LCLs showed consistent imprinting in fewer loci than primary cells. Random monoallelic expression has previously been observed in LCLs and we show that random monoallelic expression in LCLs can be partly explained by aberrant methylation in the genome., Conclusions: Our results indicate that widespread parent-of-origin-dependent expression observed recently in rodents is unlikely to be captured by assessment of human cells derived from adult tissues where genome-wide assessment of both primary and immortalized cells yields few new imprinted loci.

Cis-acting variants altering gene expression are a source of phenotypic differences. The cis-acting components of expression variation can be identified through the mapping of differences in allelic expression (AE), which is the measure of relative expression between two allelic transcripts. We generated a map of AE associated SNPs using quantitative measurements of AE on Illumina Human1M BeadChips. In 53 lymphoblastoid cell lines derived from donors of European descent, we identified common cis variants affecting 30% (2935/9751) of the measured RefSeq transcripts at 0.001 permutation significance. The pervasive influence of cis-regulatory variants, which explain 50% of population variation in AE, extend to full-length transcripts and their isoforms as well as to unannotated transcripts. These strong effects facilitate fine mapping of cis-regulatory SNPs, as demonstrated by dissection of heritable control of transcripts in the systemic lupus erythematosus-associated C8orf13-BLK region in chromosome 8. The dense collection of associations will facilitate large-scale isolation of cis-regulatory SNPs.

Cellular signal transduction pathways modify gene expression programs in response to changes in the environment, but the mechanisms by which these pathways regulate populations of genes under their control are not entirely understood. We present evidence that most mitogen-activated protein kinases and protein kinase A subunits become physically associated with the genes that they regulate in the yeast (Saccharomyces cerevisiae) genome. The ability to detect this interaction of signaling kinases with target genes can be used to more precisely and comprehensively map the regulatory circuitry that eukaryotic cells use to respond to their environment.

Eukaryotic genomes are packaged into nucleosomes whose position and chemical modification state can profoundly influence regulation of gene expression. We profiled nucleosome modifications across the yeast genome using chromatin immunoprecipitation coupled with DNA microarrays to produce high-resolution genome-wide maps of histone acetylation and methylation. These maps take into account changes in nucleosome occupancy at actively transcribed genes and, in doing so, revise previous assessments of the modifications associated with gene expression. Both acetylation and methylation of histones are associated with transcriptional activity, but the former occurs predominantly at the beginning of genes, whereas the latter can occur throughout transcribed regions. Most notably, specific methylation events are associated with the beginning, middle, and end of actively transcribed genes. These maps provide the foundation for further understanding the roles of chromatin in gene expression and genome maintenance.

Chromatin regulators play fundamental roles in the regulation of gene expression and chromosome maintenance, but the regions of the genome where most of these regulators function has not been established. We explored the genome-wide occupancy of four different chromatin regulators encoded in Saccharomyces cerevisiae. The results reveal that the histone acetyltransferases Gcn5 and Esa1 are both generally recruited to the promoters of active protein-coding genes. In contrast, the histone deacetylases Hst1 and Rpd3 are recruited to specific sets of genes associated with distinct cellular functions. Our results provide new insights into the association of histone acetyltransferases and histone deacetylases with the yeast genome, and together with previous studies, suggest how these chromatin regulators are recruited to specific regions of the genome.

DNA-binding transcriptional regulators interpret the genome's regulatory code by binding to specific sequences to induce or repress gene expression. Comparative genomics has recently been used to identify potential cis-regulatory sequences within the yeast genome on the basis of phylogenetic conservation, but this information alone does not reveal if or when transcriptional regulators occupy these binding sites. We have constructed an initial map of yeast's transcriptional regulatory code by identifying the sequence elements that are bound by regulators under various conditions and that are conserved among Saccharomyces species. The organization of regulatory elements in promoters and the environment-dependent use of these elements by regulators are discussed. We find that environment-specific use of regulatory elements predicts mechanistic models for the function of a large population of yeast's transcriptional regulators.

We have systematically explored the in vivo occupancy of promoters and open reading frames by components of the RNA polymerase II transcription initiation and elongation apparatuses in yeast. RNA polymerase II, Mediator, and the general transcription factors (GTFs) were recruited to all promoters tested upon gene activation. RNA polymerase II, TFIIS, Spt5, and, unexpectedly, the Paf1/Cdc73 complex, were found associated with open reading frames. The presence of the Paf1/Cdc73 complex on ORFs in vivo suggests a novel function for this complex in elongation. Elongator was not detected under any conditions tested, and further analysis revealed that the majority of elongator is cytoplasmic. These results suggest a revised model for transcription initiation and elongation apparatuses in living cells.

Expression of the Escherichia coli leuV operon, which contains three tRNA(1)(Leu) genes, is regulated by several mechanisms including growth-rate-dependent control (GRDC) and stringent control (SC). Structural variants of the leuV promoter which differentially affect these regulatory responses have been identified, suggesting that promoter targets for GRDC and SC may be different and that GRDC of the leuV promoter occurs in the absence of guanosine 3', 5'-bisdiphosphate. To determine the mechanisms of the leuV promoter regulation, we have examined the stability of promoter open complexes and the effects of nucleotide triphosphate (NTP) concentration on the efficiency of the leuV promoter and its structural variants in vitro and in vivo. The leuV promoter open complexes were an order of magnitude more stable to heparin challenge than those of rrnBp(1). The major initiating nucleotide GTP as well as other NTPs increased the stability of the leuV promoter open complexes. When the cellular level of purine triphosphates was increased at slower growth rates by pyrimidine limitation, a 10% reduction in leuV promoter activity was seen. It therefore appears that transcription initiation from the leuV promoter is less sensitive to changes in intracellular NTP concentration than that from rrnBp(1). Comparative analysis of regulation of the leuV promoter with and without upstream activating sequences (UAS) demonstrated that the binding site for factor of inversion stimulation (FIS) located in UAS is essential for maximal GRDC. Moreover, the presence of UAS overcame the effects of leuV promoter mutations, which abolished GRDC of the leuV core promoter. However, although the presence of putative FIS binding site was essential for optimal GRDC, both mutant and wild-type leuV promoters containing UAS showed improved GRDC in a fis mutant background, suggesting that FIS protein is an important but not unique participant in the regulation of the leuV promoter.

Membrane lymphotoxin (LT) complex is a trimer composed of two subunits , LT-alpha and LT-beta of which the latter is a 33-kDa transmembrane protein. The LT-beta gene is expressed in lymphoid cells and organs, but little is known about its inducible regulation. Previously, the surface expression of LT-beta in Jurkat cells has been shown to increase in response to PMA. In this report, we used this model to study the transcriptional control of the human and murine LT-beta genes. PMA strongly induced the expression of LT-beta mRNA, and the level of induction was not changed markedly by cycloheximide (CHX) treatment. The LT-beta promoter region contains conserved Egr-1, nuclear factor (NF)-kappaB, and Ets binding sites, and PMA-inducible factors bound to these sites were detected by the gel-retardation technique (electrophoretic mobility shift assay (EMSA)). To identify sequences involved in transcriptional control, sets of human and mouse promoter-chloramphenicol acetyltransferase (CAT) constructs were generated and assayed by transient transfections. The PMA response was lost after deletion of the distal Ets binding site at -110. Mutations at either the Ets or NF-kappaB sites that prevented factor binding dramatically reduced PMA-inducible promoter activity, suggesting cooperative interaction between corresponding transcription factors in PMA activation. Mutation at the Egr-1 site also resulted in substantial loss of promoter activity, and the residual activity may be attributed to binding of constitutively expressed Sp-1 to the same site. We propose that the interaction between the members of NF-kappaB and Ets families of transcription factors and their cognate sites in the promoter is the major determinant of inducible expression of the LT-beta gene in Jurkat cells.

Prolonged treatment of AIDS patients with azidothymidine results in the development of resistance to the drug which correlates with the appearance of point mutations in the reverse transcriptase (RT) coding region within the HIV-1 pol gene. Kinetic studies of interactions of wild type RT and its mutants harbouring the above mutations with substrates and azidothymidine 5'-triphosphate (AZTTP) have been carried out. The complete mutant containing all the above described mutations possess the highest resistance on all the templates tested. Significant increases in resistance for mutants 67,70,215 and 67,215 on all the templates have also been observed. Inhibition of mutant enzymes by AZTTP depends on the template used.

Tumor necrosis factor alpha (TNF-alpha) and soluble lymphotoxin (LT) (also called LT-alpha or TNF-beta) are cytokines with similar biological activities that are encoded by related and closely linked genes. TNF-alpha, a mediator of the inflammatory response, exists in soluble and transmembrane forms. LT-alpha can be secreted or retained at the cell surface by binding to a 33-kDa transmembrane subunit, LT-beta. The recently cloned human LT-beta gene encodes another TNF family member and is linked to the TNF/LT locus within the major histocompatibility complex locus. The cell surface LT is a heterotrimer consisting of LT-alpha and LT-beta, whose physiological function is not yet clearly defined. We now report the sequence analysis of the genomic region and cDNA of murine LT-beta gene, which is closely associated with the TNF-alpha and LT-alpha genes within the murine major histocompatibility complex locus. Unlike the TNF-alpha, LT-alpha, and human LT-beta genes, which contain four exons, the murine LT-beta contains three exons and encodes a 244-amino acid polypeptide with a 66-amino acid insert that is absent from the human homologue. In situ hybridization demonstrates constitutive expression of LT-beta in lymphoid and hematopoietic tissues. LT-beta transcription is maximal in the thymic medulla and in splenic white pulp. LT-beta mRNA is also detected in the skin and in specific regions of the brain. The LT-beta promoter region contains putative Ets-binding sites, suggesting that the expression of LT-beta may be regulated in part by Ets transcription factors whose pattern of lymphoid expression overlaps that of LT-beta.

The interactions of HIV-I reverse transcriptase with some alkaloids were studied. Among nine compounds tested three--berberine, palmatine and sanguiritrine--inhibited RT. The dependence of the inhibition on the type of template-primer was also demonstrated. The kinetic analysis as well as circular dichroism experiments suggest the complex mechanism of RT inhibition by alkaloids. This mechanism includes both enzyme-alkaloid and alkaloid-template interactions; the latter effect also results in RT inhibition.

Prolonged therapy of AIDS patients with azidothymidine results in the development of resistance to the drug. This phenomenon is accompanied by the appearance of point mutations in the pol gene coding for reverse transcriptase (RT). Kinetic studies of interactions of wild type RT and its forms containing the above-mentioned mutations with substrates and azidothymidine 5'-triphosphate have been carried out. Considerable differences in the affinities of RT and the mutants for RNA and DNA heteropolymer templates were established. The mutations did not affect the RT affinity for dNTP; however, its location influenced considerably the inhibition of the reaction with azidothymidine 5'-triphosphate.

To investigate the biochemical basis of the HIV-1 resistance to AZT we obtained the RT mutant containing four amino acid substitutions by an oligonucleotide-directed mutagenesis technique. Enzymatic properties of the wild type and mutant RTs were compared. 'AZT-resistant' mutations in RT were shown to be associated with the reduced capability of AZT-TP to block the DNA- but not RNA-directed DNA synthesis.

Lys-172 residue of bacteriophage T7 RNA polymerase (T7RP) was substituted for Leu and Gly and Lys-172, Arg-173 were deleted by the site-directed mutagenesis using synthetic oligonucleotides. The specific activity of all mutant enzymes did not differ significantly from that of the wild-type (w.t.) T7RP while for Gly-172 mutant (G172) it was somewhat lower. Leu-172 (L172) and deletion (DEL172-3) mutants were able to direct RNA synthesis on the templates lacking the T7 promoter. DEL172-3 was not able to synthesize extraneous RNA sequences in addition to the expected run-off transcripts. L172 and DEL172-3 mutants revealed altered template specificity toward various DNA templates and showed the lower stability of enzyme-promoter complexes. The possible role of Lys-172 likely belonging to an interdomain "stretch" is discussed.