Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.2.1.23 (beta-galactosidase)
14,648 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Functional elements of a vaccinia virus early promoter were characterized by making a complete set of single nucleotide substitutions, as well as more complex mutations, and assaying their effects on gene expression. Synthetic oligonucleotides, based primarily on the sequence of the 7.5-kD early promoter, were inserted into a plasmid vector containing the lacZ gene of Escherichia coli flanked by sequences from the thymidine kinase (TK) gene of vaccinia virus. The lacZ gene, under control of the synthetic promoter, was introduced into the vaccinia virus genome at the TK locus by homologous recombination, and each of the 331 different recombinant viruses thus obtained was assayed for beta-galactosidase expression. The relative amounts and precise 5' ends of lacZ mRNAs specified by a subset of the recombinants were determined by primer extension. Many promoters were tested for their ability to direct specific transcription in vitro. A generally good correlation was noted between measurements of promoter strength estimated by beta-galactosidase expression, primer extension of in vivo mRNA and transcription in vitro. A relatively simple picture emerged from the analysis. The early promoter consists of a 16 base-pair critical region, in which most single nucleotide substitutions have a major effect on expression, separated by 11 base-pairs of a less critical T-rich sequence from a seven base-pair region within which initiation with a purine usually occurs. For the critical region of the 7.5-kD promoter, AAAAgTaGAAAataTA, any substitution of an upper-case nucleotide reduced expression, usually drastically, whereas certain substitutions of lower-case nucleotides maintained or significantly enhanced expression. On the basis of this analysis, the wide range of activities of natural promoters could be attributed to the presence of one or more non-optimal nucleotides in the critical region. Moreover, single nucleotide substitutions in such promoters had the predicted enhancing effects. Most mutations in the critical region of the 7.5-kD promoter behaved independently, but some nucleotide substitutions compensated for potentially detrimental nucleotides at other positions. Promoters substantially stronger than any natural ones examined were constructed by combining several up-mutations within the critical region of the 7.5-kD promoter and by repeating the critical region sequence. Like the TATA box of eukaryotic RNA polymerase II promoters, the critical region specifies the site of transcriptional initiation.
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PMID:Structure of vaccinia virus early promoters. 251 86

Promoters were isolated at random from the genome of Saccharomyces cerevisiae by using a plasmid that contains a divergently arrayed pair of promoterless reporter genes. A comprehensive library was constructed by inserting random (DNase I-generated) fragments into the intergenic region upstream from the reporter genes. Simple in vivo assays for either reporter gene product (alcohol dehydrogenase or beta-galactosidase) allowed the rapid identification of promoters from among these random fragments. Poly(dA-dT) homopolymer tracts were present in three of five randomly cloned promoters. With two exceptions, each RNA start site detected was 40 to 100 base pairs downstream from a TATA element. All of the randomly cloned promoters were capable of activating reporter gene transcription bidirectionally. Interestingly, one of the promoter fragments originated in a region of the S. cerevisiae rDNA spacer; regulated divergent transcription (presumably by RNA polymerase II) initiated in the same region.
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PMID:Properties of promoters cloned randomly from the Saccharomyces cerevisiae genome. 284 31

Genomic sequences for the large subunit of human RNA polymerase II corresponding to a part of the fifth exon were inserted into an expression vector at the carboxy-terminal end of the beta-galactosidase gene. The in-frame construct produced a 125-kilodalton fusion protein, containing approximately 10 kilodaltons of the large subunit of RNA polymerase II and 116 kilodaltons of beta-galactosidase. The purified bacterially produced fusion protein inhibited specific transcription from the adenovirus type 2 major late promoter, while beta-galactosidase had no effect. This effect of the fusion protein was during RNA elongation, not at the level of initiation, resembling the faithfully initiated but incomplete transcripts produced with purified factors in the absence of SII. Similarly, monoclonal antibody 2-7B, which reacts with the RNA polymerase II region represented in the fusion protein, inhibited specific transcription at the level of elongation in a whole-cell extract. Both monoclonal antibody 2-7B and the fusion protein, although unable to inhibit purified RNA polymerase II in a nonspecific transcription assay, selectively blocked the stimulation elicited by transcription elongation factor SII on the activity of the purified enzyme in vitro. This suggests that the fusion protein traps the SII in nonstimulatory interactions and that antibody 2-7B inhibits SII binding to RNA polymerase II. Thus, this suggests that an SII-binding contact required for specific RNA elongation resides within the fifth exon region of the largest RNA polymerase II subunit.
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PMID:Transcription elongation factor SII interacts with a domain of the large subunit of human RNA polymerase II. 314 7

The effects of cis-diamminedichloroplatinum(II) (cis-DDP) and trans-DDP adducts on mammalian transcription in vivo have been investigated. A plasmid containing the beta-galactosidase (beta-gal) reporter gene was modified with either of the two platinum compounds and transfected into human or hamster cell lines. A 2-3 fold higher level of transcription was observed in both cell lines from plasmids containing trans-DDP adducts as compared to plasmids modified by cis-DDP. This difference in transcriptional activity was not decreased in human and rodent nucleotide excision repair deficient cell lines, indicating that more efficient excision repair of the trans-DDP adducts was not the cause of its lower ability to block transcription in this assay. For this conclusion to be valid, it is assumed that trans-DDP adducts are repaired primarily by the nucleotide excision repair pathway, as is the case with the adducts of cis-DDP. The possibility that trans-DDP adducts are preferentially bypassed by RNA polymerase was examined by monitoring the elongation of beta-gal mRNA on damaged templates in vivo. Nascent beta-gal mRNA transcripts were recovered from excision repair deficient xeroderma pigmentosum A cells transfected with platinated plasmids, and the extent of RNA synthesis was measured by using ribonuclease protection. Fourfold more trans-DDP than cis-DDP adducts were required to inhibit transcription elongation by 63%. RNA polymerase II bypassed cis- and trans-DDP DNA adducts with efficiencies of 0-16% and 60-70%, respectively. These data provide insight into the differential toxicity of the two platinum isomers.
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PMID:DNA adducts of cis-diamminedichloroplatinum(II) and its trans isomer inhibit RNA polymerase II differentially in vivo. 757 87

The SKI2 gene is part of a host system that represses the copy number of the L-A double-stranded RNA (dsRNA) virus and its satellites M and X dsRNA, of the L-BC dsRNA virus, and of the single-stranded replicon 20S RNA. We show that SKI2 encodes a 145-kDa protein with motifs characteristic of helicases and nucleolar proteins and is essential only in cells carrying M dsRNA. Unexpectedly, Ski2p does not repress M1 dsRNA copy number when M1 is supported by aN L-A cDNA clone; nonetheless, it did lower the levels of M1 dsRNA-encoded toxin produced. Since toxin secretion from cDNA clones of M1 is unaffected by Ski2p, these data suggest that Ski2p acts by specifically blocking translation of viral mRNAs, perhaps recognizing the absence of cap or poly(A). In support of this idea, we find that Ski2p represses production of beta-galactosidase from RNA polymerase I [no cap and no poly(A)] transcripts but not from RNA polymerase II (capped) transcripts.
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PMID:Evidence that the SKI antiviral system of Saccharomyces cerevisiae acts by blocking expression of viral mRNA. 832 Dec 35

Polycyclic aromatic hydrocarbons such as benzo(a)pyrene diol-epoxide (BPDE-I) cause hepatocellular carcinoma. To identify short-term carcinogen effects, we studied hepatocytes transfected with nonreplicating plasmids, adducted covalently with BPDE-I, varying in promoter structure and encoded reporter gene (beta-galactosidase or luciferase). BPDE inactivated gene expression as a first-order function of BPDE concentration in adduction reactions. No evidence of cytotoxicity, diminished coprecipitation and availability, enhanced nicking of supercoiled forms and reduced cellular uptake, or instability of adducted plasmids was observed. At low BPDE:plasmid ratios, inactivation occurred with 1 adduct/plasmid within a target 23-27% of plasmid bases. Using nuclear extracts and BPDE-adducted G-free cassette-encoding plasmids, the fraction of full-length RNA polymerase II-initiated transcripts also declined as a first-order function of BPDE concentration when approximately 3 adducts were distributed among 48% of plasmid bases. These observations suggest that carcinogens such as BPDE block mRNA transcription along DNA templates by forming limited numbers of persistent adducts at coding or noncoding sites.
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PMID:Inactivation of plasmid reporter gene expression by one benzo(a)pyrene diol-epoxide DNA adduct in adult rat hepatocytes. 848 14

The yeast two-hybrid system was used to isolate a clone from a 17-day-old mouse embryo cDNA library that codes for a novel 812-aa long protein fragment, glucocorticoid receptor-interacting protein 1 (GRIP1), that can interact with the hormone binding domain (HBD) of the glucocorticoid receptor. In the yeast two-hybrid system and in vitro, GRIP1 interacted with the HBDs of the glucocorticoid, estrogen, and androgen receptors in a hormone-regulated manner. When fused to the DNA binding domain of a heterologous protein, the GRIP1 fragment activated a reporter gene containing a suitable enhancer site in yeast cells and in mammalian cells, indicating that GRIP1 contains a transcriptional activation domain. Overexpression of the GRIP1 fragment in mammalian cells interfered with hormone-regulated expression of mouse mammary tumor virus-chloramphenicol acetyltransferase gene and constitutive expression of cytomegalovirus-beta-galactosidase reporter gene, but not constitutive expression from a tRNA gene promoter. This selective squelching activity suggests that GRIM can interact with an essential component of the RNA polymerase II transcription machinery. Finally, while a steroid receptor HBD fused with a GAL4 DNA binding domain did not, by itself, activate transcription of a reporter gene in yeast, coexpression of this fusion protein with GRIP1 strongly activated the reporter gene. Thus, in yeast, GRIP1 can serve as a coactivator, potentiating the transactivation functions in steroid receptor HBDs, possibly by acting as a bridge between HBDs of the receptors and the basal transcription machinery.
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PMID:GRIP1, a novel mouse protein that serves as a transcriptional coactivator in yeast for the hormone binding domains of steroid receptors. 864 9

Few promoters are active at high levels in all cells. Of these, the majority encode structural RNAs transcribed by RNA polymerases I or III and are not accessible for the expression of proteins. An exception are the small nuclear RNAs (snRNAs) transcribed by RNA polymerase II. Although snRNA biosynthesis is unique and thought not to be compatible with synthesis of functional mRNA, we have tested these promoters for their ability to express functional mRNAs. We have used the murine U1a and U1b snRNA gene promoters to express the Escherichia coli lacZ gene and the human alpha-globin gene from either episomal or integrated templates by transfection, or infection into a variety of mammalian cell types. Equivalent expression of beta-galactosidase was obtained from < 250 nucleotides of 5'-flanking sequence containing the complete promoter of either U1 snRNA gene or from the 750-nt cytomegalovirus promoter and enhancer regions. The mRNA was accurately initiated at the U1 start site, efficiently spliced and polyadenylylated, and localized to polyribosomes. Recombinant adenovirus containing the U1b-lacZ chimeric gene transduced and expressed beta-galactosidase efficiently in human 293 cells and airway epithelial cells in culture. Viral vectors containing U1 snRNA promoters may be an attractive alternative to vectors containing viral promoters for persistent high-level expression of therapeutic genes or proteins.
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PMID:Efficient expression of protein coding genes from the murine U1 small nuclear RNA promoters. 879 16

To analyze transcriptional control regions of Drosophila melanogaster housekeeping genes, we have characterized the promoter of the gene coding for the second-largest subunit of RNA polymerase II (DmRP140). Upstream of DmRP140 the genomic region harbors a gene which is transcribed in the opposite direction (DmRP140up). By determination of the transcription start sites of both genes we found a short non-transcribed intergenic region of 220 bp. Functional analysis of various promoter reportergene constructs by transient transfection of cultured cells revealed that sequences important for transcription of DmRP140 are located in the untranslated leader of the upstream gene. The onset of DmRP140 transcription during embryonic development was studied in transgenic flies using beta-galactosidase as reportergene. To distinguish between the maternally provided DmRP140 transcripts and the embryonically transcribed RNA the offspring of nontransformed females and male transformants was examined. The development of a sensitive detection assay based on a chemiluminescent substrate for beta-galactosidase allowed us to determine the onset of DmRP140 transcription to between 8-10 h after oviposition. Thus, DmRP140 transcription does not start following the transcriptional transition period between 2-3 h of development but occurs much later in embryogenesis coinciding with decreasing DNA synthesis and cell division rates.
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PMID:Transcription of DmRP140, the gene coding for the second-largest subunit of RNA polymerase II. 906 Oct 24

DNA topoisomerase I (topo I) from Drosophila melanogaster contains a nonconserved, hydrophilic N-terminal domain of about 430 residues upstream of the conserved core domains. Deletion of this N terminus did not affect the catalytic activity of topo I, while further removal of sequences into the conserved regions inactivated its enzymatic activity. We have investigated the cellular function of the Drosophila topo I N-terminal domain with top1-lacZ transgenes. There was at least one putative nuclear localization signal within the first 315 residues of the N-terminal domain that allows efficient import of the large chimeric proteins into Drosophila nuclei. The top1-lacZ fusion proteins colocalized with RNA polymerase II (pol II) at developmental puffs on the polytene chromosomes. Either topo I or the top1-lacZ fusion protein was colocalized with RNA pol II in some but not all of the nonpuff, interband loci. However, the fusion proteins as well as RNA pol II were recruited to heat shock puffs during heat treatment, and they returned to the developmental puffs after recovery from heat shock. By immunoprecipitation, we showed that two of the largest subunits of RNA pol II coprecipitated with the N-terminal 315-residue fusion protein by using antibodies against beta-galactosidase. These data suggest that the topo I fusion protein can be localized to the transcriptional complex on chromatin and that the N-terminal 315 residues were sufficient to respond to cellular processes, especially during the reprogramming of gene expression.
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PMID:Targeting to transcriptionally active loci by the hydrophilic N-terminal domain of Drosophila DNA topoisomerase I. 963 19


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