Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.1.30.1 (S1 nuclease)
 3,660 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Studies with molecular and immunological techniques identified and mapped the transcript encoding glycoprotein D (gD) of equine herpesvirus 1 KyA, as well as two continuous gD antigenic determinants. Three mRNA species of 5.5, 3.8, and 1.7 kb overlap the gD open reading frame and are transcribed from the DNA strand encoding gD. Northern (RNA) blot hybridization with both DNA clones and riboprobes, as well as S1 nuclease analyses, showed the 3.8-kb mRNA to encode gD and to be synthesized as a late (beta-gamma) transcript. The 3.8-kb gD mRNA initiates within the US segment 91 and 34 nucleotides downstream of the CCAAT and TATA elements, respectively, and encodes a potential polypeptide of 392 amino acids. The termination site of this transcript maps within the terminal repeat at a site also used by the 5.5-kb mRNA and the IR6-encoded 1.2-kb mRNA, such that these three transcripts form a 3'-coterminal nested set. The extended size (2,250 nucleotides) of the 3' untranslated region of the gD transcript and its termination within the terminal repeat may result from the deletion of 3,859 bp, which eliminates two consensus polyadenylation signals downstream of the gD open reading frame of EHV-1 KyA. Use of antisera to synthetic peptides of 19 amino acids (residues 4 to 22) and 20 amino acids (residues 267 to 285) in Western immunoblot analyses revealed that gD is present in EHV-1 virions as a 55-kDa polypeptide. In addition, these antisera detected the 55-kDa protein as well as 58- and 47-kDa polypeptides in infected-cell extracts at late times of infection. Residues 4 to 22 make up a continuous neutralizing epitope of gD, since incubation of equine herpesvirus 1 with the anti-19-mer serum prior to infection results in reduced numbers of plaques and reduced levels of virus-encoded thymidine kinase. Complement is not required for neutralization mediated by the anti-19-mer serum.
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PMID:Equine herpesvirus 1 glycoprotein D: mapping of the transcript and a neutralization epitope. 138 65

To investigate the regulatory processes involved in the expression of the D2 dopamine receptor gene, a rat genomic clone was isolated using a 21-mer oligonucleotide probe made of exon 1 sequences. A 1.3-kb region including all of exon 1, its 5'-flanking region, and part of intron 1 was sequenced. S1 nuclease analysis indicated three consecutive nucleotides as the main transcription start sites; several weaker sites were also noted between 321 and 363 nucleotides upstream from the 3' end of exon 1. The promoter region lacks TATA and CAAT boxes and is rich in G+C content with several putative Sp1 binding sites. Transient expression assays using chimeric constructs of D2 promoter deletion mutants-chloramphenicol acetyl-transferase gene in the neuroblastoma cell line NB41A3 which expresses D2 binding sites indicated strong transcription enhancing activity between nucleotides -75 and -30 and silencing activity between nucleotides -217 and -76. DNase I footprinting studies using nuclear extract from NB41A3 suggested Sp1 binding to its consensus sequence at nucleotide -48 but inhibition of Sp1 binding at nucleotide -86 by the extract. The D2 promoter could not induce transcription of the heterologous CAT gene in C6 glioma, embryonal NIH 3T3, or hepatic Hep G2 cells. It is concluded that the rat D2 gene shares with the human D1A dopamine receptor gene several features typical of "housekeeping" genes but they are both tissue-specific, regulated genes. Unlike the D1A gene, however, the D2 gene has a strong preference for transcription initiation to three consecutive nucleotides.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Analysis of the promoter region of the rat D2 dopamine receptor gene. 139 Jun 23

Antisense oligodeoxynucleotide (ODN), which are directed against the splice acceptor site of exon II of the regulatory gene tat of the human immunodeficiency virus type 1 (HIV-1), have been described. These 20-mer ODN's displayed moderate anti-HIV activity in vitro. Using the same antisense ODN (termed ODN-2), which was additionally modified and protected both at the 3'- and the 5'-terminus by two phosphorothioate internucleotide linkages, a strong anti-HIV activity (EC50: 2.7 micrograms/ml) could be measured in the HIV-1/CEM- and HIV-1/HeLa-T4+ cell system. The analogous ODNs which were protected only at one end were either inactive (up to 10 micrograms/ml) or displayed a low antiviral activity. Time kinetic studies revealed that the antisense ODN-2 reduced the release of HIV-1 already after an incubation time of 1 h. By applying S1 nuclease protection procedures, it could be established that the antisense ODN-2 inhibited splicing of high molecular weight transcript to the 2-kb tat mRNA in HIV-1-infected CEM cells. Transfection experiments with pU3R-III chloramphenicol acetyltransferase expression vector in HeLa-T4+ cells revealed that the antisense ODN-2 blocked the Tat protein-mediated transactivation process. In co-transfection experiments using pSV2tat72 or scrape loading studies with purified Tat, the transactivation was restored. These data indicate that the selected antisense ODN-2 displays its anti-HIV effect by blocking the splicing process leading to the functional 2-kb tat mRNA.
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PMID:Antisense oligodeoxynucleotide: inhibitor of splicing of mRNA of human immunodeficiency virus. 156 36

DNA oligonucleotides with the sequence corresponding to the plus strand origin of replication of the filamentous bacteriophage M13 are studied. Biochemical structure probing and UV melting studies, supplemented with initial NMR experiments, are used to investigate structural features of a 51-nucleotides long synthetic oligonucleotide and two oligonucleotides that are integral parts of this latter molecule. The results demonstrate the feasibility and complementarity of the use of methidiumpropyl.EDTA-Fe(II) and nuclease S1 in the structural analysis of small oligonucleotides. The bacteriophage origin region appears to comprise two hairpins. The first hairpin, which contains a cleavage site for the bacteriophage gene II protein, has a large and probably flexible loop. NMR as well as UV melting studies demonstrate that the second hairpin contains a stable three-membered loop. Both hairpins are present in the 51-mer, which forms a stable tertiary structure.
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PMID:Biochemical and biophysical studies on the folding of the core region of the origin of replication of bacteriophage M13. 239 37

Using two 33-mer synthetic oligonucleotides derived from the amino acid sequence of the catalytic site of estradiol 17 beta-dehydrogenase (E2DH) and polyclonal antibodies raised against the enzyme purified from human placenta, clones were isolated from a lambda gt11 human placental cDNA library. A 327-amino acid sequence was deduced from cDNA sequencing. Two mRNA species have been identified in poly(A)+ RNA from human placenta, a major species migrating at 1.3 kb while a minor one is found at approx. 2.2 kb. Primer extension and S1 nuclease analysis indicate that the major mRNA species starts 9-10 nucleotides while the minor mRNA starts 971 nucleotides upstream from the ATG initiating codon, respectively. Sequence analysis of the longest cDNA clone (2092 bp) shows that it possesses identical coding and non-coding sequences in the regions of overlap with the shorter cDNA clones. The 32P-labeled 5' non-coding fragment hybridizes only to the 2.2 kb band, thus providing evidence for the existence of two distinct mRNA species which differ in their 5' noncoding regions. Using hp E2DH-36 cDNA as a probe for in situ hybridization of translocated chromosomes, the human E2DH gene was localized to the q11-q12 region of chromosome 17.
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PMID:Characterization of two mRNA species encoding human estradiol 17 beta-dehydrogenase and assignment of the gene to chromosome 17. 248 19

Eubacterium sp. strain VPI 12708 is an anaerobic intestinal bacterium that has inducible bile acid 7-dehydroxylation activity. At least four new polypeptides were synthesized after addition of primary bile acids to the growth medium. One of these, of molecular weight 27,000 (P-27), was shown to be involved in the 7-dehydroxylation reaction sequence. The gene coding for P-27 was cloned, and the entire DNA sequence for the protein-coding region was determined. In addition, sequence information was obtained for 294 bases upstream from the translational start codon and 329 bases downstream from the stop codon. Induction studies with a synthetic oligonucleotide probe (16-mer) revealed the presence of a cholic acid-inducible mRNA species approximately 900 bases long. A 5' terminus of this mRNA was detected by primer extension analysis, and the location of the 3' terminus of the mRNA was estimated by using S1 nuclease mapping. The 3' terminus of the mRNA contained a large element with dyad symmetry of unknown function. The open reading frame contained 249 codons, and the corresponding polypeptide had a calculated molecular weight of 26,745. The amino acid sequence of P-27 showed significant homology to several previously described alcohol-polyol dehydrogenases ("nonzinc" dehydrogenases), especially in the region believed to contain a pyridine nucleotide-binding domain. The implications of this homology and the possible function of P-27 in bile acid 7-dehydroxylation are discussed.
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PMID:Nucleotide sequence and regulation of a gene involved in bile acid 7-dehydroxylation by Eubacterium sp. strain VPI 12708. 283 20

We isolated and characterized LP1.2, a mouse myeloma mutant with a deletion of at least 4 kilobases (kb) immediately 3' of the alpha gene and introduction of at least 5 kb of novel (nonimmunoglobulin) sequence in its place. A 6.2-kb genomic EcoRI fragment from the mutated allele was cloned, and a subfragment was sequenced. The deletion begins 11 base pairs (bp) beyond the normal site of cleavage and polyadenylation for the secreted form of alpha mRNA. A short direct repeat, eight copies of the 17-mer GCCT ATAGAAGTAAGGA, is located at the junction of the alpha and novel sequences. The first 4 bp of the 17-mer are identical to the last 4 bp of the alpha sequence. Novel sequences downstream of the direct repeats in LP1.2 include a low-copy-number sequence flanked by two distinct, highly repetitive elements. The low-copy-number portion of the novel sequence appears on a single 30-kb EcoRI fragment in several myelomas and in liver DNA; one copy of this fragment has rearranged in cell line W3129, and this allele has rearranged a second time in LP1.2. LP1.2 contains low levels of apparently normal alpha protein and mRNA. The S1 nuclease protection of nuclear and cytoplasmic RNAs shows that cleavage and polyadenylation are efficient and accurate and that they occur without the accumulation of aberrant transcripts. Alpha transcription in isolated nuclei is decreased sevenfold in LP1.2 relative to its parent, which accounts for the low steady-state levels of cytoplasmic alpha mRNA and protein in LP1.2. Decreased alpha transcription could result either from the deletion of a positive regulator in the 3' flanking region or from the introduction of novel sequences which exert a negative effect.
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PMID:Myeloma mutant with a novel 3' flanking region: loss of normal sequence and insertion of repetitive elements leads to decreased transcription but normal processing of the alpha heavy-chain gene products. 302 10

P450c17 is the single enzyme mediating both 17 alpha-hydroxylase (steroid 17 alpha-monooxygenase, EC 1.14.99.9) and 17,20 lyase activities in the synthesis of steroid hormones. It has been suggested that different P450c17 isozymes mediate these activities in the adrenal gland and testis. We sequenced 423 of the 509 amino acids (83%) of the porcine adrenal enzyme; based on this partial sequence, a 128-fold degenerate 17-mer was synthesized and used to screen a porcine adrenal cDNA library. This yielded a 380-base cloned cDNA, which in turn was used to isolate several human adrenal cDNAs. The longest of these, lambda hac17-2, is 1754 base pairs long and includes the full-length coding region, the complete 3'-untranslated region, and 41 bases of the 5'-untranslated region. This cDNA encodes a protein of 508 amino acids having a predicted molecular weight of 57,379.82. High-stringency screening of a human testicular cDNA library yielded a partial clone containing 1303 identical bases. RNA gel blots and nuclease S1-protection experiments confirm that the adrenal and testicular P450c17 mRNAs are indistinguishable. These data indicate that the testis possesses a P450c17 identical to that in the adrenal. The human amino acid sequence is 66.7% homologous to the corresponding regions of the porcine sequence, and the human cDNA and amino acid sequences are 80.1 and 70.3% homologous, respectively, to bovine adrenal P450c17 cDNA. Both comparisons indicate that a central region comprising amino acid residues 160-268 is hypervariable among these species of P450c17. Comparison of the amino acid sequence of P450c17 with two other human steroidogenic cytochromes P450 show much greater homology with P450c21 (28.9%), another microsomal enzyme, than with P450scc (12.3%), a mitochondrial enzyme.
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PMID:Cytochrome P450c17 (steroid 17 alpha-hydroxylase/17,20 lyase): cloning of human adrenal and testis cDNAs indicates the same gene is expressed in both tissues. 302 70

Eukaryotic protein synthesis initiation factor 4A (eIF-4A), a 46-kDa polypeptide, is involved both in mRNA cap recognition and in the binding of mRNA to 40S ribosomal subunits. A 41-mer oligodeoxynucleotide probe was synthesized complementary to a portion of the published coding sequence of eIF-4A mRNA [Nielsen et al., Nucleic Acids Res. 13 (1985) 6867-6870] and used to screen a mouse genomic library. We have isolated and characterized a full-length clone from that library. The eIF-4A sequence is contained in eleven exons. The eleventh exon also has the 3'-nontranslated sequence and two separate polyadenylation sites. Northern-blot analysis of mouse poly(A)+RNA indicates that there are several distinct mRNA species coding for eIF-4A. Two of these contain the same coding sequence and differ only in the length of the 3'-nontranslated region. Two of the eIF-4A mRNAs are therefore likely to be the result of differential processing at the 3'-end. We have used a fragment of the genomic clone to measure the steady-state levels of eIF-4A mRNA during the induced differentiation of murine erythroleukemia cells. S1 nuclease protection experiments demonstrated that by the fourth day after induction eIF-4A mRNA declined to 25% of its steady-state level in uninduced cells. In contrast, the steady-state level of beta-globin mRNA increased dramatically during differentiation. In vitro transcription assays using nuclei isolated from uninduced and induced cells show that the rate of transcription of eIF-4A mRNA was 40% greater in differentiated cells, indicating a posttranscriptional component is involved in the regulation of the steady-state mRNA level.
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PMID:Isolation and mapping of a gene for protein synthesis initiation factor 4A and its expression during differentiation of murine erythroleukemia cells. 321 17

Endogenous oligonucleotides were found in trichloroacetic acid extracts of hamster lung fibroblasts and Tetrahymena cells. Peaks of radioactivity that eluted with retention times similar to oligonucleotide markers (5- to 50-mer) were found by HPLC in cells labeled briefly with 32Pi. Only minute amounts of UV-absorbing material were detected, consistent with a rapid turnover of phosphate groups. The 32P-labeled material also migrated as oligonucleotides on 20% polyacrylamide gels; it was not hydrolyzed by alkaline phosphatase but was digested by snake venom phosphodiesterase, S1 nuclease, and pancreatic RNase and was phosphorylated by T4 polynucleotide kinase. The 32P-labeled material isolated by HPLC was alkali labile and the hydrolyzate ran as nucleotides on paper chromatography. It is concluded that the oligonucleotides are mainly oligoribonucleotides, but it is possible that oligodeoxynucleotides are also present.
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PMID:Oligonucleotides with rapid turnover of the phosphate groups occur endogenously in eukaryotic cells. 347 Jul 67


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