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:3.1.30.1 (S1 nuclease)
3,660 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The control of ribosomal RNA (rRNA) gene expression during development can be productively studied by examination of the relationship between promoter structure and function as well as the processing of primary transcripts. Toward this end total cell RNA was extracted from embryos at various stages and probed with cloned rRNA genes using the "dot blot" method. This exercise showed that rRNA gene expression is a stage-specific process and is thus under developmental control. S1 nuclease protection experiments localized fourteen different upstream DNA sites encoding 5'-termini of pre-rRNAs during this synthetic phase of development. There is no indication of any spacer fail-safe terminator function. The S1 approach contributed to the sequencing of several of the sites. Comparative sequence alignments reveal short conserved regions in DNAs corresponding to these sites, which are shown to fall into two structural classes. Sites 3, 4, 6 and 9 are proposed to function in transcription initiation and are found to have the consensus sequence 5'...T-A-T-A-T-Pu-Pu-Pu-G-Pu-Pu-G-T-C-A 3'. Sites 1, 2, 5 and 8 which are proposed to function in 5'-processing have the consensus sequence; 5'...Pu-G-T-Pu-T-T-G 3'. These short sequence conserved regions are hypothesized to serve as recognition signals for proteins within the rDNA transcription initiation complex and for 5'-processing enzymes, respectively. Sequencing of the intergenic spacer region from which a model for spacer evolution is derived shows that tandem ca 600 bp subrepeats explain much of the multiplicity observed within control sites.
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PMID:In vivo transcription from multiple spacer rRNA gene promoters during early development and evolution of the intergenic spacer in the brine shrimp Artemia. 303 91

The genomic location of the gene(s) which provides vaccinia virus (VV) alpha-amanitin-resistant mutants with a drug-resistant phenotype have been mapped to the HindIII N/M region of the genome by the use of marker rescue techniques [E. C. Villarreal and D. E. Hruby (1986) J. Virol. 57, 65-70]. Nucleotide sequencing of a 2356-bp HindIII-Sau3A fragment of the vaccinia virus genome encompassing this region reveals the presence of two complete leftward-reading open reading frames (ORFs, N2 and M1) and two incomplete ORFs (N1 and M2). By computer analysis the N2 and M1 ORFs would be predicted to encode soluble VV polypeptides with molecular weights of approximately 20 and 48 kDa, respectively. The N2 and M1 ORFs have extremely A-T-rich 5'-proximal sequences, consistent with previous data regarding the location and A-T-richness of viral early promoters. Likewise, the consensus signal believed to be involved in terminating VV early gene transcription, TTTTTNT, was evident at the 3'-boundary of both the N2 and M1 ORFs suggesting that these genes may be VV early genes. The in vivo transcriptional activity, orientation, and limits of these putative transcriptional units were investigated by Northern blot, nuclease S1, and primer extension analysis. Both N2- and M1-specific transcripts were detected in the cytoplasm of VV-infected cells, suggesting that these loci are bonafide viral genes. Time-course nuclease S1 experiments revealed that the N2 gene was transcribed exclusively prior to VV DNA replication. In contrast, the M1 gene was transcribed throughout infection, although different start sites were used at early versus late times postinfection. These results are discussed in relation to the drug-resistant phenotype and future experiments to identify the viral gene product responsible.
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PMID:Nucleotide sequence and molecular genetic analysis of the vaccinia virus HindIII N/M region encoding the genes responsible for resistance to alpha-amanitin. 338 67

We have isolated a human gastrin gene from a genomic library by employing a human gastrin cDNA clone as a hybridization probe. The total length of the gene is approximately 4.0 kilobase pairs, and the gene is separated into three exons and two introns. A 130-base-pair intron interrupts the coding region and a 3.0-kilobase-pair intron is located in the 5' untranslated region. Nucleotide sequence analysis showed that all of the exon-intron boundaries follow the A-G/G-T consensus sequences. A putative transcription initiation site is assigned to the adenine 60 nucleotides upstream from the exon-intron junction on the basis of S1 nuclease protection mapping. A possible "TATA" equivalent sequence T-T-A-T-A-A is located 28 base pairs upstream from the transcription initiation site. A "CAT box" sequence, C-A-T-T, is located 99 nucleotides upstream of the transcription initiation site. A poly(A)-addition signal, A-A-U-A-A-A, is located 80 base pairs downstream from the termination codon. Comparison of the nucleotide sequences of the human cDNA and the genomic clone revealed that the aspartic acid codon at position 71 of preprogastrin is interrupted by the small intron (130 base pairs). The 3' region of the large intron contains a sequence of 300 nucleotides that is flanked by 15-nucleotide direct repeats. This sequence exhibits a striking homology to the human Alu-type sequence.
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PMID:Structural analysis of the gene encoding human gastrin: the large intron contains an Alu sequence. 608 40

The mom gene of bacteriophage Mu encodes a DNA modification function. Expression of this modification requires the host Escherichia coli Dam (DNA-adenine methylase) function and the transacting phage Mu Dad function. The mom gene was subcloned into a variety of sites on plasmid pBR322. Insertions were made into the HincII and PvuI sites within the amp gene and into the ClaI site of the tet gene promoter. The only clones found were those in which the orientation of the mom gene prevents its transcription from the vector promoter(s), suggesting that constitutive expression of mom from a foreign promoter can occur independently of Dad function but is lethal for the cell. Employing S1 nuclease mapping, we have identified two Mu mRNA transcripts: (1) the gin transcript extends into the gin-mon intercistronic divide and terminates downstream from the BclI site; and (2) the mom transcript appears to initiate about 74 bp upstream from the BclI site, 12 bp downstream from a promoter-like sequence. Production of the mom transcript is dependent on the host Dam activity and on Dad transactivation. In contrast, the gin transcript is produced independently of Dam and Dad functions; the gin transcript may extend into the mom gene, but it appears to be either degraded at the 3' end or differentially terminated. We propose that regulation of mom gene transcription involves both positive and negative regulatory proteins, and that binding of the Dad protein (a "late" Mu protein) is required for transcription initiation by the host RNA polymerase. However, Dad protein action may be inhibited by prior binding of a repressor to the mom operator, located farther upstream. We propose that this repressor (encoded by a phage or host gene) binds to the operator only when there is no active Dam enzyme present, i.e., when there is no methylation of (or methylase binding to) the G-A-T-C sites within the mom operator.
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PMID:S1 nuclease mapping of the phage Mu mom gene promoter: a model for the regulation of mom expression. 609 20

Six phage clones that contain sequences hybridizable with the small nuclear RNA U2 were isolated from a rat gene library. Of these clones, one which includes a candidate for a functional U2 RNA gene was selected and characterized. The sequence within the clone which hybridizes with rat U2 RNA was completely co-linear with that of the RNA. A T-A-T-A box was not found in the region of more than 400 base-pairs which lies upstream of the gene. However, several block homologies were found with the upstream sequences of a rat U1 RNA gene candidate cloned in our laboratory. An "identifier sequence", which was reported to be an element of gene regulation related to differentiation, was found downstream of the coding region at the same distance and with the same orientation as the identifier sequence located downstream of the U1 RNA gene candidate. We detected a presumed U2 RNA precursor elongated by about 11 nucleotides at the 3' end by S1 nuclease mapping using a fragment from the clone. A potential termination signal for transcription was found within the elongated region of the presumed precursor. Southern blot analysis suggests that families of U2 RNA genes that have conserved flanking sequences are present in the genomes of rat, mouse, man, calf and chicken.
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PMID:Molecular cloning and characterization of a gene for rat U2 small nuclear RNA. 619 79

Total steady-state RNA was extracted from nuclei of HeLa cells late after infection with adenovirus serotype 2. Most of the nuclear RNA is transcribed from the major late transcription unit (16.2 to 100.0 map units). To study the cleavage reactions involved in the splicing of leaders 1 and 2, we have used the S1 nuclease mapping technique with restriction fragments located in the region of intron 1 as DNA probes. The S1 mapping data showed that in total nuclear RNA, RNA species accumulate from which the 5' part of intron 1 has been excised, but which still contain the 3' part of the intron. This indicates that intron 1 can be removed in a stepwise fashion following the 5' to 3' direction. We have compared the nucleotide sequences from the ends of the putative processing intermediates. The internal cleavage sites do not resemble the consensus 5' or 3' splice site sequences. However, they show considerable homology to the sequence 5' A-T-G-A-T-G-G-C-A-T 3', which may act as a signal for internal cleavage. The intermediates are present in both the poly(A)+ and poly(A)- RNA fractions, although with different relative intensities. Primer extension experiments have been performed in which a primer, located with its left end in leader 2, is extended into intron 1. The results show that there may be a cleavage site as short as 35 nucleotides before the 3' splice site. Cleavage at the 3' splice site seems to be rapidly followed by ligation of leader 1 to leader 2. A model for RNA splicing based on these findings and data from the literature is presented.
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PMID:A model for the excision of introns 1 and 2 from adenoviral major late pre-messenger RNAs. 620 3

The nucleotide sequence of the yeast gene TRP5 and its 5' and 3' flanking regions was determined. The deduced coding sequence for tryptophan synthase contains 2,127 base pairs. The protein chain has a calculated molecular weight of 76,544. Yeast tryptophan synthase, a bifunctional protein, has a primary structure which corresponds to an Escherichia coli tryptophan synthase alpha chain-beta chain fusion. An NH2-terminal 239 amino acid segment of yeast tryptophan synthase is homologous with E. coli tryptophan synthase alpha subunit, while a distal 389 amino acid residue segment is homologous to the E. coli tryptophan synthase beta chain. This order of segments of the yeast enzyme is the reverse of the chromosomal order characteristic of all prokaryotes that have been examined. The two segments are joined by a connecting region of 28 residues in the yeast enzyme which is not homologous to either the alpha or beta chains of the bacterial enzyme. A portion of the connecting region of yeast tryptophan synthase exhibits nucleotide sequence similarity to the 3' terminus of E. coli trpC and the trpC-trpB intercistronic region. Active site cysteine, histidine, and lysine residues in the beta 2 subunit of E. coli tryptophan synthase are conserved in the yeast enzyme. Also conserved in the yeast enzyme are 6/8 amino acid residues having an important role in maintaining the structure and function of the E. coli tryptophan synthase alpha subunit. S1 nuclease mapping was used to identify three major mRNA transcripts with different 5' termini. Potential T-A-T-A sites for transcription initiation were identified, as well as other sequences that occur frequently in yeast genes. A 5' flanking region of TRP5 was shown by DNA/DNA hybridization to be present in multiple copies in the yeast genome. TRP5 mRNA levels, measured by RNA/DNA hybridization, increased 2- to 7-fold in response to starvation for either tryptophan or histidine, indicating transcriptional regulation.
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PMID:Yeast gene TRP5: structure, function, regulation. 627 87

The gene structure of a phenobarbital-inducible rat liver cytochrome P-50 was elucidated by sequence analysis of two isolated genomic clones. The total length of the gene was approximately 14 kilobases and was separated into nine exons by eight intervening sequences. Nucleotide sequences of all exon/intron boundaries follow the G-T/A-G rule. A putative transcription initiation site was assigned to an A, 30 base pairs upstream from the initiation codon by S1 nuclease protection mapping. A possible "TATA" equivalent sequence, C-A-T-A-A-A, was found 27 base pairs further upstream from this initiation site. A poly(A) attachment site was determined to be 386 or 387 base pairs downstream from the termination codon by comparison with the cDNA sequence. Detailed comparison with two cDNA sequences determined previously showed the coding nucleotide sequence of the genomic clones to concur with that of the pcP-450pb2 cDNA clone coding for cytochrome P-450e except for three neutral base substitutions. Therefore, we conclude that the gene sequence determined here is for the cytochrome P-450e gene or a similar gene. On the other hand, 40 base substitutions were found in about 1,900 base pairs compared between the sequences of the genomic clone and the other cDNA clones (pcP-450pbl and 4) coding for cytochrome P-450b, and 15 of them result in 14 amino acid replacements in the total 491 amino acid residues. These base substitutions occur in relatively limited regions of the sequences. Most of them are found in exons 6, 7, 8, and 9; most frequently in exon 7.
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PMID:Gene structure of a phenobarbital-inducible cytochrome P-450 in rat liver. 630 54

The chicken alpha 2 type I collagen gene is 38 kilobases long and its coding information is subdivided into more than 50 exons. In the current study, we used primer extension and S1 nuclease mapping to determine the sequence of the 5' end of alpha 2 collagen mRNA and to locate the start site for transcription of the alpha 2 collagen gene. The DNA sequence around the start site for transcription shows a typical Goldberg-Hogness sequence, 5' T-A-T-A-A-A-T 3', between -33 and -26 and a 5' G-C-C-C-A-T-T 3' sequence ("CAT" box) between -84 and -78. Three AUGs are found in the initial portion of the mRNA, the first from +54 to +56, the second from +117 to +119, and the third from +134 to +136. The first two AUGs are followed by short coding sequences that could specify a hexapeptide a tetrapeptide, respectively. Only the third AUG is followed by an open reading frame coding for a sequence that presents considerable homology with the previously determined amino acid sequence of prepro alpha 1 collagen. In the promoter region sequence there are several extensive dyads of symmetry. Three of these inverted repeats which precede the start site for transcription overlap each other and may have a role in the developmental regulation of this gene.
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PMID:Structure of the promoter for chicken alpha 2 type I collagen gene. 694 74

Sea urchin (psammechinus miliaris) H2A histone genes shown to be promoter mutants from oocyte injection experiments were tested for their ability to initiate transcription in vitro. Circular templates were transcribed with HeLa cell extracts, and the transcripts were assayed by mung bean or S1 nuclease mapping of the 5' ends. The transcripts of the H2A mutants produced in vitro were qualitatively similar and, in most cases, identical to those seen in oocyte injection experiments, but quite large quantitative differences were observed for some H2A mutant genes. Both the T-A-T-A box and far upstream sequences residing in the modulator segment E [Grosschedl, R. & Birnstiel, M. L. (1980) Proc. Natl. Acad. Sci. USA 77, 7102--7106] were found to be essential for maximal transcription in vitro. Deletion of either of these sequence elements reduced transcription to 20%. A similar reduction in the amount of H2a transcripts was found when a T-A-T-A-to-T-A-G-A point mutant was tested in vitro. Essential far upstream sequences were mapped between nucleotides -139 and -111, 5' to the initiation site of transcription. In the standard run-off transcription test using restriction fragments, the effects of these sequences could be mimicked by free DNA ends, suggesting that the function of this in vitro upstream sequence might be to provide an entry side for RNA polymerase II.
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PMID:Delimitation of far upstream sequences required for maximal in vitro transcription of an H2A histone gene. 695 85


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