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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 CDC9 gene of Saccharomyces cerevisiae encodes a DNA ligase, and we have determined the nucleotide sequence of a 3.85 kb fragment of DNA which encompasses the convergently transcribed CDC9 and CDC36 genes. S1 nuclease mapping has revealed a major 5' end for the CDC9 mRNA, and one major and one minor site for 3' polyadenylation. These two sites lie within the C-terminal coding region of the CDC36 gene, implying that these two genes are transcribed from overlapping sequences. An interesting structural feature of the CDC9 gene is a series of 6 hexanucleotide repeats (ATGATT) which occur within the 650 bp immediately upstream from the site of transcription initiation. These repeat elements may be implicated in the cell division cycle regulated expression of CDC9. Comparison of the predicted amino acid sequence of the yeast DNA ligase (Mr 84,806) with the sequences of the T4 and T7 bacteriophage DNA ligases reveals little similarity except for a stretch of approximately 45 amino acids, comprising 3 short homologous segments. This region may represent an ATP-binding domain common to polynucleotide ligases.
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PMID:The nucleotide sequence of the DNA ligase gene (CDC9) from Saccharomyces cerevisiae: a gene which is cell-cycle regulated and induced in response to DNA damage. 390 3

The major 5'-termini of human adenovirus type 2 early gene block 4 mRNA were sequenced. Poly(A+) polyribosomal RNA was isolated from Ad2 early infected cells, the 5'-terminal m7GPPP removed and the 5'-OH of the penultimate 2'-0-methylated nucleotide labeled with [gamma-32P]ATP using polynucleotide kinase. Ad2 E4 mRNA was purified by hybridization to the Ad2 EcoRI-C fragment and was digested with RNase T1. The resulting oligonucleotides were resolved by two dimensional paper electrophoresis-homochromatography. Four major and 3-4 minor 5'-terminal sequences were identified and characterized. The sequence of the 5'-terminal structures of the major four termini are: (1) m7GpppUmU(m)UUACACUGp, (2) m7GpppUmU(m)UACACUGp, (3) m7GpppUmU(m)ACACUGp, and (4) m7Gppp(m6)AmC(m)ACUGp. These major 5'-terminal sequences were aligned with nucleotide 325, 326, 327, and 329 from the righthand end of the known Ad2 DNA sequence (1) in the region mapped as the 5'-terminus of E4 mRNA by electron microscopy (2,3) and S1 nuclease-gel (4) mapping. Two potential ribosomal binding sites and an initiator codon were found at 40 to 65 nucleotides and about 80 nucleotides, respectively, from these heterogenous 5'-termini. Ad2 E4 major mRNA species appear to be unique since mRNA molecules initiate at a pyrimidine, perhaps by RNA polymerase stuttering, or they are products of an unusual type of RNA processing.
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PMID:Nucleotide sequences and mapping of novel heterogenous 5'-termini of adenovirus 2 early region 4 mRNA. 616 92

The in vitro initiation site for RNA polymerase I on the mouse rRNA gene was identified using a new method that is generally applicable to the study of other eukaryotic transcripts. First, the 5' end of mouse rRNA was located to an ApC . . . by high resolution S1 nuclease mapping. Dinucleotide primers were then used in transcription reactions to demonstrate that this position is the actual de novo initiation site, and not a rapid RNA processing site. For this analysis, initiation was inhibited by reduced rXTP concentration, and, upon supplementation with various dinucleotides, only ApC stimulated correct synthesis. Independently confirming its role as the initiating nucleotide, ATP was shown to be required at a much higher concentration than the other rXTPs for RNA initiation, but not for elongation. These results also demonstrate a marked sequence conservation of rRNA initiation sites between the mouse and frog, two species that violate the general rule of species specificity in RNA polymerase I initiation. Extending these studies to RNA polymerase III, the initiation site for 5 S RNA can be similarly located by dinucleotide analysis and confirmed from the concentration requirements of each rXTP. In addition to allowing initiation at suboptimal rXTP concentration, dinucleotide primers can also circumvent the need for a factor normally required for initiation, suggesting their potential value in dissecting the mechanism of eukaryotic transcription.
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PMID:Dinucleotide primers facilitate convenient identification of the mouse ribosomal DNA transcription initiation site. A general method for analysis of transcription by RNA polymerases I and III. 631 12

A cDNA complementary to the mRNA of the ADP/ATP carrier from Neurospora crassa was identified among ordered cDNA clones by hybridizing total polyadenylated RNA to pools of 96 cDNA recombinant plasmids and subsequent cell-free translation of hybridization-selected mRNA. Further carrier cDNAs were found by colony filter hybridization at a frequency of 0.2-0.3%. The gene of the carrier was cloned and isolated on a 4.6-kbp EcoRI fragment of total Neurospora DNA, and the start of the mRNA was determined by S1 nuclease mapping. From the nucleotide sequence of the cDNA and the genomic DNA, the primary structure of the gene, of the mRNA and of the ADP/ATP carrier protein could be deduced. The gene occurs in a single copy in the genome and related genes are absent. It contains two short introns, and a pyrimidine-rich promoter region. The mRNA has a 46-bp 5' end and a 219-bp 3' end. There is an open reading frame coding for the 313 amino acid residues of the Neurospora carrier protein. The amino acid sequence is homologous in 148 positions with the established primary structure of the beef heart carrier.
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PMID:Nucleotide sequence of the cloned mRNA and gene of the ADP/ATP carrier from Neurospora crassa. 632 69

Various plant viral RNAs possess a 3' terminus with tRNA-like properties. These viral RNAs are charged with an amino acid upon incubation with the cognate aminoacyl-tRNA synthetase and ATP. We have studied the structure of end-labelled 3'-terminal fragments of turnip yellow mosaic virus RNA and brome mosaic virus RNA 2 with chemical modifications of the adenosine and cytidine residues and with enzymatic digestions using RNase T1, nuclease S1 and the double-strand-specific ribonuclease from cobra venom. The data indicate that the 3' termini of these plant viral RNAs lack a cloverleaf structure as found in classical tRNA. The three-dimensional folding, however, reveals a striking resemblance with classical tRNA. The models proposed are supported by phylogenetic data. Apparently distinct three-dimensional solutions have evolved to meet the requirements for faithful recognition by tRNA-specific enzymes. The way in which the aminoacyl acceptor arms of these tRNA-like structures are constructed reveal novel features in RNA folding which may have a bearing on the secondary and tertiary structures of RNA in general. The dynamic behaviour of brome mosaic virus RNA 2 in solution presumably is illustrative of conformational transitions, which RNAs generally undergo on changing the ionic conditions.
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PMID:Three-dimensional models of the tRNA-like 3' termini of some plant viral RNAs. 662 63

The specificity of transcription of Euglena gracilis Z chloroplast DNA by chloroplast DNA-dependent RNA polymerase in a transcriptionally active chromosome (Hallick, R.B., Lipper, C., Richards, O.C., and Rutter, W.J. (1976) Biochemistry 15, 3039-3045) has been studied. RNA molecules are both initiated and elongated in vitro. The RNA transcripts have been characterized as to their size, nuclease sensitivity, 5'-terminal oligonucleotides, and coding locus on the chloroplast genome. RNA labeled in vitro at the 5' end with [gamma-32P]ATP was digested with RNase T1, RNase A, and S1 nuclease. The resulting 5'-gamma-32P-oligonucleotides were fractionated by gel electrophoresis. In each case, one or two discrete products were obtained, consistent with initiation in vitro only at defined loci. RNA labeled in vitro with [alpha-32P]ATP or CTP has been hybridized to Southern (Southern, E.M. (1975) J. Mol. Biol. 98, 503-517) transfers of restriction endonuclease fragments of chloroplast DNA. The most abundant in vitro transcripts hybridize to chloroplast DNA fragments coding for 23 S, 16 S, and 5 S rRNAs. Only the coding strands of the rRNA genes are transcribed. Non-rDNA sequences of chloroplast DNA are also selectively transcribed but at much lower levels. The transcriptionally active chromosome has proved to be an ideal biochemical preparation for the study of selective transcription of cell organelle DNA.
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PMID:Selective in vitro transcription of Euglena chloroplast ribosomal RNA genes by a transcriptionally active chromosome. 676 27

By making operon fusions with lambda placMu53, we identified, cloned, and analyzed the phoH gene belonging to the phosphate (pho) regulon. We mapped the phoH gene at 23.6 min in the Escherichia coli genomic library (Y. Kohara, K. Akiyama, and K. Isono, Cell 50:495-508, 1987). Its nucleotide sequence revealed an open reading frame of 354 amino acids which contains sequences for nucleotide-binding motifs. From comparison of the DNA sequences, phoH was found to be identical to psiH, which had been identified as a phosphate starvation-inducible gene (W.W. Metcalf, P.M. Steed, and B.L. Wanner, J. Bacteriol. 172:3191-3200, 1990). The PhoH protein was overproduced by the T7 promoter system, identified as a protein of about 39 kDa, and purified. The amino-terminal amino acid sequence of the PhoH protein agreed with the one deduced from the DNA sequence. We demonstrated that PhoH has an ATP-binding activity by a photoaffinity labeling experiment. Two transcriptional initiation sites (P1 and P2) were identified by S1 nuclease mapping. The upstream P1 promoter contains a pho box, the conserved sequence shared by the pho regulon genes. The region containing the pho box was bound by PhoB protein, the transcriptional activator of the pho regulon, as revealed by footprinting. Regulation of phoH expression in vivo was studied by constructing plasmids containing transcriptional fusions of the phoH promoters with a promoterless gene for chloramphenicol acetyltransferase. Transcription from the P1 promoter required the phoB function and was induced by phosphate limitation, while transcription from the P2 promoter was independent of phoB and constitutive under tested conditions.
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PMID:Molecular analysis of the phoH gene, belonging to the phosphate regulon in Escherichia coli. 844 94

In order to determine the relative quantities of different HLA-A and -B mRNAs in cells, we have developed a simple and reliable method by using reverse transcription-polymerase chain reaction (RT-PCR), denaturing gradient gel electrophoresis (DGGE) and phosphor imaging analysis. Cytoplasmic RNA from lymphoblastoid cell lines with well-characterized HLA phenotypes are reversely transcribed with a primer specific for all HLA-A and -B antigens. The first-strand cDNA is used as template for quantitative PCR. The primer pair used for quantitative PCR are specific for all class I HLA and one of the primers is labeled with [gamma-32P]ATP. The amplified sequences include parts of exon 2 and exon 3 which contain most polymorphic residues in class I HLA molecules. The RT-PCR products containing the amplified HLA-A and -B sequences are separated by DGGE. The radioactivities of different DNA bands separated in denaturing gradient polyacrylamide gels are measured by phosphor imaging and used to determine the relative amounts of HLA-A and -B mRNAs. This approach is validated by using samples containing known quantities of different HLA-A and -B mRNA transcripts and confirmed by S1 nuclease protection assay. The combined RT-PCR/DGGE approach therefore provides a simple and reliable method for quantitation of relative amounts of different HLA-A and -B mRNAs in cells. This method should also be useful for studying the expression of other highly conserved and duplicated genes.
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PMID:Measurement of relative quantities of different HLA-A and -B mRNAs in cells by reverse transcription-polymerase chain reaction and denaturing gradient gel electrophoresis. 913 31

The complete nucleotide sequence for the aot operon of Pseudomonas aeruginosa PAO1 was determined. This operon contains six open reading frames. The derived sequences for four of these, aotJ, aotQ, aotM, and aotP, show high similarity to those of components of the periplasmic binding protein-dependent ABC (ATP binding cassette) transporters of enteric bacteria. Transport studies with deletion derivatives established that these four genes function in arginine-inducible uptake of arginine and ornithine but not lysine. The aotO gene, which encodes a polypeptide with no significant similarity to any known proteins, is not essential for arginine and ornithine uptake. The sixth and terminal gene in the operon encodes ArgR, which has been recently shown to function in regulation of arginine metabolism. Studies with an aotJ::lacZ translational fusion showed that expression of the aot operon is strongly induced by arginine and that this effect is mediated by ArgR. S1 nuclease and primer extension experiments showed the presence of two promoters, P1 and P2. The downstream promoter, P2, is induced by arginine and appears to be subject to carbon catabolite repression. The upstream promoter, P1, is induced by glutamate. Footprinting experiments established the presence of a 44-bp ArgR binding site that overlaps the -35 region for P2, as was shown to be the case for the arginine-inducible aru promoter, and the -10 region for P1, as was shown to be the case for arginine-repressible operons in P. aeruginosa. Sequence alignment confirms the architecture and the consensus sequence of the ArgR binding sites, as was previously reported.
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PMID:Molecular characterization and regulation of an operon encoding a system for transport of arginine and ornithine and the ArgR regulatory protein in Pseudomonas aeruginosa. 979 Nov 3

To gain insights into inefficient allele exchange in mycobacteria, we compared homologous pairing and strand exchange reactions promoted by RecA protein of Mycobacterium tuberculosis to those of Escherichia coli RecA protein. The extent of single-stranded binding protein (SSB)-stimulated formation of joint molecules by MtRecA was similar to that of EcRecA over a wide range of pH values. In contrast, strand exchange promoted by MtRecA was inhibited around neutral pH due to the formation of DNA networks. At higher pH, MtRecA was able to overcome this constraint and, consequently, displayed optimal strand exchange activity. Order of addition experiments suggested that SSB, when added after MtRecA, was vital for strand exchange. Significantly, with shorter duplex DNA, MtRecA promoted efficient strand exchange without network formation in a pH-independent fashion. Increase in the length of duplex DNA led to incomplete strand exchange with concomitant rise in the formation of intermediates and networks in a pH-dependent manner. Treatment of purified networks with S1 nuclease liberated linear duplex DNA and products, consistent with a model in which the networks are formed by the invasion of hybrid DNA by the displaced linear single-stranded DNA. Titration of strand exchange reactions with ATP or salt distinguished a condition under which the formation of networks was blocked, but strand exchange was not significantly affected. We discuss how these results relate to inefficient allele exchange in mycobacteria.
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PMID:RecA protein of Mycobacterium tuberculosis possesses pH-dependent homologous DNA pairing and strand exchange activities: implications for allele exchange in mycobacteria. 1007 73


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