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:2.7.7.49 (reverse transcriptase)
31,746 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

We have investigated in detail the secondary and tertiary structures of the 16 S rRNA binding site of protein S8 using a variety of chemical and enzymatic probes. Bases were probed with dimethylsulfate (at A(N-1), C(N-3) and G(N-7)), with N-cyclohexyl-N'-(2-(N-methylmorpholino)-ethyl)-carbodiimide-p- toluenesulfonate (at G(N-1) and U(N-3)) and with diethylpyrocarbonate (at A(N-7)). The involvement of phosphates in hydrogen bonds or ion co-ordination was monitored with ethylnitrosourea. RNases T1, U2 and nuclease S1 were used to probe unpaired nucleotides and RNase V1 to monitor base-paired or stacked nucleotides. The RNA region, encompassing nucleotides 582 to 656 was probed within: (1) the complete 16 S rRNA molecule; (2) a 16 S rRNA fragment corresponding to nucleotides 578 to 756 obtained by transcription in vitro; (3) the S8-16 S rRNA complex; (4) the S8-RNA fragment complex; (5) the 30 S subunit. Cleavage or modification sites were detected by primer extension with reverse transcriptase. We present a three-dimensional model derived from mapping experiments and graphic modeling. Nucleotides in area 594-599/639-645 display unusual features: a non-canonical base-pair is formed between U598 and U641; and A595, A640 and A642 are bulging out of the major groove. The resulting helix is slightly unwound. Comparative analysis of probing experiments leads to several conclusions. (1) The synthesized fragment adopts the same conformation as the corresponding region in the complete RNA molecule, thus confirming the existence of independent folding domains in RNAs. (2) A long-range interaction involving cytosine 618 and its 5' phosphate occurs in 16 S rRNA but not in the fragment. (3) The fragment contains the complete information required for S8 binding. (4) The RNA binding site of S8 is centered in the major groove of the slightly unwound helix (594-599/639-645), with the three bulged adenines appearing as specific recognition sites. (5) This same region of the 16 S RNA is not exposed at the surface of the 30 S subunit.
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PMID:Binding of Escherichia coli ribosomal protein S8 to 16 S rRNA. A model for the interaction and the tertiary structure of the RNA binding site. 332 31

The polypeptide product of the gene for the large subunit of ribulose-1,5-bisphosphate carboxylase from Euglena gracilis based on the DNA sequence of the chloroplast-encoded gene is described. The large subunit polypeptide of 475 codons is co-linear with the homologous polypeptides from other chloroplasts and cyanobacteria. The amino acid sequence is 92% homologous to that of Chlamydomonas, 84% homologous to spinach, 82% homologous to maize, and 80% homologous to that of the cyanobacterium Anabaena variabilis. Known functional domains of the protein are coded by the larger exons of the gene. Introns in the gene generally occur at coding sequences specifying hydrophilic, presumably surface exposed, regions of the polypeptide. The location of some of the introns may reflect a separation of functional domains. The 5'- and 3'-ends of the rbcL transcript were determined by primer extension sequencing using reverse transcriptase and S1 nuclease protection, respectively. The transcribed but untranslated sequences are quite distinct from those from other rbcL loci.
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PMID:The Euglena gracilis chloroplast ribulose-1,5-bisphosphate carboxylase gene. II. The spliced mRNA and its product. 393 71

The transcription initiation site of the xylDEGF operon on the TOL plasmid of Pseudomonas putida mt-2 was determined in P. putida and in Escherichia coli by S1 nuclease and reverse transcriptase mapping. The induced synthesis of mRNA started at the same start point in both P. putida and E. coli, although the amount of mRNA in E. coli cells was less than that in P. putida. The nucleotide sequence of the region surrounding the start point was also determined. The ribosome-binding site (RBS) complementary to the 3' end of the 16S rRNA of Pseudomonas aeruginosa and E. coli preceded the predicted start codon for the xylD gene. The consensus nucleotide sequence for E. coli promoters was not found in the region preceding the transcription start point. On the other hand, the sequences of the "-10" and the "-35" regions of the xylDEGF operon revealed some homology with the respective, previously determined sequences of the xylABC operon of the TOL plasmid.
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PMID:Nucleotide sequence of the promoter region of the xylDEGF operon on TOL plasmid of Pseudomonas putida. 609 37

cDNA was synthesized on Ig kappa-L-chain mRNA isolated from mouse plasmocytoma MOPC 21 using avial myeloblastosis RNA-dependent DNA polymerase. The cDNA was used as a matrix for second DNA chain synthesis using 5'-exonuclease free DNA-polymerase I (Klenoff fragment). Hairpin DNA having double length of initial cDNA was obtained without added exogenous primer. SI nuclease treatment of the hairpin DNA results in reducing the DNA length twice, as shown by electrophoresis in denaturing conditions. The fact, that S1 nuclease resistance of the hairpin DNA is 75% shows high complementarity between first and second DNA chain. The product length obtained after S1 nuclease treatment was shown to be heterogenous. The maximal length of the product, reaching at least 900 base pairs, is near to be initial mRNA length (without the poly(A)-fragment).
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PMID:[Synthesis of double-stranded DNA on light immunoglobulin chain matrix RNA]. 615 6

Poly(A+)--RNA from rat ventral prostate was isolated using oligo(dT)-cellulose chromatography. 45% of the total poly(A+)--RNA was a single peak at 10S as demonstrated by centrifugation in a 5-20% sucrose gradient containing 1% SDS. By using complementary DNA probes, it was shown that the 10S RNA contained the major abundance class of poly(A+)--RNA. Denaturing agarose-gel analysis revealed 2 major bands in the 10S poly(A+)--RNA preparation approx. 600 NT and 500 NT (NT = nucleotides) long, resp. Double-stranded 32 P-DNAs complementary to light side and heavy side of the 10S poly(A+)--RNA peak were synthesized and isolated using reverse transcriptase and hydroxyapatite (HAP) chromatography. Approx. 40% of the first strand of the cDNAs were converted to double-stranded structures with a Tm of 88 degrees C. HAP purified double-stranded material was 92% resistant to S1 nuclease. the DNA--DNA reannealing profile of double stranded 32 P-cDNA enriched for the 500 NT band gave a Cot 1/2 of approximately 7 X 10(-4) moles X sec X 1(-1) indicating a complexity for this enriched synthetic gene of 500-600 nucleotide pairs (NTP).
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PMID:Purification of major abundance class of poly(A+)-RNA from rat ventral prostate. 615 88

The 5' terminus of Saccharomyces cereviasiae 35S pre rRNA was mapped on the rDNA using two methods: 1) Suitable restriction endonuclease fragments were hybridized to total high molecular weight RNA and extended with reverse transcriptase to the 5' end of the RNA template. 2) Other restriction fragments spanning the 5' terminus of 35S pre rRNA and radioactively labeled at their ends were hybridized to high molecular weight RNA and the non hybridized nucleic acids were digested with S1 nuclease. On the basis of these experiments, the 5' terminus of 35S pre rRNA was placed approximately 670 nucleotides upstream from the 17S rRNA coding region. The exact position was determined by reverse transcription as above, but in the presence of dideoxyribonucleoside triphosphates, which served as a way of sequencing the 5' terminal region. 35S pre rRNA synthesis is initiated at a site in EcoRI restriction fragment B which is 48 nucleotides upstream from the EcoRI cleavage site in the coding strand.
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PMID:The 5' terminus of the precursor ribosomal RNA of Saccharomyces cerevisiae. 615 79

Double-stranded cDNA sequences were prepared from Xenopus laevis ovary poly A(+) RNA with AMV reverse transcriptase and nuclease S1. They were inserted into the plasmid pBR 322 after ligation with a Hind III linker and were cloned in E. coli strain X1776. Plasmid pools containing a cDNA insert were identified by Hind II restriction and hybridization of the DNA fragments with radiolabelled pBR 322 DNA. Hybridization of the positive pools with ovary RNA labelled in vitro led to the identification of cloned cDNA sequences which represent RNA species of high to intermediate abundance in the ovary. Positive clones were further challenged with in vitro labelled mitochondrial DNA and RNA from different developmental stages. One clone of mitochondrial origin has been detected. The hybridization characteristics of the cDNA sequences with the RNA probes from later developmental stages is discussed.
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PMID:Characterization of cloned cDNA sequences derived from Xenopus laevis poly A(+) oocyte RNA. 615 93

Approximately one kilobase pairs surrounding and upstream the transcription initiation site of a cloned ribosomal DNA (rDNA) of the mouse were sequenced. The putative transcription initiation site was determined by two independent methods: one nuclease S1 protection and the other reverse transcriptase elongation mapping using isolated 45S ribosomal RNA precursor (45S RNA) and appropriate restriction fragments of rDNA. Both methods gave an identical result; 45S RNA had a structure starting from ACTCTTAG---. Characteristically, mouse rDNA had many T clusters (greater than or equal to 5) upstream the initiation site, the longest being 21 consecutive T's. A pentadecanucleotide, TGCCTCCCGAGTGCA, appeared twice within 260 nucleotides upstream the putative initiation site. No such characteristic sequences were found downstream this site. Little similarity was found in the upstream of the transcription initiation site between the mouse, Xenopus laevis and Saccharomyces cerevisiae rDNA.
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PMID:The nucleotide sequence of the putative transcription initiation site of a cloned ribosomal RNA gene of the mouse. 616 56

Human thyroglobulin mRNA was isolated from Graves' goitres by size selection of total poly(A)-rich RNA in a sucrose gradient. It sedimented at 33 S, as in other mammalian species, and showed a single component of approximately 8500 bases by gel electrophoresis. cDNA was synthesized from the 33-S RNA by using reverse transcriptase in the presence of human placenta ribonuclease inhibitor and in conditions allowing the formation of long transcripts. The latter was made double-stranded using reverse transcriptase and blunt-ended with nuclease S1. After tailing with dCTP and terminal transferase, the double-stranded cDNA was annealed to pBR322 DNA that had been cleaved at the endonuclease PstI site and tailed with dGTP. The resulting plasmids were used to transform Escherichia coli C600 cells and four cloned recombinants were selected. Each plasmid DNA was shown to contain a sequence complementary to human thyroglobulin mRNA by hybridization with a labeled 33-S mRNA, visualization of cDNA . mRNA hybrids by electron microscopy and filter hybridization selection of mRNA directing the synthesis of immunologically related thyroglobulin peptides in the reticulocyte lysate. The four inserted DNA sequences were 1400 - 1800 base pairs long, two of them showing an homologous sequence of 1100 base pairs. Together, the four cloned DNA fragments represented 63% of the 8500 bases of human thyroglobulin mRNA.
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PMID:Cloning of four DNA fragments complementary to human thyroglobulin messenger RNA. 617 25

Double-stranded cDNA was prepared from prorennin-specific mRNA by sequential actions of reverse transcriptase, DNA polymerase and S1 nuclease, and inserted into the Sa/I site of pBR322 by the poly(dG)-(dC) annealing method. Transformation of Escherichia coli C600 r- m- by the hybrid plasmid yielded transformants containing prorennin cDNA. The presence of the cDNA sequence in these clones was confirmed by both colony hybridization and hybrid-arrested translation of the mRNA in vitro. The largest size of the cloned cDNA was 1,020 bp.
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PMID:Cloning in Escherichia coli of the structural gene of prorennin, the precursor of calf milk-clotting enzyme renin. 617 64


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