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)

Double-stranded DNA synthesized from the pigeon globin mRNA by the subsequent actions of avian myeloblastosis virus reverse transcriptase and E. coli DNA polymerase I was split with nuclease S1 and inserted into PstI site in the plasmid pBR322 by poly(dG) times poly(dC) homopolymer extension technique using terminal deoxynucleotidyl transferase. E. coli transformants have been shown to contain pigeon globin sequences by colony hybridization with pigeon globin [32P]cDNA. The inserted DNA fragment deleted from recombinant DNA by PstI treatment hybridizes with globin cDNA. The maximal length of the inserted fragment measured in agarose gel was found to be 550--560 base pairs. Inserted sequences subjected to analysis by hybridization with alpha- and beta-[32P]cDNA have been ascribed to the pigeon alpha globin chain. EcoRI, HindIII, BglII, SalI, BamHI, PstI restriction enzymes did not cleave the inserted DNA fragment. Pigeon DNA coding alpha-globin chain contains recognition sites for AluI, HindII and HaeIII restriction enzymes. Part of the recombinant clones remains resistant to ampicillin and therefore in some of these clones the globin gene could be expressed.
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PMID:[Enzymatic synthesis and molecular cloning of the pigeon alpha-globin structural gene]. 617 3

We had shown earlier (Gopalakrishna, Y., Langley, D., Sarkar, N. (1981) Nucleic Acid Res. 9, 3545-3554) that a substantial fraction of mRNA of various bacterial species carries 3'-terminal polyadenylate sequences. In this paper, we show that poly(A)-containing RNA from Bacillus subtilis can serve as template for the synthesis of complementary DNA by avian myeloblastosis virus reverse transcriptase, provided that oligodeoxythymidylate is added as primer. Poly(A)-RNA purified by affinity chromatography on oligo(dT)-cellulose was 20 times more effective as template for cDNA formation than total bacterial RNA, whereas rRNA was inactive. The average chain length of the cDNA was 400 nucleotides (range = 230-800 nucleotides), and 95% of the cDNA could be degraded by the single-strand specific S1 nuclease after denaturation. The small fraction (5%) that was resistant to S1 nuclease may represent duplex hairpin structures. Annealing with poly(A)-RNA protected cDNA from degradation by S1 nuclease, indicating that cDNA indeed contains nucleotide sequences complementary to poly(A)-RNA. These results constitute independent evidence that a large fraction (about 40%) of B. subtilis mRNA is polyadenylated. Moreover, the synthesis of cDNA to bacterial mRNA provides an important new tool for the study of bacterial mRNA structure.
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PMID:The synthesis of DNA complementary to polyadenylate-containing RNA from Bacillus subtilis. 617 11

The malPQ operon, one of the three operons of the maltose regulon, is positively controlled by the product of gene malT. The starting point for malPQ transcription was deduced from experiments which involved a hybridization of in vivo-synthesized malPQ mRNA with adequate DNA probes, followed either by a digestion of nonhybridized DNA (S1 nuclease mapping) or by an extension of the hybridized probe (reverse transcriptase mapping). In the wild-type strain, this starting point was 37 nucleotides upstream from the initiation codon for malP. This analysis was also performed on a double mutant which contained both a 13-base pair deletion and a 3-base pair insertion in the promoter region. This double mutant expressed the malPQ operon exactly as the wild-type strain did, in a maltose-inducible manner. In this strain, the starting point for malPQ transcription was shifted 11 nucleotides downstream from the wild-type location. An analysis of these results suggests that (i) the binding site for the malT product is located upstream from the region which is severely altered in the double mutant, i.e., upstream from position -31; and (ii) the 30-base pair sequence which precedes the transcription starting point contains very few positions which are essential for promoter activity.
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PMID:Expression of the Escherichia coli malPQ operon remains unaffected after drastic alteration of its promoter. 618 58

Poly(A)-RNA enriched for prothrombin was isolated by specific immunoprecipitation of bovine liver polysomes. Prothrombin consisted of about 8% of the cell-free translation products of this RNA. A double-stranded cDNA was synthesized by using reverse transcriptase (RNA-dependent DNA nucleotidyltransferase) and made 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 previously at the single Pst I site and similarly tailed with dGTP. The resulting plasmids were used to transform Escherichia coli strain RR1 under P3-EK1 conditions. Sixty-three tetracycline-resistant clones were obtained that hybridized to 32P-labeled cDNA synthesized from prothrombin-enriched mRNA. Recombinants containing cDNA to prothrombin mRNA sequences were screened by a solution hybridization assay with a [3H]cDNA synthesized from mRNA. This enriched mRNA was 50% prothrombin mRNA, as determined by a reticulocyte lysate translation assay. Three positive clones were identified by this assay; they contained bovine DNA inserts of 700, 500, and 400 base pairs. The DNA sequence of the 700-base-pair insert was then determined. This recombinant plasmid contained DNA coding for the carboxyl-terminal 160 residues of bovine prothrombin followed by a noncoding region of 119 base pairs and a poly(A) tail of 60 base pairs.
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PMID:Cloning and analysis of a cDNA coding for bovine prothrombin. 625 59

The four major adeno-associated virus type 2 (AAV2)-specific RNAs were mapped on the linear viral genome by a variety of biochemical techniques, including S1 nuclease and exonuclease VII mapping, RNA gel-transfer hybridization, and analysis of reverse transcriptase extension products. All the major AAV2 RNAs were derived from the minus DNA strand and had 3' termini at position 96. The nucleus-specific 4.3- and 3.6-kilobase (kb) RNAs had 5' termini at positions 6 and 19, respectively. The 5' terminus of the 2.6-kb RNA mapped to position 38.5. The predominant 2.3-kb AAV2 mRNA was spliced and contained a short leader sequence (approximately 50 nucleotides) which mapped to position 38.5, coincident with the 5' terminus of the 2.6-kb RNA. The 5' end of the body of the 2.3-kb RNA mapped to position 46.5. These results are discussed in terms of the involvement of single versus multiple promoters (for transcription) and RNA splicing mechanisms in the generation of the AAV2 RNAs.
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PMID:Transcripts of the adeno-associated virus genome: mapping of the major RNAs. 625 15

DNA complementary (cDNA) to a partially purified preparation of bovine parathyroid hormone mRNA was synthesized using avian myeloblastosis viral reverse transcriptase. The PTH cDNA contained about 750 bases and was greater than 95% sensitive to digestion by S1 nuclease. Analysis of the mRNA preparation by excess RNA hybridization to the PTH cDNA revealed one rapidly hybridizing component consisting of 50% of the PTH cDNA. Sequential incubation of the PTH mRNA with reverse transcriptase and E. coli DNA polymerase I produced near full length double-stranded PTH cDNA. Of the 22 restriction endonucleases tested, double-stranded PTH cDNA could be cleaved with Alu I, Mbo II, Sau 3A, Sst I, and Taq I. The restriction fragments corresponding to the 5' terminus of the sense strand were identified for the last three enzymes by comparing the size of fragments obtained from PTH cDNA before and after cleavage of the hairpin loop connecting the two strands by S1 nuclease. The restriction map of the cDNA was used to detect clones of bacteria containing recombinant plasmids with near full length PTH cDNA inserts.
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PMID:Synthesis, restriction analysis, and molecular cloning of near full length DNA complementary to bovine parathyroid hormone mRNA. 625 49

Poly(A)-RNA enriched for type I procollagen sequences was isolated from normal human fibroblasts and used as template to synthesize double-stranded cDNA with avian myeloblastosis virus (AMV) reverse transcriptase. After the ends had been blunted with nuclease S1 and dGMP tails had been added with terminal deoxynucleotidyltransferase, the double-stranded cDNA was annealed with pBR322 DNA that had previously been cleaved with EcoRI, blunted with AMV reverse transcriptase, and dCMP-tailed with terminal deoxynucleotidyltransferase. The chimeric molecule was used to transform Escherichia coli strain HB101. Ninety-five recombinant clones were obtained and screened by dot hybridization analysis using 32P-labeled cDNA synthesized from the original poly(A)-RNA collagen-enriched population. Three positive clones were isolated and further characterized by blot hybridization techniques and by EcoRII digestion. One clone with an insert of 2.2 kilobases was shown to contain sequences encoding for the pro-alpha 2 chain of human type I procollagen. DNA sequence analysis of a 172-nucleotide fragment demonstrated that the cloned cDNA extends from amino acid position 450 of the alpha 2 chain to the middle of the COOH-terminal propeptide.
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PMID:Cloning a cDNA for the pro-alpha 2 chain of human type I collagen. 626 97

We have used two methods to detect specific transcription of the chicken alpha 2 (type I) collagen gene in cell-free extracts derived from Rous sarcoma virus-transformed chicken embryo fibroblasts. The first method is a modification of the S1 nuclease mapping procedure which utilizes a DNA probe labeled with 32P at the 5' end of the HindIII linker originally used to clone the collagen promoter region into PBR322. The probe distinguishes newly made, specific RNA from endogenous RNA and nonspecific transcripts. Using this procedure we have found that chicken whole cell extracts support accurate initiation of transcription of the chicken alpha 2 (type I) collagen DNA template. Addition of either creatine phosphate, GTP, or UTP to concentrations of approximately 3 to 5 mM was found to stimulate RNA polymerase II transcription by 5- to 10-fold. The second method employs an avian myeloblastosis virus reverse transcriptase-catalyzed primary extension procedure, rendered in vitro-specific by use of a pBR322 fragment as primer. These two techniques should be useful for analyzing specific transcription in other types of cell-free extracts.
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PMID:Transcription of the chicken alpha 2 (Type I) collagen gene by homologous cell-free extracts. 628 36

The DNA sequence of the intragenic region of the rat 45S ribosomal RNA precursor was determined. This sequence contains 2282 nucleotides and extends from the conserved EcoR I site near the 3' terminus of 18S rRNA to 69 nucleotides downstream of the 5' terminus of 28S rRNA. The sequences corresponding to 18S and 5.8S rRNA were identified by comparison with previously published data. The 5' terminus of rat 28S rRNA was identified by S1 nuclease protection and reverse transcriptase elongation assays. The internal transcribed spacers were found to be 1066 and 765 nucleotides long and had little homology with those of Xenopus and yeast. Regions of sequence homology between rat and Xenopus were found at the junctions of the internal transcribed spacers with 18S, 5.8S and 28S rRNA. These homologies suggest that these sequences may function as recognition sites for the processing of the ribosomal precursor RNA.
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PMID:Nucleotide sequence of the region between the 18S rRNA sequence and the 28S rRNA sequence of rat ribosomal DNA. 628 18

A new cloning strategy has been developed for cloning the genomes of double-stranded RNA viruses by using bovine rotavirus as a test system. The major modification adopted was the use of denatured polyadenylated double-stranded RNA as the template for reverse transcriptase. This allowed the two complementary strands of cDNA to be synthesized in a single reaction and removed the need for S1 nuclease digestion to remove the 5' hairpin structure normally generated in cDNA synthesis.
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PMID:Molecular biology of rotaviruses. IV. Molecular cloning of the bovine rotavirus genome. 629 22


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