Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.1.30.2 (endonuclease)
 18,621 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Nucleosome formation on inverted repeats or on some alternations of purines and pyrimidines can be inhibited in vitro by DNA supercoiling through their supercoiling-induced structural transitions to cruciforms or Z-form DNA, respectively. We report here, as a result of study of single nucleosome reconstitutions on a DNA minicircle, that a physiological level of DNA supercoiling can also enhance nucleosome sequence preference. The 357 base-pair minicircle was composed of a promoter of phage SP6 RNA polymerase joined to a 256 base-pair fragment containing a sea urchin 5 S RNA gene. Nucleosome formation on the promoter was found to be enhanced on a topoisomer with in vivo superhelix density when compared to topoisomers of lower or higher superhelical densities, to the nicked circle, or to the linear DNA. In contrast, nucleosomes at other positions appeared to be insensitive to supercoiling. This observation relied on a novel procedure for the investigation of nucleosome positioning. The reconstituted circular chromatin was first linearized using a restriction endonuclease, and the linear chromatin so obtained was electrophoresed as nucleoprotein in a polyacrylamide gel. The gel showed well-fractionated bands whose mobilities were a V-like function of nucleosome positions, with the nucleosome near the middle migrating less. This behavior is similar to that previously observed for complexes of sequence-specific DNA-bending proteins with circularly permuted DNA fragments, and presumably reflects the change in the direction of the DNA axis between the entrance and the exit of the particle. Possible mechanisms for such supercoiling-induced modulation of nucleosome formation are discussed in the light of the supercoiling-dependent susceptibility to cleavage of the naked minicircle with S1 and Bal31 nucleases; and a comparison between DNase I cleavage patterns of the modulated nucleosome and of another, non-modulated, overlapping nucleosome.
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PMID:Chromatin reconstitution on small DNA rings. IV. DNA supercoiling and nucleosome sequence preference. 131 7

As a means of generating homogeneous populations of elongation complexes with the RNA polymerases encoded by phages T7 and SP6, transcription has been carried out in vitro on templates associated with the Gln-111 mutant of EcoRI endonuclease. The Gln-111 protein, as a result of a single amino acid substitution at position 111, lacks cleavage function yet shows higher than wild-type affinity for the EcoRI recognition sequence GAATTC. On a series of linear and circular templates associated with Gln-111 protein, blockage of the phage RNA polymerase elongation complex is observed. The 3' endpoint of the major blocked-length RNA species, just 3 bp upstream from the GAATTC, reveals an extremely close approach of polymerase's leading edge to essential contacts between Gln-111 protein and its binding site. In contrast to E. coli RNA polymerase, which is blocked stably and quantitatively by Gln-111 protein (Pavco, P.A. and Steege, D. A. (1990) J. Biol. Chem. 265, 9960-9969), the phage polymerases show substantial levels of readthrough transcription beyond the protein block.
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PMID:Characterization of elongating T7 and SP6 RNA polymerases and their response to a roadblock generated by a site-specific DNA binding protein. 189 55

In studies of the effects of changes in mRNA structure and sequence on the initiation of protein synthesis, we used a generally applicable approach to transcribe reconstructed genes for duck alpha A globin by the bacteriophage SP6 RNA polymerase promoter in a pGEM-2 plasmid vector. The genes were reconstructed such that the first nucleotide to be transcribed, the 5' adenosine, was placed directly adjacent to the SP6 promoter sequence. The 3' ends of the genes were constructed such that cleavage with Ssp 1 endonuclease yielded a template that directed the synthesis of mRNA terminating in a poly A tail containing 56 adenosines and a single 3' uridine. Special conditions using a Mn++ buffer were developed to enable the SP6 RNA polymerase to initiate at the 5' adenosine and synthesize the A-start transcription product. The mRNA could be capped and was subsequently used as an effective template for in vitro translation and synthesis of duck alpha A globin.
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PMID:Construction of mRNA genes for the synthesis and translation of duck alpha globin mRNA. 263 91

We report a simple and reliable protocol for nucleotide sequencing using the Sanger dideoxy technique on linearized double-stranded DNA molecules with specific oligonucleotide primers. The method is demonstrated for restriction fragments cloned into the plasmid vectors pSP64 and pSP65 using two vector-specific primers, the M13 reverse primer and a new SP6 primer, flanking the multiple cloning site. Template DNA may be prepared by a rapid alkaline lysis procedure. Mild linearization conditions with the appropriate restriction endonuclease avoid the appearance of artifact bands.
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PMID:Rapid and reliable dideoxy sequencing of double-stranded DNA. 300 96

DNA from bacteriophage SP6 grown on Salmonella typhimurium LT2 is a linear duplex with a unique DNA sequence having a molecular weight of 2.9 x 10(6) (43.500 base pairs). Restriction endonuclease cleavage maps on SP6 DNA for Ava II, Kpn I, Bgl II, Eco RI, and HindIII have been determined. SP6 DNA is transcribed selectively in vitro by Escherichia coli RNA polymerase, predominantly from three strong promoter sites located near the left end of the standard physical map, reading rightward to a termination site near 6,000 base pairs. Transcription in vitro by purified SP6-specific RNA polymerase gives rise to at least 10 discrete RNA species, all of which are read rightward. Promoter sites for these species are located throughout the rightmost 90% of the SP6 DNA molecule, although precise mapping has not yet been carried out. The general form of the SP6 transcriptional map is similar to the T7- and T3-like phages, although no gross sequence homologies are evident from DNA-RNA hybridization experiments.
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PMID:Bacteriophage SP6-specific RNA polymerase. II. Mapping of SP6 DNA and selective in vitro transcription. 627 14

The restriction-modification genes of the EcoRII system have been cloned into plasmids under control of phage-specific promoters T7 and SP6. The transcription was induced by cell infection with the recombinant M13 phages with the corresponding genes of phage RNA-polymerases under control of the Plac-promoter in the presence of IPTG. The induction yields significant amounts of EcoRII DNA-methylase for both phage-specific promoters. In both cases no increase in EcoRII endonuclease expression could be achieved. We hypothesize that the expression of the endonuclease gene is regulated on the translational level.
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PMID:Peculiarities of gene expression of the EcoRII modification-restriction system. 946 34

For applications such as sequencing, transfection, and in vitro transcription, PCR products have to be subcloned into plasmids. Many strategies are used for cloning, blunt-end ligation or the incorporation of restriction endonuclease sites into PCR primers for appropriate vectors. However, the most convenient and direct method is T/A cloning. In this study, we developed two of the pGEM-7Zf(+) phagemid T-tail vectors using AhdI-restriction endonuclease sites, and these T vectors have all the features of pGEM-7Zf(+): f1 ori, T7, and SP6 RNA polymerase promoters, the alpha-peptide coding region of beta-galactosidase for X-gal blue/white color selection, the beta-lactamase gene for recombinant colony selection, and binding sites for pUC/M13 forward and reverse sequencing primers. These AhdI-containing phagemid vectors, pGEM-NJ105 and pGEM-NJ107, are useful for the easy and inexpensive preparation of T vectors and direct cloning of PCR products.
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PMID:Construction of two pGEM-7Zf(+) phagemid T-tail vectors using AhdI-restriction endonuclease sites for direct cloning of PCR products. 1238 33