Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:2.7.7.6 (RNA polymerase)
 34,946 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The DNA-binding protein ICP8 of herpes simplex virus is a multifunctional protein which is required for viral replication. To identify the single-stranded DNA-binding domain of the protein, recombinant plasmids containing the 5' or 3' coding portion of the ICP8 gene or the intact gene were constructed and transcribed using SP6 RNA polymerase. The resulting RNA was translated in vitro to produce a 62,000-Da amino-terminal peptide, a 69,000-Da carboxyl-terminal peptide, or the intact protein. When these were analyzed by single-stranded DNA-cellulose column chromatography, large amounts of the intact ICP8 bound to the columns while small amounts of the carboxyl-terminal peptide and undetectable amounts of the amino-terminal peptide bound. The majority of the carboxyl-terminal peptide which bound eluted from the columns with the same salt concentration as the intact ICP8. The in vitro synthesized intact protein had the same affinity for single-stranded DNA-cellulose as ICP8 purified from infected cells. These results suggest that the carboxyl-terminal portion of ICP8 contains a single-stranded DNA-binding site.
 ...
PMID:A carboxyl-terminal peptide of the DNA-binding protein ICP8 of herpes simplex virus contains a single-stranded DNA-binding site. 304 18

We have used an in vitro transcription-translation system to study initial protein processing events of the rat substance P/neurokinin A gene products. cDNA clones for three different mRNA species, which are derived by differential RNA splicing, were subcloned into a plasmid, pGEM1, which contains the promoter for the bacteriophage SP6 RNA polymerase. In vitro synthesized mRNAs for alpha-, beta-, and gamma-preprotachykinin were translated in a wheat germ or rabbit reticulocyte cell-free system. When analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the translated protein products migrate consistent with the deduced molecular masses of alpha (13,035 Da)-, beta (15,003 Da)-, and gamma (13,343 Da)-preprotachykinin. The addition of dog pancreatic microsomal membranes to either cell-free translation system causes the production of a protease-resistant form of each of the three preprotachykinins which migrates with an apparent increase in molecular mass of approximately 2,000 Da. Each of these modified preprotachykinins lacks the putative signal peptide of the prepro- form, with signal peptidase cleavage occurring after the alanine residue at position 19. Both the prepro- and proforms of each tachykinin precursor molecule are recognized by antiserum R-140, an antiserum specific for the mid-portion of the undecapeptide substance P. The most likely explanation for the apparent increase in molecular mass is anomalous electrophoretic migration, since beta-preprotachykinin mRNA lacking the signal peptide encoding sequence is translated, in the absence of microsomal membranes, into a protein with the same apparent molecular mass as the modified form of beta-preprotachykinin. Therefore, each of the three preprotachykinin mRNAs are translatable, and their products are targeted to the secretory pathway by the presence of a cleavable signal peptide.
 ...
PMID:Posttranslational processing of alpha-, beta-, and gamma-preprotachykinins. Cell-free translation and early posttranslational processing events. 304 2

We have investigated the use of in vitro expression as a quick and convenient means of screening large numbers of interferon (IFN) analogs generated using in vitro mutagenesis. The IFN-alpha 1 mRNA generated from DNA template using SP6 RNA polymerase is efficiently translated in rabbit reticulocyte lysate (RRL). The antiviral specific activity of this RRL-synthesized IFN-alpha l is equivalent to the yeast-synthesized protein. In contrast with the yeast-expression system, where some IFN-alpha analogs are poorly expressed, all analogs tested were well expressed in RRL.
 ...
PMID:Efficient in vitro expression of interferon alpha analogs using SP6 polymerase and rabbit reticulocyte lysate. 305 2

We describe a method for creating a population of randomly digested, blunt-ended DNA fragments with 5'-TG... 3'-AC... (TG) at their termini. When these fragments were ligated to a blunt-ended vector that contains ...CATA-3' ...GTAT-5' at its termini, as high as 84% of the clones obtained after transformation contained plasmids with a reconstructed Nde I site ... CATATG... ...GTATAC.... When the DNA vector is prepared from an appropriate plasmid [Gross et al., Mol. Cell. Biol. 5 (1985) 1015-1024; Kotewicz et al., Gene 35 (1985) 249-258], the ATG within the restriction site corresponds to a start codon positioned downstream from a strong ribosome-binding site and controllable promoter. If a gene has been digested to the TG contained within its authentic initiation codon, the endogenous translation-initiation control sequences are deleted and expression can be controlled using the plasmid-derived promoter. In addition, a gene digested to other in-frame TGs can potentially express proteins with altered N termini. Using this method, we have placed the structural gene of SP6 RNA polymerase, trimmed precisely to its authentic start codon, under the control of the tac promoter.
 ...
PMID:Enrichment for 5'-TG termini: a method for subcloning structural genes into expression vectors. 306 14

The plastoquinone-binding polypeptide D-2 of the reaction center complex of photosystem II in barley has been expressed in vitro using the expression vector pGem3 containing the psbD gene of chloroplast DNA as template. The full length D-2 polypeptide with an apparent mol. weight of 32 kD is a major product when mRNA is transcribed with SP6 RNA polymerase from an insert containing the psbD gene with a 36 bp 5' leader sequence and a 3' tail of 99 bp downstream of the stop codon. Translation of the mRNA had to be done in a rabbit reticulocyte lysate. Translation also occurred from the internal AUG codons giving rise to truncated D-2 polypeptides with sizes of 30, 25 and 17 kD. They are immunoprecipitable with antisera either raised against amino acid residues 235 to 241 or against 345 residues of the D-2 polypeptide. A truncated translation product of 12 kD probably initiated at codon 247 is not recognized by either antiserum. An additional immunoprecipitable protein with an apparent molecular weight of 46 kD is observed by SDS-PAGE and is interpreted as a homomeric aggregate of D-2 polypeptides. Upon addition of methanol solubilized 2,6 dichloro-p-benzoquinone or other quinones a preferential translation of the full-length and truncated D-2 polypeptides is observed. The use of in vitro synthesized D-2 polypeptides for studies of binding quinones, other electron carriers and chlorophyll chromophores is discussed.
 ...
PMID:In vitro transcription and translation of the psbD gene encoding the D-2 protein of photosystem II in barley. 307 64

We describe the construction of a recombinant DNA plasmid, consisting of the vector pBR322 and full-length tissue-type plasminogen-activator (t-PA) cDNA, by using polyadenylated RNA from cultured Bowes melanoma cells as substrate. A 1280-base-pair PstI restriction fragment, covering the 3' untranslated region and part of the coding region for the t-PA L-chain, was used as a radiolabelled probe to determine the size and the number of t-PA mRNA molecules in cultured endothelial cells of different origin from the same individual. Northern blotting showed that in all these cells a t-PA mRNA is synthesized of about 2500 nucleotides, indicating that transcriptional initiation, splicing and polyadenylation is similar. The number of t-PA mRNA molecules per cell measured, by using a dot-blotting technique and t-PA mRNA made in vitro, with a plasmid DNA preparation harbouring a specific promotor of the Salmonella typhimurium bacteriophage SP6, t-PA cDNA and SP6 RNA polymerase as standard, is approx. 10,000 in all cultured endothelial cells from adult vessels. However, the amount of t-PA antigen synthesized and/or secreted differs by a factor of 6-20. Relatively large amounts of t-PA antigen secreted were detected in conditioned medium from vena-cava-derived cells, whereas low amounts were found in conditioned medium from arteria-iliaca-derived cells.
 ...
PMID:Quantification of tissue-type plasminogen activator (t-PA) mRNA in human endothelial-cell cultures by hybridization with a t-PA cDNA probe. 309 Oct 7

The regulation of ornithine decarboxylase (ODC) activity by the polyamine derivatives N1,N8-bis(ethyl)-spermidine and N1,N12-bis(ethyl)spermine was studied using a line of L1210 cells resistant to alpha-difluoromethylornithine (D-R cells), which contain very high levels of ODC, and a synthetic mRNA prepared from a plasmid containing an insert corresponding to ODC mRNA adjacent to an SP6 RNA polymerase promoter. Studies in which ODC protein was labeled in the D-R cells by exposure to [35S]methionine indicated that the polyamine derivatives and their physiological counterparts led to an increased rate of degradation of ODC and to a rapid reduction in ODC synthesis without affecting the content of ODC mRNA. Direct evidence that the polyamine derivatives act by inhibiting the translation of the ODC mRNA was obtained by studying their effects on the translation of ODC mRNA in reticulocyte lysates. This translation was strongly inhibited by the addition of N1,N8-bis(ethyl)spermidine, spermidine, N1,N12-bis(ethyl)spermine, or spermine but was not affected much by putrescine. The inhibition of the translation of ODC mRNA by either of the bis(ethyl) polyamine derivatives occurred at concentrations which stimulated total protein synthesis showing the selectivity of the reduction in ODC. The effects of polyamine derivatives and polyamines on translation of the plasmid-derived ODC mRNA were identical with those found with the D-R L1210 cell mRNA. This synthetic ODC mRNA lacks 261 bases of the 5'-leader sequences and 200 bases plus the poly(A) section from the 3'-nontranslated sequence. Therefore, these regions appear not to influence sensitivity of the ODC mRNA to inhibition of translation by polyamine derivatives.
 ...
PMID:Control of ornithine decarboxylase activity in alpha-difluoromethylornithine-resistant L1210 cells by polyamines and synthetic analogues. 313 56

Xenopus oocytes express a gene encoding bovine rhodopsin as well as its SP6 RNA polymerase-derived transcripts and total retinal mRNA. The opsin produced is in unglycosylated (30 kDa) and two glycosylated (35 kDa and 41 kDa) forms. Incubation of the cells expressing the above proteins with 11-cis-retinal generates rhodopsin, which was purified by immunoaffinity chromatography. The purified protein shows the expected UV/visible absorption spectrum and characteristic light-dependent activation of the rod outer segment GTP binding protein. Oocytes expressing rhodopsin exhibit light-dependent ionic currents that are detected by voltage-clamp techniques.
 ...
PMID:Expression of a bovine rhodopsin gene in Xenopus oocytes: demonstration of light-dependent ionic currents. 314 20

Effects of mutations around the phage SP6 transcription initiation site on SP6 RNA polymerase's selection of initiation site were studied. In the in vitro transcription reactions, the limiting concentration of a ribonucleotide causes the SP6 RNA polymerase to stall long enough only at the positions of the limited nucleotide and dissociate from the elongation complex. As a result, a series of RNA oligomers comprises a sequencing ladder, and abortive initiation cycling products up to 6-mer are made in high yield. Precise sizing of the product RNAs from the elongation pausings determined the initiation site of each mutant. When the wild-type +1 G is changed to C or A without change in the upstream sequence including TATA from -4 to -1, transcription still starts only at the +1 site. But, the mutant containing TATCC from -4 to +1 C. We propose that the phage SP6 RNA polymerase selects the initiation site precisely at a certain distance from a direct contact point in the upstream promoter sequence, regardless of the species of initiating nucleotide. It is also suggested that the sequence-dependent perturbations of DNA helical structure, for example D to B form, may shift the initiation site.
 ...
PMID:Transcription initiation site selection and abortive initiation cycling of phage SP6 RNA polymerase. 319 28

One or more of the five acidic amino-terminal residues of skeletal muscle actin have been implicated as being important in a number of actin-related processes. We have constructed a series of actins containing mutations at Asp3 and Asp11 and tested these mutant proteins for their ability to bind to DNase I-agarose, polymerize with rabbit skeletal muscle actin, undergo amino-terminal processing, and bind to the myosin-S1 subfragment. The mutant actins were expressed in vitro using a coupled transcription/translation system which involves the synthesis of mutant RNAs with SP6 RNA polymerase followed by their translation in a rabbit reticulocyte lysate. When Asp3 was changed to Ala, His, or Asn there was no difference in the tested properties as compared to wild type actin. These results suggest that an acidic residue at position 3 is not critical for the actin functions measured. When Asp11 was changed to Glu, Asn, or His or if the conserved Asp-Asn sequence at positions 11 and 12 was reversed, the mutants were able to copolymerize with rabbit skeletal muscle actin and be cross-linked to myosin-S1 to nearly the same extent as wild type actin. However, the amount of in vitro-synthesized actin capable of binding to DNase I-agarose with high affinity or undergoing amino-terminal processing was reduced significantly relative to the wild type actin synthesized in vitro. The Asp11 mutants ran anomalously on native polyacrylamide gels suggestive of a conformational change induced in the actin. Together, these results suggest that Asp11 may be important in proper actin folding and function.
 ...
PMID:Studies on the role of actin's aspartic acid 3 and aspartic acid 11 using oligodeoxynucleotide-directed site-specific mutagenesis. 319 44


<< Previous 1 2 3 4 5 6 7 8 9 10