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:3.1.30.1 (S1 nuclease)
3,660 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

cAMP stimulates the transcription of the gene for the cytosolic form of phosphoenolpyruvate carboxykinase (GTP) (EC 4.1.1.32) (PEPCK) in rat liver. We have investigated the nucleotide sequences required for regulation of PEPCK gene expression by cAMP. A chimeric gene was constructed in which a 620-base pair fragment of the 5'-end of the PEPCK gene (including 547 base pairs of 5'-flanking sequence) was ligated to the herpes simplex virus thymidine kinase (TK) structural gene. The PEPCK promoter fragment was introduced either in the proper orientation for transcription of the TK gene or in the opposite orientation. These fusion genes and the parent vector, pOPF, which contains the intact TK gene, were transfected individually into TK-deficient FTO-2B rat hepatoma cells. FTO-2B cells contain an active endogenous PEPCK gene which is stimulated by cAMP. Cells were selected in HAT medium and grown either as mass cell cultures or as individual clones. Dibutyryl cyclic AMP (Bt2cAMP) plus theophylline (16 h) stimulated TK activity 1.6-6.1-fold in cell lines transfected with the PEPCK-TK fusion gene containing the PEPCK promoter fragment in the correct orientation. However, the intact TK gene was not induced by Bt2cAMP after transfection, nor was there any expression of the PEPCK-TK fusion gene in cells which contained the PEPCK promoter fragment in the wrong transcriptional orientation. Bt2cAMP also increased the levels of TK mRNA in cells transfected with the PEPCK-TK fusion gene, but not in cells transfected with the intact TK gene. The chimeric PEPCK-TK mRNA initiated at the PEPCK start site, as determined by S1 nuclease mapping. There was no relationship between the number of copies of the PEPCK-TK gene integrated in the various cell lines and either the basal level of TK activity or its inducibility of Bt2cAMP.
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PMID:Identification of a cAMP regulatory region in the gene for rat cytosolic phosphoenolpyruvate carboxykinase (GTP). Use of chimeric genes transfected into hepatoma cells. 609 Apr 58

The Chinese hamster ovary adenine phosphoribosyl transferase gene (aprt) was reengineered to be flanked by sequences from the thymidine kinase (tk) gene of herpes simplex virus. This construct was cotransfected with DNA from herpes simplex virus type 1, and after 3 days, virus was harvested and Tk- plaques were selected after the virus was plated on Tk- cells in the presence of bromodeoxycytosine. Recombinant viruses were identified by dot-blot hybridization, and the arrangement of aprt and tk sequences were determined by Southern blot hybridization. Analysis of the recombinants revealed that acquisition of aprt sequences resulted from insertional inactivation of the tk locus as a consequence of homology-based recombination. Recombination was precise, as evidenced by the failure to detect plasmid sequences or the synthetic restriction endonuclease sites that bounded the mutant tk gene in the aprt-tk construct. Infection of Aprt- mouse or Chinese hamster ovary cells with UV-irradiated virus and selection in medium containing azaserine and adenine resulted in the survival of numerous colonies that stably express the aprt gene. Transformed cells synthesized an aprt mRNA that is identical to wild-type mRNA as determined by Northern blot and S1 nuclease analyses. Cells lytically infected with the recombinant virus do not appear to transcribe the aprt gene. Thus, infected cells differentiate between virus and foreign promoters even when a cellular gene is cis to the virus chromosome.
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PMID:Transduction of the Chinese hamster ovary aprt gene by herpes simplex virus. 609 82

Cloned complementary DNAs encoding chicken ovalbumin, chicken prelysozyme and calf preprochymosin, prochymosin and chymosin were inserted downstream from various viral promoters in modified recombinant "shuttle" vectors. Microinjection of the ovalbumin, prelysozyme and preprochymosin constructs into the nuclei of Xenopus laevis oocytes resulted in the synthesis, segregation in membranes and secretion into the extracellular medium of ovalbumin, lysozyme and prochymosin, respectively. Judging from molecular weight estimations, lysozyme and prochymosin were correctly proteolytically processed while ovalbumin, which lacks a cleavable signal sequence, was glycosylated. Injection of the DNA construct encoding prochymosin without its signal sequence resulted in synthesis of prochymosin protein that was localized exclusively in the oocyte cytoplasm. No immunospecific protein was detected after injection of the DNA encoding mature chymosin. In terms of protein expression in oocytes, the Herpes simplex thymidine kinase (TK) promoter was up to sevenfold more effective than the simian virus 40 (SV40) early promoter, and equally as effective as the Moloney murine sarcoma virus long terminal repeat element. Where tested, protein expression in oocytes was much reduced if DNA sequences encoding the SV40 small t intron and its flanking sequences were present in the constructs. S1 nuclease mapping of transcripts produced after injection of DNAs containing the TK promoter indicated that the majority of transcripts initiated at, or within, two bases of the known "cap" site. However, minor transcripts initiating upstream from this site were observed and one (or more) of these transcripts was responsible for the synthesis of an ovalbumin polypeptide containing a 51 amino acid N-terminal extension. This extended protein remained in the oocyte cytosol. When ovalbumin cDNA was inserted into the vectors with opposite polarity to the viral promoter, expression in oocytes resulted in the predominant synthesis and secretion of a variant ovalbumin with a 21 amino acid N-terminal extension, although some full-length ovalbumin was also synthesized and secreted. S1 mapping revealed the presence, in these oocytes, of transcripts of predicted polarity initiating 118 bases upstream from the wild type ovalbumin initiator ATG, at a previously unreported SV40 "promoter". No protein synthesis was detected after the injection of these reverse-orientation constructs into baby hamster kidney (BHK-21) cells.
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PMID:Efficient expression of cloned complementary DNAs for secretory proteins after injection into Xenopus oocytes. 609 86

This paper presents the nucleotide sequence of the Herpes Simplex Virus thymidine kinase (tk) gene. The position on the DNA sequence corresponding to the 5' and 3' termini of tk messenger RNA have been mapped. The mRNA termini are separated by slightly more than 1,300 nucleotides. The same 2,300 nucleotide segment of tk coding strand DNA is fully protected from S1 nuclease digestion when hybridized to tk mRNA. The location and size of the mRNA-coding segment corresponds to a region of the viral DNA that is essential for tk gene expression in microinjected frog oocytes. The nucleotide sequence of the HSV tk gene exhibits an open translational reading frame of 376 codons that extends from the methionine codon most proximal to the 5' terminus of tk mRNA to a UGA stop codon approximately 70 nucleotides from the poly-A addition site. The results of these experiments indicate that the tk gene is not interrupted by intervening DNA sequences, and that certain oligonucleotide sequences adjacent to the termini of the tk gene are homologous to similarly positioned sequences common to structural genes of eukaryotic cells.
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PMID:The nucleotide sequence and transcript map of the herpes simplex virus thymidine kinase gene. 625 56

To study the molecular basis for lack of expression of the simian virus 40 (SV40) early region genes in murine teratocarcinoma-derived stem cells, we introduced a recombinant plasmid consisting of pBR322 linked to the herpes simplex virus type 1 thymidine kinase gene and SV40 genome into thymidine kinase-deficient F9 stem cells. The resulting stem cell clone, 12-1, and a retinoic acid-induced differentiated daughter cell clone, 12-1a, each contain one copy per cell of the entire recombinant plasmid integrated into the cellular genome through a site on the pBR322 genome. Restriction endonuclease analyses indicate that there is no difference in integration site or organization of the three component parts of the plasmid genome within cellular DNA of stem and differentiated cells; yet the differentiated cells, 12-1a, express SV40 large tumor antigen whereas the stem cells, 12-1, do not. Both stem and differentiated cells produce two size classes of polyadenylylated RNA, 2900 and 2600 bases in length, homologous to the early region of the SV40 genome, detectable by RNA blotting analysis. S1 nuclease analysis of the SV40 transcripts present in stem and differentiated cells indicate that the SV40 mRNAs were identically spliced in the two cell types, in a manner consistent with that observed for spliced large and small tumor antigen mRNAs in SV40-infected monkey kidney cells. Thus, the failure of 12-1 teratocarcinoma stem cells, containing an integrated SV40 genome, to express SV40 tumor antigen is not due to a lack of transcription of the SV40 early region or to an inability to splice primary transcripts.
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PMID:Transcription of the simian virus 40 genome in DNA-transformed murine teratocarcinoma stem cells. 627 68

Enzymatically active thymidine kinase (TK) was made in reticulocyte lysates programmed with early vaccinia mRNA that hybridized to plasmid recombinants containing either of two adjacent small DNA subsegments of the viral HindIII-J fragment. The map position of an early polypeptide, with a molecular weight of 19,000 (19K), coincided precisely with that of the TK. The absence of the 19K polypeptide in cell-free translation products of hybridization-selected mRNAs from several TK-negative mutants provided an independent identification of the TK polypeptide. The small size of the TK polypeptide of vaccinia virus distinguishes it from that of procaryotes, eucaryotes, and herpesvirus. Five early mRNAs of 3,840, 2,390, 1,790, 1,070, and 590 nucleotides were mapped within the HindIII-J fragment by RNA blotting and nuclease S1 digestion of RNA-DNA hybrids. The RNAs of 590 and 2,380 nucleotides were found to have 5' coterminal ends and represent major and minor forms, respectively, of the TK message. The 3' end of the minor TK mRNA appeared to be coterminal with the 3' end of the 1,790-nucleotide transcript which encodes a 41K polypeptide. The 1,070-nucleotide RNA was identified as the message for a 21K polypeptide. All of these RNAs, including the two forms of the TK message, were made by the putative TK-negative nonsense mutants.
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PMID:Vaccinia virus thymidine kinase and neighboring genes: mRNAs and polypeptides of wild-type virus and putative nonsense mutants. 629 59

The thymidine kinase (ATP:thymidine 5'-phosphotransferase, EC 2.7.1.21) gene of vaccinia virus has previously been mapped near the middle of the viral DNA, within the 4.85-kilobase HindIII J fragment, and shown to encode a Mr 19,000 polypeptide [Hruby, D. E. & Ball, L. A. (1982) J. Virol. 43, 403-409]. To locate the gene more precisely and to determine the structure of the basic transcriptional unit, the positions of cleavage sites for several restriction endonucleases were mapped within the HindIII J DNA fragment. Four appropriate subfragments of HindIII J DNA were inserted into plasmid pBR322 derivatives and cloned in Escherichia coli. These recombinant plasmid DNAs were tested for their ability to inhibit the cell-free synthesis of active thymidine kinase and to retain the mRNA for this enzyme when immobilized on nitrocellulose filters. The data showed that the gene spanned an EcoRI cleavage site that lies 850 base pairs from the left-hand end of the HindIII J fragment (the HindIII L-J boundary). Because hybridization of vaccinia virus DNA to partially purified thymidine kinase mRNA detected only a single 670-nucleotide RNA species capable of hybridizing to this region of the genome, nuclease S1 mapping experiments were carried out with thymidine kinase mRNA to protect DNA fragments that were terminally labeled at this EcoRI site. The results indicated that the gene extended from about 550 to 1,150 base pairs from the left end of HindIII J, was transcribed in a rightward direction, and contained no intervening sequences. Hence, a 1.04-kilobase Ava II-Hpa II restriction fragment containing this region of DNA was isolated and subjected to nucleotide sequence analysis. An examination of this nucleotide sequence revealed the presence of an open reading frame of 531 nucleotides capable of encoding a protein of 177 amino acids with a Mr of 20,077.
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PMID:Fine structure analysis and nucleotide sequence of the vaccinia virus thymidine kinase gene. 630 9

Four early transcripts and the polypeptides they encode have been mapped to the vaccinia virus EcoRI F DNA fragment, which spans the vaccinia HindIII J and H fragments. In addition, two transcripts for which no encoded polypeptides have been identified have also been mapped. Elucidation of the organization of these six transcripts by hybrid selection, S1 nuclease mapping, translation of size-fractionated RNA, and by filter hybridization demonstrates that approximately 90% of this DNA fragment is transcribed during early infection. All but one of these RNAs (1.35 kilobases [kb]) are transcribed in a rightward direction. The leftmost transcript of 0.6 to 0.7 kb encodes a 19,000-dalton (19K) polypeptide that has been determined to be thymidine kinase (D.E. Hruby and L.A. Ball, J. Virol. 43:403-409, 1982; G. Bajszar et al., J. Virol. 45:62-72, 1983). Immediately following the 3' end of this RNA is the 1.7-kb transcript encoding a 36K polypeptide. The 5' end of a 1.25-kb RNA encoding a 22K polypeptide is downstream of the 5' end of the 1.7-kb RNA, and its 3' end may be coterminal with the 3' end of the 1.7-kb RNA. The 2.45-kb transcript is coterminal at its 5' end with the message encoding thymidine kinase and is coterminal at its 3' end with the adjacent 1.7-kb mRNA. This RNA was not demonstrated to encode a polypeptide. Approximately 300 nucleotides from the 3' end of the 1.7-kb RNA is a 3.6-kb transcript encoding a 110K polypeptide. The 3' end of this large RNA lies several hundred nucleotides from the 3' end of the 1.35-kb RNA which is transcribed in the opposite direction. No splicing of RNA has been detected with the S1 nuclease mapping technique of Berk and Sharp. The organization of three late messages, which overlap these early transcripts, has been determined, and these results are discussed in the accompanying paper.
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PMID:Organization of six early transcripts synthesized from a vaccinia virus EcoRI DNA fragment. 631 49

An in vitro approach has been used to study trout protamine gene expression using various recombinant plasmids containing trout protamine genes as templates in the HeLa cell lysate transcription system. The specific RNA transcript which is protected against S1 nuclease digestion by hybridization to the protamine gene sequence is alpha-amanitin sensitive (1 micrograms/mL), showing that RNA polymerase II is involved. The sizes of transcripts from templates linearized with Bam HI, Rsa I, and Hpa II (all downstream from the putative TATA box) are consistent with those predicted from the known sequence of the protamine gene. Digestion at an Alu I site only 14 base pairs (bp) upstream from TATA box has no effect on the accuracy of transcription in vitro; however, cutting at an Ava II site 9 bp downstream from the TATA box (reading from the first T) abolishes transcription. Chimeric plasmids, in which a herpes simplex virus (HSV-1) thymidine kinase (tk) promoter is tandemly inserted upstream from the trout protamine DNA sequences or as a replacement of the natural protamine promoter, were constructed. Use of these plasmids allowed an examination in a single assay of eight different putative promoter sequences (TATAAAA, TATAAA, TACAAA, TATATA, TATTTAA, CATATTA, TATATTAT, and TATTTAT) that are localized in either the protamine or the tk genes. The canonical TATAAAA promoter (the natural protamine promoter) was the strongest one and, in its presence, none of the others were used significantly for transcription. However, when this promoter was removed the weaker promoters were able to promote transcription.
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PMID:Transcription of a trout protamine gene in vitro: the effects of alteration of promoters. 632 91

Individual cloned HindIII fragments of vaccinia virus DNA were introduced into cells by DNA-mediated gene transfer. The presence of individual fragments in the different transformants was confirmed by Southern blot hybridization analysis. Several of the transformants were found to express viral sequences at various levels. The sizes of the transcripts containing vaccinia virus sequences were highly heterogeneous, with no discrete species of RNA. Positive clones contained vaccinia virus sequences in both the poly(A)+ and poly(A)- RNA fractions, although the prevalence of these sequences was variable in the two fractions. The S1 nuclease map of the 5' end of the transcripts from transformants containing the HindIII-J fragment revealed a unique 5' end, similar to RNA from virus-infected cells. In contrast, analysis of the 3' end of RNAs from these transformants showed a high degree of heterogeneity, which might explain the heterogeneity found in Northern blot patterns. In this report, it is shown for the first time that in cells transformed with vaccinia virus DNA there is proper initiation for, at least, the viral thymidine kinase gene.
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PMID:Expression of cloned vaccinia virus DNA sequences introduced into animal cells. 674 2


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