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:2.7.7.6 (RNA polymerase)
34,946 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The growth of MCF-7 cells was arrested by 24 h of isoleucine deprivation. Following replenishment of the medium, the incorporation of uridine and thymidine into trichloroacetic acid-precipitable material began to increase slowly and gradually rose to the level of cycling cells. The addition of 5 X 10(-9) M estradiol to growth-arrested cells dramatically shortened the time of onset of macromolecular synthesis and increased the overall amount of precursor incorporation 2- to 4-fold over the level obtained by arrested control cells. The increase in uridine incorporation preceded the increase in thymidine incorporation by 6 h. Inhibition of protein synthesis with cycloheximide blocked the recovery of macromolecular synthesis in both control and estrogen-treated cells. Actinomycin D was ineffective in blocking the estrogen-stimulated recovery of macromolecular synthesis at concentrations known to inhibit pre-rRNA synthesis (10(-8) M). At higher concentrations, uridine and thymidine incorporation were inhibited in a dose-dependent manner. Inhibition of RNA polymerase II activity with alpha-amanitin similarly blocked both the recovery of the cells from isoleucine starvation and the potentiation of this by estradiol. Dihydrofolate reductase and thymidine kinase activities are both stimulated by estradiol in MCF-7 cells. In cycling cells, estrogen stimulates a 2-fold increase in their messenger RNAs (mRNAs) within 24 h. The level of dihydrofolate reductase mRNA is unaffected by isoleucine starvation, and estrogen caused no change in dihydrofolate reductase mRNA levels over a 24-h period following reversal of growth arrest. Similar results were observed for the 600-nucleotide pS2 mRNA that has been identified as an estrogen-induced RNA in MCF-7 cells. In contrast, thymidine kinase mRNA was found to be increased by estrogen at 24 h, but not at 12 h, following reversal of growth arrest. This increase correlates with increases in thymidine, but not uridine incorporation. These data indicate that the estrogen-stimulated increase in thymidine incorporation following release from growth arrest is dependent on new RNA synthesis. However, the hormone did not increase the levels of three estrogen-regulated mRNAs coordinately with the increases observed in uridine incorporation.
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PMID:Relationship between the expression of estrogen-regulated genes and estrogen-stimulated proliferation of MCF-7 mammary tumor cells. 398 99

The sites of in vitro transcription initiation on the BamHI Q fragment of herpes simplex virus DNA have been compared with the sites of 5' ends of RNAs made in vivo after virus infection. S1-nuclease protection analysis of these RNAs shows that there are in vivo counterparts for each of the five previously identified in vitro transcripts. The whole-cell-extract RNA polymerase II transcription system faithfully initiates RNAs predominantly at bona fide in vivo start sites and gives few, if any, false positive start sites. Further, antiparallel, self-complementary transcripts from the BamHI Q fragment were observed in the coding region of the HSV thymidine kinase gene.
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PMID:In vitro and in vivo transcription initiation sites on the TK-encoding BamHI Q fragment of HSV-1 DNA. 609 73

mRNA's homologous to the herpes simplex virus type 1 DNA restriction endonuclease fragment BamHI p, which contains the thymidine kinase gene, have been identified and mapped by hybrid-arrested translation and mRNA selection. Such mRNA's, when translated in vitro, directed the synthesis of polypeptides of apparent molecular weights 43,000 (VI43) and 39,000 (VI39). mRNA for enzymatically active thymidine kinase was enriched by more than 20-fold after selection. Mapping was carried out with restriction endonuclease fragments of BamHI p, and locations of the 5' and 3' termini of VI43 mRNA were deduced. Analysis of nucleotide sequences around the 5' terminus revealed several consensus sequences commonly found at the start of eucaryotic mRNA's and which are presumably involved in initiation of transcription by RNA polymerase II. Translation of mRNA's for VI43, VI39, and the thymidine kinase enzyme was arrested only by a 1,170-base-pair region of BamHI p. Since this region is insufficient for adjacent genes, coding sequences for VI43 and VI39 must overlap; the possible relationship of these two polypeptides is discussed. A virus-induced product equivalent to VI39 was detected in infected cells.
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PMID:Identification and mapping of two polypeptides encoded within the herpes simplex virus type 1 thymidine kinase gene sequences. 626 30

The hybrid plasmid pTK1 consists of the herpes simplex virus type 1 (HSV-1) BamHI p fragment, which contains the thymidine kinase (TK) gene, inserted into the vector pAT 153. When pTK1 DNA was microinjected into nuclei of Xenopus laevis oocytes, functional HSV-1-specific TK was produced, showing that transcription and translation of the gene occurred. Investigation of pTK1-specific RNA by "Southern' blot hybridization revealed that all regions of the hybrid plasmid were transcribed by RNA polymerase II, but sequences present in TK mRNA were most highly represented in stable transcripts.
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PMID:Transcription and translation of the herpes simplex virus type 1 thymidine kinase gene after microinjection into Xenopus laevis oocytes. 627 Feb 58

We describe a 2560 base pair herpes simplex virus type 1 (HSV-1) DNA sequence containing the entire immediate-early mRNA-5 (IEmRNA-5) gene. The 3' and 5' termini of IEmRNA-5 were mapped within this DNA sequence by single-strand specific endonuclease protection experiments. The IEmRNA-5 gene contains DNA sequences from both the unique (Us) and reiterated (TRs/IRs) regions of the HSV-1 DNA short component and is interrupted by a single intron mapping in TRs/IRs. A search of the transcribed DNA sequence revealed no initiator codon within TRs/IRs. The first ATG was located 6 bases into Us sequences and this reading frame (316 codons) was also observed in the 3' transcribed region. The oligonucleotide sequences adjacent to the IEmRNA-5 termini are discussed in relation to those of the HSV-1 thymidine kinase gene and other genes transcribed by RNA polymerase II.
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PMID:DNA sequence of an immediate-early gene (IEmRNA-5) of herpes simplex virus type I. 627 43

We used partially purified RNA polymerase II from uninfected (Pol II) and from herpes simplex virus type 1 (HSV-1) infected HEp-2 cells (Pol II-H) to transcribe HSV-1 DNA in vitro. Gel electrophoretic analysis of the products produced from native HSV-1 DNA yielded weight average chain lengths of 4.0 and 3.5 kb for the Pol II and Pol II-H products, respectively. Blot hybridization analyses of the HSV DNA transcripts showed that both enzymes transcribed RNA from essentially all regions of the genome. However, Pol II preferentially transcribed regions coding for the immediate-early or alpha mRNAs, whereas Pol II-H preferentially copied regions coding for the early (beta) and late (gamma) gene products. Transcriptional analyses of the cloned HSV-1 Bam HI-Q fragment (containing the thymidine kinase (TK) gene) and its subfragments showed that (1) the major transcripts produced by Pol II-H were distinctly different from those produced by Pol II; (2) Pol II and Pol II-H utilized different promoters for the synthesis of major transcripts; (3) both enzymes produced three minor transcripts that were partially overlapping and in opposite direction to the TK gene; and (4) only Pol II-H initiated transcription from the TK promoter. In contrast, both Pol II and Pol II-H generated an identical set of transcripts from an adenovirus 2 early region DNA fragment. The sizes of the products suggest that RNA processing may be occurring in vitro. These results show that HSV-1 infection alters the in vitro transcriptional specificity of RNA polymerase II and demonstrate that this system should be useful for studying in vitro the regulation of gene transcription.
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PMID:Regulation of herpes simplex virus gene transcription in vitro. 629 54

Using both clones of mouse LMTK- cells cotransformed with various chimeric conalbumin promoter simian virus 40 (SV40) early gene recombinants and the herpes thymidine kinase gene, and HeLa cells transfected with the same chimeric recombinants, we show that the SV40 72 base pair (bp) repeat sequence is a bidirectional potentiator of initiation of transcription from adjacent T-A-T-A box-dependent and -independent start sites. These results are consistent with our previous model based mainly on the results of T antigen gene expression assays that the 72-bp repeat acts as a bidirectional entry site for RNA polymerase B. We also show that the conalbumin T-A-T-A box is an important element for efficient and accurate in vivo initiation of transcription.
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PMID:A novel eukaryotic promoter element: the simian virus 40 72 base pair repeat. 631 2

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

During the past two decades, the essentiality of zinc for man has been established. Deficiency of zinc in man due to nutritional factors and several diseased states has been recognized. High phytate content of cereal proteins decreases availability of zinc; thus the prevalence of zinc deficiency is likely to be high in a population subsisting mainly on cereal proteins. Alcoholism is known to cause hyperzincuria and thus may play a role in producing zinc deficiency in man. Malabsorption, cirrhosis of the liver, chronic renal disease and other chronically debilitating diseases may similarly induce zinc deficiency in human subjects. A severe deficiency of zinc has recently been recognized to occur in patients with sickle cell anemia and a beneficial effect of zinc therapy in such patients has been reported. Growth retardation, male hypogonadism, skin changes, poor appetite, mental lethargy and delayed wound healing are some of the manifestations of chronically zinc-deficient human subjects. Taste abnormalities, correctable with zinc supplementation, have been observed in uremic subjects. Recently, abnormal dark adaptation related to zinc deficiency in patients with cirrhosis of the liver and sickle cell disease has been reported. In severely zinc-deficient patients, dermatological manifestations, diarrhea, alopecia, mental disturbances and intercurrent infections predominate and if untreated the condition becomes fatal. Zinc deficiency is known to affect testicular functions adversely in man and animals. This effect of zinc is at the end organ level and it appears that zinc is essential for spermatogenesis and testosterone steroidogenesis. Zinc is involved in many biochemical functions. Several zinc metalloenzymes have been recognized in the past decade. Zinc is required for each step of cell cycle in microorganisms and is essential for DNA synthesis. Thymidine kinase, RNA polymerase, DNA-polymerase from various sources and RNA-dependent DNA polymerase from viruses have been shown to be zinc-dependent enzymes. Zinc also regulates the activity of RNase; thus the catabolism of RNA appears to be zinc-dependent. The effect of zinc on protein synthesis may be attributable to its vital role in nucleic acid metabolism. The activities of many zinc-dependent enzymes have been shown to be affected adversely in zinc-deficient tissues. Three enzymes, alkaline phosphatase, carboxypeptidase and thymidine kinase, appear to be most sensitive to zinc restriction in that their activities are affected adversely within three to six days of institution of a zinc-deficient diet to experimental animals.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Zinc deficiency in human subjects. 636 78

In mammalian cells, salvage pathway phosphorylation of thymidine is catalyzed by two thymidine kinases: the cell-cycle regulated cytoplasmic TK1 and the constitutively expressed mitochondrial TK2. Since TK1 is virtually absent in non-dividing cells, TK2 is probably the only thymidine kinase present in these cells. In cellular metabolism, TK1 and TK2 presumably serve to maintain sufficient dTTP for DNA replication and repair. TK1 purified from phytohemagglutinin-stimulated human lymphocytes is a dimer in the absence and a tetramer in the presence of ATP. In addition to the molecular weight transition, incubation with ATP at 4 degrees C or storage with ATP induces a reversible, enzyme concentration-dependent, kinetically slow transition from a low to a high affinity form of TK1, with Km values of 14 microM and 0.5 microM, respectively. This affinity difference implies that at cellular thymidine concentrations, the difference in catalytic activity between the two TK1 forms will be 3-5-fold. Calculations of cellular TK1 concentration suggested that the low affinity dimer form was dominant in G0/G1 cells and the high affinity tetramer form in S-phase cells. Hence, the transition may serve to fine-tune the cell-cycle regulation of thymidine kinase activity on the post-translational level. To study the ATP effect on the molecular level, an IPTG inducible T7 RNA polymerase-dependent expression system for the entire human TK1 polypeptide in E. coli was established. The recombinant TK1 has the same subunit mass and specific activity as the native enzyme. However, the recombinant TK1 solely displayed the kinetics of the high affinity form, with Km values of 0.3-0.4 microM regardless of pre-exposure to ATP, indicating that the ATP effect may be dependent on post-translational modifications absent in E. coli. Surprisingly, we did not observe any effect of ATP on TK1 purified from bone-marrow cells from a patient with acute monocytic leukemia (AMOL). Furthermore, the Km values of TK1 from these cells were 45 microM for the ATP-free enzyme and 65 microM for the ATP-incubated enzyme. With TK1 purified from HL-60 cells, we obtained the same pattern and kinetic values as for TK1 from lymphocytes. In the light of the results with the recombinant TK1, we presume that the lack of ATP effect and very high Km values observed for the AMOL TK1 may be due to changes in post-translational regulatory mechanisms in acute monocytic cells.
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PMID:Human thymidine kinase 1. Regulation in normal and malignant cells. 757 55


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