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At least 541 records · Page 30Linked to original sources
PubMed1999

Analysis of rpoS mRNA in Salmonella dublin: identification of multiple transcripts with growth-phase-dependent variation in transcript stability.

In Salmonella dublin, rpoS encodes an alternative sigma factor of the RNA polymerase that activates a variety of stationary-phase-induced genes, including some virulence-associated genes. In this work, we studied the regulation and transcriptional organization of rpoS during growth. We found two transcripts, 2.3 and 1.6 kb in length, that represent the complete rpoS sequence. The 2.3-kb transcript is a polycistronic message that also includes the upstream nlpD gene. It is driven by a weak promoter with increasing activity when cells enter early stationary growth. The 1.6-kb message includes 566 bp upstream of the rpoS start codon. It is transcribed from a strong sigma70 RNA polymerase-dependent promoter which is independent of growth. The decay of this transcript decreases substantially in early stationary growth, resulting in a significant net increase in rpoS mRNA levels. These levels are approximately 10-fold higher than the levels of the 2.3-kb mRNA, indicating that the 1.6-kb message is mainly responsible for RpoS upregulation. In addition to the 2.3- and 1.6-kb transcripts, two smaller 1.0- and 0.4-kb RNA species are produced from the nlpD-rpoS locus. They do not allow translation of full-length RpoS; hence their significance for rpoS regulation remains unclear. We conclude that of four transcripts arising from the nlpD-rpoS locus, only one plays a significant role in rpoS expression in S. dublin. Its upregulation when cells enter stationary growth is due primarily to an increase in transcript stability.

Bacterial Proteins↗
PubMed1991

The three major immediate-early transcripts of bovine herpesvirus 1 arise from two divergent and spliced transcription units.

Among 54 transcripts expressed in a temporal cascade during lytic infection with bovine herpesvirus 1, we have previously identified three major immediate-early (IE) RNAs, IER4.2 (4.2 kb), IER2.9 (2.9 kb), and IER1.7 (1.6 to 1.8 kb depending on the virus strain) transcribed from the HindIII C genome region (U. V. Wirth, K. Gunkel, M. Engels, and M. Schwyzer, J. Virol. 63:4882-4889, 1989). Northern (RNA) blot, S1 nuclease protection, and primer extension analysis used in the present study demonstrated that all three IE transcripts were spliced and originated from two divergent transcription units with start sites located in the inverted repeat. Transcription unit 1 encoded two alternative spliced transcripts, IER4.2 and IER2.9, with a common exon 1 located at 0.797 to 0.795 map units (m.u.) and an exon 2 for IER4.2 (0.792 to 0.762 m.u.) in the inverted repeat; exon 2 for IER2.9 (0.754 to 0.738 m.u.) was located in the unique long sequence and transcribed in antisense orientation to latency-related RNA. Transcription unit 2 (0.818 to 0.836 m.u.), further characterized by cDNA cloning, encoded the spliced IER1.7 with three exons in the inverted repeat. Additional minor IE transcripts were interpreted as unspliced precursors and splicing variants. With regard to the number and layout of IE genes, bovine herpesvirus 1 occupies an intermediate position between pseudorabies virus and equine herpesvirus 1 on the one hand and varicella-zoster virus and herpes simplex virus type 1 on the other.

Animals↗
PubMed1995

Immediate-early transcription from the channel catfish virus genome: characterization of two immediate-early transcripts.

With cDNA probes and by Northern (RNA) blot analysis, a region containing immediate-early (IE) genes in the channel catfish virus (CCV) genome was identified. IE transcription in CCV-infected cells appears to be restricted to the terminal repeat region, suggesting that CCV is most closely related to the alpha subfamily of herpesviruses. CCV DNA fragments from this region encoding IE transcripts were cloned. Northern analysis with one of these cloned fragments, a 3,927-bp EcoRI-XbaI fragment, indicates that it encodes two IE transcripts. Both transcripts (ie1 and ie2) were characterized by S1 nuclease analysis, primer extension analysis, and analysis of cDNAs. The ie2 transcript is a 1.3-kb bicistronic mRNA containing open reading frame (ORF) 8a and ORF 9. ORF 8a is a 5'-truncated version of ORF 8 which, along with ORF 9, was previously identified (A. J. Davison, Virology 186:9-14, 1992). The ie1 transcript is 0.6 kb in size, contains only ORF 9, and is expressed at a level approximately six times that of ie2 in cycloheximide-treated cells. The putative product of ORF 9 is predicted to have a basic pI and contains a potential zinc-binding domain, making it a probable transcription factor. ORF 8a encodes a putative product which is very hydrophobic, an unusual characteristic for an IE protein.

Animals↗
PubMed1998

A virus with a mutation in the ICP4-binding site in the L/ST promoter of herpes simplex virus type 1, but not a virus with a mutation in open reading frame P, exhibits cell-type-specific expression of gamma(1)34.5 transcripts and latency-associated transcripts.

The herpes simplex virus type 1 L/S junction-spanning transcripts (L/STs) are a family of multisized transcripts expressed at high levels in cells infected with mutant viruses that (i) do not express ICP4, (ii) specify forms of ICP4 unable to bind to the consensus ICP4 binding site, or (iii) contain mutations in the ICP4 binding site located at the transcriptional start site of the L/STs. By extension, the failure to detect the L/STs in wild-type virus-infected cells is due to the repressive effect of ICP4 bound to its cognate binding site upstream of the L/ST transcription initiation site. ORF-P, the first and largest open reading frame (ORF) encoded by the L/STs, overlaps >90% of the ORF encoding ORF-34.5, a putative neurovirulence factor, which is transcribed from the opposite DNA strand. Viruses with mutations in the overlapping region of ORF-P and ICP34.5 exhibit premature shutoff of infected-cell protein synthesis and are highly attenuated following intracranial inoculation of juvenile mice. To determine whether the premature protein shutoff and neuroattenuated phenotypes of ORF-P ORF-34.5 double mutants are a consequence of alterations in ORF-P, ORF-34.5, or both, viruses containing mutations only in ORF-P or only in the ICP4 binding site in the L/ST promoter were isolated and characterized. Mutant virus L/ST-n38 contains a single-base-pair transition mutation in ORF-P codon 38, resulting in translational termination of the ORF-P protein (OPP). This mutation does not alter the amino acid sequence of ICP34.5. Expression of a truncated form of OPP by mutant virus L/ST-n38 did not result in premature shutoff of infected-cell protein synthesis and produced no other observable phenotype relative to wild-type virus in in vitro tests. Moreover, the 50% lethal dose (LD50) of L/ST-n38 was comparable to that of wild-type virus following intracranial inoculation of 3-week-old mice, as were the latency and reactivation phenotypes of the virus. These properties of L/ST-n38 indicate that the attenuated phenotype of ORF-P ORF-34.5 double mutants is a consequence of mutations that affect the function of ICP34.5 and not the function of OPP. Mutant virus LST-4BS contains four single-base-pair substitutions in the ICP4 binding site in the L/ST promoter that abrogate the binding of ICP4 to this site, leading to high-level expression of the L/STs and OPP. LST-4BS induced premature shutoff of viral and cellular protein synthesis and was slightly growth restricted in cells of neural lineage (SK-N-SH human neuroblastoma cells) but was wild type for these two parameters in cells of nonneural lineage (immortalized primate Vero cells). Of particular interest was the observation that L/ST-4BS exhibited cell-type-specific expression of both the gamma(1)34.5 transcripts and the latency-associated transcripts (LATs). Thus, expression of these transcripts was barely detectable in cells of neural lineage (NB41A3 mouse neuroblastoma cells) but was wild type in Vero cells. In vivo, L/ST-4BS was reactivated from mouse trigeminal ganglia with reduced efficiency and delayed kinetics relative to wild-type virus. L/ST-4BS was completely attenuated for neurovirulence (LD50 > 10(6) PFU) relative to wild-type virus (LD50 < 900 PFU), although the four single-base-pair substitutions lie outside the coding region for the neurovirulence factor, ICP34.5. Collectively, the complex in vitro and in vivo phenotypes of L/ST-4BS can be attributed to (i) disruptions of the ICP4 binding site in the L/ST promoter and subsequent overexpression of the L/STs and OPP; (ii) alterations in ORF-O, which is also mutated in L/ST-4BS; or (iii) alterations in other cryptic genes or cis-acting elements.

Amino Acid Sequence↗
PubMed1990

Transcriptional potentiation of the vitellogenin B1 promoter by a combination of both nucleosome assembly and transcription factors: an in vitro dissection.

The Xenopus laevis vitellogenin B1 promoter was assembled into nucleosomes in an oocyte extract. Subsequent RNA polymerase II-dependent transcription from these DNA templates fully reconstituted in chromatin in a HeLa nuclear extract was increased 50-fold compared with naked DNA. Remarkably, under specific conditions, production of a high level of transcripts occurred at very low DNA (1 ng/microliter) and HeLa nuclear protein (1.6 micrograms/microliters) concentrations. When partially reconstituted templates were used, transcription efficiency was intermediate between that of fully reconstituted and naked DNA. These results implicate chromatin in the process of the transcriptional activation observed. Depletion from the oocyte assembly extract of an NF-I-like factor which binds in the promoter region upstream of the TATA box (-114 to -101) or deletion from the promoter of the region interacting with this factor reduced the transcriptional efficiency of the assembled templates by a factor of 5, but transcription of these templates was still 10 times higher than that of naked DNA. Together, these results indicate that the NF-I-like factor participates in the very efficient transcriptional potentiation of the vitellogenin B1 promoter which occurs during nucleosome assembly.

Animals↗
PubMed1991

GAL11 (SPT13), a transcriptional regulator of diverse yeast genes, affects the phosphorylation state of GAL4, a highly specific transcriptional activator.

The GAL4 protein of Saccharomyces cerevisiae is a DNA-binding transcriptional activator that is highly specific for the GAL genes. In vivo levels of GAL gene transcription are closely correlated with the phosphorylation state of GAL4. In vivo levels of GAL gene transcription are also affected by the activity of the GAL11 (SPT13) protein, a protein that has been implicated as a global auxiliary transcriptional factor. Here we examine the influence of GAL11 (SPT13) on the phosphorylation state of GAL4. Cells bearing a gal11 deletion mutation are defective in the production or maintenance of GAL4III, a phosphorylated form of GAL4 that is associated with higher levels of GAL gene transcription. In addition, the gal11 deletion cells are reduced in total GAL4 protein. However, the fivefold-reduced expression of the GAL1 gene observed in gal11 deletion cells cannot be due solely to reduced levels of total GAL4 protein, since gal11 deletion cells amplified for GAL4 production are still markedly reduced in GAL4 protein-dependent transcription. Thus, these data demonstrate that the GAL11 protein augments GAL4 protein-dependent transcription in a manner that is tightly coupled to the formation or maintenance of a phosphorylated form of GAL4.

Blotting, Western↗
PubMed1986

Identification of primary transcriptional start sites of mouse mitochondrial DNA: accurate in vitro initiation of both heavy- and light-strand transcripts.

The major transcriptional control sequences of vertebrate mitochondrial DNA lie within the displacement loop region. Transcription events initiating in the displacement loop sequence of the mouse genome were identified by 5' end mapping of primary transcripts by S1 nuclease protection and primer extension techniques. Light-strand transcription initiates at a single site, 165 nucleotides upstream of the major heavy-strand origin of replication. Transcription of the heavy strand occurs at two distinct sites, 5 and 13 nucleotides upstream of the gene for phenylalanyl-tRNA, the first heavy-strand-encoded gene. This spatial relationship of the two transcriptional start sites with each other and with the origin of heavy-strand replication and the gene for tRNAPhe is quite similar to that for human mitochondrial DNA. The predominant form of primary heavy-strand transcript in mouse is a short, ca. 75-nucleotide, RNA containing the sequences of tRNAPhe and a few additional nucleotides at the 5' end of tRNAPhe, suggesting that the processing of tRNA involves independent cleavages at the 5' and 3' ends of tRNA sequences.

Animals↗
PubMed1987

Evidence for transcriptional and post-transcriptional control of the cellular thymidine kinase gene.

We have studied the cell cycle-regulated expression of the thymidine kinase (TK) gene in mammalian tissue culture cells. TK mRNA and enzyme levels are low in resting, G0-phase cells, but increase dramatically (10- to 20-fold) during the S phase in both serum-stimulated and simian virus 40-infected cells. To determine whether an increase in the rate of TK gene transcription is responsible for this induction, nuclear run-on transcription assays were performed at various times after serum stimulation or simian virus 40 infection of growth-arrested simian CV1 cells. When assays were performed at 12-h intervals, a small (two- to threefold) but reproducible increase in TK transcription was detected during the S phase. When time points were chosen to span the G1-S interface a larger (six- to sevenfold) increase in transcriptional activity was observed in serum-stimulated cells but not in simian virus 40-infected cells. The large increase in TK mRNA levels and the relatively small increase in transcription rates in growth-stimulated cells suggest that TK gene expression is controlled at both a transcriptional and post-transcriptional level during the mammalian cell cycle. To identify the DNA sequences required for cell cycle-regulated expression, several TK cDNA clones were transfected into Rat-3 TK- cells, and their expression was examined in resting and serum-stimulated cultures. These experiments indicated that the body of the TK cDNA is sufficient to insure cell cycle-regulated expression regardless of the promoter or polyadenylation signal used.

Animals↗
PubMed1989

Development of an in vitro transcription system for Neurospora crassa mitochondrial DNA and identification of transcription initiation sites.

We have developed an in vitro transcription system for Neurospora crassa mitochondrial DNA (mtDNA) and used it to identify transcription initiation sites at the 5' ends of the genes encoding the mitochondrial small and large rRNA and cytochrome b (cob). The in vitro transcription start sites correspond to previously mapped 5' ends of major in vivo transcripts of these genes. Sequences around the three transcription initiation sites define a 15-nucleotide consensus sequence, 5'-TTAGARA(T/G)G(T/G)ARTRR-3', all or part of which appears to be an element of an N. crassa mtDNA promoter. A somewhat looser 11-nucleotide consensus sequence, 5'-TTAGARR(T/G)R(T/G)A-3', was derived by including two additional promoters identified recently. Group I extranuclear mutants, such as [poky] and [SG-3], have a 4-base-pair (bp) deletion in the consensus sequence at the 5' end of the mitochondrial small rRNA and are grossly deficient in mitochondrial small rRNA (R. A. Akins and A. M. Lambowitz, Proc. Natl. Acad. Sci. USA 81:3791-3795, 1984). We show here that the 4-bp deletion in the consensus sequence decreases in vitro transcription from this site by more than 99%. N. crassa mtDNA is similar to Saccharomyces cerevisiae mtDNA in having multiple promoters, including separate promoters for the genes encoding the mitochondrial small and large rRNAs. Our results suggest that the primary effect of the 4-bp deletion in group I extranuclear mutants is to inhibit transcription of the mitochondrial small rRNA, leading to severe deficiency of mitochondrial small rRNA and small ribosomal subunits.

Base Sequence↗
PubMed1994

Competitive reverse transcription-polymerase chain reaction analysis indicates that levels of aromatase cytochrome P450 transcripts in adipose tissue of buttocks, thighs, and abdomen of women increase with advancing age.

Circulating androstenedione is converted to estrone in adipose tissue, which is the principal site of estrogen biosynthesis in postmenopausal women. This reaction is catalyzed by a specific form of cytochrome P450 (P450arom; the product of the CYP19 gene). The fractional conversion of plasma androstenedione to estrone as well as the specific activity of aromatase in adipose stromal cells were previously shown to increase with advancing age. To determine whether this positive effect of aging on estrogen biosynthesis is due to an alteration in tissue levels of P450arom transcripts, we quantified P450arom mRNA levels in sc fat biopsy samples (n = 33) from buttocks, thighs, and abdomen of 11 women who ranged in age from 23-61 yr. Competitive polymerase chain reaction linked to reverse transcription was used to quantify P450arom transcripts in total RNA that was isolated from sc fat obtained by needle aspiration. In each sample, primer extension and coamplification of a rat P450arom cRNA as an internal standard were used to control possible differences in amplification efficiencies between samples. The results demonstrate that with advancing age in women, there is a progressive and statistically significant increase in adipose tissue P450arom transcript levels (normalized to total RNA content) in buttocks, thighs, and abdomen (correlation coefficients: r = 0.704, 0.854, and 0.933, respectively). The levels of transcripts observed in the older subjects reach 2- to 4-fold greater than those observed in the young women. Adipose tissue P450arom transcript levels were highest in the buttocks, followed by the thighs, and lowest in the abdomen. This increase in P450arom transcript levels is likely to be a major factor contributing to the increased extragonadal estrogen biosynthesis in elderly women.

Abdomen↗
PubMed2004

Temporal and spatial changes in transcription factor binding and histone modifications at the steroidogenic acute regulatory protein (stAR) locus associated with stAR transcription.

We investigated the binding of transcription factors and histone modifications associated with expression of the steroidogenic acute regulatory protein (StAR) gene in cultured MA-10 Leydig cells and in granulosa cells isolated from mouse periovulatory follicles before and after in vivo human chorionic gonadotropin administration. Quantitative chromatin immunoprecipitation assays were employed to prove association of specific transcription factors (GATA-4, steroidogenic factor 1/adrenal-4 binding protein, CCAAT/enhancer-binding protein beta, cAMP response element binding protein/cAMP response element modulator) and a coactivator (cAMP response element binding protein-binding protein) with the promoter, to define patterns of binding to test hypotheses regarding interactions among these factors, and to correlate changes in histone modification at the StAR locus with transcription. Although each of the transcription factors bound to the StAR proximal promoter, we observed cell-specific binding patterns for individual factors. From these findings we infer that associations among some of the factors can be more complex than can be explained by simple models of stable protein-protein interactions. Histone modifications were also found to exhibit cell-specific, temporal and spatial differences across the StAR locus. In MA-10 cells, these modifications included increased acetylation of histone H3, increased dimethylation of lysine 4 on histone H3 in exonic/intronic sequences (a modification that marks transcriptionally permissive chromatin), and reduced dimethylation of lysine 9 on histone H3 (a modification linked with gene silencing). In mouse granulosa cells, we observed no change in histone H3 or H4 acetylation, but a rapid loss of the dimethyl K9 histone H3 mark. Our findings demonstrate that increased StAR transcription can occur in the context of different patterns of transcription factor binding and histone modification.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed2000

[Transcriptional mapping and study of transcription regulation of the Pseudomonas aeruginosa phage-transposon D3112].

Regulation of transcription was studied in a wild-type transposable phage (TP) D3112 of Pseudomonas aeruginosa and its mutants for different genes. For this purpose, [3H]RNA-DNA hybridization with denatured fragments from various regions of the D3112 genome bound to nitrocellulose filters was used. A transcriptional map of TP D3112 was constructed based on the data obtained. The map comprised six independent transcriptional units corresponding to the modular organization of the phage genome. Only the repressor gene cI was transcribed in the lysogenic state. After repressor thermoinactivation, the cI transcription ceased, and transcription occurred in the same order as the genes (modules) were located on the D3112 phage genetic map (from left to right): cip1 (a negative regulator) and early genes A and B (controlling the replication-transposition of the phage), nonessential genome region, genes C and the locus marked with the ts47 mutation (positive regulators of late gene transcription), the genes of the head morphogenesis, gene c91 (a positive regulator responsible for the lysogenic state), and the genes of the tail morphogenesis. Similarities between nonhomologous TPs D3112 of P. aeruginosa and Mu of Escherichia coli with respect to genetic organization and transcription regulation are discussed.

DNA Transposable Elements↗
PubMed2002

Inhibition of transcription results in accumulation of Wt p53 followed by delayed outburst of p53-inducible proteins: p53 as a sensor of transcriptional integrity.

Here we investigate activation of the p53 pathway by inhibition of transcription. Comparison of cells with either mutant p53 or wt p53 indicated that inhibition of p53- dependent transcription is necessary and sufficient for wt p53 accumulation. In addition to Mdm-2, p21 is required for effective p53 degradation. Transient inhibition of transcription resulted in initial downregulation of p21 and Mdm-2 leading to accumulation of wt p53. This was followed by induction of p21 and Mdm-2, normalization of p53 levels, and p21-dependent growth arrest. Although simultaneous induction of p53 and p21 could be detected by immunoblot, levels of p53 and p21 were discordant in individual cells. By inducing p21 and Mdm-2, p53 discriminates between transient and sustained inhibition of transcription. Transient inhibition results in p21-dependent growth, while sustained inhibition of transcription leads to p53-facilitated cell death. One can envision p53 as a physiological sensor of transcriptional integrity. Transient inhibition of p53- stimulated transcription by numerous stimuli including nucleotide depletion, hypoxia, UV light may be an prevalent mechanism of activation of wt p53 and its downstream pathways.

Cell Division↗
PubMed1991

Inhibition and stimulation of c-myc gene transcription by insulin in rat hepatoma cells. Insulin alters the intragenic pausing of c-myc transcription.

One of insulin's least studied actions is its ability to induce DNA synthesis and cell division. In rat H4IIE hepatoma cells insulin, acting through its own receptor, stimulates cell division. However, little is known about the molecular mechanisms involved in this effect. The proto-oncogene c-myc is a cellular gene which when expressed at abnormal levels is often associated with the process of tumorigenesis. Expression of the normal cellular myc gene may be necessary for growth factor-induced cell cycling. In the present work, insulin was shown to regulate cellular accumulation and transcription of the c-myc gene in rat hepatoma cells. The control of c-myc by insulin was complex, with an initial-induced decrease in c-myc transcription to 50% of control values at 15 and 30 min. This was followed by an increase in transcription of about 3-fold by 60-120 min. Similar to the initial inhibitory effect of insulin, the protein synthesis inhibitors cycloheximide or anisomycin decreased c-myc transcription. However, there was no secondary induction of c-myc transcription by protein synthesis inhibitors. The effects of both insulin and protein synthesis inhibitors were shown to be through alterations in intragenic pausing of transcription of the sense mRNA, not through changes initiation of transcription.

Animals↗
PubMed1991

Cytokine-induced IL-1 beta gene expression in the human polymorphonuclear leukocyte: transcriptional and post-transcriptional regulation by tumor necrosis factor and IL-1.

We have previously demonstrated that Il-1 and TNF could rapidly, but transiently, induce gene expression of Il-1 beta in human polymorphonuclear leukocytes (PMN) at both the protein and mRNA level. Additionally, we demonstrated a cooperative effect of Il-1 and TNF on the kinetics of induction of Il-1 beta mRNA and protein. In order to better understand the molecular basis of Il-1 beta induction, we have further investigated the regulation of Il-1 and TNF-induced gene expression in the PMN. Using nuclear run-on transcription analysis, we found that within 1 h Il-1, TNF, and TNF plus Il-1 induced the transcription of the Il-1 beta gene by 33-, 61-, and 99-fold, respectively. By 2 h, the levels of transcription had been reduced to approximately 50% of peak levels for TNF- and TNF plus Il-1-treated PMN, and to near noninduced levels in Il-1-treated PMN. We also found that these cytokines induced stable mRNA, i.e., Il-1 beta mRNA t1/2 for Il-1-, TNF-, and TNF plus Il-1-induced PMN were 57, 94, and 86 min, respectively. By 2 h, when steady state levels of Il-1 beta mRNA were found to decrease, Il-1 beta mRNA t1/2 had fallen to approximately 18 min for all cytokine treatments. To determine if protein synthesis was required for induction of Il-1 beta gene expression, we treated PMN simultaneously with cytokines and cycloheximide, and found that cycloheximide enhanced the accumulation of Il-1-induced Il-1 beta mRNA, but abrogated the accumulation of Il-1 beta mRNA, by TNF- or TNF plus Il-1-treated PMN. This abrogation of Il-1 beta mRNA accumulation was not caused by inhibition of induction of Il-1 beta transcription because TNF induction of transcription of Il-1 beta was not affected by simultaneous treatment with cycloheximide. Thus, we report that Il-1 and TNF regulate IL-1 beta gene expression via both transcriptional and post-transcriptional mechanisms in vitro.

Cycloheximide↗
PubMed1988

Inhibition of RNA polymerase I-directed transcription by novobiocin. Potential use of novobiocin as a general inhibitor of eukaryotic transcription initiation.

The effect of novobiocin, a coumarin class antibiotic, on rat ribosomal gene (rDNA) transcription in a fractionated extract derived from adenocarcinoma ascites cells (fraction DE-B) was studied. This drug inhibited transcription of rDNA by blocking initiation of transcription, whereas it had no effect on the elongation of the rRNA transcript. Order of addition experiments indicated that the novobiocin effect was at a step(s) in preinitiation complex formation. Preincubation of fraction DE-B with ATP before exposure to this antibiotic prevented inhibition of rDNA transcription. Since novobiocin has been shown to inhibit RNA polymerases II- and III-directed transcription of linear DNAs by interfering with a step(s) in the initiation reaction, these data suggest that initiation of transcription of all classes of RNA is inhibited by novobiocin by a mechanism independent of its effect on DNA topoisomerase II.

Adenocarcinoma↗
PubMed1998

Detection of human papillomavirus 16 transcriptional activity in cervical intraepithelial neoplasia grade III lesions and cervical carcinomas by nested reverse transcription-polymerase chain reaction and in situ hybridization.

Continued expression of the oncogenes E6 and E7 of human papilloma virus "high risk" type 16 (HPV16) initiates neoplastic transformation and maintenance of the malignant phenotype in cervical carcinoma cells. The transcriptional activity of the HPV16 E6/E7 oncogenes was investigated in HPV16-containing cervical cell lines, cervical carcinomas, and cervical intraepithelial neoplasia grade III lesions using the techniques of reverse transcription-polymerase chain reaction (RT-PCR), Southern blotting, and in situ hybridization. To facilitate detection of the full-length HPV16 E6/E7 oncogene transcript and its characteristic splice products E6*I and E6*II in cervical tissues, a nested RT-PCR (nRT-PCR) assay was designed. Specific detection of HPV E6/E7 oncogene transcripts in clinical specimens was found to be improved by nRT-PCR, being as sensitive as the combination of conventional RT-PCR and subsequent Southern blot hybridization. Regarding the progression of premalignant lesions to cervical cancer, detection of the HPV transcriptional activity by nRT-PCR may provide additional information for risk evaluations. Moreover, improvements in the amplification of HPV oncogene transcripts may also be advantageous for monitoring the activity of HPV before and after transcript-targeted gene therapy of cervical cancer.

Carcinoma↗
PubMed1998

Evidence of multiple transcription initiation and termination sites within the rDNA intergenic spacer and rRNA readthrough transcription in the urochordate Herdmania curvata.

Analysis of the structure of the urochordate Herdmania curvata ribosomal DNA intergenic spacer (IGS) and its role in transcription initiation and termination suggests that rRNA gene regulation in this chordate differs from that in vertebrates. A cloned H. curvata IGS is 1881 bp and composed predominantly of two classes of similar repeat sequences that largely alternate in a tandem array. Southern blot hybridization demonstrates that the IGS length variation within an individual and population is largely the result of changes in internal repeat number. Nuclease S1 mapping and primer extension analyses suggest that there are two transcription initiation sites at the 3' end of the most 3' repetitive element; these sites are 6 nucleotides apart. Unlike mouse, Xenopus, and Drosophila, there is no evidence of transcription starting elsewhere in the IGS. Most sequence differences between the promoter repeat and the other internal repeats are in the vicinity of the putative initiation sites. As in Drosophila, nuclease S1 mapping of transcription termination sites suggest that there is not a definitive stop site and a majority of the pre-rRNAs read through a substantial portion of the IGS. Some transcription appears to proceed completely through the promoter repeat into the adjacent rDNA unit. Analysis of oocyte RNA by reverse transcription-polymerase chain reaction (RT-PCR) confirms that readthrough transcription into the adjacent rDNA unit is occurring in some small IGS length variants; there is no evidence of complete readthrough of IGSs larger than 1.0 kb.

Animals↗