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PubMed1999

Identification of three new alternate human kallikrein 2 transcripts: evidence of long transcript and alternative splicing.

In a search for prostate-specific genes in the human expressed sequence tag (EST) database, we identified a seemingly unique EST cluster C81. Experimental data linked C81 to the human hKLK2 gene that encodes a prostate specific serine protease-human glandular kallikrein (hK2). We uncovered a full-length hKLK2 cDNA corresponding to a 3.0 kb hKLK2 mRNA by PCR and sequence analysis. The 3.0 kb transcript accounts for about 25% of the hKLK2 transcripts as compared to the previously known 1.5 kb transcript. We also identified a third spliced form of the hKLK2 gene produced by alternative splicing between intron III and exon 4. This spliced form was detected in normal prostate, prostate cancer and the prostate adenocarcinoma cell line LNCaP. The identification of long hKLK2 transcript and an alternative spliced form of the hKLK2 gene indicates that regulation of the gene is complex.

Alternative Splicing↗
PubMed1998

Comparison of methods for identifying transcription units and transcription map of the Werner syndrome gene region.

To isolate a human disease gene by positional cloning, a critical step is the identification of candidate genes from a targeted genomic region. We used cDNA selection, exon trapping, and genomic sequencing to identify 12 transcription units from a 1.4-Mb genomic region containing the Werner syndrome gene (WRN). This included sequencing of 650 kb in the region of the WRN gene, to date, the most DNA sequenced as part of a positional cloning effort. The result of this combined method was significant overlap among the transcription units identified by each method; yet, no one method identified all of the transcription units. We present here a comparison of the effectiveness and efficiency of these methods and present a transcription map of the Werner syndrome gene region.

Blotting, Northern↗
PubMed1996

Reduced precore transcription and enhanced core-pregenome transcription of hepatitis B virus DNA after replacement of the precore-core promoter with sequences associated with e antigen-seronegative persistent infections.

Hepatitis B virus variants harboring nucleotide alterations in the preC-C promoter have been detected in fulminant hepatitis B as well as in HBeAg-seronegative persistent infection. However, it has not been demonstrated that variants with nucleotide alterations in the preC-C promoter cause various disease states. We replaced the preC-C promoter region of a wild-type genome with the most frequent naturally occurring mutated form and introduced it into HepG2 cells. The mutant with coexisting A1762T and G1764A substitutions produced less than one-fifth of the wild-type level of HBeAg. Conversely, the mutant generated 2.4 times more core particle antigen and showed a high-replicator phenotype. RNase protection and quantitative 5' RACE showed a 16- to 32-fold reduction of preC transcripts and a 4-fold induction of C transcripts of the mutant compared to wild-type. The preC transcript of the mutant had a more heterogeneous 5' end than that of the wildtype. However, the mutations did not alter the initiation sites of C transcription. When the promoter region was cloned into CAT plasmids, the mutations had dual effects on preC and C promoter activities, decreasing and increasing them, respectively. These results suggest that these mutations are responsible for the reduced HBeAg production as well as the enhanced replication and core production. Analysis of revertants with either single point mutation showed that T at 1762 is critical for the mutant phenotype.

Amino Acid Sequence↗
PubMed1996

Sequence homology requirements for transcriptional silencing of 35S transgenes and post-transcriptional silencing of nitrite reductase (trans)genes by the tobacco 271 locus.

The transgene locus of the tobacco plant 271 (271 locus) is located on a telomere and consists of multiple copies of a plasmid carrying an NptII marker gene driven by the cauliflower mosaic virus (CaMV) 19S promoter and the leaf-specific nitrite reductase Nii1 cDNA cloned in the antisense orientation under the control of the CaMV 35S promoter. Previous analysis of gene expression in leaves has shown that this locus triggers both post-transcriptional silencing of the host leaf-specific Nii genes and transcriptional silencing of transgenes driven by the 19S or 35S promoter irrespective of their coding sequence and of their location in the genome. In this paper we show that silencing of transgenes carrying Nii1 sequences occurs irrespective of the promoter driving their expression and of their location within the genome. This phenomenon occurs in roots as well as in leaves although root Nii genes share only 84% identity with leaf-specific Nii1 sequences carried by the 271 locus. Conversely, transgenes carrying the bean Nii gene (which shares 76% identity with the tobacco Nii1 gene) escape silencing by the 271 locus. We also show that transgenes driven by the figwort mosaic virus 34S promoter (which shares 63% identity with the 35S promoter) also escape silencing by the 271 locus. Taken together, these results indicate that a high degree of sequence similarity is required between the sequences of the silencing locus and of the target (trans)genes for both transcriptional and post-transcriptional silencing.

Fabaceae↗
PubMed1995

Amiloride modulates urokinase gene expression at both transcription and post-transcription levels in human colon cancer cells.

Activity of receptor-bound urokinase plasminogen activator (uPA) on the surface of colon cancer cells appears to be a function of the number of uPA receptors. The regulation of uPA therefore may determine the invasive phenotype. The effects of amiloride on the modulation of uPA mRNA and protein induced by phorbol ester (PMA) and cycloheximide (CHX) were studied in four colon cancer cell lines, HCT116, KM12SM, LIM1215 and LS123. Northern blot analyses showed that PMA induced uPA mRNA that peaked at 2-48 h in HCT116 cells. In all colon cancer cell lines tested, the expression of uPA mRNA by PMA was super-induced after the addition of the protein synthesis inhibitor CHX, suggesting that stimulation of uPA gene expression does not require de novo protein synthesis. uPA mRNA was also induced by CHX alone, indicating that there may be a labile protein which inhibits uPA mRNA processing. Amiloride profoundly inhibited uPA mRNA production at concentrations between 0.1-1 mM in the presence or absence of PMA or CHX. uPA protein levels on the colon cancer cell surface reflected PMA induction and amiloride inhibition of uPA mRNA levels. Transcriptional elongation experiments using isolated nuclei indicated that while the induction effects of PMA or CHX on uPA gene expression were mediated at the post-transcriptional level, amiloride acted at both transcription and post-transcription levels. The inhibitory effects of amiloride on uPA gene expression reported in this paper may offer the prospect of developing new therapeutic approaches to the prevention of invasion and metastasis by adenocarcinomas.

Amiloride↗
PubMed1990

Tandem promoters, tsrp1 and tsrp2, direct transcription of the thiostrepton resistance gene (tsr) of Streptomyces azureus: transcriptional initiation from tsrp2 occurs after deletion of the -35 region.

Nuclease S1 protection experiments indicated that the thiostrepton resistance gene (tsr) of Streptomyces azureus is transcribed from tandem promoters, tsrp1 and tsrp2, that initiate transcription 45 and 173 nucleotides, respectively, upstream of the presumptive translational start codon. The -10 regions of both promoters show similarity to the consensus sequence for the major class of prokaryotic promoters, but the -35 regions do not, although they show some similarity to each other. Replacement of sequences upstream of position -22 relative to the tsrp2 start site with two different DNA segments affected the levels of the tsrp2 transcript but did not alter the tsrp2 initiation site. In vitro transcription assays using RNA polymerase from Streptomyces coelicolor A3(2) also confirmed the location of tsrp2 and identified additional start sites near tsrp2 that were barely detectable with in vivo synthesised RNA. Transcripts corresponding to initiation in vitro at trsp1 could not be detected, suggesting that additional factors are required for utilisation of this promoter.

Amino Acid Sequence↗
PubMed1990

Transcript and sequence analysis of a 5.1 kb contiguous fragment of Dictyostelium discoideum plasmid Ddp1 that contains the origin of replication and codes for several transcripts.

The sequence of a 5.1 kb contiguous fragment of the Dictyostelium plasmid Ddp1 is presented. This fragment contains three long open reading frames which correspond to the developmentally regulated and cAMP-inducible transcript d-5, the growth phase specific transcript g-1 and the three overlapping transcripts g-2, g-3 and d-4. The transcripts that originate from Ddp1 resemble chromosomally-encoded ones: they are products of RNA polymerase II, are polyadenylated and accumulate at different time points during Dictyostelium development. The presented nucleotide sequence encompasses a 2,033 bp HindIII fragment that had previously been shown to carry all the information necessary for extrachromosomal replication. None of the identified genes is completely contained within this HindIII fragment.

Amino Acid Sequence↗
PubMed1991

Sequence analysis of wheat mitochondrial transcripts capped in vitro: definitive identification of transcription initiation sites.

To identify transcription initiation sites in wheat mitochondria, the nascent 5'-ends of transcripts were specifically labeled by incubation of wheat mitochondrial RNA with [alpha-32P]GTP in the presence of the enzyme guanylyltransferase. After separation of the resulting capped transcripts by electrophoresis in polyacrylamide gels, individual RNAs were recovered and directly sequenced. Four RNA sequences obtained in this way were localized upstream of the protein-coding genes atpA, coxII, coxIII and orf25. Comparison of mRNA and gene sequences allowed precise positioning of transcription initiation sites for these four genes. Sequence similarities immediately upstream of these sites define a conserved motif that we suggest as a candidate regulatory element in wheat mtDNA. The relationship between this motif and putative mitochondrial promoters in other plant species is discussed.

Base Sequence↗
PubMed1979

The occurrence of long transcription units among the X and Y ribosomal genes of Drosophila melanogaster: transcription of insertion sequences.

Most of the ribosomal transcription units (rTUs) in Drosophila melanogaster observed by electron microscopy measure about 8 kb; a length which corresponds to the size of the 38S precursor to ribosomal RNA in D. melanogaster. However, interspersed among these rTUs are transcription units that are much longer (up to 14.6 kb) than the 8 kb expected for rTUs. Some of these larger length estimates can be attributed to stretching but an important fraction is significantly larger and has up to 60 more fibers per gene.--The following evidence suggests that these larger transcription units are ribosomal genes consisting of insertion sequences. The long transcription units are within the sizes expected for rTUs containing insertion sequences as reported by other workers. Their RNP fibers cross-react with antibodies raised against ribosomal proteins in a manner similar to that observed for ribosomal RNP. They are interspersed among rTUs in the X chromosome.--These putative ribosomal genes carrying insertions are present both in the X and, although to a lesser extent, in the Y ribosomal chromatin as is indicated by their existence in nurse cells of both Oregon R females and females of the genotype sc4sc8/sc4sc8/y+ Y. Analysis of the fiber patterns of "long TUs" supports the hypothesis that the insertion region is being transcribed.--"Long TUs" are found in tandem with non-transcribed spacer regions which are heterogeneous in length with a mean of 1.53+/-0.61 micrometers (or 8.5+/-3.4 kb).

Animals↗
PubMed1999

The bi-directional transcriptional promoters for the latency-relating transcripts of the pp38/pp24 mRNAs and the 1.8 kb-mRNA in the long inverted repeats of Marek's disease virus serotype 1 DNA are regulated by common promoter-specific enhancers.

In cell lines established from Marek's disease tumors, several viral transcripts are expressed and among them the products of pp38/pp24 mRNA and 1.8 kb-mRNA have been suggested to be involved in viral oncogenicity. The long inverted repeats of Marek's Disease virus serotype 1 (MDV1) genome contain closely located transcriptional promoters for phosphorylated protein pp38/pp24 and 1.8 kb-mRNA. These promoters initiate transcription in opposite directions and are separated only by a short enhancer region, which is likely to regulate both promoters simultaneously. We have analyzed the transcription activity of these promoters in MDV1 (Md5 strain) infected CEF by transient expression of CAT reporter genes and found that the promoters were in fact active in infected cells and the promoter for 1.8 kb-mRNA was more active than the pp38/pp24 promoter. Deletion analysis of the short enhancer region revealed that the 30 bp region overlapping the enhancer elements for 1.8 kb-mRNA was important for promoter activity for pp38/pp24. The gel shift analysis revealed that nuclear factor(s) actually bound to the overlapping 30 bp region. In addition, the activity of these promoters in infected cells varied with MDV strains. These results suggest that pp38/pp24 and 1.8 kb-mRNA promoters share a common regulatory sequence but a viral or a cellular factor(s) induced by viral infection regulates the promoter by distinct mechanisms.

Antigens, Viral↗
PubMed2006

Transforming growth factor-beta1 upregulates transcription of alpha3 integrin gene in hepatocellular carcinoma cells via Ets-transcription factor-binding motif in the promoter region.

The invasive and metastatic potentials of hepatocellular carcinoma (HCC) are positively correlated with the expression level of alpha3beta1 integrin, a high-affinity adhesion receptor for laminin isoforms. Transforming growth factor (TGF)-beta1 stimulates non-invasive HCC cells to acquire invasive phenotypes in association with the enhanced expression of alpha3 integrin. In this study, we investigated the molecular mechanism underlying the upregulation of alpha3beta1 integrin by TGF-beta1 in non-invasive HepG2 HCC cells. The treatment of HepG2 cells with TGF-beta1 induced the expression of alpha3 integrin and potentiated these cells to adhere to laminin-5 and to migrate through laminin-5-coated membranes. The promoter activity was measured by luciferase assay with a series of deletion constructs of the 5'-flanking region of the mouse alpha3 integrin gene, and the results showed that the -260/-119 region (relative to the major transcription start site) contained elements responsive to TGF-beta1 stimulation. The introduction of mutations into the putative consensus binding sequence for the Ets-family of transcription factors located at -133 greatly decreased the promoter activity responding to TGF-beta1 stimulation. The nuclear proteins extracted from TGF-beta1-stimulated HepG2 cells yielded a larger amount of DNA-nuclear protein complexes than did those extracted from unstimulated cells, as determined by an electrophoretic mobility shift assay using an oligonucleotide containing the Ets-site as a probe. These results suggest that TGF-beta1 stimulates HepG2 cells to express a higher level of alpha3 integrin by transcriptional upregulation via Ets transcription factors and to exhibit a more invasive phenotype.

Base Sequence↗
PubMed1989

Effects of insulin and fructose on transcriptional and post-transcriptional regulation of malic enzyme synthesis in diabetic rat liver.

Insulin action on regulation of hepatic malic enzyme has been investigated in comparison with fructose, using streptozotocin-induced diabetic rats. Insulin-treatment caused a 2.8-fold increase in the transcriptional rate of malic enzyme (EC 1.1.1.40) after 8 h, and a 5-fold increase in the mRNA concentration of the liver. In Northern blot analysis, we demonstrated that after insulin treatment, the nuclear mRNA of malic enzyme tended to increase more rapidly than the total cellular mRNA. Therefore, it is suggested that the nuclear mRNA was primarily increased by insulin. The insulin-dependent increase of malic enzyme mRNA was blocked by cycloheximide, suggesting that synthesis of a peptide is required. On the other hand, by feeding a high-fructose diet to diabetic rats, the malic enzyme mRNA concentration was considerably increased, though with a delayed peaking in comparison with the insulin-treated animals, whereas the transcriptional rate was not significantly increased. Dietary fructose may stabilize the transcripts. Fructose increased the enzyme level far less than the mRNA level. These results suggest that insulin is required in both the translational and transcriptional regulation of malic enzyme.

Animals↗
PubMed1989

Transcription in nematodes: early Ascaris embryos are transcriptionally active.

In this report we analyze early zygotic gene expression in the parasitic nematode Ascaris lumbricoides var. suum. Using synchronous populations of early embryonic stages, nuclei were isolated, and in vitro run-off transcription assays were performed. We find transcriptional activity as early as the 4- to 8-cell stage. The percentage of RNA polymerase II activity, as measured in these assays, is greater than 80% of the total transcription at the 60-cell stage. Furthermore, we show that a specific transcript (actin) can be identified in all early stages tested.

Actins↗
PubMed1987

RNA polymerase molecules initiating transcription at tandem promoters can collide and cause premature transcription termination.

Using purified E. coli RNA polymerase we have studied the transcription in vitro of a series of DNA fragments carrying two tandemly arranged promoters, where the corresponding transcription start points were separated by 263, 138, 83 and 78 base pairs. In the case where the transcription start points are 83 base pairs apart, there is an interaction between RNA polymerase molecules transcribing from the two promoters. This interaction results in premature termination of the upstream transcript at a precise site. We propose that this is the result of RNA polymerase transcribing from the upstream promoter bumping into polymerase at the downstream promoter. The interaction between the two polymerase molecules is crucially dependent on the distance between the two promoters.

DNA-Directed RNA Polymerases↗
PubMed1982

Globin RNA transcription: a possible termination site and demonstration of transcriptional control correlated with altered chromatin structure.

The transcription unit that encodes beta-globin (major) mRNA has been examined by analysis of nascent labeled RNA from the nuclei of mouse erythroleukemia cells treated with agents that induce beta-globin formation. More than 95% of the time, transcription appears to terminate within a region 1400 +/- 100 nucleotides downstream from the poly(A) site. The nuclei of mouse erythroleukemia cells treated with agents that induce beta-globin synthesis showed a 10 to 20 fold stimulation of transcription assayed by chain elongation of beta-globin RNA sequences, including sequences downstream from the poly(A) site. Associated with the increase in transcription was a generalized increase in sensitivity to DNAase treatment of globin genomic DNA in whole nuclei. A DNAase I-hypersensitive site in the vicinity of the cap site was also found to be prominent in induced cells.

Animals↗
PubMed1983

The pattern of transcription of the human mitochondrial rRNA genes reveals two overlapping transcription units.

A detailed analysis of the mapping and kinetic properties of oligo(dT)-cellulose bound and unbound transcripts synthesized in HeLa cells has indicated that two distinct transcription events take place in the rDNA region. Of these, one appears to start approximately 25 bp upstream of the tRNAPhe gene and to terminate at or near the 3' end of the 16S rRNA gene, being responsible for the synthesis of the bulk of rRNA. The other transcription event starts near the 5' end of the 12S rRNA gene, proceeds beyond the 3' end of the 16S rRNA gene, and results in the synthesis of a polycistronic molecule corresponding to almost the entire H-strand, which is destined to be processed to yield the mRNAs and most of the tRNAs encoded in the heavy strand. The existence of two overlapping transcription units with distinct promoters is probably the basis for the differential regulation of synthesis of the rRNAs and heavy-strand-coded mRNAs.

DNA↗
PubMed1984

Transcription in vaccinia virus cores. The RNA produced during the first round of transcription does not differ from RNA transcribed in subsequent rounds.

RNA was synthesized in vitro in vaccinia virus cores for times sufficiently short that only the first round of transcription took place. This RNA was compared to RNA synthesized for longer times (which is mainly comprised of reinitiated RNA) by hybridization to fragments of vaccinia virus DNA obtained with several different restriction endonucleases. Under the conditions studied, the composition of mRNA produced during the first round of transcription was the same as that produced in subsequent (reinitiated) rounds. Thus, there is no special arrangement of polymerase molecules within the cores that would allow, in the first round of transcription, for the synthesis of mRNAs distinct from those made in subsequent rounds of transcription. Thus, attachment of polymerase molecules to DNA is likely to occur during preincubation in a way similar to that during reinitiation.

DNA, Viral↗
PubMed1990

Identification and characterization of transcripts from the neurofibromatosis 1 region: the sequence and genomic structure of EVI2 and mapping of other transcripts.

Mapping of the EVI2 gene between the translocation breakpoints of two patients with neurofibromatosis type 1 (NF1), combined with the likely role of its murine homolog in neoplastic disease, implicates EVI2 as a possible candidate for the NF1 gene. We report here the expression of a 1.6-kb EVI2 transcript in normal human brain and peripheral blood mononuclear cells. Sequencing studies predict an EVI2 protein of 232 amino acids that contains an N-terminal signal peptide, an extracellular domain with five potential glycosylation sites, a single hydrophobic transmembrane domain with a leucine zipper, and a hydrophilic cytoplasmic domain. These features are all well-conserved with respect to the mouse Evi-2 protein and are consistent with the hypothesis that EVI2 is a membrane protein that may complex with itself and/or other proteins within the membrane, perhaps to function as part of a cell-surface receptor. In the course of these studies we have also identified three other transcripts (classes of cDNAs) from the NF1 region. Two of these transcripts map between the NF1 translocation breakpoints; the remaining transcript maps just outside this region.

Amino Acid Sequence↗