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At least 397 records · Page 22Linked to original sources
PubMed2001

Repression of glucagon gene transcription by peroxisome proliferator-activated receptor gamma through inhibition of Pax6 transcriptional activity.

The nuclear receptor peroxisome proliferator-activated receptor gamma (PPARgamma) is involved in glucose homeostasis and synthetic PPARgamma ligands, the thiazolidinediones, a new class of antidiabetic agents that reduce insulin resistance and, as a secondary effect, reduce hepatic glucose output. PPARgamma is highly expressed in normal human pancreatic islet alpha-cells that produce glucagon. This peptide hormone is a functional antagonist of insulin stimulating hepatic glucose output. Therefore, the effect of PPARgamma and thiazolidinediones on glucagon gene transcription was investigated. After transient transfection of a glucagon-reporter fusion gene into a glucagon-producing pancreatic islet cell line, thiazolidinediones inhibited glucagon gene transcription when PPARgamma was coexpressed. They also reduced glucagon secretion and glucagon tissue levels in primary pancreatic islets. A 5'/3'-deletion and internal mutation analysis indicated that a pancreatic islet cell-specific enhancer sequence (PISCES) motif within the proximal glucagon promoter element G1 was required for PPARgamma responsiveness. This sequence motif binds the paired domain transcription factor Pax6. When the PISCES motif within G1 was mutated into a GAL4 binding site, the expression of GAL4-Pax6 restored glucagon promoter activity and PPARgamma responsiveness. GAL4-Pax6 transcriptional activity was inhibited by PPARgamma in response to thiazolidinedione treatment also at a minimal viral promoter. These results suggest that PPARgamma in a ligand-dependent but DNA binding-independent manner inhibits Pax6 transcriptional activity, resulting in inhibition of glucagon gene transcription. These data thereby define Pax6 as a novel functional target of PPARgamma and suggest that inhibition of glucagon gene expression may be among the multiple mechanisms through which thiazolidinediones improve glycemic control in diabetic subjects.

Cell Line↗
PubMed1990

Activation of octamer-containing promoters by either octamer-binding transcription factor 1 (OTF-1) or OTF-2 and requirement of an additional B-cell-specific component for optimal transcription of immunoglobulin promoters.

Several distinct octamer-binding transcription factors (OTFs) interact with the sequence ATTTGCAT (the octamer motif), which acts as a transcription regulatory element for a variety of differentially controlled genes. The ubiquitous OTF-1 plays a role in expression of the cell cycle-regulated histone H2b gene as well as several other genes, while the tissue-specific OTF-2 has been implicated in the tissue-specific expression of immunoglobulin genes. In an attempt to understand the apparent transcriptional selectivity of these factors, we have investigated the physical and functional characteristics of OTF-1 purified from HeLa cells and both OTF-1 and OTF-2 purified from B cells. High-resolution footprinting and mobility shift-competition assays indicated that these factors were virtually indistinguishable in binding affinities and DNA-protein contacts on either the H2b or an immunoglobulin light-chain (kappa) promoter. In addition, each of the purified factors showed an equivalent intrinsic capacity to activate transcription from either immunoglobulin promoters (kappa and heavy chain) or the H2b promoter in OTF-depleted HeLa and B-cell extracts. However, with OTF-depleted HeLa extracts, neither factor could restore immunoglobulin gene transcription to the relatively high level observed in unfractionated B-cell extracts. Restoration of full immunoglobulin gene activity appears to require an additional B-cell regulatory component which interacts with the OTFs. The additional B-cell factor could act either by facilitating interaction of OTF activation domains with components of the general transcriptional machinery or by contributing a novel activation domain.

Base Sequence↗
PubMed2002

Transcriptional activation of human CYP17 in H295R adrenocortical cells depends on complex formation among p54(nrb)/NonO, protein-associated splicing factor, and SF-1, a complex that also participates in repression of transcription.

The first 57 bp upstream of the transcription initiation site of the human CYP17 (hCYP17) gene are essential for both basal and cAMP-dependent transcription. EMSA carried out by incubating H295R adrenocortical cell nuclear extracts with radiolabeled -57/-38 probe from the hCYP17 promoter showed the formation of three DNA-protein complexes. The fastest complex contained steroidogenic factor (SF-1) and p54(nrb)/NonO, the intermediate complex contained p54(nrb)/NonO and polypyrimidine tract-binding protein-associated splicing factor (PSF), and the slowest complex contained an SF-1/PSF/p54(nrb)/NonO complex. (Bu)(2)cAMP treatment resulted in a cAMP-inducible increase in the binding intensity of only the upper complex and also activated hCYP17 gene transcription. SF-1 coimmunoprecipitated with p54(nrb)/NonO, indicating direct interaction between these proteins. Functional assays revealed that PSF represses basal transcription. Further, the repression of hCYP17 promoter-reporter construct luciferase activity resulted from PSF interacting with the corepressor mSin3A. Trichostatin A attenuated the inhibition of basal transcription, suggesting that a histone deacetylase interacts with the SF-1/PSF/p54(nrb)/NonO/mSin3A complex. Our studies lend support to the idea that the balance between transcriptional activation and repression is essential in the control of adrenocortical steroid hormone biosynthesis.

Adrenal Cortex↗
PubMed1991

The murine adenosine deaminase promoter requires an atypical TATA box which binds transcription factor IID and transcriptional activity is stimulated by multiple upstream Sp1 binding sites.

We have explored the template and factor requirements for in vitro transcription of the GC-rich promoter of the murine adenosine deaminase gene. The core promoter consists of an A-rich sequence (TAAAAAA) 27 base pairs upstream of the initiation site which binds transcription factor IID (TFIID) and a high affinity Sp1 binding site located 27 base pairs further upstream. Multiple upstream elements increased core promoter activity 20-fold and correspond to protected regions in DNase I footprinting assays with purified Sp1 protein. Internal deletion of the TA6 element alone eliminated transcription in spite of the presence of all other promoter elements including four Sp1 binding sites. Recombinant human TFIID supported weak basal transcription in heat-treated nuclear extracts whereas a partially purified TFIID fraction from HeLa cells reconstituted a maximal level of transcription. Inclusion of 12 base pairs immediately adjacent to the proximal Sp1 site resulted in a 5-fold boost in transcriptional activity and corresponds to a second Sp1 binding site. These results serve as a basis for further exploration of the factors involved in the developmental and selective high level tissue expression of the murine adenosine deaminase gene.

Adenosine Deaminase↗
PubMed1997

Subcellular partitioning of transcription factors during osteoblast differentiation: developmental association of the AML/CBF alpha/PEBP2 alpha-related transcription factor-NMP-2 with the nuclear matrix.

The subnuclear location of transcription factors may functionally contribute to the regulation of gene expression. Several classes of gene regulators associate with the nuclear matrix in a cell type, cell growth, or cell cycle related-manner. To understand control of nuclear matrix-transcription factor interactions during tissue development, we systematically analyzed the subnuclear partitioning of a panel of transcription factors (including NMP-1/YY-1, NMP-2/AML, AP-1, and SP-1) during osteoblast differentiation using biochemical fractionation and gel shift analyses. We show that nuclear matrix association of the tissue-specific AML transcription factor NMP-2, but not the ubiquitous transcription factor YY1, is developmentally upregulated during osteoblast differentiation. Moreover, we show that there are multiple AML isoforms in mature osteoblasts, consistent with the multiplicity of AML factors that are derived from different genes and alternatively spliced cDNAs. These AML isoforms include proteins derived from the AML-3 gene and partition between distinct subcellular compartments. We conclude that the selective partitioning of the YY1 and AML transcription factors with the nuclear matrix involves a discriminatory mechanism that targets different classes and specific isoforms of gene regulatory factors to the nuclear matrix at distinct developmental stages. Our results are consistent with a role for the nuclear matrix in regulating the expression of bone-tissue specific genes during development of the mature osteocytic phenotype.

Animals↗
PubMed1994

AML1 fusion transcripts in t(3;21) positive leukemia: evidence of molecular heterogeneity and usage of splicing sites frequently involved in the generation of normal AML1 transcripts.

The t(3;21)(q26;q22) is associated with chronic myelogenous leukemia in blast crisis (CML-BC), leukemia evolving from (therapy-related) myelodysplasia, and with leukemia following other hematopoietic proliferative diseases. Molecular cytogenetic analysis and cloning of a few t(3;21) cases indicate that the breakpoints are quite heterogeneous even within a specific clinical phenotype. Interestingly some of the (3;21) breakpoints involve the AML1 gene previously found rearranged in the t(8;21) associated with acute myelogenous leukemia. AML1 is related to the Drosophila gene runt and is the human counterpart of the gene for the alpha subunit of the nuclear polyoma enhancer binding protein (PEBP2) also known as the core binding factor (CBF). In the t(3;21) AML1 was found rearranged with EAP, a gene on chromosome 3 encoding a small ribosomal protein, as well as with EV11, another gene on chromosome 3. Here we report our study of six cases of t(3;21). By using fluorescence in situ hybridization (FISH) analysis and AML1 probes we could conclude that at least in two CML-BC cases the breakpoint occurred in the AML1 intron that is disrupted by the t(8;21). An AML1/EAP fusion transcript, different from the one described in a therapy-related myelodysplasia, was detected in both CML-BC cases. This transcript is expected to result in a predicted protein containing the AML1 nuclear binding domain with an attached stretch of 17 amino acids unrelated to the EAP small ribosomal protein. In the other t(3;21) patients we could not detect an AML1/EAP transcript or an AML1/EV11 transcript. This result suggests heterogeneity of the t(3;21) at the molecular level. The AML1 chimeric transcripts identified so far, both in the t(3;21) and in the t(8;21), diverge from the normal transcripts either after exon 5 or exon 6. Here we show that in normal AML1 transcripts different splicing events are seen to occur after AML1 exon 5 as well as exon 6.

Amino Acid Sequence↗
PubMed1989

A selection for mutants of the RNA polymerase III transcription apparatus: PCF1 stimulates transcription of tRNA and 5S RNA genes.

A genetic approach has been developed to study transcription by RNA polymerase III. A pair of Schizosaccharomyces pombe nonsense suppressor tRNA genes were arranged in tandem such that expression of the downstream (supS1) tRNA suppressor was dependent upon transcription initiated by the internal promoter of the upstream (sup9-e) gene. Dominant mutant strains of Saccharomyces cerevisiae were isolated that suppress in trans the effect of an A block promoter mutation (A19) in the sup9-e gene and restore supS1 suppressor activity. Fifteen mutant strains, eight of which were independently isolated, all have elevated steady-state levels of sup9-e A19 RNA consistent with an increase in gene transcription. Extracts of a strain carrying the dominant mutant gene, PCF1, show a general 6-fold stimulation in transcription of mutant (A19) and wild-type tRNA genes and increase 5S gene transcription 4-fold compared with extracts from a wild-type strain. A transcription factor exclusion assay was used to show that the PCF1 mutation affects two distinct stages in transcription: one prior to and one after stable complex formation; and that these effects are mediated by a component of the stable complex. Further evidence of an effect during complex assembly was obtained in a time-course experiment that showed a shortened lag phase in the PCF1 extract. The results indicate that PCF1 is either a component of the stable complex or a positive regulator of its activity.

Base Sequence↗
PubMed1993

Transcriptional slippage during the transcription initiation process at a mutant lac promoter in vivo.

A C.G to A.T transversion at position +10 of the lac promoter activates a nascent sigma 70-dependent promoter (the +10A promoter). The lac +10A promoter has two unusual properties; it programs a large family of transcripts with multiple 5' ends, and its sequence bears little resemblance to other sigma 70-dependent promoters. The 5' end of the +10A in vivo mRNA was determined to contain oligo(U) sequences of varying lengths suggesting that the true start site was at a run of three T.A base-pairs located 20 to 22 bp downstream of the lac wild-type promoter start site, and that the transcription initiation process involved a transcriptional slippage event (which resulted in multiple rU incorporation). Only mutations at or near the start site and those deletions that changed the location of the start site abolished this transcriptional slippage property of the transcription initiation process. This transcriptional slippage was also found to be promoter independent because changing the lac UV5 start site to a run of five T.A base-pairs (-1 to +4) resulted in similar transcriptional slippage. Saturated mutagenesis of the +10A promoter identified a potential -10-like region and indicated that sequences immediately upstream of the -10 region contributed to the promoter's activity. Decreasing the weak -35 region homology did not change promoter strength; however, introduction of the consensus -35 hexamer TTGACA increased expression tenfold. RNA polymerase bound to the +10A promoter partially protects a 20 base-pair sequence from DNase I digestion upstream of the start site. These results suggest that RNA polymerase interacts with the +10A promoter in a different manner from that for the majority of sigma 70 promoters.

Base Sequence↗
PubMed1996

Incorrect base insertion and prematurely terminated transcripts during T7 RNA polymerase transcription elongation past benzo[a]pyrenediol epoxide-modified DNA.

DNA replication and transcription are affected adversely by the presence of bulky adducts that are generated by the covalent binding of a variety of metabolically activated environmental pollutants to cellular DNA. When these lesions are not cleared by cellular repair enzymes prior to replication, mutations and ultimately tumor initiation can occur. Transcription and DNA repair appear to be intimately connected, since certain adducts are more efficiently removed from the transcribed strands of active loci than from non-transcribed strands and other quiescent domains in the genome. The mechanism by which RNA polymerases deal with bulky adducts during DNA transcription is therefore of great interest. The availability of site-specifically modified and stereochemically defined oligodeoxyribonucleotides derived from the covalent reaction of 7r, 8t-dihydroxy-9, 10t-epoxy- 7,8,9,10-tetrahydrobenzo[a]pyrene (anti-BPDE) with guanine residues prompted us to study the efficiencies of transcription past these lesions using bacteriophage T7 RNA polymerase. We show here that T7 RNA polymerase can bypass such lesions in a DNA template, providing that a cytosine residue is incorporated opposite anti-BPDE-modified guanine. However, when an incorrect base (most frequently a purine) is inserted opposite the modified site, the RNA polymerase stalls, and the complex dissociates, resulting in a truncated transcript. The ability of the T7 RNA polymerase to discriminate between a correct and an incorrect inserted base and, accordingly, to continue or terminate transcription, might constitute an important mechanism that ensures the fidelity of transcription past a modified base present on the transcribed strand of the DNA template.

Bacteriophage T7↗
PubMed1987

Human RNA polymerase II can prematurely terminate transcription of the adenovirus type 2 late transcription unit at a precise site that resembles a prokaryotic termination signal.

Premature termination of transcription has been demonstrated by eukaryotic RNA polymerase II at specific sites in the major late transcriptional unit of SV40 and in one of the transcriptional units of the parvovirus, minute virus of mice (MVM) (Y. Aloni and N. Hay, CRC Critical Reviews of Biochem., 18:327-383, 1985). In both cases the prematurely terminated (attenuated) RNA can be folded into a hairpin structure followed by U-residues that resemble a termination signal in prokaryotes. The experiments presented herein demonstrate premature termination of transcription 185 nucleotides (nt) downstream from the major late promoter of adenovirus type 2 (Ad2) in vivo, and in vitro in isolated nuclei and in HeLa whole cell extract. As in SV40 and MVM the attenuated RNA of Ad2 can be folded into a hairpin structure followed by U-residues. Transcription-termination was significantly reduced when ITP replaced GTP and when Br-UTP replaced UTP in the transcription reaction mixture, indicating that RNA secondary structure and the rU-dA interactions, respectively, are parts of the termination signal. Moreover, in isolated nuclei transcription-termination at the attenuation site occurred when the reaction mixture contained between 50-150 mM NaCl but not when it contained 300 mM NaCl. These results indicate that, at least in isolated nuclei, attenuation can be regulated. The possible involvement of termination factor(s) in the regulation of attenuation is discussed.

Adenoviridae↗
PubMed1991

Characterization of transcription initiation sites on the soybean mitochondrial genome allows identification of a transcription-associated sequence motif.

Transcription initiation sites on the soybean mitochondrial genome have been characterized by sequence analysis of in vitro-capped soybean mtRNAs and corresponding mtDNA regions. The most abundant, discrete soybean mtRNA species labeled by guanylyltransferase and [alpha-32P]GTP are shown to correspond to the major transcript of the atp9 gene and to a group of small RNAs consisting of a discrete 80 nucleotide (nt) species plus heterogeneous species ranging in size from 133 to 148 nt. The 133-148 nt RNAs represent a set of transcripts with a common 5' terminus and ragged 3' ends, while the 80 nt RNA corresponds to positions 53-133 of the 133 nt species. The major, discrete in vitro-capped RNA species thus correspond to primary transcripts originating at three sites located in two regions of the soybean mitochondrial genome. The sequences extending from 13 nucleotides upstream to 8 nucleotides downstream of the initiation sites for the atp9 and 133-148 nt transcripts are identical at 18 of 21 positions. Sequences closely resembling this motif are located at some other 5' transcript termini of dicot plant mitochondria. Less closely related sequences are found at transcription initiation sites of wheat and maize mitochondria.

Base Sequence↗
PubMed1974

Transcription of bacteriophage mu. An analysis of the transcription pattern in the early phase of phage development.

It has previously been shown that the transcription of Mu is asymmetric and takes place on the heavy DNA strand (Bade, 1972; Wijffelman et al., 1974). The direction of transcription of Mu has now been determined by RNA-DNA hybridizations between purified Mu-RNA and the separated strands of lambda-Mu hybrid phages. The direction of transcription is from the c-gene (immunity gene) end of the heavy strand to the beta-end (immunity distal end) (Fig. 1). Thermo-inducible, defective Mu lysogens, in which the prophage is deleted from the beta-end, have a normal early transcription pattern, but the increase of RNA at later times is absent. A defective lysogen, which contains only the immunity gene c and the genes A and B, still has an early transcription pattern similar to that of the wild-type. Therefore, we conclude that the early RNA is transcribed from that region of the Mu genome. The early Mu-RNA synthesis is negatively regulated with a minimum rate of transcription at 9 minutes after induction. Before the onset of the late RNA synthesis, at about 22 minutes there is a rather long period in which the rate of Mu-RNA synthesis slowly increases. Using DNA strands of lambda-Mu hybrids which contain only that part of the Mu-DNA on which the early RNA synthesis takes place, we have determined that during the first half in the intermediate phase only early genes are transcribed. The amount of Mu-RNA synthesized by a Mu prophage carrying the X-mutation, which influences the excision of Mu, is greatly reduced. Negative regulation of early transcription occurs normally in this mutant.

Coliphages↗
PubMed1993

Transcriptional analysis of the Chlamydia trachomatis plasmid pCT identifies temporally regulated transcripts, anti-sense RNA and sigma 70-selected promoters.

We analysed transcription of the DNA region immediately downstream of the origin of replication in the chlamydial plasmid pCT. This region comprises two convergent open reading frames (ORF7, ORF8), encoding putative polypeptides that are homologous to each other and with C-terminal domains typical of the phage integrase family of proteins. Northern blot and RNA 5' end mapping analyses indicated that both ORFs were transcribed in the late phase of the chlamydial replicative cycle. RNA mapping showed the presence of a transcript starting 31 nucleotides (nt) before the ATG start codon of ORF7, and two temporally regulated transcripts starting 59 and 89 nt upstream of the ATG start codon of ORF8. Two abundant RNA species of 225 and 415 nt were also identified as overlapping anti-sense transcripts (AS-RNAs), complementary to the 3' end of ORF8 mRNA, with identical 5' ends but different 3' ends. In vitro and in vivo experiments in Escherichia coli showed that the sigma 70-RNA polymerase complex was capable of initiating RNA synthesis at the same sites as observed in Chlamydia trachomatis for ORF7 and AS-RNA transcripts, but was not able to transcribe ORF8. In accord with this, sequences at -10 and -35 nt upstream of the RNA 5' ends resemble sigma 70 consensus promoters in the case of ORF7 and AS, but not in the case of the two ORF8 transcripts. Therefore, transcription of ORF7 and ORF8 is controlled by different types of promoters.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1995

Mutations causing high basal level transcription that is independent of transcriptional activators but dependent on chromosomal position in Saccharomyces cerevisiae.

Two single (bel2 and bel4) and two double (bel3 bel7 and bel5 be16) mutations causing enhanced transcription of a gene fusion, consisting of the open reading frame of PHO5 connected to the HIS5 promoter (HIS5p) integrated at the ura3 or leu2 locus, were isolated from a gcn4-disrupted mutant of Saccharomyces cerevisiae. The PHO5 gene, encoding repressible acid phosphatase, in the HIS5p-PHO5 construct was derepressed under amino acid starved conditions by the action of the transcriptional activator Gcn4p. The bel mutants showed temperature-sensitive cell growth and/or cell aggregation. All the mutants except bel4 also showed high levels of transcription of an intact PHO5 DNA integrated at the URA3 locus in the absence of the cognate transcriptional activator, Pho4p, and in the absence of upstream activating sequences of PHO5. The HIS5 and PHO5 genes at their original chromosomal positions were, however, not affected by the bel2 mutation. The BEL2 gene was found to be identical with SIN4/TSF3, mutations in which cause high levels of transcription of the HO and GAL genes in the absence of their respective transcriptional activators, Swi5p and Gal4p. The effect of the bel2/sin4/tsf3 mutation on PHO5 transcription was additive with the Pho4p function. Thus the effect of the bel2/sin4/tsf3 mutation is dependent on the position of PHO5 in the chromosome and independent of Pho4p and Gen4p activation.

Alleles↗
PubMed1990

The complete and symmetric transcription of the main non coding region of rat mitochondrial genome: in vivo mapping of heavy and light transcripts.

The experiments here reported demonstrate that the main non-coding region of rat mitochondrial DNA is symmetrically transcribed. We have identified stable heavy and light transcripts, whose pattern is rather complex, in the D-loop region of rat mitochondrial DNA. Their relative concentrations have been determined. We detected heavy transcripts which encompass the whole D-loop and more abundant heavy RNA species which we interpreted as transcripts terminating downstream of the 3' end of the last coded gene (Thr-tRNA). The processed heavy RNA species contain polyA, suggesting a strict association between cleavage and polyadenylation. The pattern of light transcripts shows a long RNA, which, starting from the light strand promoter, covers the whole segment, and shorter RNA species which seems to be actively processed at the level of the conserved sequence boxes, probably acting as primers. The symmetric transcription of the D-loop containing region of rat mitochondrial DNA, and in particular the presence of stable transcripts complementary to the putative RNA primers, suggest that mechanisms mediated by interaction between complementary transcripts (antisense RNAs) might play a role in the regulation of mitochondrial DNA replication and expression.

Amino Acid Sequence↗
PubMed1989

Effects of nutrients and insulin on transcriptional and post-transcriptional regulation of glucose-6-phosphate dehydrogenase synthesis in rat liver.

The transcriptional and post-transcriptional regulation of glucose-6-phosphate dehydrogenase induction of rat liver was investigated using a cDNA cloned in our laboratory. By feeding a carbohydrate/protein diet to fasted rats, the mRNA concentration and enzyme induction of glucose-6-phosphate dehydrogenase (EC 1.1.1.49) reached maximal levels about 10-fold those in the fasted rats at 16 h and 72 h, respectively, whereas the transcriptional rate was increased about 3-fold in 6 h. In the protein fed (without carbohydrate) group, both the mRNA concentration and enzyme induction were increased to about 60% of the levels in the carbohydrate/protein fed group and in the group fed on a carbohydrate diet (without protein) to 30-40%. Further, dietary fat significantly reduced the transcriptional rate, mRNA concentration and enzyme induction to less than half, suggesting that dietary fat primarily reduced transcription. Thus, dietary nutrients appear to be involved in the steps preceding the translation. On the other hand, in diabetic rats, the transcriptional rate was significantly decreased as compared to the normal level and restored by insulin-treatment in 4 h. The mRNA concentration was very low in diabetic rats, and was restored to the normal level by insulin treatment in 8 h, and was half restored by fructose feeding. However, the enzyme induction of glucose-6-phosphate dehydrogenase was scarcely restored by fructose, unless accompanied by insulin treatment. Thus, it is suggested that insulin is involved in translation as well as in transcription. Further, the insulin-dependent increase of glucose-6-phosphate dehydrogenase mRNA was blocked by cycloheximide, suggesting that synthesis of a peptide is required.

Animals↗
PubMed1991

Temporal aspects of major viral transcript expression in Hep G2 cells transfected with cloned hepatitis B virus DNA: with emphasis on the X transcript.

The major transcripts of hepatitis B virus (HBV) and the kinetics of their expression were studied in a transient expression system by transfecting partially duplicated copies of HBV genome into Hep G2 cells. By Northern blotting, six species of HBV-specific transcripts could be identified. They were the pregenomic (3.6 kb), the preS1 (2.6 kb), the preS2/S (2.2 kb), the X (0.8 kb), and two spliced (2.2 kb) RNAs, respectively. The preS2/S RNA and the spliced RNAs could be distinguished when a core gene-specific probe, which could not hybridize with the former, was used. Kinetic analysis of the expression of these RNAs revealed that the X transcript exhibited a pattern different from that of other viral transcripts. Amounts of all RNAs peaked at 24-48 hr post-transfection and then gradually declined. However, the X transcript became undetectable on Day 4 post-transfection while other viral RNAs persisted for at least 10 days. The unique expression profile of the X transcript suggested that it probably behaves as an early gene and this is consistent with its proposed role as a transactivator. Nevertheless, frameshift mutations within the X ORF had no obvious effects on the activities and temporal pattern of HBV transcription in this transient expression system.

Base Sequence↗
PubMed1982

Analysis of transcriptional initiation of yeast mitochondrial DNA in a homologous in vitro transcription system.

We have developed an in vitro transcription system for yeast mitochondrial rRNA genes. Using highly purified yeast mitochondrial RNA polymerase and bacterial plasmids carrying DNA segments containing the mitochondrial rRNA sites of transcriptional initiation, we have been able to demonstrate correct initiation of transcription in vitro. By directly sequencing the transcription products, we show that transcription in vitro of both the 14S and 21S rRNAs is initiated at precisely the same site as it is in vivo. Transcription of the rRNA genes is highly sensitive to ionic strength and RNA polymerase concentration. Additional factors or modified conditions may be necessary to permit accurate transcription of mitochondrial protein genes.

Base Sequence↗