Run-off Transcription Research Articles

Mechanistic Studies on the Impact of Transcription on Sequence-specific Termination of DNA Replication and Vice Versa

Since DNA replication and transcription often temporally and spatially overlap each other, the impact of one process on the other is of considerable interest. We have reported previously that transcription is impeded at the replication termini of Escherichia coli and Bacillus subtilis in a polar mode and that, when transcription is allowed to invade a replication terminus from the permissive direction, arrest of replication fork at the terminus is abrogated. In the present report, we have addressed four significant questions pertaining to the mechanism of transcription impedance by the replication terminator proteins. Is transcription arrested at the replication terminus or does RNA polymerase dissociate from the DNA causing authentic transcription termination? How does transcription cause abrogation of replication fork arrest at the terminus? Are the points of arrest of the replication fork and transcription the same or are these different? Are eukaryotic RNA polymerases also arrested at prokaryotic replication termini? Our results show that replication terminator proteins of E. coli and B. subtilis arrest but do not terminate transcription. Passage of an RNA transcript through the replication terminus causes the dissociation of the terminator protein from the terminus DNA, thus causing abrogation of replication fork arrest. DNA and RNA chain elongation are arrested at different locations on the terminator sites. Finally, although bacterial replication terminator proteins blocked yeast RNA polymerases in a polar fashion, a yeast transcription terminator protein (Reb1p) was unable to block T7 RNA polymerase and E. coli DnaB helicase.

Characterization of Yeast Protein Deg1 as Pseudouridine Synthase (Pus3) Catalyzing the Formation of Ψ38 and Ψ39 in tRNA Anticodon Loop

The enzymatic activity of yeast gene product Deg1 was identified using both disrupted yeast strain and cloned recombinant protein expressed in yeast and in Escherichia coli. The results show that the DEG1-disrupted yeast strain lacks synthase activity for the formation of pseudouridines psi 38 and psi 39 in tRNA whereas the other activities, specific for psi formation at positions 13, 27, 28, 32, 34, 35, 36, and 55 in tRNA, remain unaffected. Also, the His6-tagged recombinant yeast Deg1p expressed in E. coli as well as a protein fusion with protein A in yeast display the enzymatic activity only toward psi 38 and psi 39 formation in different tRNA substrates. Therefore, Deg1p is the third tRNA:pseudouridine synthase (Pus3p) characterized so far in yeast. Disruption of the DEG1 gene is not lethal but reduces considerably the yeast growth rate, especially at an elevated temperature (37 degrees C). Deg1p localizes both in the nucleus and in the cytoplasm, as shown by immunofluorescence microscopy. Identification of the pseudouridine residues present (or absent) in selected naturally occurring cytoplasmic and mitochondrial tRNAs from DEG1-disrupted strain points out a common origin of psi 38- and psi 39-synthesizing activity in both of these two cellular compartments. The sensitivity of Pus3p (Deg1p) activity to overall three-dimensional tRNA architecture and to a few individual mutations in tRNA was also studied. The results indicate the existence of subtle differences in the tRNA recognition by yeast Pus3p and by its homologous tRNA:pseudouridine synthase truA from E. coli (initially called hisT or PSU-I gene product).

Modulation of DNA-protein interactions in the P1 and P2 c-myc promoters by two intercalating drugs.

Regulation of transcription from the oncogene c-myc has an important role in the genesis of various tumors. Therefore, c-myc is a potential target for chemotherapy by drugs which are able to modify its activity directly. In this article, we identify the binding sites in the P1 and P2 promoter regions of c-myc for the intercalating antibiotics actinomycin D and elsamicin A. Gel retardation experiments indicate that actinomycin D or elsamicin A binding can inhibit the formation of several DNA-protein complexes. However, relatively low concentrations of elsamicin A, but not actinomycin D, appear to increase the level of binding to the P1 promoter of a protein factor. Using pure Sp1 transcription factor and an oligonucleotide containing the Sp1 putative binding site, we determined that the binding enhancement induced by small amounts of elsamicin was on the Sp1-DNA complex. Run-off transcription experiments in vitro showed that the effect of elsamicin A on Sp1 binding is followed by the maintenance or a relative rise in transcription levels from the P1 promoter of c-myc, while actinomycin D always inhibited the transcription from the P1 c-myc promoter in a concentration-dependent manner. Higher concentrations of elsamicin acted as an inhibitor of the transcription from the P1 start site but not from the P2.

Identification of the in vivo promoters of bacteriophages S13 and phi X174 and measurement of their relative activities.

Regions of bacteriophages phi X174 and S13 that contain putative promoter sequences were amplified by the polymerase chain reaction (PCR) and cloned into the reporter vector pKO-1. Assays of galactokinase activity revealed in vivo promoter activity in those constructs containing the promoter sequences with transcription initiation (+1) sites at nucleotide positions 45, 982, 1823, and 5211. These were identical in location to sequences with in vitro promoter activity and to the three known promoters PA, PB, and PD. P5211 is the location of a new, fourth, promoter. A site with a +1 position at nucleotide 4876, previously shown to initiate RNA synthesis in an in vitro run-off transcription assay, had no in vivo promoter activity. To investigate whether flanking sequences had effects on promoter activity, restriction fragments of phi X174 and S13 that encompass the in vivo promoters were cloned into the reporter vector pKO-1. The PA and P5211 promoter constructs showed dramatic effects with increases in activity of up to 7 times that shown with the PCR-generated promoter constructs. The phi X174 PB promoter construct had a 50% decrease in activity compared with the PCR-generated PB clone. While the data showed that in most instances promoter activity is affected by the flanking sequences in which the promoter is embedded, no general pattern correlating flanking sequences and promoter activity could be discerned. Additional evidence that the promoter sequence regions were active in vivo promoters was obtained by S1 nuclease mapping experiments. Initiation of RNA synthesis was shown at positions 45, 982, and 5211.

Tex261,a Novel Gene Presumably Related but Distinct from Steroidogenic Acute Regulatory (StAR) Gene, Is Regulated during the Development of Germ Cells

Tex261is a new gene cloned from a subtractive cDNA library from 10-day postnatal mouse testis. Tex261transcribes three mRNAs of 3.5, 1.6 and 1.4kb. The 3.5 kb and 1.4 kb transcripts are expressed in different gonadal and somatic tissues analyzed. However, the 1.6Kb transcript is only detected in testis and differentially regulated during development. This 1.6 kb mRNA is highly expressed in adult testis, with detection beginning at 15 days of postnatal life, which coincides with the presence of pachytene cells in prepuberal mouse. This expression was confirmed in pachytene cells by run-off transcription assay and by in situhybridization. A region of 86 amino acids from the predicted Tex261 was recently reported as a part of the steroidogenic acute regulatory protein StARgene by its sequence identity to a rat StARcloned cDNA. We demonstrate here that, in the mouse, StARand Tex261are two different genes with different expected functions, yet, a high identity (43%) at amino acid level is detected in a region of 153 amino acids corresponding to a transmembrane protein.

Effects of heterologous downstream sequences on the activity of the HIV- 1 promoter and its response to Tat

In HIV-1 infection, Tat acts at least in part to control transcriptional elongation by overcoming premature transcriptional termination. In some other genes this process is governed by DNA elements called attenuators in concert with cellular transcription factors. To understand the action of Tat more fully and explore its role as an anti-attenuator, we examined the ability of several natural and synthetic attenuation sequences to modulate transcription initiated at the HIV LTR. Fragments containing these signals were inserted downstream of the TAR element in an HIV-CAT chimera and their effects on transcription were assessed both in vitro and in vivo. Runoff transcription assays in HeLa cell extracts demonstrated that the attenuators give rise to premature termination of transcripts initiated from the heterologous HIV-LTR promoter in vitro. When transiently expressed following transfection into Cos cells, however, premature transcript termination at the attenuation site was not observed. Nevertheless, many of the inserted sequences exerted marked effects on CAT gene expression and on transactivation by Tat at both the RNA and protein levels. The nature and magnitude of the effects depended upon the identity of the attenuator and its orientation but only one of 16 sequences tested met the criteria for a Tat-suppressible attenuator in vivo. One other sequence, in contrast, severely reduced Tat-activated transcription without inhibiting basal transcription These results indicate that sequences downstream of the HIV LTR can influence its function as a promoter and its response to Tat transactivation, but lend little support to their role as attenuators in vivo.

Rescue of synthetic minireplicons establishes the absence of the NS1 and NS2 genes from avian pneumovirus.

We have determined the nucleotide sequences of the regions 3' and 5' proximal to the avian pneumovirus (APV) N and L genes, respectively. These sequences were used in the construction of a synthetic minireplicon construct in which the chloramphenicol acetyltransferase (CAT) reporter gene was flanked at its 3' end with the APV leader together with the APV N gene start signal and at its 5' end with the APV L gene end signal and the genome trailer region. The ability of T7 RNA polymerase runoff transcripts to direct the replication and expression of the CAT reporter gene in APV-infected cells demonstrated the ability of the putative leader and trailer regions to direct genome replication and gene expression. Furthermore, this confirms the absence of the NS1 and NS2 gene analogs within the APV genome. We were able to detect the expression of CAT protein from cells that had been infected with supernatants from the initially infected and transfected cells. These results have identified the cis-acting sequences of APV responsible for viral replication, gene expression, and packaging into virus-like particles.

Alternative transcription factor sigmaSB of Staphylococcus aureus: characterization and role in transcription of the global regulatory locus sar.

A homolog of the multiple-stress-responsive transcription factor sigmaB of Bacillus subtilis was predicted from the DNA sequence analysis of a region of the Staphylococcus aureus chromosome. A hybrid between the coding sequence of the first 11 amino acids of the gene 10 leader peptide of phage T7 (T7.Tag) and the putative sigB gene of S. aureus was constructed and cloned into Escherichia coli BL21(DE3)pLysS for overexpression from a T7 promoter. A homogeneous preparation of the overproduced protein was obtained by affinity chromatography with a T7.Tag monoclonal antibody coupled to agarose. The amino-terminal amino acid sequence of the first 22 residues of the purified protein matched that deduced from the nucleotide sequence. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the purified protein, designated sigmaSB, indicated that it migrated as an approximately 39-kDa polypeptide. Promoter-specific transcription from the B. subtilis sigmaB-dependent PB promoter of the sigB operon was stimulated by sigmaSB in a concentration-dependent fashion when reconstituted with the S. aureus core RNA polymerase (RNAP). Specific transcript from the predicted sigmaB-dependent PB promoter of the sigB operon of S. aureus was obtained by the reconstituted RNAP in a runoff transcription reaction. The sar operon of S. aureus contains three promoter elements (P1, P2, and P3) and is known to partly control the synthesis of a number of extracellular toxins and several cell wall proteins. Our in vitro studies revealed that transcription from the P1 promoter is dependent on the primary sigma factor sigmaSA, while that of the P3 promoter is dependent on sigmaSB. As determined by primer extension studies, the 5' end of the sigmaSB-initiated mRNA synthesized in vitro from the sar P3 promoter is in agreement with the 5' end of the cellular RNA.

Efficient Transfer of Synthetic Ribozymes into Cells Using Hemagglutinating Virus of Japan (HVJ)-Cationic Liposomes: APPLICATION FOR RIBOZYMES THAT TARGET HUMAN T-CELL LEUKEMIA VIRUS TYPE I tax/rex mRNA

We investigated the usefulness of ribozymes in inhibiting the expression of human T-cell leukemia virus type I (HTLV-I) gene. Two hammerhead ribozymes that were against HTLV-I rex (RR) and tax (TR) mRNA were synthesized. Both ribozymes were sequence-specific in the in vitro cleavage analysis of run-off transcripts from tax/rex cDNA. Intracellular activities of the ribozymes were studied in HTLV-I tax cDNA-transfected rat embryonic fibroblasts (Rat/Tax cells), which expressed the Tax but not Rex. Ribozymes were delivered into cells using anionic or cationic liposomes fused with hemagglutinating virus of Japan (HVJ). Cellular uptake of ribozymes complexed with HVJ-cationic liposomes was 15-20 times higher cellular uptake than naked ribozymes, and 4-5 times higher than that of ribozymes complexed with HVJ-anionic liposomes. HVJ-cationic liposomes promoted accumulation of ribozymes in cytoplasm and accelerated transport to the nucleus. Tax protein levels were decreased about 95% and were five times lower when the same amount of TR was introduced into the cells using HVJ-cationic, rather than HVJ-anionic liposomes. Inactive ribozyme and tax antisense oligodeoxynucleotides reduced Tax expression by about 20%, whereas RR and tax sense oligodeoxynucleotides had no effect. These results suggest that the ribozymes' effect against tax mRNA was sequence-specific, and HVJ-cationic liposomes can be useful for intracellular introduction of ribozymes.

Characterization of an ‘orthogonal’ suppressor tRNA derived from E. coli tRNA 2Gln

Background: In an effort to expand further our ability to manipulate protein structure, we have completed the first step towards a general method that allows the site-specific incorporation of unnatural amino acids into proteins in vivo. Our approach involves the construction of an ‘orthogonal’ suppressor tRNA that is uniquely acylated in vivo, by an engineered aminoacyl-tRNA synthetase, with the desired unnatural amino acid. The Escherichia coli tRNA 2 Gln-glutaminyl-tRNA synthetase (GlnRS) pair provides a biochemically and structurally well-characterized starting point for developing this methodology. To generate the orthogonal tRNA, mutations were introduced into the acceptor stem, D-loop/stem, and anticodon loop of tRNA 2 Gln. We report here the characterization of the properties of the resulting tRNAs and their suitability to serve as an orthogonal suppressor. Our efforts to generate an engineered synthetase are described elsewhere. Results: Mutant tRNAs were generated by runoff transcription and assayed for their ability to be aminoacylated by purified E. coli GInRS and to suppress an amber codon in an in vitro transcription/translation reaction. One tRNA bearing eight mutations satisfies the minimal requirements for the delivery of an unnatural amino acid: it is not acylated by any endogenous E. coli aminoacyl-tRNA synthetase, including GInRS, yet functions efficiently during protein translation. Mutations in the acceptor stem and D-loop/stem, when introduced in combination, had very different effects on the properties of the resulting tRNAs compared with the effects of the individual mutations. Conclusions: Mutations at sites within tRNA 2 Gln separated by 23–31 Å interact strongly with each other, often in a nonadditive fashion, to modulate both aminoacylation activities and translational efficiencies. The observed correlation between the effects of mutations at very distinct regions of the GlnRS-tRNA and possibly the ribosomal/tRNA complexes may contribute in part to the fidelity of protein biosynthesis.

Characterization of the rpoC gene of Streptomyces coelicolor A3(2) and its use to develop a simple and rapid method for the purification of RNA polymerase

The Streptomyces coelicolor rpoC gene, that encodes the β′ subunit of RNA polymerase, was isolated using the Escherichia coli rpoC gene as a hybridization probe. Comparision of the predicted amino acid sequence of the S. coelicolor β′ subunit to those characterized from other bacteria revealed three distinct subfamilies of β′ subunits, one of which consists of the S. coelicolor subunit and those from Mycobacterium leprae and Mycoplasma genitalium. Using site-directed mutagenesis, the carboxy terminus of the S. coelicolor β′ subunit was modified to contain six histidine residues. The histidine-tagged gene, rpoC HIS, was used to replace the wild-type allele in the chromosome of S. coelicolor and S. lividans. These strains were unaffected in growth and sporulation, demonstrating that the histidine-tagged RNA polymerase was competent to carry out all essential in-vivo functions. During a 1-day procedure, highly purified RNA polymerase was obtained by nickel–NTA agarose affinity chromatography followed by heparin–sepharose chromatography. Using in-vitro run-off transcription assays, the affinity purified RNA polymerase was shown to initiate transcription correctly from the S. lividans galP1 and galP2 promoters, and the Bacillus subtilus veg and ctc promoters. An extension of this procedure yielded highly-purified core RNA polymerase. To facilitate introduction of the rpoC HIS allele into other genetic backgrounds, a mutation in the adjacent gene, rpoB ( rifA), conferring rifampin-resistance, was isolated in S. coelicolor to provide a genetic marker to follow transfer of the rpoC HIS allele. The use of this affinity chromatography procedure, in combination with the ability to introduce the rpoC HIS allele into different Streptomyces strains by transformation, will greatly facilitate the in-vitro analysis of transcription in members of this genus.

Translation of an Uncapped mRNA Involves Scanning

tat, an essential gene of human immunodeficiency virus, when placed under the control of the RNA polymerase III promoter from the adenovirus VA RNA1 gene, is transcribed into an uncapped and nonpolyadenylated mRNA. This VA-Tat RNA is translated to produce functional Tat protein in transfected mammalian cells (Gunnery, S., and Mathews, M. B. (1995) Mol. Cell. Biol. 15, 3597-3607). The presence of an upstream open reading frame (ORF) in VA-Tat RNA is inhibitory to the translation of the Tat ORF, suggesting that the RNA is scanned during translation even though it is uncapped. Because the effect of the upstream ORF is relatively small (about 2-fold), we sought more definitive evidence of scanning by introducing secondary structures of varying stabilities into the 5'-untranslated region of VA-Tat RNA. The results of transfection experiments showed that highly stable secondary structure was inhibitory to Tat synthesis, whereas structures of lower stability were not inhibitory, confirming that uncapped mRNA is subject to scanning. Furthermore, translation of the downstream ORF was reduced but not eliminated by mutations that caused the upstream ORF to overlap the Tat ORF. Extending the overlap of the two ORFs further decreased the translation of the downstream ORF. This observation implies that ribosomes reinitiate after termination, possibly after migrating in a 3' to 5' direction through the overlap region of the mRNA. Similar results were obtained with a capped polymerase II transcript, indicating that the translation of polymerase II and polymerase III transcripts occurs through similar mechanisms.

Transcriptional strategy of closteroviruses: mapping the 5' termini of the citrus tristeza virus subgenomic RNAs.

Citrus tristeza virus (CTV) induces formation of a nested set of at least nine 3' coterminal subgenomic RNAs (sgRNAs) in infected tissue. The organization and expression of the 19,296-nucleotide (nt) CTV genome resembles that of coronaviruses, with polyprotein processing, translational frameshifting, and multiple sgRNA formation, but phylogenetically the CTV polymerase, like polymerases of other closteroviruses, belongs to the Sindbis virus-like lineage of RNA virus polymerases. Both positive-strand RNA virus supergroups, coronaviruses and Sindbis-like viruses, utilize different mechanisms of transcription. To address the mechanism of CTV transcription, 5' termini for the two most abundant sgRNAs, 1.5 and 0.9 kb, respectively, were mapped by runoff reverse transcription. The two sgRNAs were demonstrated to have 48- and 38-nt 5' untranslated regions (5'-UTRs), respectively. The 5'-UTR for the 1.5-kb RNA was cloned, sequenced, and demonstrated to be colinear with the 48-nt genomic sequence upstream of the initiator codon of the respective open reading frame 10, i.e., to be of continuous template origin. The data obtained suggest that the sgRNA transcription of CTV is dissimilar from the coronavirus transcription and consistent with the transcriptional mechanism of other Sindbis-like viruses. Thus, the Sindbis virus-like mechanism of transcription of the positive-strand RNA genomes might be successfully utilized by the closterovirus genome of up to 19.3 kb with multiple sgRNAs.

A new type of hairpin ribozyme consisting of three domains.

We have constructed a new hairpin ribozyme with three stem-loop domains. In the ribozyme, another domain (domain I') was connected to the 3'-end of domain II of the parent hairpin ribozyme, and the new ribozyme can be trimmed after transcription from the DNA template using T7 RNA polymerase. Since a mutant ribozyme containing a substitution at the essential base in domain I' lacked the 3'-trimming reaction, the autoprocessing activity was proved to be derived from the catalytic reaction, similar to the wild-type ribozyme. Furthermore, the structure of the cleavage site from the self-trimming reaction was identified as a 2',3'-cyclic phosphate, which is the same as that of the wild-type. The processed ribozyme was designed to cleave an external substrate RNA derived from the mRNA of the human inducible nitric oxide synthase and was proved to cleave at the expected, unique site. The hairpin ribozyme containing the three-domains exhibited the 3'-self-trimming activity even in a runoff transcription reaction from the plasmid harboring the ribozyme gene with the three domains. The new type of hairpin ribozyme thus obtained has three stem-loop domains and is able to act as a catalytic RNA for both cis and trans cleavage. These ribozymes are of interest from the point of the structure-function relationship of the hairpin ribozyme and provide an important insight into over understanding of the role of the domain-domain interaction in the catalytic activity.

CREB controls LAP/C/EBP beta transcription.

LAP/C/EBP beta is a member of the C/EBP family of transcription factors and is involved in hepatocyte-specific gene expression. Recently we showed that, besides its posttranscriptional regulation, LAP/C/EBP beta mRNA is modulated during liver regeneration. Therefore, in this study we investigated mechanisms which control LAP/C/EBP beta gene transcription. Deletion analysis of the 5'-flanking region, located upstream of the start site of transcription in the LAP/C/EBP beta gene, demonstrated that a small region in close proximity to the TATA box is important in maintaining a high level of transcription of the luciferase reporter gene constructs. In gel shift experiments two sites were identified which are important for specific complex formation within this region. Further analysis by cross-linking, super shift, and competition experiments was performed with liver cell nuclear extracts, hepatoma cell nuclear extracts, or recombinant CREB protein. These experiments conclusively demonstrated that CREB binds to both sites in the LAP/C/EBP beta promoter with an affinity similar to that with the CREB consensus sequence. Transfection experiments with promoter constructs where the CREB sites were mutated showed that these sites are important to maintain both basal promoter activity and LAP/C/EBP beta inducibility through CREB. Northern blot analysis and runoff transcription assays demonstrated that the protein kinase A pathway not only stimulated the activity of the luciferase reporter construct but also the transcription of the endogenous LAP/C/EBP beta gene in different cell types. Western blot analysis of rat liver cell nuclear extracts and runoff transcription assays of rat liver cell nuclei after two-thirds hepatectomy showed a functional link between the induction of CREB phosphorylation and LAP/C/EBP beta mRNA transcription during liver regeneration. These results demonstrate that the two CREB sites are important to control LAP/C/EBP beta transcription in vivo. As several pathways control CREB phosphorylation, our results provide evidence for the transcriptional regulation of LAP/C/EBP beta via CREB under different physiological conditions.

The global regulator CsrA of Escherichia coli is a specific mRNA-binding protein.

The csrA gene encodes a global regulatory protein which facilitates glgC mRNA decay in vivo. A purified recombinant CsrA protein was found to inhibit in vitro glg (glycogen biosynthesis) gene expression posttranscriptionally and bind specifically to a glgC runoff transcript without causing its decay. Our results provide further insight into the mechanism by which CsrA functions as an mRNA decay factor.

The herpes simplex virus immediate-early protein ICP0 affects transcription from the viral genome and infected-cell survival in the absence of ICP4 and ICP27.

ICP4, ICP0, and ICP27 are the immediate-early (IE) regulatory proteins of herpes simplex virus that have the greatest effect on viral gene expression and growth. Comparative analysis of viral mutants defective in various subsets of these IE genes should help elucidate how these proteins affect cellular and viral processes. This study focuses on the mutant d97, which is defective for the genes encoding ICP4, ICP0, and ICP27 and expresses the bacterial beta-galactosidase (beta-gal) gene from the ICP0 promoter. Together with the d92 virus (ICP4- ICP27-) and the ICP0-complementing cell line L7, d97 provided a unique opportunity to evaluate ICP0 function in the absence of the regulatory activities specified by ICP4 and ICP27. The pattern of protein synthesis in d97-infected cells was unique relative to other IE gene mutants in that it was similar to that seen in the absence of prior viral protein synthesis, possibly approximating the effect of cellular factors and virion components alone. Inactivation of ICP0 in the absence of ICP4 produced a significant decrease in the levels of the early mRNAs ICP6 and thymidine kinase (tk). There was also a marginal reduction in the levels of the IE ICP22 mRNA, and this was most notable at low multiplicity of infection (MOI). In d97-infected L7 cells, the levels of the viral mRNAs were mostly restored to those observed in infections with d92. Nuclear runoff transcription analysis demonstrated that the presence of ICP0 resulted in an increase in the transcription rates of the analyzed genes. The transcription rates of the early genes were dramatically reduced in the absence of ICP0. At low MOI, the transcription rates of ICP6 and tk were comparable to the rate of transcription of a cellular gene. Relevant to the potential use of d97 as a transfer vector, it was also determined that the absence of ICP0 reduced the cellular toxicity of the virus compared to that of d92. The beta-gal transgene expressed from an IE promoter was detected for up to 14 days postinfection; however, the level of beta-gal expression declined dramatically after 1 day postinfection. In the presence of ICP0, the level of expression of beta-gal was increased; however the infected monolayer was destroyed by 3 days postinfection. Therefore, deletion of ICP0 in the absence of ICP4 and ICP27 reduces toxicity and lowers the level of expression of genes from the viral genome.

Echovirus-coxsackievirus chimeras for the evaluation of the role of the echovirus 5' nontranslated region (5'NTNTR) in pathogenesis. • 758

Echoviruses (ECVs), members of the genus enterovirus(polioviruses [PV], group A and B coxsackieviruses [CAV and CBV], ECV and enteroviruses), cause syndromes ranging from febrile exanthems to myocarditis and meningitis. The PV1-3 and CBV3 5'NTR, the major viral regulatory region, has been shown to contain virulence determinants important in pathogenesis. Despite their clinical significance, few studies have explored the role of the ECV 5'NTR in pathogenesis. Murine models exist for the study of the PV and CBV. No such models exist for the ECV due to the inability of these viruses to replicate in mice. Because the ECV 5'NTR is similar to CBV, a possible approach to the study of the ECV 5'NTR, using a murine model, is to create chimeric viruses in which the 5'NTR of CBV is replaced with that of the ECV. We constructed such ECV/CBV chimeras and report their viability, growth kinetics and ability to retain parental virulence phenotype. ECV serotypes 9 and 12 were selected based on clinical virulence phenotype; ECV9 infection more frequently results in overt clinical disease, while ECV12 infection results in subclinical seroconversion. Nucleotides 62-753 of the ECV9 and 12 5'NTRs were RT-PCR amplified, directly sequenced, cloned and used to replace the homologous region of a cloned, transcriptionally controlled, full-length, noncardiovirulent CBV3. HeLa cell transfection of run-off RNA transcripts from the chimeric constructs resulted in viable virus. Single passage chimeric virus was sequenced prior to further characterization. No variation from the parental ECV 5'NTR sequence was found. One-step viral growth studies revealed that while both chimeras achieved similar final titers, the ECV12/CBV3 chimera was slower in doing so. CH3/He mice inoculated with the chimeric viruses were examined for evidence of myocarditis as a marker of virulence phenotype. The ECV9/CBV3 chimera produced myocardial lesions while ECV12/CBV3 failed to do so. These studies indicate that 5'NTR ECV/CBV3 chimeras are viable, appear to retain the parental ECV virulence phenotype and provide evidence for ECV 5'NTR virulence determinants.

In vitro transcription of human T-cell leukemia virus type 1 is RNA polymerase II dependent.

The HTLV-1 promoter directs RNA polymerase II transcription of viral genomic RNA in vivo. However, it has been reported that in vitro, a unique RNA polymerase, with characteristics of RNA polymerases II and III, is capable of HTLV-1 transcription (G. Piras, F. Kashanchi, M. F. Radonovich, J. F. Duvall, and J. N. Brady, J. Virol. 68:6170-6179, 1994). To further characterize the polymerase involved in HTLV-1 transcription in vitro, runoff transcription assays were performed with a variety of extracts and RNA polymerase inhibitors. Under all in vitro reaction conditions tested, RNA polymerase II appeared to be the only polymerase capable of correct transcriptional initiation from the HTLV-1 promoter. Synthesis of the specific HTLV-1 RNA transcript showed sensitivities to the RNA polymerase inhibitors tagetitoxin and alpha-amanitin that are consistent with RNA polymerase II transcription. Together, these data indicate that in vitro, as in vivo, the HTLV-1 promoter directs transcription by RNA polymerase II.

Disruption of transcription in vitro and gene expression in vivo by DNA adducts derived from a benzo[a]pyrene diol epoxide located in heterologous sequences.

Previous studies indicated a high affinity of the transcription factor Sp1 for DNA adducts derived from benzo[a]pyrene diol epoxide (BPDE) in sequences that are not normal binding sites for Sp1. We tested for functional effects of this phenomenon in three systems in which transcription is Sp1-dependent. In an in vitro, Sp1-dependent transcription system addition of heterologous plasmid DNA containing BPDE adducts abolished production of a specific run-off transcript. This inhibition was not seen with unmodified plasmid DNA, and could be overcome by addition of purified Sp1 protein. In SL2 insect cells, high-level expression of an Sp1-dependent reporter gene, which was dependent on co-transfection of an Sp1 expression vector, was inhibited >95% by co-transfection of heterologous DNA containing BPDE adducts. This inhibition could be partially overcome by increasing the amount of the Sp1 expression vector in the transfections. In human C33A cells, expression of a transfected reporter gene driven by a GC box containing fragment of the human E2F1 promoter was enhanced by co-transfection of an Sp1 expression plasmid. Expression was inhibited 3-6-fold by co-transfection of heterologous DNA containing BPDE-DNA adducts. A similar inhibition was seen in human SAOS-2 cells, which lack functional p53 protein. These data are consistent with functionally significant sequestration of the Sp1 transcription factor by BPDE-DNA adducts in all three systems. Altered availability of transcription factors such as Sp1 in carcinogen-treated cells may disrupt patterns of gene expression.