Thioredoxin genetics.
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Bacterial Proteins↗
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Characterization of the effects of Escherichia coli replication terminator protein (Tus) on transcription reveals dynamic nature of the tus block to transcription complex progression.
We have characterized the blocks to progression of T7 and T3 RNA polymerase transcription complexes created when a Tus protein is bound to the template. The encounter with Tus impedes the progress of the transcription complexes of either enzyme. The duration of the block depends on which polymerase is used and the orientation of Tus on the DNA. Both genuine termination (dissociation of the transcription complex) and halting followed by continued progression after the block is abrogated are observed. The fraction of complexes that terminates depends on which polymerase is used and on the orientation of the Tus molecule. The efficiency of the block to transcription increases as the Tus concentration is increased, even if the concentration of Tus is already many times in excess of what is required to saturate its binding sites on the template in the absence of transcription. The block to transcription is rapidly abrogated if an excess of a DNA containing a binding site for Tus is added to a transcription reaction in which Tus and template have been preincubated. Finally, we find that transcription will rapidly displace Tus from a template under conditions that generate persistent blocks to transcription. These observations reveal that during the encounter with the transcription complex Tus rapidly dissociates from the template but that at sufficiently high concentrations Tus usually rebinds before the transcription complex can move forward. The advantage of a mechanism which can create a persistent block to transcription or replication complex progression, which can nevertheless be rapidly abrogated in response to down regulation of the blocking protein, is suggested.
Bacterial Proteins↗
Photochemical sterilization of 3SR reactions.
The self-sustained sequence replication (3SR) reaction is an extremely efficient method for amplifying target DNA and RNA sequences that may be present in minute quantities. A serious problem often encountered in its practice is carryover contamination from products of previous 3SR reactions. A postamplification treatment of 3SR reaction products with the photoactive agent 4'-aminomethyl-4,5-dimethylisopsoralen (IP-10) was investigated as an approach for preventing carryover contamination by 3SR amplicons. Initially, inhibition of the amplification reaction by high concentrations of the reagent was observed. This problem was circumvented by developing a gel-based delivery of IP-10, and the method was found to provide highly efficient sterilization (approximately 10(6)-fold) of 3SR amplicons. Evaluation of this strategy on a number of 3SR targets has indicated that the degree of sterilization is dependent on the length of the amplified region and on the concentration of IP-10. It appears that the sterilization effect is caused by covalent modification of the pyrimidine bases of RNA and DNA, which renders them unusable as templates for the 3SR reaction. Modification of a purified RNA transcript with IP-10 was shown to prevent effectively reverse transcription by avian myeloblastosis virus reverse transcriptase (AMV RT). Similarly, treatment of a T7 RNA polymerase promoter-containing DNA template with IP-10 eliminated full-length transcription by T7 RNA polymerase. This isopsoralen method may be used to sterilize multiple 3SR reactions in a clinical assay with a convenient UV irradiation step.
Base Sequence↗
Rescue of measles virus using a replication-deficient vaccinia-T7 vector.
A system which allows the reconstitution of measles virus (MV) from cloned cDNA is described. The severely host cell restricted vaccinia vector MVA-T7 expressing bacteriophage T7 RNA polymerase was used to generate full-length antigenomic MV RNA and simultaneously the mRNAs encoding the viral N, P and L proteins in order to produce replicationally and transcriptionally active nucleocapsids. The functionality of the N, P and L proteins was demonstrated first by their ability to rescue MV specific subgenomic RNAs. Assembly and budding of reconstituted MV was shown by syncytia formation and subsequently by virus isolation. The inability of MVA-T7 to produce progeny virus in most mammalian cells circumvents the necessity to separate the reconstituted MV from the MVA-T7 helper virus. Since all components are expressed transiently, this system is especially suitable for studying the functions of N, P and L. Furthermore, it is useful for investigating later steps in the MV life cycle.
Animals↗
An endodeoxyribonuclease induced after infection with phage T7 bearing amber mutations in genes 3, 5, and 6.
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Carbon Isotopes↗
Replication-deficient vaccinia virus encoding bacteriophage T7 RNA polymerase for transient gene expression in mammalian cells.
The vaccinia virus/bacteriophage T7 hybrid transient expression system employs a recombinant vaccinia virus that encodes the T7 RNA polymerase gene, a plasmid vector with a gene of interest regulated by a T7 promoter, and any cell line suitable for infection and transfection. Although high expression in a majority of cells is achieved, the severe cytopathic effects of vaccinia virus and the safety precautions required for use of infectious agents are undesirable features of the system. Here, we report the construction of a highly attenuated and avian host-restricted vaccinia virus recombinant that encodes the T7 RNA polymerase gene (MVA/T7 pol) and demonstrate the use of the virus for transient expression in mammalian cells. MVA/T7 pol has reduced cytopathic effects compared to the previously used replication-competent vaccinia virus, while providing a high level of gene expression in multiple mammalian cell lines.
Animals↗
RNA structure and heterologous recombination in the double-stranded RNA bacteriophage phi 6.
Bacteriophage phi 6 has a genome of three segments of double-stranded RNA, designated L, M, and S. A 1.2-kbp kanamycin resistance gene was inserted into segment M but was shown to be genetically unstable because of a high recombination rate between segment M and the 3' ends of segments S and L. The high rate of recombination is due to complementary homopolymer tracts bounding the kan gene. Removal of one arm of this potential hairpin stabilizes the insertion. The insertion of a 241- or 427-bp lacZ' gene into segment M leads to a stable Lac+ phage. The insertion of the same genes bounded by complementary homopolymer arms leads to recombinational instability. A stable derivative of this phage was shown to have lost one of the homopolymer arms. Several other conditions foster recombination. The truncation of a genomic segment at the 3' end prevents replication, but such a damaged molecule can be rescued by recombination. Similarly, insertion of the entire 3-kb lacZ gene prevents normal formation of virus, but the viral genes can be rescued by recombination. It appears that conditions leading to the retardation or absence of replication of a particular genomic segment facilitate recombinational rescue.
Bacteriophage T7↗
Inhibition of hepatitis C virus RNA replication by short hairpin RNA synthesized by T7 RNA polymerase in hepatitis C virus subgenomic replicons.
RNA interference (RNAi) is a cellular process that induces gene silencing by which small duplexes of RNA specifically target a homologous sequence for cleavage by cellular ribonucleases. Here, to test the RNAi method for blocking hepatitis C virus (HCV) RNA replication, we created four short hairpin RNAs (shRNAs) targeting the HCV internal ribosome entry site/Core gene transcript using T7 RNA polymerase. shRNA suppressed the replication of HCV RNA in the HCV replicon. On the other hand, short interfering RNAs synthesized using the T7 RNA polymerase system trigger a potent induction of interferon-alpha and -beta in a variety of cells. We examined whether the shRNAs synthesized using the T7 RNA polymerase system activated double-stranded RNA-dependent protein kinase, 2'-5' oligoadenylate synthetase, or interferon-regulatory factor-3. Our results demonstrated that the T7-transcribed shRNA did not activate these proteins in Huh-7 cells and the HCV replicon. These shRNAs are a promising new strategy for anti-HCV gene therapeutics.
Base Sequence↗
Localization of Escherichia coli RNA polymerase initiation sites in T7 DNA early promoter region.
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Coliphages↗
Isolation of the gene encoding yeast DNA polymerase I.
A yeast genomic DNA expression library in lambda gt11 antibody prepared against yeast DNA polymerase I were used to isolate the gene encoding DNA polymerase I. The identity of the DNA polymerase I gene was determined by several criteria. First, the clone-encoded protein is immunologically related to DNA polymerase I. Second, cells containing the gene cloned in the high copy number plasmid YEp24 overproduce the polymerase activity 4- to 5-fold as measured in yeast extracts. Finally, insertion of the gene downstream from a bacteriophage T7 promoter allows synthesis of yeast DNA polymerase I in Escherichia coli. Gene disruption and Southern hybridization experiments show that the polymerase is encoded by an essential, single copy gene. Examination of the germinated spores containing the disrupted gene reveals a defect in nuclear division and a terminal phenotype typical of replication mutants.
Cell Cycle↗
Asymmetric repair of bacteriophage T7 heteroduplex DNA.
Heteroduplex DNA molecules were prepared in vitro using one strand of DNA carrying a point mutation and one strand of the corresponding wild-type DNA. The heteroduplex DNA was transfected into competent bacteria and the progeny genotypes in the resulting infective centers were determined. From the results we conclude that about 80% of all transfected DNA molecules are repaired before DNA replication starts. This fraction of repaired DNA is independent of the location of the mismatched nucleotide pair. However, mismatch correction occurs preferentially on the H strand of the heteroduplex DNA. The repair does not depend on a known phage coded function but requires the active bacterial genes mutU, mutH, mutS and probably mutL.
Base Sequence↗
Virus promoters determine interference by defective RNAs: selective amplification of mini-RNA vectors and rescue from cDNA by a 3' copy-back ambisense rabies virus.
Typical defective interfering (DI) RNAs are more successful in the competition for viral polymerase than the parental (helper) virus, which is mostly due to an altered DI promoter composition. Rabies virus (RV) internal deletion RNAs which possess the authentic RV terminal promoters, and which therefore are transcriptionally active and can be used as vectors for foreign gene expression, are poorly propagated in RV-infected cells and do not interfere with RV replication. To allow DI-like amplification and high-level gene expression from such mini-RNA vectors, we have used an engineered 3' copy-back (ambisense) helper RV in which the strong replication promoter of the antigenome was replaced with the 50-fold-weaker genome promoter. In cells coinfected with ambisense helper virus and mini-RNAs encoding chloramphenicol acetyltransferase (CAT) and luciferase, mini-RNAs were amplified to high levels. This was correlated with interference with helper virus replication, finally resulting in a clear predominance of mini-RNAs over helper virus. However, efficient successive passaging of mini-RNAs and high-level reporter gene activity could be achieved without adding exogenous helper virus, revealing a rather moderate degree of interference not precluding substantial HV propagation. Compared to infections with recombinant RV vectors expressing CAT, the availability of abundant mini-RNA templates led to increased levels of CAT mRNA such that CAT activities were augmented up to 250-fold, while virus gene transcription was kept to a minimum. We have also exploited the finding that internal deletion model RNAs behave like DI RNAs and are selectively amplified in the presence of ambisense helper virus to demonstrate for the first time RV-supported rescue of cDNA after transfection of mini-RNA cDNAs in ambisense RV-infected cells expressing T7 RNA polymerase.
3' Untranslated Regions↗
Escherichia coli mutants thermosensitive for deoxyribonucleic acid gyrase subunit A: effects on deoxyribonucleic acid replication, transcription, and bacteriophage growth.
Temperature-sensitive nalA mutants of Escherichia coli have been used to investigate the structure and functions of deoxyribonucleic acid (DNA) gyrase. Extracts of one such mutant (nalA43) had thermosensitive DNA gyrase subunit A activity but normal gyrase subunit B activity, proving definitively that nalA is the structural gene for subunit A. Extracts of a second nalA (Ts) mutant (nalA45) had a 50-fold deficiency of gyrase subunit A activity. The residual DNA supertwisting was catalyzed by the mutant DNA gyrase rather than by a novel supertwisting enzyme. The nalA45(Ts) extract was also deficient in the nalidixic acid target, which is defined as the protein necessary to confer drug sensitivity to in vitro DNA replication directed by a nalidixic acid-resistant mutant extract. Thus, gyrase subunit A and the nalidixic acid target are one and the same protein, the nalA gene product. Shift of the nalA43(Ts) mutant to a nonpermissive temperature resulted in a precipitous decline in the rate of [(3)H]thymidine incorporation, demonstrating an obligatory role of the nalA gene product in DNA replication. The rates of incorporation of [(3)H]uridine pulses and continuously administered [(3)H]uracil were quickly reduced approximately twofold upon temperature shift of the nalA43(Ts) mutant, and therefore some but not all transcription requires the nalA gene product. The thermosensitive growth of bacteriophages phiX174 and T4 in the nalA43(Ts) host shows that these phages depend on the host nalA gene product. In contrast, the growth of phage T7 was strongly inhibited by nalidixic acid but essentially unaffected by the nalA43(Ts) mutation. The inhibition of T7 growth by nalidixic acid was, however, eliminated by temperature inactivation of the nal43 gene product. Therefore, nalidixic acid may block T7 growth by a corruption rather than a simple elimination of the nalidixic acid target. Possible mechanisms for such a corruption are considered, and their relevance to the puzzling dominance of drug sensitivity is discussed.
Coliphages↗
ColIb plasmid genes that inhibit the replication of T5 and T7 bacteriophage.
The colicin Ib (ColIb) plasmid genes that inhibit the replication of the T5-like and T7 bacteriophage have been cloned on an approximately 7200-bp ClaI fragment and their sites relative to each other and to the colicin immunity (imm) gene have been mapped. The inhibition of wild-type T7 by the clone is shown to be caused by the same gene or genes (pic) that cause the inhibition of T7 kinase-negative mutants and is a different gene than the one that causes inhibition of T5 (ibf or abi). The pic gene does not hybridize to the pif genes of the F plasmid that also cause the replication of T7 to be inhibited. The abi gene and the pic gene map very closely together but are under the control of different promoters. The abi gene has a maximum size of 900 bp and lies approximately 3000 bp away from the immunity gene, distal to the colicin gene. A site which maps in or near the gene binds very tightly to Escherichia coli RNA polymerase. The pic gene or genes lie between the abi gene and the imm gene and are contiguous with abi. Promoters for pic have been mapped and hypotheses to explain the inhibition of T7 by a cloned gene but not the whole ColIb plasmid are presented.
Bacteriocin Plasmids↗
Infectious RNA transcripts derived from full-length DNA copies of the genomic RNAs of cowpea mosaic virus.
A set of full-length DNA copies of both M and B RNA of cowpea mosaic virus (CPMV) was cloned downstream of a phage T7 promoter. Upon in vitro transcription using T7 RNA polymerase, M and B RNA-like transcripts were obtained from these DNA copies with only two additional nucleotides at the 5' end and five extra nucleotides at the 3' end in comparison to natural viral RNA. In cowpea protoplasts the transcripts of several cDNA clones of B RNA were able to replicate leading to detectable synthesis of viral RNA and proteins. Transcripts of M cDNA clones inoculated together with these B RNA transcripts were also expressed, although the number of protoplasts in which both transcripts were expressed was very low. Preliminary infectivity tests with mutagenized RNA transcripts indicate essential roles of the B RNA-encoded 24K and 32K polypeptides in viral RNA replication.
Base Sequence↗
The functions of rho in T7-DNA transcription in vitro.
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Bacterial Proteins↗
Ligase-defective bacteriophage T4. II. Physiological studies.
The timing of the suppression of gene 30 (deoxyribonucleic acid ligase) mutations by rII mutations was studied by temperature shift-down experiments with a temperature-sensitive rII mutation. The rII function must remain inactivated for about 5 to 8 min at 37 C for suppression to occur, thus making suppression an early function. This result is in agreement with the timing of expression of other rII functions. A gene 30 defect can also be overcome by replacing the Na(+) cation in the growth medium with the Mg(2+) cation, a result similar to the relief of the lethality of rII mutations in lambda lysogens. Prior infection with bacteriophages T3 or T7, which produce their own deoxyribonucleic acid ligases, can also partially overcome the lethality of gene 30 mutations.
Coliphages↗
The relationship between sequence-specific termination of DNA replication and transcription.
In Escherichia coli and Bacillus subtilis replication fork arrest occurs in the terminus at sequence-specific sites by the binding of replication terminator proteins to the fork arrest sites. The protein-DNA complex causes polar arrest of the replication forks by inhibiting the activity of the replicative helicases in only one orientation of the terminus with respect to the replication origin. This activity has been named as polar contrahelicase. In this paper we report on a second novel activity of the terminator proteins of E.coli and B.subtilis, namely the ability of the proteins to block RNA chain elongation by several prokaryotic RNA polymerases in a polar mode. The replication terminator proteins ter and RTP of E.coli and B.subtilis respectively, impeded RNA chain elongation catalyzed by T7, SP6 and E.coli RNA polymerases in a polar mode at the replication arrest sites. The RNA chain anti-elongation and the contrahelicase activities were isopolar. Whereas one monomer of ter was necessary and sufficient to block RNA chain elongation, two interacting dimers of RTP were needed to effect the same blockage. The biological significance of the RNA chain anti-elongation activity is manifested in the functional inactivation of a replication arrest site by invasion of RNA chains from outside, and the consequent need to preserve replication arrest activity by restricting the passage of transcription through the terminus-terminator protein complex.
Bacillus subtilis↗