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PubMed1994

Molecular epidemiology of enterovirus outbreaks in Canada during 1991-1992: identification of echovirus 30 and coxsackievirus B1 strains by amplicon sequencing.

The relatedness of enteroviral isolates associated with two recent outbreaks in Canada was assessed using direct sequencing of amplicons derived from a large portion of the 5' nontranslated region (NTR) of the viral genome. The amplicons of 60 echovirus 30 isolates originating from seven different provinces in 1991 were found to share 99% or greater sequence identity. Recent coxsackievirus B1 isolates characterised in the same manner were identical to each other. When the 5' NTR sequence of these isolates was compared to prototype strains a difference of 11-15% in nucleotide composition was observed. These results indicate that the variability of nucleotide sequence found in 5' NTRs can be utilized to identify rapidly enteroviral strains associated with particular outbreaks and distinguish them from other strains and serotypes.

Animals↗
PubMed1995

Rho-independent terminators without 3' poly-U tails from the early region of actinophage øC31.

Previous work has identified three intergenic regions from the early region of actinophage øC31 where transcription was either terminated or the mRNA was processed. Here we show using in vivo and in vitro approaches that these regions contain rho-independent terminators designated eta, etb and etc. Transcripts through eta-c would be expected to form stable RNA stem-loops but would lack poly-U tails. Eta-c contained part or all of the conserved sequences 5' AGCCCC and 5' GGGGCTT. A Streptomyces 'terminator probe' vector, pUGT1, was constructed and used to assay the efficiency of termination of transcription by eta-c from the thiostrepton-inducible tipA promoter by measuring the expression of a downstream reporter gene (aphII). In pUGT1 etb was at best a minor terminator in vivo whilst eta and etc exhibited strong termination activity. In vitro termination was assayed using templates containing a synthetic promoter recognised by E.coli RNA polymerase and fragments containing eta-c inserted downstream. All three terminators stimulated the formation of 3' ends in the promoter-distal arm of the inverted repeats with efficiencies eta > etc > etb. As all three terminators either overlap with or lie close to sequences which interact with phage repressor proteins (conserved inverted repeats, CIRs) and these can potentially form stem-loop structures in RNA, the effect of CIRs on termination was also investigated. Termination at etb was unaffected by the presence or absence on the transcription template of CIR3. CIR4 forms the central 17 bp of etc and a 37 nt deletion which eliminated this stem-loop abolished termination in vivo and in vitro. Eta was investigated using an antisense oligonucleotide interference assay; an oligo designed to bind the 5' arm of eta inhibited termination whilst an oligo antisense to CIR5 was ineffective and an oligo targeted further upstream enhanced termination. Taken together these data show that eta-c are intrinsic, rho-independent terminators of varying efficiencies despite the absence of a poly-U tail.

Bacteriophages↗
PubMed1989

A novel technique for the rapid preparation of mutant RNAs.

We have developed a novel in vitro mutagenesis technique that allows us to introduce mutations at the level of double-stranded DNA and then transcribe the mutant DNA directly. The technique is useful for those wishing to produce recombinant RNA, particularly if the desired recombinant is the result of an insertion or deletion. It is also useful for the preparation of 3'-truncated RNAs with a defined end. The technique is not dependent on the presence of a convenient restriction site within the target gene, and does not involve construction of a clone or amplification of the mutant DNA within a bacterial host. It is intended as a simple and rapid method for the preparation of roughly 100-200 pmol of mutant RNA, which would be sufficient for obtaining sequence information and assessing the functional consequences of the mutation.

Animals↗
PubMed1988

Studies of UV-inducible promoters from Clostridium perfringens in vivo and in vitro.

Expression of a 4 kb segment of the bacteriocinogenic plasmid, pIP404, from Clostridium perfringens is inducible by UV-irradiation. DNA sequence analysis revealed that this region contains three genes: uviA, uviB and bcn encoding the bacteriocin BCN5. Biochemical studies with mRNAs showed that expression was controlled at the transcriptional level and that the genes were organized in two independent transcriptional units, uviAB and bcn, both directed by tandem promoters inducible by UV light. The bcn gene is transcribed from three promoters (P1, P2, P3) while transcription of uviAB is directed by two promoters (P4, P5). With the exception of P4, which bears some resemblance to the consensus eubacterial promoter sequence, none of these promoters was recognized in vitro by the major forms of RNA polymerase from C. perfringens, Bacillus subtilis or Escherichia coli. Promoters P1, P3 and P5, which show striking homology with each other, contain unusual sequences in the '-35' and '-10' regions known to be recognized by RNA polymerase and this might indicate positive control.

Amino Acid Sequence↗
PubMed1983

Relationship between promoter structure and template specificities exhibited by the bacteriophage T3 and T7 RNA polymerases.

To explore the basis for the template specificities of the bacteriophage T3 and T7 RNA polymerases (EC 2.7.7.6), we determined the nucleotide sequences of six promoters recognized by the T3 RNA polymerase and compared them with the previously determined promoter sequences recognized by the bacteriophage T7 RNA polymerase. Recombinant plasmids containing random Hpa II and Taq I fragments of T3 DNA were screened for T3 promoter activity in vitro in a transcription assay using purified T3 RNA polymerase. Five promoters for the T3 RNA polymerase were identified in this manner and their sequences were determined; the sequence of an additional promoter was determined directly from a genomic DNA fragment. In five of the T3 promoters an identical 16-base-pair sequence (A-C-C-C-T-C-A-C-T-A-A-A-G-G-G-A) extends from -12 to +4 (initiation occurring with GTP at +1); this sequence is preceded by a 6-base-pair A + T region. The remaining promoter contains an inserted C at position -1 and an A at the +1 position. The sequence of the 5' end of the RNA transcript from the latter promoter confirms that transcription is initiated with ATP at the +1 position. Previously, late T3 or T7 transcripts had not been found to initiate with ATP. The highly conserved T3 promoter sequence was compared to the T7 promoter consensus sequence. The fundamental difference between the two kinds of phage promoters is the occurrence of G-A at positions -11 and -10 in the T7 promoter, whereas there is a single C at position -10 in the T3 promoter.

Base Sequence↗
PubMed1992

Mutational analysis of the "slippery-sequence" component of a coronavirus ribosomal frameshifting signal.

The ribosomal frameshift signal in the genomic RNA of the coronavirus IBV is composed of two elements, a heptanucleotide "slippery-sequence" and a downstream RNA pseudoknot. We have investigated the kinds of slippery sequence that can function at the IBV frameshift site by analysing the frameshifting properties of a series of slippery-sequence mutants. We firstly confirmed that the site of frameshifting in IBV was at the heptanucleotide stretch UUUAAAC, and then used our knowledge of the pseudoknot structure and a suitable reporter gene to prepare an expression construct that allowed both the magnitude and direction of ribosomal frameshifting to be determined for candidate slippery sequences. Our results show that in almost all of the sequences tested, frameshifting is strictly into the -1 reading frame. Monotonous runs of nucleotides, however, gave detectable levels of a -2/+1 frameshift product, and U stretches in particular gave significant levels (2% to 21%). Preliminary evidence suggests that the RNA pseudoknot may play a role in influencing frameshift direction. The spectrum of slip-sequences tested in this analysis included all those known or suspected to be utilized in vivo. Our results indicate that triplets of A, C, G and U are functional when decoded in the ribosomal P-site following slippage (XXXYYYN) although C triplets were the least effective. In the A-site (XXYYYYN), triplets of C and G were non-functional. The identity of the nucleotide at position 7 of the slippery sequence (XXXYYYN) was found to be a critical determinant of frameshift efficiency and we show that a hierarchy of frameshifting exists for A-site codons. These observations lead us to suggest that ribosomal frameshifting at a particular site is determined, at least in part, by the strength of the interaction of normal cellular tRNAs with the A-site codon and does not necessarily involve specialized "shifty" tRNAs.

Base Sequence↗
PubMed1979

Contacts between Escherichia coli RNA polymerase and thymines in the lac UV5 promoter.

I have identified those 5 positions of thymines in the lac UV5 promoter that lie close to bound Escherichia coli RNA polymerase (nucleosidetriphosphate:RNA nucleotidyltransferase, EC 2.7.7.6). Although ultraviolet irradiation of DNA with 5-bromouracil substituted in place of thymine normally cleaves the DNA at the bromouracils, a protein bound to the DNA can perturb these cleavages at those locations at which the protein lies close to the bromine. In the lac promoter most of these contacts lie in three regions. Four contacts lie in the region where transcription initiates; four lie in the "Pribnow box," which is located about 10 base pairs upstream from the initiation site; and three more lie in the "-35 region," located about 35 base pairs upstream from the initiation site. The "Pribnow box" and the "-35 region" are regions whose sequences are partially conserved between promoters and in which most promoter mutations are located; thus, contacts in these two regions probably represent sites of sequence-specific recognition by RNA polymerase.

Base Sequence↗
PubMed1996

DNA diagnosis of pulmonary infections: particular emphasis on Mycobacterium tuberculosis.

The past two decades have brought significant changes to the clinical laboratory. Microbiologists now have highly sensitive, rapid and specific molecular methods of identifying infectious agents by the direct detection of DNA or RNA sequences unique to a particular organism. Advanced DNA technology such as nucleic-acid hybridization, PCR and DNA fingerprinting have been used in the direct detection of causative organisms in clinical specimens, with resultant benefits such as increased sensitivity and specificity of the diagnostic approach and reduction of turnaround time. This review outlines a brief description of the various DNA diagnostic tools used in the detection of pulmonary infections with emphasis on their applications in the diagnosis of Mycobacterium tuberculosis.

DNA Fingerprinting↗
PubMed1998

Mutational analysis of a satellite phage activator.

The late gene activator, Delta, of satellite phage P4 is more efficient than the Delta of satellite phage phiR73 in utilizing a P2 helper prophage that lacks an activator (ogr) gene. Analysis of P4 Delta is complicated by the fact that this protein contains two tandem phiR73 Delta-like domains. We performed a mutational analysis of phiR73 Delta, in order to select mutations that might not be found using P4 Delta. The host RNA polymerase alpha subunit mutation rpoA155 (L289F) blocks the growth of P2, P4, and P4 carrying the delta gene of phiR73. A mutant of this latter phage that can grow in the presence of rpoA155 carries a V19M mutation in phiR73 Delta. This suggests that aa 19 contacts RNA polymerase, in addition to the aa residues 13, 42 and 44, that have been implicated in interactions with RNA polymerase by previous mutational analyses of P2 ogr and P4 delta. In corroboration of the proposed role of the regions at aa residues 19, 42, and 44, we found phiR73 Delta mutations in these regions that showed a reduced activation of late gene expression, but a normal ability to bind to late gene promoters. All activators in the Delta class contain four Cys residues that bind Zn2+. Mutation of these aa residues in phiR73 Delta eliminated late gene activation. Spectroscopic analysis of these mutant proteins revealed that they were unable to bind Zn2+. Histidine residues were substituted for two of the Cys residues (C30 and C35), changing a C2C2 type Zn-binding motif to a C2H2 motif. Although His residues are used in coordinating Zn2+ in other proteins, these His substitutions resulted in complete loss of activity and the inability to bind Zn2+.

Bacteriophages↗
PubMed2001

Escherichia coli RNA polymerase subunit omega and its N-terminal domain bind full-length beta' to facilitate incorporation into the alpha2beta subassembly.

The omega subunit of Escherichia coli RNA polymerase, consisting of 90 amino acids, is present in stoichiometric amounts per molecule of core RNA polymerase (alpha2betabeta'). The presence of omega is necessary to restore denatured RNA polymerase in vitro to its fully functional form, and, in an omega-less strain of E. coli, GroEL appears to substitute for omega in the maturation of RNA polymerase. The X-ray structure of Thermus aquaticus core RNA polymerase suggests that two regions of omega latch on to beta' at its N-terminus and C-terminus. We show here that omega binds only the intact beta' subunit and not the beta' N-terminal domain or beta' C-terminal domain, implying that omega binding requires both these regions of beta'. We further show that omega can prevent the aggregation of beta' during its renaturation in vitro and that a V8-protease-resistant 52-amino-acid-long N-terminal domain of omega is sufficient for binding and renaturation of beta'. CD and functional assays show that this N-terminal fragment retains the structure of native omega and is able to enhance the reconstitution of core RNA polymerase. Reconstitution of core RNA polymerase from its individual subunits proceeds according to the steps alpha + alpha --> alpha2 + beta --> alpha2beta + beta' --> alpha2betabeta'. It is shown here that omega participates during the last stage of enzyme assembly when beta' associates with the alpha2beta subassembly.

Amino Acid Sequence↗
PubMed1989

Two transcribing activities are involved in expression of the Streptomyces galactose operon.

The Streptomyces galactose operon is transcribed from two independently regulated promoters: galP1, located at the 5' end of the operon and responsible for galactose-dependent transcription of the operon, and galP2, an internal constitutive promoter. We identified and partially separated two distinct transcribing activities involved in expression of this operon. Using RNA polymerase from Streptomyces lividans and Streptomyces coelicolor partially purified by chromatography on heparin-agarose and DNA-cellulose, we detected activities capable of initiating transcription in vitro specifically from either galP1 or galP2. Circumstantial evidence suggests that the activity for galP2 transcription is a holoenzyme species associated with the previously described sigma 28 protein (referred to here as sigma C). The galP1-transcribing activity is more difficult to evaluate. This activity may correspond to a holoenzyme species associated with sigma A (formerly sigma 35), although other possibilities are discussed. This would be the second reported example of a catabolite-controlled gene in Streptomyces species expressed from multiple promoters recognized by different holoenzyme forms. This may indicate that the involvement of RNA polymerase heterogeneity in gene expression in Streptomyces species is a more general strategy for regulation than the specialized gene expression seen in Escherichia coli.

Base Sequence↗
PubMed1999

Model for polymerase access to the overlapped L gene of respiratory syncytial virus.

The last two genes of respiratory syncytial virus (RSV), M2 and L, overlap by 68 nucleotides, an arrangement which has counterparts in a number of nonsegmented negative-strand RNA viruses. Thus, the gene-end (GE) signal of M2 lies downstream of the L gene-start (GS) signal, separated by 45 nucleotides. Since RSV transcription ostensibly is sequential and unidirectional from a single promoter within the 3' leader region, it was unclear how the polymerase accesses the L GS signal. Furthermore, it was previously shown that 90% of transcripts which are initiated at the L GS signal are polyadenylated and terminated at the M2 GE signal, yielding a short, truncated L mRNA as the major transcription product of the L gene. Despite these apparent down-regulatory features, we show that the accumulation of full-length L mRNA during RSV infection is only sixfold less than that of its upstream neighbor, M2. We used cDNA-encoded genome analogs in an intracellular transcription assay to investigate the mechanism of transcription of the overlapped genes. Expression of L was found to be dependent on sequential transcription from the 3' end of the genome. Apart from the L GS signal, the only other strict requirement for initiation at L was the M2 GE signal. This implies that the polymerase accesses the L GS signal only following arrival at the M2 GE signal. Thus, polymerase which terminates at the M2 GE signal presumably scans upstream to initiate at the L GS signal. This also would provide a mechanism whereby polymerase which terminates prematurely during transcription of L could recycle from the M2 GE signal to the L GS signal, thereby accounting for the unexpectedly high level of synthesis of full-length L mRNA. The sequence and spacing between the two signals were not critical. Furthermore, the polymerase also was capable of efficiently transcribing from an L GS signal placed downstream of the M2 GE signal, implying that the overlapping arrangement is not obligatory. When copies of the L GS signal were placed concurrently upstream and downstream of the M2 GE signal, both were utilized. This finding indicates that a polymerase situated at a GE signal is capable of scanning for a GS signal in either the upstream or downstream direction and thereafter initiating transcription.

Base Sequence↗
PubMed1996

Insusceptibility of members of the class Mollicutes to rifampin: studies of the Spiroplasma citri RNA polymerase beta-subunit gene.

In order to study the mechanism of insusceptibility of Spiroplasma citri to rifampin, we have cloned and sequenced its rpoB gene, which encodes the beta subunit of RNA polymerase. By comparison of the deduced amino acid sequence with sequences of beta subunits from susceptible and resistant bacteria, it was possible to identify several differences in the so-called Rif region (encompassing rpoB codons 500 to 575 in the Escherichia coli sequence). We constructed a chimeric rpoB gene made of the E. coli rpoB gene in which the Rif region was replaced by the equivalent region from S. citri. E. coli cells harboring this chimeric gene were resistant to rifampin. Subsequent experiments involving site-directed mutagenesis demonstrated that a single amino acid substitution (asparagine at position 526) was able to provide high-level rifampin resistance in E. coli.

Amino Acid Sequence↗
PubMed1995

In vitro transcription of pathogenesis-related genes by purified RNA polymerase from Staphylococcus aureus.

The RNA polymerase (RNAP) holoenzyme of Staphylococcus aureus was purified by DNA affinity, gel filtration, and ion-exchange chromatography. This RNAP contained four major subunits with apparent molecular masses of 165, 130, 60, and 47 kDa. All four subunits of the RNAP were serologically related to the subunits of Escherichia coli E sigma 70 holoenzyme by Western immunoblot analysis. The 60-kDa subunit was subsequently isolated and found to react with a monoclonal antibody specific to the E. coli sigma 70 subunit. This sigma 70-related protein allowed E. coli core RNAP promoter-specific initiation and increased transcription by S. aureus RNAP that is unsaturated with sigma. We therefore suggest that this 60-kDa protein is a sigma factor. Purified S. aureus RNAP transcribed from the promoters of several important S. aureus virulence genes (sea, sec, hla, and agr P2) in vitro. The in vitro transcription start sites of the sea, sec, and agr P2 promoters, mapped by primer extension, were similar to those identified in vivo. The putative promoter hexamers of these three genes showed strong sequence similarity to the E. coli sigma 70 consensus promoter, and transcription by E sigma 70 from some of these promoters has been observed. Conversely, S. aureus RNAP does not transcribe from all E. coli sigma 70-dependent promoters. Taken together, our results indicate that the promoter sequences recognized by purified S. aureus RNAP are similar but not identical to those recognized by E. coli E sigma 70.

Bacterial Proteins↗
PubMed1998

Mutational analysis of the rous sarcoma virus DR posttranscriptional control element.

The direct repeat (DR) sequences flanking the src gene in Rous sarcoma virus are essential posttranscriptional control elements; at least one copy of this sequence is necessary for cytoplasmic accumulation of unspliced viral RNA. These sequences promote Rev-independent human immunodeficiency virus type 1 expression, suggesting they act as constitutive transport elements (CTEs). To determine which regions of this sequence are critical for CTE function, mutations in the downstream DR were generated and tested in a viral deletion construct lacking src and the upstream DR. Two single-point mutations and three different clustered mutations caused substantial reductions in reverse transcriptase activity, Gag protein levels, and unspliced viral RNA in the cytoplasm. Three conserved regions of the CTE, including nucleotides 8844 to 8847, 8862 to 8864, and 8868 to 8870, were most sensitive to inactivation by mutagenesis.

Animals↗
PubMed1989

Deoxynucleotide-containing oligoribonucleotide duplexes: stability and susceptibility to RNase V1 and RNase H.

Oligoribonucleotide duplexes containing one to four 2'-deoxynucleotide residues were used as substrates for ribonuclease V1 and RNase H. Either deoxyadenosine and/or deoxythymidine were incorporated into the duplex, 5'GGCCGGAUCCGCGC3'-5'GCGCGGAUCCGGCC3' by substitution of the appropriate deoxynucleoside triphosphate into a transcription reaction with T7 RNA polymerase. The melting temperature, Tm, of the duplex (1.8 microM in strands in 50 mM NaCl) containing only ribonucleotides was 79.9 degrees C. Substitution of deoxyadenosine in both strands of the duplex lowered the Tm by 2.4 degrees C. Substitution of deoxythymidine had no measurable effect on the Tm. Comparison of RNase V1 digestion patterns of fully ribonucleotide and deoxy-substituted duplexes suggest that any distortion is localized to the site of the substitution. An oligoribonucleotide containing two deoxy residues directs specific cleavage of RNA by E. coli RNase H. Structural requirements for cleavage are proposed for RNase V1 and RNase H.

Base Sequence↗
PubMed2004

Distinctive protein signatures provide molecular markers and evidence for the monophyletic nature of the deinococcus-thermus phylum.

The Deinococcus-Thermus group of species is currently recognized as a distinct phylum solely on the basis of their branching in 16S rRNA trees. No unique biochemical or molecular characteristics that can distinguish this group from all other bacteria are known at present. In this work, we describe eight conserved indels (viz., inserts or deletions) in seven widely distributed proteins that are distinctive characteristics of the Deinococcus-Thermus phylum but are not found in any other group of bacteria. The identified signatures include a 7-amino-acid (aa) insert in threonyl-tRNA synthetase, 1- and 3-aa inserts in the RNA polymerase beta' subunit, a 5-aa deletion in signal recognition particle (Ffh/SR54), a 2-aa insert in major sigma factor 70 (sigma70), a 2-aa insert in seryl-tRNA synthetase (SerRS), a 1-aa insert in ribosomal protein L1, and a 2-aa insert in UvrA homologs. By using PCR primers for conserved regions, fragments of these genes were amplified from a number of Deinococcus-Thermus species, and all such fragments (except SerRS in Deinococcus proteolyticus) were found to contain the indicated signatures. The presence of these signatures in various species from all three known genera within this phylum, viz., Deinococcus, Thermus, and Meiothermus, provide evidence that they are likely distinctive characteristics of the entire phylum which were introduced in a common ancestor of this group. The signature in SerRS, which is absent in D. proteolyticus, was likely introduced after the branching of this species. Phylogenetic studies as well as the nature of the inserts in some of these proteins (viz., sigma70 and SerRS) also support a sister group relationship between the Thermus and the Meiothermus genera. The identified signatures provide strong evidence for the monophyletic nature of the Deinococcus-Thermus phylum. These molecular markers should prove very useful in the identification of new species related to this group.

Amino Acid Sequence↗