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PubMed2007

DNA sequence variability in isolates recovered from patients with postvaccination rash or herpes zoster caused by Oka varicella vaccine.

Little is known about the pathogenic potential of individual strains in the varicella vaccine. We analyzed genomic variation among specimens obtained from vaccine recipients with postvaccination rash or herpes zoster (HZ), focusing on polymorphisms between live attenuated varicella vaccine virus and wild-type varicella-zoster virus. Eleven of 18 postvaccination HZ specimens contained >1 strain, and 7 of 18 appeared to be clonal. All 21 postvaccination rash specimens contained mixtures of vaccine strains. Four single-nucleotide polymorphisms (SNPs) consistently occurred in every isolate; all were polymorphisms in open-reading frame (ORF) 62, and 2 confer amino acid substitutions in the immediate-early protein 62. Four wild-type SNPs occurred in every isolate: one each occurred in ORF 10, ORF 21, ORF 62, and a noncoding region upstream of ORF 64. The frequencies of the remaining wild-type SNPs were variable, with the SNPs uniformly expressed (even in mixtures) in 20.5%-97.4% of isolates (mean frequency, 67.7%). No 2 clinical isolates had identical SNP profiles; as such, vaccine latency usually involves >1 strain.

Adolescent↗
PubMed1989

Molecular cloning, sequencing and expression in Escherichia coli of the bean yellow mosaic virus coat protein gene.

The sequence of 1015 nucleotides from the 3' poly(A) tract of the potyvirus bean yellow mosaic virus (BYMV) RNA has been determined from two cDNA clones. This sequence contained a single long open reading frame (ORF) starting upstream of the cloned region. The ORF was expressed as a fusion protein in Escherichia coli, and the product was detected by antibodies specific for the coat protein of BYMV. The predicted length of the coat protein gene was 822 nucleotides, corresponding to a 273 amino acid coat protein of Mr 30910. The deduced amino acid sequence of the BYMV coat protein was compared to the chemically determined amino acid composition of purified virion protein, and of protein prepared from trypsin-treated virions. The nucleotide and deduced amino acid sequences were compared to the sequences of the coat protein genes of other potyviruses. The BYMV coat protein gene was found to be 50 to 61% homologous to those of other potyviruses at both the nucleotide and amino acid levels; the greatest variation was between the 5'-proximal one-fifth of the genes. Amino acid sequences and hydrophilicity plots of the different potyvirus coat proteins showed similarities which indicated that the structure of the coat protein is highly conserved; a non-terminal region of variability was predicted to be exposed on the exterior of the virion. A putative cleavage site at a glutamine-serine dipeptide was identified by similarity in context to the cleavage sites of tobacco etch virus and tobacco vein mottling virus coat proteins from the viral polyproteins. The BYMV 3'-terminal non-coding region of 166 nucleotides is followed by a poly(A) tract.

Amino Acid Sequence↗
PubMed1992

Mechanism of translation of monocistronic and multicistronic human immunodeficiency virus type 1 mRNAs.

We have used a panel of cDNA clones expressing wild-type and mutant human immunodeficiency virus type 1 (HIV-1) mRNAs to study translation of these mRNAs in eucaryotic cells. The tat open reading frame (ORF) has a strong signal for translation initiation, while rev and vpu ORFs have weaker signals. The expression of downstream ORFs is inhibited in mRNAs that contain the tat ORF as the first ORF. In contrast, downstream ORFs are expressed efficiently from mRNAs that have rev or vpu as the first ORF. All env mRNAs contain the upstream vpu ORF. Expression of HIV-1 Env protein requires a weak vpu AUG, which allows leaky scanning to occur, thereby allowing ribosomes access to the downstream env ORF. We concluded that HIV-1 mRNAs are translated by the scanning mechanism and that expression of more than one protein from each mRNA was caused by leaky scanning at the first AUG of the mRNA.

Base Sequence↗
PubMed1990

Modulation of translation by the 5' leader sequence of the mRNA encoding murine tissue inhibitor of metalloproteinases.

Murine tissue inhibitor of metalloproteinases (TIMP) is a Mr 28,000 secreted glycoprotein. The nucleotide sequence has been determined previously, and evidence for a multiplicity of TIMP transcripts presented. The longest TIMP transcript contains a small open reading frame (ORF) which is upstream of and overlaps the TIMP ORF, whereas the shorter inducible transcripts lack the AUG start codon of the upstream putative ORF. Serum stimulation of quiescent fibroblasts selectively enhances the abundance of the shorter transcripts (Edwards, D. R., Waterhouse, P., Holman, M. L., and Denhardt, D. T. (1986) Nucleic Acids Res. 14, 8863-8878). Here we have used an in vitro transcription/translation system to determine the effect of the upstream region on the efficiency of TIMP translation. TIMP transcripts either containing or lacking a segment including the AUG of the 5' ORF were generated by SP6 polymerase, and equal amounts of these transcripts were translated in reticulocyte lysates. TIMP synthesized in vitro was immunoprecipitable by an antiserum raised against a recombinant TIMP protein. TIMP was translated 3-fold more efficiently from the 5'-truncated transcript, consistent with the possibility that induction of shorter, more efficiently translated, TIMP transcripts are an important means of amplifying TIMP expression in response to serum.

Animals↗
PubMed1986

Amino acid sequence homology in gag region of reverse transcribing elements and the coat protein gene of cauliflower mosaic virus.

A nucleic acid binding protein (NBP) derived from the gag gene of retroviruses that is thought to interact with genomic RNA in virion cores, contains a highly conserved arrangement of cysteine residues. A search of available nucleic acid and protein sequences has revealed that the motif CysX2CysX4HisX4Cys (NBPcys) is invarient in all replication competent retroviruses, a Syrian hamster intracisternal A-particle gene, the Drosophila retrotransposon copia and in cauliflower mosaic virus (CaMV). In each case, NBPcys is located in that part of the 'gag-pol' region just preceding a conserved protease amino acid sequence. This is of special significance for CaMV as NBPcys is in the coat protein gene (ORF IV) upstream of the putative reverse transcriptase gene (ORF V) and demonstrates that the gag-pol arrangement of reverse transcribing elements is preserved in CaMV. Moreover, CaMV differs from all other known NBPcys-containing elements in that it packages a DNA genome in virions.

Amino Acid Sequence↗
PubMed1994

Transcriptional mapping of the varicella-zoster virus regulatory genes encoding open reading frames 4 and 63.

Four of the 68 varicella-zoster virus (VZV) unique open reading frames (ORFs), i.e., ORFs 4, 61, 62, and 63, encode proteins that influence viral transcription and are considered to be positional homologs of herpes simplex virus type 1 (HSV-1) immediate-early (IE) proteins. In order to identify the elements that regulate transcription of VZV ORFs 4 and 63, the encoded mRNAs were mapped in detail. For ORF 4, a major 1.8-kb and a minor 3.0-kb polyadenylated [poly(A)+] RNA were identified, whereas ORF 63-specific probes recognized 1.3- and 1.9-kb poly(A)+ RNAs. Probes specific for sequences adjacent to the ORFs and mapping of the RNA 3' ends indicated that the ORF 4 RNAs were 3' coterminal, whereas the RNAs for ORF 63 represented two different termination sites. S1 nuclease mapping and primer extension analyses indicated a single transcription initiation site for ORF 4 at 38 bp upstream of the ORF start codon. For ORF 63, multiple transcriptional start sites at 87 to 95, 151 to 153, and (tentatively) 238 to 243 bp upstream of the ORF start codon were identified. TATA box motifs at good positional locations were found upstream of all mapped transcription initiation sites. However, no sequences resembling the TAATGARAT motif, which confers IE regulation upon HSV-1 IE genes, were found. The finding of the absence of this motif was supported through analyses of the regulatory sequences of ORFs 4 and 63 in transient transfection assays alongside those of ORFs 61 and 62. Sequences representing the promoters for ORFs 4, 61, and 63 were all stimulated by VZV infection but failed to be stimulated by coexpression with the HSV-1 transactivator Vmw65. In contrast, the promoter for ORF 62, which contains TAATGARAT motifs, was activated by VZV infection and coexpression with Vmw65. These results extend the transcriptional knowledge for VZV and suggest that ORFs 4 and 63 contain regulatory signals different from those of the ORF 62 and HSV-1 IE genes.

Base Sequence↗
PubMed1991

Translation of a polycistronic mRNA in the presence of the cauliflower mosaic virus transactivator protein.

Polycistronic mRNAs containing an upstream beta-glucuronidase (GUS) and a downstream chloramphenicol acetyltransferase (CAT) reporter open reading frame (ORF) were expressed in transfected plant protoplasts. CAT expression could be strongly induced by coexpression of the cauliflower mosaic virus encoded translation transactivator. Transactivation was abolished when an upstream ORF overlapped the CAT ORF for a long distance. No specific sequence elements were required for transactivation but the presence of a short ORF upstream of the GUS ORF strongly enhanced the process. The inhibitory effect of additional presumed stem structures inserted into various regions of the reporter mRNAs indicates that both ORFs are translated by ribosomes that associate with the RNA at the 5' end and reach the ORFs by a linear migration mechanism.

Base Sequence↗
PubMed1991

Improved vectors for stable expression of foreign genes in mammalian cells by use of the untranslated leader sequence from EMC virus.

Dicistronic mRNA expression vectors efficiently translate a 5' open reading frame (ORF) and contain a selectable marker within the 3' end which is inefficiently translated. In these vectors, the efficiency of translation of the selectable 3' ORF is reduced approximately 100-fold and is highly dependent on the particular sequences inserted into the 5' cloning site. Upon selection for expression of the selection marker gene product, deletions within the 5' ORF occur to yield more efficient translation of the selectable marker. We have generated improved dicistronic mRNA expression vectors by utilization of a putative internal ribosomal entry site isolated from encephalomyocarditis (EMC) virus. Insertion of the EMC virus leader sequence upstream of an ORF encoding either a wildtype or methotrexate resistant dihydrofolate reductase (DHFR) reduces DHFR translation up to 10-fold in a monocistronic DHFR expression vector. However, insertion of another ORF upstream of the EMC leader to produce a dicistronic mRNA does not further reduce DHFR translation. In the presence of the EMC virus leader, DHFR translation is not dependent on sequences inserted into the 5' end of the mRNA. We demonstrate that stable high level expression of inserted cDNAs may be rapidly achieved by selection for methotrexate resistance in DHFR deficient as well as DHFR containing cells. In contrast to previously described dicistronic expression vectors, these new vectors do not undergo rearrangement or deletion upon selection for amplification by propagation in increasing concentrations of methotrexate. The explanation may be either that the EMC virus leader sequence allows internal initiation of translation or that cryptic splice sites in the EMC virus sequence mediate production of monocistronic mRNAs. These vectors may be generally useful to rapidly obtain high level expression of cDNA genes in mammalian cells.

Amino Acid Sequence↗
PubMed1992

Transcriptional analysis of the isopenicillin N synthase-encoding gene of Streptomyces clavuligerus.

The gene (pcbC) encoding isopenicillin N synthase of Streptomyces clavuligerus is separated from an upstream open reading frame (ORF) by a 31-bp intergenic region. Inspection of the sequence of this intergenic region did not identify a promoter sequence. The promoter probe plasmid, pIJ4083, which contains the promoter-less catechol-2,3-dioxygenase (C23O)-encoding gene (xylE) as a reporter gene, was used to analyze the sequence upstream from the pcbC gene for promoter activity. Introduction of an SphI site at the start codon of pcbC by site-directed mutagenesis allowed the cloning of a 335-bp fragment (-334 to +1 in relation to the pcbC start codon) immediately upstream from xylE in pIJ4083. C23O activity was detected in both Streptomyces lividans and S. clavuligerus cultures that contained the upstream fragment, suggesting the presence of a promoter sequence. Northern analysis of total RNA extracted from S. clavuligerus identified a monocistronic 1.2-kb transcript hybridizing to a pcbC-specific probe. When RNA was isolated at various times during growth in liquid culture, the presence of a transcript was first detected during stationary phase. Analysis of the pcbC transcript by primer extension located the transcription start point to a C residue within the upstream ORF, 91 bp upstream from the pcbC start codon.

Blotting, Northern↗
PubMed1999

The E2 protein of human papillomavirus type 16 is translated from a variety of differentially spliced polycistronic mRNAs.

The major regulation protein of human papillomavirus (HPV) transcription is the viral E2 protein. Previous studies have identified a variety of alternatively spliced mRNAs containing multiple open reading frames (ORFs) encoding the E2 protein of HPV type 16. In these mRNAs the E2 ORF is contained as an internal ORF. In the present study, the translational capacities of three mRNA species starting at the p97 promoter and containing the 880/2581, 880/2708 and 226/2708 splice junctions upstream of the E2 ORF were investigated. Partial cDNAs spanning the E2 ORF and the related upstream ORFs were synthesized and assessed for E2 protein translation in vivo, in COS cells, and in vitro, in cell-free systems. Results of these analyses indicated that E2 protein was translated from all three mRNAs. Translation efficiency of E2 from the natural polycistronic templates was lower compared with that from a synthetic monocistronic control. Translation from the d-type bicistronic template (226/2708) was more efficient than that from the a-type (880/2708) and a'-type (880/2561) polycistronic templates. Further investigation of the translation of proteins encoded by the ORFs preceding the E2 ORF showed that a- and a'-type templates served for translation mainly of E7 but also of E61, while the d-type template served for translation of E6IV. Overall, the translation data support the suggestion that the corresponding mRNAs may function as polycistronic transcripts.

Alternative Splicing↗
PubMed1990

Complete cDNA sequence of a South American isolate of potato virus X.

The complete cDNA sequence corresponding to the genomic RNA of a South American strain of potato virus X (PVXc) is reported. The sequence (6432 nucleotides) contains five open reading frames coding for polypeptides with molecular weights of 165.3, 24.3, 12.3, 7.6 and 25.0 and displays an overall homology of 77.4% with those previously reported for two European isolates. Comparison of amino acid sequences shows an average homology of 87%. Two major domains of variability, located between amino acids 476-615 of ORF 1 and 64-100 of ORF 5, are identified. Sequence similarities between RNA stretches lying upstream of ORFs 2, 4 and 5, and at the 3'-non coding regions of PVX and other plus-strand RNA viruses are described.

Amino Acid Sequence↗
PubMed2006

Regulation of class D beta-lactamase gene expression in Ralstonia pickettii.

Ralstonia pickettii, an environmental bacterium that may also be responsible for human infections, produces two unrelated, inducible and chromosomally encoded oxacillinases, OXA-22 and OXA-60. In order to study the molecular basis of the induction process of these oxacillinase genes, the induction kinetics, the promoter/operator regions necessary for expression and induction, and the role of several ORFs located upstream and downstream of the bla(OXA) genes were investigated. The beta-lactamase production reached a maximal level after 1 h induction, returned to its basal level within the following 3 h and was then again inducible. Using 5'RACE experiments, the promoter sequences of both oxacillinases were determined. These sequences showed weak promoter activities, which could, however, be increased approximately 200-fold by mutating the -35 promoter sequence. Deletion of the sequences located upstream of the promoter regions did not modify the basal beta-lactamase expression in R. pickettii, but resulted in the lack of induction. A minimum of 240 and 270 bp upstream of the transcription initiation sites was required for inducible expression of the bla(OXA-22) and bla(OXA-60) genes, respectively. Analysis of the genetic environment of both bla(OXA) genes revealed several ORFs that were inactivated by homologous recombination. Disruption of ORF-RP3, located 190 bp upstream of bla(OXA-60) and divergently transcribed, abolished induction of both beta-lactamases. ORF-RP3, which encoded a polypeptide of 532 aa with an estimated molecular mass of 58.7 kDa, displayed no obvious sequence homology with known regulatory proteins. Trans-complementation of ORF-RP3 restored the basal and inducible expression of both oxacillinase genes, indicating that the induction of both enzymes was related to the presence of ORF-RP3. In addition to the loss of induction, inactivation of the ORF-RP3 in R. pickettii resulted in a complex pleiotropic phenotype, with increased lag phase and reduced survival after heat exposure, suggesting that ORF-RP3 might be a global regulator involved in unrelated regulatory pathways.

Amino Acid Sequence↗
PubMed1999

Intramolecular transposition of insertion sequence IS91 results in second-site simple insertions.

A series of plasmids carrying an IRL-kan-IRR transposable cassette, in which IRL and IRR are the left- and right-terminal sequences of IS91, have been constructed. These cassettes could be complemented for transposition with similar efficiency when IS91 transposase was provided either in cis or in trans. A total of 87% of IS91 transposition products were simple insertions of the element, while the remaining 13% were plasmid fusions and co-integrates. When transposase expression was induced from an upstream lac promoter, transposition frequency increased approximately 100-fold. An open reading frame (ORF) present upstream of the transposase gene, ORF121, could be involved in target selection, as mutations affecting this ORF were altered in their insertion specificity. Intramolecular rearrangements were analysed by looking at transposition events disrupting a chloramphenicol resistance gene (cat ) located outside the transposable cassette. Plasmid instability resulting from insertion of an extra copy of IRL-kan-IRR within the cat gene was observed; transposition products contained a second copy of the cassette inserted either as a direct or as an inverted repeat. No deletion or inversion of the intervening DNA was observed. These results could be explained as a consequence of intramolecular transposition of IS91 according to a model of rolling-circle transposition.

Chloramphenicol Resistance↗
PubMed1995

An archaeal gene upstream of grpE different from eubacterial counterparts.

In some eubacteria with a dnaK locus in which grpE is close upstream of dnaK, grpE is preceded by an open reading frame (orf) believed to be a heat-shock gene. We also found an orf, orf16, upstream of grpE in the archaeon Methanosarcina mazei S-6, but this gene differs from the eubacterial counterpart: it is shorter, does not respond to a temperature upshift as heat-shock genes do, and the deduced protein Orf16, does not resemble the proteins coded by the eubacterial equivalents. orf16 is expressed monocistronically, with a transcription initiation site 24 bases upstream of the translation start codon, 22 bases downstream of a putative promoter identical to the consensus promoter for genes in methanogens. This initiation site is used by heat-shocked and non-heat-shocked cells in the two morphologic stages of M. mazei S-6 tested, i.e., packets and single cells. Three transcription termination sites were identified, one of which is detectable only in non-heat-shocked cells. Data from comparative analyses of the Orf16 deduced amino acid sequence and those of other known proteins, as well as the apparent biochemical characteristics of Orf16, suggest that the latter is a membrane molecule.

Amino Acid Sequence↗
PubMed1989

Maize streak virus genes essential for systemic spread and symptom development.

The entire genome of single component geminiviruses such as maize streak virus (MSV) consists of a single-stranded circular DNA of ~2.7 kb. Although this size is sufficient to encode only three average sized proteins, the virus is capable of causing severe disease of many monocots with symptoms of chlorosis and stunting. We have identified viral gene functions essential for systemic spread and symptom development during MSV infection. Deletions and gene replacement mutants were created by site-directed mutagenesis and insertion between flanking MSV or reporter gene sequences contained in Agrobacterium T-DNA derived vectors. Following Agrobacterium-mediated inoculation of maize seedlings, the mutated MSV DNAs were excised from these binary vectors by homologous recombination within the flanking sequences. Our analyses show that the capsid gene of MSV, while not required for replication, is essential for systemic spread and subsequent disease development. The ;+' strand open reading frame (ORF) located immediately upstream from the capsid ORF and predicted to encode a 10.9 kd protein was also found to be dispensable for replication but essential for systemic spread. By this analysis, MSV sequences that support autonomous replication were localized to a 1.7 kb segment containing the two viral intergenic regions and two overlapping complementary ;-' strand ORFs. Despite the inability of the gene replacement mutants to spread systemically, both inoculated and newly developed leaves displayed chlorotic patterns similar to the phenotype observed in certain developmental mutants of maize. The similarity of the MSV mutant phenotype to these developmental mutants is discussed.

Journal Article↗
PubMed1993

Cloning and sequencing of a gene encoding acidophilic amylase from Bacillus acidocaldarius.

Two starch-degrading enzymes produced by Bacillus acidocaldarius (renamed as Alicyclobacillus acidocaldarius) were identified. According to SDS-PAGE, the apparent molecular masses of the enzymes were 90 and 160 kDa. Eight peptide fragments and the N-terminal end of the 90 kDa polypeptide were sequenced. An oligonucleotide, based on the amino acid sequence of a peptide fragment of the 90 kDa protein, was used to screen a lambda gt10 bank of B. acidocaldarius, and the region encoding the 90 kDa protein was cloned. Unexpectedly, the ORF continued upstream of the N terminus of the 90 kDa protein. The entire ORF was 1301 amino acids (aa) long (calculated molecular mass 140 kDa) and it was preceded by a putative ribosomal binding site and a promoter. Computer analysis showed that the 1301 aa protein was closely related to an alpha-amylase-pullulanase of Clostridium thermohydrosulfuricum. We suggest that the starch-degrading 160 kDa protein of B. acidocaldarius is an alpha-amylase-pullulanase, and the 90 kDa protein is a cleavage product of the 160 kDa protein. Another ORF, apparently in the same transcription unit, was found downstream from the amylase gene. It encoded a protein that was closely related to the maltose-binding protein of Escherichia coli.

ATP-Binding Cassette Transporters↗
PubMed1989

Development of a DNA probe from the deoxyribonucleotide sequence of a 3-N-aminoglycoside acetyltransferase [AAC(3)-I] resistance gene.

The aacC1 gene encoding the 3-N-aminoglycoside acetyltransferase [AAC(3)-I] was cloned from enteric plasmid pJR88, and its deoxyribonucleotide sequence was determined. Significant nucleotide homology was noted in the region extending from the proposed -35 sequences through the first 59 base pairs of the aacC1 gene open reading frame (ORF) and the upstream flanking regions and ORFs of several other antibiotic resistance genes. Sequences were noted to be homologous with the 6'-N-aminoglycoside acetyltransferase [AAC(6')-I], 2''-O-aminoglycoside adenylyltransferase [AAD(2'')], and 3''-O-aminoglycoside adenylyltransferase [AAD(3'')] resistance genes; the OXA-1, OXA-2, and PSE-2 beta-lactamase genes; and several dihydrofolate reductase genes. Small regions of homology were noted in the 3'-flanking regions of these resistance genes as well. A DNA probe for the aacC1 gene was selected from the nucleotide sequence information and was tested against a series of genetically and enzymatically defined strains. The probe, which proved specific for the aacC1 gene, was then tested against a series of 58 gentamicin-susceptible and 219 gentamicin-resistant gram-negative bacilli isolated from patients at the Seattle Veterans Administration Medical Center. Only six clinical isolates were noted to carry the aacC1 gene. Each was resistant to gentamicin but susceptible to kanamycin, tobramycin, and amikacin. The presence of homologous regions of DNA at both the 3' and 5' ends of the aacC1 gene reinforces the importance of choosing probes from within the ORFs of genes and of avoiding flanking sequences. When the homology with other sequences extends into the ORF, as it does with the aacC1 gene, development of a specific probe may require determination of the nucleotide sequence.

Acetyltransferases↗
PubMed1997

Molecular characterization and cellular localization of TpLRR, a processed leucine-rich repeat protein of Treponema pallidum, the syphilis spirochete.

Automated Edman degradation was used to obtain N-terminal and internal amino acid sequences from a 26-kDa protein in isolated Treponema pallidum outer membranes (OMs). The resulting sequences enabled us to PCR amplify from T. pallidum DNA a 275-bp fragment of the corresponding gene. The complete nucleotide sequence of the gene was determined from fragments amplified by long-distance PCR. Primer extension verified the assigned translational start of the open reading frame (ORF) and putative upstream promoter elements. The ORF encoded a highly basic (pI 9.6) 26-kDa protein which contained an N-terminal 25-amino-acid leader peptide terminated by a signal peptidase I cleavage site. The mature protein contained seven tandemly spaced copies (as well as an eighth incomplete copy) of a leucine-rich repeat (LRR), a motif previously identified in a number of prokaryotic and eukaryotic proteins. Accordingly, the polypeptide was designated T. pallidum leucine-rich repeat protein (TpLRR). Although Triton X-114 phase partitioning showed that TpLRR was hydrophilic, cell localization studies showed that most of the antigen was associated with the peptidoglycan-cytoplasmic membrane complex rather than being freely soluble in the periplasmic space. Immunoblot studies showed that syphilis patients develop a weak antibody response to the antigen. Lastly, the lrr(T. pallidum) gene was mapped to a 60-kb SfiI-SpeI fragment of the T. pallidum chromosome which also contains the rrnA and flaA genes. The function(s) of TpLRR is currently unknown; however, protein-protein and/or protein-lipid interactions mediated by its LRR motifs may facilitate interactions between components of the T. pallidum cell envelope.

Amino Acid Sequence↗