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PubMed1995

The varicella-zoster virus (VZV) open reading frame 47 (ORF47) protein kinase is dispensable for viral replication and is not required for phosphorylation of ORF63 protein, the VZV homolog of herpes simplex virus ICP22.

To investigate the role of varicella-zoster virus (VZV) open reading frame 47 (ORF47) protein kinase during infection, a VZV mutant was generated in which two contiguous stop codons were introduced into ORF47, thus eliminating expression of the ORF47 kinase. ORF47 kinase was not essential for the growth of VZV in cultured cells, and the growth rate of the VZV mutant lacking ORF47 protein was indistinguishable from that of parental VZV. Nuclear extracts from cells infected with parental VZV contained several phosphorylated proteins which were not detected in extracts from cells infected with the ORF47 mutant. The herpes simplex virus type 1 (HSV-1) UL13 protein (the homolog of VZV ORF47 protein) is responsible for the posttranslational processing associated with phosphorylation of HSV-1 ICP22 (the homolog of VZV ORF63 protein). Immunoprecipitation of 32P-labeled proteins from cells infected with parental virus and those infected with ORF47 mutant virus yielded similar amounts of the VZV phosphoproteins encoded by ORF4, ORF62, ORF63, and ORF68 (VZV gE), and the electrophoretic migration of these proteins was not affected by the lack of ORF47 kinase. Therefore, while the VZV ORF47 protein is capable of phosphorylating several cellular or viral proteins, it is not required for phosphorylation of the ORF63 protein in virus-infected cells.

Cell Line↗

PubMed2006

Development of a rapid strain differentiation method for methicillin-resistant Staphylococcus aureus isolated in Japan by detecting phage-derived open-reading frames.

AIMS: To develop a rapid genotyping method for investigating outbreaks of methicillin-resistant strains of Staphylococcus aureus (MRSA) isolated in Japan. METHODS AND RESULTS: Isolates were genotyped by detecting the keeping pattern of 16 open-reading frames (ORFs), a process we call phage ORF typing (POT). Thirteen of the ORFs were selected from phage genomes and one from a genomic island SaGIm in the genome of strain Mu50. The other two ORFs, one from Tn554 and one from staphylococcal cassette chromosome mec (SCCmec) type II, were used as strain markers. Three hundred and sixty-eight isolates from five hospitals were classified into 133 types by POT, whereas they were classified into 139 types by pulsed-field gel electrophoresis (PFGE) subtyping. The discriminatory power of POT (D=0.989) was equal to that of PFGE subtyping (D=0.986). CONCLUSIONS: MRSA isolates collected in Japan can be genotyped by detecting the keeping pattern of phage-derived ORFs with a discriminatory power equal to that of PFGE subtyping. SIGNIFICANCE AND IMPACT OF THE STUDY: MRSA isolates can be genotyped rapidly by detecting phage-derived ORFs. As particular pandemic clones can be found in a specific region, a typing method localized to a pandemic clone may be effective for the rapid genotyping of MRSA during outbreaks.

Bacterial Typing Techniques↗

PubMed1992

The complete sequence of a 10.8 kb segment distal of SUF2 on the right arm of chromosome III from Saccharomyces cerevisiae reveals seven open reading frames including the RVS161, ADP1 and PGK genes.

We have entirely sequenced a 10,835 bp segment of the right arm from chromosome III contained in the J11D and J11D-K3B GF clones. The segment contains seven open reading frames longer then 100 amino acids. Three of them, RVS161 (Urdaci et al., 1990; Crouzet et al., 1991), ADP1 (Purnelle et al., 1991) and PGK1 (Hitzeman et al., 1982) have been described previously. YCR10C encodes a putative membrane protein. YCR8W (encoding a putative protein kinase) and YCR14C extend inside the D10H (Skala et al., 1991) and 62B5-2D clones respectively. Four ARS elements previously reported by Palzkill et al. (1986) are located between RVS161 and YCR10C.

Amino Acid Sequence↗

PubMed2000

In vitro and in vivo characterization of a murine cytomegalovirus with a transposon insertional mutation at open reading frame M43.

We have recently generated a pool of murine cytomegalovirus (MCMV) mutants by using a Tn3-based transposon mutagenesis approach. In this study, one of the MCMV mutants, RvM43, which contained the transposon inserted in open reading frame M43, was characterized. Our results provide the first direct evidence to suggest that M43 is not essential for viral replication in vitro in NIH 3T3 cells. Moreover, RvM43 exhibited a titer similar to that of the wild-type virus in the lungs, livers, spleens, and kidneys of both BALB/c and SCID mice and was as virulent as the wild-type virus in killing SCID mice that had been intraperitoneally infected with the viruses. In contrast, titers of the mutant virus in the salivary glands of the infected animals at 21 days postinfection were significantly (100 to 1,000-fold) lower than those of the wild-type virus and a rescued virus that restored the M43 region and its expression. Thus, M43 appears to be not essential for viral growth in vivo in the lungs, livers, spleens, and kidneys of infected animals and is also dispensable for virulence in killing SCID mice. Moreover, our results suggest that M43 is an MCMV determinant for growth in the salivary glands. Studies of viral genes required for replication in the salivary glands are important in understanding the mechanism of viral tropism for the salivary glands and shedding in saliva, which is believed to be one of the major routes of CMV transmission among healthy human populations.

3T3 Cells↗

PubMed1996

The sequence of a 16,691 bp segment of Saccharomyces cerevisiae chromosome IV identifies the DUN1, PMT1, PMT5, SRP14 and DPR1 genes, and five new open reading frames.

As part of the European BIOTECH programme, the nucleotide sequence of a 16691 bp fragment from the left arm of chromosome IV of Saccharomyces cerevisiae has been deduced. Analysis of the sequence reveals the presence of 13 open reading frames (ORFs) larger than 100 codons. five of these were previously identified as genes DUN1, PMT1, PMT5, SRP14 and DPR1. One putative protein, D2371p, contains an ATP-GTP binding site, and shares homology to the ArsA component of an Escherichia coli arsenical pump. No significant homology to any known protein has been found for the other ORFs. D2378p contains a zinc finger domain.

Amino Acid Sequence↗

PubMed2000

Construction and characterization of murine cytomegaloviruses that contain transposon insertions at open reading frames m09 and M83.

A transposon derived from Escherichia coli Tn3 was introduced into the genome of murine cytomegalovirus (MCMV) to generate a pool of viral mutants, including two recombinant viruses that contained the transposon sequence within open reading frames m09 and M83. Our studies provide the first direct evidence to suggest that m09 is not essential for viral replication in mouse NIH 3T3 cells. Studies in cultured cells and in both BALB/c-Byj and CB17 severe combined immunodeficient (SCID) mice indicated that the transposon insertion is stable during viral propagation both in vitro and in vivo. Moreover, the virus that contained the insertion mutation in m09 exhibited a titer similar to that of the wild-type virus in the salivary glands, lungs, livers, spleens, and kidneys of both the BALB/c and SCID mice and was as virulent as the wild-type virus in killing the SCID mice when these animals were intraperitoneally infected with these viruses. These results suggest that m09 is dispensable for viral growth in these organs and that the presence of the transposon sequence in the viral genome does not significantly affect viral replication in vivo. In contrast, the virus that contained the insertion mutation in M83 exhibited a titer of at least 60-fold lower than that of the wild-type virus in the organs of the SCID mice and was attenuated in killing the SCID mice. These results demonstrate the utility of using the Tn3-based system as a mutagenesis approach for studying the function of MCMV genes in both immunocompetent and immunodeficient animals.

Animals↗

PubMed1998

Expression of foreign proteins by poliovirus polyprotein fusion: analysis of genetic stability reveals rapid deletions and formation of cardioviruslike open reading frames.

Using a strategy developed by R. Andino, D. Silvera, S. D. Suggett, P. I. Achacoso, C. J. Miller, D. Baltimore, and M. B. Feinberg (Science 265:1448-1451, 1994), we constructed recombinant polioviruses by fusing the open reading frame (ORF) of the green fluorescent protein gene (gfp) of Aequorea victoria or the gag gene (encoding p17-p24) of human immunodeficiency virus type 1 (HIV-1) to the N terminus of the poliovirus polyprotein. All poliovirus expression vectors constructed by us and those obtained from Andino et al. were found to be severely impaired in viral replication and genetically unstable. Upon replication, inserted sequences were rapidly deleted as early as the first growth cycle in HeLa cells. However, the vector viruses did not readily revert to the wild-type sequence but rather retained some of the insert plus the artificial 3Cpro/3CDpro cleavage site, engineered between the heterologous sequence and the poliovirus polyprotein, to give rise to genotypes reminiscent of cardioviruses. These virus variants that carry a small leader polypeptide were now relatively stable, and they grew better than their progenitor strains. Reverse transcription followed by PCR and sequence analysis of the genomic RNAs reproducibly revealed a few preferred genotypes among the isolated deletion variants. The remaining truncated inserts were retained through subsequent passages. In the immediate vicinity of the deletion borders, we observed short direct sequence repeats that we propose are involved in aligning RNA strands for illegitimate (nonhomologous) RNA recombination during minus-strand synthesis. On the basis of our results, which are at variance with published data, the utility of poliovirus vectors to express proteins > 10 kDa in size through fusion with the polyprotein needs to be reevaluated.

Amino Acid Sequence↗

PubMed1997

Open reading frame 3, which is adjacent to the mycocerosic acid synthase gene, is expressed as an acyl coenzyme A synthase in Mycobacterium bovis BCG.

The aim of this study was to test for expression of a 900-bp open reading frame (ORF), ORF3, located at the 5' end of the mycocerosic acid synthase gene in Mycobacterium bovis BCG and to determine the nature of the ORF3 protein. ORF3 was expressed as a 61-kDa C-terminal fusion protein with glutathione S-transferase in Escherichia coli. Polyclonal rabbit antiserum, prepared against this fusion protein, cross-reacted with a 65-kDa protein in M. bovis BCG crude extracts. Since this protein was larger than that predicted from the nucleotide sequence (32 kDa), ORF3 was resequenced, revealing an ORF of 1,749 bp that encodes a 64.8-kDa protein containing 583 amino acids. Reverse transcription-PCR revealed that ORF3 is expressed in M. bovis BCG. The ORF3 product has a high degree of similarity to the acyladenylate family of enzymes. Immunoaffinity absorption chromatography was used to isolate the 65-kDa cross-reacting protein from M. bovis BCG. This purified protein catalyzed coenzyme A (CoA) ester synthesis of n-C10 to n-C18 fatty acids but not mycocerosic acids. ORF3 antibodies severely inhibited acyl-CoA synthase activities of the purified protein and extracts of M. bovis BCG, Mycobacterium smegmatis, and E. coli. They also showed immunological cross-reactivity with proteins in these extracts. Both the ORF3 protein and the acyl-CoA synthase activity were located in the cell cytosol or were loosely associated with the cell membrane. These results indicate that ORF3 encodes an acyl-CoA synthase-like protein.

Acyltransferases↗

PubMed2000

Prediction of a novel RNA 2'-O-ribose methyltransferase subfamily encoded by the Escherichia coli YgdE open reading frame and its orthologs.

The amino acid sequence of the RNA 2'-O-ribose methyltranserase RrmJ was used as a probe for detecting putative homologs through iterative searches of genomic databases. We found a previously unannotated YgdE open reading frame (ORF) in the genome sequences of Escherichia coli and other gamma-Proteobacteria, which shares key features with RrmJ, despite the mutual sequence similarity of these proteins is relatively low. The predicted structural compatibility and the conservation of all functionally important residues between RrmJ and YgdE strongly suggests that the newly identified methyltranserase also modifies 2'-OH groups of ribose. The N-terminal region of YgdE, which has no counterpart in RrmJ, is predicted to form an independent domain, possibly involved in target recognition.

Amino Acid Sequence↗

PubMedDate not supplied

An open reading frame downstream of Rhizobium meliloti nodQ1 shows nucleotide sequence similarity to an Agrobacterium tumefaciens insertion sequence.

We sequenced a small uncharacterized region in the Rhizobium meliloti nod gene cluster downstream of nodQ1. We found the beginning of a large open reading frame (260 amino acids) in this fragment. The sequence reported here has striking similarity to IS66 (Y. Machida, M. Sakurai, S. Kiyokawa, A. Ubasawa, and S. Yasushiro, 1984, Proc. Natl. Acad. Sci. USA 81:7495-7499), an insertion element found in an Agrobacterium tumefaciens mutant.

Agrobacterium tumefaciens↗

PubMed2000

Efficient homologous RNA recombination and requirement for an open reading frame during replication of equine arteritis virus defective interfering RNAs.

Equine arteritis virus (EAV), the prototype arterivirus, is an enveloped plus-strand RNA virus with a genome of approximately 13 kb. Based on similarities in genome organization and protein expression, the arteriviruses have recently been grouped together with the coronaviruses and toroviruses in the newly established order Nidovirales. Previously, we reported the construction of pEDI, a full-length cDNA copy of EAV DI-b, a natural defective interfering (DI) RNA of 5.6 kb (R. Molenkamp et al., J. Virol. 74:3156-3165, 2000). EDI RNA consists of three noncontiguous parts of the EAV genome fused in frame with respect to the replicase gene. As a result, EDI RNA contains a truncated replicase open reading frame (EDI-ORF) and encodes a truncated replicase polyprotein. Since some coronavirus DI RNAs require the presence of an ORF for their efficient propagation, we have analyzed the importance of the EDI-ORF in EDI RNA replication. The EDI-ORF was disrupted at different positions by the introduction of frameshift mutations. These were found either to block DI RNA replication completely or to be removed within one virus passage, probably due to homologous recombination with the helper virus genome. Using recombination assays based on EDI RNA and full-length EAV genomes containing specific mutations, the rates of homologous RNA recombination in the 3'- and 5'-proximal regions of the EAV genome were studied. Remarkably, the recombination frequency in the 5'-proximal region was found to be approximately 100-fold lower than that in the 3'-proximal part of the genome.

Animals↗

PubMed1997

An 18.3 kb DNA fragment from yeast chromosome VII carries four unknown open reading frames, the gene for an Asn synthase, remnants of Ty and three tRNA genes.

An 18.3 kb DNA segment from yeast Saccharomyces cerevisiae VII encompasses the previously characterized MEP1, NUP57 and PPT1 genes as well as seven new open reading frames (ORFs) of at least 100 residues. G6358 is an ubiquitous glutamine-dependent asparagine synthase. G6362 is membrane protein highly homologous to a protein of unknown function in the yeast Schizosaccharomyces pombe. Three ORFs (G6324, G6335 and G6365) have no significant homology with previously reported proteins of characteristic motifs. G6321 and G6359, enclosed in longer ORFs, are not likely to be coding. The segment also contains tRNA genes for Asn, Arg and Ile as well as sigma element and two solo deltas. ORFs and genetic elements are named according to a preliminary working nomenclature.

Arabidopsis Proteins↗

PubMed2000

Improved identification and differentiation of varicella-zoster virus (VZV) wild-type strains and an attenuated varicella vaccine strain using a VZV open reading frame 62-based PCR.

A new method was developed to identify and differentiate varicella-zoster virus (VZV) wild-type strains from the attenuated varicella Oka vaccine strain. The PCR technique was used to amplify a VZV open reading frame (ORF) 62 region. A single specific amplicon of 268 bp was obtained from 71 VZV clinical isolates and several laboratory strains. Subsequent digestion of the VZV ORF 62 amplicons with SmaI enabled accurate strain differentiation (three SmaI sites were present in amplicons of vaccine strain VZV, compared with two enzyme cleavage sites for all other VZV strains tested). This method accurately differentiated the Oka vaccine strain from wild-type VZV strains circulating in countries representing all six populated continents. Moreover, the assay more reliably distinguished wild-type Japanese strains from the vaccine strain than did previously described methods.

Chickenpox↗

PubMed1991

The sequence of a 6.3 kb segment of yeast chromosome III reveals an open reading frame coding for a putative mismatch binding protein.

We report the sequence of a 6.3 kb segment of DNA mapping near the end of the right arm of chromosome III of Saccharomyces cerevisiae. The sequence reveals a major open reading frame coding for a putative protein of 1047 amino acids with a striking similarity to the bacterial proteins involved in recognition of mismatched DNA base pairs. This is particularly interesting as the existence of a yeast mismatch repair system similar to that of bacteria has been postulated for some years, but a yeast protein homologous to the bacterial mismatch binding protein had not been identified. The results of a comparison of the putative yeast mismatch binding protein with the bacterial mismatch binding proteins and with two cognate mammalian sequences, support the idea that a similar mismatch repair system may be present also in mammalian cells. The possibility that all of these proteins may have evolved from a common ancestral gene is also discussed.

Amino Acid Sequence↗

PubMed1995

Sequences 5' of the first upstream open reading frame in GCN4 mRNA are required for efficient translational reinitiation.

Translation of yeast GCN4 mRNA occurs by a reinitiation mechanism that is modulated by amino acid levels in the cell. Ribosomes which translate the first of four upstream open reading frames (uORFs) in the mRNA leader resume scanning and can reinitiate downstream. Under non-starvation conditions reinitiation occurs at one of the remaining three uORFs and GCN4 is repressed. Under starvation conditions, in contrast, ribosomes bypass the uORFs and reinitiate at GCN4 instead. The high frequency of reinitiation following uORF1 translation depends on an adequate distance to the next start codon and particular sequences surrounding the uORF1 stop codon. We present evidence that sequences 5' to uORF1 also strongly enhance reinitiation. First, reinitiation was severely inhibited when uORF1 was transplanted into the position of uORF4, even though the native sequence environment of the uORF1 stop codon was maintained, and this effect could not be accounted for by the decreased uORF1-GCN4 spacing. Second, insertions and deletions in the leader preceding uORF1 greatly reduced reinitiation at GCN4. Sequences 5' to uORF1 may influence the probability of ribosome release following peptide termination at uORF1. Alternatively, they may facilitate rebinding of an initiation factor required for reinitiation prior to resumption of the scanning process.

Alleles↗

PubMed1994

Translation start site multiplicity of the CCAAT/enhancer binding protein alpha mRNA is dictated by a small 5' open reading frame.

The CCAAT/enhancer binding proteins (C/EBP) alpha and beta of the bZIP family of transcription factors each occur as multiple forms due to translation initiation at different in-frame AUG codons from the same messenger RNA. The C/EBP alpha mRNAs of chicken, rat and Xenopus all contain a small 5' open reading frame (5'ORF) whose size (18 nucleotides) and distance (seven nucleotides) to the C/EBP alpha cistron has been conserved in vertebrate evolution. The present studies shows that the small 5'ORF is crucial to the leaky scanning mechanism of ribosomes causing a fraction of them to ignore the first C/EBP alpha AUG codon and to start at internal AUGs. Our data challenge the view that translational start site multiplicity is mainly governed by the sequence context of the potential initiation codons. Western analysis showed that the two major chicken C/EBP alpha translation products, the full-length cC/EBP alpha-42 which acts a trans-activator in liver and the N-terminally truncated cC/EBP alpha-29 which lacks transcription activation potential, occur in a fixed ratio which is similar in different expressing tissues, like liver, lung and small intestine. The presence of a similar, thusfar unnoticed, small ORF 5' to the major initiation codon of C/EBP beta mRNA suggests that start site multiplicity from this mRNA may be governed by the same mechanism.

Animals↗

PubMed1996

The DNA sequence of cosmid 14-5 from chromosome XIV reveals 21 open reading frames including a novel gene encoding a globin-like domain.

In this paper is described the DNA sequence of cosmid 14-5 from chromosome XIV of Saccharomyces cerevisiae. The sequence is 38 855 bases long and contains 21 open reading frames (ORFs) plus three internal ORFs. Six ORFs correspond to known yeast genes (SLA2, ZWF1, BLH1, KEX2, SIN4 and URE2); two other ORFs had already been sequenced because they are adjacent to known genes; the remaining 12 ORFs are novel genes. Of these, one ORF (NII42) is particularly interesting since it shows a significant similarity to mammalian globin. Another ORF (N1254) displays two zinc finger motifs as well as a DNAJ motif.

Animals↗

PubMed1996

Long RT-PCR of the entire 8.5-kb NF1 open reading frame and mutation detection on agarose gels.

Previous approaches to mutation detection in mRNA from the neurofibromatosis 1 (NF1) locus have required the PCR amplification of five or more overlapping cDNA segments to screen the entire 8.5-kb open reading frame (ORF). Systematically, these assays do not detect deletions that span the region of overlap (usually 1-3 exons) of any two consecutive target segments. In such cases, amplification from the mutant region of the disease-causing allele fails because binding sites for the PCR primers are missing, but amplification from the normal allele proceeds, yielding only the normal product. To alleviate this problem, we have developed a protocol to reverse transcribe and amplify the entire protein-coding sequence of NF1 as a single PCR product, starting with total RNA from lymphoblast cell lines or from whole blood. The 8.7-kb RT-PCR product was prepared from nine NF1 patients with known deletions or insertions, ranging in size from a 30-bp deletion within 1 exon to a 2.4-kb deletion that removes 12 exons. Agarose gel analysis of the initial products detected deletions as small as 341 bp. Restriction endonuclease digestion with Asel and Fspl, followed by agarose gel electrophoresis, revealed the predicted abnormal bands in all nine patients. All mutant bands were identified readily by observers with no knowledge of the patients' mutations. This simple assay should detect a great variety of insertion/deletion mutations in the NF1mRNA internal to the primer binding sites, including all possible single and multiple exon dropouts and approximately 30% of all previously reported NF1 mutations.

Base Sequence↗