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PubMed2006

Identification of the cleavage sites of sapovirus open reading frame 1 polyprotein.

Sapovirus (SaV), a member of the family Caliciviridae, is a causative agent of acute gastroenteritis in humans and swine and is currently divided into five genogroups, GI-GV. The proteolytic processing of the SaV open reading frame 1 (ORF1) polyprotein with a human GII SaV Mc10 strain has recently been determined and the products are arranged in the following order: NH(2)-p11-p28-p35 (NTPase)-p32-p14 (VPg)-p70 (Pro-Pol)-p60 (VP1)-COOH. The cleavage site between p14 (VPg) and p70 (Pro-Pol) was identified as E(1055)/A(1056) by N-terminal amino acid sequencing. To identify other cleavage sites, a series of GII SaV Mc10 full-length clones containing disrupted potential cleavage sites in the ORF1 polyprotein were constructed and used to generate linear DNA templates for in vitro coupled transcription-translation. The translation products were analysed by SDS-PAGE or by immunoprecipitation with region-specific antibodies. N-terminal amino acid sequencing with Escherichia coli-expressed recombinant proteins was also used to identify the cleavage site between p32 and p14. These approaches enabled identification of the six cleavage sites of the Mc10 ORF1 polyprotein as E(69)/G(70), Q(325)/G(326), Q(666)/G(667), E(940)/A(941), E(1055)/A(1056) and E(1722)/G(1723). The alignment of the SaV full-length ORF1 amino acid sequences indicated that the dipeptides used for the cleavage sites were either E or Q at the P1 position and A, G or S at the P1' position, which were conserved in the GI, GII, GIII, GIV and GV SaV ORF1 polyprotein.

Binding Sites↗

PubMed2002

Varicella-zoster virus open reading frame 2 encodes a membrane phosphoprotein that is dispensable for viral replication and for establishment of latency.

Varicella-zoster virus (VZV) encodes six genes that do not have homologs in herpes simplex virus. One of these genes, VZV open reading frame 2 (ORF2), was expressed as a 31-kDa phosphoprotein in the membranes of infected cells. Unlike equine and bovine herpesvirus type 1 ORF2 homologs that are associated with virions, VZV virions contained no detectable ORF2 protein. The ORF2 deletion mutant established a latent infection in cotton rats at a frequency and with a number of VZV genomes similar to that of the parental virus. ORF63 transcripts, a hallmark of latent infection, were present in ganglia latently infected with both the ORF2 deletion mutant and parental VZV. Thus, ORF2 is the first VZV gene shown to be dispensable for establishment of latent infection in an animal model.

Animals↗

PubMed2006

Evolutionary changes in the fungal carbamoyl-phosphate synthetase small subunit gene and its associated upstream open reading frame.

The Neurospora crassa arg-2 and the Saccharomyces cerevisiae ortholog CPA1 encode the arginine-specific carbamoyl-phosphate synthetase (CPS-A) small subunit. Arginine decreases synthesis of this subunit through the action of a 5' upstream open reading frame in the mRNA that encodes a cis-regulatory element, the arginine attenuator peptide (AAP), which stalls ribosomes in response to arginine. We performed a comparative analysis of the genomic structure and predicted peptide sequence of the AAP and CPS-A small subunit across many fungi. Differences at the genomic level included variation in intron number and position within the AAP and CPS-A coding regions and differences in known regulatory motifs. Although differences exist in AAP sequence, there were three absolutely conserved amino acid residues in the predicted peptide, including an aspartic acid crucial for arginine-dependent regulation of arg-2 and CPA1. A diverged Basidiomycete AAP was shown to retain function as an Arg-specific negative regulator of translation.

Amino Acid Motifs↗

PubMed2005

The open reading frame 3 gene of hepatitis E virus contains a cis-reactive element and encodes a protein required for infection of macaques.

An infectious cDNA clone of hepatitis E virus was mutated in order to prevent synthesis of either open reading frame 2 (ORF2) protein or ORF3 protein. HuH-7 cells transfected with an ORF2-null mutant produced ORF3, and those transfected with an ORF3-null mutant produced ORF2. Silent mutations introduced into a highly conserved nucleotide sequence in the ORF3 coding region eliminated the synthesis of both ORF2 and ORF3 proteins, suggesting that it comprised a cis-reactive element. A mutant that was not able to produce ORF3 protein did not produce a detectable infection in rhesus macaques. However, a mutant that encoded an ORF3 protein lacking a phosphorylation site reported to be critical for function was able to replicate its genome in cell culture and to induce viremia and seroconversion in rhesus monkeys, suggesting that phosphorylation of ORF3 protein was not necessary for genome replication or for production of infectious virions.

Amino Acid Sequence↗

PubMed1995

Analysis of a 42.5 kb DNA sequence of chromosome X reveals three tRNA genes and 14 new open reading frames including a gene most probably belonging to the family of ubiquitin-protein ligases.

We have sequenced a 42,500 bp stretch located on chromosome X of Saccharomyces cerevisiae between the genes MET3 and CDC8. This stretch contains 24 open reading frames (ORFs) of at least 100 amino acids. Ten of these correspond to previously published sequences, whereas of the 14 remaining ORFs, only one, GTD892, has significant similarity to proteins from yeast or other organisms. It may belong to the family of ubiquitin-protein ligases and be involved in the ubiquitin-dependent proteolytic pathway. In addition, three tRNA genes were recognized, two of which had not been hitherto localized.

Amino Acid Sequence↗

PubMed1995

The DNA sequence of a 7941 bp fragment of the left arm of chromosome VII of Saccharomyces cerevisiae contains four open reading frames including the multicopy suppressor gene of the pop2 mutation and a putative serine/threonine protein kinase gene.

We report the sequence of a 7941 bp DNA fragment from the left arm of chromosome VII of Saccharomyces cerevisiae which contains four open reading frames (ORFs) of greater than 100 amino acid residues. ORF biC834 shows 100% bp identity with the recently identified multicopy suppressor gene of the pop2 mutation (MPT5); its deduced protein product carries an eight-repeat domain region, homologous to that found in the hypothetical regulatory YGL023 protein of S. cerevisiae and the Pumilio protein of Drosophila. ORF biE560 protein exhibits patterns typical of serine/threonine protein kinases, with which it shares high degrees of homology.

Amino Acid Sequence↗

PubMed2000

Distribution of open reading frames of plasticity region of strain J99 in Helicobacter pylori strains isolated from gastric carcinoma and gastritis patients in Costa Rica.

The plasticity region of Helicobacter pylori strain J99 is a large chromosomal segment containing 33 strain-specific open reading frames (ORFs) with characteristics of a pathogenicity island. To study the diversity of the plasticity region, 22 probes corresponding to 20 ORFs inside the plasticity region and two ORFs on its boundaries were hybridized to genomic DNA isolated from clinical strains of H. pylori from patients with gastritis or gastric adenocarcinoma. Highly variable hybridization patterns were observed. The majority of the clinical strains presented a hybridization profile similar to that of J99; thus, these ORFs are not J99 strain specific. No association was found between a particular hybridization pattern and the clinical origin of the strain. Nevertheless, two single ORFs (JHP940 and JHP947) were more likely to be found in gastric cancer strains. They may be new pathogenicity markers. An in vitro expression study of these ORFs was also performed for the J99 strain, under different conditions. Thirteen ORFs were consistently expressed, six were consistently shut off, and three were expressed differentially. Most of the constitutionally expressed genes were located on the 3' part of the plasticity region. Our results show that the plasticity region, rather than being considered a pathogenicity island per se, should be considered a genomic island, which represents a large fragment of foreign DNA integrated into the genome and not necessarily implicated in the pathogenic capacity of the strain.

Blotting, Southern↗

PubMed2002

Lack of canonical E6 and E7 open reading frames in bird papillomaviruses: Fringilla coelebs papillomavirus and Psittacus erithacus timneh papillomavirus.

Determination and analyses of the complete sequence of Fringilla coelebs papillomavirus and Psittacus erithacus timneh papillomavirus indicate that they represent a distinct and distant lineage of papillomaviruses. The lack of canonical E6-E7 open reading frames suggests that they serve adaptive functions during papillomavirus evolution.

Amino Acid Sequence↗

PubMed1992

Cobalamin (vitamin B12) repression of the Cob operon in Salmonella typhimurium requires sequences within the leader and the first translated open reading frame.

Expression of the Cob operon in Salmonella typhimurium is repressed by cobalamin (Cbl). Here it is shown that Cbl repression is mediated by a post-transcriptional regulatory mechanism that requires sequences within the leader and the first translated open reading frame, the cbiA gene. Transcriptional and translational Cob::lacZ fusions containing various lengths of Cob DNA were analysed. In a translational Cob::lacZ fusion 407 bp of leader sequence (+69 to +476) was sufficient to confer normal repression. However in a transcriptional Cob::lacZ fusion a 618 bp region (+69 to +687) was required for normal repression. This 618 bp region included sequences in the leader as well as sequences within the cbiA gene. Point mutations which resulted in loss of repression control were isolated and shown to be clustered in the leader sequence (+257 to +380). This region contains a putative hairpin-loop structure which we propose functions as an RNA operator site for a vitamin B12-responsive repressor protein.

Base Sequence↗

PubMed1995

The sequence of 24.3 kb from chromosome X reveals five complete open reading frames, all of which correspond to new genes, and a tandem insertion of a Ty1 transposon.

We have determined the complete nucleotide sequence of a 24.3 kb segment from chromosome X carried by the cosmid pEJ103. The sequence encodes five complete open reading frames (ORFs), none of which correspond to previously described genes; however, four of these ORFs display interesting similarities with sequences present in the databanks. The sequence also contains a tandem insertion of a Ty1 element. An investigation of the Ty1 polymorphism in other strains has revealed that the original insertion occurred within an ORF. Finally, the structure of the Ty1 repeat suggests a mechanism by which it may have been generated.

Amino Acid Sequence↗

PubMed2000

Murine cytomegalovirus containing a mutation at open reading frame M37 is severely attenuated in growth and virulence in vivo.

A pool of murine cytomegalovirus (MCMV) mutants was generated by using a Tn3-based transposon mutagenesis procedure. One of the mutants, RvM37, which contained the transposon sequence at open reading frame M37, was characterized both in tissue culture and in immunocompetent BALB/c and immunodeficient SCID mice. Our results provide the first direct evidence to suggest that M37 is not essential for viral replication in vitro in NIH 3T3 cells. Compared to the wild-type strain and a rescued virus that restored the M37 region, the viral mutant was severely attenuated in growth in both BALB/c and SCID mice after intraperitoneal infection. Specifically, titers of the Smith strain and rescued virus in the salivary glands, lungs, spleens, livers, and kidneys of the SCID mice at 21 days postinfection were about 5 x 10(5), 2 x 10(5), 5 x 10(4), 5 x 10(3), and 1 x 10(4) PFU/ml of organ homogenate, respectively; in contrast, titers of RvM37 in these organs were less than 10(2) PFU/ml of organ homogenate. Moreover, the virulence of the mutant virus appeared to be significantly attenuated because none of the SCID mice infected with RvM37 had died by 120 days postinfection, while all animals infected with the wild-type and rescued viruses had died by 26 days postinfection. Our results suggest that M37 probably encodes a virulence factor and is required for MCMV virulence in SCID mice and for optimal viral growth in vivo.

3T3 Cells↗

PubMed1995

Identification and transcriptional analysis of a 3'-coterminal gene cluster containing UL1, UL2, UL3, and UL3.5 open reading frames of bovine herpesvirus-1.

We have identified and sequenced 3113 nucleotides located at the right end of the HindIII L fragment of the bovine herpesvirus-1 genome from map units 0.712 to 0.734. Analysis of the sequence identified four open reading frames (ORFs) which are designated UL1, UL2, UL3, and UL3.5 based on their homology with proteins of herpes simplex virus-1 (HSV-1), pseudorabies virus (PRV), equine herpesvirus-1, and varicella-zoster virus. The UL1 ORF of 158 amino acids exhibits limited homology with UL1 (glycoprotein gL) of HSV-1 (27%) and PRV (21%). The UL2 ORF of 204 amino acids shows significant homology to UL2 (uracil-DNA glycosylase) of HSV-1 (68%) and PRV (75%). The UL3 ORF of 204 amino acids shows significant homology to UL3 (nuclear phosphoprotein) of HSV-1 (62%) and PRV (53%). The UL3.5 ORF of 126 amino acids shows limited homology to the UL3.5 ORF of PRV (31%). The homolog of this gene is absent in HSV-1. Nucleotide sequence analyses also revealed potential TATA boxes located upstream of each ORF. However, only one polyadenylation signal was detected downstream of the UL3.5 ORF. Northern (RNA) blot analyses revealed four transcripts of 2.4, 1.9, 1.3, and 0.7 kb, which are transcribed in the same direction and are 3'-coterminal transcripts. These mRNAs appear to yield proteins encoded by UL1 (2.4 kb), UL2 (1.9 kb), UL3 (1.3 kb), and UL3.5 (0.7 kb) ORFs.

Amino Acid Sequence↗

PubMed1996

Nucleotide sequence analysis of a 40 kb segment on the right arm of yeast chromosome XV reveals 18 open reading frames including a new pyruvate kinase and three homologues to chromosome I genes.

We have determined the nucleotide sequence of a 40 kb fragment from the right arm of chromosome XV of Saccharomyces cerevisiae. Subsequent analysis revealed 18 non-overlapping open reading frames (ORFs) numbered from 06257 to 06357, an ARS, two tRNA genes and a Ty2 with its flanking elements. Ten ORFs have been sequenced previously: TEA1, RPA43, RPA190, SGC1 (also called TYE7) REV1, PUT4, CIN1, MNE and MRE4 (also called MEK1). Among the others, two seem to code for a new pyruvate kinase and for a new ubiquitin-conjugating enzyme; three have interesting homology with genes located on the left arm of chromosome I. This similarity with chromosome I extends to the left of the sequence presented here (Parle et al., submitted to Yeast). The homologous genes on the two chromosomes are placed in the same relative order.

Amino Acid Sequence↗

PubMed1994

Open reading frames in a 4556 nucleotide sequence within MDV-1 BamHI-D DNA fragment: evidence for splicing of mRNA from a new viral glycoprotein gene.

A DNA segment of the MDV-1 BamHI-D fragment was sequenced, and the open reading frames (ORFs) present in the 4556 nucleotide fragment were analyzed by computer programs. Computer analysis identified 19 putative ORFs in the sequence ranging from a coding capacity of 37 amino acids (aa) (ORF-1a) to 684aa (ORF-1). The special properties of four ORFs (1a, 1, 2, and 3) were investigated. Two adjacent ORFs, ORF-1a and ORF-1, were found by computer analysis to have the properties of two introns encoding a glycoprotein: ORF-1a encodes an aa sequence with the properties of a signal peptide, and ORF-1 encodes a polypeptide with a membrane anchor domain and putative N-glycosylation sites in the aa sequence. ORF-1a and ORF-1 were found to be transcribed in MDV-1-infected cells. Two RNA transcripts were detected: a precursor RNA and its spliced form. Both are transcribed from a promoter located 5' to ORF-1a, and splice donor and acceptor sites are used to splice the mRNA after cleavage of a 71-nucleotide sequence. This finding suggest that ORF-1a and ORF-1 are two introns of a new MDV-1 glycoprotein gene. The DNA sequence containing ORF-1 was transiently expressed in COS-1 cells, and the viral protein produced in these cells was found to react with anti-MDV serotype-1 Antigen B-specific monoclonal antibodies. These studies indicate that the protein encoded by ORF-1 has antigenic properties resembling Antigen B of MDV-1. A gene homologous to ORF-1 was detected in the genome of both MDV-2(SB1) and MDV-3(HVT), which serve as commercial vaccine strains. Two additional ORFs were noted in the 4556 nucleotide sequence: ORF-2, which encodes a 333 aa polypeptide initiating in the UL and terminating in the TRL prior to the putative origin of replication, and ORF-3, which encodes a 155 aa polypeptide that is partly homologous to the phosphoprotein pp38 encoded by the BamHI-H sequence. The 65 N-terminal aa of the two gene products are identical, both being derived from the nucleotide sequences in the TRL and IRL, respectively. Additional homologous aa sequences are the hydrophobic aa domain in the middle of both proteins. The functions of ORF-2, ORF-3, and additional ORFs are under study.

Amino Acid Sequence↗

PubMed1992

Sequence of a 12.7 kb segment of yeast chromosome II identifies a PDR-like gene and several new open reading frames.

A 12,684 bp DNA fragment, between FUS3 and the centromere, from the left arm of chromosome II of Saccharomyces cerevisiae was sequenced as part of the European project to sequence the whole chromosome. This segment contains at least five complete new open reading frames (ORFs) and the beginning (191 first 5' codons) of an ORF whose putative translational product is highly similar to the multidrug resistance PDR1 gene previously characterized by Balzi et al. (1987) on chromosome VII.

Amino Acid Sequence↗

PubMed1994

Analysis of an 11.7 kb DNA fragment of chromosome XI reveals a new tRNA gene and four new open reading frames including a leucine zipper protein and a homologue to the yeast mitochondrial regulator ABF2.

We report the nucleotide sequence of an 11.7 kb fragment from the left arm of Saccharomyces cerevisiae chromosome XI. Analysis reveals a new tRNA for valine and four unknown open reading frames among which YKL245 shows homology with a yeast mitochondrial regulatory protein and YKL244, YKL246 and YKL247 are unknown.

Amino Acid Sequence↗

PubMed1993

DNA sequence analysis of a 17 kb fragment of yeast chromosome XI physically localizes the MRB1 gene and reveals eight new open reading frames, including a homologue of the KIN1/KIN2 and SNF1 protein kinases.

We report in this paper the sequence of a part of chromosome XI of Saccharomyces cerevisiae. This 17 kbp nucleotide sequence represents the right half of cosmid pUKG151 and contains nine open reading frames, YKL453, 450, 449, 448, 445, 443, 442, 441 and the 5' part of YKL440. YKL440 was previously identified as the MBR1 gene and plays a role in mitochondrial biogenesis. YKL443 is a homologue of the yeast serine-rich protein (SRP1), while YKL453 presents strong homologies with the KIN1/KIN2/SNF1 kinase family. It must be pointed out that the size of this gene is well above average for yeast.

Amino Acid Sequence↗

PubMed1994

The molecular cloning and sequence of an open reading frame encoding for non-structural proteins of feline calicivirus F4 strain isolated in Japan.

The nucleotide sequence of the 5'-end of feline calicivirus (FCV) Japanese F4 strain genome was determined. This region had 5311 bases and contained a large open reading frame (ORF1) encoding the non-structural proteins. The nucleotide sequence of the ORF1 region was highly conserved as compared with that of FCV F9 strain. When the deduced amino acid sequence of the ORF1 was compared with those of FCV F9 and CFI strains, the sequence was also highly conserved (88.9% and 88.8%, respectively). Functional motifs of the non-structural proteins were common to these strains. There were 2C polypeptide-, 3C cysteine protease- and 3D RNA-dependent RNA polymerase-like regions. The N-terminal region of 2C-like region continued upstream from the region identified by Neill [Virus Res. 17: 145-160]. Furthermore, the presence of 2B-like region was suggested in the upper stream of the 2C-like region, although the function of the region is unknown. When Kyte and Dolittle hydrophobicity profiles of the predicted amino acid sequences of the ORF1s of FCV F4 and F9 were computed and compared, both the profiles had striking similarities. In the region between residues 950-1000, there was a high rate of basic amino acid residues, suggesting that the polypeptide in this region of FCV may have a nucleic acid-binding function.

Amino Acid Sequence↗