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PubMed1995

Sequencing analysis of a 24.7 kb fragment of yeast chromosome XIV identifies six known genes, a new member of the hexose transporter family and ten new open reading frames.

The DNA sequence of a 24.7 kb region covering the left arm of chromosome XIV from Saccharomyces cerevisiae was determined. This region contains 17 open reading frames (ORFs) which code for proteins of more than 100 amino acids. Five ORFs correspond to the KRE1, ATP11, DAL82, RFA2 and MCK1 loci, described previously. Two ORFs present high similarity to known proteins: NO345 with the hexose transporter family, and NO351 with the yeast chorismate mutase/prephenate dehydratase enzyme encoded by PHA2. Six ORFs show limited similarity with known proteins or some specific features: NO339 presents 11 potential transmembrane domains. NO343, which is internal to NO345, presents a putative signal sequence and a potential transmembrane domain. NO348 shows similarity with YCW2, TUP1 and SEC13. NO364 reveals a signature for a pyridoxal-phosphate attachment site. Finally, NO384 and NO388 present a biased amino acid composition, being rich in Asn or Glu/Lys/Arg, respectively. Four other ORFs (NO342, NO376, NO381 and NO397) show no similarity to proteins within the databases screened.

Amino Acid Sequence↗

PubMed1995

Deletion in the L1 open reading frame of human papillomavirus type 6a genomes associated with recurrent laryngeal papilloma.

Human papillomavirus (HPV) type 6a genomes with a large deletion in their L1 open reading frames (ORF) were found in two of five recurrent cases of laryngeal papilloma. One of these mutant HPVs had a 186 base pair (bp) deletion near the N-terminus end of the L1 ORF, which encodes a major capsid protein. The other had a 454 bp deletion at the C-terminus end of L1 at which is located a nuclear localising signal (NLS). No other large deletion or insertion was found in the remaining regions of all five HPV6a genomes. The laryngeal papillomas which harboured the mutant viruses showed typical hyperplasia and pathological changes as observed in tumours induced by the wild-type virus. The biological significance of the two large deletions in the late region of HPV6a associated with laryngeal papilloma is discussed.

Amino Acid Sequence↗

PubMed2005

A complete set of Escherichia coli open reading frames in mobile plasmids facilitating genetic studies.

To facilitate genetic studies of Escherichia coli, we constructed a complete set of mobile plasmid clones of intact open reading frames (ORFs). Their expression is strictly controlled by Ptac / lacI(q). The plasmids carrying each ORF were introduced into an F+ recA strain and stored in 96-well microtiter plates. In this way, 96 clones can be transferred simultaneously to F- bacteria using the conjugative system. This provides a convenient procedure for systematic identification of ORFs that suppress or complement mutations. We created two types of clone sets: the original set contained individual clones in 45 microtiter plates, and a second set contained pools of 48 clones stored in a single microtiter plate. Using these clone sets, we have identified 403 genes that can correct in trans the temperature-sensitive defect of cell division mutants, which would suggest multiple global regulators for bacterial cell division.

Base Sequence↗

PubMed2004

[Identification of the open reading frame coding transposase of Bordetella pertussis RS-element].

A computer-aided analysis of the repeating sequence of Bordetella pertussis chromosome (RSBP3) revealed 3 open reading frames, one of whose (ORF1) can code a protein whose structure and properties are similar to those of transposasas, i.e. enzymes in charges for the traveling of migrating genetic elements of pro- and eukaryote. Mutants of the RSBP3 insertion sequence with the affected and unaffected ORF1 sequence were constructed in order to substantiate the above assumption. Two independent experimental models (formation of inter-plasmid co-integrates and of co-integrates between plasmid and E. coli chromosome) were used to show that the RSBP3-stimulated formation of co-integrates is only true for plasmids containing RSBP3 with the unaffected ORF1 sequence. An activity of the Hpr protein (a component of the phosphoenolpyruvate-dependent phosphotransferase) was proven to influence the formation process of inter-plasmid co-integrates.

Bacterial Proteins↗

PubMed2002

Mass spectrometric characterization of full-length rat selenoprotein P and three isoforms shortened at the C terminus. Evidence that three UGA codons in the mRNA open reading frame have alternative functions of specifying selenocysteine insertion or translation termination.

Selenoprotein P is an abundant extracellular glycoprotein. Its mRNA contains 10 UGAs in an open reading frame terminated by a UAA. This predicts that full-length selenoprotein P will contain 10 selenocysteine residues. Full-length selenoprotein P and three smaller isoforms that have identical N termini have been demonstrated. Selenoprotein P was purified from rat plasma, and the four isoforms were separated by heparin chromatography and SDS-PAGE. Mass spectrometric peptide analysis of the full-length isoform verified 357 of its 366 predicted amino acid residues, including its C terminus and all 10 selenocysteines. The C termini of the smaller isoforms were characterized by mass spectrometry. The shortened isoforms terminated where the second, third, and seventh selenocysteine residues were predicted to be. This suggests that all isoforms arise from the same mRNA and that the UGAs that specify the second, third, and seventh selenocysteines in full-length selenoprotein P can alternatively serve to terminate translation, producing the shorter isoforms.

Amino Acid Sequence↗

PubMed1994

A coupled one-step reverse transcription PCR procedure for generation of full-length open reading frames.

A one-step (all reactants added simultaneously) reverse transcription and polymerase chain reaction (RT-PCR) procedure for amplification of full-length open reading frames (ORFs) of relatively rare transcripts was developed. It was applied for cloning rat luteinizing hormone/chorionic gonadotropin receptor cDNA isoforms larger than two kb. In the procedure developed, manual work is minimized, thus large numbers of samples can be handled, since after denaturation of template RNA and the primers and addition of other reagents, no further manual steps are needed. No inhibitory effect of avian myeloblastosis virus (AMV) reverse transcriptase (RT) on Thermus aquaticus (Taq) DNA Polymerase was found. This was because, under the conditions described, Taq DNA Polymerase effectively amplified picogram amounts of plasmid DNA or template reverse transcribed from nanograms of total ovarian RNA in the presence of AMV-RT. Even a large excess of AMV-RT did not inhibit Taq DNA Polymerase. Thus, our coupled one-step RT-PCR procedure amplifies fast and reproducibly full-length ORFs from nanogram amounts of total RNA.

Animals↗

PubMed1994

The complete sequencing of a 24.6 kb segment of yeast chromosome XI identified the known loci URA1, SAC1 and TRP3, and revealed 6 new open reading frames including homologues to the threonine dehydratases, membrane transporters, hydantoinases and the phospholipase A2-activating protein.

We report the entire sequence of a 26.4 kb segment of chromosome XI of Saccharomyces cerevisiae. Identification of the known loci URA1, TRP3 and SAC1 revealed a translocation compared to the genetic map. Additionally, six unknown open reading frames have been identified. One of them is similar to catabolic threonine dehydratases. Another one contains characteristic features of membrane transporters. A third one is homologous in half of its length to the prokaryotic hydantoinase HyuA and in the other half to hydatoinase HyuB. A fourth one is homologous to the mammalian phospholipase A2-activating protein. A fifth one, finally, is homologous to the hypothetical open reading frame YCR007C of chromosome III. The sequence has been deposited in the EMBL data library under Accession Number X75951.

Amidohydrolases↗

PubMed2004

Sequence variability of human cytomegalovirus UL144 open reading frame in low-passage clinical isolates.

OBJECTIVE: To explore the relationship between human cytomegalovirus (HCMV) UL144 sequence variability and clinical disease. METHODS: HCMV UL144 open reading frame (ORF) was amplified by PCR assay in 72 low-passage isolates [65 congenitally infective children and 7 healthy children who were HCMV-DNA positive by quantitative PCR (qPCR)]. All positive PCR products were analyzed by heteroduplex mobility assay and single-stranded conformation polymorphism (HMA-SSCP) and 32 of them were sequenced. RESULTS: Fifty-five patient isolates and five healthy children isolates were HCMV-UL144 positive by PCR. Sequencing and HMA-SSCP analysis showed that significant strain-specific variability was present in the UL144 ORF. Phylogenetic analysis indicated that the nucleotide sequences could be separated into 3 major genotypes. Comparing between UL144 sequences and the corresponding symptoms showed that genotype 2 did not exist in megacolon isolates. And genotype 1 and 3 were the major types among microcephaly and jaundice isolates respectively. CONCLUSIONS: HCMV-UL144 existed in most of low passage isolates and sequences were hypervariable. The UL144 ORF and its predicted product with the high level of sequence variability in different kinds of isolates suggest that UL144 ORF might play a role in HCMV infectivity and subsequent diseases.

Amino Acid Sequence↗

PubMed1989

N-terminal sequencing of low-molecular-mass components in cyanobacterial photosystem II core complex. Two components correspond to unidentified open reading frames of plant chloroplast DNA.

We recently reported the presence of several low-molecular-mass protein components in the PS II O2-evolving core complex from the thermophilic cyanobacterium, Synechococcus vulcanus [(1989) FEBS Lett. 244, 391-396]. Here we have characterized the three components (4.1, 4.7, 5 kDa) of the same cyanobacterial core complex by N-terminal sequencing. There were two components in the 4.7 kDa region, both having a blocked N-terminus. One has a sequence highly homologous to open reading frame 34 of plant chloroplast DNA (tentatively designated psbM), while the other has a sequence partially homologous to open reading frame 43 of chloroplast DNA (designated psbN), although neither of the two gene products has yet been confirmed in chloroplasts. The cyanobacterial 4.1 kDa protein partially corresponds to the 4.1 kDa nuclear-encoded core component of higher plant PS II. The cyanobacterial 5 kDa component, however, shows a sequence that is unrelated to any other known proteins.

Amino Acid Sequence↗

PubMed1995

Expression of human papillomavirus type 16 E6-E7 open reading frame varies quantitatively in biopsy tissue from different grades of cervical intraepithelial neoplasia.

The proteins encoded by the human papillomavirus type 16 E6-E7 open reading frame are essential for transformation of the host cell. Two mRNA species, E6*I and E6*II, generated by alternative splicing of a polycistronic pre-mRNA, encode truncated E6 proteins and the E7 protein. Our investigation assessed whether or not the level of expression of E6*I and E6*II varies quantitatively in relation to the grade of cervical intraepithelial neoplasia (CIN). We used a quantitative reverse transcription PCR assay to quantify these transcripts in concurrently collected biopsy tissue and exfoliated cervical cells from 22 women with a normal cervix or various grades of CIN. We evaluated transcription profiles in relation to CIN grade and specimen type. The expression levels of E6*I and E6*II in exfoliated cervical cells did not vary significantly in relation to the grade of CIN. However, expression of E6*II was significantly diminished or absent in biopsy tissue obtained from CIN grade II and III lesions (P = 0.014). Our findings suggest that quantification of E6*I and E6*II expression in biopsy tissue may be more clinically relevant than analysis of exfoliated cells. The identification of distinct patterns of expression in association with low- and high-grade CIN suggests that quantification of E6*I and E6*II expression in biopsy tissue may have prognostic value as an indicator of CIN progression.

Base Sequence↗

PubMed1995

Translation of the human papillomavirus type 16 E7 oncoprotein from bicistronic mRNA is independent of splicing events within the E6 open reading frame.

In this study we investigated the translational capacities of bicistronic and spliced mRNAs originating from the E6 and E7 regions of the high-risk genital human papillomavirus type 16 (HPV-16) and the low-risk HPV-11. For HPV-16 it was found, unexpectedly, that E7 protein could be translated from full-length bicistronic E6-E7 mRNAs. E6*I and E6*II splicing events were not required for E7 synthesis, nor did splicing increase the efficiency of E7 translation significantly. In cells, E7 synthesis from all known naturally occurring mRNA structures was very inefficient compared with that from synthetic monocistronic controls, suggesting that HPV-16 employs translational mechanisms to restrict E7 protein levels. For HPV-11, only RNAs initiated at the P264 promoter, located within the E6 open reading frame, were capable of providing an efficient template for E7 synthesis. P264-initiated mRNAs were as efficient in vivo as monocistronic controls, suggesting that the low-risk HPV-11 does not limit E7 synthesis by translational mechanisms. A detailed analysis of HPV-16 templates by using site-directed mutagenesis showed that the majority of ribosomes which ultimately translate E7 have not reinitiated after translating some or all of the upstream open reading frames. The data support a model in which the failure of 40S ribosomal initiation complexes to recognize the E6 AUG renders them capable of proceeding efficiently to translate E7.

Animals↗

PubMed1994

Varicella-zoster virus (VZV) open reading frame 10 protein, the homolog of the essential herpes simplex virus protein VP16, is dispensable for VZV replication in vitro.

Varicella-zoster virus (VZV) open reading frame 10 (ORF10) protein in the homolog of the herpes simplex virus type 1 (HSV-1) protein VP16. VZV ORF10 transactivates the VZV IE62 gene and is a tegument protein present in the virion. HSV-1 VP16, a potent transactivator of HSV-1 immediate-early genes and tegument protein, is essential for HSV-1 replication in vitro. To determine whether VZV ORF10 is required for viral replication in vitro, we constructed two VZV mutants which were unable to express ORF10. One mutant had a stop codon after the 61st codon of the ORF10 gene, and the other mutant was deleted for all but the last five codons of the gene. Both VZV mutants grew in cell culture to titers similar to that of the parental virus. To determine whether HSV-1 VP16 alters the growth of VZV, we constructed a VZV mutant in which VP16 was inserted in place of ORF10. Using immune electron microscopy, we found that HSV-1 VP16 was present in the tegument of the recombinant VZV virions. The VZV VP16 substitution mutant produced smaller plaques and grew to a lower titer than parental virus. Thus, VZV ORF10 is not required for growth of the virus in vitro, and substitution of HSV-1 VP16 for VZV ORF10 impairs the growth of VZV.

Base Sequence↗

PubMed1996

Expression of a continuous open reading frame encoding subunits 1 and 2 of cytochrome c oxidase in the mitochondrial DNA of Acanthamoeba castellanii.

We have investigated the expression of a continuous open reading frame (ORF) present in the mitochondrial genome of Acanthamoeba castellanii and specifying the two largest subunits (COX1 and COX2) of the cytochrome c oxidase complex. Northern hybridization and primer extension analysis demonstrated that this ORF (cox1/2, 873 codons) is transcribed as part of a 4.7 kb RNA that also includes the upstream small subunit rRNA sequence. Between the cox1 and cox2 portions of the transcript, RNA sequence exactly matches gene sequence, excluding the possibility that a standard cox1 termination codon is created by post-transcriptional RNA processing or editing. Western analysis revealed an A. castellanii COX2 protein with a mobility matching that of mature COX2 from yeast (Saccharomyces cerevisiae) mitochondria. These observations indicate that although A. castellanii COX1 and COX2 are apparently translated from the same ORF, they do not exist in mature form as a COX1-COX2 "fusion" protein. Whereas translation of COX2 could potentially be initiated from an internal AUG codon in the cox1/2 ORF, COX1 must be generated either through an unusual translation termination mechanism acting between the cox1 and cox2 coding regions of the cox1/2 mRNA, or by co-translational or post-translational proteolytic processing of a translation product whose synthesis continues into the cox2 coding region. Because the cox2 nucleotide sequence predicts a COX2 protein considerably larger than that observed by Western analysis, A. castellanii COX2 may undergo additional post-translational processing to its final form.

Acanthamoeba↗

PubMed1995

Orthopoxvirus fusion inhibitor glycoprotein SPI-3 (open reading frame K2L) contains motifs characteristic of serine proteinase inhibitors that are not required for control of cell fusion.

The cowpox virus (CPV) SPI-3 gene (open reading frame K2L in vaccinia virus) is one of three orthopoxvirus genes whose products are members of the serpin (serine proteinase inhibitor) superfamily. The CPV SPI-3 gene, when overexpressed by using the vaccinia virus/T7 expression system, synthesized two proteins of 50 and 48 kDa. Treatment with the N glycosylation inhibitor tunicamycin converted the two SPI-3 proteins to a single 40-kDa protein, close to the size of 42 kDa predicted from the DNA sequence, suggesting that the SPI-3 protein, unlike the other two orthopoxvirus serpins, is a glycoprotein. Immunoblotting with an anti-SPI-3 antibody showed that the SPI-3 protein is synthesized early in infection prior to DNA replication. SPI-3 inhibits cell-cell fusion during infections with both CPV and vaccinia virus. A transfection assay was devised to test engineered mutants of SPI-3 for the ability to inhibit fusion. Two mutants with C-terminal deletions of 156 and 70 amino acids were completely inactive in fusion inhibition. Site-directed mutations were constructed near the C terminus of SPI-3, in or near the predicted reactive-site loop which is conserved in inhibitory serpins. Substitutions within the loop at the P1 to P1' positions and P5 to P5' positions, inclusive, did not result in any loss of activity, nor did changes at the P17 to P10 residues in the stalk of the reactive loop. Therefore, SPI-3 does not appear to control cell fusion by acting as a serine proteinase inhibitor.

Amino Acid Sequence↗

PubMed1996

Bovine herpesvirus 1 U(s) open reading frame 4 encodes a glycoproteoglycan.

Sequence analysis of the short unique (Us) segment of the bovine herpesvirus 1 (BHV-1) genome predicted that the Us open reading frame (ORF) 4 encodes a protein with homology to glycoprotein G (gG) of other alpha-herpesviruses (P. Leung-Tack, J.-C. Audonnet, and M. Riviere, Virology 199:409-421, 1994). RNA analysis showed that the Us ORF4 is contained within two transcripts of 3.5 and 1.8 kb. The 3.5 kb RNA represents a structurally bicistronic RNA which encompasses the Us ORF3 and Us ORF4, whereas the 1.8-kb RNA constitutes the monocistronic Us ORF4 mRNA. To identify the predicted BHV-I gG, recombinant vaccinia virus expressing the Us ORF4 was used to raise specific antibodies in rabbits. The antiserum recognized a 65-kDa polypeptide and a very diffusely migrating species of proteins with an apparent molecular mass of between 90 and greater than 240 kDa in supernatants of BHV-1-infected cells which was also precipitated together with 61- and 70-kDa polypeptides from cell-associated proteins. The specificity of the reaction was demonstrated by the absence of these proteins from the supernatant of cells infected with the Us ORF4 deletion mutant BHV-l/gp1-8. Treatment of the immunoprecipitated proteins with glycosidases and chondroitinase AC showed that the 65-kDa protein constitutes gG, which contains both N- and O-linked carbohydrates, and that the high-molecular-mass proteins contain glycosaminoglycans linked to a 65-kDa glycoprotein that is antigenically related to gG. These molecules were therefore named glycoproteoglycan C (gpgG). Pulse chase experiments indicated that gG and gpgG were processed from a common precursor molecule with an apparent molecular mass of 61 kDa via a 70-kDa intermediate. Both gG and gpgG could not be found associated with purified virions. In summary, our results identify the BHV-I gG protein and demonstrate the presence of a form of posttranslational modification, glycosamino-glycosylation, that has not yet been described for a herpesvirus-encoded protein.

Animals↗

PubMed2003

Determination of the sequence of the complete open reading frame and the 5'NTR of the Paderborn isolate of classical swine fever virus.

The classical swine fever (CSF) epidemic in the Netherlands in 1997-1998 lasted 14 months, during which 429 infected and 1300 at risk herds were culled, at an estimated economical cost of 2 billion US dollars. Despite the overwhelming scale of the epizootic, the CSF virus (CSFV) strain causing the outbreak has remained largely uncharacterized. The Dutch epizootic is epidemiologically linked to a small CSF outbreak in 1997, in Paderborn in Germany. E2 and partial 5' NTR sequencing has shown that the index Paderborn isolate, and several Dutch isolates taken during the 1997-1998 epizootic, are virtually identical, confirming that the Paderborn isolate triggered the Dutch outbreak, and furthermore showing that this single isolate was stable throughout the whole Dutch outbreak (the above reviewed in [C. Terpstra, A. J. de Smit, Veterinary Microbiol. 77 (2000) 3-15]). We determined the nucleotide sequence of the 5' NTR (by 5' RACE) and the complete open reading frame of the Paderborn isolate (GenBank AY072924). Our sequence was identical to previously published partial 5'NTR and E2 sequences for the index Paderborn 1997 and Dutch 1997 (Venhorst) isolates, confirming the identity of the virus we sequenced. Phylogenetic analysis based on the complete open reading frame showed that Paderborn is genetically very different from common European laboratory reference strains. Neutralization studies showed that Paderborn is also antigenically very different from common laboratory strains such as Alfort 187. Paderborn is the only recent European CSFV field isolate for which a complete sequence is available, and given Paderborns genetic and antigenic uniqueness, the Paderborn sequence may have practical use for diagnostic and vaccine antigen development.

Amino Acid Sequence↗

PubMed1996

Identification of a differentiation-inducible promoter in the E7 open reading frame of human papillomavirus type 16 (HPV-16) in raft cultures of a new cell line containing high copy numbers of episomal HPV-16 DNA.

Gene expression of human papillomaviruses (HPV) is tightly linked to differentiation processes within the pluristratified epithelium. To analyze changes in the transcription pattern of HPV-16 during epithelial cell differentiation, we established a permanently growing HPV-16 positive cell line, designated KG, from a vulvar intraepithelial neoplasm. KG cells of early passages harbored multiple copies of the HPV-16 DNA as episomes and were able to form a stratified epithelium in an organotypic raft culture system. Analysis of viral gene expression revealed the known transcription pattern of the early region of HPV-16 with the exception of a so far undefined mRNA class with start sites in the E7 open reading frame. Quantitative analysis of primer extension experiments with RNA from KG cells grown in monolayer and raft culture showed a strong induction of this transcript in differentiated KG cells, whereas the level of the mRNAs initiated at the early promoter P97 remained almost constant. Primer extension analyses with four different primers and direct sequencing of the extension product revealed that the differentiation-inducible transcript initiated at a novel promoter with a major start site around nucleotide position 670 (P670) in the E7 open reading frame of HPV-16. Sequence analysis of cDNAs derived from RNA of KG cells grown in raft culture suggested that the transcripts initiated at P670 have a coding potential for an E1E4 fusion protein and for the E5 protein.

3T3 Cells↗

PubMed1989

Characterization of EcoRI mutation in fucosidosis patients: a stop codon in the open reading frame.

Recently, a subset of fucosidosis patients was identified in which the single EcoRI site in the open reading frame of the human cDNA encoding alpha-L-fucosidase was obliterated. We have employed the polymerase chain reaction technique to amplify alpha-L-fucosidase DNA from the five patients known to carry the EcoRI abnormality as well as four patients and two additional fucosidosis patients who do not carry the EcoRI abnormality. Sequence analysis of the amplified DNA has determined that the EcoRI site was destroyed by a C-T transition in the last position of the EcoRI site. This single base change results in the generation of a stop codon 120 base pairs upstream of the normal stop codon. In addition, we have determined that EcoRI cleavage of amplified DNA may be a useful diagnostic tool in the diagnosis of heterozygotes and in prenatal diagnosis of fetuses at risk for this disease.

Amino Acid Sequence↗