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PubMed1985

An active variant of the prokaryotic transposable element IS903 carries an amber stop codon in the middle of an open reading frame.

The prokaryotic mobile genetic element IS903.B is an active variant of IS903. It differs from IS903 and IS102 by 34 and 61 nucleotide substitutions, respectively. The large open reading frame (ORFI) which probably encodes the transposase is conserved in all three IS elements, whereas the smaller open reading frame (ORFII), which codes on the opposite DNA strand and entirely overlaps ORFI, contains an amber stop codon past the middle of ORFII in IS903.B. Experiments using Escherichia coli K12 strains permissive or non-permissive for amber mutations revealed no difference in the cointegration frequency mediated by IS903.B. Therefore, a possible peptide encoded by ORFII on the IS903-related element is unlikely to be necessary for transposition.

Base Sequence↗

PubMed1990

The complete nucleotide sequence of PEBV RNA2 reveals the presence of a novel open reading frame and provides insights into the structure of tobraviral subgenomic promoters.

The 3374 nucleotide sequence of RNA2 from the British PEBV strain SP5 has been determined. The RNA includes three open reading frames flanked by 5' and 3' noncoding regions of 509 and 480 nucleotides. The open reading frames specify coat protein, a 29.6K product homologous to the 29.1K product of TRV(TCM) RNA2 and a 23K product not homologous to any previously described protein. The homology demonstrated between the coat proteins of PRV, TRV and PEBV indicates a common evolutionary origin for these proteins. Upstream of each ORF are located sequences homologous to those with which subgenomic RNAs of other tobraviruses start. Subgenomic RNAs for the expression of the three ORFs may start at these points. On all five tobraviral RNA2 molecules sequenced to date, these sequences were found upstream of the coat protein ORF in association with a strongly-conserved potential secondary structural element. Similar potential structures were identified upstream of other tobraviral ORFs. These structures may contribute to the activity of the tobraviral subgenomic promoter.

Amino Acid Sequence↗

PubMed1986

Expression of the protein encoded by the 3' open reading frame of human T-cell lymphotropic virus type III in bacteria: demonstration of its immunoreactivity with human sera.

The genome of human T-cell lymphotropic virus type III/lymphadenopathy-associated virus (HTLV-III/LAV), the infectious agent etiologically associated with the acquired immunodeficiency syndrome, contains, in addition to the genes for the polymerase, core, and envelope proteins, several open reading frames. To investigate whether the 3' open reading frame (3' orf) located between the envelope gene and the 3' long terminal repeat is a gene expressed in vivo in infected individuals, we inserted a fragment of 3' orf in a prokaryotic expression vector. The protein product synthesized in bacteria was purified and allowed to react with sera from individuals infected with human T-cell lymphotropic virus type III as indicated by seropositivity for other viral proteins. Two-thirds of the sera, regardless of the clinical status of the individuals, reacted with the purified protein indicating that 3' orf is a viral gene the product of which is immunogenic in vivo. A polyclonal rabbit antiserum reacting against the 3' orf gene product was obtained by serial injection of rabbits with the purified bacterial protein. The antiserum recognized a 27-kDa protein in the human T-cell lymphotropic virus type III-infected lymphocytes.

Acquired Immunodeficiency Syndrome↗

PubMed1998

On the presence and organization of open reading frames of the nonmotile pathogen Brucella abortus similar to class II, III, and IV flagellar genes and to LcrD virulence superfamily.

Brucellae are pathogenic, nonmotile bacteria that are facultative intracellular parasites. Little is known about the genetics of these bacteria. Open reading frames from Brucella abortus with similarity to the flagellin, M-ring, and hook of related bacteria were discovered. The open reading frames encode proteins of three of the four flagellum gene classes, namely II, III, and IV. A homolog of the LcrD virulence superfamily was also found. This superfamily is involved in type III protein secretion. B. abortus has the potential for motility and type III secretion.

Amino Acid Sequence↗

PubMedDate not supplied

[Investigation of the amino acid sequences of open reading frames of the Mycobacterium tuberculosis complete genome with the use of the protein family pattern bank Prof_Pat].

The new version of protein family patterns bank Prof_Pat 1.16 was used to investigate 3924 amino acid sequences of open reading frames of Mycobacterium tuberculosis strain H37Rv. The similarity to proteins with the known function was confirmed for 2772 amino acid sequences. Possible functions for 44 thus far unidentified sequences of open reading frames were determined.

Algorithms↗

PubMed1998

Requirement of cauliflower mosaic virus open reading frame VI product for viral gene expression and multiplication in turnip protoplasts.

Cauliflower mosaic virus (CaMV) open reading frame (ORF) VI product (P6) has been shown to be the major constituent of viral inclusion body, to function as a post-transcriptional transactivator, and to be essential for infectivity on whole plants. Although these findings suggest that P6 has an important role in viral multiplication, it is unknown whether P6 is required for viral multiplication in a single cell. To address this question, we transfected turnip protoplasts with an ORF VI frame-shift (4 bp deletion) mutant (pCaFS6) of an infectious CaMV DNA clone (pCa122). The mutant was uninfectious. Co-transfection of plasmids expressing P6 complemented the mutant. Overexpression of P6 elevated the infection rate in co-transfection experiments with either pCa122 or pCaFS6. This would have been achieved by elevating the level of pregenomic 35S RNA, a putative polycistronic mRNA for ORFs I, II, III, IV and V, and by enhancing the accumulation of these five viral gene products. When CaMV ORFs I, II, III, IV and V were expressed from monocistronic constructs in which each of the ORFs was placed just downstream of the 35S promoter, the accumulation of ORF III, IV and V products depended on the co-expression of P6. The accumulation of ORF I and II products was not detected, even in the presence of P6. These results suggest that P6 is involved in the stabilization of other viral gene products as well as in the activation of viral gene expression, and thus, is a prerequisite for CaMV multiplication.

Blotting, Northern↗

PubMed2006

Novel gene and gene model detection using a whole genome open reading frame analysis in proteomics.

BACKGROUND: Defining the location of genes and the precise nature of gene products remains a fundamental challenge in genome annotation. Interrogating tandem mass spectrometry data using genomic sequence provides an unbiased method to identify novel translation products. A six-frame translation of the entire human genome was used as the query database to search for novel blood proteins in the data from the Human Proteome Organization Plasma Proteome Project. Because this target database is orders of magnitude larger than the databases traditionally employed in tandem mass spectra analysis, careful attention to significance testing is required. Confidence of identification is assessed using our previously described Poisson statistic, which estimates the significance of multi-peptide identifications incorporating the length of the matching sequence, number of spectra searched and size of the target sequence database. RESULTS: Applying a false discovery rate threshold of 0.05, we identified 282 significant open reading frames, each containing two or more peptide matches. There were 627 novel peptides associated with these open reading frames that mapped to a unique genomic coordinate placed within the start/stop points of previously annotated genes. These peptides matched 1,110 distinct tandem MS spectra. Peptides fell into four categories based upon where their genomic coordinates placed them relative to annotated exons within the parent gene. CONCLUSION: This work provides evidence for novel alternative splice variants in many previously annotated genes. These findings suggest that annotation of the genome is not yet complete and that proteomics has the potential to further add to our understanding of gene structures.

Alternative Splicing↗

PubMed2006

Protection against heartwater by DNA immunisation with four Ehrlichia ruminantium open reading frames.

We have reported previously that a recombinant DNA vaccine consisting of four Ehrlichia ruminantium (Welgevonden) open reading frames (ORFs) known as the 1H12 cocktail provided protection against a virulent E. ruminantium (Welgevonden) needle challenge in sheep. In this study, we have investigated the vaccine effectiveness of two other cocktails of E. ruminantium (Welgevonden) ORFs, as well as single ORFs from the 1H12 cocktail, to protect sheep against a virulent needle challenge with the homologous strain. Each individual 1H12 ORF provided protection, but all the animals vaccinated with the other cocktails succumbed to the challenge.

Animals↗

PubMed1995

Intron-encoded open reading frame of the GIY-YIG subclass in a plastid gene.

Group-I introns, containing open reading frames (ORFs) that code for homing endonucleases, are widely distributed amongst eukaryotic organellar genomes. However, endonucleases of the GIY-YIG subclass have a restricted distribution in mitochondria and bacteriophages, and have never been observed in plastids. We have found the GIY-YIG motif in an intronic ORF within the previously published psbA gene sequence from Chlamydomonas reinhardtii chloroplasts. Based on phylogenetic analysis and an evaluation of amino-acid substitutions, this ORF is not closely related to any of the other GIY-YIG ORFs. These results suggest that GIY-YIG ORFs have a longer evolutionary history than previously assumed.

Amino Acid Sequence↗

PubMed2001

Rat cytomegalovirus open reading frame R44 is an early-late gene that encodes a nuclear protein.

Rat cytomegalovirus (RCMV) open reading frame R44 is the homolog of human cytomegalovirus gene UL44, which encodes the DNA polymerase accessory protein. Here, we show that R44 is transcribed as a 3.6-kb mRNA within the early and late phases of infection in vitro. In order to find potential monoclonal antibodies (MoAbs) directed against the R44-encoded protein (pR44), a panel of anti-RCMV MoAbs was screened for binding to pR44 recombinant proteins. Thus, an anti-pR44 MoAb, termed RCMV8, was identified. By using this MoAb, pR44 could be detected as early as 8 h after RCMV infection in vitro. The pR44 protein was determined to have a molecular mass of approximately 55 kDa and was found to be localized to the nucleus of RCMV-infected cells.

Animals↗

PubMed1995

The vaccinia virus J5L open reading frame encodes a polypeptide expressed late during infection and required for viral multiplication.

A number of open reading frames (ORFs) are found in the vaccinia virus (VV) genome whose activities in the viral life cycle have not yet been determined. This report examines one such ORF, designated J5L, which was demonstrated to be essential for viral multiplication. Stable inactivation of the J5L ORF by insertion of a lacZ ORF was impossible unless another copy of the J5L ORF was present in the VV genome. Fusion genes between the J5L ORF and either the lacZ gene or the VV K1L gene were employed to study its temporal expression as well as its protein product. These experiments showed that J5L is transcribed late in infection and gives rise to a protein product which migrates by SDS-PAGE with the expected molecular weight (16 kDa). Numerous unsuccessful attempts to establish a stable cell line expressing J5L suggest that the J5L gene product could be cytotoxic.

Animals↗

PubMed2005

Ribosome occupancy of the yeast CPA1 upstream open reading frame termination codon modulates nonsense-mediated mRNA decay.

Saccharomyces cerevisiae CPA1 mRNA contains an upstream open reading frame (uORF) encoding the arginine attenuator peptide (AAP). Negative translational regulation of CPA1 occurs when the nascent AAP responds to arginine (Arg) by stalling ribosomes at the uORF termination codon. CPA1 expression is also controlled by nonsense-mediated mRNA decay (NMD). Using wild-type and decay-defective strains expressing CPA1-LUC, we determined how this uORF contributes to NMD control. Arg addition to media rapidly destabilized the CPA1 transcript in wild-type but not upf1delta cells. The wild-type uORF exerted translational control and induced NMD of CPA1-LUC; the mutated D13N uORF, which eliminates stalling and regulation, did not. Thus, regulation by NMD was not governed simply by ribosomes encountering the uORF terminator but appeared dependent on the AAP's ribosome-stalling ability. Improving the D13N uORF initiation context also promoted NMD. Hence, NMD appears to be triggered by increased ribosomal occupancy of the uORF termination codon.

Carbamoyl-Phosphate Synthase (Glutamine-Hydrolyzin↗

PubMed1995

Zinc-binding of the cysteine-rich domain encoded in the open reading frame of 1B of the RNA polymerase gene of coronavirus.

We cloned and sequenced the second open reading frame of the RNA polymerase gene, ORF1b, of bovine coronavirus. In the region representing nucleotide positions 4919-5677 upstream from the initiation codon of the 32K non-structural protein gene, we identified two putative functional domains. One of these domains contained four leucine residues repeated exactly in every seventh position, and the other domain represented a cluster of cysteine and histidine residues. The DNA sequence representing these domains was cloned and expressed in Escherichia coli as fusion proteins with glutathione S-transferase from Schistosoma japonicum. A high level expression of the cysteine-rich domain was achieved as a fusion protein when the bacterial culture was induced with IPTG. In a solid phase zinc binding assay using the recombinant fusion protein, we found that the protein containing the cysteine-rich domain was able to bind to radioactive zinc in vitro, demonstrating that the polypeptide encoded by the ORF1b of coronavirus is a zinc-binding protein.

Animals↗

PubMed2003

What determines whether mammalian ribosomes resume scanning after translation of a short upstream open reading frame?

If the 5'-proximal AUG triplet in a mammalian mRNA is followed by a short open reading frame (sORF), a significant fraction of ribosomes resume scanning after termination of sORF translation, and reinitiate at a downstream AUG. To examine the underlying mechanism, we examined reinitiation in vitro using a series of mRNAs that differed only in the 5'-untranslated region (UTR). Efficient reinitiation was found to occur only if the eIF4F complex, or at a minimum the central one-third fragment of eIF4G, participated in the primary initiation event at the sORF initiation codon. It did not occur, however, when sORF translation was driven by the classical swine fever virus or cricket paralysis virus internal ribosome entry sites (IRESs), which do not use eIF4A, 4B, 4E, or 4G. A critical test was provided by an mRNA with an unstructured 5'-UTR, which is translated by scanning but does not absolutely need eIF4G and eIF4A: There was efficient reinitiation in a standard reticulocyte lysate, when initiation would be largely driven by eIF4F, but no reinitiation in an eIF4G-depleted lysate. These results suggest that resumption of scanning may depend on the interaction between eIF4F (or the eIF4G central domain) and the ribosome being maintained while the ribosome translates the sORF.

Animals↗

PubMed1997

Human cytomegalovirus open reading frame UL11 encodes a highly polymorphic protein expressed on the infected cell surface.

Human cytomegalovirus (HCMV) open reading frame (ORF) UL11 locates within a polymorphic region of the viral genome identified previously by a restriction-fragment-length-polymorphism. We report here that ORF UL11 encodes a polymorphic protein expressed on the surface of HCMV-infected cells. First, we determined the nucleotide sequence of ORF UL11 from ten strains and compared it among the strains. Out of 205 amino acids consisting of the predicted N-terminal region beside the putative transmembrane stretch in strain AD169, 88 residues were divergent on more than one strain. In contrast, the predicted C-terminal side including the putative transmembrane domain was identical at the amino acid sequence level. In addition, the number and location of predicted cysteine residues were also conserved. Next, we screened a cDNA library from HCMV-infected cells and obtained a cDNA clone containing the full-length ORF UL11. Finally, we identified the gene product of UL11 on the surface of HCMV-infected cells by FACS analysis with polyclonal antibodies generated against a glutathione S-transferase/UL11 fusion protein. The fusion protein contained a region within the N-terminal side next to the predicted transmembrane stretch. These results indicate that the N-terminal side of UL11 protein containing variable amino acid residues protrudes from the infected cell surface.

Amino Acid Sequence↗

PubMedDate not supplied

Identification and characterization of human herpesvirus 8 open reading frame K9 viral interferon regulatory factor by a monoclonal antibody.

OBJECTIVE: To identify and characterize an open reading frame (ORF) in association with its mRNA and a coding protein recognized by anti-human herpesvirus 8 (HHV-8) monoclonal antibody B291. STUDY DESIGN/METHODS: The antigen detected by B291 was characterized by immunofluorescence method, Western blot analysis, laser confocal microscopy, and immunohistology cDNA of the protein was identified by immunoscreening the HHV-8 cDNA library with B291 and sequenced. Structure and kinetics of the mRNA expression was investigated by 5',3' rapid amplification of cDNA ends (RACE) and Northern blot analysis. RESULTS: Viral interferon regulatory factor (vIRF; approximately 50 kd) was a protein recognized by B291. It was detected in 12-O-tetradecanoyl phorbol-13 acetate (TPA)-induced, but not uninduced, HHV-8-infected cell lines (BCBL-1 and BCP-1) and the tumor cells obtained from a SCID mouse injected with uninduced BCBL-1. The expression of vIRF was predominantly in the nucleus and was partially diminished by phosphonoformic acid (PFA) treatment, whereas the mRNA (approximately 2.4 kb) was accumulated. CONCLUSIONS: vIRF is an early protein expressed in the nucleus and may be involved in Kaposi's sarcoma tumor formation.

Amino Acid Sequence↗

PubMed1989

Deletion mutants of herpesvirus saimiri define an open reading frame necessary for transformation.

Analysis of a 5,549-base-pair sequence at the left end of herpesvirus saimiri unique (L-) DNA revealed two open reading frames and genes for five small nuclear U RNAs (herpesvirus saimiri U RNAs). Replication-competent deletion mutants were constructed in order to assess the importance of these genetic features for transformation by this oncogenic herpesvirus. Although not required for replication, one of the open reading frames was found to be required for immortalization of marmoset T lymphocytes into continuously growing cell lines. The protein predicted by this reading frame (STP; saimiri transformation-associated protein) has a highly hydrophobic stretch of 26 amino acids sufficient for a membrane-spanning domain near its carboxy terminus; this domain is immediately preceded by a sequence appropriate for formation of a metal-binding domain (His X2 His X6 Cys X2 Cys, where Xs are other amino acids). One of two poly(A)+ RNAs that could encode STP is bicistronic, while the other has a long 5' untranslated region (approximately 1.5 kilobases). Although some analogies can be drawn between STP and LMP (lymphocyte membrane protein) of Epstein-Barr virus, STP is not related in sequence to LMP.

Amino Acid Sequence↗

PubMed2001

Kaposi's sarcoma-associated herpesvirus vCyclin open reading frame contains an internal ribosome entry site.

We have previously examined the transcription and splicing of open reading frames (ORFS) 71 (K13), 72, and 73 of Kaposi's sarcoma-associated herpesvirus (KSHV) in the primary effusion lymphoma cell line BCP-1 (latently infected with KSHV) (45). The three genes encoded by these ORFs (for vFLIP, vCyclin, and latency-associated nuclear antigen [LANA]) are transcribed from a common transcription start site in BCP-1 cells during both latency and the lytic cycles. The resulting transcript is spliced to yield a 5.32-kb message encoding LANA, vCyclin, and vFLIP and a 1.7-kb bicistronic message encoding vCyclin and vFLIP. To investigate whether the vFLIP protein could be expressed from this vCyclin/vFLIP message, we utilized a bicistronic luciferase reporter system. The genes for Renilla and firefly luciferases (which utilize different substrates) were cloned in tandem downstream from a T7 RNA polymerase promoter. Fragments of DNA immediately upstream from the initiating codon of vFLIP were cloned between the two luciferase genes. The relative expression of the two luciferases, one directed by the putative internal ribosome entry site (IRES) sequences and the other by cap-dependent ribosome scanning, was used to compare the activities of the different DNA fragments. A minimum fragment of 233 bp within the coding region of vCyclin was found to direct efficient expression of the downstream cistron (firefly luciferase). The activity of this IRES was orientation dependent and unaffected by methods used to inhibit cap-dependent translation. This is the first demonstration of an IRES element encoded by a DNA virus and may represent a novel mechanism through which KSHV controls protein expression.

Base Sequence↗