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PubMed2006

tRNA properties help shape codon pair preferences in open reading frames.

Translation elongation is an accurate and rapid process, dependent upon efficient juxtaposition of tRNAs in the ribosomal A- and P-sites. Here, we sought evidence of A- and P-site tRNA interaction by examining bias in codon pair choice within open reading frames from a range of genomes. Three distinct and marked effects were revealed once codon and dipeptide biases had been subtracted. First, in the majority of genomes, codon pair preference is primarily determined by a tetranucleotide combination of the third nucleotide of the P-site codon, and all 3 nt of the A-site codon. Second, pairs of rare codons are generally under-used in eukaryotes, but over-used in prokaryotes. Third, the analysis revealed a highly significant effect of tRNA-mediated selection on codon pairing in unicellular eukaryotes, Bacillus subtilis, and the gamma proteobacteria. This was evident because in these organisms, synonymous codons decoded in the A-site by the same tRNA exhibit significantly similar P-site pairing preferences. Codon pair preference is thus influenced by the identity of A-site tRNAs, in combination with the P-site codon third nucleotide. Multivariate analysis identified conserved nucleotide positions within A-site tRNA sequences that modulate codon pair preferences. Structural features that regulate tRNA geometry within the ribosome may govern genomic codon pair patterns, driving enhanced translational fidelity and/or rate.

Bacillus subtilis↗
PubMed2000

Restriction fragment length polymorphism analysis of open reading frame 5 gene of porcine reproductive and respiratory syndrome virus isolates in Korea.

The genetic variability of porcine reproductive and respiratory syndrome virus (PRRSV) was studied by restriction fragment length polymorphism (RFLP) of polymerase chain reaction (PCR)-amplified fragments among 50 Korean isolates from open reading frame 5. All Korean PRRSVs were isolated from the field cases after the marketing of an U.S. ATCC VR2332-derived modified live PRRSV vaccine. Combining the restriction enzyme digestion patterns obtained with MluI, HincII, SacII, and HaeIII, we observed 19 distinct RFLP patterns. Seventeen out of 50 PRRSV isolates (34%) exhibited the modified live PRRSV vaccine RFLP pattern. The genomic variations that have been identified in the present study seemed to represent characteristic features of the Korean PRRSV isolates. PCR-based RFLP analysis using several restriction enzymes provides a good genetic estimate for isolate differentiation.

Animals↗
PubMed1997

Sequence analysis of a 37.6 kbp cosmid clone from the right arm of Saccharomyces cerevisiae chromosome XII, carrying YAP3, HOG1, SNR6, tRNA-Arg3 and 23 new open reading frames, among which several homologies to proteins involved in cell division control and to mammalian growth factors and other animal proteins are found.

The nucleotide sequence of 37,639 bp of the right arm of chromosome XII has been determined. Twenty-five open reading frames (ORFs) longer than 300 bp were detected, two of which extend into the flanking cosmids. Only two (L2931 and L2961) of the 25 ORFs correspond to previously sequenced genes (HOG1 and YAP3, respectively). Another ORF is distinct from YAP3 but shows pronounced similarity to it. About half of the remaining ORFs show similarity to other genes or display characteristic protein signatures. In particular, ORF L2952 has striking homology with the probable cell cycle control protein crn of Drosophila melanogaster. L2949 has significant similarity to the human ZFM1 (related to a potential suppressor oncogene) and mouse CW17R genes, though it lacks the carboxy-terminal oligoproline and oligoglutamine stretches encoded by these mammalian genes. The small ORF L2922 is similar to part of the much larger yeast flocculation gene FLO1. Other sequences found in the 37639 bp fragment are one delta and one solo-sigma element, the tRNA-Arg3 gene, the small nuclear RNA gene SNR6 and three ARS consensus sequences.

Amino Acid Sequence↗
PubMed2004

Human T-cell lymphotropic virus type 1 open reading frame II-encoded p30II is required for in vivo replication: evidence of in vivo reversion.

Human T-cell lymphotropic virus type 1 (HTLV-1) causes adult T-cell leukemia/lymphoma and exhibits high genetic stability in vivo. HTLV-1 contains four open reading frames (ORFs) in its pX region. ORF II encodes two proteins, p30(II) and p13(II), both of which are incompletely characterized. p30(II) localizes to the nucleus or nucleolus and has distant homology to the transcription factors Oct-1, Pit-1, and POU-M1. In vitro studies have demonstrated that at low concentrations, p30(II) differentially regulates cellular and viral promoters through an interaction with CREB binding protein/p300. To determine the in vivo significance of p30(II), we inoculated rabbits with cell lines expressing either a wild-type clone of HTLV-1 (ACH.1) or a clone containing a mutation in ORF II, which eliminated wild-type p30(II) expression (ACH.30.1). ACH.1-inoculated rabbits maintained higher HTLV-1-specific antibody titers than ACH.30.1-inoculated rabbits, and all ACH.1-inoculated rabbits were seropositive for HTLV-1, whereas only two of six ACH.30.1-inoculated rabbits were seropositive. Provirus could be consistently PCR amplified from peripheral blood mononuclear cell (PBMC) DNA in all ACH.1-inoculated rabbits but in only three of six ACH.30.1-inoculated rabbits. Quantitative competitive PCR indicated higher PBMC proviral loads in ACH.1-inoculated rabbits. Interestingly, sequencing of ORF II from PBMC of provirus-positive ACH.30.1-inoculated rabbits revealed a reversion to wild-type sequence with evidence of early coexistence of mutant and wild-type sequence. Our data provide evidence that HTLV-1 must maintain its key accessory genes to survive in vivo and that in vivo pressures select for maintenance of wild-type ORF II gene products during the early course of infection.

Animals↗
PubMed1996

Nucleotide sequence and molecular analysis of the rhesus cytomegalovirus immediate-early gene and the UL121-117 open reading frames.

Cytomegalovirus (CMV) has been isolated from many nonhuman primates, including rhesus macaques (Macaca mulatta). To better understand the molecular biology of rhesus CMV (RhCMV), a 9.2-kb DNA restriction fragment spanning the immediate-early (IE) gene has been molecularly cloned and sequenced. Open reading frames (ORF) have been identified and transcripts mapped for regions corresponding to exons 1, 2, 3, and 4 of the IE1 protein of human CMV (HCMV) and to exons 1, 2, 3, and 5 of IE2. The predicted RhCMV IE1 protein was 29 and 40% identical with the HCMV and African green monkey (AGM) CMV IE1 proteins, respectively, and the predicted RhCMV IE2 protein was 48 and 65% identical with the HCMV and AGM CMV IE2 proteins, respectively. Five additional ORF 3' to the RhCMV IE gene were identified which contained significant homologies with the HCMV UL121-UL117 ORF. The predicted translation products ranged from 29 to 47% identical with, and 52 to 66% similarity to the corresponding ORF of HCMV. Conservation of nucleic and amino acid sequences, and colinearity of genes, between primate CMV genomes contribute to a better understanding of primate CMV evolution, regulation, and pathogenesis.

Amino Acid Sequence↗
PubMed1999

Open reading frame 1a-encoded subunits of the arterivirus replicase induce endoplasmic reticulum-derived double-membrane vesicles which carry the viral replication complex.

The replicase of equine arteritis virus (EAV; family Arteriviridae, order Nidovirales) is expressed in the form of two polyproteins (the open reading frame 1a [ORF1a] and ORF1ab proteins). Three viral proteases cleave these precursors into 12 nonstructural proteins, which direct both genome replication and subgenomic mRNA transcription. Immunofluorescence assays showed that most EAV replicase subunits localize to membranes in the perinuclear region of the infected cell. Using replicase-specific antibodies and cryoimmunoelectron microscopy, unusual double-membrane vesicles (DMVs) were identified as the probable site of EAV RNA synthesis. These DMVs were previously observed in cells infected with different arteriviruses but were never implicated in viral RNA synthesis. Extensive electron microscopic analysis showed that they appear to be derived from paired endoplasmic reticulum membranes and that they are most likely formed by protrusion and detachment of vesicular structures with a double membrane. Interestingly, very similar membrane rearrangements were observed upon expression of ORF1a-encoded replicase subunits nsp2 to nsp7 from an alphavirus-based expression vector. Apparently, the formation of a membrane-bound scaffold for the replication complex is a distinct step in the arterivirus life cycle, which is directed by the ORF1a protein and does not depend on other viral proteins and/or EAV-specific RNA synthesis.

Animals↗
PubMed1991

Mutagenesis of the AC3 open reading frame of African cassava mosaic virus DNA A reduces DNA B replication and ameliorates disease symptoms.

Small insertions were made independently at each of four unique restriction sites on African cassava mosaic virus (ACMV) DNA A to disrupt the three overlapping complementary-sense open reading frames (ORFs) herein designated AC1, AC2 and AC3. The DNA A mutants were assayed for their infectivity by agroinoculation of monomeric constructs to Nicotiana benthamiana plants containing chromosomal insertions of ACMV DNA B. Disruption of the AC3 ORF alone resulted in a delay and amelioration of disease symptoms which correlated with reduced replication of DNA B. Normal replication of DNA A still carrying the AC3 ORF mutation was found in extracts from these plants. No ACMV DNA or symptoms were observed in corresponding inoculations with either the simultaneous disruption of the overlapping AC2 and AC3 ORFs or disruption of the AC1 ORF. Complementation by the inoculation of different mutant pairs produced a delay in disease symptoms followed by repair of mutated sites. A DNA A construct with the virus-sense AV1 (coat protein) ORF deleted was infectious producing typical ACMV disease symptoms. A similar construct with a larger deletion encompassing the complementary-sense AC3 ORF produced symptomless infections. The DNA recovered from plants revealed DNA A of normal size where the position of the deleted ORF was replaced with cloning vector DNA. Significantly reduced DNA B replication was observed for the AC3 deletion construct.

Blotting, Southern↗
PubMed2001

A mammalian sequence-dependent upstream open reading frame mediates polyamine-regulated translation in yeast.

In mammals, control of S-adenosylmethionine decarboxylase (AdoMetDC) translation is one component of a feedback network that regulates intracellular levels of the polyamines, spermidine, and spermine. AdoMetDC mRNA from mammals contains a highly conserved upstream open reading frame (uORF) within its leader sequence that confers polyamine-regulated suppression of translation on the associated downstream cistron. This regulation is mediated through an interaction that depends on the amino acid sequence of the uORF-encoded hexapeptide. It remains to be shown whether polyamines participate directly in this interaction or indirectly through a specialized signal transduction pathway. We show that Saccharomyces cerevisiae does not have a uORF associated with its AdoMetDC gene (SPE2) and that ribosome loading on the SPE2 mRNA is not positively influenced by polyamine depletion, as it is in mammalian cells. Nevertheless, the mammalian AdoMetDC uORF, when introduced into a polyamine auxotroph of yeast, conferred polyamine regulation of both translational efficiency and ribosome loading on the associated mRNA. This regulatory activity depended on the amino acid sequence encoded by the fourth and fifth codons of the uORF, as in mammalian cells. The fact that the regulatory properties of this mammalian translational control element are quite similar in both mammalian and yeast cells suggests that a specialized signal transduction pathway is not required. Rather, it seems likely that polyamines may be directly participating in an interaction between the uORF-encoded peptide and a constitutive component of the translation machinery, which leads to inhibition of ribosome activity.

Adenosylmethionine Decarboxylase↗
PubMed2004

Murine gammaherpesvirus 68 open reading frame 31 is required for viral replication.

Murine gammaherpesvirus 68 (MHV-68) is genetically related to the human gammaherpesviruses, Kaposi's sarcoma-associated herpesvirus (KSHV/HHV-8) and Epstein-Barr virus (EBV). It has been proposed as a model for gammaherpesvirus infection and pathogenesis. Open reading frame 31 (ORF31) is conserved among the Beta- and Gammaherpesvirinae subfamily, and there is no known mammalian homologue of this protein. The function of MHV-68 ORF31 and its viral homologues has not yet been determined. We described here a primary characterization of this protein and its requirement for lytic replication. The native MHV-68 ORF31 was detected at peak levels by 24 h postinfection, and the FLAG-tagged and green fluorescent protein fusion ORF31 were localized in the cytoplasm and nucleus in a diffuse pattern. Two independent experimental approaches were then utilized to demonstrate that ORF31 was required for lytic replication. First, small interfering RNA generated against ORF31 expression blocked protein expression and virus production in transfected cells. Then, two-independent bacterial artificial chromosome-derived ORF31-null MHV-68 mutants (31STOP) were generated and found to be defective in virus production in fibroblast cells. This defect can be rescued in trans by MHV-68 ORF31 and importantly by its KSHV homologue. A repair virus of 31STOP was also generated by homologous recombination in fibroblast cells. Finally, we showed that the defect in ORF31 blocked late lytic protein expression. Our results demonstrate that MHV-68 ORF31 is required for viral lytic replication, and its function is conserved in its KSHV homologue.

Animals↗
PubMed2001

Murine cytomegalovirus open reading frame M27 plays an important role in growth and virulence in mice.

Using a Tn3-based transposon mutagenesis approach, we have generated a pool of murine cytomegalovirus (MCMV) mutants. In this study, one of the mutants, RvM27, which contained the transposon sequence at open reading frame M27, was characterized both in tissue culture and in immunocompetent BALB/c mice and immunodeficient SCID mice. Our results suggest that the M27 carboxyl-terminal sequence is dispensable for viral replication in vitro. Compared to the wild-type strain and a rescued virus that restored the M27 region, RvM27 was attenuated in growth in both BALB/c and SCID mice that were intraperitoneally infected with the viruses. Specifically, the titers of RvM27 in the salivary glands, lungs, spleens, livers, and kidneys of the infected SCID mice at 21 days postinfection were 50- to 500-fold lower than those of the wild-type virus and the rescued virus. Moreover, the virulence of the mutant virus appeared to be attenuated, because no deaths occurred among SCID mice infected with RvM27 for up to 37 days postinfection, while all the animals infected with the wild-type and rescued viruses died within 27 days postinfection. Our observations provide the first direct evidence to suggest that a disruption of M27 expression results in reduced viral growth and attenuated viral virulence in vivo in infected animals. Moreover, these results suggest that M27 is a viral determinant required for optimal MCMV growth and virulence in vivo and provide insight into the functions of the M27 homologues found in other animal and human CMVs as well as in other betaherpesviruses.

3T3 Cells↗
PubMed1994

The peptide synthetase catalyzing cyclosporine production in Tolypocladium niveum is encoded by a giant 45.8-kilobase open reading frame.

Cyclosporin A, a potent and clinically-important immunosuppressive drug (SandimmunR), is synthesized from its precursor amino acids by cyclosporin synthetase, a single multi-functional enzyme. In this study we report the cloning of the corresponding coding region of this synthetase. It contains an open reading frame of 45.8 kb which encodes a peptide with a calculated M(r) of 1,689,243. The predicted gene product contains 11 amino-acid-activating domains that are very similar to one another and to the domains of other peptide synthetases. Seven of these domains harbour N-methyltransferase functions. This is the largest genomic ORF described so far.

Amino Acid Sequence↗
PubMed1998

The porphyromonas gingivalis prtP/kgp homologue exists as two open reading frames in strain 381.

P. gingivalis is considered to be a major pathogen of adult periodontitis. Among its cadre of putative virulence factors are hemagglutinins (adhesins) and proteases. We here report the cloning, sequencing and characterization of two genes, designated kgp(381) and hagD. Kgp(381), an open reading frame (ORF) of 1095 bp encoding a 40.1 kda protein, has high homology to the proteolytic domain of cysteine protease/hemagglutinin genes. HagD, an ORF of 4077 bp encoding a 147.1 kda protein, contains one HArep sequence which establishes it as an additional member of the HArep multigene family. Although similar in sequence to kgp and prtP which were identified from strains HG66 and W12, respectively, the kgp(381)-hagD genes have several characteristics which distinguish them from kgp and prtP. Foremost among these is a single base difference which produces a termination codon and an immediate frame shift resulting in two ORFs in strain 381 as compared to one ORF in strains HG66 and W12. In addition, a 172 amino acid sequence near the C-terminal end of hagD has very low identity (20.5-27.8%) to the corresponding region of kgp and prtP. These demonstrate that the homologue of kgp and prtP in strain 381 occurs as two separate genes which may genetically separate the adhesive and enzymatic domains of Kgp and PrtP proteins. Reverse polymerase chain reaction (PCR) analysis indicates that hagD expression is regulated by hemin concentration.

Amino Acid Sequence↗
PubMed1992

Open reading frames 1 and 2 of adenovirus region E4 are conserved between human serotypes 2 and 5.

The E4 region of human adenovirus type 2 is predicted to encode seven proteins as judged from its nucleotide sequence and the pattern of differential splicing of its transcript. Two of the open reading frames (ORFs), ORF1 and ORF2, had been identified as being disrupted in the recently published sequence of the related serotype 5 virus. These ORFs were resequenced and found to be intact in the wt300 strain of adenovirus type 5.

Adenovirus E4 Proteins↗
PubMed1994

The sequence of 29.7 kb from the right arm of chromosome II reveals 13 complete open reading frames, of which ten correspond to new genes.

We have determined the complete nucleotide sequence of a 29.7 kb segment from the right arm of chromosome II carried by the cosmid alpha 61. The sequence encodes the 3' region of the IRA1 gene and 13 complete open reading frames, of which ten correspond to new genes and three (CIF1, ATPsv and CKS1) have been sequenced previously. The density of protein coding sequences is particularly high and corresponds to 84% of the total length. Two new genes encode membrane proteins, one of which is particularly large, 273 kDa. In one case (ATPsv), the comparison of our sequence and the published sequence reveals significant differences.

Adaptor Proteins, Signal Transducing↗
PubMed1995

The acidic amino-terminal region of varicella-zoster virus open reading frame 4 protein is required for transactivation and can functionally replace the corresponding region of herpes simplex virus ICP27.

Both varicella-zoster virus open reading frame 4 (ORF4) protein and its herpes simplex virus type 1 homolog ICP27 have highly acidic amino-terminal regions and cysteine-rich carboxy-terminal regions. To investigate the functional domains of these proteins, mutants were constructed and their transregulatory functions were tested in transient expression assays using two reporter plasmids, pTK-CAT-SV40A and pTK-CAT-synA, containing the same promoter sequences but different mRNA processing signals. ORF4 transactivates both pTK-CAT-SV40A and pTK-CAT-synA, while ICP27 transrepresses pTK-CAT-SV40A and transactivates pTK-CAT-synA. Deletion of the ORF4 amino-terminal region abolished most of the transactivating activity for pTK-CAT-synA but retained most of the transactivating activity for pTK-CAT-SV40A. Construction of chimeric ORF4-ICP27 molecules indicated that the ORF4 amino-terminal region was able to replace the corresponding region of ICP27 which is required for both transrepression of pTK-CAT-SV40A and transactivation of pTK-CAT-synA. Similarly, the ICP27 amino-terminal region was able to partially replace the corresponding region of ORF4 which is required for transactivation of pTK-CAT-synA Thus, while ORF4 and ICP27 have different properties in transient expression assays, the amino-terminal regions of ORF4 and ICP27 are functionally homologous to each other and are important in regulating gene expression.

Amino Acid Sequence↗
PubMed1988

Mutational analysis of the open reading frames in the transposable element IS1.

IS1 is one of the smallest transposable elements found in bacteria (768 bp). It contains eight overlapping open-reading-frames (ORFs) greater than 50 codons, designated insA to insG and insB'. To determine which of the ORFs actually code for proteins involved in transposition, we have introduced amber codons into each ORF by site-directed mutagenesis which make neutral changes in the overlapping ORFs. Each mutant IS1 was then tested for its ability to mediate cointegrate formation in Su+ and Su- backgrounds. The mutant elements were also tested for trans-complementation in an IS1-free Salmonella background. Our results show that the products of the insA and insB genes are the only ones essential for cointegrate formation. We suggest that other ORFs may, however, encode accessory proteins.

Amino Acid Sequence↗
PubMed1985

Transcription of novel open reading frames of AIDS retrovirus during infection of lymphocytes.

The retrovirus frequently isolated from patients with the acquired immune deficiency syndrome (AIDS) has two novel open reading frames previously designated "A" and "B." The "A" region was found to be specifically expressed as polyadenylated RNA's of 5.5 and 5.0 kilobases in infected cells. The "B" region was expressed as 1.8- to 2.0-kilobase RNA species. Additional full-length and spliced messenger RNA's of the env region were also identified.

Acquired Immunodeficiency Syndrome↗
PubMed1989

Open reading frames E6 and E7 of bovine papillomavirus type 1 are both required for full transformation of mouse C127 cells.

A series of mutations in open reading frames (ORFs) E6 and E7 of bovine papillomavirus type 1 (BPV1) was constructed to analyze the roles of these ORFs in transformation of mouse C127 cells. The mutations were designed to prevent synthesis of specific proteins encoded by these genes. None of the mutations caused a decrease in the focus-forming activity of the full-length viral genome or in the ability of the viral DNA to replicate as a high-copy-number plasmid. Analysis of these mutants in the absence of a functional BPV1 E5 gene revealed a weak focus-forming activity encoded by ORF E6. Mutations preventing synthesis of the E6 protein did cause defects in anchorage-independent growth and tumorigenicity of transfected and transformed cells. However, a frameshift mutation between the first and second ATG codons of ORF E6 did not inhibit induction of colony formation, suggesting that translation from the first methionine codon is not required. Mutations that inactivated ORF E7 or E6/E7 individually did not inhibit induction of colony formation in agarose. However, a defect in this activity was caused by simultaneous disruption of both ORF E7 and ORF E6/E7 when they were expressed from the full-length viral genome but not when they were expressed under the control of a retrovirus long terminal repeat. These results suggest that translation of both ORF E6 and the 3' end of ORF E7 is required for efficient induction of anchorage-independent growth by the intact BPV1 genome.

Animals↗