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Transcript processing internal to a mitochondrial open reading frame is correlated with fertility restoration in male-sterile sorghum.

A chimeric mitochondrial DNA (mtDNA) configuration of the cytoplasmic male-sterile (cms) sorghum line IS1112C includes a 321 bp open reading frame designated orf107, encoding a predicted polypeptide product of 11.85 kDa. The open reading frame, similar to several other genes associated with cms, consists of amino-terminal sequences derived from an obligate gene. Unlike other examples to date, however, the carboxy-terminal sequences are highly similar to the carboxy terminus of an open reading frame implicated in cms of rice, orf79. The amino-terminal 31 residues of orf107 are 84% similar to atp9, and the carboxy-terminal 49 residues are 57% identical and 80% similar to the carboxy terminus of orf79. Transcripts of orf107 are edited, with four C-to-U changes that alter amino acids. Sorghum lines partially or fully restored to fertility exhibit a high-efficiency internal-orf107 transcript processing activity, precluding abundant whole-length transcripts, while male-sterile lines exhibit only a trace of the activity. Previous data on the abundance of a 12kDa in organello-synthesized polypeptide in male-sterile versus male-fertile lines are correlated with differential orf107 transcript processing activity of these lines. Examinations of backcross and F2 lines suggest a gametophytic mode of restoration, and indicate that enhanced transcript processing activity is necessary, but not sufficient, to restore full fertility. These novel observations indicate that mitochondrial open reading frames associated with cms in different species can include highly similar motifs, and that fertility restoration could involve a mechanism by which synthesis of a cms-associated gene product may be precluded through internal transcript cleavage.

Amino Acid Sequence↗

Proteins and cis-acting elements associated with transactivation of the varicella-zoster virus (VZV) immediate-early gene 62 promoter by VZV open reading frame 10 protein.

Varicella-zoster virus (VZV) open reading frame 10 (ORF10) protein, the homolog of herpes simplex virus type 1 (HSV-1) VP16, is a virion-associated transactivator of the VZV immediate-early (IE) gene 62 (IE62) promoter. VP16 forms a complex with cellular factors (Oct1 and host cell factor [HCF]) and TAATGARAT elements (found in all HSV-1 IE promoter/enhancer sequences) to mediate stimulation of IE transcription. The VZV IE62 promoter also contains three TAATGARAT-like elements. Mutagenesis studies of the VZV IE62 promoter indicated that TAATGARAT-like elements contribute to transactivation of the VZV IE62 promoter by ORF10 protein. Other cis-acting elements such as GA-rich and cyclic AMP-responsive elements were also needed for full transactivation by ORF10 protein. In mobility shift assays, ORF10 protein formed a complex with either of two TAATGARAT-like elements that lack an overlapping octamer-binding motif (octa-/TAATGARAT) but not with a TAATGARAT element with an overlapping octamer-binding motif (octa+/TAATGARAT). In contrast, VP16 formed a high-affinity ternary complex with an octa+/TAATGARAT element and a low-affinity complex with octa-/TAATGARAT elements. Addition of antibodies to ORF10 protein, Oct1, or HCF disrupted the complexes, demonstrating that ORF10 protein interacts with Oct1 and HCF. These results suggest that transactivation of the VZV IE62 gene by ORF10 protein and HSV IE genes by VP16 require similar cellular proteins but distinct cis-acting elements.

Base Sequence↗

Sendai virus P gene produces multiple proteins from overlapping open reading frames.

The Sendai virus P gene contains overlapping open reading frames (ORFs), and several unusual mechanisms are used to produce multiple proteins from all three ORFs of this gene. These include the use of a non-AUG start codon, leaky ribosomal scanning, what appears to be scanning-independent ribosomal initiation and/or ribosomal jumping, and a form of mRNA editing.

DNA-Directed RNA Polymerases↗

The open reading frames UL3, UL4, UL10, and UL16 are dispensable for the replication of herpes simplex virus 1 in cell culture.

By means of insertion and deletion mutagenesis, we have constructed four herpes simplex virus 1 recombinants, each lacking most sequences encoding a different open reading frame. The deleted genes are located in the unique sequences of the long component and include those designated UL3, UL4, UL10, and UL16. The recombinant virus R7211 lacks 579 of the 696 bp of UL3. The recombinant virus R7217 lacks 307 of the 597 bp of the UL4 open reading frame. R7216 contains a 972-bp deletion within the 1,419-bp open reading frame of UL10, whereas R7210 lacks 988 bp of the 1,119-bp UL16 open reading frame. Growth curves indicated that the yields of these viruses in Vero and BHK cell cultures were only slightly reduced from or in some instances equivalent to that of the parent virus. The function of the gene products is not known. It is of interest to note that (i) the UL16 open reading frame maps entirely within the single intron of UL15 and (ii) on the basis of the extent and size of hydrophobic domains, the UL3 and UL10 gene products were predicted to be membrane proteins.

Animals↗

Nucleotide and peptide sequences of the open reading frame encoding a truncated toxin A gene of Clostridium difficile strain CCUG 20309.

The open reading frame encoding a putative truncated toxin A gene of Clostridium difficile in strain CCUG 20309 (ATCC 8864), a strain that produces toxin B but not toxin A, was sequenced by cycle sequencing method. The coding region contains 2097 base pairs and has a GC content of 26.4%. The deduced polypeptide is 50 kDa and is generally hydrophilic. Although strain CCUG 20309 of C. difficile was reported not to produce toxin A, it is enterotoxic, an inherent property of toxin A in pathogenic strains of C. difficile. It therefore remains to be determined whether a truncated form of toxin A could actually be expressed from the open reading frame by this strain of C. difficile.

Amino Acid Sequence↗

A mutation in the short 5'-proximal open reading frame on Rous sarcoma virus RNA alters virus production.

The 5'-proximal open reading frame on Rous sarcoma virus RNA encodes a seven-amino-acid peptide and is conserved in all avian sarcoma-leukosis retroviruses. Ribosome-binding site analysis in intact chick cells showed that the 5'-proximal AUG codon is a strong site for initiation of translation in vivo. Removal of the 5'-proximal AUG codon by site-specific mutagenesis resulted in a virus with a reduced ability either to replicate or to transform a population of chicken embryo fibroblasts. These results establish a procedure for determining sites of ribosome binding and initiation of translation on mRNAs in intact eucaryotic cells and strongly suggest that the 5'-proximal open reading frame (or its AUG codon) on Rous sarcoma virus RNA has an important role in regulating viral activity.

Animals↗

Chromosomal assignment of human endogenous retrovirus K (HERV-K) env open reading frames.

The haploid human genome contains at least one human endogenous retrovirus K (HERV-K) env gene displaying an open reading frame, as evidenced by the high antibody titers against HERV-K Env in germ cell tumor patients at the time of tumor detection. However, the chromosomal assignment of an intact HERV-K env sequence is complicated by the existence of 25-30 HERV-K copies per haploid human genome. Recently, we reported the application of the protein truncation test (PTT) to chromosomally assign HERV-K gag open reading frames. Here, we set out to determine the chromosomal distribution of full-length HERV-K env genes. As demonstrated by somatic hybrid mapping and the PTT, HERV-K env open reading frames were found on human chromosomes 7, 19, and Y. By sequencing chromosome-specific HERV-K env subregions, we assigned two recently reported intact HERV-K sequences on human chromosomes 7 and 19, respectively. Chromosomes 7 and 19 and the Y are furthermore candidates for harboring a putative completely intact HERV-K locus.

Chromosome Mapping↗

Expression of rotavirus proteins encoded by alternative open reading frames of genome segment 11.

The nucleotide sequence of rotavirus genome segment 11 shows that this gene contains three potential open reading frames. We used several approaches to determine whether any polypeptides other than NS26, the primary protein product, are expressed. In particular, we sought to determine whether the strong out-of-phase start codon present at nucleotides 80-82, which would encode a protein of 92 amino acids, is used in vivo or in cell-free systems. Several modifications of gene 11 were made and found to produce proteins from the different initiation codons in cell-free transcription-translation systems. The protein from the out-of-phase open reading frame was shown to be expressed in rotavirus-infected MA104 cells; this was demonstrated using monospecific sera prepared to this protein expressed in Spodoptera frugiperda insect cells infected with a baculovirus recombinant containing only the out-of-phase open reading frame. The origin of some of the lower-molecular-weight bands serologically related to the primary product of gene 11, NS26, was also studied by selective immunoprecipitation using two different sera made from recombinant baculovirus lysates. All of these polypeptides are present in infected cells in a complex which is still incompletely defined.

Amino Acid Sequence↗

Kaposi's sarcoma-associated herpesvirus open reading frame 57 encodes a posttranscriptional regulator with multiple distinct activities.

Open reading frame (ORF) 57 of Kaposi's sarcoma-associated herpesvirus (KSHV) encodes a homolog of known posttranscriptional regulators that are essential for replication in other herpesviruses. Here, we examined the expression of this gene and the function(s) of its product. KSHV ORF 57 is expressed very early in infection from a 1.6-kb spliced RNA bearing several in-frame initiation codons. Its product is a nuclear protein that, in transient assays, has little effect on the expression of luciferase reporter genes driven by a variety of KSHV and heterologous promoters. However, ORF 57 protein enhances the accumulation of several viral transcripts, in a manner suggesting posttranscriptional regulation. These transcripts include not only known cytoplasmic mRNAs (e.g., ORF 59) but also a nuclear RNA (nut-1) that lacks coding potential. Finally, ORF 57 protein can also modulate the effects of the ORF 50 gene product, a classical transactivator known to be required for lytic induction. The expression from some (e.g., nut-1) but not all (e.g., tk) ORF 50-responsive promoters can be synergistically enhanced by coexpression of ORF 50 and ORF 57. This effect is not due to upregulation of ORF 50 expression but rather to a posttranslational enhancement of the transcriptional activity of ORF 50. These data indicate that ORF 57 is a powerful pleiotropic effector that can act on several posttranscriptional levels to modulate the expression of viral genes in infected cells.

Base Sequence↗

The prediction of exons through an analysis of spliceable open reading frames.

We have developed a computer program which predicts internal exons from naive genomic sequence data and which will run on any IBM-compatible 80286 (or higher) computer. The algorithm searches a sequence for 'spliceable open reading frames' (SORFs), which are open reading frames bracketed by suitable splice-recognition sequences, and then analyzes the region for codon usage. Potential exons are stratified according to the reliability of their prediction, from confidence levels 1 to 5. The program is designed to predict internal exons of length greater than 60 nucleotides. In an analysis of 116 genes of a training set, 384 out of 441 such exons (87.1%) are identified, with 280 (63.5%) of predictions matching the true exon exactly (at both 5' and 3' splice junctions and in the correct reading frame), and with 104 (23.6%) exons matching partially. In a similar analysis of 14 genes in a test set unrelated to the genes used to generate the parameters of the program, 70 out of 80 internal exons greater than 60 bp in length are identified (87.5%), with 47 completely and 23 partially matched. SORFs that partially match true internal exons share at least one splice junction with the exon, or share both splice junctions but are interpreted in an incorrect reading frame. Specificity (the percentage of SORFs that correspond to true exons) varies from 91% at confidence level 1 to 16% at confidence level 5, with an overall specificity of 35-40%. The output displays nucleotide position, confidence level, reading frame phase at the 5' and 3' ends, acceptor and donor sequences and scoring statistics and also gives an amino acid translation of the potential exon. SORFIND compares favourably with other programs currently used to predict protein-coding regions.

3',5'-Cyclic-GMP Phosphodiesterases↗

Conservation and loss of the ERV3 open reading frame in primates.

The human endogenous retrovirus ERV3 possesses an open reading frame for a truncated envelope, which is expressed as mRNA and protein. Here we examine the env sequence in primates for evidence of evolutionary conservation. ERV3 sequences were amplified by PCR from genomic DNA of great ape and Old World primates but not from New World primates or gorilla, suggesting an integration event more than 30 million years ago with a subsequent loss in one species. In the chimpanzee, the protein sequence of Env is 98.18% identical to that of human. In other species the identity falls (93.71% in rhesus macaque) in proportion to the separation from the human lineage. Start and stop codons and domains of functional significance in the envelope protein are conserved. The evolutionary conservation of the ERV3 envelope suggests a beneficial function, though the loss from gorilla shows that it is not essential for survival or reproduction.

Amino Acid Sequence↗

[Gene chip analysis of mutation of HBV-DNA open reading frame].

OBJECTIVE: To detect the mutation of HBV-DNA open reading frame (ORF), provide evidences for clinical diagnosis and treatment. METHODS: By using gene chip technique, HBV-DNA was amplified by PCR, incorporated with fluorescence, hybridized with oligonucleotide to detect the gene sequence of DNA by computer analysis and observe the natural mutation of HBV-DNA ORF. RESULTS: The mutations of HBV-DNA ORF existed widespread. The rates of mutation on Pre-C 1896, PreC 1814, BCP 1762, BCP 1764, P 528, P 552MI, P 552MV were 23.5%, 3.9%, 55.9%, 53.9%,39.2%, 38.2%, 10.8%, respectively. CONCLUSION: The gene chip technique possesses extremely high sensitivity and reliability,it is one of the effective methods to detect gene mutation. The mutation of HBV-DNA has important influence on the stability and progress of the disease, and on the judgement of prognosis.

DNA, Viral↗

Variola and camelpox virus-specific sequences are part of a single large open reading frame identified in two German cowpox virus strains.

A large open reading frame (ORF) has been identified in two German cowpox virus strains. The ORFs (5676 and 5679 nt, respectively) differ in 10 nucleotides, resulting in an amino acid homology of 99.8%. In searching GenBank nucleotide sequences (>90% identity) were present in several small ORFs in variola major, variola minor and camelpox virus genomes. Alignments revealed that these small ORFs are fragments of a large ORF. However, sequences of the ORF described here are entirely absent in the two cowpox virus reference strains. Databank analysis revealed amino acid identities (ranging from 25 to 39%) with so-called B22R-like poxviral proteins with unknown function encoded by several chordopoxviruses. Further sequencing of one cowpox virus strain under study identified an ORF (5790 nt) which displays high levels of nucleotide identity to ORFs present in several orthopoxvirus species. Taken together, the two cowpox viruses analyzed here contain one large ORF which is conserved within the genus Orthopoxvirus and a unique, more distantly related ORF of similar size, which is conserved in the subfamily Chordopoxvirinae.

Chordopoxvirinae↗

Purification of a soluble hepatitis E open reading frame 2-derived protein with unique antigenic properties.

The second open reading frame (ORF2) of hepatitis E virus (HEV) is predicted to encode a 73-kDa capsid protein (1). When full-length ORF2 was expressed in insect cells (Spodoptera frugiperda (Sf9)) using a recombinant baculovirus, two distinct HEV polypeptides were observed: a full-length insoluble 73-kDa protein, and a soluble 56.5-kDa protein. Following purification and sequence analysis, it was determined that the 56.5-kDa protein was derived from endoproteolytic cleavage site that was between the Thr and Ala residues located at amino acids 111 and 112 in the ORF2 sequence with the carboxy terminus corresponding to residue 636 of the ORF2 sequence. Comparative ELISA data using human acute-phase antisera demonstrated that the 56.5-kDa protein served as a highly reactive antigen in detecting anti-HEV antibodies. These data suggest that the 56.5-kDa protein may serve as a particularly useful antigen for both diagnostic and vaccine purposes.

Amino Acid Sequence↗

Hydrophobic cluster analysis predicts an amino-terminal domain of varicella-zoster virus open reading frame 10 required for transcriptional activation.

Varicella-zoster virus open reading frame 10 (ORF10) protein, the homolog of the herpes simplex virus protein VP16, can transactivate immediate-early promoters from both viruses. A protein sequence comparison procedure termed hydrophobic cluster analysis was used to identify a motif centered at Phe-28, near the amino terminus of ORF10, that strongly resembles the sequence of the activating domain surrounding Phe-442 of VP16. With a series of GAL4-ORF10 fusion proteins, we mapped the ORF10 transcriptional-activation domain to the amino-terminal region (aa 5-79). Extensive mutagenesis of Phe-28 in GAL4-ORF10 fusion proteins demonstrated the importance of an aromatic or bulky hydrophobic amino acid at this position, as shown previously for Phe-442 of VP16. Transactivation by the native ORF10 protein was abolished when Phe-28 was replaced by Ala. Similar amino-terminal domains were identified in the VP16 homologs of other alphaherpesviruses. Hydrophobic cluster analysis correctly predicted activation domains of ORF10 and VP16 that share critical characteristics of a distinctive subclass of acidic activation domains.

Amino Acid Sequence↗

Epigenetic regulation of varicella-zoster virus open reading frames 62 and 63 in latently infected human trigeminal ganglia.

Open reading frames (ORFs) 21, 29, 62, 63, and 66 of varicella-zoster virus (VZV) are transcribed during latency in human ganglia. ORF 63 is the most frequently expressed gene, and ORF 62 encodes a transcriptional activator. The mechanisms regulating the expression of these genes are not well understood, although analyses of other alphaherpesviruses indicate a role for chromatin in virus gene regulation during latent infection. Using chromatin immunoprecipitation (ChIP) assays to analyze the euchromatic state of ORFs 62 and 63 compared to the centromere from human chromosome 4 (heterochromatic) and the human glyceraldehyde-3-phosphate dehydrogenase promoter (euchromatic), we show that the promoters of ORFs 62 and 63 are associated with the histone protein H3K9(Ac) and thus maintained in a euchromatic state during latency. Conversely, the promoters of ORF 36 (thymidine kinase) and ORF 14 (glycoprotein C), genes expressed during lytic but not latent infection, were not enriched in the fraction of latently infected ganglia that bound to anti-H3K9(Ac) antibody. A ChIP assay using productively infected MeWo cells revealed that VZV ORFs 62, 63, 36, and 14 are all euchromatic. Together, these data indicate that the expression of the two latency-related VZV genes, ORFs 62 and 63, is regulated epigenetically through chromatin structure.

Acetylation↗

Enrichment of open reading frames presented on bacteriophage M13 using hyperphage.

The enrichment of open reading frames (ORFs) from large gene libraries and the presentation of the corresponding polypeptides on filamentous phage M13 (phage display) is frequently used to identify binding partners of unknown ORFs. In particular phage display is a valuable tool for the identification of pathogen-related antigens and a first step for the development of new diagnostics and therapeutics. Here, we introduce a significant improvement of phage-based ORF enrichment by using Hyperphage, a helperphage with a truncated gIII. The methods allow both the enrichment of ORFs from cDNA libraries and the display of the corresponding polypeptides on phage, thus combining ORF enrichment with a screening for binding in one step without any further subcloning steps. We demonstrated the benefits of the method by isolating the sequences encoding two predicted immunogenic epitopes of the outer membrane protein D encoding gene (ompD) of Salmonella typhimurium. Here, we showed that when using a mixture of three constructs with only one containing an ORF solely this correct construct could be reisolated in phage particles. Further; both epitopes were detected by enzyme-linked immunosorbent assay (ELISA), demonstrating correct translation of fusion proteins. Furthermore, the enrichment system was evaluated by the enrichment of ORFs from total cDNA of lymphocytes. Here, we could show that 60% of the phage contained ORFs, which is an increase of an order of magnitude compared with conventional phage expression system. Together these data show that the Hyperphage-based enrichment system significantly improves the enrichment of ORFs and directly allows the display of the corresponding polypeptide on bacteriophage M13.

Amino Acid Sequence↗

Molecular cloning of the closed circular provirus of human T cell leukaemia virus type I: a new open reading frame in the gag-pol region.

A DNA clone of human T cell leukaemia virus type I (HTLV-I) was isolated from extrachromosomal closed circular copies in chronically infected promyelocytic leukaemia HL60 cells. The new HTLV-I isolate had an intact reading frame in the gag-pol region which could encode protein of 234 amino acids. This open reading frame has not been observed in previous HTLV-I isolates, although similar open reading frames have been reported in the corresponding locations in the related bovine leukaemia virus and HTLV type II. We consider that this open reading frame codes for the virus-encoded protease, on the basis of the homology of the predicted amino acid sequence with those of previously identified retrovirus proteases.

Amino Acid Sequence↗