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A set of new algorithms is described for the fast prediction of post-splicing operational reading frames in acceptor sequences, or for predicting expression vectors expressing the correct reading frame in a potential insert.
The products of six unidentified reading frames of human mitochondrial DNA are precipitated from a mitochondrial lysate by antibodies against highly purified native beef heart NADH-ubiquinone oxidoreductase (complex I). These products are enriched greatly in a human submitochondrial fraction enriched in NADH-Q1 and NADH-K3Fe(CN)6 oxidoreductase activities. We conclude that the six reading frames encode components of the respiratory-chain NADH dehydrogenase.
It is known that 5'-untranslated sequences of eukaryotic mRNA often contain AUG triplets, which may serve as the sites of translation initiation. It is thought that these leader open reading frames can fulfil the regulatory functions and encode functionally active proteins. However, they have been incompletely characterized. The article deals with the context organization of leader reading frames of eukaryotic mRNAs. It has been shown that their characteristics correlate with the position relative to the protein-encoding sequence, which may be associated with the efficiency of initiation of translation.
The identification of a nucleic acid sequence's correct reading frame has important implications for homology-independent protein engineering techniques such as incremental truncation and SCRATCHY. We report the development and experimental implementation of a general in-frame selection system, pSALect, a plasmid vector that utilizes two marker sequences flanking the DNA of interest. This dual selection approach overcomes inconsistencies observed with traditional C-terminally fused reporter proteins. In the pSALect vector, sequences of interest are positioned between an N-terminal Tat-signal sequence and a C-terminal beta-lactamase reporter. In-frame selection of the resulting three-domain protein is performed by growing colonies on ampicillin-containing plates, requiring full-length translation in order to link covalently the signal sequence to the lactamase for export into the periplasm. This dual selection scheme has been validated successfully using defined sequences of glycinamide ribonucleotide formyltransferases (GARTs) from Escherichia coli and human and, in contrast to C-terminal fusion systems, proved effective when applied towards the selection of in-frame constructs in an incremental truncation library.
We have determined the nucleotide sequence of a 12.8 kb fragment from the left arm of chromosome XIV carried by the cosmid 14-16d. An analysis of the sequence reveals the presence of a sigma element, a pro-tRNA gene and eight open reading frames, six of which are complete. All of the eight open reading frames correspond to new genes. Of the eight new genes, two show strong similarities to a pair of new genes from chromosome IX, suggesting an ancestral duplication, and one gene encodes a protein similar to mammalian leukotriene A4 hydrolase.
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.
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.
Messenger RNA's are translated in successive three-nucleotide steps (a reading frame), therefore decoding must proceed in only one of three possible frames. A molecular model for correct propagation of the frame is presented based on (i) the measured translational properties of transfer RNA's (tRNA's) that contain an extra nucleotide in the anticodon loop and (ii) a straightforward concept about anticodon loop structure. The model explains the high accuracy of reading frame maintenance by normal tRNA's, as well as activities of all characterized frameshift suppressor tRNA's that have altered anticodon loops.
The DNA sequence of an 8079 bp ClaI fragment located at 40 kb from the centromere on the left arm of chromosome II from Saccharomyces cerevisiae has been determined. Sequence analysis reveals five new open reading frames, tRNA(Gly) and tRNA(Leu) genes as well as sigma and truncated delta elements. The disruption of the three larger open reading frames shows that they are not essential for mitotic growth.
We have recently generated a pool of murine cytomegalovirus (MCMV) mutants by using a Tn3-based transposon mutagenesis approach. In this study, one of the mutants, Rvm166, which contained the transposon sequence at open reading frame m166, was characterized both in tissue culture and in immunocompetent BALB/c mice and immunodeficient SCID mice. The viral mutant replicated as well as the wild-type Smith strain in vitro in NIH 3T3 cells, whereas the transposon insertion precluded the expression of >65% of the m166 open reading frame. Compared to the wild-type strain and a rescued virus that restored the m166 region, the viral mutant was significantly attenuated in growth in both BALB/c and SCID mice that were intraperitoneally infected with the viruses. At 21 days postinfection, the titers of the viral mutant in the salivary glands, lungs, spleens, livers, and kidneys of the infected SCID mice were lower than the titers of the Smith strain and the rescued virus by about 30000-, 10000-, 1000-, 300-, and 800-fold, respectively. Moreover, the virulence of the mutant virus appears to be severely attenuated because no death was found in SCID mice infected with the viral mutant up to 90 days postinfection, whereas all of the animals infected with the wild-type and rescued viruses died at 27 days postinfection. Our results suggest that m166 probably encodes a virulence factor and is required for MCMV virulence in killing SCID mice and for optimal viral growth in vivo.
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.
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.
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.
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.
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.
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.
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.
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.