Oenothera mitochondrial orf454, a gene involved in cytochrome c biogenesis corresponds to orf169 and orf322 of Marchantia

We have characterized a mitochondrial gene in Oenothera, designated orf454, capable of encoding a component of the cytochrome c biogenesis system. This open reading frame is interrupted by an intron of 941 nucleotides showing high similarity to a group II intron residing in the rpl2 gene. RNA editing, which is observed at 18 cytidine positions within the orf454 reading frame, improves the similarity to protein-coding sequences in bacteria and higher plants and removes the last 16 amino acids. orf454 also shows high sequence similarity to two overlapping reading frames (orf169 and orf322) of Marchantia mitochondria. These ORFs belong to an operon-like cluster of genes in the liverwort that is not conserved in Oenothera mitochondria. However, in bacteria these reading frames are organized like the Marchantia gene cluster. It has been shown by genetical analysis in Rhodobacter capsulatus that these genes are essential for cytochrome c biogenesis. Genes of bacterial operons — ccl1 in Rhodobacter and yejR and nrfE in Escherichia coli — show high sequence similarity to the mitochondrial reading frames orf577 and orf454 of Oenothera. orf454, which we describe here, is homologous to the C-terminal region of these bacterial genes, while the previously described orf577 is homologous to the N-terminal region.     

Analysis of BmNPV orf101 disruption: orf101 is essential for mediating budded virus production

In our previous study, Orf101 (Bm101) of Bombyx mori nucleopolyhedrovirus (BmNPV) was identified as a component of the budded virions important for viral late gene expression. In this study we demonstrate that Bm101 is actually a previously unrecognized core gene and that it is essential for mediating budded virus production. To determine the role of Bm101 in the baculovirus life cycle, a Bm101 knockout bacmid containing the BmNPV genome was generated through homologous recombination in Escherichia coli. Furthermore, a Bm101 repair bacmid was constructed by transposing the Bm101 open reading frame with its native promoter region into the polyhedrin locus of the Bm101 knockout bacmid. Bacmid DNA transfection assay revealed that the Bm101 knockout bacmid was unable to produce the infectious budded virus, while the Bm101 repair bacmid rescued this defect, allowing budded-virus titers to reach wild-type levels. Real time PCR analysis indicated that the viral DNA genome in the absence of Bm101 was unaffected in the first 24 h p.t. Thus, studies of a Bm101-null BACmid indicate that Bm101 is required for viral DNA replication during the infection cycle.     

Characterization of the wheat mitochondrial orf25 gene

The wheat mitochondrial orf25 nucleotide sequence of 576 pb has been determined. Its derived protein sequence shares 88% and 75% amino acid identity with those of maize and tobacco mitochondria, respectively. The wheat and tobacco orf25 sequences lack four inserts, of 6 bp to 36 bp, that are present in the maize homologue. The wheat orf25 gene is actively transcribed and is preceded by a regulatory sequence block very similar to those located upstream of the wheat coxII and atp6 genes. Our observations support the view that orf25 sequences encode a functional polypeptide in plant mitochondria.     

Molecular Dissection of Bombyx mori Nucleopolyhedrovirus orf8 Gene

Viruses including baculoviruses are obligatory parasites, as their genomes do not encode all the proteins required for replication. Therefore, viruses have evolved to exploit the behavior and the physiology of their hosts and often eoevolved with their hosts over millions of years. Recent comparative analyses of complete genome sequences of baculoviruses revealed the patterns of gene acquisitions and losses that have occurred during baculovirus evolution. In addition, knowledge of virus genes has also provided understanding of the mechanism of baculovirus infection including replication, species-specific virulence and host range. The Bm8 gene of Bombyx mori nucleopolyhedrovirus (NPV) and its homologues are found only in group I NPV genomes. The Autographa californica NPV Acl6 gene is a homologue of Bm8 and, encodes a viral structural protein. It has been shown that Bm8/Ac 16 interacts with baculoviral and cellular proteins. Bm8/Ac 16 interacts with baculoviral IE1 that is facilitated by coiled coil domains, and the interaction with IE1 is important for Bin8 function. Ac16 also forms a complex with viral FP25 and cellular actin and associates with membranes via palmitoylation. These data suggested that this gene family encodes a multifunctional protein that accomplishes specific needs of group INPVs.     

猪繁殖与呼吸综合征病毒orf7和orf5双基因的原核表达研究

根据PRRSV已知序列设计两对引物,采用RT-PCR方法分别扩增orr7和orf5基因片段.利用EcoRI、SpeⅠ和Hind Ⅲ位点将orf7和orf5基因片段依次克隆到pMD-18T载体,构建成重组质粒pMD18NE,并比较所克隆基因序列的同源性.将串联的orf7和orf5基因亚克隆到原核表达载体pGEX-KG,构建重组原核融合表达质粒pGEX-KGNE,并转化BL21,SDS-PAGE和Western-blot分析表明orf7和orf5基因与GST获得了融合表达,并且表达的融合蛋白GST-NE具有免疫学反应活性,这为猪繁殖与呼吸综合征血清学诊断方法的建立及疫苗研究打下基础.     

Fusion proteins encoded by orf 129L of ectromelia and orf A30L of smallpox viruses cross-react with neutralizing monoclonal antibodies

Open reading frame (orf) 129L of ectromelia (EV) and orf A30L of smallpox viruses (SPV) encoding fusion proteins were cloned and expressed in E. coli cells. The recombinant polypeptides (prA30L H pr129L) were purified from cell lysates by Ni-NTA chromatography. Recombinant polypeptides were able to form trimers in buffered saline and they destroyed under treatment with SDS and 2-mercaptoethanol. Reactivity of prA30L, pr129L and orthopoxvirus proteins was analyzed by ELISA and Western blotting with panel of 22 monoclonal antibodies (MAbs) against orthopoxviruses (19 against EV, 2 MAbs against vaccinia virus and 1 Mabs against cowpox virus). This data allowed us to conclude that there are 12 EV-specific epitopes of pr129L and EV fusion proteins, ten orthopox-specific epitopes of EV, VV, CPV fusion proteins, from them 9 orthopox-specific epitopes of prA30L and SPV fusion proteins. Five Mabs, which cross-reacted with orthopox-specific epitopes, were able to neutralize the VV on Vero cells and from them two MAbs has neutralizing activity against smallpox virus. Our findings demonstrate that 129L fusion protein have EV-specific epitopes, that EV 129L and SPV A30L fusion proteins have a several orthopox-specific epitopes to induce a neutralizing antibodies against human pathogenic orthopoxviruses.     

猪繁殖与呼吸综合征病毒orf7和orf5双基因的原核表达研究

根据PRRSV已知序列设计两对引物,采用RT-PCR方法分别扩增orf7和orf5基因片段。利用EcoRI、SpeI和HindIIl位点将orf7和orf5基因片段依次克隆到pMD-18T载体,构建成重组质粒pMDl8NE,并比较所克隆基因序列的同源性。将串联的orf7和orf5基因亚克隆到原核表达载体pGEX．KG,构建重组原核融合表达质粒pGEX．KGNE,并转化BL21,SDS．PAGE和Western．blot分析表明：orf7和orf5基因与GST获得了融合表达,并且表达的融合蛋白GST-NE具有免疫学反应活性,这为猪繁殖与呼吸综合征血清学诊断方法的建立及疫苗研究打下基础。     

Identification of envelope protein orf10 of channel catfish herpesvirus

Channel catfish virus (CCV) is a viral pathogen of fry and fingerling channel catfish and can cause significant commercial loss. Previous studies have shown that the CCV virion contains at least 25 predicted structural proteins, including viral protein 10, which is encoded by the orf10 gene of the CCV. In this paper, the orf10 gene was expressed in Escherichia coli and used to produce a specific antibody. Western blot analysis confirmed that open reading frame 10 is an envelope protein. A viral neutralization assay demonstrated that open reading frame 10 antiserum was able to inhibit CCV infection of channel catfish ovary cells, suggesting that viral protein 10 is likely to play an important role in the CCV infection of channel catfish ovary cells.     

Characterization of the orf1glnKamtB operon of Herbaspirillum seropedicae

Herbaspirillum seropedicae is an endophytic nitrogen-fixing bacterium that colonizes economically important grasses. In this organism, the amtB gene is co-transcribed with two other genes: glnK that codes for a PII-like protein and orf1 that codes for a probable periplasmatic protein of unknown function. The expression of the orf1glnKamtB operon is increased under nitrogen-limiting conditions and is dependent on NtrC. An amtB mutant failed to transport methylammonium. Post-translational control of nitrogenase was also partially impaired in this mutant, since a complete switch-off of nitrogenase after ammonium addition was not observed. This result suggests that the AmtB protein is involved in the signaling pathway for the reversible inactivation of nitrogenase in H. seropedicae.     

Induction of apoptosis by adenovirus E4orf4 protein

Adenovirus E4orf4 protein is a multifunctional viral regulator that induces p53-independent apoptosis in transformed cells, but not in normal cells. E4orf4-induced apoptosis can occur without activation of known caspases, although E4orf4 induces caspase activity in some cell lines. The interaction of E4orf4 with a specific subpopulation of protein phosphatase 2A (PP2A) molecules that contain B subunits, but not with those that contain B subunits, is required for induction of apoptosis. This review suggests the potential use of E4orf4 in cancer therapy, and discusses whether E4orf4-induced apoptosis plays a role in the viral life cycle. Future research directions are also highlighted.     

Effects of the Deletion of Early Region 4 (E4) Open Reading Frame 1 (orf1), orf1-2, orf1-3 and orf1-4 on Virus-Host Cell Interaction,Transgene Expression,and Immunogenicity of Replicating Adenovirus HIV Vaccine Vectors

The global health burden engendered by human immunodeficiency virus (HIV)-induced acquired immunodeficiency syndrome (AIDS) is a sobering reminder of the pressing need for a preventative vaccine. In non-human primate models replicating adenovirus (Ad)-HIV/SIV recombinant vaccine vectors have been shown to stimulate potent immune responses culminating in protection against challenge exposures. Nonetheless, an increase in the transgene carrying capacity of these Ad vectors, currently limited to approximately 3000 base pairs, would greatly enhance their utility. Using a replicating, E3-deleted Ad type 5 host range mutant (Ad5 hr) encoding full-length single-chain HIV_BaLgp120 linked to the D1 and D2 domains of rhesus macaque CD4 (rhFLSC) we systematically deleted the genes encoding early region 4 open reading frame 1 (E4orf1) through E4orf4. All the Ad-rhFLSC vectors produced similar levels of viral progeny. Cell cycle analysis of infected human and monkey cells revealed no differences in virus-host interaction. The parental and E4-deleted viruses expressed comparable levels of the transgene with kinetics similar to Ad late proteins. Similar levels of cellular immune responses and transgene-specific antibodies were elicited in vaccinated mice. However, differences in recognition of Ad proteins and induced antibody subtypes were observed, suggesting that the E4 gene products might modulate antibody responses by as yet unknown mechanisms. In short, we have improved the transgene carrying capacity by one thousand base pairs while preserving the replicability, levels of transgene expression, and immunogenicity critical to these vaccine vectors. This additional space allows for flexibility in vaccine design that could not be obtained with the current vector and as such should facilitate the goal of improving vaccine efficacy. To the best of our knowledge, this is the first report describing the effects of these E4 deletions on transgene expression and immunogenicity in a replicating Ad vector.     

Roles for the E4 orf6, orf3, and E1B 55-Kilodalton Proteins in Cell Cycle-Independent Adenovirus Replication

Adenoviruses bearing lesions in the E1B 55-kDa protein (E1B 55-kDa) gene are restricted by the cell cycle such that mutant virus growth is most impaired in cells infected during G(1) and least restricted in cells infected during S phase (F. D. Goodrum and D. A. Ornelles, J. Virol. 71:548-561, 1997). A similar defect is reported here for E4 orf6-mutant viruses. An E4 orf3-mutant virus was not restricted for growth by the cell cycle. However, orf3 was required for enhanced growth of an E4 orf6-mutant virus in cells infected during S phase. The cell cycle restriction may be linked to virus-mediated mRNA transport because both E1B 55-kDa- and E4 orf6-mutant viruses are defective at regulating mRNA transport at late times of infection. Accordingly, the cytoplasmic-to-nuclear ratio of late viral mRNA was reduced in G(1) cells infected with the mutant viruses compared to that in G(1) cells infected with the wild-type virus. By contrast, this ratio was equivalent among cells infected during S phase with the wild-type or mutant viruses. Furthermore, cells infected during S phase with the E1B 55-kDa- or E4 orf6-mutant viruses synthesized more late viral protein than did cells infected during G(1). However, the total amount of cytoplasmic late viral mRNA was greater in cells infected during G(1) than in cells infected during S phase with either the wild-type or mutant viruses, indicating that enhanced transport of viral mRNA in cells infected during S phase cannot account for the difference in yields in cells infected during S phase and in cells infected during G(1). Thus, additional factors affect the cell cycle restriction. These results indicate that the E4 orf6 and orf3 proteins, in addition to the E1B 55-kDa protein, may cooperate to promote cell cycle-independent adenovirus growth.     

Deletion of the Candida albicans G-protein-coupled receptor, encoded by orf19.1944 and its allele orf19.9499, produces mutants defective in filamentous growth

Filamentous growth of Candida albicans occurs in response to a variety of environmental signals. The C. albicans gene orf19.1944 and its allele orf19.9499 are identical and are predicted to encode an 823-residue, 7-transmembrane-domain protein that has all the expected features of a G-protein-coupled receptor. The protein is 20.9% identical to the Saccharomyces cerevisiae Gpr1p receptor that signals both glucose availability and nitrogen limitation. Deletion of both copies of the gene in C. albicans abolished filamentation by colonies embedded in rich media (YPS, YPGal, and YPGlu), whereas mutants carrying a single copy of the gene were indistinguishable from the parental strain under these conditions. On medium containing low concentrations of ammonia (SLAD and SLAM media), surface colonies of both the homozygous deletion mutants and the mutants carrying a single copy of the gene were defective in filamentation. Serum-induced germ tube formation was unaffected by deletion of this gene, as was filamentation of the mutants growing on the surface of solid Spider medium at 37 degrees C or embedded in solid Spider medium at 25 degrees C. The protein encoded by orf19.1944 and orf19.9499 has a role in filamentation by both surface and embedded colonies, presumably as a sensor of environmental cues.     

Purification and functional motifs of the recombinant ATPase of orf virus

Our previous study showed that the recombinant ATPase encoded by the A32L gene of orf virus displayed ATP hydrolysis activity as predicted from its amino acids sequence. This viral ATPase contains four known functional motifs (motifs I-IV) and a novel AYDG motif; they are essential for ATP hydrolysis reaction by binding ATP and magnesium ions. The motifs I and II correspond with the Walker A and B motifs of the typical ATPase, respectively. To examine the biochemical roles of these five conserved motifs, recombinant ATPases of five deletion mutants derived from the Taiping strain were expressed and purified. Their ATPase functions were assayed and compared with those of two wild type strains, Taiping and Nantou isolated in Taiwan. Our results showed that deletions at motifs I-III or IV exhibited lower activity than that of the wild type. Interestingly, deletion of AYDG motif decreased the ATPase activity more significantly than those of motifs I-IV deletions. Divalent ions such as magnesium and calcium were essential for ATPase activity. Moreover, our recombinant proteins of orf virus also demonstrated GTPase activity, though weaker than the original ATPase activity.     

Physical mapping of bchG, orf427, and orf177 in the photosynthesis gene cluster of Rhodobacter sphaeroides: functional assignment of the bacteriochlorophyll synthetase gene

The purple photosynthetic bacterium Rhodobacter sphaeroides has within its genome a cluster of photosynthesis-related genes approximately 41 kb in length. In an attempt to identify genes involved in the terminal esterification stage of bacteriochlorophyll biosynthesis, a previously uncharacterized 5-kb region of this cluster was sequenced. Four open reading frames (ORFs) were identified, and each was analyzed by transposon mutagenesis. The product of one of these ORFs, bchG, shows close homologies with (bacterio)chlorophyll synthetases, and mutants in this gene were found to accumulate bacteriopheophorbide, the metal-free derivative of the bacteriochlorophyll precursor bacteriochlorophyllide, suggesting that bchG is responsible for the esterification of bacteriochlorophyllide with an alcohol moiety. This assignment of function to bchG was verified by the performance of assays demonstrating the ability of BchG protein, heterologously synthesized in Escherichia coli, to esterify bacteriochlorophyllide with geranylgeranyl pyrophosphate in vitro, thereby generating bacteriochlorophyll. This step is pivotal to the assembly of a functional photosystem in R. sphaeroides, a model organism for the study of structure-function relationships in photosynthesis. A second gene, orf177, is a member of a large family of isopentenyl diphosphate isomerases, while sequence homologies suggest that a third gene, orf427, may encode an assembly factor for photosynthetic complexes. The function of the remaining ORF, bchP, is the subject of a separate paper (H. Addlesee and C. N. Hunter, J. Bacteriol. 181:7248-7255, 1999). An operonal arrangement of the genes is proposed.