Hypothetical ORF Research Articles

Development of single nucleotide polymorphism (SNP) application for detection and genotyping of RSIV-type megalocytiviruses.

Red sea bream iridovirus (RSIV) belonging to the genus Megalocytivirus of the family Iridoviridae is the cause of serious mass mortality of cultured marine fishes. RSIV-type megalocytiviruses show extremely high nucleotide sequence identities. Thus, epidemiological studies on this virus are limited. This study developed two primer sets amplifying the regions possessing single nucleotide polymorphism (SNP) to determine the relationships and divergence of RSIV-type megalocytiviruses isolated from cultured marine fishes in Japan. The two regions were designed according to the genome sequences of the representative RSIV genotype II of megalocytivirus members in GenBank. The SNP 1 and 2 regions have sequences homologous to hypothetical protein ORF 24 and ORF 31, respectively, of RSIV (accession no. AP017456.1). By sequencing the regions, 53 polymorphic sites were identified. The phylogenetic analysis of 25 RSIV-type megalocytivirus isolates, classified into RSIV cluster, was clustered into eight haplotypes (seven haplotypes from Oita, two haplotypes from Ehime, and one haplotype shared between Oita and Ehime). These findings suggested that SNP in the RSIV genome is a powerful application for the detection and identification of RSIV-type megalocytiviruses.

Capacity to Elicit Cytotoxic CD8 T Cell Activity Against Mycobacterium avium subsp. paratuberculosis Is Retained in a Vaccine Candidate 35 kDa Peptide Modified for Expression in Mammalian Cells.

Studies focused on development of an attenuated vaccine against Mycobacterium avium subsp. paratuberculosis (Map), the causative agent of paratuberculosis (Ptb) in cattle and other species, revealed that deletion of relA, a global gene regulator, abrogates the ability of Map to establish a persistent infection. In the absence of relA, cattle develop CD8 cytotoxic T cells (CTL) with the ability to kill intracellular bacteria. Analysis of the recall response to a relA mutant, Map/ΔrelA, with cells from a vaccinated steer demonstrated that a 35-kDa membrane peptide (MMP) is one of the targets of the response. This observation suggested that it might be possible to develop a peptide-based vaccine. As reported here, the gene encoding the hypothetical MMP ORF, MAP2121c, was modified for expression in mammalian cells as a first step in developing an expression cassette for incorporation into a mammalian expression vector. The modified sequence of MMP, tPA-MMP, was mutated to generate two additional sequences for the study, one with substitutions to replace five potential residues that could be glycosylated, tPA-MMP-5mut, and one with substitutions to replace the first two potential residues that could be glycosylated, tPA-MMP-2mut. The sequences were placed in an expression cassette to produce peptides for analysis. An ex vivo platform was used with flow cytometry and a bacterium viability assay to determine if modifications in the gene encoding MMP for expression in mammalian cells altered its capacity to elicit development of CD8 CTL, essential for its use in a peptide-based vaccine. Monocyte-depleted PBMC (mdPBMC) were stimulated with antigen-presenting cells (APC) pulsed with different MMP constructs. CD4 and CD8 T cells proliferated in response to stimulation with MMP (control) expressed in Escherichia coli (eMMP), tPA-MMP, and tPA-MMP-2mut. CD8 T cells retained the capacity to kill intracellular bacteria. The tPA-MMP-5mut failed to elicit a proliferative response and was not included in further studies. The data show that the expression cassettes containing MMP and MMP-2mut can be used to screen and select a mammalian expression vector for the development of an efficacious peptide-based vaccine against Ptb.

ExoMeg1: a new exonuclease from metagenomic library.

DNA repair mechanisms are responsible for maintaining the integrity of DNA and are essential to life. However, our knowledge of DNA repair mechanisms is based on model organisms such as Escherichia coli, and little is known about free living and uncultured microorganisms. In this study, a functional screening was applied in a metagenomic library with the goal of discovering new genes involved in the maintenance of genomic integrity. One clone was identified and the sequence analysis showed an open reading frame homolog to a hypothetical protein annotated as a member of the Exo_Endo_Phos superfamily. This novel enzyme shows 3′-5′ exonuclease activity on single and double strand DNA substrates and it is divalent metal-dependent, EDTA-sensitive and salt resistant. The clone carrying the hypothetical ORF was able to complement strains deficient in recombination or base excision repair, suggesting that the new enzyme may be acting on the repair of single strand breaks with 3′ blockers, which are substrates for these repair pathways. Because this is the first report of an enzyme obtained from a metagenomic approach showing exonuclease activity, it was named ExoMeg1. The metagenomic approach has proved to be a useful tool for identifying new genes of uncultured microorganisms.

Screening of early antigen genes of adult-stage Trichinella spiralis using pig serum from different stages of early infection

The goal of this work was to identify novel, early antigens present in Trichinella spiralis. To this end, a cDNA library generated from 3-day old adult worms (Ad3) was immunologically screened using serum from a pig infected with 20,000 muscle larvae. The serum was obtained from multiple, time course bleeds coinciding with early worm development. Seventeen positive clones were isolated using serum obtained at 20 days post infection (dpi). All clones corresponded to one gene that exhibited high sequence identity with the T. spiralis ATP-dependent RNA helicase DDX19B which is involved in parasite growth and development. In addition, nine additional positive clones representing 5 unique genes were identified when the library was screened with 30dpi serum; four of these five genes displayed high similarity with members of a putative T. spiralis serine protease family known to be involved in host invasion and host–parasite interactions. The remaining gene aligned with the T. spiralis hypothetical ORF 11.30. The identification of these antigens provides potential candidates for the early diagnosis of trichinellosis and for the development of a vaccine against this parasite.

Chlamydia trachomatis secretion of hypothetical protein CT622 into host cell cytoplasm via a secretion pathway that can be inhibited by the type III secretion system inhibitor compound 1

Using antibodies raised with C. trachomatis fusion proteins, we localized a hypothetical protein encoded by the ORF ct622 in the cytoplasm of C. trachomatis-infected mammalian cells. The detection was specific since the antibody labelling of CT622 protein was removed by preabsorption with CT622 but not other fusion proteins. We similarly confirmed that CT621, a known secretion protein encoded by a hypothetical ORF downstream of ct622, was secreted into host cell cytosol. Proteins CT622 and CT621 displayed a similar secretion pattern, with both intra-inclusion and host cell cytosol localization, that was distinct from that of CPAF (chlamydial protease/proteasome-like activity factor). However, the expression and secretion kinetics differed significantly between CT622 and CT621: CT622 mRNA was detected at 2 h, protein at 6 h and secretion of protein into host cell cytoplasm at 36 h post-infection, while CT621 mRNA was detected at 8 h, protein at 16 h and secretion at 24 h. The secretion of both CT622 and CT621 was blocked by N'-(3,5-dibromo-2-hydroxybenzylidene)-4-nitrobenzohydrazide (compound 1), an inhibitor known to target the type III secretion system of bacteria. These results suggest that CT621 and CT622 may fulfil different functions during chlamydial intracellular growth. Further characterization of these proteins may generate important information for understanding chlamydial pathogenesis.

Ureaplasma antigenic variation beyond MBA phase variation: DNA inversions generating chimeric structures and switching in expression of the MBA N-terminal paralogue UU172

Phase variation of the major ureaplasma surface membrane protein, the multiple-banded antigen (MBA), with its counterpart, the UU376 protein, was recently discussed as a result of DNA inversion occurring at specific inverted repeats. Two similar inverted repeats to the ones within the mba locus were found in the genome of Ureaplasma parvum serovar 3; one within the MBA N-terminal paralogue UU172 and another in the adjacent intergenic spacer region. In this report, we demonstrate on both genomic and protein level that DNA inversion at these inverted repeats leads to alternating expression between UU172 and the neighbouring conserved hypothetical ORF UU171. Sequence analysis of this phase-variable ‘UU172 element’ from both U. parvum and U. urealyticum strains revealed that it is highly conserved among both species and that it also includes the orthologue of UU144. A third inverted repeat region in UU144 is proposed to serve as an additional potential inversion site from which chimeric genes can evolve. Our results indicate that site-specific recombination events in the genome of U. parvum serovar 3 are dynamic and frequent, leading to a broad spectrum of antigenic variation by which the organism may evade host immune responses.

1084. A Screen for Host Cellular Proteins That Interact with Adeno-Associated Virus Capsid Proteins

Top of pageAbstract Vectors from adeno-associated virus (AAV) appear very promising for use in some gene therapy applications, and have even shown success in animal models. Different serotypes of AAV have been found, each having different properties. For example, it has been shown that AAV-8 uncoats more readily than AAV-2 in the nuclei of hepatocytes in mouse liver, possibly explaining the more robust AAV-8 |[ndash]|transduction efficiencies (Thomas et al., Journal of Virology, 2004, 78 (6) p. 3110|[ndash]|3224). Our goal is to better understand the mechanisms of AAV capsid uncoating in vivo. One approach is to identify cellular proteins that interact with the capsid, and perhaps establish if certain proteins interact with the capsids in a serotype-specific manner. Towards this end, we used a yeast two-hybrid screen to identify cellular proteins that interact with different domains of the AAV-8 capsid. In one of the screens, using the middle portion of the AAV capsid protein VP3 as bait against a mouse liver cDNA library, we found that over half of the library clones recovered encoded part of the same hypothetical ORF AK014478. These clones did interact with the corresponding portion of the AAV-2 capsid, indicating this interaction is not serotype-specific. However, they did not interact with the C- or N-terminal portions of the capsid protein. Homologues for this hypothetical protein have been found only in certain vertebrates and insects, and the human homologue has been shown to localize to the mitochondria. Additional clones isolated from these and related screens are currently being identified, and their association with AAV-capsid proteins are being verified by independent assays. By identifying these cellular proteins such as the one encoded by AK014478 we can better understand the mechanisms behind AAV entry and uncoating in the cell, and design better vectors for gene transfer.

Expression and characterization of Rv2430c, a novel immunodominant antigen of Mycobacterium tuberculosis

About 10% of the coding sequence of Mycobacterium tuberculosis corresponds to hitherto unknown members of the PE and PPE protein families which display significant sequence and length variation at their C-terminal region. It has been suggested that this could possibly represent a rich source of antigenic variation within the pathogen. We describe the purification and biophysical characterization of the recombinant PPE protein coded by hypothetical ORF Rv2430c, a member of the PPE gene family that was earlier shown to induce a strong B cell response. Expression of the recombinant PPE protein in Escherichia coli led to its localization in inclusion bodies and subsequent refolding using dialysis after its extraction from the same resulted in extensive precipitation. Therefore, an on-column refolding strategy was used, after which the protein was found to be in the soluble form. CD spectrum of the recombinant protein displayed predominantly α helical content (81%) which matched significantly with in silico and web-based secondary structure predictions. Furthermore, fluorescence emission spectra revealed that aromatic amino acids are buried inside the protein, which are exposed to aqueous environment under 8 M urea. These results, for the first time, provide evidence on the structural features of PPE family protein which, viewed with its reported immunodominant characteristics, have implications for other proteins of the PE/PPE family.

Characterization of three novel snoRNAs from Schizosaccharomyces pombe implies the structural and functional diversity of box H/ACA snoRNA family

Site-specific pseudouridylations of rRNAs and snRNAs are directed by small guide RNAs that usually possess conserved H and ACA motifs and the consensus secondary structure consisting of hairpin-hinge-hairpin-tail. In this study, through screening cDNA library, three novel box H/ACA snoRNAs, termed SP12, SP16 and SP20, have been identified from fisson yeast Schizosacchromyces pombe. Each of the small RNAs possesses an ACA-like motif and exhibits secondary structures containing two or three large hairpinlike domains. Although the box H is noncanonical, SP20 can be potentially folded into a typical box H/ACA snoRNA-like structure and is predicted to guide two pseudouridylations, U1208 and U1053, on 18S rRNA. In contrast, SP12 and SP16 display different structures from SP20 and have no antisense elements to rRNAs or snRNAs. Obviously, they are new members of orphan guide snoRNAs whose functions remain elusive. The genes encoded for SP12 and SP16 are both located in long intergenic regions between protein-coding genes. A post-transcriptional processing for SP16 precursor was demonstrated. Interestingly, SP20 gene is nested inside a very hypothetical ORF, but transcribed in a reverse sense. Northern and RT-PCR analyses could not detect the transcript from the hypothetical ORF, and therefore exclude the expression of the spurious gene. This is the first example that a genetic locus predicted for protein-coding gene actually encodes a small non-messenger RNA in S. pombe.

SSP2, a sporulation-specific gene necessary for outer spore wall assembly in the yeast Saccharomyces cerevisiae.

Sporulation in yeast consists of two highly coordinated processes. First, a diploid cell that is heterozygous at the mating-type locus undergoes meiosis, in which one round of DNA replication is followed by two rounds of nuclear division. Second, the meiotic products are packaged into spore cells that remain within the mother cell. A large number of genes are induced specifically during sporulation, and their products carry out different sporulation-specific events. Expression of these sporulation-specific genes is controlled by several regulators which function at different stages of the sporulation program, resulting in a cascade of gene expression following induction of meiosis. Here we describe one sporulation-specific gene, SSP2, which is induced midway through meiosis. Ssp2 shows significant homology to the predicted product of a hypothetical ORF in Candida albicans. Homozygous mutant ssp2 diploid cells fail to sporulate. In the mutant background, meiotic recombination and nuclear divisions remain normal; however, viability declines rapidly. Following meiosis, ssp2 cells form the prospore membrane, but fail to form the outer layer of the spore wall. The Ssp2 protein localizes to the spore wall after meiosis II. In addition, the ssp2 defect is also associated with delayed and reduced expression of late sporulation-specific genes. Our results suggest that SSP2 function is required after meiosis II and during spore wall formation.

Functional cloning of the adenylate cyclase gene ofCandida albicans inSaccharomyces cerevisiae within a genomic fragment containing five other genes, including homologues ofCHS6 andSAP185

We have cloned a genomic fragment of Candida albicans by complementation of a Saccharomyces cerevisiae cyr1 mutant. This fragment contains the two-thirds C-terminal part of the adenylate cyclase CaCYR1. The complete gene has been sequenced from PCR fragments amplified from genomic DNA, and contains an ORF of 1690 amino acids closely related to other fungal adenylate cyclases. Adjacent to the adenylate cyclase gene, we have sequenced six other putative genes. CaCHS6, CaYNL191 and CaYJL098 are named on the basis of their close similarity with S. cerevisiae genes. ORFs CaYJL097a and CaYJL097b represent two repeated homologues of the S. cerevisiae YJL097w, which probably arose from an ancient duplication. The last one is a hypothetical ORF, CaYKR049, which presents only a very weak similarity with YKR049. The S. cerevisiae homologues of three of these genes are co-localized on chromosome X but with a different order and orientation.

Identification and Characterization of HU Protein fromMycoplasma gallisepticum

A hypothetical ORF ofMycoplasma gallisepticumwith a putative 99-amino-acid product (ORF99) was noted previously in the upstream region from the type II topoisomerase gene. The amino acid sequence shows weak homology with theEscherichia colihistone-like protein HU. To identify and characterize the protein product of ORF99, we prepared mouse antiserum against recombinant GST-ORF99 fusion protein. The antiserum reacted with an 11-kDa peptide in the crude cell extract ofM. gallisepticum,indicating that this protein is an ORF99 product. ORF99 protein binds to DNA, although its binding affinity is weaker than that ofE. coliHU. When ORF99 was cloned in a plasmid and expressed inE. colicells depleted of HU, Mu phage growth was strongly promoted in the cells, showing the presence of HU activity. The effect of IHF mutation was suppressed when a high level of ORF99 protein was expressed in anE. colimutant deficient in IHF.

The nucleotide sequence of the bacteriophage T5 ltf gene

The nucleotide sequence of the bacteriophage T5 Bg/II- BamHI fragment (4,835 bp in length) known to carry a gene encoding the LTF protein which forms the phage L-shaped tail fibers was determined. It was shown to contain an open reading frame for 1,396 amino acid residues that corresponds to a protein of 147.8 kDa. The coding region of ltf gene is preceded by a typical Shine-Dalgarno sequence. Downstream from the ltf gene there is a strong transcription terminator. Data bank analysis of the LTF protein sequence reveals 55.1% identity to the hypothetical protein ORF 401 of bacteriophage λ in a segment of 118 amino acids overlap.

A large deletion in the plastid DNA of the holoparasitic flowering plant Cuscuta reflexa concerning two ribosomal proteins (rpl2, rpl23), one transfer RNA (trnI) and an ORF 2280 homologue.

We have determined the nucleotide sequence of a 5.3-kb region of the plastid DNA (ptDNA) from the heterotrophic holoparasitic plant Cuscuta reflexa. The cloned area contains genes for the D1-protein (32-kDa protein; psbA), tRNA(His) (trnH), ORF 740 (homologous to ORF 2280 from Nicotiana tabacum), ORF 77 (homologous to ORF 70), tRNA(Leu) (trnL) and a hypothetical ORF 55 which has no homology to any known gene among higher plants. This 5.3-kb area is colinear with a 12.4-kb region of tobacco ptDNA and has therefore undergone several deletions totalling 7.1 kb. Most of the missing nucleotides belong to one large deletion in the ptDNA of C. reflexa of approximately 6.5 kb. This deletion involves two ribosomal protein genes, rpl2 and rpl23, as well as the transfer RNA for Isoleucin (trnI) and a region encoding 1540 amino-acid residues of an ORF 2280 homologue, as compared to tobacco chloroplast DNA. This is remarkable since the remaining genes, especially the psbA gene, are highly conserved in C. reflexa. Furthermore, we found that the expression of the psbA gene is in the same range as in the autotrophic Ipomoea purpurea which belongs to the same family as Cuscuta (Convolvulaceae). Here we hypothesize a total loss of rpl2 and rpl23 in the entire genome of C. reflexa. The phylogenetic position of, and the evolutionary change of ptDNA from, Cuscuta are discussed.