The three-dimensional structure of cocksfoot mottle virus at 2.7 A resolution

Cocksfoot mottle virus is a plant virus that belongs to the genus Sobemovirus. The structure of the virus has been determined at 2.7 A resolution. The icosahedral capsid has T = 3 quasisymmetry and 180 copies of the coat protein. Except for a couple of stacked bases, the viral RNA is not visible in the electron density map. The coat protein has a jelly-roll beta-sandwich fold and its conformation is very similar to that of other sobemoviruses and tobacco necrosis virus. The N-terminal arm of one of the three quasiequivalent subunits is partly ordered and follows the same path in the capsid as the arm in rice yellow mottle virus, another sobemovirus. In other sobemoviruses, the ordered arm follows a different path, but in both cases the arms from three subunits meet and form a similar structure at a threefold axis. A comparison of the structures and sequences of viruses in this family shows that the only conserved parts of the protein-protein interfaces are those that form binding sites for calcium ions. Still, the relative orientations and position of the subunits are maintained.     

Identification and characterization of genes encoding the human transferrin-binding proteins from Haemophilus influenzae.

Haemophilus influenzae, a strict human pathogen, acquires iron in vivo through the direct binding and removal of iron from human transferrin by an as yet uncharacterized process at the bacterial cell surface. In this study, the tbpA and tbpB genes of H. influenzae, encoding the transferrin-binding proteins Tbp1 and Tbp2, respectively, were cloned and sequenced. Alignments of the H. influenzae Tbp1 and Tbp2 protein sequences with those of related proteins from heterologous species were analyzed. On the basis of similarities between these and previously characterized proteins, Tbp1 appears to be a member of the TonB-dependent family of outer membrane proteins while Tbp2 is lipid modified by signal peptidase II. Isogenic mutants deficient in expression of Tbp1 or Tbp2 or both proteins were prepared by insertion of the Tn903 kanamycin resistance cassette into cloned sequences and reintroduction of the interrupted sequences into the wild-type chromosome. Binding assays with the mutants showed that a significant reduction in transferrin-binding ability resulted from the loss of either of the Tbps and a complete loss of binding was evident when neither protein was expressed. Loss of either Tbp2 or both proteins correlated with an inability to grow on media supplemented with transferrin-bound iron as the sole source of iron, whereas the Tbp1+ Tbp2- mutant was able to grow only at high transferrin concentrations.     

Analysis of the introns in genes encoding small G proteins

Because all small G proteins (SGPs) possess a very similar array of structural and functional domains, they are obvious candidates for examining the relationships postulated to exist between the exon-intron structure of genes and the domain structure of the encoded proteins. To address this issue, and to possibly gain insight into the evolution of their introns, we have analyzed positions, sizes, and sequences of 125 introns from 28 SGP genes. These introns were found to be distributed in 60 different locations throughout the aligned sequences, with a preference for the 5-half of the genes. More than 50% of the positions were found to be shared by two or more genes, and genes encoding SGPs of very similar amino acid sequence (i.e., isotypes) in quite closely related species tend to have most, or all, of their introns in identical locations, indicating a common evolutionary origin (homologous introns). However, with few exceptions, no statistically significant sequence similarity or common folding motif was found between homologous intron pairs. Only three intron positions are shared between members of distantly related SGP subfamilies. These three potentially ancient intron locations fall between regions encoding -helices or -sheets, but two of them interrupt regions encoding known functional (guanosine-nucleotide-binding) modules. Intron positions that are occupied only in single genes, or in genes encoding very similar SGPs, do not show any preferential distribution with respect to regions encoding structural or functional motifs. This discordance between exon modules and structural and/or functional protein domains suggests that most, if not all, introns in modern SGP genes arose by independent insertion events after diversification of the various SGP subfamilies, and therefore probably did not participate in the early evolution of these genes.     

Identification of Francisella tularensis genes encoding exported membrane-associated proteins using TnphoA mutagenesis of a genomic library

Francisella tularensis, the causative agent of tularemia, is a highly infectious pathogen of humans and animals, yet little is known about the surface proteins of this organism that mediate mechanisms of pathogenicity. lambdaTnphoA was used to generate random alkaline phosphatase gene fusions in a F. tularensis subsp. tularensis (strain Schu S4) genomic library to identify genes encoding exported extracytoplasmic proteins. Eleven genes encoding membrane-associated proteins were identified by this method and their respective signal peptides were characterized. Three of the genes encoded conserved 'housekeeping' enzymes, while the other eight genes were unique to F. tularensis, encoding proteins with molecular masses ranging from 11 to 78kDa as deduced from the amino acid sequences. Two genes putatively encoded lipoproteins based on the presence of characteristic signal peptidase II cleavage sites. Four selected proteins were found associated with outer membranes from Schu S4 and LVS strains by Western blotting. Indirect immunofluorescence of strain Schu S4 cells also showed evidence of protein localization to the outer membrane. Protein database searches produced significant alignments with proteins from other bacteria involved in carbohydrate transport, lipid metabolism, and cell envelope biogenesis, thereby providing clues for putative functions. These findings demonstrated that TnphoA mutagenesis can be used in conjunction with F. tularensis genome sequence data to provide a foundation for studies to identify and define cellular surface protein virulence factors of this pathogen.     

Paralogous genes encoding transport proteins in microbial genomes

The largest superfamilies of prokaryotic genes encode transport proteins, but many transporters are encoded by orphan genes or those that comprise very small families. We have analyzed eighteen completely sequenced prokaryotic genomes for paralogous transport systems and have thereby identified 76 permease families. In this short review, we present and discuss the paralogues in some of these families and interpret the most prominent results, particularly those relevant to the largest permease superfamilies.     

Identification and characterization of Raspberry mottle virus, a novel member of the Closteroviridae

Raspberry mosaic is one of the most important viral diseases of raspberry. Four virus and virus-like agents, two of which are poorly characterized, have been implicated in the disease complex based on symptom development in Rubus indicators. Three novel viruses were identified in a red raspberry plant that caused typical raspberry mosaic symptoms when grafted onto indicators. This communication focuses on one of these viruses, Raspberry mottle virus (RMoV), a new member of the family Closteroviridae. The complete nucleotide sequence of RMoV has been determined and exceeds 17 kilobases encoding 10 genes. The genome organization of RMoV is similar to that of Beet yellows virus, the type member of the Closterovirus genus, and phylogenetic analysis using the polymerase conserved motifs and the heat shock protein 70 homolog revealed a close relationship of RMoV with Strawberry chlorotic fleck associated virus and Citrus tristeza virus, which suggests the possibility of an aphid vector. The virus was detected in symptomatic raspberry plants in production areas in mixed infections with several other viruses, indicating that RMoV may impact raspberry production.     

Evidence for three separate genes encoding the proteins of the mycobacterial antigen 85 complex.

The secreted Mycobacterium bovis BCG antigen 85 complex, which is known to bind to human fibronectin, consists of three closely related cross-reacting antigens. Amino-terminal sequence analysis of the purified proteins showed distinct differences. Data are presented to show that the three components are produced by individual cells, which indicates that three separate genes are involved.     

Patterns of expression of sperm flagellar genes: early expression of genes encoding axonemal proteins during the spermatogenic cycle and shared features of promoters of genes encoding central apparatus proteins

Mol. Hum. Reprod.,     

Identification of the V genes encoding xenoantibodies in non-immunosuppressed rhesus monkeys

The major immunological barrier that prevents the use of wild-type pig xenografts as an alternative source of organs for human xenotransplantation is antibody-mediated rejection. In this study, we identify the immunoglobulin variable region heavy (IgV(H)) chain genes encoding xenoantibodies to porcine heart and fetal porcine islet xenografts in non-immunosuppressed rhesus monkeys. We sought to compare the IgV(H) genes encoding xenoantibodies to porcine islets and solid organ xenografts. The immunoglobulin M (IgM) and IgG xenoantibody response was analysed by enzyme-linked immunosorbent assay and cDNA libraries from peripheral blood lymphocytes were prepared and sequenced. The relative frequency of IgV(H) gene usage was established by colony filter hybridization. Induced xenoantibodies were encoded by the IGHV3-11 germline progenitor, the same germline gene that encodes xenoantibodies in humans mounting active xenoantibody responses. The immune response to pig xenografts presented as solid organs or isolated cells is mediated by identical IgV(H) genes in rhesus monkeys. These animals represent a clinically relevant model to identify the immunological basis of pig-to-human xenograft rejection.     

In situ hybridization screen in zebrafish for the selection of genes encoding secreted proteins.

An in situ hybridization expression screen using a signal sequence trap system has been conducted in zebrafish to isolate cDNAs that encode secreted proteins. Random clones (secreted expressed sequence tags; sESTs) were sequenced from zebrafish embryonic (18-24 hr postfertilization) and adult kidney libraries. From the two RNA sources, 627 random sEST cDNAs were identified as being homologous or identical to known genes and 166 clones encode currently unidentified genes. The sESTs represent a broad range of enzymes and other regulatory molecules. Whole-mount in situ hybridization analysis was carried out by using antisense probes generated from 244 selected sESTs, and a range of expression patterns was obtained. Genetic mapping undertaken with sEST sequences demonstrated that assignment of map position was attainable by using 5' primers. The signal sequence trap system used in this work has yielded a range of cDNAs that encode secreted proteins and, together with analysis of patterns of expression and genetic mapping, has the potential to facilitate analysis of signaling pathways central to development and physiology.     

Identification and quantitative analysis of genes encoding odorant binding proteins in Aedes albopictus (Diptera: Culicidae)

Odorant binding proteins (OBPs) play a critical role in mediating mosquito behaviors. In the current study, four AealOBP genes were cloned and sequenced. Basic Local Alignment Search Tool Program and phylogenetic analysis of the deduced amino acid sequences identified a unique putative ortholog in Aedes aegypti (L.) for each Aedes albopictus (Skuse) OBP. Quantitative analysis showed some AealOBPs with a strong female/male expression ratio, which we proposed to be involved in the host seeking of female mosquitoes. Other OBPs are expressed at higher levels in male antennae and are considered candidates for the detection of floral volatiles. The current study provides basis for further detailed molecular characterization of Ae. albopictus olfactory systems.     

Cloning and sequence analysis of the genes encoding the nonstructural proteins of Langat virus and comparative analysis with other flaviviruses.

Langat virus, a member of the family Flaviviridae is antigenically very similar to highly pathogenic tick-borne encephalitis viruses. We cloned and sequenced the complete nonstructural gene-coding region of Langat virus (strain TP21) and compared the deduced amino acid sequences of each nonstructural protein to those of other flaviviruses. By alignment with the reported amino acid sequences of the nonstructural proteins of several flaviviruses, we were able to predict proteolytic cleavage sites and identify sequence motifs, which are highly conserved among flaviviruses. Sequence similarity calculations revealed that the NS3 and NS5 proteins are the most highly conserved of the flavivirus nonstructural proteins. The NS3 and NS5 proteins of Langat virus contained specific peptide sequences that have been demonstrated to be associated with helicase or polymerase activities, respectively. The NS1 protein of Langat virus displayed complete homology of potential N-linked glycosylation sites and cysteine residues with the NS1 proteins of other tick-borne flaviviruses, suggesting a highly conserved NS1 protein structure. The data presented in this report serve to complete the entire sequence of the Langat virus-coding region and provide the basis for comparison of this naturally attenuated virus to the other highly virulent tick-borne flaviviruses.     

The synthesis and processing of the proteins of bean pod mottle virus in rabbit reticulocyte lysates

Translation of the two RNA components (B-RNA and M-RNA) of bean pod mottle virus (BPMV) in rabbit reticulocyte lysates resulted in the formation of a specific set of proteins. Among the products formed was a viral protease that catalyzed the cleavage of two precursor proteins coded by M-RNA. The protease showed some specificity for BPMV proteins in that it did not cleave the proteins of cowpea mosaic virus (CPMV), which is related to BPMV. Conversely, the synthesized BPMV precursor proteins were not cleaved by a CPMV protease.     

Identification of sequences encoding the equine infectious anemia virus tat gene

Equine infectious anemia virus (EIAV), a lentivirus, encodes a trans-activator (tat) which stimulates gene expression directed by the viral long terminal repeat (LTR). This function has been previously shown by us and others to be encoded by sequences within the middle region of the EIAV genome in which two short open reading frames, S1 and S2, reside. In the present study, by using in vitro mutagenesis, we show that disruption of S1, but not S2, completely abolished trans-activation. Addition of oligonucleotides complementary to S1 to cells transfected with a tat expression vector resulted in inhibition of trans-activation. EIAV cDNAs were isolated from a library of EIAV-infected cells constructed by using a eukaryotic expression vector. One cDNA clone which contained S1 sequences was able to trans-activate the EIAV LTR. Sequence analysis of this cDNA clone revealed that, in addition to S1, two other open reading frames were present. The cDNA still retained its activity when the latter two sequences were deleted.