Expression and hemocyte-targeting of a Campoletis sonorensis polydnavirus cysteine-rich gene in Heliothis virescens larvae

The polydnavirus associated with the parasitic wasp Campoletis sonorensis is injected into the lepidopteran insect, Heliothis virescens, during parasitization, after which viral gene products suppress the cellular immune system of the hosts. Four related cysteine-rich polydnavirus gene have been identified in parasitized H. virescens larvae and grouped into a family. In this study, we investigated the expression and hemocyte targeting of the cysteine-rich VHv1.4 protein. Full-length and truncated VHv1.4 proteins were produced in a bacterial expression system, and the purified proteins were used to raise polyclonal antisera. In immunoblots the VHv1.4 protein was detected in parasitized insects as early as 6 h and throughout the entire course of parasitism. The VHv1.4 protein appeared predominantly in the plasma fraction of hemolymph from parasitized larvae, suggesting that this protein is secreted. The VHv1.4 protein expressed from a recombinant baculovirus was secreted in two lepidopteran cell lines and in larvae injected with the recombinant virus. Digestion with endoglycosidases suggests that the VHv1.4 protein is glycosylated at multiple N-glycosylation sites. Immunofluorescence assays showed that the VHv1.4 protein binds to the hemocytes, most notably the granulocytes, in H. virescens larvae. After binding, the VHv1.4 protein was internalized, probably by endocytosis. Specific binding of the VHv1.4 to granulocytes implies an important function in the suppression of host cellular encapsulation response.     

Elderly hospitalized patients'' perceptions of the interaction with the social worker during discharge planning

During parasitization of Heliothis virescens, Campoletis sonorensis deposits an egg along with venom, polydnavirus particles (CsPDV), and ovarian proteins (OPs). Oviposited eggs are not encapsulated, while washed eggs are encapsulated rapidly by H. virescens. Early protection from encapsulation is afforded by a group of 29-36 kD OP glycoforms. These glycoforms are endocytosed by host hemocytes within 30 min post-parasitization (pp) and disrupt hemocyte spreading behavior and egg encapsulation through at least 24 h p.i. Purified CsPDV does not protect eggs from encapsulation early, but disrupts hemocyte spreading and egg encapsulation from 24 h through at least 5 days p.i. Functional activity of CsPDV appears to be correlated with time-dependent accumulation of virus-specific proteins in parasitized insects. We propose that the fast-acting 29-36 kD OP protects Campoletis eggs from encapsulation until accumulation of CsPDV proteins which sustain immunosuppression.     

Near-haploidy in two malignant fibrous histiocytomas

In order to isolate proteinase genes from parasitic nematodes by polymerase chain reaction (PCR) techniques, we employed a pair of consensus oligonucleotide primers designed to anneal to the active site cysteine (primer ncpC) and asparagine (primer ncpN) coding regions of cysteine proteinases. The primers were biased toward the nucleotide and codon usages of cysteine proteinase genes of nematodes and were based on the consensus nucleotide sequences flanking the active site residues of genes from Haemonchus contortus, Caenorhabditis elegans, and Ostertagia ostertagi. We employed 'touchdown' PCR conditions and were able to amplify novel cysteine proteinase gene fragments from the rodent parasite Strongyloides ratti, the human pathogen S. stercoralis, the canine hookworm Ancylostoma caninum, and from C. elegans. These clones are gene homologs of cathepsin B-like (lysosomal associated) proteases and will facilitate screening of both cDNA and genomic DNA libraries.     

Gene rearrangements in snake mitochondrial genomes: highly concerted evolution of control-region-like sequences duplicated and inserted into a tRNA gene cluster

Mitochondrial DNA (mtDNA) regions corresponding to two major tRNA gene clusters were amplified and sequenced for the Japanese pit viper, himehabu. In one of these clusters, which in most vertebrates characterized to date contains three tightly connected genes for tRNA(Ile), and tRNA(Gln), and tRNA(Met), a sequence of approximately 1.3 kb was found to be inserted between the genes for tRNA(Ile) and tRNA(Gln). The insert consists of a control-region-like sequence possessing some conserved sequence blocks, and short flanking sequences which may be folded into tRNA(Pro), tRNA(Phe), and tRNA(Leu) genes. Several other snakes belonging to different families were also found to possess a control-region-like sequence and tRNA(Leu) gene between the tRNA(Ile)and tRNA(Gln) genes. We also sequenced a region surrounded by genes for cytochrome b and 12S rRNA, where the control region and genes for tRNA(Pro) and tRNA(Phe) are normally located in the mtDNAs of most vertebrates. In this region of three examined snakes, a control-region-like sequence exists that is almost completely identical to the one found between the tRNA(Ile) and tRNA(Gln) genes. The mtDNAs of these snakes thus possess two nearly identical control-region-like sequences which are otherwise divergent to a large extent between the species. These results suggest that the duplicate state of the control-region-like sequences has long persisted in snake mtDNAs, possibly since the original insertion of the control-region-like sequence and tRNA(Leu) gene into the tRNA gene cluster, which occurred in the early stage of the divergence of snakes. It is also suggested that the duplicated control-region-like sequences at two distant locations of mtDNA have evolved concertedly by a mechanism such as frequent gene conversion. The secondary structures of the determined tRNA genes point to the operation of simplification pressure on the T psi C arm of snake mitochondrial tRNAs.     

Periplasmic location of the pesticin immunity protein suggests inactivation of pesticin in the periplasm

The pesticin activity and immunity genes on plasmid pPCP1 of Yersinia pestis were sequenced. They encoded proteins of 40 kDa (pesticin) and 16 kDa (immunity protein); the latter was found in the periplasm. The location of the immunity protein suggests that imported pesticin is inactivated in the periplasm before it hydrolyzes murein. Pesticin contains a TonB box close to the N-terminal end that is identical to the TonB box of colicin B. The DNA sequences flanking the pesticin determinant were highly homologous to those flanking the colicin 10 determinant. It is proposed that through these highly homologous DNA sequences, genes encoding bacteriocins may be exchanged between plasmids by recombination. In the case of pesticin, recombination may have destroyed the lysis gene, of which only a rudimentary fragment exists on pPCP1.     

The short 3''-end region of complementary DNAs as PCR-based polymorphic markers for an expression map of the mouse genome

All of the protein-encoding exons and the 3' flanking region of the human decorin gene have been cloned and partially sequenced. The locations of the intron-exon junctions within the coding portion of the gene were identical to those found for the homologous human gene, biglycan. The sizes of the introns in the decorin gene, however, were substantially larger than those of the same introns of the biglycan gene. Portions of introns 1, 2, and 3 as well as exon 1 were not found during our extensive screening process. The 5' end of intron 2 was found to have an AG-rich region followed immediately by a CT-rich region. Furthermore, the 5' end of intron 3 was very rich in thymidine, whereas the 3' end of intron 7 was rich in adenosine. Several cDNA clones constructed from cultured human bone cell mRNA were found to contain a different sequence at the 5' end compared to that previously published for mRNA from a human embryonic fibroblast cell line. We were also unable to find the alternate 3' flanking region of the previously published cDNA sequence. We have mapped the human decorin gene by in situ methods to chromosome 12q21.3.     

What makes dreams positive or negative: relations to fundamental dimensions of positive and negative mood

The coding sequence at the boundaries of exons flanking nuclear introns shows some degree of conservation. To the extent that such sequences might be recognized by the splicing machinery, this conservation may be a derived result of evolution for efficient splicing. Alternatively, such conserved sequences might be remnants of proto-splice sites, which might have existed early in eukaryotic genes and served as the targets for the insertion of introns, as has been proposed by the introns-late theory. The distribution of intron phases, the position of the intron within a codon, is biased with an over-representation of phase 0 introns. Could any distribution of proto-splice sites account for today's intron phase distribution? Here, we examine the dicodon usage in six model organisms, based on current sequences in the GenBank database, and predict the phase distribution that would be expected if introns had been inserted into proto-splice sites. However, these predictions differ between the various model organisms and disagree with the observed intron phase distributions. Thus, we reject the hypothesis that introns are inserted into hypothetical proto-splice sites. Finally, we analyze the sequences around the splice sites of introns in all six of the species to show that the actual conservation of sequence in exon regions near introns is very small and differs considerably between these species, which is inconsistent with a general proto-splice sites model.     

College students'' attitudes and knowledge of AIDS

Specialized cells within the aphid, Schizaphis graminum, contain intracellular, vesicle-enclosed eubacterial endosymbionts (Buchnera aphidicola). Using oligonucleotide probes derived from conserved sequences of the ATP synthase beta-subunit and glyceraldehyde-3-phosphate dehydrogenase, and the polymerase chain reaction (PCR), we have amplified, cloned, and sequenced three DNA fragments. Amino acid sequence similarity indicated that two of these fragments corresponded to endosymbiont and host genes potentially coding for the beta-subunit of ATP synthase. The host gene fragment contained two putative introns. The third DNA fragment corresponded to a portion of a gene coding for a glyceraldehyde-3-phosphate dehydrogenase that was highly related to one of the enzymes from Escherichia coli (GapA). These results indicate that B. aphidicola may have an ATP synthase and consequently could synthesize ATP from a proton motive force generated within the intracellular vesicles of host cells containing the endosymbionts. The detection of a gene fragment coding for a protein similar to glyceraldehyde-3-phosphate dehydrogenase suggests the presence of this glycolytic enzyme in the endosymbiont and its involvement in energy-yielding metabolism.     

Structural analysis of Arabidopsis thaliana chromosome 5. VII. Sequence features of the regions of 1,013,767 bp covered by sixteen physically assigned P1 and TAC clones

Sixteen P1 and TAC clones assigned to Arabidopsis thaliana chromosome 5 were sequenced, and their sequence features were analyzed using various computer programs. The total length of the sequences determined was 1,013,767 bp. Together with the nucleotide sequences of 109 clones previously reported, the regions of chromosome 5 sequenced so far now total 9,072,622 bp, which presumably covers approximately one-third of the chromosome. A similarity search against the reported gene sequences predicted the presence of a total of 225 protein-coding genes and/or gene segments in the newly sequenced regions, indicating an average gene density of one gene per 4.5 kb. Introns were identified in 72.4% of the potential protein genes for which the entire gene structure was predicted, and the average number per gene and the average length of the introns were 3.3 and 163 bp, respectively. These sequence features are essentially identical to those in the previously reported sequences. The sequence data and gene information are available on the World Wide Web database KAOS (Kazusa Arabidopsis data Opening Site) at http://www.kazusa.or.jp/arabi/.