Structure of the glycoprotein gene of sonchus yellow net virus, a plant rhabdovirus

The nucleotide sequence of the glycoprotein (G) gene of sonchus yellow net virus (SYNV), a plant rhabdovirus, was determined from viral genomic and mRNA cDNA clones. The G cistron is 2045 nucleotides (nt) long and the G protein mRNA open reading frame (ORF), as determined from the cDNA sequence, contains 1896 nt and encodes a protein of 632 amino acids. Immunoblots with antibodies elecited against the purified glycoprotein from virus particles reacted with a fusion protein produced in Escherichia coli, indicating that the cloned ORF encodes the G protein. The 5' end of the G protein mRNA corresponds to nt 5111, relative to the 3' end of the viral (minus sense) genome, as determined by primer extension from mRNA isolated from infected plants, and extends to nt position 7155 on the genomic RNA. A 34-nt untranslated 5' leader sequence and a 115-nt untranslated 3' end flank the ORF on the mRNA. The gene junctions on either side of the G gene on the genomic RNA are identical to those previously described for other SYNV genes and are similar to sequences separating genes of animal rhabdoviruses. The predicted molecular weight of the G protein is 70,215 Da, a value less than the 77,000 Da estimated for the glycosylated G protein from virus particles. The deduced amino acid sequence of the SYNV G protein shares little direct relatedness with the G proteins of other rhabdoviruses, but appears to contain a similar signal sequence, a transmembrane anchor domain, and glycosylation signals. In addition, the SYNV G protein contains a putative nuclear targeting site near the carboxy terminus, which may be involved in transit to the nuclear membrane prior to morphogenesis.     

Expression and characterization of the human YWK-II gene, encoding a sperm membrane protein related to the alzheimer betaA4-amyloid precursorprotein

The YWK-II cDNA, RSD-2, encoding a sperm membrane protein was isolated from a rat testis cDNA expression library. Using the RSD-2 insert in combination with rapid amplification of cDNA ends (RACE), the corresponding human gene was isolated from a human testis cDNA expression library. The human testis cDNA, HSD-2, is 3654 bp in length and contains an open reading frame of 763 codons. Hydropathicity analysis showed that the deduced polypeptide is a single strand transmembrane protein. The deduced polypeptide has partial homology with the amyloid precursor protein (APP) and high homology with the amyloid precursor homologue, APLP2/APPH. The YWK-II gene was mapped and assigned to human chromosome locus: 11q24-25. Northern blotting of various human tissue RNAs using the HSD-2 cDNA as a probe showed that the gene is transcribed ubiquitously. The cytoplasmic domain of HSD-2 was expressed in Escherichia coli. In-vitro studies showed that the recombinant polypeptide bound to a GTP-binding protein (G(o)) and was phosphorylated by protein kinase C and cdc2 kinase. In mammalian F11 cells, the recombinant polypeptide was found to be coupled to G(o). Thus, the YWK-II component has the characteristics of a G(o)-coupled receptor and may be involved in G(o)-mediated signal transduction pathway. Protein kinase C and cdc2 kinase may regulate this pathway in spermatozoa by phosphorylating the cytoplasmic domain of the YWK-II component.     

Molecular characterization of the TCP11 gene which is the human homologue of the mouse gene encoding the receptor of fertilization promoting peptide.

A human testis-specific gene was isolated by subtractive hybridization between the cDNA pools of adult and fetal testes, followed by rapid amplification of cDNA ends (RACE). This gene sequence is highly homologous to a large portion of the mouse Tcp11 gene which is important in sperm function because it encodes the receptor for fertilization-promoting peptide (FPP). The gene was mapped to human chromosome band 6p21 by fluorescence in-situ hybridization. The 9 exon gene spans a 22.8 kp genomic DNA sequence. The mature processed message encodes a 441 amino acid protein that is highly homologous to the mouse 566 amino acid protein after the first 142 amino acids. Results of Northern blot and RT-PCR analyses of RNA extracted from human tissues revealed that the gene is only expressed in fertile adult testes, but not in azoospermic testes, fetal testes nor in other human tissues. Taken together, our results along with the mouse Tcp11 function suggest that TCP11 gene is important in sperm function and fertility.     

Characterisation of the putative nucleoprotein gene of infectious salmon anaemia virus (ISAV)

A gene encoding the putative nucleoprotein (NP) of infectious salmon anaemia virus (ISAV), a commercially important salmonid Orthomyxovirus, has been identified. cDNA obtained from a subtractive cDNA library bound specifically to RNA extracted from ISAV-infected SHK-1 cell cultures. The 5' and 3' ends of the gene were amplified using RACE PCR and a full length open reading frame (ORF) of 1851 nt identified encoding a predicted protein of 616 amino acids. No significant homology of this sequence with any other orthomyxovirus nucleoprotein was identifiable using BLAST or FASTA-based database searches. The ISAV-protein was however identified as a nucleoprotein based on its characteristic amino-acid composition. Furthermore the conserved sequence 5' GCAAAGA 3' was identified preceding the ORF, as has been identified in all other ISAV-genes characterised to date.     

Cloning of Tabby, the murine homolog of the human EDA gene: evidence for a membrane-associated protein with a short collagenous domain

X-Linked hypohidrotic ectodermal dysplasia (XLHED) is a human congenital disorder resulting in abnormal tooth, hair and sweat gland development. A candidate gene for the disorder has been cloned, but the function and full size of its putative protein product is unclear. We have identified a candidate cDNA for the mouse Tabby gene (Ta), which, based on phenotype and syntenic mapping, is postulated to represent the analogous murine disorder. Mutations have been identified in three different Ta alleles and Northern analysis indicates that the gene is expressed at increasing levels during embryogenesis (11-17 days p.c.), the period when affected structures develop. The putative protein product encoded by exon 1 is highly homologous (87% identical) to the predicted EDA protein product (135 amino acids), including the presence of a single transmembrane domain. However, the murine cDNA also encodes an additional 246 amino acids, which contains a short collagenous domain (Gly-X-Y)19. This predicted structure is similar to a number of membrane-associated proteins with either single or multiple collagenous domains in their extracellular C-terminal regions. Since mutations can only be identified in 10-15% of families with XLHED, it is likely that additional homologous exons exist for the human EDA gene. Hybridization of YACs from the EDA region with the Ta cDNA support this hypothesis. The predicted extracellular collagenous domain of this membrane protein may play a key role in epithelial-mesenchymal interactions, defects of which are thought to underlie the Ta/XLHED phenotype.      

A unique amino-terminal sequence predicted for the chicken proto-ets protein

Avian cellular sequences homologous to the ets domain of the avian leukemia retrovirus, E26, have been characterized, and shown to contain nine discrete regions within a single locus of about 60 kb. The structure of the viral homologous and nonhomologous domains of this chicken ets-1 gene is characterized and seen to define the unique amino acid sequences of the ets protein. We have isolated and sequenced an avian ets-1 cDNA clone obtained from chicken thymus. This cDNA clone contains an open reading frame (ORF) encoding the normal cellular product of 441 amino acids. This product is significantly smaller than that encoded by the v-ets domain of the E26 transforming protein, p135, which contains 491 amino acids. The open reading frame predicted by our sequence data results in a protein calculated to be 50 kDa, which is slightly smaller than that actually observed in chicken cells. The presence of termination codons 5' to this ORF demonstrates that the cDNA characterized contains the entire coding region for the chicken ets gene.     

Plasmodium vivax merozoite surface protein-3 contains coiled-coil motifs in an alanine-rich central domain.

Plasmodium merozoites are covered with a palisade layer of proteins that are arranged as organized bundles or appear as protruding spikes by electron microscopy. Here we present a third Plasmodium vivax merozoite surface protein, PvMSP-3, which is associated with but not anchored in the merozoite membrane. Serum from a P. vivax immune squirrel monkey was used to screen a lambdagt11 P. vivax genomic DNA (gDNA) library. Plaque-selected antibodies from clone no. 6.1, and rabbit antisera against its encoded protein, produced a pattern in immunofluorescence assays (IFAs) that is consistent with a localization at the surface of mature schizonts and free merozoites. Specific antisera also agglutinated merozoites and recognized a protein of 150 000 Da by SDS-PAGE. The complete msp-3 gene and flanking sequences were cloned from a P. vivax lambda Dash II gDNA library and also partly characterized by RACE (rapid amplification of cDNA ends). The immediate upstream sequence contains non-coding repeats and a putative protein encoding open reading frame (ORF), which are also present on the msp-3 5'RACE gene product. Pvmsp-3 encodes a protein with a calculated mass of 89 573 Da, which has a potential signal peptide and a major central alanine-rich domain (31%) that exhibits largely alpha-helical secondary structure and is flanked by charged regions. The protein does not have a putative transmembrane domain or a consensus sequence for a glycosylphosphatidylinositol (GPI) anchor modification. However, the alanine-rich domain has heptad repeats that are predicted to form coiled-coil tertiary structures, which mediate protein-protein interactions. PvMSP-3 is structurally related to P. falciparum MSP-3 and the 140000 Da MSP of P. knowlesi. Characterization of PvMSP-3, thus, also begins to define a new interspecies family of evolutionarily related Plasmodium merozoite proteins.     

The genomic organization of the mouse CD94 C-type lectin gene.

The mouse natural killer (NK) gene complex is located on chromosome 6 and contains a number of genes encoding C-type lectin receptors which have been found to regulate NK cell function. Among these are CD94 and four NKG2 genes. Like its human counterpart, the mouse CD94 protein associates with different NKG2 isoforms and recognizes the atypical MHC class I molecule Qa-1b. Here, the genomic organization of the mouse CD94 gene was determined by analysing a BAC clone containing the CD94 gene. The mouse CD94 gene contains six exons separated by five introns. Exons I and II encode the 5' untranslated region (UTR) and the transmembrane domain. Exon III encodes the stalk region and exons IV-VI encode the carbohydrate recognition domain (CRD). Furthermore, we cloned and sequenced the CD94 promoter region, and putative regulatory DNA elements were identified. Further studies on the CD94 promoter region may help to elucidate the restricted expression pattern of CD94 in NK cells and a subpopulation of T cells.     

Beilong virus, a novel paramyxovirus with the largest genome of non-segmented negative-stranded RNA viruses

During a subtraction study on gene expression in human kidney mesangial cells (HMCs), cDNA clones with sequence homology to paramyxovirus P, M and F genes were isolated. Subsequent investigation revealed that this particular HMC line was infected with a previously unknown paramyxovirus. Here, we report the isolation and genome characterization of this new virus, now named Beilong virus (BeV). The genome of BeV is 19,212 nucleotides (nt) in length and is the largest among all known members of the order Mononegavirales. The BeV genome contains eight genes in the order 3'-N-P/V/C-M-F-SH-TM-G-L-5'. The SH and TM genes code for a small hydrophobic protein of 76 aa and a transmembrane protein of 254 aa, respectively. The BeV G gene, at 4527 nt, codes for an attachment protein of 734 aa and contains two additional open reading frames (ORFs) in the 3' half of the gene, coding for putative proteins of 299 and 394 aa in length. Although the exact origin of BeV is presently unknown, we provide evidence indicating that BeV was present in a rat mesangial cell line used in the same laboratory prior to the acquisition of the HMC line, suggesting a potential rodent origin for BeV.     

A SPGY copy homologous to the mouse gene Dazla and the Drosophila gene boule is autosomal and expressed only in the human male gonad

We have isolated a series of human testis poly(A) cDNA clones by cross- hybridization to SPGY1, a Y gene homologous to DAZ. Their sequence analysis revealed an identical nucleotide composition in different 'full-length' clones, suggesting that all were encoded by the same gene. We mapped this gene to the short arm of chromosome 3 and designated it SPGYLA (SPGY like autosomal). Comparison of the SPGYLA cDNA sequence with the cDNA sequences of DAZ and SPGY1 revealed two prominent differences. The tandem repetitive structure of 72 bp sequence units (DAZ repeats) is absent. SPGYLA contains only one 72 bp sequence unit. Downstream of it, a specific 130 bp sequence domain is present which is absent in DAZ and SPGY1 but present in the mouse gene Dazla and in the Drosophila gene boule. SPGYLA encodes an RNA binding protein expressed only in the human male gonad. The data presented give strong evidence that not DAZ but SPGYLA is the functional human homologue of Dazla and boule.