cDNA cloning and expression of a family of UDP-N-acetyl-D-galactosamine:polypeptide N-acetylgalactosaminyltransferase sequence homologs from Caenorhabditis elegans

The initiation of mucin-type O-glycosylation is catalyzed by a family of UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferases (ppGaNTase) (EC 2.4.1.41). By screening two mixed-stage Caenorhabditis elegans cDNA libraries, a total of 11 distinct sequence homologs of the ppGaNTase gene family were cloned, sequenced, and expressed as truncated recombinant proteins (gly-3, gly-4, gly-5a, gly-5b, gly-5c, gly-6a, gly-6b, gly-6c, gly-7, gly-8, and gly-9). All clones encoded type II membrane proteins that shared 60-80% amino acid sequence similarity with the catalytic domain of mammalian ppGaNTase enzymes. Two sets of cDNA clones (gly-5 and gly-6) contained variants that appeared to be produced by alternative message processing. gly-6c contained a reading frameshift and premature termination codon in the C-terminal lectin-like domain found in most other ppGaNTase proteins, and a second clone (gly-8) lacked the typical C-terminal region completely. Homogenates of nematodes and immunopurified preparations of the recombinant GLY proteins demonstrated that worms express functional ppGaNTase enzymes (GLY-3, GLY-4, GLY-5A, GLY-5B, and GLY-5C), which can O-glycosylate mammalian apomucin peptide sequences in vitro. In addition to demonstrating the existence of ppGaNTase enzymes in a nematode organism, the substantial diversity of these isoforms in C. elegans suggests that mucin O-glycosylation is catalyzed by a complex gene family, which is conserved among evolutionary-distinct organisms.     

A secretory cellulose-binding protein cDNA cloned from the root-knot nematode (Meloidogyne incognita)

A cDNA encoding a secretory cellulose-binding protein was cloned from the root-knot nematode (Meloidogyne incognita) with RNA fingerprinting. The putative full-length cDNA, named Mi-cpb-1, encoded a 203 amino acid protein containing an N-terminal secretion signal peptide. The C-terminal sequence of the putative MI-CBP-1 was similar to a bacterial-type cellulose-binding domain, whereas the N-terminal sequence did not show significant similarity to any proteins in data bases. Recombinant MI-CBP-1 lacked cellulase activity, but bound to cellulose and plant cell walls. In Southern blot hybridization, Mi-cbp-1 hybridized with genomic DNA from M. incognita, M. arenaria, and M. javanica, but not M. hapla, Heterodera glycines, or Caenorhabditis elegans. Polyclonal antibodies raised against recombinant MI-CBP-1 strongly labeled secretory granules in subventral gland cells of second-stage juveniles in indirect immunofluorescence microscopy. Enzyme-linked immunosorbent assay detection of MI-CBP-1 in stylet secretions of second-stage juveniles with the polyclonal antibodies indicated MI-CBP-1 could be secreted through the nematodes' stylet, suggesting that the cellulose-binding protein may have a role in pathogenesis.     

Isolation of AF2 (KHEYLRFamide) from Caenorhabditis elegans: evidence for the presence of more than one FMRFamide-related peptide-encoding gene

Numerous FMRFamide-related peptides (FaRPs) have been isolated and sequenced from extracts of free-living and parasitic nematodes. The most abundant FaRP identified in ethanolic/methanolic extracts of the parasitic forms, Ascaris suum and Haemonchus contortus and from the free-living nematode, Panagrellus redivivus, was KHEYLRFamide (AF2). Analysis of the nucleotide sequences of cloned FaRP-precursor genes from C. elegans and, more recently, Caenorhabditis vulgaris identified a series of related FaRPs which did not include AF2. An acid-ethanol extract of Caenorhabditis elegans was screened radioimmunometrically for the presence of FaRPs using a C-terminally directed FaRP antiserum. Approximately 300 pmols of the most abundant immunoreactive peptide was purified to homogeneity and 30 pmols was subjected to Edman degradation analysis and gas-phase sequencing. The unequivocal primary structure of the heptapeptide, Lys-His-Glu-Tyr-Leu-Arg-Phe-NH2 (AF2) was determined following a single gas-phase sequencing run. The molecular mass of the peptide was determined using a time-of-flight mass spectrometer and was found to be 920 (MH+)+, which was consistent with the theoretical mass of C-terminally amidated AF2. These results indicate that C. elegans possesses more than one FaRP gene.     

Molecular cloning and characterization of an alpha1,3 fucosyltransferase, CEFT-1, from Caenorhabditis elegans

We report on the identification, molecular cloning, and characterization of an alpha1,3 fucosyltransferase (alpha1,3FT) expressed by the nematode, Caenorhabditis elegans . Although C. elegans glycoconjugates do not express the Lewis x antigen Galbeta1-->4[Fucalpha1-->3]GlcNAcbeta-->R, detergent extracts of adult C.elegans contain an alpha1,3FT that can fucosylate both nonsialylated and sialylated acceptor glycans to generate the Lexand sialyl Lexantigens, as well as the lacdiNAc-containing acceptor GalNAcbeta1-->4GlcNAcbeta1-->R to generate GalNAcbeta1-->4 [Fucalpha1-->3]GlcNAcbeta1-->R. A search of the C.elegans genome database revealed the existence of a gene with 20-23% overall identity to all five cloned human alpha1,3FTs. The putative cDNA for the C.elegans alpha1,3FT (CEFT-1) was amplified by PCR from a cDNA lambdaZAP library, cloned, and sequenced. COS7 cells transiently transfected with cDNA encoding CEFT-1 express the Lex, but not sLexantigen. The CEFT-1 in the transfected cell extracts can synthesize Lex, but not sialyl Lex, using exogenous acceptors. A second fucosyltransferase activity was detected in extracts of C. elegans that transfers Fuc in alpha1,2 linkage to Gal specifically on type-1 chains. The discovery of alpha-fucosyltransferases in C. elegans opens the possibility of using this well-characterized nematode as a model system for studying the role of fucosylated glycans in the development and survival of C.elegans and possibly other helminths.     

Golgi localization and functional expression of human uridine diphosphatase

A full-length E(ecto)-ATPase (Plesner, L. (1995) Int. Rev. Cytol. 158, 141-214) cDNA was cloned from a human brain cDNA library; it encodes a 610-amino acid protein that contains two putative transmembrane domains. Heterologous expression of this protein in COS-7 cells caused a significant increase in intracellular membrane-bound nucleoside phosphatase activity. The activity was highest with UDP as substrate and was stimulated by divalent cations in the following order: Ca2+ > Mg2+ > Mn2+. The results of immunofluorescence staining indicate that this protein is located in the Golgi apparatus. UDP hydrolysis was increased in the presence of Triton X-100 or alamethicin, an ionophore that facilitates movement of UDP across the membrane, suggesting that the active site of this UDPase is on the luminal side of the Golgi apparatus. This is the first identification of a mammalian Golgi luminal UDPase gene. Computer-aided sequence analysis of the EATPase superfamily indicates that the human UDPase is highly similar to two hypothetical proteins of the nematode Caenorhabditis elegans and to an unidentified 71.9-kDa yeast protein and is less related to the previously identified yeast Golgi GDPase.     

Caenorhabditis elegans is a nematode

Caenorhabditis elegans is a rhabditid nematode. What relevance does this have for the interpretation of the complete genome sequence, and how will it affect the exploitation of the sequence for scientific and social ends? Nematodes are only distantly related to humans and other animal groups; will this limit the universality of the C. elegans story? Many nematodes are parasites; can knowledge of the C. elegans sequence aid in the prevention and treatment of disease?     

Molecular cloning and functional expression of a human cDNA encoding translation initiation factor 6

Eukaryotic translation initiation factor 6 (eIF6) binds to the 60S ribosomal subunit and prevents its association with the 40S ribosomal subunit. In this paper, we devised a procedure for purifying eIF6 from rabbit reticulocyte lysates and immunochemically characterized the protein by using antibodies isolated from egg yolks of laying hens immunized with rabbit eIF6. By using these monospecific antibodies, a 1.096-kb human cDNA that encodes an eIF6 of 245 amino acids (calculated Mr 26,558) has been cloned and expressed in Escherichia coli. The purified recombinant human protein exhibits biochemical properties that are similar to eIF6 isolated from mammalian cell extracts. Database searches identified amino acid sequences from Saccharomyces cerevisiae, Drosophila, and the nematode Caenorhabditis elegans with significant identity to the deduced amino acid sequence of human eIF6, suggesting the presence of homologues of human eIF6 in these organisms.     

New insights into the negative regulation of hematopoiesis by chemokine platelet factor 4 and related peptides

Platelet factor 4 (PF4) has been recognized as an inhibitor of myeloid progenitors. However, the mechanism of action of this chemokine remains poorly understood. The present study was designed to determine its structure/function relationship. A series of peptides overlapping the C-terminal and central regions of PF4 were analyzed in vitro for their action on murine hematopoietic progenitor growth to assess the minimal sequence length required for activity. The peptides p17-58 and p34-58 possessed an increased hematopoietic inhibitory activity when compared with PF4, whereas the shorter peptides p47-58 and p47-70 were equivalent to the native molecule and the peptide p58-70 was inactive. The PF4 functional motif DLQ located in 54-56 was required for the activity of these peptides. The peptide p34-58 impaired to a similar extent the growth of colony-forming unit-megakaryocyte (CFU-MK) as well as burst-forming unit-erythroid (BFU-E) and colony-forming unit-granulocyte-macrophage (CFU-GM), whereas PF4 was more active on CFU-MK. In the experiments using purified murine CD34(+) marrow cells, statistically significant inhibition induced by p34-58 was shown at concentrations of 2.2 nmol/L or greater for progenitors of the three lineages, whereas that induced by PF4 was seen at 130 nmol/L for CFU-MK and 650 nmol/L for CFU-GM and BFU-E, indicating that the p34-58 acts directly on hematopoietic progenitors and its activity is approximately 60- to 300-fold higher than PF4. The p34-58, unlike PF4, lacked affinity for heparin and its inhibitory activity could not be abrogated by the addition of heparin. In addition, an antibody recognizing p34-58 neutralized the activity of p34-58 but not whole PF4 molecule. These results demonstrate that PF4 contains a functional domain in its central region, which is independent of the heparin binding properties, and provide evidence for a model of heparin-dependent and independent pathways of PF4 in inhibiting hematopoiesis.     

Multiple isoforms of eukaryotic protein synthesis initiation factor 4E in Caenorhabditis elegans can distinguish between mono- and trimethylated mRNA cap structures

The rate-limiting step for cap-dependent translation initiation in eukaryotes is recruitment of mRNA to the ribosome. An early event in this process is recognition of the m7GTP-containing cap structure at the 5'-end of the mRNA by initiation factor eIF4E. In the nematode Caenorhabditis elegans, mRNAs from 70% of the genes contain a different cap structure, m32,2,7GTP. This cap structure is poorly recognized by mammalian elF4E, suggesting that C. elegans may possess a specialized form of elF4E that can recognize m32,2,7GTP. Analysis of the C. elegans genomic sequence data base revealed the presence of three elF4E-like genes, here named ife-1, ife-2, and ife-3. cDNAs for these three eIF4E isoforms were cloned and sequenced. Isoform-specific antibodies were prepared from synthetic peptides based on nonhomologous regions of the three proteins. All three eIF4E isoforms were detected in extracts of C. elegans and were retained on m7GTP-Sepharose. One eIF4E isoform, IFE-1, was also retained on m32,2,7GTP-Sepharose. Furthermore, binding of IFE-1 and IFE-2 to m7GTP-Sepharose was inhibited by m32,2,7GTP. These results suggest that IFE-1 and IFE-2 bind both m7GTP- and m32,2, 7GTP-containing mRNA cap structures, although with different affinities. In conjunction with IFE-3, these eIF4E isoforms would permit cap-dependent recruitment of all C. elegans mRNAs to the ribosome.     

The DevBCA exporter is essential for envelope formation in heterocysts of the cyanobacterium Anabaena sp. strain PCC 7120

Ceramide glucosyltransferase (EC 2.4.1.80) catalyzes the first glycosylation step of glycosphingolipid (GSL) synthesis, the transfer of glucose from UDP-Glucose to hydrophobic ceramide and generate glucosylceramide (GlcCer). We have cloned mouse ceramide glucosyltransferase cDNA from a brain cDNA library by PCR based homology cloning. The nucleotide sequence determination revealed that mouse ceramide glucosyltransferase cDNA encodes 394 amino acids with a calculated molecular mass of 45 kDa. The amino acid sequence of mouse ceramide glucosyltransferase showed 98% identity with the human sequence. Homology searches against currently available databases identified three homologous proteins in Caenorhabditis elegans and one homologous protein in Cyanobacteria. Highly conserved sequences of ceramide glucosyltransferases and the homologs among a wide variety of organisms suggest biological significance of the lipid glucosylation system.     

Identification of cDNA clones for the large subunit of eukaryotic translation initiation factor 3. Comparison of homologues from human, Nicotiana tabacum, Caenorhabditis elegans, and Saccharomyces cerevisiae

Initiation of translation in eukaryotes is mediated by a set of initiation factors. Mammalian initiation factor 3 is composed of at least 8 subunits, with the largest being about 180 kDa in size. Here we report the cloning of the p180 subunit of human eukaryotic translation initiation factor (eIF) 3. The amino acid sequence deduced from the cDNA agrees with the sequences of CNBr fragments of eIF-3, confirming the identity of the clone. The 1382 amino acid open reading frame contains a high percentage of charged residues (48%) and an unusual repetitive domain near the carboxyl terminus composed of 25 repeats of 10 amino acids each. Data base searches identified related sequences found in members of the plant and fungal kingdoms as well as in other mammals and the nematode Caenorhabditis elegans. These sequences share significant identity with the human clone and probably represent the homologues of the p180 subunit in these organisms. This is the first report identifying the sequence of the large subunit of eIF-3.     

Isolation of an RNA-directed RNA polymerase-specific cDNA clone from tomato

A 3600-bp RNA-directed RNA polymerase (RdRP)-specific cDNA comprising an open reading frame (ORF) of 1114 amino acids was isolated from tomato. The putative protein encoded by this ORF does not share homology with any characterized proteins. Antibodies that were raised against synthetic peptides whose sequences have been deduced from the ORF were shown to specifically detect the 127-kD tomato RdRP protein. The immunoresponse to the antibodies correlated with the enzymatic activity profile of the RdRP after chromatography on Q-, poly(A)-, and poly(U)-Sepharose, hydroxyapatite, and Sephadex G-200 columns. DNA gel blot analysis revealed a single copy of the RdRP gene in tomato. RdRP homologs from petunia, Arabidopsis, tobacco, and wheat were identified by using polymerase chain reaction. A sequence comparison indicated that sequences homologous to RdRP are also present in the yeast Schizosaccharomyces pombe and in the nematode Caenorhabditis elegans. The previously described induction of RdRP activity upon viroid infection is shown to be correlated with an increased steady state level of the corresponding mRNA. The possible involvement of this heretofore functionally elusive plant RNA polymerase in homology-dependent gene silencing is discussed.     

Expression of human MRP6, a homologue of the multidrug resistance protein gene MRP1, in tissues and cancer cells

Arachidonic acid and eicosapentaenoic acid are important precursors for the production of prostaglandins and other hormone-like eicosanoid molecules. These fatty acids are synthesized by animals by elongating and desaturating precursor fatty acids such as linoleic acid (18:2Delta9,12) and alpha-linolenic acid (18:3Delta9, 12,15). We have identified a Delta5 fatty acid desaturase gene (fat-4) from the nematode Caenorhabditis elegans. We have expressed this gene product in Saccharomyces cerevisiae and demonstrate that it readily converts di-homo-gamma-linolenic acid (20:3Delta8,11,14) to arachidonic acid (20:4Delta5,8,11,14). The FAT-4 Delta5-desaturase also acts on a number of other substrates, including fatty acids that do not contain a double bond at the Delta8 position.     

Molecular cloning of a new unc-33-like cDNA from rat brain and its relation to paraneoplastic neurological syndromes

Anti-CV2-autoantibodies from patients with paraneoplastic neurological syndromes were used to purify protein(s) related to this disease. A novel cDNA, c-22, was obtained by PCR with primers based on amino-acid sequence of peptides obtained from this protein and rat brain cDNA as template. The deduced amino-acid sequence of c-22 shows homology to the Unc-33 gene from C. elegans in which mutations lead to defects in neuritic outgrowth and axonal guidance and cause uncoordinated movements of the nematode. Several consensus sites for putative protein kinase C phosphorylation were found, suggesting that the c-22 gene product may be a phosphoprotein. Northern hybridizations show that the apparently unique 3.8-kb mRNA of c-22 is present in rat brain tissue and its expression is developmentally regulated: the levels of C-22 mRNA, detectable in brain at embryonic day 17 (E17), increase up to post-natal day 7 (P7) and decline rapidly to an almost undetectable level in adult.     

A gene for a low density lipoprotein receptor-related protein in the nematode Caenorhabditis elegans

A >23-kb gene that encodes a large integral membrane protein with a predicted structure similar to that of the low density lipoprotein (LDL) receptor-related protein (LRP) of mammals has been isolated and sequenced from the free-living nematode Caenorhabditis elegans. The 4753-amino acid predicted C. elegans product shares a nearly identical number and arrangement of amino acid sequence motifs with human LRP, and several exons of the C. elegans LRP gene correspond to exons of related parts of the human LDL receptor gene. The existence of an apparent homolog of LRP in C. elegans offers the possibility of genetic analysis of the in vivo roles of LRP and of the relationship between protein structure and function in a simple model organism.     

Exon 4, and in particular codon 152 of the LDL receptor gene, is a hot spot for point mutations

Chlorpromazine inhibited the hatching of eggs of the parasitic nematode Haemonchus contortus and the free-living nematode Caenorhabditis elegans. In both species, hatching occurred at a concentration of 100 microg/ml but was almost totally blocked at 400 microg/ml. In the case of C. elegans, the effect was shown to be reversible by removal of chlorpromazine after exposure of the eggs to the drug for 1 hr. Caenorhabditis elegans larvae that hatched in a chlorpromazine concentration of 100 microg/ml were killed, but those that hatched in a concentration of 6.25 microg/ml were not. Taken together with data published by others, these observations indicate that the first-stage larva of C. elegans is less sensitive to chlorpromazine than is the adult worm.     

Radiofrequency catheter ablation for idiopathic right ventricular tachycardia with special reference to morphological variation and long-term outcome

We have cloned and sequenced a beta subunit of integrin from a cDNA library of crayfish hemocytes. This beta integrin shows great similarity to beta integrin subunits from other animals; the highest is towards beta pat-3 from Caenorhabditis elegans followed by beta PS from Drosophila melanogaster. By immunoblotting with antibodies raised towards a synthetic peptide corresponding to a part of the cytoplasmic region of the deduced protein sequence, it was shown that the integrin is present in the membrane of the hemocytes. This is the first integrin found in hemocytes of an invertebrate animal and this finding opens the door for further investigations on integrins and their role in the invertebrate immune system.