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.     

Insertion into Aspergillus nidulans of functional UDP-GlcNAc: alpha 3-D- mannoside beta-1,2-N-acetylglucosaminyl-transferase I, the enzyme catalysing the first committed step from oligomannose to hybrid and complex N-glycans

Filamentous fungi are capable of secreting relatively large amounts of heterologous recombinant proteins. Recombinant human glycoproteins expressed in this system, however, carry only carbohydrates of the oligomannose type limiting their potential use in humans. One approach to the problem is genetic engineering of the fungal host to permit production of complex and hybrid N-glycans. UDP-GlcNAc:alpha 3-D-mannoside beta- 1,2-N-acetylglucosaminyltransferase I (GnT I) is essential for the conversion of oligomannose to hybrid and complex N-glycans in higher eukaryotic cells. Since GnT I is not produced by fungi, we have introduced into the genome of Aspergillus nidulans the gene encoding full-length rabbit GnT I and demonstrated the expression of GnT I enzyme activity at levels appreciably higher than occurs in most mammalian tissues. All the GnT I activity in the Aspergillus transformants remains intracellular suggesting that the rabbit trans-membrane sequence may be capable of targeting GnT I to the fungal Golgi apparatus.     

Cloning of total mRNA populations from adult and embryonic mice

Total clone banks of cDNAs synthesized from poly(A)-RNA obtained from three stages of the developing mouse were constructed. The stages chosen were 13-day-old embryo, neonatal, and fully grown adult. To have as complete a bank as possible, large numbers of individual clones were generated approximately 400,000 for the 13th day embryo and neonatal mouse and approximately 610,000 for the adult bank. In each case the clone bank was constructed by inserting double stranded cDNA into the PstI site of pBR322 by the "G-C tailing" method. Sequences cloned in this way could be separated from the plasmid host DNA by treatment of the resultant total chimeric plasmid population with PstI. Aliquots of the cloned cDNA material were labeled with 32P by "nick translation" using Escherichia coli DNA polymerase I for the preparation of hybridization probes. Back-hybridization of these probes to the total clone banks allowed the determination of the sequence diversity among the above three very different developmental stages. The use of such clone banks should allow the identification of developmental stage specific mRNAs.     

cDNA cloning and gene mapping of human homologs for Schizosaccharomyces pombe rad17, rad1, and hus1 and cloning of homologs from mouse, Caenorhabditis elegans, and Drosophila melanogaster

Mutations in DNA repair/cell cycle checkpoint genes can lead to the development of cancer. The cloning of human homologs of yeast DNA repair/cell cycle checkpoint genes should yield candidates for human tumor suppressor genes as well as identifying potential targets for cancer therapy. The Schizosaccharomyces pombe genes rad17, rad1, and hus1 have been identified as playing roles in DNA repair and cell cycle checkpoint control pathways. We have cloned the cDNA for the human homolog of S. pombe rad17, RAD17, which localizes to chromosomal location 5q13 by fluorescence in situ hybridization and radiation hybrid mapping; the cDNA for the human homolog of S. pombe rad1, RAD1, which maps to 5p14-p13.2; and the cDNA for the human homolog of S. pombe hus1, HUS1, which maps to 7p13-p12. The human gene loci have previously been identified as regions containing tumor suppressor genes. In addition, we report the cloning of the cDNAs for genes related to S. pombe rad17, rad9, rad1, and hus1 from mouse, Caenorhabditis elegans, and Drosophila melanogaster. These include Rad17 and Rad9 from D. melanogaster, hpr-17 and hpr-1 from C. elegans, and RAD1 and HUS1 from mouse. The identification of homologs of the S. pombe rad checkpoint genes from mammals, arthropods, and nematodes indicates that this cell cycle checkpoint pathway is conserved throughout eukaryotes.     

Circulatory arrest

UDP-GlcNAc: alpha-3-D-mannoside beta-1,2-N-acetylglucosaminyltransferase I (GnTI, EC 2.4.1.101) plays an essential role in the conversion of oligomannose to complex and hybrid N-glycans. Rabbit GnTI is 447 residues long and has a short four-residue N-terminal cytoplasmic tail, a 25-residue putative signal-anchor hydrophobic domain, a stem region of undetermined length and a large C-terminal catalytic domain, a structure typical of all glycosyltransferases cloned to date. Comparison of the amino acid sequences for human, rabbit, mouse, rat, chicken, frog and Caenorhabditis elegans GnTI was used to obtain a secondary structure prediction for the enzyme which suggested that the location of the junction between the stem and the catalytic domain was at about residue 106. To test this hypothesis, several hybrid constructs containing GnT I with N- and C-terminal truncations fused to a mellitin signal sequence were inserted into the genome of Autographa californica nuclear polyhedrosis virus (AcMNPV), Sf9 insect cells were infected with the recombinant baculovirus and supernatants were assayed for GnTI activity. Removal of 29, 84 and 106 N-terminal amino acids had no effect on GnTI activity; however, removal of a further 14 amino acids resulted in complete loss of activity. Western blot analysis showed strong protein bands for all truncated enzymes except for the construct lacking 120 N-terminal residues indicating proteolysis or defective expression or secretion of this protein. The data indicate that the stem is at least 77 residues long.     

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.     

Human herpesvirus 6 open reading frame U12 encodes a functional beta-chemokine receptor

Human herpesvirus 6 (HHV- 6), which belongs to the betaherpesvirus subfamily and infects mainly T cells in vitro, causes acute and latent infections. HHV- 6 contains two genes (U12 and U51) that encode putative homologs of cellular G-protein-coupled receptors (GCR), while three other betaherpesviruses, human cytomegalovirus, murine cytomegalovirus, and human herpesvirus 7, have three, one, and two GCR-homologous genes, respectively. The U12 gene is expressed late in infection from a spliced mRNA. The U12 gene was cloned, and the protein was expressed in cells and analyzed for its biological characteristics. U12 functionally encoded a calcium-mobilizing receptor for beta-chemokines such as regulated upon activation, normal T expressed and secreted (RANTES), macrophage inflammatory proteins 1alpha and 1beta (MIP-1alpha and MIP-1beta) and monocyte chemoattractant protein 1 but not for the alpha-chemokine interleukin-8, suggesting that the chemokine selectivity of the U12 product was distinct from that of the known mammalian chemokine receptors. These findings suggested that the product of U12 may play an important role in the pathogenesis of HHV- 6 through transmembrane signaling by binding with beta-chemokines.     

Histamine receptor-independent muscle relaxation elicited by a series of histamine H2-receptor agonists on the isolated guinea pig duodenum: a study into the mechanism of action

To assess the dolphin's capacity for color vision and determine the absorption maxima of the dolphin visual pigments, we have cloned and expressed the dolphin opsin genes. On the basis of sequence homology with other mammalian opsins, a dolphin rod and long-wavelength sensitive (LWS) cone opsin cDNAs were identified. Both dolphin opsin cDNAs were expressed in mammalian COS-7 cells. The resulting proteins were reconstituted with the chromophore 11-cis-retinal resulting in functional pigments with absorption maxima (lambdamax) of 488 and 524 nm for the rod and cone pigments respectively. These lambdamax values are considerably blue shifted compared to those of many terrestrial mammals. Although the dolphin possesses a gene homologous to other mammalian short-wavelength sensitive (SWS) opsins, it is not expressed in vivo and has accumulated a number of deletions, including a frame-shift mutation at nucleotide position 31. The dolphin therefore lacks the common dichromatic form of color vision typical of most terrestrial mammals.     

Occurrence of two somatostatin variants in the frog brain: characterization of the cDNAs, distribution of the mRNAs, and receptor-binding affinities of the peptides

In tetrapods, only one gene encoding a somatostatin precursor has been identified so far. The present study reports the characterization of the cDNA clones that encode two distinct somatostatin precursors in the brain of the frog Rana ridibunda. The cDNAs were isolated by using degenerate oligonucleotides based on the sequence of the central region of somatostatin to screen a frog brain cDNA library. One of the cDNAs encodes a 115-amino acid protein (prepro-somatostatin-14; PSS1) that exhibits a high degree of structural similarity with the mammalian somatostatin precursor. The other cDNA encodes a 103-amino acid protein (prepro-[Pro2, Met13]somatostatin-14; PSS2) that contains the sequence of the somatostatin analog (peptide SS2) at its C terminus, but does not exhibit appreciable sequence similarity with PSS1 in the remaining region. In situ hybridization studies indicate differential expression of the PSS1 and PSS2 genes in the septum, the lateral part of the pallium, the amygdaloid complex, the posterior nuclei of the thalamus, the ventral hypothalamic nucleus, the torus semicircularis and the optic tectum. The somatostatin variant SS2 was significantly more potent (4-6 fold) than somatostatin itself in displacing [125I-Tyr0, D-Trp8] somatostatin-14 from its specific binding sites. The present study indicates that the two somatostatin variants could exert different functions in the frog brain and pituitary. These data also suggest that distinct genes encoding somatostatin variants may be expressed in the brain of other tetrapods.     

Temporal and spatial expression of distinct troponin T genes in embryonic/larval tail striated muscle and adult body wall smooth muscle of ascidian

During development of the ascidian Halocynthia roretzi, the tadpole larva hatched from the tailbud embryo metamorphoses to the adult with a body wall muscle. Although the adult body wall muscle is morphologically nonsarcomeric smooth muscle, it contains a troponin complex consisting of three subunits (T, I, and C) as do vertebrate striated muscles. Different from vertebrate troponins, however, the smooth muscle troponin promotes actin-myosin interaction in the presence of high concentration of Ca2+, and this promoting property is attributable to troponin T. To address whether the embryonic/larval tail striated muscle and the adult smooth muscle utilize identical or different regulatory machinery, we cloned troponin T cDNAs from each cDNA library. The embryonic and the adult troponin Ts were encoded by distinct genes and shared only < 60% identity with each other. These isoforms were specifically expressed in the embryonic/larval tail striated muscle and the adult smooth muscle, respectively. These results may imply that these isoforms regulate actin-myosin interaction in different manners. The adult troponin T under forced expression in mouse fibroblasts was unexpectedly located in the nuclei. However, a truncated protein with a deletion including a cluster of basic amino acids colocalized with tropomyosin on actin filaments. Thus, complex formation with troponin I and C immediately after the synthesis is likely to be essential for the protein to properly localize on the thin filaments.     

Alternative splicing of a Caenorhabditis elegans gene produces two novel inhibitory amino acid receptor subunits with identical ligand binding domains but different ion channels

Two full-length cDNAs, gbr-2A and gbr-2B, encoding inhibitory amino acid receptor subunits have been amplified and cloned from Caenorhabditis elegans mRNA. The 5' 732 bp of the two cDNAs, encoding 237 amino acids, are identical. The 3' 758 bp of the gbr-2B cDNA are present within the 3' untranslated region of the gbr-2A clone. As a result, the two cDNAs are predicted to encode subunits which share a common extracellular N-terminal sequence of 237 amino acids, but different, though closely related, C-terminal sequences which include four predicted membrane-spanning regions. A search of the EMBL database revealed that the sequences of the two subunits are most closely related to the alpha-subunit of the C. elegans avermectin receptor. Northern blot analysis showed the presence of two related mRNAs of approximately 2.2 and 1.5 kb in a developmentally mixed population of C. elegans. The genomic DNA sequence confirms that both mRNAs were transcribed from the same gene, gbr-2, suggesting that the closely related 3' sequences have arisen as a result of a partial gene duplication event. We propose that C. elegans is utilising alternative splicing to generate receptor subunits with identical extracellular, ligand-binding domains but different transmembrane, channel forming domains.     

Nature and regulation of pistil-expressed genes in tomato

The specialized reproductive functions of angiosperm pistils are dependent in part upon the regulated activation of numerous genes expressed predominantly in this organ system. To better understand the nature of these pistil-predominant gene products we have analyzed seven cDNA clones isolated from tomato pistils through differential hybridization screening. Six of the seven cDNAs represent sequences previously undescribed in tomato, each having a unique pistil- and/or floral-predominant expression pattern. The putative protein products encoded by six of the cDNAs have been identified by their similarity to sequences in the database of previously sequenced genes, with a seventh sequence having no significant similarity with any previously reported sequence. Three of the putative proteins appear to be targeted to the endomembrane system and include an endo-beta-1,4-glucanase which is expressed exclusively in pistils at early stages of development, and proteins similar in sequence to gamma-thionin and miraculin which are expressed in immature pistils and stamens, and in either sepals or petals, respectively. Two other clones, similar in sequence to each other, were expressed primarily in immature pistils and stamens and encode distinct proteins with similarity to leucine aminopeptidases. An additional clone, which encodes a protein similar in sequence to the enzyme hyoscyamine 6-beta-hydroxylase and to other members of the family of Fe2+/ascorbate-dependent oxidases, was expressed at high levels in pistils, stamens and sepals, and at detectable levels in some vegetative organs. Together, these observations provide new insight into the nature and possible functional roles of genes expressed during reproductive development.