Ovine submaxillary mucin. Primary structure and peptide substrates of UDP-N-acetylgalactosamine:mucin transferase

Tryptic digests of ovine submaxillary apomucin were fractionated by gel filtration and ion exchange chromatography to give 14 peptide fractions. Three purified tryptic peptides, representing 106 of the 650 residues in apomucin, were submitted to automated sequence analysis. The NH2-terminal 50 of the 74 residues in one peptide and the entire sequence of the other two hexadecapeptides were established. These studies suggest that purified ovine submaxillary, mucin is chemically homogeneous, containing a unique primary structure without substantial repeating sequences in its polypeptide chain. The sequences adjacent to 28 known O-glycosidically substituted seryl and threonyl residues were compared. No homologies were apparent around the glycosylated seryl and threonyl residues which might define the specificity of the UDP-N-acetylgalactosaminyl:mucin polypeptide transferase that incorporates N-acetylgalactosamine into O-glycosidic linkage in glycoproteins. However, there appears to be a minimum size requirement for glycosylation, because the transferase catalyzes glycosylation of tryptic peptides efficiently, while chymotryptic and thermolytic peptides were much poorer substrates for the transferase.     

The complete nucleotide sequence of the human immunoglobulin heavy chain variable region locus

The complete nucleotide sequence of the 957-kb DNA of the human immunoglobulin heavy chain variable (VH) region locus was determined and 43 novel VH segments were identified. The region contains 123 VH segments classifiable into seven different families, of which 79 are pseudogenes. Of the 44 VH segments with an open reading frame, 39 are expressed as heavy chain proteins and 1 as mRNA, while the remaining 4 are not found in immunoglobulin cDNAs. Combinatorial diversity of VH region was calculated to be approximately 6,000. Conservation of the promoter and recombination signal sequences was observed to be higher in functional VH segments than in pseudogenes. Phylogenetic analysis of 114 VH segments clearly showed clustering of the VH segments of each family. However, an independent branch in the tree contained a single VH, V4-44.1P, sharing similar levels of homology to human VH families and to those of other vertebrates. Comparison between different copies of homologous units that appear repeatedly across the locus clearly demonstrates that dynamic DNA reorganization of the locus took place at least eight times between 133 and 10 million years ago. One nonimmunoglobulin gene of unknown function was identified in the intergenic region.     

Cloning and nucleotide sequence of mouse immunoglobulin epsilon chain cDNA

cDNA corresponding to mouse IgE heavy (epsilon) chain mRNA was cloned from mouse IgE-secreting hybridoma cells. A clone containing the epsilon cDNA insert was identified by hybridization to epsilon mRNA and subsequent translation in vitro to unprocessed epsilon chain reactive with anti-mouse IgE antibodies. This clone was used to select 20 other epsilon cDNA clones by colony hybridization. The clone containing the longest insert was selected and the epsilon cDNA insert was subjected to sequence analysis. The determined sequence is 1,279 nucleotides long and contains the coding regions for part of the constant region (C epsilon) I and all of the C epsilon 2, C epsilon 3, and C epsilon 4 domains and also the entire 3' untranslated region of epsilon mRNA. When the amino acid sequence determined from the nucleotide sequence is compared to that of human epsilon chain, significant homologies between corresponding domains of the two epsilon chains are found, including conservations in cysteine and tryptophan residues and carbohydrate attachment sites.     

Sequence of a second gene encoding bovine submaxillary mucin: implication for mucin heterogeneity and cloning

Secreted epithelial mucins are extremely large and heterogeneous glycoproteins. We report the 5 kilobase DNA sequence of a second gene, BSM2, which encodes bovine submaxillary mucin. The determined nucleotide and deduced amino acid sequences of BSM2 are 95.2% and 92. 2% identical, respectively, to those of the previously described BSM1 gene isolated from the same cow. Further, the five predicted protein domains of the two genes are 100%, 94%, 93%, 77%, and 88% identical. Based on the above results, we propose that expression of multiple homologous core proteins from a single animal is a factor in generating diversity of saccharides in mucins and in providing resistance of the molecules to proteolysis. In addition, this work raises several important issues in mucin cloning such as assembling sequences from seemingly overlapping clones and deducing consensus sequences for nearly identical tandem repeats.     

Ig heavy chain of the sturgeon Acipenser baeri: cDNA sequence and diversity

To investigate the gene organization of the IGH locus, and the VH diversity of the Siberian sturgeon, a cDNA library was constructed and screened with VH-specific probes from two holostean fish. Isolated clones were analyzed and domain-specific probes used in rescreening of the library, Southern blot analysis, and northern blots. It was concluded that the Siberian sturgeon has one IGH locus with a translocon type of organization. Two allelic variants of the mu gene were found, with identities ranging from 80 to 100% for the different domains (highest for CH4 and lowest for CH2). Sturgeon CH sequences are most closely related to those of holostean fish. There are three distinct VH families, VHI grouping with mammalian clan III, VHII grouping with the teleost clan, and VHIII grouping with the archaic clan. The variability of the CDR 3 region is substantial, and we identified a number of conserved motifs in the D segment. Further, we deduced that there are at least nine different JH segments in the locus, contributing to the antibody repertoire of the sturgeon. The variable segments of the three families can be associated with any of the D or JH segments in the rearrangement. Sturgeon, in addition to the random rearrangement of VH, D, and JH segments, have exonuclease activity, and an introduction of N and probably P nucleotides at the site of rearrangement.     

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.     

Identification of the half-cystine residues in porcine submaxillary mucin critical for multimerization through the D-domains. Roles of the CGLCG motif in the D1- and D3-domains

Plasmids encoding the amino-terminal region of porcine submaxillary mucin were modified by site-specific mutagenesis to assess the roles of individual half-cystine residues in the assembly of disulfide-linked multimers of mucin. COS-7 cells with the plasmid containing C1199A expressed primarily monomers, suggesting that half-cystine 1199 in the D3-domain is involved in forming mucin multimers. This residue is in the sequence C1199SWRYEPCG, which is highly conserved in the D3-domain of other secreted mucins and human prepro-von Willebrand factor. In contrast, cells with the plasmid containing C1276A expressed trimers like those with unmutated plasmid, suggesting that half-cystine 1276 is not involved in formation of disulfide-bonded multimers. The roles of the half-cystines in the CGLCG motifs in the assembly of disulfide-bonded multimers of mucin were also assessed. Cells with plasmids in which both half-cystines in the motif in the D1- or D3-domain of mucin are replaced by alanine expressed proteins that were poorly secreted, suggesting that these mutations impair normal folding of the expressed proteins. A plasmid with a mutant D1-domain motif expressed monomers, whereas one with a mutant D3-domain motif expressed monomers and trimers. However, the trimers expressed by the latter plasmid were assembled in non-acidic compartments, as judged by expression studies in the presence of monensin, which inhibits trimer formation by unmutated plasmid, but not by the mutant plasmid. These results suggest that the CGLCG motif in the D1-domain is required for multimerization in the trans-Golgi complex. However, the CGLCG motif in the D3-domain appears to prevent formation of mucin multimers in non-acidic compartments of the cell. Plasmids encoding the D1- and D2-domains, the D1- and D3-domains, or only the D3-domain also expressed oligomers in the presence of monensin, suggesting that the three D-domains must be contiguous to avoid multimerization in non-acidic compartments. It is possible that these motifs in mucins are engaged in the thiol-disulfide interchange reactions during the assembly of disulfide-bonded multimers of mucin.     

The complete genome sequence of the gram-positive bacterium Bacillus subtilis

Bacillus subtilis is the best-characterized member of the Gram-positive bacteria. Its genome of 4,214,810 base pairs comprises 4,100 protein-coding genes. Of these protein-coding genes, 53% are represented once, while a quarter of the genome corresponds to several gene families that have been greatly expanded by gene duplication, the largest family containing 77 putative ATP-binding transport proteins. In addition, a large proportion of the genetic capacity is devoted to the utilization of a variety of carbon sources, including many plant-derived molecules. The identification of five signal peptidase genes, as well as several genes for components of the secretion apparatus, is important given the capacity of Bacillus strains to secrete large amounts of industrially important enzymes. Many of the genes are involved in the synthesis of secondary metabolites, including antibiotics, that are more typically associated with Streptomyces species. The genome contains at least ten prophages or remnants of prophages, indicating that bacteriophage infection has played an important evolutionary role in horizontal gene transfer, in particular in the propagation of bacterial pathogenesis.     

The complete sequence of four major structural proteins of African horse sickness virus serotype 6: evolutionary relationships within and between the orbiviruses

This paper examines the assumption that male homosexuality has a natural affinity with femininity and that male heterosexuality has a natural affinity with masculinity. An analysis of the relationship between people's disclosure or concealment of their homosexual practice or identity, particularly as it relates to notions of hegemonic masculinity and femininity provides the focus of this paper. It is argued that everyday understandings of homosexuality tend to be resolved in such as way as to press homosexuality into the service of privileging a male, masculine, and heterosexual subjectivity. This privileging is achieved, in part, as a result of the everyday social practices of homosexually active men's witting and unwitting deference to the hegemonic presumption that masculine men are naturally heterosexual, and its inverse, that feminine men are homosexual and are a perturbation of the natural order. We argue that this correlation is manufactured in everyday life in the world of appearances, but that the appearance of things is not reflected at a level of practice, which is to say, male homosexual practice is not necessarily feminine, just as male heterosexual practice is not necessarily masculine. Realities that conflict with the hegemonic realities are masked in the public world, for a variety of reasons. What we have called closet dynamics are the various discourses through which homosexuality is concealed and disclosed, and the various subject positions people take up in relation to those discourses.     

The complete sequence of the genomic RNAs of spinach latent virus

We describe the sequence for the complete genome of spinach latent virus (SpLV). Comparisons of this genome with that of the only other complete genome described for a species within the genus Ilarvirus (citrus leaf rugose virus-CiLRV) indicate that while there are marked differences between the RNA 3 of the two viruses, their respective RNAs 1 and 2 share many similarities. However, the putative 2a protein of SpLV contains a C2H2 type "zinc finger"-like motif located towards the carboxy terminal of the protein which is absent in CiLRV and has not been reported for other members of the family Bromoviridae. A second open reading frame (2b), located at a similar position to that described for the cucumoviruses, occurs in the RNA 2 of both SpLV and CiLRV. The putative coat protein of SpLV is similar to that of citrus variegation virus (CVV) and asparagus virus 2 (AV-2), both members of subgroup 2 of the ilarviruses. We have subsequently demonstrated a serological relationship between SpLV and other viruses in subgroup 2 and suggest that SpLV should be included in this subgroup rather than remain in a separate group (subgroup 6). However, while the putative movement protein of SpLV is remarkably similar to that of AV-2, it shows little relationship with the corresponding protein of CVV and the lack of similarity suggests that a recombination event may have occurred in the past. The relationship between the genera Alfamovirus and Ilarvirus is discussed in the light of the data for the genome of SpLV and recently published information for other members of the genus Ilarvirus.     

The canine factor VIII cDNA and 5'' flanking sequence

Photoreceptors which use a phospholipase C-mediated signal transduction cascade harbor a signaling complex in which the phospholipase Cbeta (PLCbeta), the light-activated Ca2+ channel TRP, and an eye-specific protein kinase C (ePKC) are clustered by the PDZ domain protein INAD. Here we investigated the function of ePKC by cloning the Calliphora homolog of Drosophila ePKC, by precipitating the TRP signaling complex with anti-ePKC antibodies, and by performing phosphorylation assays in isolated signaling complexes and in intact photoreceptor cells. The deduced amino acid sequence of Calliphora ePKC comprises 685 amino acids (MW = 78 036) and displays 80.4% sequence identity with Drosophila ePKC. Immunoprecipitations with anti-ePKC antibodies led to the coprecipitation of PLCbeta, TRP, INAD and ePKC but not of rhodopsin. Phorbolester- and Ca2+-dependent protein phosphorylation revealed that, apart from the PDZ domain protein INAD, the Ca2+ channel TRP is a substrate of ePKC. TRP becomes phosphorylated in isolated signaling complexes. TRP phosphorylation in intact photoreceptor cells requires the presence of extracellular Ca2+ in micromolar concentrations. It is proposed that ePKC-mediated phosphorylation of TRP is part of a negative feedback loop which regulates Ca2+ influx through the TRP channel.     

cDNA sequence and expression of bovine prodynorphin

An enzyme with alpha-L-rhamnosidase activity was purified by anion exchange chromatography from an Aspergillus niger commercial preparation. The alpha-L-rhamnosidase was shown to be N-glycosylated, and had a molecular mass of 85 kD on sodium dodecylsulfate-polyacrylamide gel electrophoresis of which approximately 12% was contributed by carbohydrate. The enzyme was optimally active at pH 4.5 and 65 degrees C. When tested towards p-nitrophenyl-alpha-L-rhamnopyranoside it showed Km and Vmax values of 2.9 mM and 20.6 U mg-1, respectively whereas it was inhibited competitively by L-rhamnose (Ki 3.5 mM). Substrate specificity studies showed alpha-L-rhamnosidase to be active both on alpha-1,2 and alpha-1,6 linkages to beta-D-glucose. Moreover, the enzyme was able to release L-rhamnose from geranyl-beta-D-rutinoside and 2-phenylethyl-beta-D-rutinoside.     

The complete mitochondrial DNA sequence of the pig (Sus scrofa)

Familial adenomatous polyposis (FAP) is an autosomal dominant hereditary disorder caused by a germline inactivating mutation of the adenomatous polyposis coli (APC) gene. Patients with FAP sometimes develop various extracolonic manifestations including adrenocortical neoplasms. We present a 14-year-old boy with FAP who had an adrenocortical tumor with atypical histopathologic features, ie, sex-cord-like differentiation. Immunohistochemical studies of adrenal 4 binding protein (Ad4BP) and steroidogenic enzymes showed the capacity of these tumor cells to produce steroids. Genetic analysis of the tumor disclosed a two-hit mutation in APC: a germline 5-base pair deletion accompanied by a loss of the normal allele. Because there were no reports of genetic alterations in adrenocortical tumors developed in FAP patients, we examined 10 sporadic adrenal tumors (four carcinomas and six adenomas) for mutations in APC. However, no mutations were found in these 10 sporadic adrenal tumors. These results suggest that mutation of APC is also responsible for some fraction of the adrenocortical tumors: the tumor in this case is included.     

The cDNA sequence of proton-pumping nicotinamide nucleotide transhydrogenase from man and mouse

cDNA clones for the human and mouse nicotinamide nucleotide transhydrogenases have been isolated and their sequences have been determined. Multiple alignments show that the functional proteins are encoded by single mRNAs. The deduced amino acid sequences are approximately 95% identical for the previously known bovine, and the human and mouse proteins. The major variable region is located in the presequence. It is proposed that all mammalian transhydrogenases have a similar structure.     

Development of a PCR-restriction fragment length polymorphism assay using the nucleotide sequence of the Helicobacter hepaticus urease structural genes ureAB

Infection with Helicobacter hepaticus causes chronic active hepatitis in certain strains of mice and is associated with hepatocellular carcinoma in A/JCr mice. Like the gastric helicobacters, H. pylori and H. mustelae, H. hepaticus possesses a high level of urease activity. However, the H. hepaticus urease structural gene sequences have not been previously determined, and the role of the urease enzyme in colonization and in pathogenesis is not known. PCR was used to amplify a portion of the urease structural genes from H. hepaticus genomic DNA. Amplified DNA fragments were cloned, and the nucleotide sequence was determined. The deduced amino acid sequence of the partial H. hepaticus ureA gene product was found to exhibit 60% identity and 75% similarity to the predicted H. pylori UreA. The deduced amino acid sequence of a partial H. hepaticus ureB gene product exhibited 75% identity and 87% similarity to the predicted H. pylori UreB. Diversity among H. hepaticus isolates was evaluated by means of a restriction fragment length polymorphism (RFLP) assay. The 1.6-kb fragments within the ureAB open reading frames, amplified from 11 independent isolates, were digested with the restriction endonuclease HhaI. Three distinct RFLP patterns were observed. Identical RFLP profiles were noted in sequential isolates of one strain of H. hepaticus during an 18 month in vivo colonization study, suggesting that the urease genes of H. hepaticus are stable. The urease genes among H. hepaticus strains were also well conserved, showing 98.8 to 99% nucleotide sequence identity among three isolates analyzed. These findings indicate that H. hepaticus has urease structural genes which are homologous to those of the gastric Helicobacter species and that these gene sequences can be used in a PCR and RFLP assay for diagnosis of this important murine pathogen.     

cDNA sequence analysis of the human brain insulin receptor

Brain tissue mRNA was amplified using polymerase chain reaction (PCR) with eight overlapping sets of primers that span the cDNA coding sequence for the human placental insulin receptor. Only the A isoform (lacking exon 11) of the receptor was detected. No difference was found in the predicted amino acid sequence of brain derived insulin receptor cDNA compared with the receptor from human placenta. A silent polymorphism was detected at nucleotide position 1698 (amino acid 523), confirming that mRNA corresponding to both alleles of the human brain receptor was sequenced. Our findings indicate that the unique glycosylation properties of brain insulin receptors do not stem from differences in primary structure, but rather are due to tissue-specific differences in post-translational processing.     

Nucleotide sequence of the murine leukaemia virus amphotropic strain 4070A integrase (IN) coding region and comparative structural analysis of the inferred polypeptide

The complete nucleotide sequence of the integrase (IN) protein coding region of the murine leukaemia virus (MLV) amphotropic strain 4070A is presented. The sequence comprises 1,224 nucleotides, encoding a 408-residue polypeptide of M(r) 46,312. Alignment of the inferred 4070A IN amino acid sequence with the IN proteins of other MLV showed that substitutions are confined largely to segments within the N- and C-terminal domains. In the N-terminal domain the majority of substitutions occur as contiguous 2- to 6-residue blocks, whereas in the C-terminal domain they occur as isolated entities except within a short segment characterized by deletions/insertions. Selection appears to act on the C-terminal 19 residues of IN rather than on the N-terminal residues of ENV (encoded by overlapping reading frames), suggesting a functional role for this segment. Phylogenetic analyses grouped the sequences into two clusters, one comprising IN from the amphotropic strain 4070A and three ecotropic MLV (CAS-BR-E, Moloney and Friend), the other consisting of IN from three ecotropic MLV (two radiation-induced viruses and AKV) and a mink cell focus-forming (MCF) MLV virus. The same dichotomy and cluster composition was obtained from analysis of the long terminal repeat (LTR) regions from these viruses (consistent with the functional interrelationship of IN and LTR) but not from analysis of envelope protein sequences (consistent with the functional independence of ENV proteins from both IN and LTR). Secondary structure predictions supported features determined from the catalytic domain of human immunodeficiency virus and avian sarcoma virus IN, and identified probable structures within the relatively long N- and C-terminal domains of MLV IN proteins.