Human mucosal addressin cell adhesion molecule-1 (MAdCAM-1) demonstrates structural and functional similarities to the alpha 4 beta 7-integrin binding domains of murine MAdCAM-1, but extreme divergence of mucin-like sequences

The mucosal vascular addressin, mucosal addressin cell adhesion molecule-1 (MAdCAM-1), is an Ig family adhesion receptor preferentially expressed by venular endothelial cells at sites of lymphocyte extravasation in murine mucosal lymphoid tissues and lamina propria. MAdCAM-1 specifically binds both human and mouse lymphocytes that express the homing receptor for Peyer's patches, the integrin alpha 4 beta 7. Functional expression cloning was used to isolate a cDNA from a macaque mesenteric lymph node library that encodes the homologue to murine MAdCAM-1. The macaque cDNA was subsequently used to clone the human homologue as well. Expression of human MAdCAM-1 RNA is restricted to mucosal tissues, gut-associated lymphoid tissues and spleen. Human MAdCAM-1 selectively binds both murine and human lymphocyte cell lines expressing alpha 4 beta 7. Human and macaque MAdCAM-1 have two Ig-like domains that are similar to the two amino-terminal integrin binding domains of murine MAdCAM-1. The conservation of sequences within the region homologous to the mucin/IgA domain of murine MAdCAM-1 is, however, much less apparent. These receptors exhibit considerable variation from murine MAdCAM-1 with respect to the length of the mucin-like sequence and the lack of a membrane proximal Ig (IgA-like) domain. The isolation of these different species of MAdCAM-1 demonstrates greater seleoffctive pressure for maintenance of amino acids involved in alpha 4 beta 7 binding than those sequences presumably involved in the presentation of carbohydrates for selectin binding.     

Biochemical characterization and molecular cloning of a novel endothelial-specific sialomucin

We have generated rat monoclonal antibodies (MoAbs) against cell surface antigens of the mouse endothelioma cell line bEND.3. Three antibodies (V.1A7, V.5C7, and V.7C7) were selected, all of which recognize a 75-kD antigen on bEND.3 cells and bind selectively to endothelial cells in cryostat sections of mouse tissues. A cDNA for the antigen was isolated from a bEND.3 pCDM8 expression library by using transient expression in COS-7 cells and immunoselection with the three MoAbs. This cDNA coded for a novel, type I membrane protein of 248 amino acids with an extracellular domain rich in threonine and serine residues (35%). The protein is sensitive to O-sialoglycoprotein endopeptidase, indicating that it belongs to the class of sialomucin-like proteins. Therefore, we suggest the name endomucin. Treatment of isolated endomucin by sialidase and O-glycosidase reduced the apparent molecular weight to 45 kD and abolished binding of all three antibodies, indicating that carbohydrates are directly or indirectly involved in the formation of the antibody epitopes. Immunohistological analysis of all examined mouse tissues showed that endomucin is an endothelial antigen found in venous endothelium as well as in capillaries, but not on arterial endothelium. Interestingly, high endothelial venules of peripheral and mesenteric lymph nodes as well as of Peyers's patches were negative for staining with the three MoAbs.     

L-selectin-mediated lymphocyte rolling on MAdCAM-1

The L-selectin, a cell surface C-type lectin, directs lymphocyte traffic to lymph nodes, and contributes to lymphocyte homing to Peyer's patches and to leukocyte interactions with inflamed venules. Here we report that the mucosal vascular addressin MAdCAM-1, a mucosal endothelial adhesion molecule with immunoglobulin- and mucin-like domains, is a facultative ligand for L-selectin. MAdCAM-1 isolated from mesenteric lymph nodes, but not from cultured endothelioma cells, bears N-glycanase-resistant sialic acid-containing carbohydrate which supports adhesion of L-selectin-transfected lymphoid cells under shear. Interacting lymphoid cells display a 'rolling' behaviour similar to the selectin-dependent rolling of neutrophils observed in inflamed venules. MAdCAM-1 is also a ligand for the lymphocyte integrin homing receptor for Peyer's patches, alpha 4 beta 7 (ref. 7), and may be uniquely adapted to support both selectin-mediated lymphocyte rolling and integrin-mediated adhesion and arrest in vivo.     

Association of protein kinase CK2 with nuclear matrix: influence of method of preparation of nuclear matrix

The cDNA for the mouse bone morphogenetic protein type II receptor (BMPR-II) was isolated using the human counterpart as a probe and its genomic structure was determined. The cDNA encodes a protein of 1,038 amino acids with a single transmembrane domain, a serine/threonine kinase domain, and a long carboxy-terminal tail. The overall amino acid sequence identity between the mouse and the human BMPR-II is 96.6%. mRNA is widely distributed in various adult tissues. The gene is encoded by 13 exons spanning over 80 kb. Two large introns (intron 1 and 3) contribute to the majority of the gene size, as in the mouse activin type II receptor gene. The intron/exon boundaries were sequenced. The results suggest that alternative splicing can yield a shorter form of BMPR-II of 530 amino acids, as reported previously. Knowledge of the structure of the BMPR-II gene is essential for the understanding of the role of bone morphogenetic proteins in the developmental and physiological processes of animals.     

Cloning and sequencing an ovine interleukin-4-encoding cDNA

We have cloned a cDNA containing the complete coding sequence of ovine(ov) interleukin 4 (IL4) by the polymerase chain reaction using primers based on the 5' and 3' untranslated regions of the human IL4 gene. RNA was isolated from phorbol myristate acetate- and calcium ionphore A23187-stimulated mesenteric lymph node cells. The ovIL4 cDNA is 535 bp in length and contains an open reading frame of 408 nucleotides (nt) coding for a 15.1-kDa IL4 precursor of 135 amino acids (aa). Cleavage of the putative signal peptide of 22 aa yields the mature form of 13.2 kDa. Analysis of the mature aa sequence shows two potential N-linked glycosylation sites and six Cys residues. Ovine and bovine IL4 are shorter than human, mouse and rat IL4, because of a 51-nt deletion in the coding region. Comparison of the predicted aa sequence shows that ovIL4 shares 92, 57, 37 and 42% identity with the bovine, human, mouse and rat IL4s, respectively.     

Expression of cytokine receptors in the placenta in term and preterm labour

A cDNA encoding the brushtail possum immunoglobulin A heavy chain constant region (C alpha) was isolated by screening a mesenteric lymph node cDNA library with a porcine C alpha exon 3 probe. The larger of the two positive clones isolated (Tv4a) consisted of 1325 bp of possum cDNA that included an open reading frame of 1191 bp. Its deduced amino acid sequence had a high degree of sequence identity with known eutherian C alpha sequences. This clone appears to encode the entire possum IgA heavy chain constant region. The possum C alpha sequence had a nucleotide sequence identity of 57.7% with porcine C alpha, 51% with mouse C alpha, 46.7% with dog C alpha and 45.9% with human C alpha2. The corresponding amino acid identities were 46.7, 45.6, 49.4 and 49%, respectively.     

Identification and characterization of galectin-9, a novel beta-galactoside-binding mammalian lectin

A 36-kDa beta-galactoside mammalian lectin protein, designated as galectin-9, was isolated from mouse embryonic kidney by using a degenerate primer polymerase chain reaction and cloning strategy. Its deduced amino acid sequence had the characteristic conserved sequence motif of galectins. Endogenous galectin-9, extracted from liver and thymus, as well as recombinant galectin-9 exhibited specific binding activity for the lactosyl group. It had two distinct N- and C-terminal carbohydrate-binding domains connected by a link peptide, with no homology to any other protein. Galectin-9 had an alternate splicing isoform, exclusively expressed in the small intestine with a 31-amino acid insertion between the N-terminal domain and link peptide. Sequence homology analysis revealed that the C-terminal carbohydrate-binding domain of mouse galectin-9 had extensive similarity to that of monomeric rat galectin-5. The presence of galectin-5 in the mouse could not be demonstrated by polymerase chain reaction or by Northern or Southern blot genomic DNA analyses. Sequence comparison of rat galectin-5 and rat galectin-9 cDNA did not reveal identical nucleotide sequences in the overlapping C-terminal carbohydrate-binding domain, indicating that galectin-9 is not an alternative splicing isoform of galectin-5. However, galectin-9 had a sequence identical with that of its intestinal isoform in the overlapping regions in both species. Southern blot genomic DNA analyses, using the galectin-9 specific probe derived from the N-terminal carbohydrate-binding domain, indicated the presence of a novel gene encoding galectin-9 in both mice and rats. In contrast to galectin-5, which is mainly expressed in erythrocytes, galectin-9 was found to be widely distributed, i.e. in liver, small intestine, thymus > kidney, spleen, lung, cardiac and skeletal muscle > reticulocyte, brain. Collectively, these data indicate that galectin-9 is a new member of the galectin gene family and has a unique intestinal isoform.     

A single mouse glutathione synthetase gene encodes six mRNAs with different 5' ends

To understand more about the role of glutathione (GSH) in metabolism, we have cloned both cDNA and genomic sequences for mouse glutathione synthetase (GSH syn), the enzyme that catalyzes the last step in the synthesis of glutathione. The mouse cDNA contains an open reading frame (ORF) of 474 aa and shares 64 and 95% deduced amino acid sequence identity with Xenopus cDNA and rat cDNA, respectively. The cDNA complements Schizosaccaromyces pombe strains deficient in GSH syn. The gene is a single-copy gene spanning approximately 30 kb and is composed of at least 15 exons. Steady-state RNA levels and enzyme activity levels are highest in kidney, about 3-fold lower in liver, and 8- to 10-fold lower in lung and brain. We have identified six different GSH syn RNAs: three, termed types A1, A2, and A3, have different 5' ends that localize to different sites in the gene, but appear to encode the same protein (474 aa). Types B, C1, and C2 all have unique 5' ends and type-specific ORFs, which are shorter than that for types A1, A2, and A3. In liver only type A1 GSH syn RNA is detectable, while in kidney 90% of GSH syn RNA is type A1 and types B and C account for about 10%.     

Cloning of cDNA and genomic structure of the mouse gamma-glutamyl transpeptidase-encoding gene

We have isolated and characterized cDNA and genomic clones containing the coding region for the mouse gamma-glutamyl transpeptidase (GGT). The sequences of the full-length cDNAs for three of the seven known mouse Ggt RNAs (types I, II and III) were determined and found to be identical in the coding region. Comparisons of the deduced amino-acid sequence of mouse GGT with that of rat and human reveal 95 and 79% overall identities, respectively. The mouse Ggt gene has 12 coding exon and spans approx. 12 kb. We have also re-analyzed rat genomic Ggt clones previously isolated by us and found that the rat and mouse genes share the same intron/exon boundaries. Our findings are of interest because they define the structure of the mouse and rat Ggt genes and will allow comparison with human GGT genes which, recent findings suggest, have diverged substantially from rodents.     

Molecular cloning and nucleotide sequences of cDNA and gene encoding endo-inulinase from Penicillium purpurogenum

A cDNA and a gene encoding endo-inulinase from Penicillium purpurogenum were isolated, and were cloned for the first time. Two oligonucleotide probes, which were synthesized based on the partial amino acid sequences of the purified endo-inulinase, were used to screen a cDNA library. A 1.7-kb DNA fragment encoding endo-inulinase was isolated and analyzed. A single open reading frame, consisting of 1548-bp, was found to encode a polypeptide that comprised a 25-amino acid signal peptide and 490-amino acid mature protein. All the partial amino acid sequences of the purified enzyme were discovered in the deduced ones. The deduced amino acid sequences of endo-inulinase had similar sequences to those of fructan hydrolases. A 3.5-kb chromosomal DNA fragment encoding endo-inulinase was also isolated and analyzed. The same ORF with cDNA clone as identified. There were no introns in the endo-inulinase gene.     

Cloning and nucleotide sequence of the alkaline protease gene from Fusarium sp. S-19-5 and expression in Saccharomyces cerevisiae

We have cloned a genomic DNA encoding the alkaline protease (Alp) of Fusarium sp. S-19-5 from a genomic DNA library and sequenced the nucleotides. Complementary DNA encoding Alp was also isolated from the cDNA library after amplifying the gene by PCR using partial sequences of the Alp genomic DNA as primers. The Alp gene has an open reading frame of 1137 nucleotides containing three introns. A TATA box (TAAATA) was observed 112 base pairs upstream from the translation initiation codon in the 5'-non coding region. The Alp protein has a pre region consisting of 14 amino acids and a pro region of 85 amino acids preceding the mature region, which consists of 280 amino acids. The amino acid sequence of Fusarium Alp has 52% homology with that of Aspergillus oryzae and 51% homology with that of Acremonium chrysogenum. The entire cDNA encoding Fusarium Alp was introduced into Saccharomyces cerevisiae, which then secreted enzymatically active Alp into the culture medium.     

Structural organization of the mouse and human GALR1 galanin receptor genes (Galnr and GALNR) and chromosomal localization of the mouse gene

The neuropeptide galanin elicits a range of biological effects by interaction with specific G-protein-coupled receptors. Human and rat GALR1 galanin receptor cDNA clones have previously been isolated using expression cloning. We have used the human GALR1 cDNA in hybridization screening to isolate the gene encoding GALR1 in both human (GALNR) and mouse (Galnr). The gene spans approximately 15-20 kb in both species; its structural organization is conserved and is unique among G-protein-coupled receptors. The coding sequence is contained on three exons, with exon 1 encoding the N-terminal end of the receptor and the first five transmembrane domains. Exon 2 encodes the third intracellular loop, while exon 3 encodes the remainder of the receptor, from transmembrane domain 6 to the C-terminus of the receptor protein. The mouse and human GALR1 receptor proteins are 348 and 349 amino acids long, respectively, and display 93% identity at the amino acid level. The mouse Galnr gene has been localized to Chromosome 18E4, homoeologous with the previously reported localization of the human GALNR gene to 18q23 in the same syntenic group as the genes encoding nuclear factor of activated T-cells, cytoplasmic 1, and myelin basic protein.     

Interferon treatment of chronic hepatitis C in human immunodeficiency virus infected patients]

There are no reports to date of entire gene sequences coding for chitinolytic enzymes from entomopathogenic fungi, even though these enzymes act synergistically with proteolytic enzymes to solubilize insect cuticle during the key step of host penetration, having considerable importance in the biological control of some insect pests. This paper reports the complete nucleotide sequence and analysis of the chromosomal and full-length cDNA copies of the regulated gene (chit1) coding one of the chitinases produced by the biocontrol agent Metarhizium anisopliae. Degenerated primers, encompassing conserved regions of other fungal chitinases, were used to amplify a 650-bp DNA fragment, which was used to isolate genomic and cDNA clones from M. anisopliae. Albeit at least two different chitinases are characterized in this fungus, only one chit gene was isolated. The chit1 gene is interrupted by three short typical fungal introns and has a 1,521-bp ORF, which encodes a protein of 423 amino acids with a stretch of 35 amino acid residues displaying characteristics of signal peptide. The deduced sequence of the mature protein predicts a 42-kDa protein with pI of 5.8. Southern analysis of genomic DNA indicates a single copy of chit1 in the M. anisopliae genome.