Isolation and characterization of a cDNA that encodes mouse fibroblast tropomyosin isoform 2.

We isolated and characterized a cDNA clone encoding tropomyosin isoform 2 (TM2) from a mouse fibroblast cDNA library. TM2 was found to contain 284 amino acids and was closely related to the rat smooth and skeletal muscle alpha-TMs and the human fibroblast TM3. The amino acid sequence of TM2 showed a nearly complete match with that of human fibroblast TM3 except for the region from amino acids 189 to 213, the sequence of which was identical to those of rat smooth and skeletal muscle alpha-TMs. These results suggest that TM2 is expressed from the same gene that encodes the smooth muscle alpha-TM, the skeletal muscle alpha-TM, and TM3 via an alternative RNA-splicing mechanism. Comparison of the expression of TM2 mRNA in low-metastatic Lewis lung carcinoma P29 cells and high-metastatic D6 cells revealed that it was significantly less in D6 cells than in P29 cells, supporting our previous observations (K. Takenaga, Y. Nakamura, and S. Sakiyama, Mol. Cell. Biol. 8:3934-3937, 1988) at the protein level.     

Discovery of new human beta-defensins using a genomics-based approach

Epithelial beta-defensins are broad-spectrum cationic antimicrobial peptides that also act as chemokines for adaptive immune cells. In the human genome, all known defensin genes cluster to a <1 Mb region of chromosome 8p22-p23. To identify new defensin genes, the DNA sequence from a contig of large-insert genomic clones from the region containing human beta-defensin-2 (HBD-2) was analyzed for the presence of defensin genes. This sequence survey identified a novel beta-defensin, termed HBD-3. The HBD-3 gene contains two exons, is located 13 kb upstream from the HBD-2 gene, and it is transcribed in the same direction. A partial HBD-3 cDNA clone was amplified from cDNA derived from IL-1beta induced fetal lung tissue. The cDNA sequence encodes for a 67 amino acid peptide that is approximately 43% identical to HBD-2 and shares the beta-defensin six cysteine motif. By PCR analysis of two commercial cDNA panels, HBD-3 expression was detected in adult heart, skeletal muscle, placenta and in fetal thymus. From RT-PCR experiments, HBD-3 expression was observed in skin, esophagus, gingival keratinocytes, placenta and trachea. Furthermore, in fetal lung explants and gingival keratinocytes, HBD-3 mRNA expression was induced by IL-1beta. Additional sequence analysis identified the HE2 (human epididymis secretory protein) gene 17 kb upstream from the HBD-3 gene. One splice variant of this gene (HE2beta1) encodes a beta-defensin consensus cysteine motif, suggesting it represents a defensin gene product. HE2beta1 mRNA expression was detected in gingival keratinocytes and bronchial epithelia using RT-PCR analysis. The discovery of these novel beta-defensin genes may allow further understanding of the role of defensins in host immunity at mucosal surfaces.     

Isolation and characterization of non-muscle tropomyosin cDNAs of human and mouse origin

Several cDNA clones of human and mouse non-muscle tropomyosin have been isolated. All the human clones possess a common 23 bp sequence immediate 5' of the initiation codon. However, in the further upstream regions, the nucleotide sequences diverge. Two of the mouse cDNA clones pPSI-8 and pPSI-14 have identical nucleotide sequence in the coding region sequenced. However, 5' of the initiation codon these clones have only 40 identical nucleotides and further upstream the nucleotide sequences diverge. Analysis of the genomic DNAs of mouse cells indicated the possibility of a common gene giving rise to both the tropomyosin cDNAs differing in their 5' ends.     

Molecular cloning and high-level expression of human polymerase beta cDNA and comparison of the purified recombinant human and rat enzymes

The cDNA encoding the human polymerase beta from HeLa cells was PCR amplified and cloned, and its nucleotide sequence determined. The DNA sequence is identical to the polymerase beta cDNA sequence from Tera-2 cells. Three expression strategies were employed that were designed to maximize translation initiation of the polymerase beta mRNA in Escherichia coli and all yielded a high level of human polymerase beta. The recombinant protein was purified and its properties were compared with those of the recombinant rat enzyme. The domain structure and kinetic parameters (k(cat) and K(m)) were nearly identical. A mouse IgG monoclonal antibody to the rat enzyme (mAb-10S) was approximately 10-fold less reactive with the human enzyme than with the rat enzyme as determined by ELISA.     

Cloning and characterization of a cDNA encoding the beta subunit of human casein kinase II

Previous studies [Summercorn et al. (1987) Proc. Natl. Acad. Sci. U.S.A. 84, 8834-8838; Klarlung & Czech (1988) J. Biol. Chem. 263, 15872-15875] have indicated that Balb/c 3T3 cells and 3T3-L1 adipocytes incubated with insulin show increased casein kinase II activity within minutes, implicating this serine/threonine kinase as an early step in an insulin signaling pathway. We recently reported the isolation of a cDNA encoding an alpha subunit of human casein kinase II [Meisner et al. (1989) Biochemistry 28, 4072-4076] as an initial step toward examining the regulation of this enzyme. We now describe a HepG2 cell casein kinase II beta subunit cDNA of 2.57 kb containing 96 bases of 5' untranslated sequence, 645 bases of open reading frame, and 1832 bases of 3' untranslated sequence with two polyadenylation consensus signal sequences and two poly(A) stretches. The open reading frame of the human beta subunit cDNA was 77% and 87% identical with the Drosophila sequence at the nucleotide and amino acid levels, respectively, and 99% identical with the bovine amino acid sequence. RNA analysis of HepG2 cell RNA utilizing HepG2 beta subunit cDNA fragments as probes revealed one major band migrating at 1.2 kb and two minor bands migrating at 3.0 and 4.2 kb. Results from DNA analysis of HepG2 genomic DNA, consistent with results utilizing Drosophila genomic DNA, suggest the presence of a single gene for the beta subunit of casein kinase II.     

Tissue specific expression and chromosomal mapping of a human UDP-N-acetylglucosamine: alpha1,3-d-mannoside beta1, 4-N-acetylglucosaminyltransferase

A human cDNA for UDP- N -acetylglucosamine:alpha1,3-d-mannoside beta1,4- N- acetylglucosaminyltransferase (GnT-IV) was isolated from a liver cDNA library using a probe based on a partial cDNA sequence of the bovine GnT-IV. The cDNA encoded a complete sequence of a type II membrane protein of 535 amino acids which is 96% identical to the bovine GnT-IV. Transient expression of the human cDNA in COS7 cells increased total cellular GnT-IV activity 25-fold, demonstrating that this cDNA encodes a functional human GnT-IV. Northern blot analysis of normal tissues indicated that at least five different sizes of mRNA (9.7, 7.6, 5.1, 3.8, and 2.4 kb) forGnT-IV are expressed in vivo. Furthermore, these mRNAs are expressed at different levels between tissues. Large amounts of mRNA were detected in tissues harboring T lineage cells. Also, the promyelocytic leukemia cell line HL-60 and the lymphoblastic leukemia cell line MOLT-4 revealed abundant mRNA. Lastly, the gene was mapped at the locus on human chromosome 2, band q12 by fluorescent in situ hybridization.     

Isolation and characterization of cDNA clones encoding a low molecular weight nonmuscle tropomyosin isoform

cDNA clones encoding rat fibroblast tropomyosin 4 (TM-4) were isolated and characterized. DNA sequence analysis was carried out to determine the sequence of the protein. The derived amino acid sequence revealed that rat fibroblast TM-4 was found to contain 248 amino acids. The amino acid sequence of rat fibroblast TM-4 was compared with two other low molecular weight TM isoforms, equine platelet beta-TM and a human fibroblast TM. Rat TM-4 exhibited 98% sequence identity with the equine platelet TM but only 75% identity with the human fibroblast TM isoform. The high degree of conservation between the rat and equine proteins indicates that they belong to the same isotype of TM. Comparison of the amino acid sequences of the three low molecular TM isoforms along the length of the proteins reveals regions that are strongly conserved and regions that have considerably diverged. In the regions from amino acid residues 1 to 148 and 176 to 221, amino acid substitutes are moderate. The most variant regions in the sequence are in the middle part of the proteins from amino acids 149 to 175 and at the carboxyl-terminal region of the proteins from amino acids 222 to 248. The differences in the sequence of the rat and platelet TMs compared to the human TM may define distinct functional domains among the low molecular weight TMs. In addition, expression of tropomyosin was studied in a variety of tissues and transformed cells. We also demonstrate that at least three separate genes encode tropomyosins expressed in rat fibroblasts.     

Chicken cardiac tropomyosin and a low-molecular-weight nonmuscle tropomyosin are related by alternative splicing

We have isolated and characterized complementary DNAs (cDNAs) encoding chicken cardiac muscle tropomyosin and a low-molecular-weight nonmuscle tropomyosin. The cardiac muscle cDNA (pCHT-4) encodes a 284-amino acid protein that differs from chicken skeletal muscle alpha- and beta-tropomyosins throughout its length. The nonmuscle cDNA (pFT-C) encodes a 248-amino acid protein that is most similar (93-94%) to the tropomyosin class including rat fibroblast TM-4, equine platelet tropomyosin, and human fibroblast TM30pl. The nucleotide sequences of the cardiac and nonmuscle cDNAs are identical from the position encoding cardiac amino acid 81 (nonmuscle amino acid 45) through cardiac amino acid 257 (nonmuscle amino acid 221). The sequences differ both 5' and 3' of this region of identity. These comparisons suggest that the chicken cardiac tropomyosin and low-molecular-weight "platelet-like" tropomyosin are derived from the same genomic locus by alternative splicing. S1 analysis suggests that this locus encodes at least one other tropomyosin isoform.     

Identification and characterization of beta V spectrin, a mammalian ortholog of Drosophila beta H spectrin

Four mammalian beta-spectrin genes are currently recognized, all encode proteins of approximately 240-280,000 M(r) and display 17 triple helical homologous approximately 106-residue repeat units. In Drosophila and Caenorhabditis elegans, a variant beta spectrin with unusual properties has been recognized. Termed beta heavy (beta(H)), this spectrin contains 30 spectrin repeats, has a molecular weight in excess of 400,000, and associates with the apical domain of polarized epithelia. We have cloned and characterized from a human retina cDNA library a mammalian ortholog of Drosophila beta(H) spectrin, and in accord with standard spectrin naming conventions we term this new mammalian spectrin beta 5 (betaV). The gene for human betaV spectrin (HUBSPECV) is on chromosome 15q21. The 11, 722-nucleotide cDNA of betaV spectrin is generated from 68 exons and is predicted to encode a protein with a molecular weight of 416,960. Like its fly counterpart, the derived amino acid sequence of this unusual mammalian spectrin displays 30 spectrin repeats, a modestly conserved actin-binding domain, a conserved membrane association domain 1, a conserved self-association domain, and a pleckstrin homology domain near its COOH terminus. Its putative ankyrin-binding domain is poorly conserved and may be inactive. These structural features suggest that betaV spectrin is likely to form heterodimers and oligomers with alpha spectrin and to interact directly with cellular membranes. Unlike its Drosophila ortholog, betaV spectrin does not contain an SH3 domain but displays in repeat 5 a 45-residue insertion that displays 42% identity to amino acids 85-115 of the E4 protein of type 75 human papilloma virus. Human betaV spectrin is expressed at low levels in many tissues. By indirect immunofluorescence, it is detected prominently in the outer segments of photoreceptor rods and cones and in the basolateral membrane and cytosol of gastric epithelial cells. Unlike its Drosophila ortholog, a distinct apical distribution of betaV spectrin is inapparent in the epithelial cell populations examined, although it is confined to the outer segments of photoreceptor cells. The complete cDNA sequence of human betaV spectrin is available from GenBank(TM) as accession number.     

Tumor necrosis factor-alpha gene is expressed in stimulated retinal pigment epithelial cells in culture.

Expression of TNF-alpha gene in the retinal pigment epithelial (RPE) cells was studied by using polymerase chain reaction (PCR). PCR for human TNF-alpha gene using complementary DNA (cDNA) of control RNA prepared from non-stimulated RPE cells failed to show expression of TNF-alpha gene. However, PCR by using cDNA prepared from interleukin-1 beta (IL-1 beta)-stimulated RPE cells revealed expression of the gene, suggesting in vivo production of TNF-alpha from RPE cells in response to IL-1 beta.     

Expression cloning of human globoside synthase cDNAs. Identification of beta 3Gal-T3 as UDP-N-acetylgalactosamine:globotriaosylceramide beta 1,3-N-acetylgalactosaminyltransferase

By using a eukaryocytic cell expression cloning system, we have isolated cDNAs of the globoside synthase (beta1, 3-N-acetylgalactosaminyltransferase) gene. Mouse fibroblast L cells transfected with SV40 large T antigen and previously cloned Gb3/CD77 synthase cDNAs were co-transfected with a cDNA library prepared from mRNA from human kidney together with Forssman synthase cDNA, and Forssman antigen-positive cells were panned using an anti-Forssman monoclonal antibody. The isolated cDNAs contained a single open reading frame predicting a type II membrane protein with 351 amino acids. Surprisingly, the cDNA clones turned out to be identical with previously reported beta3Gal-T3, which had been cloned by sequence homology with other galactosyltransferases. Substrate specificity analysis with extracts from cDNA-transfected L cells confirmed that the gene product was actually beta1, 3-N-acetylgalactosaminyltransferase that specifically catalyzes the transfer of N-acetylgalactosamine onto globotriaosylceramide. Results of TLC immunostaining of neutral glycolipids from the cDNA-transfected cells also supported the identity of the newly synthesized component as globoside. The results show that glycosyltransferases apparently belonging to a single glycosyltransferase family do not necessarily catalyze reactions utilizing the same acceptor or even the same sugar donor. The globoside synthase gene was expressed in many tissues, such as heart, brain, testis, etc. We propose the designation beta3GalNAc-T1 for the cloned globoside synthase gene.     

Cloning, primary structure and properties of a novel human integrin beta subunit.

The originally described integrin beta subunits that define the three subfamilies of integrin heterodimers are beta 1, beta 2 and beta 3. In this paper, we describe the isolation of a cDNA coding for a novel human integrin beta subunit, designated as beta 5. The beta 5 cDNA was isolated from a human thymic epithelial cell library, using oligonucleotide probes that were designed from a region highly conserved among the known beta 1, beta 2 and beta 3 sequences. The beta 5 cDNA codes for 799 (or 796) amino acids, including a 23 amino acid leader sequence. There are 776 (or 773) amino acids in the mature protein, which includes a long extracellular domain of 696 amino acids, a transmembrane domain and an intracellular C-terminal domain of 57 amino acids. The beta 5 sequence resembled the known beta 3, beta 1 and beta 2 sequences by 55, 43 and 38%, respectively, including conservation of 56/56 cysteines. Rabbit antiserum was prepared against a 20 amino acid synthetic peptide predicted from the beta 5 C-terminal sequence. This serum immunoprecipitated a beta 5 protein that was 100,000 Mr (reduced) and 95,000 Mr (nonreduced). Only a single alpha subunit was detected in association with beta 5, and that alpha subunit was immunochemically indistinguishable from the alpha v subunit previously found as part of the vitronectin receptor complex. By immunoprecipitation, beta 5 was most prevalent on carcinoma cell lines, was also present on hepatoma and fibroblast cell lines, and was absent from lymphoblastoid cells and platelets.     

Nucleotide sequence of cDNA for nonmuscle tropomyosin 5 of mouse fibroblast

A cDNA clone for mouse fibroblast tropomyosin (TM) 5 was obtained from a cDNA library using human TM pseudogene as a probe. Sequence analysis of the clone revealed that the deduced amino acid sequence is different only at the 4th position from the human counterpart and that both the 5' and 3' untranslated regions are highly conserved.     

Partial nucleotide sequence of a bovine major histocompatibility class II DR beta-like gene

A genomic clone containing a bovine DR beta-like gene, BoDR beta II, was isolated from a bovine genomic library and characterized by restriction enzyme mapping and nucleotide sequencing of exon regions. Alignment of this sequence with the human DR beta cDNA sequence allowed identification of exon/intron boundaries. The clone contains a 13.3-kilobase (kb) insert, and includes 1.3 kb 5' of the beta 1 exon and 6.7 kb 3' of the transmembrane (TM) exon. Open reading frames were present in the BoDR beta exons sequenced. Nucleotide identities of the bovine beta 1, beta 2 and TM exons with the corresponding human DR beta exons were 73, 91 and 83%, respectively. Nucleotide identities of these exons with those of a previously described bovine DR beta-like pseudogene, BoDR beta I, were 69, 95 and 81%, respectively. Although a limited amount of sequence data was obtained for the intron regions, a 71% identity was found within a 514-nucleotide region immediately 3' to the beta 2 exons in BoDR beta I and BoDR beta II. A series of GT residues followed by a longer series of GA residues began about 35 nucleotides 3' of the beta 1 exon in both BoDR beta I and BoDR beta II.