Molecular cloning and sequence analysis of a chicken ornithine decarboxylase cDNA

A chicken embryo cDNA library was screened with a mouse probe for ornithine decarboxylase (ODC) and 14 positively hybridizing clones isolated. The longest of these (1.7 kb) was sub-cloned and sequenced. It is estimated that the clone comprises approximately 98% of the coding region for chicken ODC. The DNA sequence shows 78% identity with the human ODC cDNA sequence and the deduced amino acid sequence is almost 90% homologous to mouse and human. Both the peptide and cDNA sequences show interesting potential regulatory features which are discussed here.     

Structure of immunoglobulin gamma 2b heavy chain gene cloned from mouse embryo gene library.

A mouse DNA clone containing the constant part of the immunoglobulin gamma 2b heavy chain was isolated from a mouse gene library. The library was constructed in Charon 4A from a partial EcoRI digest of mouse embryo DNA and was screened with a plasmid (p gamma (11)7) containing a cDNA insert of the heavy chain constant region of the plasmacytoma MPC-11 (1). The Charon 4A clone contains a 14 kb insert which is cleaved by EcoRI into a 6.8 kb and 7.2 kb fragments, of which only the 6.8 kb contains the sequence for gamma 2b heavy chain. Restriction analysis and partial sequence of the insert in p gamma (11) 7 enabled us to obtain three fragments corresponding to the 5' (amino acid 161-302) middle (amino acid 302-443) and 3' (mostly non coding 107 bp) regions of the constant region. Restriction analysis of the Charon 4A clone and hybridisation to these nick translated fragments revealed that the gamma 2b constant region gene contains about 1.5 kb and has three intervening sequences.     

Differential expression of the "B" subunit of the vacuolar H(+)-ATPase in bovine tissues.

The B subunit is one of two nucleotide-binding polypeptides found in all members of the vacuolar class of H(+)-translocating ATPases. We have isolated aDNA clone encoding the bovine brain B (58 kDa) subunit and have deduced its amino acid sequence. The bovine brain amino acid sequence is 99% identical to a partial cDNA reported from human brain. Northern blot analysis of RNA isolated from bovine tissues and a bovine kidney cell line reveals that two messages of approximately 3.2 and 2.0 kilobases (kb) are expressed in all tissues examined except brain, where only the 3.2-kb message can be detected. Northern blotting of RNA isolated from human fibroblast and human lung tumor cell lines reveals that three messages of approximately 6.0, 3.2, and 2.0 kb are expressed, whereas only the 3.2-kb message is expressed in a human brain tumor cell line. This is the first demonstration of tissue-specific expression of multiple forms of a vacuolar H(+)-ATPase subunit. We have also isolated a partial cDNA clone from bovine brain which appears to encode an isoform of the B subunit. The deduced amino acid sequence is 82% identical to the major bovine brain B subunit sequence; it does not hybridize with either the 3.2- or 2.0-kb message on Northern blot. Southern blot analysis of bovine genomic DNA with probes derived from both isolated cDNAs indicates that the bovine B subunit is encoded by a multigene family.     

Molecular cloning of the human transmembrane secretory component (poly-Ig receptor) and its mRNA expression in human tissues

A 2.5 kilobase (kb) cDNA clone containing 92% of the coding region for human transmembrane secretory component (SC) or poly-Ig receptor, was isolated from a mammary gland cDNA library. The cDNA clone encoded a protein of 693 amino acids which showed 99% homology with the primary amino acid sequence of human free SC as reported by Eiffert et al. (1), and 54% homology with the deduced amino acid sequence of rabbit transmembrane SC for which cDNA was cloned by Mostov et al. (2). Northern blot analysis showed mRNA expression in various human exocrine tissues in good agreement with our previous immunohistochemical studies of SC.     

Mouse cathepsin F: cDNA cloning, genomic organization and chromosomal assignment of the gene

A murine cysteine protease of the papain family was identified by dbEST-database search. A 1.87kb full-length cDNA encoding a predicted polypeptide of 462 amino acids was sequenced. Since the encoded polypeptide shows more than 80% sequence identity with human cathepsin F, it is most likely that this cDNA represents the murine homologue of cathepsin F, and it was therefore named accordingly. Murine cathepsin F exhibits a domain structure typical for papain-like cysteine proteases, a 20 amino acid N-terminal hydrophobic signal sequence followed by an extraordinarily long propeptide of 228 amino acids and the domain of the mature protease comprising 214 amino acids. The mature region contains all features characteristic of a papain-like cysteine protease, including the highly conserved cysteine, histidine and asparagine residues of the 'catalytic triad'. Genomic clones covering the murine cathepsin F gene were isolated. The mouse cathepsin F gene consists of 14 exons and 13 introns and spans 5.8kb. Murine cathepsin F was mapped to chromosome 19, a region with synteny homology to a region of human chromosome 11 to which human cathepsin F has been mapped previously. Northern blot analysis of RNA from multiple tissues revealed a ubiquitous expression of cathepsin F in mouse and man.     

Cloning of human heparan sulfate proteoglycan core protein, assignment of the gene (HSPG2) to 1p36.1----p35 and identification of a BamHI restriction fragment length polymorphism.

We have isolated a cDNA coding for the core protein of the large basement membrane heparan sulfate proteoglycan (HSPG) from a human fibrosarcoma cell (HT1080) library. The library was screened with a mouse cDNA probe and one clone obtained, with a 1.5-kb insert, was isolated and sequenced. The sequence contained an open reading frame coding for 507 amino acid residues with a 84% identity to the corresponding mouse sequence. This amino acid sequence contained several cysteine-rich internal repeats similar to those found in component chains of laminin. The HSPG cDNA clone was used to assign the gene (HSPG2) to the p36.1----p35 region of chromosome 1 using both somatic cell hybrid and in situ hybridization. In the study of the polymorphisms of the locus, a BamHI restriction fragment length polymorphism was identified in the gene. This polymorphism displayed bands of 23 and 12 kb with allele frequencies of 76 and 24%, respectively.     

Isolation of a full-length cDNA encoding mouse aromatase P450

A full-length cDNA clone for aromatase P450 has been isolated from a pregnant mouse ovarian cDNA library. The insert of this clone (2394 bp) contains a 1509-bp open reading frame encoding 503 amino acid residues together with a 46-bp 5'-untranslated stretch and an 839-bp 3'-untranslated region to which a poly(A) tract is attached. Northern blot analysis of ovarian RNA from pregnant mice reveals a major mRNA band of 2.5 kb with a minor band of 2.1 kb. Comparison of mouse aromatase P450 with that of rat, human, and chicken shows 91, 81, and 69% identity in the nucleotide sequence and 92, 79, and 69% identity in the deduced amino acid sequence, respectively. The membrane-spanning domain of mouse aromatase P450 is estimated to be an extremely hydrophobic segment located within the N-terminal region of the molecule. Furthermore, a highly conserved heme-binding domain is noticed.     

Identification of a novel fibroblast growth factor, FGF-23, preferentially expressed in the ventrolateral thalamic nucleus of the brain

We isolated mouse cDNA encoding a novel FGF (251 amino acids). As this is the 23rd documented FGF, we termed it FGF-23. FGF-23 has a hydrophobic amino terminus ( approximately 24 amino acids), which is a typical signal sequence. As expected, recombinant mouse FGF-23 was efficiently secreted by High Five insect cell-infected recombinant baculovirus containing the cDNA, indicating that FGF-23 is a secreted protein. We also isolated human cDNA encoding FGF-23 (251 amino acids), which is highly identical ( approximately 72% amino acid identity) to mouse FGF-23. Of human FGF family members, FGF-23 is most similar to FGF-21 and FGF-19 ( approximately 24% and approximately 22% amino acid identities, respectively). Human FGF-23 gene was localized on the chromosome 12p13 and found to be tandem linked (within 5.5 kb) to human FGF-6 gene. The expression of FGF-23 mRNA in mouse adult tissues was examined by real-time quantitative polymerase chain reaction. FGF-23 mRNA was mainly expressed in the brain and thymus at low levels. The localization of FGF-23 mRNA in the brain was examined by in situ hybridization. FGF-23 mRNA in the brain was found to be preferentially expressed in the ventrolateral thalamic nucleus. Therefore, FGF-23 is expected a unique FGF that plays roles in the function of the ventrolateral thalamic nucleus.     

Molecular cloning, primary structure, and expression of the human growth factor-activatable Na+/H+ antiporter

We present the complete sequence of a cDNA encoding the human amiloride-sensitive Na+/H+ antiporter. After functional complementation of a mouse fibroblast mutant by gene transfer, we isolated a 0.8 kb genomic probe from a third-cycle mouse transformant. The probe detects gene amplification in Na+/H+ antiporter "overexpressers" and a single class of mRNA of ca. 5.6 kb in human, mouse, and hamster cells. With this probe we isolated a 4 kb cDNA from a library constructed from a mouse transformant in which the transfected human gene was amplified. This cDNA includes a noncoding leader of 407 bp, a 2682 bp open reading frame, and a 3' noncoding sequence containing a mouse B1 repeated element. The amino acid sequence predicts a protein of Mr = 99,354 with an N-terminal amphipathic domain that contains 10 putative transmembrane-spanning segments and two potential glycosylation sites, followed by a hydrophilic stretch of 395 residues, presumably cytoplasmic. Stable expression of the transfected cDNA in Na+/H+ antiporter-deficient cells restored the key functional features of this transporter: H+i-activated Na+ influx, amiloride sensitivity, and pHi regulation.     

Cloning and characterization of a murine macrophage lipoxygenase

We have isolated a murine macrophage cDNA encoding a 12-lipoxygenase, that represents the homolog of the human 15-lipoxygenase. The predicted amino acid sequence of this lipoxygenase is highly similar to the rat 12-lipoxygenase isolated from brain and human 15-lipoxygenase. The recombinant enzyme expressed in Cos-7 cells oxidizes arachidonic acid to 12- and 15-HETE with a profile similar to that obtained from peritoneal macrophages. A polyclonal antibody generated against a putative peptide recognizes a 75 kDa protein in cell extracts from mouse peritoneal macrophages and transfected Cos-7 cells. The lipoxygenase cDNA hybridizes to a 2.5 kb mRNA present in peritoneal macrophages, lung, spleen, heart and liver. RT-PCR analysis indicates that the same lipoxygenase is expressed in mouse reticulocytes. A partial genomic clone for this lipoxygenase has also been characterized. Southern blot analysis of mouse genomic DNA indicates that this is a single copy gene.