Cloning and characterization of a cDNA that encodes a 70-kDa novel human thyroid autoantigen

cDNA clones were isolated by screening a human thyroid carcinoma lambda gt11 library with immunoglobulins purified from serum of a patient with autoimmune Graves' disease. One clone (ML8) containing a 1.25-kilobase (kb) insert hybridized with a single 2.0-kb poly(A+) mRNA in human thyroid and lymphocytes but not in human brain, liver, kidney, or muscle. In addition, this probe also hybridized with a single 2.0-kb poly(A+) mRNA from a rat thyroid cell line (FRTL-5). An apparently full length 2,074-base pair (bp) human cDNA was obtained and sequenced. The nucleotide sequence of the 2,074-bp cDNA includes a 5'-noncoding sequence of 17 bp, a 1827-bp open reading frame, and a 222-bp 3'-noncoding sequence. The canonical polyadenylation signal AATAAA is present 18 bp upstream of the poly(A) tail. This cDNA encodes a 69,812-dalton protein with two potential N-linked glycosylation sites and at least one potential membrane spanning domain. Immunoprecipitation of the in vitro translated protein by sera from several patients with Graves' disease argues that the 69,812-dalton protein is an autoantigen.     

Angiopoietin-3, a novel member of the angiopoietin family

A cDNA clone encoding angiopoietin-3 protein (Ang3), a novel member of the angiopoietin family, was identified. Ang3 cDNA was cloned from a human aorta cDNA library. Ang3 is a 503 amino acid protein having 45.1% and 44.7% identity with human angiopoietin-1 and human angiopoietin-2, respectively. Ang3 mRNA is expressed in lung and cultured human umbilical vein endothelial cells (HUVECs). Ang3 mRNA expression in HUVECs was slightly decreased by vascular endothelial cell growth factor treatment, suggesting that the regulation of Ang3 mRNA expression is different from that of Ang2.     

The human tyrosine hydroxylase gene

1. Tyrosine hydroxylase (TH) is a rate-limiting enzyme for catecholamine biosynthesis, and it is a pterin-requiring monooxygenase. Both cDNAs and genomic DNA of human TH have been cloned and the nucleotide sequence has been determined. 2. Four similar but distinct mRNAs encode human TH. The results of Southern blot analysis and the nucleotide sequence of the human TH genomic DAN indicate that the four types of human TH mRNA are produced through alternative splicing from a single gene. 3. The human TH gene was split into 4 exons and 13 introns. The 12-bp insertion sequence is encoded by the 3'-terminal portion of the first exon. The 81-bp insertion sequence corresponds to the second exon. Two kinds of alternative splicing are involved: the alternative use of two donor sites in the first exon and the inclusion/exclusion of the second exon. 4. The four types (type 1-4) were expressed in COS cells, and all had enzymatic activities. The type 1 enzyme had the highest homospecific activity (activity per enzyme protein), the values for the other enzymes ranging from 30 to 40%. The Km values of the four types for L-tyrosine and 6-methyl-5,6,7,8-tetrahydropterin were similar.     

cDNA cloning and chromosomal localization (4q11-13) of a gene for statherin, a regulator of calcium in saliva.

On the basis of the known amino acid sequence of statherin, a human salivary protein, mixed synthetic oligonucleotides were synthesized and used to screen a cDNA library constructed from human parotid-gland mRNA. A cDNA clone coding for statherin was isolated from this library and has been completely sequenced. The cDNA represents a full-length (or nearly full-length) copy of an approximately 640-bp statherin mRNA. Statherin appears to be coded by a single-copy gene that maps to chromosome 4q11-4q13 when somatic-cell hybrids are used.     

Structure of the human tyrosine hydroxylase gene: alternative splicing from a single gene accounts for generation of four mRNA types

Tyrosine hydroxylase (TH) is a rate-limiting enzyme for catecholamine biosynthesis. Recently, Grima et al. (Nature (1987) 326, 707-711) and we (Biochem. Biophys. Res. Commun. (1987) 146, 971-975; Nucleic Acids Res. (1987) 15, 6733) reported four similar but distinct mRNAs that encode human TH. These mRNAs are constant for the major part, but are distinguishable from one another as to the insertion/deletion of 12-bp and 81-bp sequences near the N-terminus. We isolated genomic clones encoding the human TH gene and determined the nucleotide sequence. The human TH gene is split into 14 exons. The 12-bp insertion sequence is encoded by the 3'-terminal portion of the first exon. The 81-bp insertion sequence corresponds to the second exon. Taking into consideration also the results of Southern blot analysis of human genomic DNA, we concluded that the four types of human TH mRNA are produced through alternative splicing from a single gene. Two kinds of alternative splicing are involved: the alternative use of two donor sites in the first exon, and the inclusion/exclusion of the second exon. We propose a possible secondary structure for the latter alternative splicing pathway.     

Isolation and expression in Escherichia coli of a cDNA clone encoding human beta-glucuronidase

Mucopolysaccharidosis type VII is a lysosomal storage disease resulting from a deficiency of beta-glucuronidase (BG) activity. To facilitate the investigation of mutation in the disease and provide molecular diagnostic tools for affected families, we have isolated human BG cDNA clones. The SV40-transformed human fibroblast cDNA library of Okayama and Berg [Mol. Cell. Biol. 3 (1982) 280-289] was screened with a fragment of a murine BG cDNA clone (pGUS-1). The 17 human cDNA clones (pHUG) isolated were identical by restriction mapping, varying only in length. The pHUG clones show 80% DNA sequence homology with pGUS-1 in a 198-bp PvuII-SstI restriction fragment. Both pGUS-1 and the pHUG clones contained an open reading frame (ORF) throughout the sequenced region with a predicted amino acid sequence homology of 73%. Expression in Escherichia coli of a 1150-bp fragment of pHUG-1 subcloned in pUC9 resulted in an isopropyl-thio-beta-galactoside (IPTG)-inducible 35-kDal fusion protein which was specifically immunoprecipitated by goat anti-human BG immunoglobulin G (IgG). This evidence provides direct confirmation that the pHUG cDNA clones correspond to human BG.     

Cloning and sequence analysis of cDNA encoding thiamin-binding proteins from sesame seeds

The amino acid sequences of the large polypeptides of thiamin-binding proteins (TBPs) from sesame ( Sesamum indicum L.) seeds (STBP-I, -II and -III) were analyzed. The large polypeptides of STBP-I, -II and -III had the same amino acid sequences as did their small polypeptides. The peptide sequence information obtained from STBPs was used to synthesize DNA primers for amplification of the gene(s) encoding STBPs. A 200-bp fragment was amplified from cDNA synthesized from RNA from sesame seeds 4 weeks after flowering. The 200-bp fragment was used to clone full-length cDNA(s) encoding STBP(s) with RACE techniques. A 644-bp fragment was amplified, cloned and sequenced. The cDNA was a full-length clone encoding STBP(s). It contained an open reading frame, which defined a 143-residue polypeptide. The identified small and large polypeptide sequences of STBPs exactly matched the sequence encoded within the cDNA clone. These results indicated that the small and large polypeptides of STBPs were encoded on the mRNA as a single large proprotein precursor and that the final mature forms were generated by post-translational processing in the same manner as the other 2S albumins of plant seeds.     

Human lysosomal acid phosphatase: cloning, expression and chromosomal assignment.

A 2112-bp cDNA clone (lambda CT29) encoding the entire sequence of the human lysosomal acid phosphatase (EC 3.1.3.2) was isolated from a lambda gt11 human placenta cDNA library. The cDNA hybridized with a 2.3-kb mRNA from human liver and HL-60 promyelocytes. The gene for lysosomal acid phosphatase was localized to human chromosome 11. The cDNA includes a 12-bp 5' non-coding region, an open reading frame of 1269 bp and an 831-bp 3' non-coding region with a putative polyadenylation signal 25 bp upstream of a 3' poly(A) tract. The deduced amino acid sequence reveals a putative signal sequence of 30 amino acids followed by a sequence of 393 amino acids that contains eight potential glycosylation sites and a hydrophobic region, which could function as a transmembrane domain. A 60% homology between the known 23 N-terminal amino acid residues of human prostatic acid phosphatase and the N-terminal sequence of lysosomal acid phosphatase suggests an evolutionary link between these two phosphatases. Insertion of the cDNA into the expression vector pSVL yielded a construct that encoded enzymatically active acid phosphatase in transfected monkey COS cells.     

Connexin43: a protein from rat heart homologous to a gap junction protein from liver

Northern blot analysis of rat heart mRNA probed with a cDNA coding for the principal polypeptide of rat liver gap junctions demonstrated a 3.0- kb band. This band was observed only after hybridization and washing using low stringency conditions; high stringency conditions abolished the hybridization. A rat heart cDNA library was screened with the same cDNA probe under the permissive hybridization conditions, and a single positive clone identified and purified. The clone contained a 220-bp insert, which showed 55% homology to the original cDNA probe near the 5' end. The 220-bp cDNA was used to rescreen a heart cDNA library under high stringency conditions, and three additional cDNAs that together spanned 2,768 bp were isolated. This composite cDNA contained a single 1,146-bp open reading frame coding for a predicted polypeptide of 382 amino acids with a molecular mass of 43,036 D. Northern analysis of various rat tissues using this heart cDNA as probe showed hybridization to 3.0-kb bands in RNA isolated from heart, ovary, uterus, kidney, and lens epithelium. Comparisons of the predicted amino acid sequences for the two gap junction proteins isolated from heart and liver showed two regions of high homology (58 and 42%), and other regions of little or no homology. A model is presented which indicates that the conserved sequences correspond to transmembrane and extracellular regions of the junctional molecules, while the nonconserved sequences correspond to cytoplasmic regions. Since it has been shown previously that the original cDNA isolated from liver recognizes mRNAs in stomach, kidney, and brain, and it is shown here that the cDNA isolated from heart recognizes mRNAs in ovary, uterus, lens epithelium, and kidney, a nomenclature is proposed which avoids categorization by organ of origin. In this nomenclature, the homologous proteins in gap junctions would be called connexins, each distinguished by its predicted molecular mass in kilodaltons. The gap junction protein isolated from liver would then be called connexin32; from heart, connexin43.     

Studies on hst, a transforming gene which belongs to a new superfamily of growth factors

The hst gene was originally identified in surgically obtained human gastric mucosae as a transforming gene which could transform NIH3T3 cells morphologically. The hst cDNA clone was synthesized from mRNA of one of the NIH3T3 transformants. A human leukocyte genomic library was screened with this cDNA clone, and an hst genomic fragment was obtained. This genomic fragment itself had transforming activity, and the protein coding sequences were proved to be completely identical to those of the cDNA clone prepared from mRNA of the NIH3T3 transformant. This fact suggests that rearrangement or other structural alterations in the coding sequence are not required for the activation of the hst gene. The predicted hst protein consists of 206 amino acids and has a significant homology (40-50%) to fibroblast growth factors and int-2 protein. They together make up a new superfamily of growth factors and transforming genes.     

Human gamma-glutamyl transpeptidase cDNA: comparison of hepatoma and kidney mRNA in the human and rat

gamma-Glutamyl transpeptidase (GGT) is a glutathione-metabolizing enzyme that has been extensively studied in relation to hepatocarcinogenesis. Using a cDNA for rat kidney GGT as a probe, we have isolated a full-length cDNA for human GGT from a hepatoma cell-line library. Nucleotide sequence analysis of the clone revealed a 2326-bp insert that includes a 5'-untranslated region of 487 nucleotides (nt), an open reading frame (ORF) of 1707 nt, and a 3'-untranslated region of 132 nt. The ORF encodes a protein with an amino acid sequence that is highly similar to that of the rat GGT precursor peptide, with an overall identity of 79%. The cDNA clone was used to probe Northern blots of hepatoma and kidney RNA from both human and rat. In both species, the GGT mRNA is longer in hepatoma than in kidney. In addition, the human mRNAs were longer than their counterparts in the rat. None of three human hepatocellular carcinomas examined showed a marked elevation in GGT mRNA levels relative to surrounding liver tissue.     

Rat cholesterol side-chain cleavage enzyme (P-450scc): use of a cDNA probe to study the hormonal regulation of P-450scc mRNA levels in ovarian granulosa cells

A rat ovarian cDNA library was constructed and screened by differential colony hybridization to detect cDNA clones specific for mRNA induced by follicle-stimulating hormone (FSH). The cDNA clone which demonstrated the greatest degree of induction contained a 766-bp insert which was characterized and sequenced. We conclude that this cDNA is specific for the rat gene coding for cholesterol side-chain cleavage enzyme (P-450scc) by virtue of nucleotide sequence homology to the bovine and human P-450scc cDNA sequences. Southern blotting of rat genomic DNA suggests the presence of a single P-450scc gene. Northern blot analysis indicates that P-450scc mRNA is present in steroidogenic tissues (ovary, adrenal, testis), but not in brain, kidney, liver, lung, or heart. The rat P-450scc mRNA is induced by FSH or pregnant mare's serum gonadotropin in ovaries of estrogen-treated immature rats in vivo. In cultured granulosa cells, estradiol treatment alone did not increase P-450scc mRNA levels, but in combination with FSH or 8-Br-cAMP resulted in three- to four-fold increase in this mRNA.     

Molecular cloning of cDNA for the catalytic subunit of rat liver type 2A protein phosphatase, and detection of high levels of expression of the gene in normal and cancer cells

A cloned cDNA encoding a catalytic subunit of type 2A protein phosphatase from a rat liver cDNA library was obtained by use of a synthetic oligonucleotide corresponding to the tryptic peptide sequence of the purified enzyme. There was only a single amino acid difference between the deduced amino acid sequence of the clone obtained and those of the catalytic subunits, 2A alpha, of the rabbit skeletal muscle, porcine kidney and human liver enzymes, suggesting that this clone was a rat 2A alpha cDNA. On Northern blot analysis using a cDNA fragment as a probe, three mRNA species were detected in rat liver: a major mRNA of 2.0 kb and a minor one of 2.7 kb under high stringency conditions, and also a 1.1 kb mRNA under low stringency conditions. The 2A alpha gene was found to be highly expressed in various tissues of rat, especially the brain. High levels of expression of the gene were also detected in mouse NIH3T3 cells and their transformants, and in human cancer cell lines as well as a human immortalized cell line.