Molecular cloning and sequencing of a cDNA encoding a human alpha 1A adrenergic receptor.

DNA from a rat hippocampus cDNA library and sets of highly degenerate oligonucleotide primers directed toward conserved regions of previously cloned G-protein receptors were used in the polymerase chain reaction to selectively amplify and clone new members of this gene family. A human hippocampus cDNA library was screened with a 610 base pair fragment generated by PCR and a cDNA clone, H318/3, was isolated. The deduced amino acid sequence of this clone encoded a protein of 501 amino acids that showed strong sequence homology to previously cloned G-protein receptors. Nucleotide sequence analysis revealed clone H318/3 was 78% homologous to a rat alpha 1A adrenergic receptor with homology being 95% when comparisons were made in the region that lies between the first to the seventh transmembrane domains. Based on this high degree of sequence homology, we conclude that clone H318/3 represents a cDNA for a human alpha 1A adrenergic receptor.     

Cloning of a Drosophila cDNA encoding a polypeptide similar to the human insulin receptor precursor

A Drosophila cDNA clone was obtained using the human insulin receptor cDNA sequence as a probe. The 3586 bp nucleotide sequence predicted a single polypeptide of 1095 amino acid residues which showed considerable homology (35.2%) with the human insulin receptor precursor. Although the cDNA was incomplete at its 5'-terminal region, it encodes a transmembrane glycoprotein as a single precursor of a two subunit molecule having a structural architecture similar to that of the human insulin receptor precursor. The presumptive beta subunit carries a well conserved Tyr kinase domain which showed 63.5% homology with that of human insulin receptor; however the protein of the alpha subunit is only weakly conserved (25%).     

Isolation of cDNA clones of human argininosuccinate lyase and corrected amino acid sequence

In the present study, we isolated clones of human argininosuccinate lyase (ASL) cDNA from a liver cDNA library using a clone of rat ASL cDNA and analyzed human ASL cDNA nucleotide sequence. The results reveal that the sequence of human ASL cDNA published by O'Brien et al. in 1986 [Proc. Natl. Acad. Sci USA 83, 7211-7215] had one-base deletions at three independent positions in the coding regions near the COOH-terminus, which caused frame-shift variations in the amino acid sequence. Amino acid sequencing of peptides prepared from purified human liver ASL showed our predicted amino acid sequence to be correct.     

Amino acid sequence of rat apolipoprotein A-II deduced from the nucleotide sequence of cloned cDNA

A rat apolipoprotein A-II cDNA clone was isolated from a rat liver cDNA library by in situ hybridization of bacteriophage plaques using a 32P-labeled human apoA-II cDNA as a probe. The cDNA insert from this clone was characterized by DNA sequencing. The amino acid composition derived from the DNA sequence data matched well with that of rat apoA-II reported earlier (Herbert et al. 1974. J. Biol Chem. 249: 5718-5724), indicating that the cDNA insert coded for rat apoA-II. Further evidence was provided by a comparison of the amino acid sequence of rat apoA-II obtained here with that of human apoA-II (Brewer et al. 1972. Proc. Natl. Acad. Sci. USA. 69: 1304-1308). While the rat apoA-II cDNA insert did not code for the entire presegment, it had the same COOH-terminal residues of the presegment as well as the same prosegment (Ala-Leu-Val-Arg-Arg) as in human preproapoA-II, suggesting that rat apoA-II was also synthesized initially as preproapoA-II. Mature rat apoA-II contains 79 amino acids. Residue 6 of mature rat apoA-II is Asp, while it is Cys in human apoA-II, and this would account for the absence of dimeric forms of rat apoA-II in plasma. While the overall amino acid sequence homology between rat and human apoA-II is about 50%, the amphipathic alpha-helical structures, which are responsible for lipid-binding, seem to be conserved in the two proteins. The size of rat apoA-II mRNA was estimated to be about 600 nucleotides.(ABSTRACT TRUNCATED AT 250 WORDS)     

A cDNA from human bone marrow encoding a protein exhibiting homology to the ATP1gamma1/PLM/MAT8 family of transmembrane proteins

A cDNA clone, IWU-1, was cloned from human bone marrow. Its putative open reading frame encoded a protein of 115 amino acids with a calculated molecular mass of 12.9 kDa. The deduced amino acid sequence exhibited high homology (>68%) to members of the ATP1gamma1/PLM/MAT8 family of single transmembrane proteins, primarily in the region containing the putative transmembrane domain. The sequence at the amino-terminal side exhibited high homology (>61%) to the cytoplasmic region of the angiotensin II type 1 receptors.     

Molecular cloning and sequence analysis of human Na,K-ATPase beta-subunit.

We have isolated a cDNA clone for the beta-subunit of HeLa cell Na,K-ATPase, containing a 2208-base-pair cDNA insert covering the whole coding region of the beta-subunit. Nucleotide sequence analysis revealed that the amino acid sequence of human Na,K-ATPase exhibited 61% homology with that of Torpedo counterpart (Noguchi et al. (1986) FEBS Lett. in press). A remarkable conservation in the nucleotide sequence of the 3' non-coding region was detected between the human and Torpedo cDNAs. RNA blot hybridization analysis revealed the presence of two mRNA species in HeLa cells. S1 nuclease mapping indicated that they were derived from utilization of two distinct polyadenylation signals in vivo. Total genomic Southern hybridization indicated the existence of only a few, possibly one set of gene encoding the Na,K-ATPase beta-subunit in the human genome.     

Cloning and sequencing of cDNA encoding human sepiapterin reductase--an enzyme involved in tetrahydrobiopterin biosynthesis

A full-length cDNA clone for sepiapterin reductase, an enzyme involved in tetrahydrobiopterin biosynthesis, was isolated from a human liver cDNA library by plaque hybridization. The nucleotide sequence of hSPR 8-25, which contained an entire coding region of the enzyme, was determined. The clone encoded a protein of 261 amino acids with a calculated molecular mass of 28,047 daltons. The predicted amino acid sequence of human sepiapterin reductase showed a 74% identity with the rat enzyme. We further found a striking homology between human SPR and carbonyl reductase, estradiol 17 beta-dehydrogenase, and 3 beta-hydroxy-5-ene steroid dehydrogenase, especially in their N-terminal region.     

Amino acid sequence of silkworm (Bombyx mori) hemolymph antitrypsin deduced from its cDNA nucleotide sequence: confirmation of its homology with serpins

A cDNA clone of silkworm (Bombyx mori) larval hemolymph antitrypsin (sw-AT) has been isolated from a fat body cDNA library. The cDNA has an open reading frame which codes a 392-amino acid residue polypeptide comprising a 16-residue signal peptide and a 376-residue mature sw-AT of Mr 41,805. The reactive site of sw-AT for inhibition of bovine trypsin [Sasaki, T. et al. (1987) J. Biochem. 102, 433-441] was identified as Lys343-Val344. Alignment of the sw-AT amino acid sequence with those of 11 members of the serpin superfamily of proteins clearly confirmed the homology of sw-AT with serpins. The amino acid sequence of sw-AT is 56% identical with that of the proteinase inhibitor from a lepidopteron, Manduca sexta [Kanost, M.R. et al. (1989) J. Biol. Chem. 264, 965-972], but the sequence around the reactive site shows no homology and the inhibitory specificity for proteinases is very different.     

Cloning and nucleotide sequence of human liver cDNA encoding for cystathionine gamma-lyase.

We have cloned and sequenced a full-length cDNA (1083 bp) encoding the human liver cystathionine-gamma-lyase enzyme (cystathionase). The human cystathionase sequence presented a substantial deletion of 132 bases (44 amino acids) compared to that reported for rat cystathionase, and of 135 bases (45 amino acids) compared to that reported for yeast cystathionase. After re-alignment for the missing nucleotides, the human cDNA sequence shows significant amino acid homology to that for the rat enzyme (85%) and the yeast enzyme (50%). A search for an undeleted cDNA, by the polymerase chain reaction, yielded a second clone which contained the missing 132 bases. Flanking nucleotides in the latter clone were identical to those in the cDNA clone containing the deletion. The two forms of human cystathionase deduced from the two cDNA clones may be derived from two different genes or may be splice variants.     

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.     

Nucleotide sequence of a full-length complementary DNA clone and amino acid sequence of human phenylalanine hydroxylase

A full-length human phenylalanine hydroxylase complementary DNA (cDNA) clone was isolated from a human liver cDNA library, and the nucleotide sequence encoding the entire enzyme was determined. The cDNA clone contains an inserted DNA fragment of 2448 base pairs, including 19 base pairs of poly(A) at the 3' end. The first methionine codon occurs at nucleotide position 223, followed by an open reading frame of 1353 base pairs, encoding 451 amino acids. Translation of the nucleotide sequence in the open reading frame predicts the amino acid sequence of human phenylalanine hydroxylase. The human protein shows a 96% amino acid sequence homology with the corresponding rat enzyme. The determination of the complete primary structure for phenylalanine hydroxylase represents the first among mixed-function oxidases.     

Molecular cloning and nucleotide sequence of the cDNA for human liver glutamate dehydrogenase precursor

Two cDNA clones (lambda GDHh1 and lambda GDHn61) for glutamate dehydrogenase (GDH) were isolated from a human liver cDNA library in lambda gt11. The clone, lambda GDHh1, was isolated from the library using a synthetic 45mer oligodeoxy-ribonucleotide, the sequence of which was derived from the known amino acid sequence near the NH2-terminus of human liver GDH. Subsequently, lambda GDHn61 was isolated from the same library using lambda GDHh1 as a probe. The inserts of both clones contained an overlapping cDNA sequence for human liver GDH, consisting of a 5'-untranslated region of 70 bp, an open reading frame of 1677 bp, a 3'-untranslated region of 1262 bp and a 15 base poly(A) tract. The predicted amino acid sequence revealed that the human liver GDH precursor consisted of a total of 558 amino acid residues including the NH2-terminal presequence of 53 amino acids. The sequence deduced for the mature enzyme showed 94% homology to the previously reported amino acid sequence of human liver GDH.     

The complete amino acid sequence of the shorter form of human basic fibroblast growth factor receptor deduced from its cDNA

We have isolated a full-length cDNA for human basic fibroblast growth factor (bFGF) receptor-like protein from a human placenta cDNA library. Determination of the nucleotide sequence of the cDNA allows elucidation of the complete amino acid sequence of the receptor (731 amino acids) which has two extracellular immunoglobulin-like domains, a transmembrane domain and an intracellular tyrosine kinase domain. The receptor has remarkable amino acid similarity (98% identity) to the shorter form of murine bFGF receptor reported recently (H.H.Reid et al. (1990) Proc.Natl.Acad.Sci. USA 87, 1596-1600). The receptor described here is expected to be the shorter form of human bFGF receptor.     

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.     

Characterization of cDNA clones encoding rabbit and human serum paraoxonase: the mature protein retains its signal sequence.

Serum paraoxonase hydrolyzes the toxic metabolites of a variety of organophosphorus insecticides. High serum paraoxonase levels appear to protect against the neurotoxic effects of organophosphorus substrates of this enzyme [Costa et al. (1990) Toxicol. Appl. Pharmacol. 103, 66-76]. The amino acid sequence accounting for 42% of rabbit paraoxonase was determined by (1) gas-phase sequencing of the intact protein and (2) peptide fragments from lysine and arginine digests. From these data, two oligonucleotide probes were synthesized and used to screen a rabbit liver cDNA library. A clone was isolated and sequenced, and contained a 1294-bp insert encoding an open reading frame of 359 amino acids. Northern blot hybridization with RNA isolated from various rabbit tissues indicated that paraoxonase mRNA is synthesized predominately, if not exclusively, in the liver. Southern blot experiments suggested that rabbit paraoxonase is coded by a single gene and is not a family member of closely related genes. Human paraoxonase clones were isolated from a liver cDNA library by using the rabbit cDNA as a hybridization probe. Inserts from three of the longest clones were sequenced, and one full-length clone contained an open reading frame encoding 355 amino acids, four less than the rabbit paraoxonase protein. Each of the human clones appeared to be polyadenylated at a different site, consistent with the absence of the canonical polyadenylation signal sequence. Of potential significance with respect to the paraoxonase polymorphism, the derived amino acid sequence from one of the partial human cDNA clones differed at two positions from the full-length clone. Amino-terminal sequences derived from purified rabbit and human paraoxonase proteins suggested that the signal sequence is retained, with the exception of the initiator methionine residue [Furlong et al. (1991) Biochemistry (preceding paper in this issue)]. Characterization of the rabbit and human paraoxonase cDNA clones confirms that the signal sequences are not processed, except for the N-terminal methionine residue. The rabbit and human cDNA clones demonstrate striking nucleotide and deduced amino acid similarities (greater than 85%), suggesting an important metabolic role and constraints on the evolution of this protein.