Structures of mouse Rep-8 cDNA and genomic clones

A mouse homologue of the human Rep-8 gene was cloned by PCR methods using degenerate oligonucleotide primers corresponding to highly conserved regions between human and mouse genes, and by the Marathon–Ready cDNA amplification method. The full-length mouse Rep-8 contains 1422 nucleotides and codes for a protein of 277 amino acids with a calculated mol. wt. of 31,519. The overall amino acid sequence homology between mouse and human Rep-8 proteins was 73%, and the overall nucleic acid sequence similarity was 65%. The predicted amino acid sequence of mouse Rep-8 has leucine zipper-like motifs in the N-terminal region, similar to the human Rep-8 protein. Rep-8 exists as a single-copy gene and is expressed in both the early and late embryonic stages of mouse development, suggesting that the Rep-8 gene product has an important role in embryogenesis. The genomic structure of the mouse Rep-8 gene was characterized extensively so that a gene targeting strategy could be used to develop an understanding of the biological function(s) of this interesting gene and its product.     

cDNA structures and regulation of two interferon-induced human Mx proteins.

Human cells treated with interferon synthesize two proteins that exhibit high homology to murine Mx1 protein, which has previously been identified as the mediator of interferon-induced cellular resistance of mouse cells against influenza viruses. Using murine Mx1 cDNA as a hybridization probe, we have isolated cDNA clones originating from two distinct human Mx genes, designated MxA and MxB. In human fibroblasts, expression of MxA and MxB is strongly induced by alpha interferon (IFN-alpha), IFN-beta, Newcastle disease virus, and, to a much lesser extent, IFN-gamma, MxA and MxB proteins have molecular masses of 76 and 73 kilodaltons, respectively, and their sequences are 63% identical. A comparison of human and mouse Mx proteins revealed that human MxA and mouse Mx2 are the most closely related proteins, showing 77% sequence identity. Near their amino termini, human and mouse Mx proteins contain a block of 53 identical amino acids and additional regions of very high sequence similarity. These conserved sequences are also present in a double-stranded RNA-inducible fish gene, which suggests that they may constitute a functionally important domain of Mx proteins. In contrast to mouse Mx1 protein, which accumulates in the nuclei of IFN-treated mouse cells, the two human Mx proteins both accumulate in the cytoplasm of IFN-treated cells.     

Cloning and characterization of human pancreatic lipase cDNA

Pancreatic lipase (triacylglycerol acylhydrolase, EC 3.1.1.3) hydrolyzes dietary long chain triacylglycerol to free fatty acids and monoacylglycerols in the intestinal lumen. In the presence of bile acids, the activity of lipase is stimulated by colipase. As a prelude to studying the relationship of the protein structures to the functional properties of lipase and colipase, a cDNA encoding human pancreatic lipase was isolated from a lambda gt11 cDNA library screened with a rabbit polyclonal anti-human pancreatic lipase antibody. The full length cDNA clone of 1477 base pairs contained an open reading frame encoding a 465-amino acid protein, including a 16-amino acid signal peptide. The nucleotide sequence was 69% identical to the dog pancreatic lipase cDNA. The predicted NH2-terminal protein sequence agreed with the published NH2-terminal sequence of human pancreatic lipase and the predicted protein sequence was 85 and 70% identical to the protein sequences of pig and dog pancreatic lipase, respectively. A region of homology around Ser-153 is conserved in a number of lipid-binding proteins. Human hepatic lipase and lipoprotein lipase share extensive homology with pancreatic lipase, suggesting that the three proteins are members of a small gene family. In vitro translation of mRNA transcribed from the cDNA resulted in a protein of the expected molecular size that could be processed by microsomal membranes to yield a glycolated protein with proper signal peptide cleavage. RNA blot analysis demonstrated tissue specificity for pancreatic lipase. Thus, for the first time, a full length human pancreatic lipase cDNA has been isolated and characterized. The demonstrated regions of homology with other lipases will aid definition of interactions with substrate and colipase through site-specific mutagenesis.     

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.     

Construction of a human full-length cDNA bank

We aimed to construct a full-length cDNA bank from an entire set of human genes and to analyze the function of a protein encoded by each cDNA. To achieve this purpose, a multifunctional phagemid shuttle vector, pKAl, was constructed for preparing a high-quality cDNA library composed of full-length cDNA clones which can be sequenced and expressed in vitro and in mammalian cells without subcloning the cDNA fragment into other vectors. Using this as a vector primer, we have prepared a prototype of the bank composed of full-length cDNAs encoding 236 human proteins whose amino acid sequences are identical or similar to known proteins. Most cDNAs contain a putative cap site sequence, some of which show a pyrimidine-rich conserved sequence exhibiting polymorphism. It was confirmed that the vector permits efficient in vitro translation, expression in mammalian cells and the preparation of nested deletion mutants.     

Mouse "protective protein". cDNA cloning, sequence comparison, and expression

The "protective protein" is the glycoprotein that forms a complex with the lysosomal enzymes beta-galactosidase and neuraminidase. Its deficiency in man leads to the metabolic storage disorder galactosialidosis. The primary structure of human protective protein, deduced from its cloned cDNA, shows homology to yeast serine carboxypeptidases. We have isolated a full-length cDNA encoding murine protective protein. The nucleotide sequences as well as the predicted amino acid sequences are highly conserved between man and mouse. Domains important for the protease function are completely identical in the two proteins. Both human and mouse mature protective proteins covalently bind radiolabeled diisopropyl fluorophosphate. Transient expression of the murine cDNA in COS-1 cells yields a protective protein precursor of 54 kDa, a size characteristic of the glycosylated form. This cDNA-encoded precursor, endocytosed by human galactosialidosis fibroblasts, is processed into a 32- and a 20-kDa heterodimer and corrects beta-galactosidase and neuraminidase activities. A tissue-specific expression of protective protein mRNA is observed when total RNA from different mouse organs is analyzed on Northern blots.     

Isolation of cDNA clones for human erythrocyte membrane sialoglycoproteins alpha and delta.

We have isolated almost full-length cDNA clones corresponding to human erythrocyte membrane sialoglycoproteins alpha (glycophorin A) and delta (glycophorin B). The predicted amino acid sequence of delta differs at two amino acid residues from the sequence determined by peptide sequencing. The sialoglycoprotein delta clone we have isolated contains an interrupting sequence within the region that gives rise to the cleaved N-terminal leader sequence for the protein and represents a product that is unlikely to be inserted into the erythrocyte membrane. Comparison of the cDNA sequences of alpha and delta shows very strong homology at the DNA level within the coding regions. The two mRNA sequences are closely related and differ by a number of clearly defined insertions and deletions.     

Characterization of long cDNA clones from human adult spleen. II. The complete sequences of 81 cDNA clones.

To accumulate information on the coding sequences (CDSs) of unidentified genes, we have conducted a sequencing project of human long cDNA clones. Both the end sequences of approximately 10,000 cDNA clones from two size-fractionated human spleen cDNA libraries (average sizes of 4.5 kb and 5.6 kb) were determined by single-pass sequencing to select cDNAs with unidentified sequences. We herein present the entire sequences of 81 cDNA clones, most of which were selected by two approaches based on their protein-coding potentialities in silico: Fifty-eight cDNA clones were selected as those having protein-coding potentialities at the 5'-end of single-pass sequences by applying the GeneMark analysis; and 20 cDNA clones were selected as those expected to encode proteins larger than 100 amino acid residues by analysis of the human genome sequences flanked by both the end sequences of cDNAs using the GENSCAN gene prediction program. In addition to these newly identified cDNAs, three cDNA clones were isolated by colony hybridization experiments using probes corresponding to known gene sequences since these cDNAs are likely to contain considerable amounts of new information regarding the genes already annotated. The sequence data indicated that the average sizes of the inserts and corresponding CDSs of cDNA clones analyzed here were 5.0 kb and 2.0 kb (670 amino acid residues), respectively. From the results of homology and motif searches against the public databases, functional categories of the 29 predicted gene products could be assigned; 86% of these predicted gene products (25 gene products) were classified into proteins relating to cell signaling/communication, nucleic acid management, and cell structure/motility.     

The cDNA sequence and the deduced amino acid sequence of human transcobalamin II show homology with rat intrinsic factor and human transcobalamin I.

The cellular uptake of cobalamin (Cbl, vitamin B12) is mediated by transcobalamin II (TCII), a plasma protein that binds Cbl and is secreted by human umbilical vein endothelial (HUVE) cells. These cells synthesize and secrete TCII and, therefore, served as the source of the complementary DNA (cDNA) library from which the TCII cDNA was isolated. This full-length cDNA consists of 1866 nucleotides that code for a leader peptide of 18 amino acids, a secreted protein of 409 amino acids, a 5'-untranslated segment of 37 nucleotides, and a 3'-untranslated region of 548 nucleotides. A single 1.9-kilobase species of mRNA corresponding to the size of the cDNA was identified by Northern blot analysis of the RNA isolated from HUVE cells. TCII has 20% amino acid homology and greater than 50% nucleotide homology with human transcobalamin I (TCI) and with rat intrinsic factor (R-IF). TCII has no homology with the amino-terminal region of R-IF that has been reported to have significant primary as well as secondary structural homology with the nucleotide-binding domain of NAD-dependent oxidoreductases. The regions of homology that are common to all three proteins are located in seven domains of the amino acid sequence. One or more of these conserved domains is likely to be involved in Cbl binding, a function that is common to all three proteins. However, the difference in the affinity of TCII, TCI, and R-IF for Cbl and Cbl analogues indicates, a priori, that structural differences in the ligand-binding site of these proteins exist and these probably resulted from divergence of a common ancestral gene.     

The complete sequence of a full length cDNA for human liver glyceraldehyde-3-phosphate dehydrogenase: evidence for multiple mRNA species.

A recombinant M13 clone (O42) containing a 65 b.p. cDNA fragment from human fetal liver mRNA coding for glyceraldehyde-3-phosphate dehydrogenase has been identified and it has been used to isolate from a full-length human adult liver cDNA library a recombinant clone, pG1, which has been subcloned in M13 phage and completely sequenced with the chain terminator method. Besides the coding region of 1008 b.p., the cDNA sequence includes 60 nucleotides at the 5'-end and 204 nucleotides at the 3'-end up to the polyA tail. Hybridization of pG1 to human liver total RNA shows only one band about the size of pG1 cDNA. A much stronger hybridization signal was observed using RNA derived from human hepatocarcinoma and kidney carcinoma cell lines. Sequence homology between clone 042 and the homologous region of clone pG1 is 86%. On the other hand, homology among the translated sequences and the known human muscle protein sequence ranges between 77 and 90%; these data demonstrate the existence of more than one gene coding for G3PD. Southern blot of human DNA, digested with several restriction enzymes, also indicate that several homologous sequences are present in the human genome.     

Molecular cloning and nucleotide sequence of human pancreatic secretory trypsin inhibitor (PSTI) cDNA

We have isolated and sequenced a cDNA clone coding for the human pancreatic secretory trypsin inhibitor (PSTI) from a human pancreatic cDNA library. The predicted product consists of 79 amino acids, and contains no apparent functional polypeptide other than PSTI. Southern blot analysis suggests that there is one copy of PSTI gene per haploid genome. This gene seems to be expressed not only in pancreas, but also in gastric mucosa, since a Northern blot analysis demonstrated the presence of a poly(A) RNA of the same size as in the pancreas. A comparison of sequences between human PSTI mRNA and mouse epidermal growth factor (EGF) mRNA revealed a high homology, suggesting that they share a common ancestral DNA sequence.     

Mouse and human CD14 (myeloid cell-specific leucine-rich glycoprotein) primary structure deduced from cDNA clones

cDNA clones complementary to MS7-4 (Setoguchi et al. (1988) Somat. Cell Mol. Genet. 14, 427-438) from a mouse macrophage cDNA library were separated. Sequence analysis of these clones demonstrated that the longest cDNA clone, MS7X, had a 1366 bp insert and high homology with that of the human CD14 gene (Ferrero and Goyert (1988) Nucleic Acids Res. 16, 4173). Using the MS7X cDNA probe, cDNA clones were separated from cDNA libraries constructed from a human macrophage cell line and macrophages. The total cDNA sequence was 1364 bp in length, with an open reading frame of 1125 nucleotides matching that of the human CD14 gene except for one nucleotide difference. The amino-acid sequence (mouse CD14), deduced from the nucleotide sequence of the MS7X insert consisted of 351 amino-acid residues with a high leucine content (17.66%) and five putative N-glycosylation sites, and in vitro translation predicted a protein of molecular mass of 37.5 kDa. Human CD14 had 356 amino-acid residues, with high leucine content (15.5%), and contained four putative N-glycosylation sites. Mouse CD14 showed 13 building blocks, of which internal nine blocks have a conserved leucine motif and significant homology with human leucine-rich alpha 2-glycoprotein.     

Nucleotide sequences of the cDNA and an intronless pseudogene for human lactate dehydrogenase-A isozyme

Eight cDNA clones for lactate dehydrogenase-A isozyme (LDH-A) were isolated from a human fibroblast cDNA library, characterized, and no sequence heterogeneity was found. Four cDNA clones appear to contain nearly full-length cDNA inserts and the complete nucleotide sequence of 1710 base pairs consists of the protein-coding sequence (999 base pairs), the 5' (97 base pairs) and 3' (565 base pairs) untranslated regions and poly(dA) tail (49 base pairs). The predicted amino acid sequence of the human LDH-A polypeptide shows 92% homology (27 differences out of 331 amino acids compared) with that of the pig LDH-A subunit determined by direct protein sequencing [Kiltz et al. (1977) Hoppe-Seyler's Z. Physiol. Chem. 358, 123-127]. Human genomic clones containing an LDH-A pseudogene were isolated and the nucleotide sequence of 1635 base pairs from an intronless pseudogene was determined. The presence of two termination codons, two deletions of three nucleotides each and the replacement of three arginine residues at the active site (nos 98, 105 and 168) by other amino acids renders its coding region incapable of producing a functional LDH-A protein. A comparison between human LDH-A cDNA and the pseudogene sequences reveals 12.9% differences (114 transitions, 65 transversions and 36 deletions/insertions). Further, only four out of the 25 dCpdG dinucleotides present in the cDNA sequence remain unchanged, although the sequences possess 87.1% homology.     

cDNA sequence of human class III alcohol dehydrogenase

A human placental cDNA library was screened using oligonucleotide probes based on the peptide sequence of the human class III alcohol dehydrogenase. An incomplete cDNA clone covering most of the coding sequence of class III alcohol dehydrogenase was isolated from a human placental cDNA library. This was subsequently used as a probe to obtain a full-length clone from a human testicular library. The cDNA sequence codes for a protein that is identical to the enzyme purified from human liver. Southern analysis of human genomic DNA suggests that it may contain more than a single copy per haploid genome.     

Feline arylsulfatase B (ARSB): isolation and expression of the cDNA, comparison with human ARSB, and gene localization to feline chromosome A1.

Arylsulfatase B (ARSB) is the lysosomal enzyme that catalyzes the hydrolysis of 4-sulfate groups from N-acetylgalactosamine 4-sulfate moieties on the glycosaminoglycans, dermatan sulfate and chondroitin sulfate A. In man, a deficiency of this enzymatic activity causes the lysosomal storage disorder, Maroteaux-Lamy disease (mucopolysaccharidosis Type VI; MPS VI). MPS VI in Siamese cats also has been described, and the comparative pathologic and biochemical abnormalities of the human and feline disorders have been well characterized. The present study describes the isolation and expression of cDNAs encoding feline ARSB and the assignment of the feline ARSB gene to feline chromosome A1. The full-length feline ARSB cDNA sequence is 1939 bp, including 3 and 328 bp of 5' and 3' untranslated sequences, respectively, and a 1608-bp open reading frame encoding 535 amino acids. The predicted human and feline ARSB proteins are 91% identical and 94% similar. However, despite this high homology, the predicted feline ARSB polypeptide has nine cysteine residues, while the human enzyme has eight. The presence of the extra cysteine residue at position 451 in the feline enzyme may explain why feline ARSB is a homodimer and the human enzyme is a monomer. To facilitate comparative structure/function studies of the human and feline enzymes and to initiate somatic gene therapy trials in the MPS VI cats, a full-length feline ARSB cDNA was reconstructed from a 1440-bp partial cDNA and an ARSB fragment amplified from feline first-strand cDNA by the polymerase chain reaction. The functional integrity of this cDNA was demonstrated by transient expression in human embryonic kidney cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

Sequence analysis of the large and small subunits of human ribonucleotide reductase.

We have isolated and sequenced overlapping cDNA clones from a breast carcinoma cDNA library containing the entire coding region of both the R1 and R2 subunits of the human ribonucleotide reductase gene. The coding region of the human R1 subunit comprises 2376 nucleotides and predicts a polypeptide of 792 amino acids (calculated molecular mass 90,081). The sequence of this subunit is almost identical to the equivalent mouse ribonucleotide reductase subunit with 97.7% homology between the mouse and human R1 subunit amino acid sequences. The coding region of the human R2 subunit of ribonucleotide reductase comprises 1170 nucleotides and predicts a polypeptide of 389 amino acids (calculated molecular mass 44,883), which is one amino acid shorter than the equivalent mouse subunit. The human and mouse R2 subunits display considerable homology in their carboxy-terminal amino acid sequences, with 96.3% homology downstream of amino acid 68 of the human and mouse R2 proteins. However, the amino-terminal portions of these two proteins are more divergent in sequence, with only 69.2% homology in the first 68 amino acids.     

Cloning and sequencing of a full length cDNA corresponding to human cellular retinol-binding protein

We have isolated and sequenced a cDNA clone corresponding to the human cellular retinol-binding protein (CRBP). The deduced amino acid sequence, which encompasses 134 amino acid residues, shows significant homology with several low molecular weight proteins which bind hydrophobic ligands. No homology to the plasma retinol-binding protein was observed. Southern and Northern blot analyses suggest that the CRBP gene is present in a single copy in the haploid genome and that it is transcribed in a single mRNA species.     

Characterization and chromosomal mapping of a cDNA encoding tryptophan hydroxylase from a mouse mastocytoma cell line

A cDNA library was constructed from RNA prepared from P815 mouse mastocytoma cells and screened for tryptophan hydroxylase. An essentially full-length clone that recognizes a major mRNA species of 1.9 kb in mastocytoma cell lines and in pineal gland, duodenum, and brainstem of the mouse was obtained. The predicted amino acid sequence of this mouse mastocytoma clone showed 97 and 87% identity, respectively, with tryptophan hydroxylase clones isolated from rat and rabbit pineal glands, but the mouse clone contains an unusual 3-amino-acid duplication near the N-terminus and lacks a phosphorylation site. A fragment of the cDNA produced an enzymatically active protein when expressed in Escherichia coli, thus demonstrating that the catalytic domain is included in the C-terminal 380 amino acids. The mouse tryptophan hydroxylase locus, termed Tph, was mapped by Southern blot analysis of somatic cell hybrids and by an interspecific backcross to a position in the proximal half of chromosome 7. Because TPH has been mapped to human chromosome 11, this assignment further defines regions of homology between these mouse and human chromosomes.