Cloning and expression of a cDNA encoding mouse kidney -amino acid oxidase

A cDNA encoding -amino acid oxidase (DAO;EC 1.4.3.3) has been isolated from a BALB/c mouse kidney cDNA library by hybridization with the cDNA for the porcine enzyme. Analysis of the nucleotide (nt) sequence of the clone revealed that it has a 1647-nt sequence with a 5′-terminal untranslated region of 68 nt that encodes 345 amino acids (aa), and a 3′-terminal untranslated region of 544 nt that contains the polyadenylation signal sequence ATTAAA. The deduced aa sequence showed 77 and 78% aa identity with the porcine and human enzymes, respectively. Two catalytically important aa residues, Tyr²²⁸ and His³⁰⁷, of the porcine enzyme, were both conserved in these three species. RNA blot hybridization analysis indicated that a DAO mRNA, of 2 kb, exists in mouse kidney and brain, but not liver. Synthesis of a functional mouse enzyme in Escherichia coli was achieved through the use of a vector constructed to insert the coding sequence of the mouse DAO cDNA downstream from the tac promoter of plasmid pKK223-3, which was designed so as to contain the lac repressor gene inducible by isopropyl-β- -thiogalactopyranoside. Immunoblot analysis confirmed the synthesis and induction of the mouse DAO protein, and the molecular size of the recombinant mouse DAO was found to be identical to that of the mouse kidney enzyme. Moreover, the maximum activity of the mouse recombinant DAO was estimated to be comparable with that of the porcine DAO synthesized in E. coli cells.     

Molecular cloning and nucleotide sequence of a bovine alpha-lactalbumin cDNA

A cDNA clone for the bovine milk protein, alpha-lactalbumin (alpha LA), has been identified using a rat cDNA probe. The bovine cDNA clone is 703 nucleotides (nt) long, contains 8 nt of 5'-untranslated sequence and 269 nt of 3'-untranslated sequence. When compared with previously reported sequences, the bovine alpha LA mRNA sequence has 74% similarity with rat alpha LA mRNA, 79% similarity with human mRNA and 74% similarity with guinea pig mRNA.     

Cloning and functional expression of a cDNA encoding coffee bean α-galactosidase

Purified coffee bean α-galactosidase (αGal) has been used for removing terminal α-galactose residues from the glycoconjugates at the red cell surface, in studies of blood group conversion. Here, we report the isolation and sequence of the full-length clone for coffee bean αGal by using the polymerase chain reaction (PCR) and rapid amplification of cDNA ends (RACE) techniques. The cDNA clone (1.4 kb) contains a single open reading frame which encodes a protein of 378 amino acids (aa). Its authenticity is confirmed by perfect alignment of aa sequences obtained from purified coffee bean αGal, and by immune reaction with the antibody raised against the enzyme. Furthermore, the protein produced in insect cells shows enzymatic activity towards a synthetic αGal substrate, p-nitro-phenyl-α-galactopyranoside.     

Cloning of a urochordate cDNA featuring mammalian short consensus repeats (SCR) of complement-control protein superfamily

Mammalian tumor necrosis factor (TNF)-α degenerate polymerase chain reaction (PCR) primers were used to amplify a probe from Botryllus schlosseri (colonial ascidian) allogeneic rejection-cDNA library. A PCR product (269 bp) was cloned and sequenced encoding an open reading frame (ORF) of 89 amino acids (aa). This clone, which revealed no similarity to TNF-α, but a substantial similarity to mammalian proteins featuring short consensus repeats (SCRs) of the complement control superfamily, was used to probe the rejection-cDNA library. Two partial cDNA clones were isolated and sequenced (Bs. 1, 846 bp; Bs.2, 712 bp). The longest ORF in clone Bs. 1 (which lacks the 5' end of the cDNA) predicts a protein of 251 aa, which differs from Bs.2 at six nucleotides and four aa. We compare the as similarity (up to 50.5%) of Bs.l with the SCR-region of mammalian complement factor H, apolipoprotein H, selectins, and complement receptors type 1 and type 2. A somatomedin B-like domain at the C-terminus of Bs. 1 deduced protein was also recorded. We propose that this mosaic and polymorphic botryllid sequence, featuring mammalian-like SCRs, might be an ancestral molecule in the evolution of the chordate's complement-control protein superfamily.     

Cloning, sequencing and functional expression of a cDNA encoding porcine pancreatic preprocarboxypeptidase A1.

A full-length cDNA clone coding for porcine pancreatic preprocarboxypeptidase A1 (prePCPA1) was isolated from a cDNA library. The open reading frame (ORF) of the nucleotide sequence was 1260 nt in length and encoded a protein of 419 amino acids (aa). The cDNA included a short signal peptide of 16 aa and a 94 aa-long activation segment. The calculated molecular mass of the mature proenzyme was 45561 Da, in accordance with that of the purified porcine pancreatic PCPA1. The deduced aa sequence of the corresponding enzyme differed from that predicted by the three-dimensional structure by 40 aa, and showed 85% identity and 55% identity to that of procarboxypeptidases A1 and A2, respectively. Moreover the sequence was identical to that of several independent cDNA clones, suggesting that it is the major transcribed gene. No evidence for a second variant was observed in the cDNA library and PCPA2 is apparently absent from the porcine pancreas. The cDNA was expressed in Saccharomyces cerevisiae under the control of the yeast triose phosphate isomerase promoter. The signal peptide of the PCPA protein efficiently directed its secretion into the culture medium (1.5 mg.L-1) as a protein of the predicted size. The recombinant proenzyme was analyzed by immunological and enzymological methods. Its activation behavior was comparable with that of the native form and led to a 35-kDa active enzyme.     

Cloning and sequencing of a full-length rat sucrase-isomaltase-encoding

Sucrase-isomaltase (SI) has been widely used as a marker enzyme to study cellular differentiation in the small intestine. We isolated a 6.1-kb SI cDNA clone (GC1.4) from a size-fractionated cDNA library from rat intestine. Sequencing of this cDNA clone showed 6066 nucleotides (nt) with an open reading frame (ORF) of 1841 amino acids (aa). The nt sequence correctly predicts several known aa stretches in the protein. The deduced aa sequence showed 78 and 75% overall identity with the rabbit and human SI, respectively. At the active sites of both S and I, the rat nt sequence encodes stretches of 14 and 16 aa, respectively, which show 100% identity to rabbit and human SI. In the region immediately beyond the transmembrane domain, the rat sequence encodes an extra 10 aa, as compared to rabbit and human. This 10-aa insertion consists almost entirely of Pro, Ser and Thr, and may be responsible for additional 0-glycosylations of rat SI. The cDNA contains a 3'-UTR (untranslated region) of 499 nt with polyadenylation signal sequence and a poly(A) tract. The ATG start codon was found 41 nt downstream from the 5' end of the cDNA. Primer extension experiments showed the cap site to be 61 nt upstream from the start codon. The results indicate that our cDNA clone lacks only 20 nt in the 5'-UTR. Given that this cDNA encodes the entire coding region of SI, it should be useful in elucidating the regulatory mechanisms of SI biosynthesis, localization and targeting during rat intestinal development and differentiation.     

黄瓜花叶病毒山东株（CMV—SD）RNA2全和cDNA克隆的构建及全序列分析

分别利用５’ＲＡＣＥ和３’ＲＡＣＥ确定了ＣＭＶ－ＳＤＲＮＡ２的５’和３’未端序列,在此基础上,利用ＲＴ－ＰＣＲ得到了ＲＮＡ２的５’端一半的ｃＤＮＡＤＱ ＢＴＧ Ｐｃ２５和３’端一半的ｃＤＮＡ克隆ＰＣ２３,并通过拼接构建了ＲＮＡ２全长ｃＤＮＡ克隆ＰＣ２Ｆ。     

The nucleotide sequence of a cDNA clone containing the entire coding region for mouse X-chromosome-linked phosphoglycerate kinase

A clone containing cDNA for X chromosome-linked phosphoglycerate kinase (PGK-1) was isolated from a mouse myeloma cDNA library. The nucleotide (nt) sequence of the cDNA has been determined, and the amino acid (aa) sequence of the enzyme thereby deduced. At the nt level, the coding region of mouse PGK cDNA has 93% homology with human X-linked cDNA and 60% homology with the yeast gene. Mouse PGK-1 protein contains 416 aa and is 98%, 96% and 64% homologous with human, horse, and yeast enzyme sequences, respectively.     

Comprehensive cloning of Schizosaccharomyces pombe genes encoding translation elongation factors

In the course of the Schizosaccharomyces pombe cDNA project, we succeeded in cloning all the genes encoding translation elongation factors EF-1α, EF-1β, EF-1γ, EF-2 and EF-3. With the exception of the EF-1γ gene, the nucleotide (nt) sequence of S. pombe elongation factors has not been previously reported. For EF-1α, we found three genes whose amino acid (aa) sequences are quite homologous each other (99.5%), but whose 3′ untranslated regions (UTRs) are completely different. Southern blot indicated that those three EF-1α genes are located at different loci. Northern analysis indicated that one of three EF-1α genes was inducible with UV-irradiation, while the level of expression for another of three EF-1α genes was repressed by UV and heat-shock (HS) treatments. The aa sequence predicted from the nt sequence of the S. pombe EF-1β cDNA clone covered almost all the coding sequence (CDS) of EF-1β except the first methionine which has 55.4% identity with that of S. cerevisiae. We also identified two copies of S. pombe EF-2 genes. Their aa sequences deduced from nt sequences are identical (100%), but they have different 3′ UTRs. The location of these two EF-2 genes in different loci was proved by Southern analysis. The S. pombe EF-3 cDNA clone encoded only a third of the CDS from the C-terminal and its deduced aa sequence has a 76% identity with those of other yeasts and fungi.     

Localization of the human Rh blood group gene structure to chromosome region 1p34.3–1p36.1 by in situ hybridization

Summary A cDNA clone, RhIXb (1384 bp), encoding the entire protein sequence of a human blood group Rh polypeptide has been used to map the Rh locus, by in situ hybridization, to the region p34.3–p36.1 of chromosome 1. Two other unrelated cDNA clones, pUCA2 (750bp) and pUCIII (1600 bp), isolated during the cloning procedure of the Rh cDNA were investigated simultaneously, and assigned to chromosome 3p21.1–3p22 (clone pUCA2) and to chromosome 22q12.1–22q13.1 (clone pUCIII).     

Sequence and expression of the Drosophila phenylalanine hydroxylase mRNA

We report the cloning, nucleotide (nt) sequence and expression of the cDNA (pah) encoding phenylalanine hydroxylase (PAH) of Drosophila melanogaster. The strong hybridization signals observed in genomic blots when D. melanogaster DNA was probed with 32P-labeled human pah cDNA, indicated the existence of a high degree of sequence similarity between the pah genes of both species. The length of the pah genomic fragment is about 30 to 40 kb. The cDNA contains 84 bp of the 5'-untranslated region, 1359 bp of the protein-coding region and 87 bp of the 3' region, with only one polyadenylation signal. The isolated cDNA is probably full-length, since the size of the D. melanogaster PAH mRNA is 1.5 kb. At the nt level, the similarity of the D. melanogaster cDNA with human and rat pah cDNAs is 57.9% and 58.1%, respectively. The highest similarities are restricted to the nt sequence coding for the presumed hydroxylation domain. There is no nt sequence similarity between the first three exons of the human pah gene and an equivalent fraction of the D. melanogaster pah gene. At the amino acid (aa) level, the similarity in the presumed hydroxylation domain is 88.5%, in which two motifs of the structure AGLLSSXXXL are found, where X represents any aa. It was interesting to notice the conservation of aa 408, 311 and 280, where mutations are associated with phenylketonuria in humans. We observed, moreover, that, as it occurs in humans and rats, the expression of the D. melanogaster pah gene is tissue-specific and temporally regulated.     

The Human β-Defensin-1 and α-Defensins Are Encoded by Adjacent Genes: Two Peptide Families with Differing Disulfide Topology Share a Common Ancestry

We cloned a novel human β-defensin gene and determined its full-length cDNA sequence. The entire gene spanned more than 7 kb and included a large 6962-bp intron. The 362-bp cDNA encoded a prepropeptide that corresponded precisely to the recently identified human β-defensin HBD-1, an antimicrobial peptide implicated in the resistance of epithelial surfaces to microbial colonization. By two-color fluorescencein situhybridization on both metaphase chromosome and released chromatin fiber, HBD-1 gene (DEFB1 in HUGO/GDB nomenclature) mapped to chromosomal region 8p23.1–p23.2 in close proximity (within 100–150 kb) to the gene for the human neutrophil α-defensin HNP-1 (DEFA1). Thus, despite a complete lack of DNA sequence similarity and despite differences in their disulfide-pairing pattern, the α- and β-families appear to have evolved from a common premammalian defensin gene.     

Cloning and sequence analysis of a cDNA for the α-globin mRNA of carp, Cyprinus carpio

A cDNA for α-globin mRNA of the carp, Cyprinus carpio, was cloned by the method of Okayama and Berg (Mol. Cell. Biol. 2 (1982) 161–170) and its complete nucleotide sequence was determined. The 5′ non-coding region contained 23 nucleotides. Following this region, there was an open reading frame encoded with an α-globin polypeptide consisting of 142 amino acids. The 3′ non-coding region was 88 nucleotides in length, including two copies of the hexanucleotide AATAAA and a poly(A) site of the GC dinucleotide. There were 16 discrepancies between the reported amino acid sequence of the carp α-globin chain and the amino acid sequence predicted from the DNA sequence of the clone. The possible explanations for these differences in amino acid sequence are discussed.     

Bovine corneal protein 54K (BCP54) is a homologue of the tumor-associated (class 3) rat aldehyde dehydrogenase (RATALD)

Amino acid (aa) sequence data from Staphylococcus areas V8 protease-digested bovine corneal 54-kDa protein (BCP54) fragments were utilized to derive mixed oligodeoxyribonucleotide (oligo) primers complementary to the reverse translation products of these sequences. These degenerate oligo primers were used to prime the amplification of BCP54 sequence from bovine corneal epithelial cell cDNA. The cDNA probe generated by this mixed oligo-primed amplification of cDNA was cloned and dideoxy-sequenced. A search of the GenBank database (version 63.0) revealed extensive sequence similarity to the cDNA encoding tumor-associated rat liver (class 3) aldehyde dehydrogenase (RATALD). Nucleotide (nt) and aa sequence alignment of the BCP54 translation product reveals it is 78% and 84% homologous with RATALD at the nt and aa levels, respectively. Conservation of aa sequence elements common to the aldehyde dehydrogenase family thought to be of structural/functional significance is further substantiated by this analysis. Included in the discussion is the likelihood that gene sharing (genes encoding metabolic enzymes and other stable proteins) may extend to the cornea.     

Simple high-performance liquid chromatographic purification procedure for porcine plasma haptoglobin

Haptoglobin is an acute-phase protein and its plasma levels increase consistently in response to infection and inflammation. Some evidence has demonstrated that haptoglobin is involved in the immune responses. In this study, we established a novel high-performance liquid chromatographic purification procedure for porcine plasma haptoglobin. The procedure required an ammonium sulfate fractionation and a HPLC Superose 12 gel-permeation chromatography. Purified porcine haptoglobin possessed one heavy (β) and light chain (α) on sodium dodecyl sulfate–polyacrylamide gel electrophoresis, under reducing conditions, with a M_r (molecular mass) of about 42 000 and 14 000 for heavy (β) and light chains (α), respectively. Although the N-terminal amino acid sequence of porcine heavy chain of haptoglobin has never been reported previously, the analyses of N-terminal amino acid sequence showed a great sequence similarity to that of human and other animal species. In addition, Western blot using our specific antibody prepared against porcine M_r 42 000 chain did react with human haptoglobin and likewise, the antibody against human haptoglobin also cross-reacted with purified porcine M_r 42 000 chain. Thus, it confirmed that the identity of the porcine protein purified from our procedures was as haptoglobin.     

Cloning and sequence analysis of porcine myoglobin cDNA

Porcine myoglobin cDNA clones have been isolated from a cDNA library prepared from enriched heart-myoglobin mRNA. Sequence analysis revealed 59 nucleotides (nt) in the 5'-untranslated, 462 nt in the amino acid (aa)-coding, and 590 nt in the 3'-untranslated regions. The myoglobin cDNA showed a high G + C content (60%). When the nt sequence of the porcine myoglobin cDNA is compared with those of seal and human myoglobin cDNAs deduced from the corresponding genomic myoglobin genes [Blanchetot et al., Nature 301 (1983) 732-734; Weller et al., EMBO J. 3 (1984) 439-446; Akaboshi, Gene 33 (1985) 241-249], a high degree of homology is observed in the 5'-untranslated region and in parts of the 3'-untranslated region, as well as in the coding region.