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PubMed1983

Cloning of human purine-nucleoside phosphorylase cDNA sequences by complementation in Escherichia coli.

We have obtained cDNA clones that contain the entire coding region of the human purine-nucleoside phosphorylase (PNP; EC 2.4.2.1) mRNA. The cDNA sequences were generated by reverse transcription of PNP-enriched mRNA obtained by immunoadsorption of HeLa cell polyribosomes with monospecific antibody to human PNP. cDNA molecules that were close in length to PNP mRNA were separated by agarose gel electrophoresis and inserted into the Pst I site of the plasmid pBR322. Plasmid DNA from the pooled clones was used to transform PNP-deficient Escherichia coli cells, and those transformants that phenotypically expressed PNP were isolated on selective media. The presence of human PNP in the selected bacterial cells was detected by immunoprecipitation with human PNP antibody.

Base Sequence↗
PubMed1991

Complete cDNA sequence of human complement pro-C5. Evidence of truncated transcripts derived from a single copy gene.

Two truncated human C5 clones, pHC5A and pHC5B, were isolated from an adult human liver cDNA library, and contained inserts of 2930 and 2181 bp, respectively. Both clones were polyadenylated and encoded the 5'-end of the C5 pro-molecule, thereby completing the human pro-C5 cDNA sequence. However, near the 3'-ends, at exon/intron boundaries, the nucleotide sequences of pHC5A and pHC5B diverged from each other and from the full-length 6.0-kb C5 cDNA sequence. Clone pHC5A, which overlapped the first human C5 clone described (J-16), encoded most of the C5 signal peptide, the complete beta-chain, the linker peptide, 177 amino acids of the alpha-chain, and contained 144 bp of Alu family consensus sequence encoding 48 amino acids of divergent protein sequence in an open reading frame. Clone pHC5B encoded the entire C5 signal peptide, the beta-chain, the linker peptide, nine amino acids of the alpha-chain, and six amino acids of divergent protein sequence in an open reading frame. Northern blot experiments demonstrated the presence of a 3.0-kb truncated C5 mRNA in adult human liver and a 4.8-kb truncated C5 mRNA in HepG2 cells in addition to the 6.0-kb full-length transcript. Truncated C5 mRNA were not detected in Raji, MOLT-4, human fibroblast or U937 cells, although the full-length 6.0-kb transcript was seen in MOLT-4 cells. Southern blot analyses indicated that the human C5 structural gene is large, complex, and is present in the human genome in a single copy, thereby demonstrating that the truncated C5 clones and mRNA are derived from a single C5 gene by alternative processing events.

Amino Acid Sequence↗
PubMed2006

Mucin gene cDNA sequence characterization in chinchilla middle ear epithelium.

OBJECTIVES: To identify mucin genes in chinchilla middle ear epithelium and characterize complimentary deoxyribonucleic acid (cDNA) sequences to facilitate further investigations into mucin physiology and pathophysiology on a molecular level using the chinchilla model. METHODS: Chinchilla mucin gene exploration and cDNA characterization was accomplished using reverse transcriptase-polymerase chain reactions (RT-PCR). Forward and reverse primer pairs were designed using consensus sequences available for human and rodent species. Chinchilla middle ear epithelium was harvested and primary cell cultures (CMEEC) were established. The CMEEC were explored for the expression of chinchilla mucin genes 1, 2, 4 and 5AC (cMuc1, cMuc2, cMuc4 and cMuc5AC). Identified cDNA amplicons for each of these genes was sequenced and homology compared to previously published human and rodent sequences. RESULTS: CMEEC express all four of the mucin genes cMuc1, cMuc2, cMuc4 and cMuc5AC. cDNA amplicons for each of the genes were able to be sequenced with lengths ranging from 66 to 362 base pairs. Each of the chinchilla cDNA sequences expressed significant homology with published human and rodent cDNA for these mucin genes. A cDNA sequence for the housekeeping gene, beta-actin, was also identified. CONCLUSIONS: Chinchilla middle ear epithelium grown in culture expresses the mucin genes 1, 2, 4 and 5AC, which have been identified as important in mucin regulation in the middle ear. cDNA sequences corresponding to these mucin genes were identified and may serve as important molecular tools in future studies of otitis media using the chinchilla model.

Actins↗
PubMed1988

Derivation of the sequence of the signal peptide in human C4b-binding protein and interspecies cross-hybridisation of the C4bp cDNA sequence.

A 5' cDNA clone coding for human C4b-binding protein (C4bp) was isolated, characterised and sequenced to complete the cDNA sequence coding for residues 1-32 thus confirming the protein sequence data of Chung et al. [(1985) Biochem. J. 230, 133-141]. The sequence extended to allow derivation of the putative leader peptide sequence which was 32 residues in length and showed a high of hydrophobicity typical of other documented leader sequences. Cross hybridisation was detected between the human C4bp cDNA probes and genomic DNA isolated from various species on Southern blots suggesting that genomic sequence homologous to that coding for C4bp has been conserved during evolution.

Amino Acid Sequence↗
PubMed1999

Identification and expression of the TREX1 and TREX2 cDNA sequences encoding mammalian 3'-->5' exonucleases.

The 3'-->5' exonucleases catalyze the excision of nucleoside monophosphates from the 3' termini of DNA. We have identified the cDNA sequences encoding two 3'-->5' exonucleases (TREX1 and TREX2) from mammalian cells. The TREX1 and TREX2 proteins are 304 and 236 amino acids in length, respectively. Analysis of the TREX1 and TREX2 sequences identifies three conserved motifs that likely generate the exonuclease active site in these enzymes. The specific amino acids in these three conserved motifs suggest that these mammalian exonucleases are most closely related to the proofreading exonucleases of the bacterial replicative DNA polymerases and the RNase T enzymes. Expression of TREX1 and TREX2 in Escherichia coli demonstrates that these recombinant proteins are active 3'-->5' exonucleases. The recombinant TREX1 protein was purified, and exonuclease activity was measured using single-stranded, partial duplex, and mispaired oligonucleotide DNA substrates. The greatest activity of the TREX1 protein was detected using a partial duplex DNA containing five mispaired nucleotides at the 3' terminus. No activity was detected using single-stranded RNA or an RNA-DNA partial duplex. Identification of the TREX1 and TREX2 cDNA sequences provides the genetic tools to investigate the physiological roles of these exonucleases in mammalian DNA replication, repair, and recombination pathways.

Amino Acid Sequence↗
PubMed1996

The heterogeneity of bovine IgG2--VIII. The complete cDNA sequence of bovine IgG2a (A2) and an IgG1.

The complete cDNA sequences of two bovine IgGs were obtained by RT-PCR cloning. The first-strand cDNA was prepared from an animal homozygous (A2/A2) for IgG2a; the resulting sequences of the two bovine IgGs reported here were identified as IgG2a(A2) and IgG1. These sequences, and their deduced amino acid sequences, are compared to the previously reported partial protein sequences of IgG2a(A2) and IgG2(A1), two genomic DNA sequences of IgG2a and one genomic DNA sequence of IgG1. Data show that the two IgG2a allotypes (A1 and A2) differ in four regions: (a) region I-the site of the L-H bond in CH1; (b) region II-the middle hinge; (c) region III-a seven amino acid region at the beginning of the intradomain loop in CH3; and (d) region IV-an Arg-to-Glu exchange at the end of the same intradomain loop. The A1 allotype, which so remarkably distinguishes these allotypic variants, must result from differences in regions III and IV. The IgG1 sequence differs in the hinge region from the sequence reported previously and may represent an allotypic variant. We found no evidence to support the hypothesis that similarities between the CH3 domains of IgG2a(A2) and IgG1 result from gene conversion in the C-region of the bovine heavy chain locus.

Amino Acid Sequence↗
PubMed2004

cDNA sequence and spatio-temporal expression of prolactin in the orange-spotted grouper, Epinephelus coioides.

The cDNA sequence encoding the prolactin (PRL) gene of the orange-spotted grouper was obtained by randomly sequencing its pituitary cDNA library. The full-length cDNA of orange-spotted grouper PRL consists of 1342 bp. The open reading frame encodes a protein of 212 amino acids, which consists of a putative signal peptide of 24 residues and a mature protein of 188 amino acids. Northern blot analysis identified a single transcript of 1.4 kb in the pituitary gland, indicating perhaps the presence of only one form of PRL in the orange-spotted grouper. The expression of PRL gene in different tissues and during embryonic development of orange-spotted grouper was determined using one-step RT-PCR followed by Southern blot analysis. In addition to the pituitary gland, high levels of PRL gene expression were detected in the olfactory bulb, forebrain, midbrain, hypothalamus, hindbrain, medulla, gill, spleen, and adipose tissue. Very low levels of expression were detected in the kidney, liver, and blood cells. The PRL gene was also expressed in the gonads, with the higher level of expression observed in the stage 3 as compared to stage 2 ovary. During embryonic development, mRNA for PRL was detected at all time points examined except at 2h 15min post-fertilization, and its expression peaked at 9h post-fertilization, at the time of neurulation. Results from the current study suggest that PRL may have important paracrine or autocrine roles in diverse tissue types, including the brain and the gonads, and during embryonic development of the orange-spotted grouper.

Amino Acid Sequence↗
PubMed1987

Primary structures of two types of alpha-subunit of rat brain Na+,K+,-ATPase deduced from cDNA sequences.

A rat brain cDNA library was screened by using as a probe a fragment of cDNA encoding the alpha-subunit of human Na+,K+-ATPase. Two different cDNA clones were obtained and analyzed. One of them was concluded to be a cDNA encoding the alpha-subunit of the weakly ouabain-sensitive rat kidney-type Na+,K+-ATPase. The deduced amino acid sequence consists of 1,018 amino acids. The alpha-subunit of the rat kidney-type Na+,K+-ATPase shows 97% homology in amino acid sequence with the alpha-subunit of human, sheep, or pig enzyme and 87% with that of Torpedo. Based on a comparison of the amino acid sequence at the extracellular domain of the alpha-subunit between weakly ouabain-sensitive rat kidney-type enzyme and the ouabain-sensitive human, sheep, pig, or Torpedo enzyme, it was proposed that only two significant amino acid replacements are unique to the rat kidney-type alpha-subunit. Another cDNA clone obtained showed 72% homology in nucleotide sequence with the former cDNA coding the alpha-subunit of the rat kidney-type Na+,K+-ATPase and the deduced amino acid sequence exhibited 85% homology with that of the alpha-subunit of rat kidney-type Na+,K+-ATPase.

Amino Acid Sequence↗
PubMed1994

Complete cDNA sequence of a human dioxin-inducible mRNA identifies a new gene subfamily of cytochrome P450 that maps to chromosome 2.

Previously, levels of a novel human mRNA, detected by a recombinant cDNA designated clone 1, were shown to be increased 50-fold in response to treatment of a keratinocyte cell line with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), in part as a function of increased rates of gene transcription (Sutter, T.R., Guzman, K., Dold, K.M., and Greenlee, W.F. (1991) Science 254, 415-418). Here we report the complete corresponding 5.1-kilobase cDNA sequence. A single open reading frame that predicts a protein of 543 amino acid residues was determined by computer-assisted analysis of the cDNA sequence. This predicted protein identifies a new gene subfamily of cytochrome P450, cytochrome P4501B1 (CYP1B1), that maps to human chromosome 2. Southern blot analysis of genomic DNA indicates that the human CYP1B subfamily is likely to contain only this single gene. Northern blot analysis of RNA isolated from primary cultures of normal human epidermal keratinocytes showed approximately 100-fold increased levels of the CYP1B1 mRNA after treatment with 10 nM TCDD for 24 h. Low levels of constitutive CYP1B1 mRNA were detected in 15 different human tissue samples. These results indicate that CYP1B1 is expressed in many normal human tissues and advance our understanding of the complexity of a gene family of cytochromes P450 whose expression is altered by TCDD.

Amino Acid Sequence↗
PubMedDate not supplied

cDNA sequence of two distinct pituitary proteins homologous to Kex2 and furin gene products: tissue-specific mRNAs encoding candidates for pro-hormone processing proteinases.

Based on the concept of sequence conservation around the active sites of serine proteinases, polymerase chain reaction applied to mRNA amplification allowed us to obtain a 260-bp probe which was used to screen a mouse pituitary cDNA library. The primers used derived from the cDNA sequence of active sites Ser* and Asn* of human furin. Two cDNA sequences were obtained from a number of positive clones. These code for two similar but distinct structures (mPC1 and mPC2), each being homologous to yeast Kex2 and human furin. In situ hybridization (mPC1) and Northern blots (mPC1 = 3.0 kb and mPC2 = 2.8 and 4.8 kb) demonstrated tissue and cellular specificity of expression, only within endocrine and neuroendocrine cells. These data suggest that mPC1 and mPC2 represent prime candidates for tissue-specific pro-hormone converting proteinases.

Amino Acid Sequence↗
PubMed2000

Isolation and Identification of cDNA Sequences Differentially Expressed in Laryngeal Carcinoma.

The isolation of the genes related to laryngeal carcinoma(LC) is necessary for revealing mechanisms of carcinogenesis and genetic predisposition to LC. The mRNA differential display method was used to compare and analyze mRNAs prepared from two adult laryngeal carcinoma tissues and paired tumor-adjacent normal tissues. A total of twenty-two differential display experiments was performed and thirty-five cDNA fragments differentially expressed in normal or malignant laryngeal epithelial tissues were identified. Differential expression of six of these thirty-five cDNA fragmens was confirmed by reverse northern dot blot. Subsequent cloning of six differentially expressed cDNA fragments and sequencing and BLASTn analysis resulted in the identification of twelve distinct cDNA sequences. Four of these were shown to be novel gene sequences that have not been reported. Eight of the remaining cDNA sequences showed sequence homology to those previously reported. The differential expression of these twelve cDNA sequences in the carcinoma or normal tissue of the larynx were confirmed by fixing the twelve cDNA sequences on the membrane, followed by the hybridization with the total cDNA probes from laryngeal carcinoma or normal tumor-adjacent tissues and by differential RT-PCR. These results suggest that these cDNA sequences might be involved in carcinogenesis of laryngeal carcinoma.

Journal Article↗
PubMed1997

Identification of members of gene families in Arabidopsis thaliana by contig construction from partial cDNA sequences: 106 genes encoding 50 cytoplasmic ribosomal proteins.

Partial cDNA sequencing to obtain expressed sequence tags (ESTs) has led to the identification of tags to about 8,000 of the estimated 20,000 genes on Arabidopsis thaliana. This figure represents four to five times the number of complete coding sequences from this organism available in international databases. In contrast to mammals, many proteins are encoded by multigene families in A. thaliana. Using ribosomal protein gene families as an example, it is possible to construct relatively long sequences from overlapping ESTs which are of sufficiently high quality to be able to unambiguously identify tags to individual members of multigene families, even when the sequences are highly conserved. A total of 106 genes encoding 50 different cytoplasmic ribosomal protein types have been identified, most proteins being encoded by at least two and up to four genes. Coding sequences of members of individual gene families are almost always very highly conserved and derived amino acid sequences are almost, if not completely, identical in the vast majority of cases. Sequence divergence is observed in untranslated regions which allows the definition of gene-specific probes. The method can be used to construct high-quality tags to any protein.

Amino Acid Sequence↗
PubMed2001

Human xanthine dehydrogenase cDNA sequence and protein in an atypical case of type I xanthinuria in comparison with normal subjects.

To investigate the properties of xanthine dehydrogenase/xanthine oxidase (XDH/XO) deficiency in a patient with atypical type I xanthinuria, as indicated by oxypurine data, a cDNA sequence encoding XDH, XDH/XO immunoblot analysis and a competitive PCR assay were performed, and the results were compared with those of normal subjects. The xanthine dehydrogenase cDNA sequence of the patient was consistent with the controls, while immunologically reactive 150 kD XDH/XO protein was not present in the xanthinuric duodenal mucosa, unlike the control duodenal mucosa. In addition, a decrease in XDH/XO messenger RNA was found by competitive PCR. These results suggest that atypical type I xanthinuria is due to a decrease in messenger RNA of XDH/XO. Furthermore, it was considered that this decrease could explain the normal plasma level and near normal urinary excretion of hypoxanthine seen in this case of xanthinuria, though XDH/XO activity and protein were not detected spectrophotometrically and immunologically, respectively.

Base Sequence↗
PubMed2000

cDNA sequence of two sheep mast cell tryptases and the differential expression of tryptase and sheep mast cell proteinase-1 in lung, dermis and gastrointestinal tract.

BACKGROUND: Mast cell tryptases are a family of serine proteinases which are implicated in the proliferation of smooth muscle cells and fibroblasts, upregulation of interleukin-8 synthesis by endothelial cells, and recruitment of neutrophils and eosinophils. Trials in sheep showed that administration of a specific tryptase inhibitor reduced the late-phase response to inhaled allergen. OBJECTIVES: The aim of this study was to characterize the sequence and distribution of sheep tryptase(s), to validate the sheep model of allergic lung disease. METHODS: Reverse transcriptase PCR cloning was used to obtain cDNA sequences for two sheep tryptases. Lung and gut extracts were used as a source of tryptase for partial purification and characterization of the protein. The distribution of tryptase in skin, lung and gut was determined by immunohistochemistry, and compared with the distribution of sheep mast cell proteinase-1 (sMCP-1). RESULTS: Two highly similar cDNA sequences encoding sheep tryptase were found, indicating the presence of a 28 amino acid leader sequence, and a mature peptide of 245 amino acids. Partial purification of a putative sheep tryptase from lung and gut extracts was achieved using heparin-Sepharose affinity chromatography. Rabbit antihuman skin tryptase antiserum recognized the putative sheep tryptase on Western blot (approximate Mr 32-34 000) and paraformaldehyde-fixed tissue sections. Tryptase was detected in all lung, skin and gut mast cells by this antibody, and transcripts for tryptase were detected in all three tissues by RT PCR. Sheep mast cell proteinase-1, detected by a specific monoclonal antibody, was present in all intestinal and gastric mucosal mast cells, but was not found in mast cells of the muscularis, thus defining at least two mast cell phenotypes in the gut. Whereas all dermal and pulmonary mast cells were tryptase positive, only a low proportion in the lung, and almost none in the dermis, were positive for sMCP-1. CONCLUSION: In view of the structural and functional similarities of sheep and human tryptases, and their similarity in tissue distribution in normal sheep, the sheep lung appears to be a good model for in vivo studies relating to human tryptase.

Amino Acid Sequence↗
PubMed1995

Molecular cloning of a group of mouse pancreatic islet beta-cell-related genes by random cDNA sequencing.

To understand the molecular basis of glucose concentration-responsive insulin synthesis and secretion from pancreatic islet beta cells, a group of pancreatic islet beta-cell-related cDNAs was cloned. A pair of cDNA libraries was constructed from a mouse pancreatic islet beta-cell line of MIN6, which was cultured in either high glucose or low glucose media. By applying a random cDNA sequencing approach, 503 and 395 independent species were obtained from a total of 1,011 and 762 clones in the high glucose and low glucose library, respectively. The unknown genes comprised the majority of about 70% independent clones in both libraries. In Northern blot analysis, 311 (69.4%) of 448 independent clones showed positive signals within 72 h of autoradiographic exposure. Surprisingly, 150 (48.2%) out of 311 positive clones showed positive signals to MIN6 cells, but not to NIH/3T3 fibroblasts. The expression level of three unknown clones were glucose-concentration dependent. Combination of a random cDNA sequencing approach and Northern blot analysis is useful to obtain a large number of novel genes and islet beta-cell-related genes.

Animals↗
PubMed1998

Molecular cloning and expression of a cDNA sequence encoding histidinol phosphate aminotransferase from Nicotiana tabacum.

A Nicotiana tabacum cDNA sequence encoding histidinol phosphate aminotransferase (HPA) was isolated by functional complementation of an Escherichia coli histidine auxotroph (UTH780). The enzymatic assay has confirmed that the isolated cDNA encodes a functional HPA protein. Amino acid sequence alignment of the HPA protein from N. tabacum, Saccharomyces cerevisiae and E. coli revealed that, despite the low degree of identity, some residues were found to be highly conserved. The predicted protein contains a transit peptide sequence at the amino-terminal end, suggesting a chloroplastic localization of the HPA enzyme. Western blot analysis demonstrated that the deduced HPA protein and the mature HPA protein have an apparent molecular mass of about 45 kDa and 40 kDa respectively. Gene copy number estimation by Southern analysis indicates the presence of at least two genes per haploid genome coding for this protein in Nicotiana sp. From northern analysis results, the gene seems to be highly expressed in green tissues and the detected transcript showed a single band of expected molecular size.

Amino Acid Sequence↗
PubMed1994

A Taenia crassiceps cDNA sequence encoding a putative immunodiagnostic antigen for bovine cysticercosis.

A cDNA expression library was constructed in lambda gt11 using poly A mRNA from the metacestode stage of Taenia crassiceps. The library was screened with rabbit antiserum to a previously defined protein fraction from Taenia hydatigena immunodiagnostic for bovine cysticercosis and with sera from cattle with experimentally induced cysticercosis. One clone (lambda TcA2) containing a 279-bp cDNA insert, reacted strongly with both antisera. A second clone (lambda TcA5.5) revealed the full-length cDNA sequence to be 361 bp. Data from Southern blots and enzymatically amplified genomic DNA segments were consistent with multiple copies or a gene family within the genome. The lambda TcA2 cDNA insert was subcloned into the plasmid pPR987 which generated a 47-kDa maltose-binding fusion protein (TcA2-MBP). Affinity-purified TcA2-MBP antigen reacted positively by ELISA with sera from cattle with experimentally induced T. saginata infections but not with sera from cattle with Fasciola hepatica or common gastrointestinal parasite infections. Rabbit polyclonal, monospecific antisera to TcA2-MBP recognized a 10-kDa protein in the cyst fluid, body wall and excretory/secretory products of the metacestode stage of T. crassiceps and immonolocalized this protein to organelles within the matrix of the cyst wall.

Animals↗
PubMed2000

Cloning and characterization of a cDNA sequence encoding the precursor of a chlorotoxin-like peptide from the Chinese scorpion Buthus martensii Karsch.

A full-length cDNA sequence encoding the precursor of a venom peptide with homology to chlorotoxin (named BmKCT) was isolated from a cDNA library made from the venom glands of the Chinese Scorpion Buthus martensii Karsch. The encoded precursor of BmKCT was 59 amino acid residues long including a signal peptide of 24 residues and a mature toxin of 35 residues with four disulfide bridges. The sequence of BmKCT is similar (68% identities) to that of chlorotoxin isolated from Leiurus quinguestriatus quinquestriatus. BmKCT is the first report of the cDNA sequences encoding four-disulfide-bridged short-chain toxins from Buthus martensuii Karsch so far.

Amino Acid Sequence↗