The thiol-specific antioxidant protein from human brain: gene cloning and analysis of conserved cysteine regions

The complete cDNA encoding human thiol-specific antioxidant protein (PRP) was isolated from a human brain cDNA library in the λZap expression vector. An open reading frame (ORF) was identified and found to encode a polypeptide of 197 aa with a M_r of 21 729. The cDNA contained 98 bp of 5′-untranslated sequence (UTR) and 259 bp of 3′-UTR containing a poly(A) signal, AATAAA. Expression of the human PRP cDNA in Escherichia coli yielded a functionally active protein. The observed local sequence homologies between human PRP and other homologous proteins whose functions have not yet been defined give important insight into elucidating the biochemical function of a new protein family which has highly conserved regions containing cysteine.     

NRDRiso酶cDNA的序列测定及生物信息学分析

通过鉴定分析人肝组织中辅酶II依赖性视黄醇脱氢酶不同剪接体全长cDNA核苷酸序列与氨基酸序列的结构特征,为今后进一步研究体内维甲酸的代谢情况奠定基础。根据人、小鼠NRDR编码区的一致性序列,设计一对引物,应用RTPCR方法从人肝组织中得到一条377bp的新的cDNA片段。采用RACE法得到了NRDR新亚型cDNA,并以生物信息学软件分析其生物学特征。 测序得知该cDNA长为1003bp,以NADP-dependent retinol dehydrogenase/reductase short isoform（NRDRiso）登录GenBank。其读码框为525bp,拟编码174个氨基酸的蛋白。     

A discordance between the human muscle NCAM sequence and those seen in other NCAM cDNA clones.

A cDNA clone, designated NC7, has been isolated from human foetal kidney that partially codes for the 140 kDa isoform of human NCAM. This clone contains a 6 bp insert that is not present in the human muscle cDNA clone lambda 4.4. This same sequence has also been found in both a cDNA clone obtained from a human Small Cell Lung Carcinoma (SCLC) line and in human genomic DNA. Furthermore, an equivalent sequence to the 6 bp region identified in the above samples is present in mouse, rat and chicken NCAM. The 6 bp insertion does not lie at a predicted intron/exon boundary as extrapolated by homology studies with the chicken and, therefore, the mechanism by which the sequence is deleted from the human muscle clone lambda 4.4 remains unclear.     

Isolation and characterisation of cDNA clones for the A alpha- and gamma-chains of human fibrinogen.

cDNA clones coding for the A alpha- and gamma-chains of human fibrinogen have been isolated from an adult liver cDNA library. Clones were identified by hybridisation with mixtures of synthetic oligonucleotides 17 bases long, predicted using amino acid sequence data for each chain. The cDNA insert sizes are 1,950bp for A alpha-fibrinogen and 950bp for gamma-fibrinogen. The clones do not show any cross-hybridisation. Each cDNA hybridises to a unique sequence in the human genome. In adult human liver, Northern blots give an estimated messenger RNA size of 2.6kb for A alpha-fibrinogen and 1.8kb for gamma-fibrinogen.     

Cloning, structure and expression of a cDNA encoding the human androgen receptor

A cDNA clone has been isolated from a library prepared of mRNA of human breast cancer T47D cells with an oligonucleotide probe homologous to part of the region encoding the DNA-binding domain of steroid receptors. The clone has a size of 1505 bp and sequence analysis revealed an open reading frame of 1356 bp. The deduced amino acid sequence displays two highly conserved regions identified as the putative DNA-binding and hormone binding domains respectively of steroid receptors. Expression of this cDNA clone in COS cells produces a nuclear protein with all the binding characteristics of the human androgen receptor (hAR). The gene encoding the cDNA is assigned to the human X-chromosome. High levels of three hybridizing mRNA species of 11, 8.5 and 4.7 kb respectively are found in the human prostate cancer cell line (LNCaP), which contains elevated levels of hAR. The present data provide evidence that we have isolated a cDNA that encodes a major part of the human androgen receptor.     

Membrane-associated peptidylglycine alpha-amidating monooxygenase in the heart

Recent investigations have shown that the heart atrium is an endocrine tissue. In the present studies, high levels of peptidylglycine alpha-amidating monooxygenase (PAM), which catalyzes the formation of bioactive alpha-amidated peptides from their glycine-extended precursors, have been found in particulate fractions from bovine and rat heart atrium; only low levels of PAM activity were present in soluble fractions. Corresponding fractions from the ventricles contained 20-fold less activity. Immunocytochemical studies demonstrated that PAM was localized primarily to atrial cardiocytes, with a distribution resembling that of atriopeptin. Following differential centrifugation of rat atrial homogenates, most of the PAM activity was associated with crude granule fractions, with lesser amounts of activity associated with crude microsomal fractions. Upon further subcellular fractionation, PAM activity in the rat atrium was found primarily with immunoactive atriopeptin in fractions enriched in secretory granules. Following sodium dodecyl sulfate-polyacrylamide gel electrophoresis, antisera to purified bovine pituitary PAM identified a 113,000-dalton protein in bovine atrial microsomes and secretory granules; the protein predicted from the sequence of the cDNA encoding bovine pituitary PAM is of similar size (Eipper, B. A., Park, L. P., Dickerson, I. M., Keutmann, H. T., Thiele, E. A., Rodriguez, H., Schofield, P. R., and Mains, R. E. (1987) Mol. Endocrinol. 1, 777-790). Northern blot analysis using cDNA probes encoding bovine pituitary PAM demonstrated higher levels of PAM mRNA in heart atrium than in anterior pituitary. Rat heart contains PAM mRNA species of 3.6 and 3.8 kilobases, the smaller mRNA species corresponding in size to the PAM mRNA expressed in rat anterior pituitary.     

Involvement of sialoadhesin in entry of porcine reproductive and respiratory syndrome virus into porcine alveolar macrophages

Porcine reproductive and respiratory syndrome virus (PRRSV) shows a very restricted tropism for cells of the monocyte/macrophage lineage. It enters cells via receptor-mediated endocytosis. A monoclonal antibody (MAb) that is able to block PRRSV infection of porcine alveolar macrophages (PAM) and that recognizes a 210-kDa protein (p210) was described previously (MAb41D3) (X. Duan, H. Nauwynck, H. Favoreel, and M. Pensaert, J. Virol. 72:4520-4523, 1998). In the present study, the p210 protein was purified from PAM by immunoaffinity using MAb41D3 and was subjected to internal peptide sequencing after tryptic digestion. Amino acid sequence identities ranging from 56 to 91% with mouse sialoadhesin, a macrophage-restricted receptor, were obtained with four p210 peptides. Using these peptide data, the full p210 cDNA sequence (5,193 bp) was subsequently determined. It shared 69 and 78% amino acid identity, respectively, with mouse and human sialoadhesins. Swine (PK-15) cells resistant to viral entry were transfected with the cloned p210 cDNA and inoculated with European or American PRRSV strains. Internalized virus particles were detected only in PK-15 cells expressing the recombinant sialoadhesin, demonstrating that this glycoprotein mediated uptake of both types of strains. However, nucleocapsid disintegration, like that observed in infected Marc-145 cells as a result of virus uncoating after fusion of the virus with the endocytic vesicle membrane, was not observed, suggesting a block in the fusion process. The ability of porcine sialoadhesin to mediate endocytosis was demonstrated by specific internalization of MAb41D3 into PAM. Altogether, these results show that sialoadhesin is involved in the entry process of PRRSV in PAM.     

Cloning, expression, and sequence homologies of cDNA for human carbonic anhydrase II

A cDNA clone for human carbonic anhydrase (CA) II was isolated from a kidney lambda gt10 library. Expression of the cDNA insert in Cos-7 cells produced an immunoprecipitable product and enzymatically active carbonic anhydrase. The cDNA insert is 1551 bp in length and contains an open reading frame which encodes a 260-amino-acid polypeptide. The deduced amino acid sequence is identical to that reported for human CA II. The protein coding region of this cDNA for human CA II shows 81 and 70% nucleotide identity with cDNAs for CA II from mouse and chick, respectively. Even the long 3'-untranslated region of the cDNA for human CA II (703 bp) is 64 and 42% identical to those of CA II from mouse and chick, showing remarkable conservation of the CA II cDNAs in amniotes. The protein coding region of the human CA II cDNA is 64 and 65% identical with those of human CA I and CA III, which are thought to have arisen from a common precursor by gene duplication.     

Molecular cloning of a novel human PAPS synthetase which is differentially expressed in metastatic and non-metastatic colon carcinoma cells.

Subtractive hybridisation was used to select for genes which are differentially expressed between a highly metastatic human colon carcinoma cell line, KM12SM, and the isogenetic non-metastatic cell line, KM12C. This led to the isolation of cDNA clones for a novel human adenosine 5'-phosphosulphate kinase/ATP sulphurylase (PAPS synthetase). Northern hybridisation revealed a single 4.2 kb mRNA species which showed an approximately 20-fold higher level of expression in the non-metastatic cell line than in the metastatic cell line. The overlapping cDNA clones together covered 3,774 bp including the entire coding region of 1,842 bp encoding a protein of 614 amino acids (calculated molecular mass of 69,496 Da). The protein contains consensus sequences for APS kinase and ATP sulphurylase, in its amino- and carboxy-terminal regions, respectively, as well as other sequences that are highly conserved amongst ATP sulphurylases and APS kinases. Interestingly, consensus sequences for GTPase activity were also identified, indicating that enzyme activity may be regulated by an intrinsic GTPase mechanism. Overall the new protein is 78% homologous with a previously described human PAPS synthetase (PAPSS1) indicating that we have identified the second member of a gene family which we have provisionally named PAPSS2. The gene locus for PAPSS2 was identified on chromosome 10 at 10q23.1-q23.2. This locus has synteny with the mouse brachymorphic gene recently identified as a PAPS synthetase (SK2). PAPSS2 appears to be the human homologue of this gene and thus PAPSS2 is likely to be important in human skeletogenesis.     

The sedlin gene for spondyloepiphyseal dysplasia tarda escapes X-inactivation and contains a non-canonical splice site

Mutations in the sedlin gene cause spondyloepiphyseal dysplasia tarda (SEDT), a rare X-linked chondrodysplasia. Affected males suffer short stature, deformation of the spine and hips, and deterioration of intervertebral discs with characteristic radiographic changes in the vertebrae. We have sequenced two full-length cDNA clones corresponding to the human sedlin gene. The longest cDNA is 2836 bp, containing a 218 bp 5' untranslated region, a 423 bp coding region, and a 2195 bp 3' untranslated region. The second cDNA does not contain exon 2, suggesting alternative splicing. Sedlin was finely mapped in Xp22.2 by Southern blot analysis on a yeast artificial chromosome/bacterial artificial chromosome map. Comparison of the cDNA sequence and genomic sequence identified six sedlin exons of 67, 142, 112, 147, 84, and 2259 bp. The corresponding introns vary in size from 339 to 14,061 bp. Splice site sequences for four of the five introns conform to the GT/AG consensus sequences, however, the splice site between exons 4 and 5 displays a rare non-canonical splice site sequence, AT/AC. Northern blot analysis showed expression of the sedlin gene in all human adult and fetal tissues tested, with the highest levels in kidney, heart, skeletal muscle, liver, and placenta. Four mRNA sizes were detected with the major band being 3 kb and minor bands of 5, 1.6, and 0.9 kb (the smallest product may reflect a sedlin pseudogene). Sedlin is expressed from both the active and the inactive human X chromosomes helping to explain the recessive nature and consistent presentation of the disease. Human sedlin shows homology to a yeast gene, which conditions endoplasmic reticulum/golgi transport. Characterization of the human sedlin cDNA and determination of the sedlin gene structure enable functional studies of sedlin and elucidation of the pathogenesis of SEDT.     

Sequence of human DNA polymerase beta mRNA obtained through cDNA cloning

A cDNA library from polyA+ RNA of a human teratocarcinoma cell line in phage lambda gt11 was screened with a fragment of the rat beta-polymerase cDNA, lambda pol beta-10, as probe. Five positive phage were identified and plaque purified. The cDNA of one positive clone selected for detailed study was 1257 bp. This insert was sequenced and found to contain the coding region for beta-polymerase, as well as 163 bp and 137 bp from the 5' and 3' untranslated regions, respectively. The primary structure of human beta-polymerase (318 amino acids, Mr = 36, 133) deduced from the cDNA was similar to rat beta-polymerase (95% matched residues). The greatest difference between the sequences of the human and rat cDNAs was in the 3' untranslated regions (64% matched base residues). These results provide necessary sequence information for study of the human beta-polymerase gene.     

Molecular biology of the alpha-L-fucosidase gene and fucosidosis

Human alpha-L-fucosidase, a lysosomal enzyme, hydrolyzes alpha-L-fucose from glycolipids and glycoproteins. Its activity is deficient in human fucosidosis an autosomal recessive disease. In order to understand the molecular basis of this lysosomal storage disorder we have cloned several cDNAs coding for human alpha-L-fucosidase from a human hepatoma and a human liver cDNA library constructed in lambda gt11. Compiling the cDNA sequences of these clones we have identified 1,829 base pairs (bp) encoding human alpha-L-fucosidase. This includes an open reading frame of 1,172 bp, a consensus polyadenylation signal AAT AAA and a poly(A)+ tail. The sequence is incomplete at the 5'-end, and clones encoding the amino terminus of the native protein, the propeptide and leader signal have not yet been isolated. The open reading frame encodes for 390 amino acids with a calculated Mr of 45,557. This represents 78-95% of the mature processed alpha-L-fucosidase. The availability of these cDNA clones has enabled us to map the structural gene for alpha-L-fucosidase to chromosome 1p34.1-1p36.1 by Southern blot analysis of DNA from human-rodent somatic cell hybrids and by in situ hybridization. Furthermore, a Pvu II restriction fragment length polymorphism (RFLP) has been identified at the human alpha-L-fucosidase gene locus. Analysis of mRNA by Northern blotting gives a major species of 2.25 kb. In 4 patients with fucosidosis no mRNA signal was detected and Western blots gave no immunoreactive enzyme. Southern blotting after Eco RI digestion in two fucosidosis families revealed a banding abnormality (extra 6-kb band).(ABSTRACT TRUNCATED AT 250 WORDS)     

Molecular cloning of LMO41, a new human LIM domain gene

We have identified a new human LIM domain gene by isolating an autoantigenic cDNA clone from a human breast tumor cDNA library. The predicted amino acid sequence of the cDNA clone's 495 bp open reading frame contains two tandem LIM domain motifs, and within the LIM domain region there is 62% identity with the analogous region of the LIM-only gene LMO1. The homology to LMO1 is restricted to the 360 bp region encoding the tandemly repeated LIM domains, the rest of the open reading frame as well as the extensive, GC-rich 5' untranslated region, and 3' region of the 2 kb cDNA sequence are unrelated to any known genes. This gene has been designated LMO4.     

Alternative splicing and endoproteolytic processing generate tissue-specific forms of pituitary peptidylglycine alpha-amidating monooxygenase (PAM).

The pituitary is a rich source of peptidylglycine alpha-amidating monooxygenase (PAM). This bifunctional protein contains peptidylglycine alpha-hydroxylating monooxygenase (PHM) and peptidyl-alpha-hydroxyglycine alpha-amidating lyase (PAL) catalytic domains necessary for the two-step formation of alpha-amidated peptides from their peptidylglycine precursors. In addition to the four forms of PAM mRNA identified previously, three novel forms of PAM mRNA were identified by examining anterior and neurointermediate pituitary cDNA libraries. None of the PAM cDNAs found in pituitary cDNA libraries contained exon A, the 315-nucleotide (nt) segment situated between the PHM and PAL domains and present in rPAM-1 but absent from rPAM-2. Although mRNAs of the rPAM-3a and -3b type encode bifunctional PAM precursors, the proteins differ significantly. rPAM-3b lacks a 54-nt segment encoding an 18-amino acid peptide predicted to occur in the cytoplasmic domain of this integral membrane protein; rPAM-3a lacks a 204-nt segment including the transmembrane domain and encodes a soluble protein. rPAM-5 is identical to rPAM-1 through nt 1217 in the PHM domain; alternative splicing generates a novel 3'-region encoding a COOH-terminal pentapeptide followed by 1.1 kb of 3'-untranslated region. The soluble rPAM-5 protein lacks PAL, transmembrane, and cytoplasmic domains. These three forms of PAM mRNA can be generated by alternative splicing. The major forms of PAM mRNA in both lobes of the pituitary are rPAM-3b and rPAM-2. Despite the fact that anterior and neurointermediate pituitary contain a similar distribution of forms of PAM mRNA, the distribution of PAM proteins in the two lobes of the pituitary is quite different. Although integral membrane proteins similar to rPAM-2 and rPAM-3b are major components of anterior pituitary granules, the PAM proteins in the neurointermediate lobe have undergone more extensive endoproteolytic processing, and a 75-kDa protein containing both PHM and PAL domains predominates. The bifunctional PAM precursor undergoes tissue-specific endoproteolytic cleavage reminiscent of the processing of prohormones.     

Molecular cloning of a human co-beta-glucosidase cDNA: evidence that four sphingolipid hydrolase activator proteins are encoded by single genes in humans and rats

Authentic cDNAs encoding the activator protein for acid beta-glucosidase (EC3.2.1.45), co-beta-glucosidase, were cloned from the pCD and lambda gt11 human cDNA libraries. Initial screening with oligonucleotide mixtures encoding amino acid sequences of co-beta-glucosidase identified partial cDNAs which were used to obtain a potentially full-length cDNA from the lambda gt11 library. This clone (2767 bp), EGTISI, contained 5' (38 bp) and 3' (1157 bp) noncoding sequences, a translation initiation site, and an open reading frame encoding 524 amino acids which included a typical hydrophobic signal sequence (16 amino acids). Computer analyses identified three regions of high similarity to co-beta-glucosidase encoded by tandem sequences in EGTISI. Searches revealed that two of these regions encoded peptides of known function; SAP1 (sphingolipid activator protein 1) and protein C (a new sphingolipid activator protein) were encoded by EGTISI sequences 5' and 3', respectively, to those for co-beta-glucosidase. The third region of similarity, encoding a theoretical peptide (undefined function), was located most 5' in the cDNA. EGTISI and its encoded polypeptide had high similarity (77% nucleotide identity and about 80% amino acid similarity) to a rat Sertoli cell cDNA and its encoded sulfated glycoprotein-1. These results indicate that a single highly conserved gene encodes the precursor for four potential sphingolipid activator proteins in rat and man.