Sequence of full length cDNA for human S-adenosylhomocysteine hydrolase.

Two cDNA clones for human S-adenosylhomocysteine hydrolase isolated from a placental cDNA library were sequenced. Each contained a sequence of 1299 nucleotides encoding a 432 amino-acide protein of MW 47660. Clone 16-1 contained 47 nucleotides 5' of the coding region, and a 780 nucleotide 3' flanking region terminating in apoly A tail. In addition, a 101 nucleotide unprocessed intron interrupted the coding sequence at nucleotide 854 (second base of codon 285). Clone 20-1 contianed 43 nucleotides 5' and 742 nucleotides 3' flanking the uninterrupted coding region. Besides the intron, the clones differed in one position of the coding sequence and at two positions of the 3' non-coding region. The cDNAs for human and rat S-adenosylhomocysteine hydrolase were identical at 91.5% of position in the coding sequence and showed 70% homology in the 3' non-coding regions. Human and rat S-adenosylhomocysteine hydrolases are identical at 97% of amino-acid residues, and the Dictyostelium and human enzymes at 75%.     

Screening of a HUVEC cDNA library with transplant-associated coronary artery disease sera identifies RPL7 as a candidate autoantigen associated with this disease.

A HUVEC cDNA library was screened with sera from two patients who had developed transplant-associated coronary artery disease (TxCAD) following cardiac transplantation. A total of six positive clones were isolated from a primary screen of 40 000 genes. Subsequent DNA sequence analysis identified these to be lysyl tRNA synthetase, ribosomal protein L7, ribosomal protein L9, beta transducin and TANK. Another gene whose product could not be identified showed homology to a human cDNA clone (DKFZp566M063) derived from fetal kidney. Full-length constructs of selected genes were expressed as his-tag recombinant fusion proteins and used to screen a wider patient base by ELISA to determine prevalence and association with TxCAD. Of these ribosomal protein L7 showed the highest prevalence (55.6%) with TxCAD sera compared to 10% non-CAD.     

The 40-kilodalton allergen of Candida albicans is an alcohol dehydrogenase: molecular cloning and immunological analysis using monoclonal antibodies

To characterize the 40-kilodalton (kD) major allergen of Candida albicans (C. albicans), six monoclonal antibodies (MoAbs) against this allergen were generated. In SDS-polyacrylamide gel electrophoresis and immunoblot analysis, these MoAbs showed four different reaction patterns to antigens of six different Candida species. With the exception of one MoAb, other MoAbs were resistant to periodate treatment indicating non-carbohydrate epitopes were probably being recognized by these MoAbs. These MoAbs were used in the molecular cloning and immunological analysis of the gene coding for the 40-kD allergen. Nucleotide sequence determination of the two lambda gt11 cDNA clones obtained showed that the 40-kD allergen is an alcohol dehydrogenase (ADH) which shares a 70% amino acid sequence homology with the ADH isozyme I of Saccharomyces cerevisiae. This finding was confirmed by positive immunological response of the lysates of the clones obtained and a preparation of ADH of Saccharomyces cerevisiae to various MoAbs and to IgE antibodies in sera of allergic patients.     

A survey of expressed genes in the leukocytes of Japanese flounder, Paralichthys olivaceus, infected with Hirame rhabdovirus

We constructed a cDNA library of Japanese flounder, Paralichthys olivaceus, leukocytes that were infected with Hirame rhabdovirus (HRV) in order to analyze some of the genes that are induced and expressed by virus infection in the immune system. Four hundred and fifty-two partial sequences representing 300 cDNA clones were obtained from the 5' and/or 3' ends of inserts derived from the Japanese flounder leukocyte cDNA library. About three-quarters of the 300 cDNA clones (217 clones, 72.3%) represented known genes in the public databases, whereas the remaining 83 (27.7%) of the clones did not show any significant homology with the sequences in the public databases. Clones matching known genes were classified into 12 categories according to their function or distribution. Only 40 (18.4%) of the 217 known genes showed homology with fish genes deposited in the database. Thirty (10%) of the clones, encoding 21 different sequences, and representing several categories, were identified as putative biodefense genes or genes associated with the immune response. Nineteen of the 21 putative biodefense or immune response-related cDNAs have not been previously reported in fish genes or cDNAs.     

Isolation of the Taenia crassiceps antigens from a phage display cDNA library and evaluation of their use for diagnosis of neurocysticercosis

A novel cDNA cloning strategy consisting in elimination of non-coding DNA sequences from 3' regions of cDNAs was applied to construct the Taenia crassiceps phage displayed cDNA expression library. After biopanning using immune sera, three phage clones expressing T. crassiceps-derived antigens specifically recognizing antibodies present in cerebrospinal fluid and plasma samples from neuroimaging-confirmed neurocysticercosis patients were selected. This novel cloning strategy may be applied to other pathogens allowing rapid identification of peptides/proteins for immunodiagnostic tests.     

Expression, cloning, and characterization of a Candida albicans gene, ALA1, that confers adherence properties upon Saccharomyces cerevisiae for extracellular matrix proteins.

Adherence of Candida albicans to host tissues is a necessary step for maintenance of its commensal status and is likely a necessary step in the pathogenesis of candidiasis. The extracellular matrix (ECM) proteins are some of the host tissue and plasma proteins to which C. albicans adheres through adhesins located on the fungal cell surface. To isolate genes encoding ECM adhesins, an assay was developed based on the ability of yeast cells to adhere to magnetic beads coated with the ECM protein fibronectin, type IV collagen, or laminin. A C. albicans genomic library was constructed by cloning XbaI-partially-digested and size-selected fragments into pAUR112, an Escherichia coli-yeast low-copy-number shuttle vector. The C. albicans library was transformed into Saccharomyces cerevisiae YPH 499, and clones capable of adherence were selected by using ECM protein-coated magnetic beads. A plasmid containing an approximately 8-kb insert was isolated from 29 adherent clones. These clones exhibited adherence to all ECM protein-coated magnetic beads and to human buccal epithelial cells. The ALA1 gene (for agglutinin-like adhesin) was localized by subcloning it into a 5-kb XbaI fragment which retained the adherence phenotype in both orientations. The complete DNA sequence of the 5-kb insert was determined, and an open reading frame (ORF) encoding 1,419 amino acid residues was identified. Deletions from the 5' and 3' ends extending into the DNA sequence encoding the 1,419-amino-acid ORF product inactivated the adherence phenotype, suggesting that it is the coding region of the ALA1 gene. A database search identified ALA1 to be similar to the C. albicans ALS1 (for agglutinin-like sequence 1) protein and the S. cerevisiae agglutinin protein (AG alpha1), although the homology at the primary amino acid sequence level is limited to the first half of each of these proteins. ALA1 contains a central domain of six tandem repeats of 36 amino acids. We discuss the significance of various predicted ALA1 structural motifs and their relationships to function in the adherence process.     

Detection of candidal antigens in autoimmune polyglandular syndrome type I.

Autoimmune polyglandular syndrome type I (APS I) is associated with chronic mucocutaneous candidiasis. To characterize the antibody responses in this subgroup of Candida albicans infections, we screened a candidal cDNA expression library with patient sera and found four cDNA clones encoding the immunopositive proteins enolase, heat shock protein 90, pyruvate kinase, and alcohol dehydrogenase. The reactivity to these antigens was studied further by immunoprecipitation assays with in vitro-transcribed and -translated proteins. Analysis of sera from 44 APS I patients showed that the highest antibody reactivity was found with enolase (80% of patients reactive), but significant serological responses were also found with heat shock protein 90 (67%), pyruvate kinase (62.5%), and alcohol dehydrogenase (64%). Overall, 95.5% of patients had detectable antibodies to at least one of these proteins. The cDNAs of enolase and heat shock protein 90 were also expressed in Escherichia coli and studied by immunoblotting. Again, 84% of sera reacted with enolase, whereas 44% of sera reacted with heat shock protein 90. A good correlation between the two methods was found for both enolase (r = 0.86; n = 58; P < 0.001) and heat shock protein 90 (r = 0.71; n = 56; P < 0.001). Our results indicate that the four abundant candidal proteins are the major antigens and can be used as accurate markers of candidiasis in APS I patients. The immunoprecipitation assay described here is particularly useful for the rapid analysis of a large number of samples.     

Identification of immunogenic proteins from Paracoccidioides brasiliensis antigenic fractions F0, FII and FIII

Paracoccidioides brasiliensis causes a chronic granulomatous mycosis prevalent in South America, and cell-mediated immunity is the principal mode of protection against this fungal infection. In this context, one of the strategies to discover proteins that are target of an effective immune response against P. brasiliensis is the partial sequencing of cDNA from an expression library previously screened with immunoglobulins (Ig) to generate antigen sequence tags (AST). In the present work, a P. brasiliensis yeast cDNA expression library was screened with affinity chromatography-purified IgG from rabbit sera immunized with P. brasiliensis antigenic fractions (F0, FII or FIII) or from paracoccidioidomycosis (PCM) patient sera by indirect ELISA. From 119 clones selected by the immunoscreening procedure, 40% were recognized by IgG from PCM patients, 25% were recognized by anti-F0, 8% were selected by anti-FII and 11% recognized by FIII specific antibodies. The remaining clones presented cross-reaction to all anti-sera tested. The AST homologies with previously reported sequences in the nonredundant GenBank at NCBI revealed high significant homology to fungal proteins of known function. One of them matched calcineurin B of Neurospora crassa with 35% identity and 55% similarity in amino acid sequence. We also identified an AST homologous to a Kinesin like protein from Ustilagus maydis and other fungi with 86% identity and 91% similarity. On the other hand, the vast majority of selected cDNA clones are new genes and represent 60% of the total. Prediction of transmembrane regions with the prediction transmembrane protein topology with a hidden markov model (TMHMM) revealed consensus sequences representing structural membrane segments in 28 encoded proteins.     

The Drosophila gene collection: identification of putative full-length cDNAs for 70% of D. melanogaster genes

Collections of full-length nonredundant cDNA clones are critical reagents for functional genomics. The first step toward these resources is the generation and single-pass sequencing of cDNA libraries that contain a high proportion of full-length clones. The first release of the Drosophila Gene Collection Release 1 (DGCr1) was produced from six libraries representing various tissues, developmental stages, and the cultured S2 cell line. Nearly 80,000 random 5' expressed sequence tags (5' expressed sequence tags [ESTs]from these libraries were collapsed into a nonredundant set of 5849 cDNAs, corresponding to ~40% of the 13,474 predicted genes in Drosophila. To obtain cDNA clones representing the remaining genes, we have generated an additional 157,835 5' ESTs from two previously existing and three new libraries. One new library is derived from adult testis, a tissue we previously did not exploit for gene discovery; two new cap-trapped normalized libraries are derived from 0-22-h embryos and adult heads. Taking advantage of the annotated D. melanogaster genome sequence, we clustered the ESTs by aligning them to the genome. Clusters that overlap genes not already represented by cDNA clones in the DGCr1 were analyzed further, and putative full-length clones were selected for inclusion in the new DGC. This second release of the DGC (DGCr2) contains 5061 additional clones, extending the collection to 10,910 cDNAs representing >70% of the predicted genes in Drosophila.     

IgA肾病肾阴虚证cDNA文库的构建

目的应用抑制性消减杂交（suppression subtractive hybridization,SSH）技术构建汉族人IgA肾病肾阴虚证cDNA消减文库。方法选择IgA肾病且中医辨证为肾阴虚证的患者以及正常人作为其对照组,进行正向和反向消减杂交。采用Trizol BD法提取总RNA,用SMART技术逆转录并扩增总cDNA,用RsaⅠ酶切基因组cDNA成大小不等的片段,分别与两种不同的接头连接,进行2次消减杂交及2次抑制性PCR,然后将PCR产物与U载体连接,经蓝白斑筛选后,再用PCR方法插入片段筛选出阳性重组质粒,构建IgA肾病肾阴虚证消减文库。结果用SSH方法筛选出了IgA肾病肾阴虚证的差异cDNA片段,其中正向消减文库共获得325个阳性克隆．反向消减文库获得306个阳性克隆,从而成功地构建了IgA肾病肾阴虚证的cDNA消减文库。结论SSH技术能够快速有效地分离差异cDNA片段,成功构建了IgA肾病肾阴虚证的cDNA文库,为进一步克隆肾阴虚证的相关基因奠定了基础。     

Cloning of cDNA for the bovine IL-2 receptor (bovine Tac antigen).

We have cloned the Tac analog of the bovine IL-2 receptor (IL-2R) cDNA. Using mouse and human cDNA probes, we isolated five bovine IL-2R clones from a lambda gt11 bovine long-term lymphocyte cDNA library. Three of the clones had inserts of 2600 base pairs (bp), the same size as the bovine IL-2R mRNA visualized on Northern blots. The full-length cDNA contain a 190-bp 5' untranslated region, followed by a 825-bp coding region, and a 3' untranslated region that contain 1600 bp. Comparison of the bovine and human IL-2R-coding sequences revealed 71% homology at the nucleotide level. The 3' and 5' non-coding regions were not as homologous, apart from a specific site in the 5'-untranslated region that contained a 5'-upstream start codon. In this region, 24 of 26 nucleotides were identical for the human and bovine cDNAs. Further analysis of the bovine IL-2R sequence also revealed the following: (i) the hydrophobic domains of the IL-2R protein were more conserved between species than the hydrophilic domains, (ii) the predominant site of intracellular IL-2R phosphorylation in mouse and human was a conserved Ser which was not conserved in the bovine sequence, and (iii) there exists a statistically significant amino acid homology with the AIDS gag protein.     

Evidence for presence in the cell wall of Candida albicans of a protein related to the hsp70 family.

We have previously reported the isolation of several clones from a cDNA expression library from Candida albicans, one of which was associated with a constitutively expressed 70-kDa protein. The moiety was present in the beta-mercaptoethanol extracts of cell walls from both blastoconidia and germ tubes. The surface expression of this moiety was revealed by an indirect immunofluorescence assay using affinity-purified antibody to the fusion protein produced by the clone. The 0.68-kb cDNA insert was sequenced. A database search revealed extensive homology with the 70-kDa family of stress or heat shock proteins (hsps). The 77% homology with another C. albicans HSP70 sequence suggested that this fragment represented a second member of the HSP70 family in this organism. Homology ranging from 65 to 76% was observed with members of four subfamilies (SSA, SSB, SSC, and SSD) of the Saccharomyces cerevisiae HSP70 gene family. The nucleic acid sequence and the deduced amino acid sequence of the open reading frame showed greatest homology with SSA1 and SSA2 sequences, and the gene corresponding to the cDNA clone was designated C. albicans SSA2. The relationship with the SSA family was supported by reactivity of the 70-kDa component with antibody recognizing the Ssa proteins of S. cerevisiae. The presence of an hsp70 in the cell wall was confirmed by two additional methods. Cell wall proteins were biotinylated with a non-membrane-permeable derivative to distinguish extracellular from cytosolic proteins. Biotinylated hsp70 was detected by Western blotting (immunoblotting) among the biotinylated components affinity purified by chromatography on streptavidin, thereby establishing its presence in the cell wall. Immunoelectron microscopy showed that the 70-kDa component was present at the cell surface as well as the outer surface of the plasma membrane and extended through the cell wall, occasionally appearing to reach the cell surface through channels. Northern (RNA) blot analysis showed that the gene was expressed in yeast cells growing in yeast extract-peptone medium at both 25 and 37 degrees C and in Lee medium at 25 degrees C and during formation of germ tubes in Lee medium 37 degrees C. No obvious increase in the expression level was detected after the temperature shift. Members of the hsp70 family have been reported to be immunoreactive. The fusion protein produced by the cDNA clone was recognized by serum from healthy individuals and patients with candidiasis. Since members of the hsp70 family of eucaryotic proteins are associated with chaperone and translocation functions, in addition to being immunogenic, this protein may play a role in the assembly and function of other cell wall proteins.     

Gene cloning of the human cytomegalovirus (HCMV) antigen reactive with the serum from a HCMV-infected patient.

The human cytomegalovirus(HCMV) gene encoding the protein reactive with the sera of HCMV-infected patient was cloned and characterized. A reactive phage clone was screened from a lambda gt11 expression library of cDNA of HCMV AD169 strain using HCMV-infected patient sera. The recombinant protein was expressed as 138 kDa-fusion protein with beta-galactosidase, which was reactive with IgM or IgG HCMV antibody-positive sera, but not with anti-HCMV antibody-negative sera. A homology search of the DNA sequence of the cloned gene with HCMV AD169 sequences revealed that it was composed of 709 base pairs spanning between 0.174 and 0.177 map units of the UL32 region of the HCMV AD169 strain genome. This position corresponded to a part of the gene encoding 150 kDa phosphoprotein-(pp150), a major tegument protein, which was reported as an immunogenic protein which evoked strong and longstanding antibody response and had no sequence homology with the proteins of other herpesviruses. These results suggested that pp150 was an immunogenic protein in natural HCMV infection and therefore this clone was regarded as a useful candidate for developing an antigen for the serodiagnosis of HCMV.     

Cloning, expression, and cDNA sequence of surface antigen P22 from Toxoplasma gondii

Immunoblot, immunofluorescence, and complement-mediated cytolytic assays revealed that two new monoclonal antibodies raised against a membrane-enriched fraction of Toxoplasma gondii tachyzoites recognize protein P22 on the surface of the parasite. Using these monoclonal antibodies to screen a cDNA expression library in lambda gt11, several clones expressing recombinant fusion proteins were isolated. Subsequent screening of the library with a synthetic oligonucleotide derived from the 5' end of one of these cDNAs permitted the isolation of additional nonexpressing clones containing the entire translated sequence. Blots of parasite RNA and DNA suggested that the corresponding gene occurs as a single copy in the tachyzoite genome. The amino acid sequence deduced from the composite cDNA indicates a primary translation product with a theoretical molecular weight of 18,959. As expected for surface protein P22, the putative polypeptide contains a predicted N-terminal signal sequence and a C-terminal hydrophobic region characteristic of proteins attached to the membrane by a glycophospholipid anchor. Recombinant fusion proteins produced by the expressing clones were recognized on immunoblots by IgG antibodies in the sera of humans with acute and chronic T. gondii infection. Antibodies selected by the fusion protein reacted predominantly with a 22-kDa antigen on immunoblots of parasite lysate.     

The identification of a Clonorchis sinensis gene encoding an antigenic egg protein

The cDNA library of Clonorchis sinensis was screened for genes encoding antigenic proteins by using sera from clonorchiasis patients. A gene of 888 bp encoding a 28-kDa protein (Cs28) was cloned and found to contain a high percentage of glycine (20%), tyrosine (11%), and lysine (11%). The amino acid sequence of Cs28 showed 60% homology with the vitelline B precursor protein of Opisthorchis viverrini and of 33% homology with the vitelline B1 and B2 proteins of Fasciola hepatica. A strong positive reaction was observed in the intrauterine eggs of adult C. sinensis by immunohistochemical analysis using specific immune sera against recombinant Cs28 protein (rCs28). By immunoblot analysis, rCs28 displayed an antigenic reaction with 73% of the serum samples from 115 cases of clonorchiasis. In addition, it cross-reacted with the sera of 77.5% of 40 opisthorchiasis cases, 90% of 20 schistosomiasis cases, and 50% of 10 paragonimiasis cases. However, no cross-reactions were observed with the sera of sparganosis or cysticercosis patients. In conclusion, the Cs28 protein was identified as an egg protein of C. sinensis and as an antigen common to the trematode species examined.     

日本血吸虫尾蚴cDNA文库的免疫学筛选及EST序列的分析

目的为获得日本血吸虫（Schistosoma japonicura，Sj）转化生长因子β1（TGF—β1）基因的部分或全长cDNA序列。同时验证用针对某一蛋白的抗体筛选表达文库是否可以得到相应蛋白对应的基因序列。方法采用兔抗鼠TGF—β1血清，对Sj尾蚴cDNA表达文库进行免疫学筛选，对阳性克隆进行PCR扩增后，将大于500bp的克隆进行复筛，对复筛阳性的克隆进行测序和生物信息学鉴定。结果对大约10^6个噬菌斑进行了初筛。共获得7个阳性克隆；经过PCR扩增后，其中有4个克隆大于500bp，复筛获得3个持续阳性克隆；对测序后的阳性克隆进行生物信息学鉴定，得到的三个克隆均与日本血吸虫辅酶Q10氧化还原酶（SjCHGC）基因具有高的同源性（Bhata分值都大于200）。结论SjCHGC蛋白与兔抗鼠TGF—β1抗体的反应为交叉反应，用抗某一蛋白的抗体筛选表达文库得到相应蛋白的基因序列的方法不一定可行。     

Cloning of a major developmentally regulated gene expressed in mature females of Schistosoma mansoni

A cDNA library constructed from RNA isolated from adult Schistosoma mansoni has been screened by differential hybridization to identify clones corresponding to genes highly expressed by female worms. Several such cDNAs encoding the same highly abundant mRNA species were identified. Studies with one of these (pSF10) which contained a 500 base pair insert demonstrated that this gene was not expressed in immature females or eggs and encoded a polypeptide of approximately 35 000 daltons. Quantitation of the levels of RNA showed that 10% of the total RNA of female parasites was homologous to pSF10. A single gene corresponding to pSF10 was identified in Southern blotting experiments using adult worm DNA. The cloning of this gene will facilitate study of the molecular and genetic events controlling female schistosome maturation.     

E. coli selection of human genes encoding secreted and membrane proteins based on cDNA fusions to a leaderless beta-lactamase reporter

Although several signal peptide-trapping methods have been devised and used to detect signal sequences, none have relied on using E.coli to identify eukaryotic proteins with signal peptides. Here, we describe a system for selecting human secreted and membrane proteins in E. coli followed by the direct validation of secretion in human cells. The method is based on cDNA fusions to a leaderless beta-lactamase reporter gene to isolate clones encoding signal peptides of human genes. We found that beta-lactamase fusion proteins carrying a eukaryotic signal peptide at its N-terminus were able to direct their export into the periplasm in E. coli to confer survival upon challenge with carbenicillin. When libraries constructed from 5' end-enriched cDNAs fused to beta-lactamase were screened in E.coli, approximately 0.5%-1% of the cDNAs are selected, and over half of the surviving clones were found to encode for secreted fusion proteins when tested in human cells. These clones were sequenced and shown to represent human genes encoding signal peptides of secreted and membrane proteins. We conclude that this is an efficient and effective strategy to easily enrich cDNA libraries for the identification of novel genes likely to encode secreted enzymes, growth factors, and receptors.     

Isolation of cDNA clones and genomic DNA clones of beta-subunit of chicken prolyl 4-hydroxylase

Prolyl 4-hydroxylase (EC 1.14.11.2) is a key enzyme in collagen biosynthesis. The active enzyme is a tetramer composed of two pairs of non-identical subunits, alpha and beta. Sheep antiserum directed against chicken proly 4-hydroxylase was initially used to screen two cDNA expression libraries. The cDNA was prepared from chicken smooth muscle mRNA and cloned into the plasmids pUC8- and pUC9. Antibodies identified twenty-five clones among the approximately 2 x 10(5) clones in the libraries. Ten clones were isolated pure and used in the subsequent analysis. Monospecific antibodies directed against beta subunit of the enzyme were used in Western-blot analyses of extracts of bacteria carrying the cDNA clones. The results indicated that the clone CPH 9-10B encodes a portion of beta-subunit. The cDNA from CPH 9-10B was used to screen another cDNA library prepared from mRNA from chicken skeletal muscle. Several overlapping clones were isolated. Together the cDNAs correspond to 2.4 kb which is the same as the corresponding mRNA. Three regions of the amino acid sequence deduced from the cDNA sequence matched with that of the NH2-terminus of beta-subunit and two CNBr peptides derived from beta-subunit. The cDNA of CPH 9-10B was also used to screen a genomic DNA library constructed with lambda EMBL-3. Two overlapping genomic clones lambda gCPH beta-22 and beta-50 were isolated and characterized by restriction enzyme analysis. The results indicate that lambda gCPH beta-22 contains the portion of the beta-subunit gene that is transcribed into the 5' portion of beta-subunit mRNA, whereas lambda gCPH beta-50 contains the 3' portion.