Lambda ZAPII Research Articles

Identification, Bioinformatics Analyses, and Expression of Immunoreactive Antigens of Mycoplasma haemofelis

Mycoplasma haemofelis infection frequently causes anemia in cats. Despite an intense immune response and/or antibiotic treatment, cats often remain asymptomatic carriers following infection. Our hypothesis is that detection of antibodies to M. haemofelis is a sensitive approach for identifying infected cats, particularly carriers. To date, no immunoassay has been developed. This is due largely to the inability to culture M. haemofelis in vitro; hence, a source of antigen is not readily available. The objective of this study was to identify, express, and purify immunogenic proteins of M. haemofelis. To accomplish this, two whole-genomic expression libraries were created in the Lambda ZapII vector and immunoscreened with preimmune plasma, plasma from specific-pathogen-free cats, and pooled acute- and convalescent-phase plasma from experimentally infected cats. The inserts from 21 immunoreactive clones were sequenced, resulting in the identification of 60 genes coding for putative proteins necessary for diverse cellular functions, along with several novel genes of M. haemofelis. Fragments of selected genes based on bioinformatic analyses were PCR amplified, cloned into a high-level protein expression system, and subsequently expressed in Escherichia coli as a His(6)-fusion protein. The recombinant fusion proteins of M. haemofelis were purified and evaluated as an antigen in a Western blot to verify the findings of previous immunoscreening. Together with bioinformatics analyses of individual genes, this approach provided several putative candidate antigens. Five antigens of M. haemofelis were reactive by Western blotting against the immune plasma and negative against nonimmune plasma; these antigens might be useful serologic or even vaccine targets.

Insights from the GC content analysis of 76genome survey sequences (GSS) from Elaeisoleifera.

South American oil-palm (Elaeis oleifera) is not cultivated in tropical countries like Malaysia on large scale due to low yield of palm oil derived from its fruit mesocarp. However, its fruit mesocarp oil contains about 68.6 % oleic acid (C(18:1)) which is more than double in comparison to commercially cultivated oilpalm, E. guineensis Jacq Tenera (hybrid of Dura (♀) x Pisifera (♂)). It is also known that E. oleifera is a good source of tocotrienols and carotenoids. Therefore, it is of interest to know the genome sequence of E. oleifera. The objective of this study is to generate genome survey sequences (GSS) to get GC content insight in the E. oleifera genome. The nuclear genomic DNA isolated from young leaf-tissues was digested with EcoRI and NdeI/DraI restriction enzymes; and three genomic DNA libraries were constructed using Lambda ZAP-II, pGEM®-T Easy, and pDONR 222™ as cloning vectors. Generated 76 GSSs were analyzed by using Bioinformatics tools. The analysis result indicates that the adenine, cytosine, guanine and thymine content in generated GSSs are 30%, 20%, 20%, and 30% respectively. In conclusion, based on the precise GC content analysis of the randomly isolated 76 GSSs by using Bioinformatics tools we hypothesize that GC content in E. oleifera genome is 40%. The hypothesized 40% GC content in E. oleifera genome is expected to remain close to the GC content based on the whole genome analysis.(ψ)The nucleotide sequence data reported in this paper have been submitted to dbGSS division of the international DNA database (GenBank/DDBJ/EMBL) under accession numbers: DX575945- DX575972 and EI798032-EI798079. gDNA - Nuclear genomic DNA, GSSs - Genome survey sequences K12, SAOP - South American oil-palm Db1.

Chlamydomonas Flagellar Outer Row Dynein Assembly Protein Oda7 Interacts with Both Outer Row and I1 Inner Row Dyneins

We previously found that a mutation at the ODA7 locus in Chlamydomonas prevents axonemal outer row dynein assembly by blocking association of heavy chains and intermediate chains in the cytoplasm. We have now cloned the ODA7 locus by walking in the Chlamydomonas genome from nearby molecular markers, confirmed the identity of the gene by rescuing the mutant phenotype with genomic clones, and identified the ODA7 gene product as a 58-kDa leucine-rich repeat protein unrelated to outer row dynein LC1. Oda7p is missing from oda7 mutant flagella but is present in flagella of other outer row or inner row dynein assembly mutants. However, Oda7 levels are greatly reduced in flagella that lack both outer row dynein and inner row I1 dynein. Biochemical fractionation and rebinding studies support a model in which Oda7 participates in a previously uncharacterized structural link between inner and outer row dyneins.

Preparación de genotecas de ADNc de Plasmodium falciparum para la búsqueda de los genes de proteínas de unión a calmodulina, GOGAT y Pfmyo A.

A cDNA library of Plasmodium falciparum (Colombian strain FCB2) asexual stage was constructed in the lambda ZipLox vector. The lambda ZipLox library and a lambda ZAPII (Dd2 strain) were screened for genes coding for proteins that bind with or are related to calmodulin (CaM). Screening was accomplished with Hot start PCR assays and hybridization with radiolabeled probes. Actin I, CaM, glutamate synthase (GOGAT) and the three myosin clones--Pfmyo A, Pfmyo B and Pfmyo C--were identified. The clones coding for actin I, CaM and GOGAT were retrieved from the lambda ZipLox library, and the GOGAT and Pfmyo A clones from the lambda ZAP II library. The GOGAT clone contained an insert of 2,413 base pairs corresponding to 24.8% of the reported sequence. The Pfmyo A insert was 2,457 base pairs long, and represented the complete mRNA coding for this gene. Finally, the first report of a complete cDNA clone containing the P. falciparum myosin A is presented.

Microsatellite markers isolated in olive ( Olea europaea L.) are suitable for individual fingerprinting and reveal polymorphism within ancient cultivars.

We have isolated and sequenced 52 microsatellites or simple sequence repeats (SSRs) from nearly 60 positive clones obtained from two 'Frantoio' olive genomic libraries enriched in (AC/GT) and (AG/CT) repeats, respectively. The repeat-containing fragments obtained from genomic DNA restricted with Tsp509I were separated using a biotinylated probe bound to streptavidin-coated paramagnetic beads. Fragments were then cloned into lambda ZAPII vector and sequenced. Thirty of the 36 primer pairs which gave correct re-amplification in the source genome were used to assay the polymorphism of 12 olive cultivars, namely four well-known cultivars ('Coratina', 'Frantoio', 'Leccino', 'Pendolino') and eight ancient cultivars grown locally near Lake Garda ('Casaliva', 'Favarol', 'Fort', 'Grignan', 'Less', 'Raza', 'Rossanel', 'Trep'). The local cultivars were each re- presented by two to four long-lived individuals. The analysis was carried out using (33)P-labelled primers and 6% polyacrylamide sequencing gels. All except two microsatellites showed polymorphism, the number of alleles varying from 1 to 5. The average genetic diversity ( H) was 0.55. The power of discrimination ( PD) was 0.60. All cultivars, including the local ones, were easily separated from each other. Variations in the SSR pattern were observed among individual plants of the same cultivar in four out of the eight local cultivars analysed. Several primer pairs (17%) amplified more than one locus.

Sequence analysis and expression of a cDNA clone encoding a 98-kDa allergen in Dermatophagoides farinae.

The important dust mite allergens identified to date are of molecular weights ranging from 14 to 60 kDa. Our previous protein study indicated that the 98-kDa native paramyosin in Dermatophagoides farinae mite showed IgE reactivity with 82% of the mite-sensitive asthmatic patients suggesting that it is a novel major mite allergen. This study described the isolation and characterization of the cDNA clone encoding the 98-kDa mite allergen. A Dermatophagoides farinae cDNA library was constructed in lambda ZAPII vector and the library was immunoscreened with a monoclonal antibody 642. The cDNA insert was sub-cloned into M13 sequencing vector for single-stranded sequencing. The whole cDNA insert was expressed in pGEX-2T Escherichia coli expression system as a fusion protein with GST. The allergenicity of the recombinant peptides was tested by skin tests and IgE immunoassay. The IgE and IgG immunoassays were performed with sera from 20 mite-allergic patients. The cDNA clone Df642 was 2134 bp long, coding for a polypeptide of 711 amino acid residues. Protein sequence analysis and alignment confirmed that the deduced polypeptide is a mite paramyosin which is truncated slightly at the N- and C-terminuses. In vivo skin tests and in vitro IgE-binding study showed that 62% (13/21) and 50% (10/20) of the mite-sensitive asthmatic patients reacted positively with the recombinant Dermatophagoides farinae paramyosin, respectively. The study indicated that 98-kDa mite paramyosin is an important allergen.

Serological diagnosis of human cysticercosis by use of recombinant antigens from Taenia solium cysticerci.

A Taenia solium metacestode cDNA expression library in the lambda ZAPII vector was screened with pooled sera from patients with neurocysticercosis. Sixty primary clones were identified and shown to belong to two classes. The clones NC-3 and NC-9 did not reveal any significant homologies to sequences deposited in the databases and were further characterized. Both recombinant antigens were expressed as glutathione S-transferase fusion proteins and applied for serological diagnosis of human cysticercosis. An enzyme-linked immunosorbent assay was established and evaluated with 27 serum samples of La Réunion and Madagascar patients with cysticercosis. Diagnosis in these patients was established with radiological and serological procedures. For antigen NC-3 a sensitivity of 96.3% and a specificity of 91.5% for the serodiagnosis were achieved. In contrast, the sensitivity of antigen NC-9 was only 33.3%.

Complementary DNA Encoding nm23/NDP Kinase Gene from the Korean Tiger Shark Scyliorhinus torazame.

: A new tumor suppressor gene, snm23, homologous to the gene for human nucleoside diphosphate kinase nm23/NDP was first cloned from Korean tiger shark (Scyliorhinus torazame) skin lambda ZAP-II complementary DNA library. The gene (named snm23) containing the tumor metastasis suppressor protein was sequenced. The nucleotide and deduced amino acid sequences of snm23 revealed an open reading frame of 450 bp that corresponded to a protein of 150 amino acid residues, with a calculated molecular mass of 16.8 kDa. Sequence comparison of snm23 with nm23/NDP kinases was performed. In order to determine tissue specificity, reverse transcription-polymerase chain reaction was used. The expression of snm23/NDP kinase was detected in tissues from skin, cartilage, and liver of Korean tiger shark.

Molecular cloning and characterization of a novel testis-specific nucleoporin-related gene.

A 20-kDa sperm membrane protein cDNA, designated as RSD-1, was isolated by epitope selection from a rat testis lambda gtll expression library. RSD-1 was used as a probe to screen a human testis lambda ZAPII cDNA expression library. A cDNA designated as BS-63 was isolated and found to consist of 1933 bp with an open reading frame of 1824 bp and assigned the accession number U64675 by GenBank. The deduced polypeptide consisted of 608 amino acid residues containing XFXFG or FG motifs that are characteristic of nuclear pore complex (NPC) proteins and act as potential binding sites for Ran. The N-terminal region has high homology with RanBP2/Nup358, a nucleoporin component, showing that BS-63 is a member of the NPC family. Northern blot analysis of mRNAs prepared from various human tissues shows that BS-63 is transcribed in two forms: 6.0 and 8.5 kb. The 8.5-kb transcript was present in low amounts in several somatic tissues, whereas the 6.0-kb transcript is expressed only in testis. In situ hybridization analysis of human testis sections showed that BS-63 mRNA is expressed only in germ cells at all stages of spermatogenesis. Sertoli cells did not transcribe the gene.

Molecular cloning and characterization of mouse ficolin-A.

A novel ficolin-related gene was isolated from the mouse liver lambda ZAPII cDNA library. The protein encoded by this gene consists of both collagen- and fibrinogen-like domains, which are common features of the ficolin family, and was named mouse ficolin-A. The amino acid sequence of mouse ficolin-A is 60.2, 59.8, 59.8, and 59.6% identical to those of porcine ficolin-alpha, -beta, human ficolin-1, and EBP-37/P35, respectively. Northern blot analysis showed that mRNA of mouse ficolin-A is highly expressed in liver and spleen. Immunoblot analysis using an anti-mouse ficolin-A antiserum showed that mouse ficolin-A is a plasma protein with binding activity to elastin and GlcNAc.

Cloning and sequencing of the pyrG encoding cytidine 5'-triphosphate synthetase from Mycobacterium bovis BCG.

The pyrG gene encoding cytidine 5'-triphosphate (CTP) synthetase which catalyzes the terminal reaction for de novo synthesis of pyrimidine nucleotides was cloned and sequenced from Mycobacterium bovis BCG. The BCG pyrG was screened in lambda ZAPII library employing a DNA probe which was constructed from PCR based on a consensus pattern of glutamine amidotransferase type I. The BCG CTP synthetase deduced from a nucleotide sequence consisting of 586 amino acid residues with a calculated molecular weight of 63,642 daltons. Alignment of BCG CTP synthetase with all sequences available in GenBank indicates that the residues suggested as a catalytic triad for glutamine hydrolysis at the carboxyl terminal domain and an amino terminal segment are completely conserved among all CTP synthetases.

The gene coding for the DOPA dioxygenase involved in betalain biosynthesis in Amanita muscaria and its regulation.

Genomic and cDNA clones derived from the gene (dodA) coding for DOPA dioxygenase, a key enzyme in the betalain pathway, were obtained from the basidiomycete Amanita muscaria. A cDNA library was established in the phage lambda ZapII and dodA clones were isolated using polyclonal antibodies raised against the purified enzyme. Their identity was confirmed by comparison of the deduced amino acid sequence with the sequence of several tryptic peptide fragments of DOPA dioxygenase. The gene coded for a 228-amino acid protein that showed no homology to published sequences. The coding region was interrupted by five short introns. Regulation was shown to occur at the transcriptional level; the mRNA accumulated to high levels only in the coloured cap tissue. dodA was found to be a single-copy gene in A. muscaria. To our knowledge, this is the first gene from the betalain pathway to be cloned. It encodes a type of aromatic ring-cleaving dioxygenase that has not been previously described.

Helicobacter pylori ABC transporter: effect of allelic exchange mutagenesis on urease activity.

Helicobacter pylori urease requires nickel ions in the enzyme active site for catalytic activity. Nickel ions must, therefore, be actively acquired by the bacterium. NixA (high-affinity nickel transport protein)-deficient mutants of H. pylori retain significant urease activity, suggesting the presence of alternate nickel transporters. Analysis of the nucleotide sequence of the H. pylori genome revealed a homolog of NikD, a component of an ATP-dependent nickel transport system in Escherichia coli. Based on this sequence, a 378-bp DNA fragment was PCR amplified from H. pylori genomic DNA and used as a probe to identify an H. pylori lambda ZAPII genomic library clone that carried these sequences. Four open reading frames of 621, 273, 984, and 642 bp (abcABCD) were revealed by sequencing and predicted polypeptides of 22.7, 9.9, 36.6, and 22.8 kDa, respectively. The 36.6-kDa polypeptide (AbcC) has significant homology (56% amino acid sequence identity) to an E. coli ATP-binding protein component of an ABC transport system, while none of the other putative proteins are significantly homologous to polypeptides in the available databases. To determine the possible contribution of these genes to urease activity, abcC and abcD were each insertionally inactivated with a kanamycin resistance (aphA) cassette and allelic exchange mutants of each gene were constructed in H. pylori UMAB41. Mutation of abcD resulted in an 88% decrease in urease activity to 27 +/- 31 mumol of NH3/min/mg of protein (P < 0.0001), and a double mutant of nixA and abcC resulted in the near abolishment of urease activity (1.1 +/- 1.4 mumol of NH3/min/mg of protein in the double mutant versus 228 +/- 92 mumol of NH3/min/mg of protein in the parent [P < 0.0001]). Synthesis of urease apoenzyme, however, was unaffected by mutations in any of the abc genes. We conclude that the abc gene cluster, in addition to nixA, is involved in production of a catalytically active urease.

Cca1 is required for formation of growth-arrested confluent monolayer of rat 3Y1 cells.

A novel cDNA fragment, named cca1 (confluent 3Y1 cell-associated 1), was previously isolated on the basis of preferential accumulation of the corresponding mRNA in growth-arrested confluent but not in growing subconfluent rat 3Y1 cells. The cca1 cDNA was found to consist of 5022 nucleotides with an open reading frame of 1905 nucleotides, encoding a protein of 635 amino acids. Unlike the 3Y1 cell case, cca1 mRNA was not detected in confluent 3Y1 BU, 3Y1 BU/pTK, 3Y1-16E6, or F2408 cells, whose growth patterns monitored by phalloidin staining and bromodeoxyuridine incorporation were different from those of the confluent 3Y1 cells. A restoration of the confluent 3Y1-type growth pattern was observed in the cca1 cDNA-introduced 3Y1 BU and 3Y1 BU/pTK cells after reaching confluence but not in the cDNA-introduced 3Y1-16E6 or F2408 cells. The results allow us to conclude that cca1 is required but not sufficient for formation of growth-arrested confluent monolayer of 3Y1 cells.

Cloning and characterization of a dextranase gene (dexS) from Streptococcus suis.

A gene (dexS) coding for a Streptococcus suis capsular type 2 dextranase (DexS) was detected in a recombinant gene library constructed in phage lambda ZapII, and its nucleotide sequence was determined. Sequence comparison showed that the dexS gene product had significant similarities with enzymes which hydrolyze glucose polymers. Moreover, conserved amino acids that are suggested to be part of the active site of the glucosidases are also found in DexS. The dexS gene, adjacent to the gene encoding a S. suis IgG-binding protein, encoded a protein of approximately 62 kDa which exhibited DexS activity.

Arabinanase A from Pseudomonas fluorescens subsp. cellulosa exhibits both an endo- and an exo- mode of action.

Pseudomonas fluorescens subsp. cellulosa expressed arabinanase activity when grown on media supplemented with arabinan or arabinose. Arabinanase activity was not induced by the inclusion of other plant structural polysaccharides, and was repressed by the addition of glucose. The majority of the Pseudomonas arabinanase activity was extracellular. Screening of a genomic library of P. fluorescens subsp. cellulosa DNA constructed in Lambda ZAPII, for recombinants that hydrolysed Red-dyed arabinan, identified five arabinan-degrading plaques. Each of the phage contained the same Pseudomonas arabinanase gene, designated arbA, which was present as a single copy in the Pseudomonas genome. The nucleotide sequence of arbA revealed an open reading frame of 1041 bp encoding a protein, designated arabinanase A (ArbA), of Mr 39438. The N-terminal sequence of ArbA exhibited features typical of a prokaryotic signal peptide. Analysis of the primary structure of ArbA indicated that, unlike most Pseudomonas plant cell wall hydrolases, it did not contain linker sequences or have a modular structure, but consisted of a single catalytic domain. Sequence comparison between the Pseudomonas arabinanase and proteins in the SWISS-PROT database showed that ArbA exhibits greatest sequence identity with arabinanase A from Aspergillus niger, placing the enzyme in glycosyl hydrolase Family 43. The significance of the differing substrate specificities of enzymes in Family 43 is discussed. ArbA purifed from a recombinant strain of Escherichia coli had an Mr of 34000 and an N-terminal sequence identical to residues 32-51 of the deduced sequence of ArbA, and hydrolysed linear arabinan, carboxymethylarabinan and arabino-oligosaccharides. The enzyme displayed no activity against other plant structural polysaccharides, including branched sugar beet arabinan. ArbA produced almost exclusively arabinotriose from linear arabinan and appeared to hydrolyse arabino-oligosaccharides by successively releasing arabinotriose. ArbA and the Aspergillus arabinanase mediated a decrease in the viscosity of linear arabinan that was associated with a significant release of reducing sugar. We propose that ArbA is an arabinanase that exhibits both an endo- and an exo- mode of action.

The isolation, localization and characterization of the QM homolog in Drosophila melanogaster.

A fragment of 443 bp was amplified from a lambda ZAPII Drosophila central nervous system (CNS) cDNA library using minimally degenerate primers to very conserved regions of the QM gene. This fragment was used as a probe to screen the lambda ZAPII Drosophila CNS cDNA library. Two clones of the Drosophila QM homolog (pDQM-7A1 and pDQM-2B1), each containing the complete coding region, were isolated. The 5'-UTR of this gene was obtained by RACE PCR and ligated to the coding sequence to produce a the full-length copy of the Drosophila QM homolog (DQM) cDNA. The DQM cDNA measures 746 nucleotides in length and encodes a polypeptide of 218 residues. The amino acid sequence shows 76.1 percent identity with human QM and 69.1 percent identity with QSR1, the yeast homolog of QM. Unlike the human or mouse genome which contains multiple copies of the QM gene, the Drosophila genome has only a single copy as indicated by genomic Southern blot analysis. In situ hybridization confirms the presence of a single copy of DQM in the Drosophila genome and localizes it to the left arm of the third chromosome at the end of region 80A (80A-4).

Genes expressed in Brugia malayi infective third stage larvae

We have used a tag sequencing approach to survey genes expressed in the third stage infective larvae of the human filarial nematode parasite Brugia malayi. RNA was isolated from late vector-stage L3 larvae after days 9 or 10 of infection in mosquitos, and converted to cDNA by reverse transcriptase. Double-stranded cDNA was produced by either conventional methods (non-SL cDNA library) or by PCR using the nematode spliced leader (SL1) and oligo(dT) primers (SL cDNA library). Two clone libraries (one from SL and one from non-SL cDNAs) were constructed in lambda ZapII. A set of these full-length clones was selected and 596 inserts were sequenced from the 5′ end. We have identified 364 B. malayi genes (the majority of which are new) that encode housekeeping proteins, structural proteins, proteins of immediate immunological or drug-discovery interest as well as a large class of novel sequences which may prove to have significant involvement in host invasion. Extensive, genome-wide approaches to the analysis of larval gene expression are now possible for B. malayi. We present several examples of this approach.

Recombinant antigen-based avidin-biotin microtiter enzyme-linked immunosorbent assay for serodiagnosis of invasive amebiasis.

An immunoscreening approach was used to isolate a strongly positive cDNA clone from an Entamoeba histolytica HK-9 cDNA expression library in the phage vector lambda ZAP-II. The 1.85-kb cDNA insert was found to be truncated and encoded the cysteine-rich, immunodominant domain of the antigenic 170-kDa subunit of the amebal galactose-N-acetylgalactosamine binding lectin. This domain was expressed as a glutathione S-transferase fusion protein in Escherichia coli. Inclusion bodies of the recombinant protein were solubilized with Sarkosyl, and the protein was enriched from the crude bacterial extract by thiol-affinity chromatography. The recombinant protein was used to develop a rapid, sensitive, and specific avidin-biotin microtiter enzyme-linked immunosorbent assay (ELISA) for invasive amebiasis. Sera from 38 individuals suffering from invasive amebiasis, 12 individuals with noninvasive amebiasis, 44 individuals with other infections, and 27 healthy subjects were screened by the recombinant antigen-based ELISA. The sensitivity and specificity of the assay were 90.4 and 94.3%, respectively, which correlated well with those of an ELISA developed with crude amebal antigen (r = 0.94; P < 0.0001), as well as with those of a commercially available serodiagnostic ELISA (r = 0.92; P < 0.0001). Thus, the bacterially expressed recombinant lectin can replace the crude amebal extract as an antigen in the serodiagnosis of invasive amebiasis by using avidin-biotin microtiter ELISA.

Molecular characterization, genomic arrangement, and expression of bmpD, a new member of the bmp class of genes encoding membrane proteins of Borrelia burgdorferi.

An expression library made with Borrelia burgdorferi DNA in the vector lambda ZapII was screened with serum from a monkey infected with the Lyme disease agent. This serum killed B. burgdorferi in vitro by an antibody-dependent, complement-mediated mechanism and contained antibodies to at least seven spirochetal antigens, none of which were the major outer surface proteins OspA or OspB. Among several positive clones, a clone containing the B. burgdorferi bmpA gene encoding the immunodominant antigen P39 was obtained. Chromosome walking and DNA sequence analysis permitted the identification of two additional upstream genes homologous to the bmpA gene and its related companion, bmpB. The first of these was the recently characterized bmpC gene, and adjacent to it was the fourth and new member of this class, which has been designated bmpD. The gene product encoded by bmpD is 34l residues long, contains a signal sequence with a potential signal peptidase II cleavage site, and has 26% identity with TmpC of Treponema pallidum. Southern blotting confirmed the tandem arrangement of all four bmp genes in the chromosome of B. burgdorferi JD1. However, Northern (RNA) blotting revealed that bmpD is expressed as a monocistronic transcript, which indicates that it is not part of an operon at the bmp locus. The bmpD gene was found to be conserved in representative members of the three species of the B. burgdorferi sensu lato complex, suggesting that it serves an important biological function in the spirochete.