Identification and cloning of a locus of serine repeat antigen (sera)-related genes from Plasmodium vivax

Polymerase chain reaction (PCR) primers based on the cysteine proteinase-like active site regions of the Plasmodium falciparum serine repeat antigen (SERA) were used to identify related sequences within the genome of P. vivax. Molecular cloning and sequence analysis of approximately 25 kb of P. vivax genomic DNA revealed a cluster of five repeated SERA-like genes (V-SERA-1-5), each encoding a cysteine proteinase-related protein. In addition to DNA sequence homology, significant similarities in deduced intron/exon organizations were also observed. The characteristic polyserine sequence found in SERA was not present in any of the deduced V-SERA sequences. Instead, in this region of the five genes, considerable sequence differences were found, suggesting the potential for antigenic variation in the V-SERA molecules. In common with SERA, however, the codon at the position corresponding to the active site cysteine residue of active mammalian and plant cysteinyl proteinases was found to be that of a serine residue in each of the V-SERA genes. Furthermore, in four of the five genes, including the expressed V-SERA-5 gene, the codon for the active site histidine residue was changed to that of a leucine residue. These critical differences reinforce the concept that a biological activity other than proteolysis is likely to be the primary function of the proteins encoded by this family of genes.     

Differentiation of Campylobacter jejuni serotype O19 strains from non-O19 strains by PCR

Guillain-Barré syndrome (GBS), a neurologic disease characterized by acute paralysis, is frequently preceded by Campylobacter jejuni infection. Serotype O19 strains are overrepresented among GBS-associated C. jejuni isolates. We previously showed that all O19 strains tested were closely related to one another by randomly amplified polymorphic DNA (RAPD) and restriction fragment length polymorphism analyses. RAPD analysis demonstrated a 1.4-kb band in all O19 strains tested but in no non-O19 strains. We cloned this O19-specific band; nucleotide sequence analysis revealed a truncated open reading frame with significant homology to DNA gyrase subunit B (gyrB) of Helicobacter pylori. PCR using the random primer and a primer specific for gyrB showed that in non-O19 strains, the random primer did not recognize the downstream gyrB binding site. The regions flanking each of the random primer binding sites were amplified by degenerate PCR for further sequencing. Although the random primer had several mismatches with the downstream gyrB binding site, a single nucleotide polymorphism 6 bp upstream from the 3' terminus was found to distinguish O19 and non-O19 strains. PCR using 3'-mismatched primers based on this polymorphism was designed to differentiate O19 strains from non-O19 strains. When a total of 42 (18 O19 and 24 non-O19) strains from five different countries were examined, O19 strains were distinguishable from non-O19 strains in each case. This PCR method should permit identification of O19 C. jejuni strains.     

Serotype-specific amplification of Rickettsia tsutsugamushi DNA by nested polymerase chain reaction

Polymerase chain reaction (PCR) with nested primer pairs was used to diagnose scrub typhus and identify the Rickettsia tsutsugamushi serotype. The primer pairs used for PCR were designed on the basis of the nucleotide sequence of the gene that encodes the 56-kDa antigen. Serotype-specific primers were used in the second PCR amplification. Five serovariants, the Gilliam, Karp, Kato, Kawasaki, and Kuroki strains of R. tsutsugamushi, were identified by nested PCR. In addition, the serotype identified by PCR with DNA from blood clots was the same as that of the strain isolated from five patients with scrub typhus. These findings indicate that this method is useful for diagnosis and identification of the rickettsial serotype in infected patients.     

Identification of hepatitis E virus in clinical specimens: amplification of hydroxyapatite-purified virus RNA and restriction endonuclease analysis

Mutants null for the cathepsin B-like cysteine proteinase gene (cpc) of Leishmania mexicana have been generated by targeted gene disruption. The gene deletion was confirmed using a polymerase chain reaction (PCR) method with cpc-specific primers and genomic DNA isolated from the mutants. cpc was re-expressed in the null mutants from an episomal vector. Re-expression of the enzyme (CPC) was detected by Western blotting with a specific anti-peptide antiserum. The cpc null mutants grew apparently normally as promastigotes and amastigotes in axenic cultures, but they showed greatly reduced infectivity to macrophages in vitro with only a low percentage of the cells being infected. Re-expression of cpc in the null mutant increased the parasite's infectivity in vitro. The null mutant parasites formed lesions in mice at a similar rate as wild type parasites, although somewhat smaller lesions were produced. The results suggest that although the cysteine proteinase encoded by cpc plays a role in the parasite's interaction with macrophages it alone is not crucial for infectivity or virulence.     

Cloning and sequencing of the gene encoding a novel lysine-specific cysteine proteinase (Lys-gingipain) in Porphyromonas gingivalis: structural relationship with the arginine-specific cysteine proteinase (Arg-gingipain)

Lys-gingipain (KGP), so termed due to its peptide cleavage specificity for lysine residues, is a cysteine proteinase produced by the Gram-negative anaerobic bacterium Porphyromonas gingivalis. Mixed oligonucleotide primers designed from the NH2-terminal sequence of the purified enzyme were used to clone the KGP-encoding gene (kgp) from the organism. The nucleotide sequence of kgp had a 5,169-bp open reading frame encoding 1,723 amino acids with a calculated molecular mass of 218 kDa. As the extracellular mature enzyme had an apparent molecular mass of 51 kDa in gels, the precursor of KGP was found to comprise at least four domains, the signal peptide, the NH2-terminal prodomain, the mature proteinase domain, and the COOH-terminal hemagglutinin domain, and to be proteolytically processed during its transport. Importantly, the COOH-terminal region contained three direct repeats of two different amino acid sequences, LKWD(or E)AP and YTYTVYRDGTKI, and the subdomains located between the two repeats exhibited strong similarity to those of Arg-gingipain (RGP), another major cysteine proteinase produced by the organism and having cleavage specificity for arginine residues, although the arrangement of the subdomains was not necessarily identical in the two enzymes. Since the KGP activity was greatly decreased in RGP-deficient mutants and since the most probable site of the propeptide cleavage was present in the homologous sequence highly susceptible to proteolysis by RGP, the precursor of KGP is likely to be processed by RGP to form the mature enzyme.     

Use of the flagellar H7 gene as a target in multiplex PCR assays and improved specificity in identification of enterohemorrhagic Escherichia coli strains

PCR products of 1.8 kb were generated with DNAs from all Escherichia coli H7 strains tested by using oligonucleotide primers which flank the fliC gene. Three RsaI digestion profiles of these PCR products were evident on agarose gels; the first occurred with serotype O55:H7, O157:H7, or nonmotile (NM) strains, the second occurred with serotype O1:H7 and O18:H7 strains, and the third occurred with serotype O?:H7, O19:H7, O121:H7, O88:H7, and O156:H7 strains. Despite these differences, the nucleotide sequences of the E. coli E32511 (O157:NM) and U5-41 (O1:H7) fliC genes were 97% homologous. Two PCR primer pairs synthesized on the basis of the E32511 H7 fliC sequence amplified specific DNA fragments from all E. coli H7 strains, but did not amplify DNA fragments from the other bacterial strains. The H7-specific primers were used in combination with other primers which target the Verotoxin 1(VT1) and VT2 genes and the E. coli O157:H7 eaeA gene in multiplex PCR assays. In these assays, vt and eaeA PCR products were observed with DNAs from the majority of EHEC strains and vt, eaeA, and fliC PCR products were observed with DNAs from E. coli O157:H7 or NM strains. Only eaeA PCR products were present with DNA from enteropathogenic E. coli, and only vt PCR products occurred with VT-producing E. coli which are not EHEC. The multiplex PCR assays described allow for the specific identification of E. coli O157:H7 or NM and other EHEC strains.     

Detection of known mutation by proof-reading PCR

Proof-reading PCR (PR-PCR) is designed to detect known mutations within genomic DNA. It differs from standard PCR approaches in that one of the two primers has its 3' end aligned with a putative mutation site, and has its 3'-OH replaced by a blocking group. Distinguishing a mutant gene from wild-type depends upon preferential removal of the blocked 3' terminal nucleotide by the polymerase proof-reading activity when it is mismatched with the template. Preferential removal of the blocked nucleotide allows subsequent extension and selective amplification, and provides the basis for distinguishing mutant from normal genes. This method has been used here to detect a transition mutation within the P53 gene of HaCaT cells with verification by direct sequencing of the selectively amplified DNA.     

Efficacy of venlafaxine and placebo during long-term treatment of depression: a pooled analysis of relapse rates

An assay was developed for the specific detection of Salmonella enterica serotype Enteritidis, using a novel application of the polymerase chain reaction (PCR). This PCR assay is based on the mismatch amplification mutation assay, an allele-specific reaction, and can discriminate Enteritidis from all other salmonella. PCR primers were selected to amplify a 351-base pair (bp) DNA fragment from the salmonella plasmid virulence A (spv A) gene of Enteritidis. A single base difference at position 272 is present between the nucleotide sequence of the spvA gene of Enteritidis and other salmonellae. The downstream PCR primer, that encompasses position 272 of the Enteritidis spvA gene, was designed to contain a single base mismatch at the penultimate position, resulting in a 1-base mismatch with Enteritidis and a 2-base mismatch with other salmonellae that harbour the virulence plasmid. The upstream primer was completely homologous with the region immediately 5' to the spvA gene. When these primers were used and the annealing and extension reactions were performed at the same temperature, the PCR assay was specific for Enteritidis; no PCR product was detected for 40 other serotypes and 28 different genera examined. In pure culture, 120 colony forming units (c.f.u.) could be detected; a PCR product was observed from template derived from a 5 h enrichment broth culture of chicken seeded with 1 c.f.u. per gram of Enteritidis. This PCR assay is specific, reproducible, and less time consuming than the standard bacteriological methods used to detect Enteritidis.     

Identification of putative sequence specific PCR primers for detection of the toxigenic fungal species Stachybotrys chartarum

The nucleotide sequence of a c 936 bp segment of the nuclear rRNA gene operon was determined for the toxigenic fungal species Stachybotrys chartarum and for other species of Stachybotrys and the related genus Memnoniella. This information was used to infer the phylogenetic relationships of these organisms and to search for sequence specific polymerase chain reaction (PCR) primers for S. chartarum in the internal transcribed spacer (ITS) regions. Searches for candidate primers were performed both by computer using the commercially available Oligo(R) v5.0 primer analysis software package and by manual inspection of the aligned sequences. Primers identified in both types of searches were evaluated for their specificities using a priming efficiency analysis algorithm available in the Oligo(R) 5.0 software. The automated computer searches were unsuccessful in finding S. chartarum-specific primers but did identify a group-specific reverse primer (designated as StacR4) for a phylogenetically related cluster of species that included S. chartarum. Manual searches led to the identification of a reverse primer (designated as StacR3) that was predicted to be specific for only S. chartarum and one other species of Stachybotrys. Experimental PCR analyses using these primers in conjunction with a universal forward primer indicated that the computer-generated amplification efficiency predictions were correct in most instances. A notable exception was the finding that StacR3 was specific only for S. chartarum. The relative merits of different PCR strategies for the detection of S. chartarum employing either one or both of the primers identified in this study are discussed.     

Differential replication timing of X-linked genes measured by a novel method using single-nucleotide primer extension

The ratio of two differentially replicating alleles is not constant during S phase. Using this fact, we have developed a method for determining allele-specific replication timing for alleles differing by at least a single base pair. Unsynchronized cells in tissue culture are first sorted into fractions based on DNA content as a measure of position in S phase. DNA is purified from each fraction and used for PCR with primers that bracket the allelic difference, amplifying both alleles. The ratio of alleles in the amplified product is then determined by a single nucleotide primer extension (SNuPE) assay, modified as described [Singer-Sam,J. and Riggs,A.D. (1993) Methods Enzymol., 225, 344-351]. We report here use of this SNuPE-based method to analyze replication timing of two X-linked genes, Pgk-1 and Xist, as well as the autosomal gene Gabra-6. We have found that the two alleles of the Gabra-6 gene replicate synchronously, as expected; similarly, the active allele of the Pgk-1 gene on the active X chromosome (Xa) replicates early relative to the silent allele on the inactive X chromosome (Xi). In contrast, the expressed allele of the Xist gene, which is on the Xi, replicates late relative to the silent allele on the Xa.     

Prenatal fluoride for growth and development: Part X

Polymerase chain reaction (PCR) with degenerate primers was utilized for partial cloning of the MADS box gene family from wheat (Triticum aestivum L.). PCR products corresponding to a part of the MADS box region were cloned and sequenced. Eleven individual clones sequenced were classified into seven types on the basis of the nucleotide sequence and five types on the deduced amino acid sequence, which included two wheat-specific MADS box protein sequences. RT-PCR analysis with degenerate primers revealed preferential expression of the MADS box genes in young spikes. Furthermore, genomic Southern blot analysis with degenerate PCR products as probes indicated that wheat MADS box genes constitute a multigene family and are dispersed throughout the genome.     

Characterization of the myotubularin dual specificity phosphatase gene family from yeast to human

X-linked myotubular myopathy (XLMTM) is a severe congenital muscle disorder due to mutations in the MTM1 gene. The corresponding protein, myotubularin, contains the consensus active site of tyrosine phosphatases (PTP) but otherwise shows no homology to other phosphatases. Myotubularin is able to hydrolyze a synthetic analogue of tyrosine phosphate, in a reaction inhibited by orthovanadate, and was recently shown to act on both phosphotyrosine and phosphoserine. This gene is conserved down to yeast and strong homologies were found with human ESTs, thus defining a new dual specificity phosphatase (DSP) family. We report the presence of novel members of the MTM gene family in Schizosaccharomyces pombe, Caenorhabditis elegans, zebrafish, Drosophila, mouse and man. This represents the largest family of DSPs described to date. Eight MTM-related genes were found in the human genome and we determined the chromosomal localization and expression pattern for most of them. A subclass of the myotubularin homologues lacks a functional PTP active site. Missense mutations found in XLMTM patients affect residues conserved in a Drosophila homologue. Comparison of the various genes allowed construction of a phylogenetic tree and reveals conserved residues which may be essential for function. These genes may be good candidates for other genetic diseases.     

Purification and characterization of cysteine proteinase from a baculovirus gene

To analyze the degradation of product proteins at the late stage of virus infection in the baculovirus expression system, a cysteine proteinase was purified from hemolymph of Bombyx mori infected with wild-type B. mori nuclear polyhedorosis virus (BmNPV). The purified cysteine proteinase preparation had two protein bands (major 35-kDa active protein and 28-kDa inactive protein) on SDS-PAGE. Based on the N-terminal amino acid sequences of them, it was found that both proteins originated in the cysteine proteinase gene of BmNPV. The purified cysteine proteinase had an optimum pH at 4.0, and also had activities at neutral pHs. When recombinant luciferase was used as a natural substrate, it was degraded rapidly by the cysteine proteinase at the physiological pH of hemolymph. These results suggest that the cysteine proteinase from a BmNPV gene participates in the degradation of foreign protein expressed by the baculovirus system.     

Monitoring genetically modified rhizobia in field soils using the polymerase chain reaction

Monitoring genetically modified (GM) bacterial inoculants after field release using conventional culture methods can be difficult. An alternative is the detection of marker genes in DNA extracted directly from soil, using specific oligonucleotide primers with the polymerase chain reaction (PCR). The PCR was used to monitor survival of two GM Rhizobium leguminosarum bv. viciae inoculants after release in the field at Rothamsted. One strain, RSM2004, is marked by insertion of transposon Tn5; the second strain, CT0370, released at the same site, is modified by chromosomal integration of a single copy of the gene from E. coli conferring GUS activity. Both GM strain provide a realistic case study for the development of PCR-based detection techniques. Specific primers were developed to amplify regions of the Tn5 and GUS genetic markers using PCR and conditions optimized for each primer set to routinely detect a signal from 10 fg of purified template DNA, the equivalent of one cell per reaction. Procedures to improve the sensitivity of detection are described, to detect fewer than 50 cells g-1 soil in soil-extracted DNA.     

Gene-specific universal mammalian sequence-tagged sites: application to the canine genome

We are developing a genetic map of the dog based partly upon markers contained within known genes. In order to facilitate the development of these markers, we have used polymerase chain reaction (PCR) primers designed to conserved regions of genes that have been sequenced in at least two species. We have refined the method for designing primers to maximize the number that produce successful amplifications across as many mammalian species as possible. We report the development of primer sets for 11 loci in detail: CFTR, COL10A1, CSFIR, CYP1A1, DCN1, FES, GHR, GLB1, PKLR, PVALB, and RB1. We also report an additional 75 primer sets in the appendices. The PCR products were sequenced to show that the primers amplify the expected canine genes. These primer sets thus define a class of gene-specific sequence-tagged sites (STSs). There are a number of uses for these STSs, including the rapid development of various linkage tools and the rapid testing of genomic and cDNA libraries for the presence of their corresponding genes. Six of the eleven gene targets reported in detail have been proposed to serve as "anchored reference loci" for the development of mammalian genetic maps [O'Brien, S. J., et al., Nat. Genet. 3:103, 1993]. The primer sets should cover a significant portion of the canine genome for the development of a linkage map. In order to determine how useful these primer sets would be for the other genome projects, we tested the 11 primer sets on the DNA from species representing five mammalian orders. Eighty-four percent of the gene-species combinations amplified successfully. We have named these primer sets "universal mammalian sequence-tagged sites" because they should be useful for many mammalian genome projects.     

Rescue of Caenorhabditis elegans pharyngeal development by a vertebrate heart specification gene

Development of pharyngeal muscle in nematodes and cardiac muscle in vertebrates and insects involves the related homeobox genes ceh-22, nkx2.5, and tinman, respectively. To determine whether the nematode and vertebrate genes perform similar functions, we examined activity of the zebrafish nkx2.5 gene in transgenic Caenorhabditis elegans. Here, we report that ectopic expression of nkx2.5 in C. elegans body wall muscle can directly activate expression of both the endogenous myo-2 gene, a ceh-22 target normally expressed only in pharyngeal muscle, and a synthetic reporter construct controlled by a multimerized CEH-22 binding site. nkx2.5 also efficiently rescues a ceh-22 mutant when expressed in pharyngeal muscle. Together, these results indicate that nkx2.5 and ceh-22 provide a single conserved molecular function. Further, they suggest that an evolutionarily conserved mechanism underlies heart development in vertebrates and insects and pharyngeal development in nematodes.     

Use of a cysteine proteinase from Carica candamarcensis as a protective agent during DNA extraction

We describe the use of a plant cysteine proteinase isolated from latex of Carica candamarcensis as a protective agent during isolation of bacterial DNA following growth in culture of these cells. Between 100 to 720 units of proteinase (1 microgram = 6 units) afforded good DNA protection when incubated with various kinds of microorganisms. Agarose gel electrophoresis showed that the resulting DNA was similar in size to DNA preparations obtained by treatment with proteinase K. The viability of the resulting material was checked by PCR amplification using species-specific primers. After standing at room temperature (25 degrees C) for 35 days, the enzyme lost 10% of its initial activity. The enzyme stability and good yield of DNA suggest the use of this proteinase as an alternative to proteinase K.     

Phylogeny of Wolbachia in filarial nematodes

Intracellular bacteria have been observed in various species of filarial nematodes (family Onchocercidae). The intracellular bacterium of the canine filaria Dirofilaria immitis has been shown to be closely related to Wolbachia, a rickettsia-like micro-organism that is widespread among arthropods. However, the relationships between endosymbionts of different filariae, and between these and the arthropod wolbachiae, appear not to have been studied. To address these issues we have examined ten species of filarial nematodes for the presence of Wolbachia. For nine species, all samples examined were PCR positive using primers specific for the ftsZ gene of Wolbachia. For one species, the examined samples were PCR negative. Sequences of the amplified ftsZ gene fragments of filarial wolbachiae fall into two clusters (C and D), which are distinct from the A and B clusters recognized for arthropod wolbachiae. These four lineages (A-D) are related in a star-like phylogeny, with higher nucleotide divergence observed between C and D wolbachiae than that observed between A and B wolbachiae. In addition, within each of the two lineages of filarial wolbachiae, the phylogeny of the symbionts is consistent with the host phylogeny. Thus, there is no evidence for recent Wolbachia transmission between arthropods and nematodes. Endosymbiont 16S ribosomal DNA sequences from a subset of filarial species support these findings.