Characterisation of Trypanosoma cruzi populations by dna polymorphism of the cruzipain gene detected by single-stranded dna conformation polymorphism (SSCP) and direct sequencing

Fifty fresh isolates of Trypanosoma cruzi from Triatoma dimidiata vectors and 31 from patients with Chagas disease were analysed for DNA polymorphisms within the 432-bp core region of the cruzipain gene which encodes the active site of cathepsin L-like cystein proteinase. The cruzipain gene showed signs of polymorphism consisting of four different DNA sequences in Central and South American isolates of T. cruzi. The PCR fragments of Guatemalan isolates could be divided into three groups, Groups 1, 2 and 3, based on different patterns of single-stranded DNA conformation polymorphism. All of the strains isolated from Brazil, Chile, and Paraguay, except for the CL strain, showed a Group 4 pattern. Two to four isolates from each group were analysed by cloning and sequencing. A silent mutation occurred between Groups 1 and 2, and five nucleotides and two aa substitutions were detected between Groups 1 and 3. The DNA sequence of Group 4 contained five nucleotides and one aa substitution from Group 1. All of the DNA sequences corresponded well with the single-stranded DNA conformation polymorphism. The Group 1 isolates, the majority in the Guatemalan population (70/81, 86.4%), were isolated from both triatomines and humans, but Group 3 were isolated only from humans. Moreover, the Group 2 isolates were detected only in triatomine vectors (9/50; 18%), but never in humans (0/32, P<0.05) suggesting that this group has an independent life-cycle in sylvatic animals and is maintained by reservoir hosts other than humans.     

Trypanosoma cruzi: exploring the nuclear genome of zymodeme 3 stocks by chromosome size polymorphism

Chagas disease is emerging in the Brazilian Amazon. We evaluated the position of eight zymodeme 3 isolates from Amazonian sylvatic vectors and one human case in relation to Trypanosoma cruzi I and II major groups and hybrid strains by chromosome size polymorphism. Nineteen isolates were analyzed by mapping nine coding sequences on chromosomal bands (0.6-3.3Mbp). Numerical analysis was based on the absolute chromosomal size difference index (aCSDI). A dendrogram was obtained applying the minimum evolution criterion and considering the aCSDI values to estimate the branch lengths. The isolates were distributed in four groups. Group A clustered hybrid isolates; Groups B and C, T. cruzi II and T. cruzi I isolates, respectively. Seven Z3 stocks were clustered in Group D, which showed low intra-group diversity and was the most divergent. The proportion of two different-sized homologous chromosomes was determined. Wild vectors harboring Z3 stocks constitute a potential reservoir of human infection in the Amazon.     

Intraspecies polymorphism of Cryptosporidium parvum revealed by PCR-restriction fragment length polymorphism (RFLP) and RFLP-single-strand conformational polymorphism analyses

A glycoprotein (Cpgp40/15)-encoding gene of Cryptosporidium parvum was analyzed to reveal intraspecies polymorphism within C. parvum isolates. Forty-one isolates were collected from different geographical origins (Japan, Italy, and Nepal) and hosts (humans, calves, and a goat). These isolates were characterized by means of DNA sequencing, PCR-restriction fragment length polymorphism (PCR-RFLP), and RFLP-single-strand conformational polymorphism (RFLP-SSCP) analyses of the gene for Cpgp40/15. The sequence analysis indicated that there was DNA polymorphism between genotype I and II, as well as within genotype I, isolates. The DNA and amino acid sequence identities between genotypes I and II differed, depending on the isolates, ranging from 73.3 to 82.9% and 62.4 to 80.1%, respectively. Those among genotype I isolates differed, depending on the isolates, ranging from 69.0 to 85.4% and 54.8 to 79.2%, respectively. Because of the high resolution generated by PCR-RFLP and RFLP-SSCP, the isolates of genotype I could be subtyped as genotypes Ia1, Ia2, Ib, and Ie. The isolates of genotype II could be subtyped as genotypes IIa, IIb, and IIc. The isolates from calves, a goat, and one Japanese human were identified as genotype II. Within genotype II, the isolates from Japan were identified as genotype IIa, those from calves in Italy were identified as genotype IIb, and the goat isolate was identified as genotype IIc. All of the genotype I isolates were from humans. The Japanese isolate (code no. HJ3) and all of the Nepalese isolates were identified as genotypes Ia1 and Ia2, respectively. The Italian isolates were identified as genotype Ib, and the Japanese isolate (code no. HJ2) was identified as genotype Ie. Thus, the PCR-RFLP-SSCP analysis of this glycoprotein Cpgp40/15 gene generated a high resolution that has not been achieved by previous methods of genotypic differentiation of C. parvum.     

Detection of polymorphism in the Trypanosoma cruzi TcP2β gene family by single strand conformational analysis (SSCA)

Single strand conformation analysis (SSCA) is a technique that has been used to detect point mutations. We explored its usefulness in the analysis of four different members of the Trypanosoma cruzi TcP2β gene family and its suitability for detection of polymorphism in different parasite strains. The availability of primers covering a 97-bp sequence at the 5′ end of the genes allowed assessment of the effect of a single base substitution, while the analysis of a 321 bp long sequence permitted the evaluation of sequences differing in several bases. PCR products were analysed under four different electrophoretic conditions: with or without the addition of 10% glycerol in a 6% polyacrylamide gel run at room temperature or at 4°C. Shifts in mobility were radically dependent on the migration condition. Both 97-bp and 321-bp amplicons were best resolved at 4°C, without glycerol. Amplification products derived from total genomic DNA showed a pattern that resembled closely a combination of the products derived from the cloned genes. The results herein demonstrate the usefulness of SSCA to differentiate forms of a complex protozoan gene family, and to scan its polymorphic nature. Furthermore, due to the remarkable sensitivity of the technique it can generate genomic markers, such as Sequence Tagged Sites (STS), of great need in the T. cruzi genome project     

Genotypic variation among lineages of Trypanosoma cruzi and its geographic aspects

Isozyme analysis with 18 enzyme loci was conducted on 146 isolates of Trypanosoma cruzi from Mexico, Guatemala, Colombia, Ecuador, Peru, Brazil, Bolivia, Paraguay and Chile. Forty-four different MLGs (groups of isolates with identical multilocus genotypes) were identified and a phylogeny was constructed. The phylogenetic tree consisted of two main groups (T. cruzi I, T. cruzi II), and the latter was further divided into two subgroups (T. cruzi IIa, T. cruzi IIb–e). Evidence of hybridization between different MLGs of T. cruzi II was found, which means that genetic exchanges seem to have occurred in South American T. cruzi. On the other hand, the persistence of characteristic T. cruzi I and T. cruzi II isozyme patterns in single small villages in Bolivia and Guatemala suggested that genetic exchange is very rare between major lineages. A significant difference in genetic diversity was shown between T. cruzi I and T. cruzi II from several indices of population genetics. Two possibilities could explain this genetic variation in the population: differences in evolutionary history and/or different tendencies to exchange genetic material. Broad-scale geographic distributions of T. cruzi I and T. cruzi IIb–e were different; T. cruzi I occurred in Central America and south to Bolivia and Brazil, while T. cruzi IIb–e occurred in the central and southern areas of South America, overlapping with T. cruzi I in Brazil and Bolivia.     

Application of single-strand conformation polymorphism and denaturing gradient gel electrophoresis for fla sequence typing of Campylobacter jejuni

Campylobacter jejuni is a frequent cause of bacterial gastroenteritis in humans all over the world. Several molecular typing methods are used to study the epidemiology of Campylobacter spp. infections. The aim of the present study was to investigate the application of single-strand conformation polymorphism (SSCP) and denaturing gradient gel electrophoresis (DGGE) analysis as rapid primary subtyping methods for C. jejuni. A variable fragment from the 3' end of the flaA to the 3' end of the intergenic region, separating the flaA and flaB genes, was subjected to SSCP and DGGE analysis. A total of 48 clinical C. jejuni isolates, 49 C. jejuni strains isolated from poultry, 2 strains isolated from ducks and 1 strain isolated from a pheasant were assigned to 24 distinct SSCP patterns. Sequence analysis of the respective DNA fragments revealed that every different fla sequence type could be distinguished by SSCP. DGGE proved to be equally discriminatory. Both methods can be applied as primary subtyping methods, because pulsed-field gel electrophoresis (PFGE) and amplified fragment length polymorphism (AFLP) analysis further differentiated isolates belonging to the same fla sequence types.     

A study of the polymorphism of mec dna in methycillin-resistant strains of Staphylococcus aureus isolated at permanent stations in different regions of Russia

Polymorphism of the chromosome staphylococcus cassette mec (SCCmec), a mobile and heterological genetic element providing resistance to beta-lactam antibiotics was studied in methycillin-resistant strains of Staphylococcus aureus (MRSA) isolated at permanent stations situated in different regions of Russia. Type SCCmec was identified using the PCR method by determining allotypes of 3 different structural genetic complexes incorporated in the cassettes mec. It was found that the isolates studied in this work contained 3 different types of SCCmec: I, III, and IVb. Both isolates containing 2 different copies of SCCmec and isolates containing defective copies of SCCmec were identified. It was demonstrated that determination of the SCC-mec type provided an opportunity to differentiate the isolates studied in this work from one another. The isolates attributed to the same genotype variant (identified by polymorphism of coagulase gene) but isolated at different hospitals located in different regions of Russia were found to contain the same type of the chromosome staphylococcus cassette mec, whereas the isolates of different coagulase groups (i.e., different genotype variants) contained different types of SCCmec. It was found that at least 2 epidemic strains circulated in the permanent hospitals of Russia. The strains differ from one another by the polymorphism of the coagulase gene and the mec DNA polymorphism. According to results of studies of several molecular markers (including mec DNA), these strains proved to be identical to the international strains EMRSA-1 and EMRSA-2. Possible mechanisms of MRSA formation and circulation in Russia and CIS countries are discussed.     

Nucleotide polymorphism in chloroplast dna of Nicotiana debneyi

Summary EcoR1 restriction endonuclease analysis of chloroplast DNA isolated from several distinct populations of Nicotiana debneyi has revealed a naturally occurring polymorphism. The chloroplast DNA of seven of the nine populations analysed possessed an additional EcoRl site. The origin of the additional restriction endonuclease fragments was confirmed by hybridisation of [³² P]-cRNA to fractionated EcoRl restricted chloroplast-DNA fragments adsorbed to nitrocellulose filters. Reciprocal f₁ hybrids between plants carrying the variant chloroplast-DNA's confirmed maternal inheritance of chloroplast-DNA.Communicated by G. Melchers and D. von Wettstein     

Differentiation of Coxiella burnetii isolates by sequence determination and PCR-restriction fragment length polymorphism analysis of isocitrate dehydrogenase gene

The isocitrate dehydrogenase (icd) gene of Coxiella burnetii was cloned and sequenced to differentiate between isolates with various geographic origins and phenotypic properties. Based on the gene sequences all 19 isolates studied could be divided into three groups. Group 1 contained isolates originating from acute cases of Q fever, ticks and cows. Groups 2 and 3 included isolates from chronic Q fever patients and a prototype strain from an aborted goat. Although the icd gene profiles were different among isolates of the latter two groups, there were two base differences common for both groups which could be used as markers to distinguish them from group 1 isolates. Based on one of the markers a simple method using PCR-restriction fragment length polymorphism analysis was developed for rapid differentiation of C. burnetii isolates as well as for direct detection and differentiation of the bacterium in human serum samples. Taken together, the study results suggest that the icd-based differentiation method may be useful in clinical investigation of Coxiella infections.     

Application of kDNA as a molecular marker to analyse Leishmania infantum diversity in Portugal

Around the Mediterranean basin Leishmania infantum is an important parasite causing canine leishmaniasis and visceral and cutaneous clinical forms in both immunocompetent and immunocompromised humans. Efficient monitoring and evaluation of epidemiology with discriminatory molecular markers are required. We investigated the genetic diversity of L. infantum in Portugal by polymerase chain amplification and restriction fragment length polymorphism analysis of kinetoplastid DNA, as molecular marker. We analysed 120 Portuguese isolates of L. infantum plus 16 other non-Portuguese isolates (as a reference group) from humans, dogs and sand flies. The Portuguese population showed a high degree of polymorphism with a total of 13 profiles identified. The predominant profile was A, which was only detected in the Portuguese samples.

The kinetoplastid DNA PCR-RFLP assay described here was suitable for use directly with biological samples and the profiles obtained were stable during long-term growth in vitro and in laboratory animals.     

Cloning and sequence analysis of the lipase and lipase chaperone-encoding genes from Acinetobacter calcoaceticus RAG-1, and redefinition of a Proteobacterial lipase family and an analogous lipase chaperone family

The genes encoding the lipase (LipA) and lipase chaperone (LipB) from Acinetobacter calcoaceticus RAG-1 were cloned and sequenced. The genes were isolated from a genomic DNA library by complementation of a lipase-deficient transposon mutant of the same strain. Transposon insertion in this mutant and three others was mapped to a single site in the chaperone gene. The deduced amino acid (aa) sequences for the lipase and its chaperone were found to encode mature proteins of 313 aa (32.5 kDa) and 347 aa (38.6 kDa), respectively. The lipase contained a putative leader sequence, as well as the conserved Ser, His, and Asp residues which are known to function as the catalytic triad in other lipases. A possible trans-membrane hydrophobic helix was identified in the N-terminal region of the chaperone. Phylogenetic comparisons showed that LipA, together with the lipases of A. calcoaceticus BD413, Vibrio cholerae El Tor, and Proteus vulgaris K80, were members of a previously described family of Pseudomonas and Burkholderia lipases. This new family, which we redefine as the Group I Proteobacterial lipases, was subdivided into four subfamilies on the basis of overall sequence homology and conservation of residues which are unique to the subfamilies. LipB, moreover, was found to be a member of an analogous family of lipase chaperones. We propose that the lipases produced by P. fluorescens and Serratia marcescens, which comprise a second sequence family, be referred to as the Group II Proteobacterial lipases. Evidence is provided to support the hypothesis that both the Group I and Group II families have evolved from a combination of common descent and lateral gene transfer.     

Detection of point mutations in the chloroplast genome by single-stranded conformation polymorphism analysis

Single-stranded conformation polymorphism (SSCP) analysis was used to examine the mutations of the chloroplast 16S rRNA locus of streptomycin-resistant mutants in Nicotiana plumbaginifolia. DNA fragments of 121, 517, 968 and 1578 bp, each possessing a known point mutation, were generated by polymerase chain reaction (PCR). The resulting fragments were denatured and separated by nondenaturing polyacrylamide gel electrophoresis. Compared to the patterns of the wild-type DNA fragments, the bands of the single-stranded DNA fragments of 121 and 517 bp with base changes were shifted. However, no pattern variations were detected from the DNA fragments of 968 and 1578 bp generated from both wild-type and mutants.     

Correlation between conformation and antibody binding: NMR structure of cross-reactive peptides from T. cruzi, human and L. braziliensis

The structure of peptides corresponding to the C-terminal residues from Trypanosoma cruzi (R13), human (H13) and Leishmania braziliensis (A13) ribosomal proteins were determined using nuclear magnetic resonance. Although there is only one amino acid difference between them, the peptides present distinct structures in solution: R13 adopts a random coil conformation while H13 and A13 form a bend. Interaction of these peptides with polyclonal antibodies from chronic Chagas’ disease patients and a monoclonal antibody raised against T. cruzi ribosomal P2β protein was probed by transferred NOE. The results show that the flexibility of R13 is fundamental for the binding to the antibody.     

Replication origin of the Bacillus subtilis chromosome determined by hybridization of the first-replicating DNA with cloned fragments from the replication origin region of the chromosome

The replication origin (ori) on the Bacillus subtilis genome was determined by the hybridization between the first-replicating DNA region and the cloned fragments from the ori region. The first-replicating DNA region was labeled specifically by [³H]thymidine in the presence of an inhibitor for DNA polymerase during a synchronous initiation of the chromosomal replication by germinating spores starved for thymine, and isolated by a sucrose density gradient centrifugation. Most of the labeled DNA molecules are small in size (up to 1000 bases long). The 45-kb ori region was cloned first in a λ Charon vector and then subcloned in pBR vectors. Restriction fragments from these cloned DNAs were purified by electrophoresis in agarose gels.

Only one region within the 45-kb ori region shows strong hybridization with the first-replicating DNA. Restriction fragments from this region were cloned in a phage M13 vector and separated into complementary strands. Hybridization of the labeled DNA with these cloned single-stranded fragments revealed that one site of the ori is located in each strand and they are some 2-kb apart from each other. Replication starts from these sites and proceeds inwards to pass each other.     

Structure and expression of three gp82 gene subfamilies of Trypanosoma cruzi

The glycoprotein gp82 is a GPI-anchored cell surface protein of Trypanosoma cruzi and is involved in cell invasion. Gp82 is encoded by multiple genes. To investigate the genetic basis of its biological function, we analyzed structure and expression of gp82 multigene family members in the Peruvian and Guatemalan strains. Three major groups of gp82 genes (A, B and C) were categorized by analyzing multiple DNA clones from the genomic PCR products. Within each group, 95–97% homology was observed, whereas between the groups, homology was 67–79%. The copy numbers of groups A, B and C as determined by real-time PCR were 18, 8 and 7 copies, respectively, in the Peru-2 strain. Significant elevation of the mRNA expression levels (5–10 times more) of all the subfamily genes was observed in the metacyclic stage compared with the epimastigote stage. When we focused on the binding motif sequence reported previously, we found substantial difference between that of A and C. However, the peptide inhibition invasion assay showed no functional difference. Taken together, we demonstrated that three subfamilies of gp82 were in the genome of T. cruzi and maintained their functional structure, and that the mRNA expressions of those genes were equally controlled in a stage-specific manner.     

Isolation of a yeast single-strand deoxyribonucleic acid binding protein that specifically stimulates yeast DNA polymerase I

We sought a protein from yeast that would bind more strongly to single-stranded DNA than to duplex DNA and would stimulate the activity of the major yeast DNA polymerase, but not polymerases from other organisms. We isolated a protein that binds about 200 times more strongly to single-stranded DNA than duplex DNA and stimulates yeast DNA polymerase I activity 4-5-fold. It inhibits synthesis catalyzed by calf thymus DNA polymerase alpha and has little effect on T4 DNA polymerase. This yeast protein, SSB-1, has a molecular weight of approximately 40 000. At apparent saturation there is one protein molecule bound per 40 nucleotides. Protein binding causes the single-stranded DNA molecule to assume a relatively extended conformation. It binds to single-stranded RNA as strongly as to DNA. SSB-1 increases the initial rate of polymerization catalyzed by yeast DNA polymerase I apparently by increasing the processivity of the enzyme. We estimate there are 7500-30 000 molecules of SSB-1 per yeast cell, enough to bind at least 400-1600 nucleotides per replication fork. Thus it is present in sufficient abundance to participate in DNA replication in vivo in the manner suggested by these in vitro experiments.     

<i>Acetobacter okinawensis</i> sp. nov., <i>Acetobacter papayae</i> sp. nov., and <i>Acetobacter persicus</i> sp. nov.; novel acetic acid bacteria isolated from stems of sugarcane, fruits, and a flower in Japan

Eleven strains of acetic acid bacteria were isolated from stems of sugarcane, fruits, and a flower in Japan. The isolates were separated into three groups, Groups I, II, and III, in the genus Acetobacter according to phylogenetic analysis based on 16S rRNA sequences. The isolates had sequence similarities of 99.8-100% within the Group, 99.3-99.6% to those of the type strains of each related Acetobacter species, and less than 98.4% to those of the type strains of other Acetobacter species. Genomic DNA G+C contents of Groups I, II, and III were 59.2-59.4, 60.5-60.7, and 58.7-58.9 mol%, respectively. The isolates in the Group showed high values of DNA-DNA relatedness to each other, but low values less than 46% to the type strains of related Acetobacter species. A good correlation was found between the three Groups and groups based on DNA G+C contents and DNA-DNA relatedness. All the strains had Q-9 as the main component, and Q-8 and Q-10 as minor components. The isolates in the three Groups did not completely match with any Acetobacter species on catalase reaction, the production of ketogluconic acids from D-glucose, growth on ammoniac nitrogen with ethanol (Hoyer-Frateur medium and Frateur modified Hoyer medium), growth on 30% (w/v) D-glucose, growth in 10% (v/v) ethanol, or DNA G+C contents. On the basis of phylogenetic relationships in the genus Acetobacter and chemosystematic and phenotypic characteristics, the three Groups were regarded as novel species in the genus Acetobacter. Acetobacter okinawensis sp. nov. is proposed for Group I, Acetobacter papayae sp. nov. for Group II, and Acetobacter persicus sp. nov. for Group III.     

Smoking-reIated DNA adducts and genetic polymorphism for metabolic enzymes in human lymphocytes

Smoking-related aromatic DNA adducts in lymphocytes were measured from smokers (n = 76), ex-smokers (n = 25) and non-smokers (n = 56) by the ³²P-postlabelling method, to clarify whether a genetic polymorphism for metabolic enzymes could explain the inter-individual variation of DNA adduct levels. Adduct levels were compared with respect to smoking status and polymorphic genotypes of cytochrome P4501A1 (CYP1A1) and glutathione S-transferase M1 (GTSM1). The mean adduct level (1.24 per 10⁸ nucleotides) in smokers was significantly higher than that (0.85 per 10⁸) in non-smokers. Although we expected higher adduct levels in the CYP1A1 variant or GSTM1 null subjects, the adduct level in 'GSN1 nulls' was significantly lower than that in 'GSTM1 presents' among smokers. DNA adduct levels had significant positive correlations with smoking indices such as number of cigarettes or smoking years in all subjects. In smokers only, however, no correlation was found, because there were negative correlations between adduct levels and smoking dose in GSTM1 null genotypes. CYP1A1 genotypes had no effects on adduct levels.     

ITS/CHS1靶位序列分析结合SRAP分子标记技术鉴定Nannizzia incurvata临床分离株

以转录间隔区（internal transcribed spacer,ITS）和几丁质合成酶1（chitinase synthase 1,CHS1）为鉴定靶位,结合种系进化分析将2007年至2016年分离自头癣或面癣患儿的12株石膏样小孢子菌复合体成员（现被归入Nannizzia菌属）鉴定到种水平,其中5株为Nannizzia gypsea（Microsporum gypseum）,另7株为Nannizzia incurvata（Microsporum incurvatum）。在此基础上,通过序列相关扩增多态性（SRAP）分子标记进一步证实二者在DNA多位点存在扩增多态性。本研究证实在我国湖北地区存在Nannizzia incurvata,为我国开展Nannizzia菌属的分子流行病学研究提供了实验室依据和支持。