RESUMO
Salmonella isolates have traditionally been classified by serotyping, the serologic identification of two surface antigens, O-polysaccharide and flagellin protein. Serotyping has been of great value in understanding the epidemiology of Salmonella and investigating disease outbreaks; however, production and quality control of the hundreds of antisera required for serotyping is difficult and time-consuming. To circumvent the problems associated with antiserum production, we began the development of a system for determination of serotype in Salmonella based on DNA markers. To identify flagellar antigen-specific sequences, we sequenced 280 alleles of the three genes that are known to encode flagellin in Salmonella, fliC, fljB, and flpA, representing 67 flagellar antigen types. Analysis of the data indicated that the sequences from fliC, fljB, and flpA clustered by the antigen(s) they encode not by locus. The sequences grouped into four clusters based on their conserved regions. Three of the four clusters included multiple flagellar antigen types and were designated the G complex, the Z4 complex, and the alpha cluster. The fourth cluster contained a single antigen type, H:z(29). The amino acid sequences of the conserved regions within each cluster have greater than 95% amino acid identity, whereas the conserved regions differ substantially between clusters (75 to 85% identity). Substantial sequence heterogeneity existed between alleles encoding different flagellar antigens while alleles encoding the same flagellar antigen were homologous, suggesting that flagellin genes may be useful targets for the molecular determination of flagellar antigen type.
Assuntos
Flagelina/genética , Genes Bacterianos , Salmonella/genética , Alelos , Sequência de Aminoácidos , Antígenos de Bactérias/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/imunologia , Sequência de Bases , Sequência Conservada , Primers do DNA/genética , DNA Bacteriano/genética , Flagelina/imunologia , Humanos , Dados de Sequência Molecular , Família Multigênica , Salmonella/classificação , Salmonella/imunologia , Homologia de Sequência de Aminoácidos , Homologia de Sequência do Ácido Nucleico , Sorotipagem , Fatores de Transcrição/genética , Fatores de Transcrição/imunologiaRESUMO
Several reports indicate geographic variation of isolates of Pneumocystis carinii hominis. We have sequenced the internal transcribed spacer (ITS) region and large subunit Group I intron of rRNA genes from P. carinii DNA obtained from two patients from Puerto Rico. Both can be subclassified as Type II, according to the sequence of the ITS region. A system capable of identifying individual isolates will be an essential tool for epidemiological studies of the organism. The amplification of DNA from fixed tissues may facilitate the processing of a large number of samples.
Assuntos
Pneumocystis/genética , Óperon de RNAr/genética , Sequência de Bases , Humanos , Dados de Sequência Molecular , Porto RicoRESUMO
Two distinct sequevars, denoted Pc1 and Pc2, of the opportunistic pathogen Pneumocystis carinii have been previously identified based on the sequence of their 26S rRNA genes, the location of group I self-splicing introns and pulsed field electrophoretic patterns of chromosomal DNA. This study shows that the sequences of 16S and 5.8S rRNA genes also vary between these sequevars, and that greater variation was seen in the internal transcribed spacer regions. Polymerase chain reaction and restriction analysis can distinguish between these sequevars.
Assuntos
DNA Ribossômico/genética , Genes Fúngicos/genética , Variação Genética/genética , Pneumocystis/genética , RNA Ribossômico 16S/genética , RNA Ribossômico 5,8S/genética , Animais , Sequência de Bases , Clonagem Molecular , DNA Fúngico/genética , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , Polimorfismo de Fragmento de Restrição , Ratos , Ratos Sprague-Dawley , Análise de Sequência de DNA , Homologia de Sequência do Ácido NucleicoRESUMO
Small portions of the 18S and the 26S rRNA genes, the entire 5.8S rRNA gene, and internal transcribed spacers ITS1 and ITS2 (located between the 18S and 5.8S rRNA genes and between the 5.8S and 26S rRNA genes, respectively) of Pneumocystis carinii that infect humans were cloned and sequenced. The nucleotide sequences of the 18S, 5.8S, and 26S rRNA genes determined in the study were approximately 90% homologous to those of P. carinii that infect rats, while the sequences of ITS1 and ITS2 of P. carinii from the two different hosts were only 60% homologous. The 18S, 5.8S, and 26S rRNA gene sequences of P. carinii from 15 patient specimens were determined and were found to be identical to each other, whereas the ITS sequences were found to be variable. With the observed sequence variation, it was possible to classify the ITS1 sequences into two types and the ITS2 sequences into three types. P. carinii strains that had the same type of ITS1 sequence could have a different type of ITS2 sequence. On the basis of the sequence types of the two ITS regions, P. carinii from the 15 patients were classified into four groups. P. carinii from three patient specimens were found to contain two different ITS sequence patterns. More surprisingly, one additional specimen was found to have one ITS sequence typical of P. carinii isolates that infect humans and another typical of P. carinii isolates that infect rats. The studies indicate that it is possible to type P. carinii strains on the basis from one patient, suggesting that coinfection with more than one strain of P. carinii may occur in the same patient.
