RESUMO
AIMS: We assessed the veracity of intergenic spacer region 1 (ITS1) ribotyping for the rapid, inexpensive and accurate identification of Brenneria goodwinii and Gibbsiella quercinecans that are associated with acute oak decline (AOD) in the UK. METHODS AND RESULTS: Agarose gel electrophoresis and polyacrylamide gel electrophoresis (PAGE) were applied for the typing of ITS1 PCR amplicons from strains of B. goodwinii, G. quercinecans and related species (n = 34). The number and length of ITS1 amplicons varied significantly between strains. ITS1 profiles generated via PAGE were used to differentiate species using a neighbour-joining phylogram. The ITS1 phylogram was compared against DNA gyrase B (gyrB) gene sequences from the same strains, demonstrating that ITS1 ribotyping is as effective as gyrB at resolving G. quercinecans and B. goodwinii to the species level. CONCLUSIONS: The ITS1 gene has been successfully employed as a novel marker to resolve newly described AOD-associated Enterobacteriaceae, B. goodwinii and G. quercinecans, to species level. SIGNIFICANCE AND IMPACT OF THE STUDY: ITS1 ribotyping of B. goodwinii and G. quercinecans provides equivalent sensitivity to the current standard method for strain identification (sequence analysis of the gyrB gene), but with reduced processing time and cost. Furthermore, the ITS1 gene is widely applicable as a rapid and inexpensive typing system for Enterobacteriaceae.
Assuntos
DNA Espaçador Ribossômico/química , Enterobacteriaceae/classificação , Enterobacteriaceae/isolamento & purificação , Quercus/microbiologia , Ribotipagem/métodos , DNA Girase/genética , Eletroforese em Gel de Ágar , Eletroforese em Gel de Poliacrilamida , Enterobacteriaceae/genética , Marcadores Genéticos , Dados de Sequência Molecular , Doenças das Plantas/microbiologia , Reação em Cadeia da PolimeraseRESUMO
The social amoebae are exceptional in their ability to alternate between unicellular and multicellular forms. Here we describe the genome of the best-studied member of this group, Dictyostelium discoideum. The gene-dense chromosomes of this organism encode approximately 12,500 predicted proteins, a high proportion of which have long, repetitive amino acid tracts. There are many genes for polyketide synthases and ABC transporters, suggesting an extensive secondary metabolism for producing and exporting small molecules. The genome is rich in complex repeats, one class of which is clustered and may serve as centromeres. Partial copies of the extrachromosomal ribosomal DNA (rDNA) element are found at the ends of each chromosome, suggesting a novel telomere structure and the use of a common mechanism to maintain both the rDNA and chromosomal termini. A proteome-based phylogeny shows that the amoebozoa diverged from the animal-fungal lineage after the plant-animal split, but Dictyostelium seems to have retained more of the diversity of the ancestral genome than have plants, animals or fungi.
Assuntos
Dictyostelium/genética , Genoma , Genômica , Comportamento Social , Transportadores de Cassetes de Ligação de ATP/genética , Animais , Composição de Bases , Adesão Celular/genética , Movimento Celular/genética , Centrômero/genética , Sequência Conservada/genética , Elementos de DNA Transponíveis/genética , DNA Ribossômico/genética , Dictyostelium/citologia , Dictyostelium/enzimologia , Dictyostelium/metabolismo , Células Eucarióticas/metabolismo , Duplicação Gênica , Transferência Genética Horizontal/genética , Humanos , Dados de Sequência Molecular , Filogenia , Proteoma , Proteínas de Protozoários/química , Proteínas de Protozoários/genética , RNA de Transferência/genética , Sequências Repetitivas de Ácido Nucleico/genética , Análise de Sequência de DNA , Transdução de Sinais/genética , Telômero/genéticaRESUMO
The gene coding for merozoite surface protein 7 has been identified and sequenced in three lines of Plasmodium falciparum. The gene encodes a 351 amino acid polypeptide that is the precursor of a 22-kDa protein (MSP7(22)) on the merozoite surface and non-covalently associated with merozoite surface protein 1 (MSP1) complex shed from the surface at erythrocyte invasion. A second 19-kDa component of the complex (MSP7(19)) was shown to be derived from MSP7(22) and the complete primary structure of this polypeptide was confirmed by mass spectrometry. The protein sequence contains several predicted helical and two beta elements, but has no similarity with sequences outside the Plasmodium databases. Four sites of sequence variation were identified in MSP7, all within the MSP7(22) region. The MSP7 gene is expressed in mature schizonts, at the same time as other merozoite surface protein genes. It is proposed that MSP7(22) is the result of cleavage by a protease that may also cleave MSP1 and MSP6. A related gene was identified and cloned from the rodent malaria parasite, Plasmodium yoelii YM; at the amino acid level this sequence was 23% identical and 50% similar to that of P. falciparum MSP7.
Assuntos
Proteínas de Membrana , Plasmodium falciparum/crescimento & desenvolvimento , Precursores de Proteínas/química , Precursores de Proteínas/metabolismo , Proteínas de Protozoários/química , Proteínas de Protozoários/genética , Sequência de Aminoácidos , Animais , Sequência Conservada , Proteína 1 de Superfície de Merozoito/química , Dados de Sequência Molecular , Plasmodium falciparum/metabolismo , Precursores de Proteínas/genética , Proteínas de Protozoários/metabolismo , Análise de Sequência de DNARESUMO
The gene coding for myristoyl-CoA:protein N-myristoyltransferase (NMT) has been cloned from the malaria parasite Plasmodium falciparum. The gene appears to be single copy and mRNA is expressed in asexual blood-stage forms. Comparison of cDNA and genomic sequences identified three small introns. The open reading frame codes for a 410-amino-acid protein and no evidence of forms with an extended N-terminal coding sequence was obtained. Residues important in substrate binding and in the catalytic mechanism in other species are conserved. The protein was expressed from a plasmid in Escherichia coli, partially purified and shown to have enzymic activity using a synthetic peptide substrate. Comparison of the malaria parasite protein with that derived from the human gene showed a different pattern of inhibition by chemical modification. Human NMT activity was inhibited by diethylpyrocarbonate and partially inhibited by iodacetamide, whereas P. falciparum NMT activity was not inhibited by either pre-treatment. Since the enzyme in infectious fungi is a target for potential chemotherapeutic drugs, it should also be investigated in the context of parasitic infections such as that responsible for malaria.