Mycobacteria mobility shift assay: a method for the rapid identification of Mycobacterium tuberculosis and nontuberculous mycobacteria

The identification of mycobacteria is essential because tuberculosis (TB) and mycobacteriosis are clinically indistinguishable and require different therapeutic regimens. The traditional phenotypic method is time consuming and may last up to 60 days. Indeed, rapid, affordable, specific and easy-to-perform identification methods are needed. We have previously described a polymerase chain reaction-based method called a mycobacteria mobility shift assay (MMSA) that was designed for Mycobacterium tuberculosis complex (MTC) and nontuberculous mycobacteria (NTM) species identification. The aim of this study was to assess the MMSA for the identification of MTC and NTM clinical isolates and to compare its performance with that of the PRA-hsp65 method. A total of 204 clinical isolates (102 NTM and 102 MTC) were identified by the MMSA and PRA-hsp65. For isolates for which these methods gave discordant results, definitive species identification was obtained by sequencing fragments of the 16S rRNA and hsp65 genes. Both methods correctly identified all MTC isolates. Among the NTM isolates, the MMSA alone assigned 94 (92.2%) to a complex or species, whereas the PRA-hsp65 method assigned 100% to a species. A 91.5% agreement was observed for the 94 NTM isolates identified by both methods. The MMSA provided correct identification for 96.8% of the NTM isolates compared with 94.7% for PRA-hsp65. The MMSA is a suitable auxiliary method for routine use for the rapid identification of mycobacteria.     

Clustering cancer gene expression data: a comparative study

Background  

The use of clustering methods for the discovery of cancer subtypes has drawn a great deal of attention in the scientific community. While bioinformaticians have proposed new clustering methods that take advantage of characteristics of the gene expression data, the medical community has a preference for using "classic" clustering methods. There have been no studies thus far performing a large-scale evaluation of different clustering methods in this context.     

Role of AREA, a regulator of nitrogen metabolism, during colonization of maize kernels and fumonisin biosynthesis in Fusarium verticillioides

Fumonisin B1 (FB(1)) biosynthesis is repressed in cultures containing ammonium as the nitrogen source and when grown on blister kernels, the earliest stages of kernel development. In this study AREA, a regulator of nitrogen metabolism, was disrupted in Fusarium verticilliodes. The mutant (DeltaareA) grew poorly on mature maize kernels, but grew similar to wild type (WT) with the addition of ammonium phosphate. FB(1) was not produced by DeltaareA under any condition or by the WT with added ammonium phosphate. Constitutive expression of AREA (strain AREA-CE) rescued the growth and FB(1) defects in DeltaareA. Growth of WT, DeltaareA, and AREA-CE on blister-stage kernels was similar. After 7 days of growth, none of the strains produced FB(1) and the pH of the kernel tissues was 8.0. Addition of amylopectin to the blister kernels resulted in a pH near 6.6 and FB(1) production by WT and AREA-CE. The results support the hypothesis that FB(1) biosynthesis is regulated by AREA. Also the failure to produce FB(1) in blister kernels is due to high pH conditions generated because of an unfavorable carbon/nitrogen environment.     

Cloning of the Aspergillus parasiticus apa-2 gene associated with the regulation of aflatoxin biosynthesis.

An Aspergillus parasiticus gene, designated apa-2, was identified as a regulatory gene associated with aflatoxin biosynthesis. The apa-2 gene was cloned on the basis of overproduction of pathway intermediates following transformation of fungal strains with cosmid DNA containing the aflatoxin biosynthetic genes nor-1 and ver-1. Transformation of an O-methylsterigmatocystin-accumulating strain, A. parasiticus SRRC 2043, with a 5.5-kb HindIII-XbaI DNA fragment containing apa-2 resulted in overproduction of all aflatoxin pathway intermediates analyzed. Specific enzyme activities associated with the conversion of norsolorinic acid and sterigmatocystin were increased approximately twofold. The apa-2 gene was found to complement an A. flavus afl-2 mutant strain for aflatoxin production, suggesting that apa-2 is functionally homologous to afl-2. Comparison of the A. parasiticus apa-2 gene DNA sequence with that of the A. flavus afl-2 gene (G. A. Payne, G. J. Nystorm, D. Bhatnagar, T. E. Cleveland, and C. P. Woloshuk, Appl. Environ. Microbiol. 59:156-162, 1993) showed that they shared > 95% DNA homology. Physical mapping of cosmid subclones placed apa-2 approximately 8 kb from ver-1.     

Cloning of the afl-2 gene involved in aflatoxin biosynthesis from Aspergillus flavus.

Aflatoxins are extremely potent carcinogens produced by Aspergillus flavus and Aspergillus parasiticus. Cloning of genes in the aflatoxin pathway provides a specific approach to understanding the regulation of aflatoxin biosynthesis and, subsequently, to the control of aflatoxin contamination of food and feed. This paper reports the isolation of a gene involved in aflatoxin biosynthesis by complementation of an aflatoxin-nonproducing mutant with a wild-type genomic cosmid library of A. flavus. Strain 650-33, blocked in aflatoxin biosynthesis at the afl-2 allele, was complemented by a 32-kb cosmid clone (B9), resulting in the production of aflatoxin. The onset and profile of aflatoxin accumulation was similar for the transformed strain and the wild-type strain (NRRL 3357) of the fungus, indicating that the integrated gene is under the same control as in wild-type strains. Complementation analyses with DNA fragments from B9 indicated that the gene resides within a 2.2-kb fragment. Because this gene complements the mutated afl-2 allele, it was designated afl-2. Genetic evidence obtained from a double mutant showed that afl-2 is involved in aflatoxin biosynthesis before the formation of norsolorinic acid, the first stable intermediate identified in the pathway. Further, metabolite feeding studies with the mutant, transformed, and wild-type cultures and enzymatic activity measurements in cell extracts of these cultures suggest that afl-2 regulates gene expression or the activity of other aflatoxin pathway enzymes. This is the first reported isolation of a gene for aflatoxin biosynthesis in A. flavus.     

Microsporidia, amitochondrial protists, possess a 70-kDa heat shock protein gene of mitochondrial evolutionary origin

An intronless gene encoding a protein of 592 amino acid residues with similarity to 70-kDa heat shock proteins (HSP70s) has been cloned and sequenced from the amitochondrial protist Encephalitozoon cuniculi (phylum Microsporidia). Southern blot analyses show the presence of a single gene copy located on chromosome XI. The encoded protein exhibits an N-terminal hydrophobic leader sequence and two motifs shared by proteobacterial and mitochondrially expressed HSP70 homologs. Phylogenetic analysis using maximum likelihood and evolutionary distances place the E. cuniculi sequence in the cluster of mitochondrially expressed HSP70s, with a higher evolutionary rate than those of homologous sequences. Similar results were obtained after cloning a fragment of the homologous gene in the closely related species E. hellem. The presence of a nuclear targeting signal-like sequence supports a role of the Encephalitozoon HSP70 as a molecular chaperone of nuclear proteins. No evidence for cytosolic or endoplasmic reticulum forms of HSP70 was obtained through PCR amplification. These data suggest that Encephalitozoon species have evolved from an ancestor bearing mitochondria, which is in disagreement with the postulated presymbiotic origin of Microsporidia. The specific role and intracellular localization of the mitochondrial HSP70-like protein remain to be elucidated.      

Identification of aflatoxin biosynthesis genes by genetic complementation in an Aspergillus flavus mutant lacking the aflatoxin gene cluster.

Aspergillus flavus mutant strain 649, which has a genomic DNA deletion of at least 120 kb covering the aflatoxin biosynthesis cluster, was transformed with a series of overlapping cosmids that contained DNA harboring the cluster of genes. The mutant phenotype of strain 649 was rescued by transformation with a combination of cosmid clones 5E6, 8B9, and 13B9, indicating that the cluster of genes involved in aflatoxin biosynthesis resides in the 90 kb of A. flavus genomic DNA carried by these clones. Transformants 5E6 and 20B11 and transformants 5E6 and 8B9 accumulated intermediate metabolites of the aflatoxin pathway, which were identified as averufanin and/or averufin, respectively.These data suggest that avf1, which is involved in the conversion of averufin to versiconal hemiacetal acetate, was present in the cosmid 13B9. Deletion analysis of 13B9 located the gene on a 7-kb DNA fragment of the cosmid. Transformants containing cosmid 8B9 converted exogenously supplied O-methylsterigmatocystin to aflatoxin, indicating that the oxidoreductase gene (ord1), which mediates the conversion of O-methylsterigmatocystin to aflatoxin, is carried by this cosmid. The analysis of transformants containing deletions of 8B9 led to the localization of ord1 on a 3.3-kb A. flavus genomic DNA fragment of the cosmid.