Detection and transcription of toxin DNA in a nontoxigenic strain of Clostridium difficile.

Genomic DNA from three Clostridium difficile strains was analyzed by PCR for DNA sequences encoding toxin A (tcdA) and toxin B (tcdB). Toxigenic control strain VPI 10463 possessed tcdA, tcdB, and an open reading frame (tcdE) between these two genes, whereas nontoxigenic control strain 85 lacked each of these genetic determinants. However, strain M90, also a nontoxigenic strain, was found to possess tcdA, tcdB, and tcdE. Normally the presence of toxin genes is associated with toxigenicity. Analysis of tcdA and tcdB mRNA revealed toxin gene transcription in strains VPI 10463, 23 (a mildly toxigenic strain), and M90, but not in strain 85. However, for strain M90, tcdA and tcdB mRNA was at the lower limit of detection, whereas mRNAs encoding tcdA and tcdB were easily detected in strains VPI 10463 and 23. Low levels of toxin gene transcription is the probable cause of M90's lack of toxigenicity.

Genetic variation in two cultures of Bradyrhizobium japonicum 110 differing in their ability to impart drought tolerance to soybean.

The polymerase chain reaction with arbitrary primers (RAPD) discriminated between two separately maintained cultures of Bradyrhizobium japonicum USDA 110 differing in symbiotic performance under drought conditions. Since strain 110 is used in inoculum production, the use of RAPD to monitor inoculum cultures could help to preserve their genetic composition and prevent the loss of important symbiotic properties. The use of RAPD could also be extended to other B. japonicum strains currently used in inoculum production.

Typing of toxic strains of Clostridium difficile using DNA fingerprints generated with arbitrary polymerase chain reaction primers.

Clostridium difficile is the causative agent for pseudomembranous colitis in humans. Toxic strains of C. difficile produce two toxins, toxin A and toxin B. A reliable and definitive method of typing the toxic strains of C. difficile is needed since nosocomial cross infection is a primary concern in hospitals and other health care facilities. A method for typing toxic strains of Clostridium difficile using arbitrary polymerase chain reaction (PCR) primers is presented in this study. The C. difficile strains were initially characterized for the toxin A genetic determinant using specific PCR primers which differentiate toxin positive from toxin negative strains. These toxic strains were then PCR typed using six arbitrary primers which generated DNA patterns that were unique for all toxic strains examined. The use of this typing scheme in clinical applications is discussed.

Simultaneous detection of toxin A and toxin B genetic determinants of Clostridium difficile using the multiplex polymerase chain reaction.

A multiplex polymerase chain reaction was developed to simultaneously detect the presence of toxin A and toxin B genes of Clostridium difficile. A 1050-bp fragment of the toxin B gene and a 1217-bp fragment of the toxin A gene were amplified from 42 toxic strains of C. difficile; however, from 10 nontoxic strains the toxin gene fragments were not amplified; these data demonstrate that this multiplex polymerase chain reaction procedure can be used to differentiate between toxic and nontoxic strains. This sensitive and specific multiplex polymerase chain reaction for C. difficile toxins may prove to be a valuable diagnostic procedure.

Molecular screening of Clostridium difficile toxins A and B genetic determinants and identification of mutant strains.

Three separate sets of polymerase chain reaction primers were designed to specifically detect the presence of a toxin A gene fragment, a toxin B gene fragment, and the entire toxin B gene. In addition toxin gene fragments that were amplified from well characterized toxic strains were tagged fluorescently and used as hybridization probes to screen C. difficile strains. A survey of 37 toxic strains and 10 non-toxic strains demonstrated that toxic strains normally contain the genetic composition for toxin A and toxin B simultaneously; whereas, non-toxic strains typically did not contain detectable toxin determinants. The only exception found was strain 39, which had the genetic composition for toxins A and B, but was not cytotoxic under the conditions tested.

Association of an isozyme locus and strawbreaker foot rot resistance derived from Aegilops ventricosa in wheat.

Thirty lines from a cross between VPM/ Moisson 421 and Selection 101 were used in the study to determine whether strawbreaker foot rot resistance derived from Aegilops ventricosa was associated with an allele for endopeptidase. The progeny examined for foot rot lesions represented F2 derived F5 lines and enzyme assays were done on F6 seedlings. The results indicate that the wheat and 'VPM/Moisson 421' endopeptidase alleles are distinctly different. The endopeptidase allele frequencies of 30 lines were compared with strawbreaker foot rot resistance as measured by the lesion severity index. The results demonstrate a close association between the gene for strawbreaker foot rot resistance and the endopeptidase allele derived from Ae. ventricosa.

Genetic regulation of the catalase developmental program in maize scutellum: Identification of a temporal regulatory gene.

Genetic and biochemical analyses suggest that the developmental program of catalase (H(2)O(2):H(2)O(2) oxidoreductase, EC 1.11.1.6) activity in maize scutella is controlled by a temporal regulatory gene (Car1) that is distinct from the structural genes thus far identified. Recombination data show that Car1 is located about 37 map units from the Cat2 structural gene on the chromosome 1S. Turnover studies indicate that Car1 may act by regulating the rate of catalase synthesis.

Duplicated cytosolic malate dehydrogenase genes in Zea mays.

Six inbred lines of Zea mays expressing different soluble (cytosolic) malate dehydrogenase (sMDH) zymogram phenotypes were analyzed genetically. sMDH was found to be coded for by unlinked duplicated loci in four of these inbred lines. The remaining two lines were found not to possess these duplicated loci. Furthermore, the duplicated loci, sMdh1 and sMdh2, have been found to be located on different chromosomes: sMdh1 on chromosome 1L linked to Amp1, and sMdh2 on chromosome 5S linked to Cat1 and Amp3. The importance of finding sMDH encoded by duplicated loci is discussed in relation to the role of chromosomal rearrangements, the relationship between the cytoplasmic and mitochondrial enzymes, and the evolution of Z. mays.

Chromosomal location of the catalase structural genes in Zea mays using B-A translocations.

B-A translocations have been used to map the catalase genes, Cat1, Cat2, and Cat3 of Zea mays. Cat1 was found to be on the short arm of chromosome 5, 9.1 map units from brittle endosperm (bt 1). Cat2 was located on chromosome 1S, while Cat3 was located on the distal half of chromosome 1L. There was no linkage between Cat2 and Cat3. The significance of mapping the catalase structural genes is discussed.

Chromosomal Location of Two Mitochondrial Malate Dehydrogenase Structural Genes in ZEA MAYS Using Trisomics and B-A Translocations.

Maize mitochondrial malate dehydrogenase is coded by four genetic loci, Mdh1, Mdh2, Mdh3 and Mdh4. Two of the four loci have been located on the long arm of chromosome 6, using trisomic analysis and B-A translocations.

Determination of the chromosomal location of a glutamate oxaloacetate transaminase structural gene using triticum-agropyron translocations.

The glutamate oxaloacetate transaminase (GOT) zymogram phenotypes of a series of 15 translocation lines, a chromosome addition line and a chromosome substitution line were determined. In each of the translocation lines a segment of the long arm of Triticum aestivum chromosome 3D has been replaced by a portion of an Agropyron elongatum homoeologue. Evidence was obtained that the products of the T. aestivum GOT-3 triplicate structural gene set randomly dimerize with the product of the homoeologous A. elongatum gene. Each translocation chromosome was found to carry either Got-D3 or Got-Ag3. By correlating the zymogram phenotype expressed by each translocation line with the observed frequency of meiotic pairing of each 3D/3Ag translocation chromosome with telocentric-3DL, it was shown that Got-D3 is located in the proximal portion of 3DL, slightly more than 4.3 crossover units from the centromere. The results of this genetic study confirm and extend earlier conclusions derived from cytogenetic studies as to the physical nature of the various 3D/3Ag chromosomes.