rDNA Restriction Fragment Length Polymorphism Research Articles

Physical mapping of rDNA loci in Brassica species

The number of major rDNA loci (the genes coding for 18S-5.8S-26S rRNA) was investigated in the economically important Brassica species and their wild relatives by in situ hybridization of an rDNA probe to metaphase chromosomes and interphase nuclei. The diploid species B. nigra (B genome) has two major pairs of rDNA loci, B. oleracea (C genome) has two major pairs and one minor pair of loci, while B. campestris (A genome) has five pairs of loci. Among the three tetraploid species arising from these three diploid ancestors, B. carinata (BBCC genomes) has four loci, B. juncea (AABB genomes) has five major pairs and one minor pair of loci, and B. napus (AACC genomes) has six pairs of loci, indicating that the number of loci has been reduced during evolution. The complexity of the known rDNA restriction fragment length polymorphism patterns gave little indication of number of rDNA loci. It is probable that chromosome rearrangements have occurred during evolution of the amphidiploid species. The data will be useful for physical mapping of genes relative to rDNA loci, micro- and macro-evolutionary studies and analysis of aneuploids including addition and substitution lines used in Brassica breeding programs.

Genetic characterization of a cryptic genospecies of Haemophilus causing urogenital and neonatal infections.

In recent years, reports originating from several areas of the world have identified biotype IV strains of Haemophilus influenzae as a cause of serious urogenital, neonatal, and mother-infant infections. Preliminary analysis of a sample of biotype IV isolates found evidence for a cryptic genospecies of Haemophilus (R. Quentin, A. Goudeau, R. J. Wallace, Jr., A. L. Smith, R. K. Selander, and J. M. Musser. J. Gen. Microbiol. 136:1203-1209, 1990). Eighteen biotype IV strains assigned to the cryptic genospecies were further characterized by their rDNA restriction fragment length polymorphism patterns and genomic DNA-DNA hybridization. Isolates of the cryptic genospecies have distinctive rDNA restriction fragment length polymorphisms that differ from those of Haemophilus haemolyticus and H. influenzae. Genomic hybridization studies show that these organisms are allied with H. influenzae and H. haemolyticus and suggest a distant trifurcation of H. influenzae, H. haemolyticus, and the cryptic genospecies.

Ribotyping of Pseudomonas aeruginosa strains isolated from surgical intensive care patients.

To elucidate the sources of Pseudomonas aeruginosa on a surgical intensive care unit, rDNA restriction fragment length polymorphism analysis (ribotyping) was applied to analyze strains isolated during a 4-month prospective study. Samples included 1635 from 153 patients, 2463 from 97 staff members, and 581 from the environment. Only 18 patients were colonized. Isolation from their animate and inanimate environment was very low, with 3 and 2 samples, respectively, being positive. Samples from tap water were negative. Ribotyping could easily distinguish 16 different digest patterns with identical follow-up isolates of the same patient. Horizontal transmission occurred only twice. The discriminatory power of ribosomal DNA in differentiating strains was dependent on the restriction enzymes used; among eight different enzymes, PvuII was the most sensitive, producing 15 different patterns. Ribotyping showed high sensitivity in typing P. aeruginosa isolates and confirmed that colonization occurs from endogenous rather than from exogenous sources.

Use of rDNA restriction fragment length polymorphisms to differentiate strains of Candida albicans in women with vulvovaginal candidiasis

Epidemiologic studies in women with recurrent Candida vaginitis have been hampered in the past by the lack of a reproducible typing system. Several molecular probes have now been developed that have the ability to differentiate strains of Candida albicans and give reproducible results. In this investigation, 24 women with Candida vaginitis were studied in a longitudinal fashion for 30 days following short-course antifungal therapy. Seven women with either recurrent vaginitis or with multiple culture-positive sites with C. albicans were included in an epidemiological study. A total of 18 isolates of C. albicans (12 vaginal and six rectal) were typed utilizing restriction fragment length polymorphisms of rDNA. This technique was able to differentiate five different strains of C. albicans. Our epidemiologic study revealed that vaginal and rectal strains recovered from the same women were usually different. None of our patients had a similar vaginal and rectal strain prior to treatment, and only one patient had the same strain isolated from both the rectum and the vagina at the time of recurrence. On the other hand, we found that the same strain of C. albicans was initially and later recovered from the vagina in four of five women who failed treatment or developed recurrent vaginitis. These results suggest that recurrent episodes of C. albicans vaginitis, following short-course antifungal therapy, are often due to relapse of the original infecting strain and not due to autoinoculation from the rectum. If further epidemiologic studies confirm these preliminary findings, then changes in current treatment regimens may be indicated for women with chronic or recurrent candida vaginitis.

Complexity ofPseudomonas aeruginosainfection in cystic fibrosis: combined results from esterase electrophoresis and rDNA restriction fragment length polymorphism analysis

Esterase electrophoretic typing and restriction fragment length polymorphism of ribosomal DNA regions (ribotyping) were used to differentiate 102 Pseudomonas aeruginosa clinical isolates obtained from chronic lung infection in 23 patients with cystic fibrosis (CF) and two reference strains (including the type strain ATCC 10145). Twenty-five zymotypes were obtained with the former method and 16 ribotypes with the latter. Combination of the two typing systems led to the finding of 30 different types. Our data highlights the physiopathological complexity of P. aeruginosa infection in CF as, in six individual cases, several types were found among isolates from a given patient. On the other hand, two unique types were found in two and three patients respectively, raising the possibility of cross-infections.

Identification of a short rDNA spacer sequence highly specific of a tomato line containing Tm-1 gene introgressed from Lycopersicon hirsutum

We studied rDNA restriction fragment length polymorphism between two tomato lines used for F1 hybrid seed production: line A, containing the Tm-1 gene responsible for tobacco mosaic virus tolerance introgressed from the wild species Lycopersicon hirsutum, and line B, a tobacco mosaic virus sensitive line. Hybridization patterns led to distinct rDNA maps with two size classes, 10.4 and 10.7 kb, in line A and a single, 8.9-kb class in line B. Size differences were located in the intergenie sequence (IGS). A highly specific 54-bp TaqI fragment was cloned from the line A IGS and used in dot blot experiments to probe total DNA from line A, line B, and their F1 hybrid. It proved capable of discriminating B from A and the hybrid. This probe could thus serve to screen inbreds in commercial seed lots where line A is used as male. This fragment showed 80-90% sequence homology with the 53-bp subrepeats previously characterized in a region of the tomato IGS close to the 25S rRNA gene. Preliminary comparison of rDNA in line A and several wild related species indicated that the L. hirsutum H2 genotype was the closest to line A. rDNA variations between line A and this wild genotype could be explained by recombination during the introgression process involving numerous backcrosses or by an important intraspecific polymorphism. Our results strongly suggest that Tm-1 and the rDNA were introgressed together into tomato from L. hirsutum through linkage drag.