Chromosomal Restriction Fragment Length Polymorphisms Research Articles

Finger printing-RFLP analysis of chromosomal IS6110 insertion sequence and PCR diagnosis of pulmonary tuberculosis, isolated from patients in El Hajeb region of Morocco

Tuberculosis (TB) is one of the contagious diseases caused by M. tuberculosis (MTB) bacteria. Prompt diagnosis is one of the active solutions to control the spread of this infection. Besides, a targeted, specific and non-complex diagnosis can prove promising in this type of epidemic. This study was designed to compare the efficiencies of a diagnosis by Ziehl-Neelsen staining (ZN) and by the polymerase chain reaction (PCR) technique. Samples presented smear-positive pulmonary TB were subjected to Chromosomal restriction fragment length polymorphism of IS6110 (IS6110-RFLP) for fingerprinting profile determination.The results showed that out of 100 sputum samples of suspected case, 53 were positive. Numbers of positive individuals for tuberculosis obtained by the different diagnostic techniques, to know, (ZN staining; culture and PCR) were respectively: 6, 25 and 22. Chromosomal RFLP fingerprinting profile revealed the presence of five different genotypes obtained from seven tested isolates.These results suggest that molecular techniques are alternative tool for fast and specific diagnosis of pulmonary MTB from sputum.

Identification of X chromosomal restriction fragment length polymorphism markers and their use in a gene localization study in Drosophila virilis and D. littoralis.

We have identified six restriction fragment length polymorphism (RFLP) markers based on unique gene sequences on the X chromosome of Drosophila virilis and D. littoralis. The markers were localized by in situ hybridization on larval polytene chromosomes, and the conjugation of the X chromosomes of the two species was studied in salivary glands of interspecific hybrid female larvae. The gene arrangement of D. virilis and D. littoralis appeared to be very different at the proximal end of the X chromosome preventing recombination between RFLP markers located in this area. Simple quantitative trait loci (QTL) analysis showed that five of our marker genes (including nonA and Dmca1A, previously found to affect male courtship song in D. melanogaster) are linked with a gene(s) having a major effect on species differences in the male courtship song between D. virilis and D. littoralis. This shows that the song gene(s) may be located inside a large X-chromosomal inversion in D. littoralis (as previously suggested), but that it may also be located on an area between this inversion and the centromere, close to nonA and Dmca1A. Localization of this gene or gene complex will be continued with the aid of our newly identified RFLP markers by making interspecific crosses between D. virilis group species with more similar X chromosomes.

Occurence of subtelomeric rearrangements in the genome of the microsporidian parasite Encephalitozoon cuniculi, as revealed by a new fingerprinting procedure based on two-dimensional pulsed field gel electrophoresis.

In Microsporidia, mitochondria-lacking eukaryotic intracellular parasites, genomic comparisons were so far based on molecular karyotyping. The mammal-infecting species Encephalitozoon cuniculi is characterized by a very low haploid genome size (approximately 2.8 Mbp) and rather high karyotype variability. Recently, we developed a two-dimensional pulsed field gel electrophoresis (2-D PFGE) fingerprinting technique useful for constructing a restriction map fo the genome of a mouse E. cuniculi isolate (karyotype variant A). The so-called karyotype and restriction display 2-D PFGE (KARD-PFGE) protocol involved 1-D chromosome separation, digestion with a rare cutter, Klenow radiolabeling of genomic DNA and 2-D separation of restriction fragments followed by autoradiography. In order to assess its suitability for detecting polymorphic loci in E. cuniculi, we applied KARD-PFGE with either BssHII or Mlul digestion to genome analysis of two rabbit isolates representative of two different karyotype variants (A and C). The 2-D spot pattern of the rabbit isolate variant A is identical to the reference mouse isolate but differs greatly from the rabbit isolate variant C. Chromosomal restriction fragment length polymorphisms (RFLPs) provide strong evidence for homologous chromosomes and frequent DNA rearrangements within subtelomeric regions just upstream of the dispersed rDNA units closely associated with each chromosomal end.

Chromosomal restriction fragment length polymorphism analysis of Escherichia coli strains causing recurrent urinary tract infections in young women.

To determine whether recurrent, symptomatic urinary tract infections (UTIs) in a given individual are due to the same or different strains, 71 Escherichia coli strains that caused recurrent UTIs were prospectively collected from 23 infection-prone young women and studied by chromosomal restriction fragment length polymorphism (RFLP) analysis using pulsed-field gel electrophoresis. Thirty-five strains from women with first-episode UTIs were also studied. Overall, 30 (68%) of 44 recurrent UTIs were caused by a strain previously identified in that person. In contrast, 32 of 35 strains from first-episode UTIs had unique RFLP profiles. Analysis of a subset of subjects established that the majority of recurrent UTIs were due to reinfection, not persistence of the pathogen within the urinary tract, and suggested that the colonic flora was the reservoir for these reinfecting strains.

Characterization of PCR-ribotyping for Burkholderia (Pseudomonas) cepacia.

Ribotyping, a method of genotyping bacterial isolates for epidemiologic study, uses rRNA as a probe to detect chromosomal restriction fragment length polymorphisms. Although ribotyping is accurate, its utility is limited by the labor and time necessary for Southern blot analysis. PCR-ribotyping uses PCR to amplify the 16S-23S intergenic spacer region of the bacterial rRNA operon. Length heterogeneity in the spacer region has previously been found to be useful as an alternative to standard ribotyping in a study of Burkholderia (Pseudomonas) cepacia. To further analyze the accuracy of PCR-ribotyping, three groups of previously characterized isolates of B. cepacia were investigated. PCR-ribotyping grouped 90 isolates recovered from seven well-defined epidemics into the correct outbreak group with a mean concordance of 93%. Both standard ribotyping and PCR-ribotyping separated 15 unrelated isolates into 14 types. In an analysis of 83 B. cepacia isolates from chronically colonized cystic fibrosis patients, the concordance of PCR-ribotyping with standard ribotyping ranged from 83 to 100%, with a mean of 98%. One isolate from a chronically colonized patient had a different type by standard ribotyping but was identical to the other isolates from this patient by PCR-ribotyping. Thus, PCR-ribotyping is a rapid and accurate method for typing B. cepacia and is less labor intensive than standard ribotyping.

Source of Pseudomonas cepacia: Ribotyping of isolates from patients and from the environment

Ribotyping, an analysis of bacterial strain identity based on chromosomal restriction fragment length polymorphisms, was performed on 68 isolates of Pseudomonas cepacia recovered from patients receiving care at the two children's hospitals in Philadelphia and from environmental samples. Twenty different ribotypes were identified. Ribotype R3 predominated among isolates from all three sources. These findings are consistent with the acquisition of P. cepacia from the environment or by person-to-person transmission.

Analysis of an outbreak of variably methicillin-resistant Staphylococcus aureus with chromosomal RFLPs and mec region probes

An outbreak of infections with multi-resistant Staphylococcus aureus with unusual methicillin resistance at a Melbourne hospital was investigated by examining restriction fragment length polymorphisms (RFLPs) of total DNA digested with the rare-cutting endonuclease SmaI. The polymorphisms were identified by pulsed-field gel electrophoresis (PFGE) and were analysed numerically to give quantitative estimates of genetic distances between isolates. The majority of the isolates were found to belong to one group, with only minor genetic differences between the isolates that showed varying resistance to methicillin, thereby suggesting development of resistant variants from one clonal type during the outbreak. These results were confirmed by DNA hybridisation analysis with specific resistance gene probes for parts of a multi-resistance gene cluster (including methicillin) in the chromosome. Analysis of the RFLP patterns of S. aureus isolates is potentially a useful procedure in clinical epidemiology.

Linkage analysis in a large family with nonspecific X-linked mental retardation.

We report on a large 5-generation family with "nonspecific" X-linked mental retardation. Nine living affected males have an IQ between 50 and 70 but have normal stature, facial appearance, and testicular volumes and no other abnormalities. Two obligate carrier females had borderline intellectual abilities and visual-psychomotor difficulties similar to those seen in affected males. Results of chromosome studies, including fragile X, were normal in males and females. Linkage analysis was undertaken, with 19 X-specific chromosomal restriction fragment length polymorphisms (RFLPs), giving a maximal LOD score of 1.60 at a 0.10 recombination fraction for F9, suggesting a localization to distal Xq for the mutant gene in this family.

Rapid field inversion gel electrophoresis in combination with an rRNA gene probe in the epidemiological evaluation of staphylococci

A rapid field inversion gel electrophoresis (FIGE) protocol was combined with an rRNA gene probe in the analysis of staphylococci that were difficult to study epidemiologically by conventional means. The following groups of clinical isolates were examined: (i) predominantly antibiotic-susceptible Staphylococcus aureus strains containing no detectable plasmids and unresponsive to bacteriophage typing and (ii) methicillin-resistant Staphylococcus epidermidis strains carrying a single plasmid ca. 30 kilobases in size. The results indicated that strain interrelationships could be established on the basis of SmaI-generated chromosomal restriction fragment length polymorphisms (RFLPs) analyzed by FIGE. RFLP analysis of strains known to be unrelated established the importance of minor differences in DNA banding patterns as indicators of strain dissimilarities. Hybridization studies with an rRNA gene probe confirmed this conclusion. These results suggest that FIGE analysis of chromosomal RFLPs (especially in combination with molecular probes) is an important addition to the armamentarium of molecular epidemiology.

Haplotype and multipoint linkage analysis in Finnish choroideremia families

Multipoint linkage analysis of choroideremia (TCD) and seven X chromosomal restriction fragment length polymorphisms (RFLPs) was carried out in 18 Finnish TCD families. The data place TCD distal to PGK and DXS72, very close to DXYS1 and DXYS5 (Zmax = 24 at theta = 0) and proximal to DXYS4 and DXYS12. This agrees with the data obtained from other linkage studies and from physical mapping. All the TCD males and carrier females studied have the same DXYS1 allele in coupling with TCD. In Northeastern Finland, 66/69 chromosomes carrying TCD had the same haplotype at loci DXS72, DXYS1, DXYS4, and DXYS12. The same haplotype is seen in only 15/99 chromosomes not carrying TCD. Moreover, in 71/104 non-TCD chromosomes, the haplotype at six marker loci is different from those seen in any of the 76 TCD chromosomes. This supports the previously described hypothesis that the large Northern Finnish choroideremia pedigrees, comprising a total of over 80 living patients representing more than a fifth of all TCD patients described worldwide, carry the same mutation. These linkage and haplotype data provide improved opportunities for prenatal diagnosis based on RFLP studies.

Limited genetic diversity of Haemophilus influenzae (type b)

170 strains of Haemophilus influenzae (serotype b), isolated largely from patients with invasive disease from differing temporal and geographic origins were characterized using the combined approaches of DNA hybridization and outer membrane protein classification. Hybridization of a DNA probe to a region of the chromosome involved in the expression of type b capsular polysaccharide revealed that 163 (96%) isolates had one of three distinct, but closely related, chromosomal restriction fragment length polymorphisms (RFLPs). Each polymorphism was associated with its own distinctive set of outer membrane protein subtypes, indicating that the majority of H. influenzae (type b) isolates have evolved from common ancestors, giving rise to globally distributed organisms that have clonal characteristics.