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PubMedDate not supplied

Restriction fragment length polymorphism in four virulence-associated genes of Listeria monocytogenes.

We observed restriction fragment length polymorphism in 4 genes of Listeria monocytogenes associated with virulence. Using the polymerase chain reaction (PCR) and primers derived from published sequences, we amplified the following genes: hlyA coding for listeriolysin O, iap coding for a putative invasion-associated factor, mpl coding for a metalloprotease, and the prfA gene that positively regulates the hylA gene. PCR-amplified DNA were cut with several restriction endonucleases, and the restriction profiles from 29 strains, representing 6 serovars (serovars 1/2a, 1/2b, 1/2c, 3a, 3b and 4b) were compared. Based on these restriction profiles, the strains were categorized into 2 subgroups: one group contained all 10 strains of serovars 1/2a, 1/2c and 3a, the other group contained all 19 strains of serovars 1/2b, 3b and 4b. This division is in complete agreement with multilocus enzyme electrophoretic analysis data which divide the species into the same 2 subgroups. Whether the differences observed in the nucleotide sequences of the 4 virulence-associated genes for the 2 subgroups of L. monocytogenes represent salient variations in pathogenic mechanisms is not known.

DNA, Bacterial↗

PubMed2003

Use of restriction fragment length polymorphism, immunoblotting, and polymerase chain reaction in the differentiation of avian poxviruses.

Restriction deoxyribonucleic acid (DNA) fragment profile analysis coupled with immunogenic protein profile analysis has provided useful information in determining the differences between vaccine strains and field isolates of fowlpox virus (FPV). The DNA of strains examined in this study clearly fell into 3 minor groups of restriction patterns similar but distinct from one another: restriction patterns exhibited by the vaccine strains except 1 vaccine strain, Vac-82; restriction profiles indicated by Vac-82 and field isolates FI-38 and FI-42; and restriction patterns indicated by field isolates FI-43, FI-51, FI-54, and FI-56. Furthermore, when the strains were analyzed and compared by immunoblotting analysis, they showed group differences similar to the differences in restriction profiles. Both techniques provided high sensitivity in verifying differences between vaccine strains and field isolates of FPV. The disparity found in restriction fragments or immunogenic protein profile between vaccine strains and field isolates does not exclude the appreciable high degree of DNA sequence conservation and homology. However, the minor disparity observed in these strains suggests a molecular basis for why vaccinated commercial flocks could have continually been infected by variant strains of FPV. A rapid and sensitive polymerase chain reaction method, which amplified a product from the 4b core protein gene of the FPV genome, was developed for identification and differentiation of members of the genus Avipoxvirus. Whereas total DNA from either vaccine strains or field isolates was used as template for amplifying a predicted product of 578 or 1409 bp, only cleavage of the amplified product (1409 bp) represented an additional detection technique for species differentiation. An attempt to distinguish between strains on the basis of amplification product was partially successful.

Animals↗

PubMed2003

Intraspecies polymorphism of Cryptosporidium parvum revealed by PCR-restriction fragment length polymorphism (RFLP) and RFLP-single-strand conformational polymorphism analyses.

A glycoprotein (Cpgp40/15)-encoding gene of Cryptosporidium parvum was analyzed to reveal intraspecies polymorphism within C. parvum isolates. Forty-one isolates were collected from different geographical origins (Japan, Italy, and Nepal) and hosts (humans, calves, and a goat). These isolates were characterized by means of DNA sequencing, PCR-restriction fragment length polymorphism (PCR-RFLP), and RFLP-single-strand conformational polymorphism (RFLP-SSCP) analyses of the gene for Cpgp40/15. The sequence analysis indicated that there was DNA polymorphism between genotype I and II, as well as within genotype I, isolates. The DNA and amino acid sequence identities between genotypes I and II differed, depending on the isolates, ranging from 73.3 to 82.9% and 62.4 to 80.1%, respectively. Those among genotype I isolates differed, depending on the isolates, ranging from 69.0 to 85.4% and 54.8 to 79.2%, respectively. Because of the high resolution generated by PCR-RFLP and RFLP-SSCP, the isolates of genotype I could be subtyped as genotypes Ia1, Ia2, Ib, and Ie. The isolates of genotype II could be subtyped as genotypes IIa, IIb, and IIc. The isolates from calves, a goat, and one Japanese human were identified as genotype II. Within genotype II, the isolates from Japan were identified as genotype IIa, those from calves in Italy were identified as genotype IIb, and the goat isolate was identified as genotype IIc. All of the genotype I isolates were from humans. The Japanese isolate (code no. HJ3) and all of the Nepalese isolates were identified as genotypes Ia1 and Ia2, respectively. The Italian isolates were identified as genotype Ib, and the Japanese isolate (code no. HJ2) was identified as genotype Ie. Thus, the PCR-RFLP-SSCP analysis of this glycoprotein Cpgp40/15 gene generated a high resolution that has not been achieved by previous methods of genotypic differentiation of C. parvum.

Amino Acid Sequence↗

PubMed1998

Genetic polymorphism of Leishmania species using kinetoplast DNA restriction fragment length polymorphism and cDNA probe of Leishmania donovani.

Leishmaniasis represents a group of diseases that range from simple cutaneous lesions through metastasizing diffused cutaneous to severe systemic infection depending upon the taxon to which the causative parasite belongs. Therefore, it is important to identify the infecting Leishmania. Methods presently being used, including immunology, biochemistry and molecular biology have one or the other limitations, leaving scope for the search for newer probes. This study reports the characterization of leishmania isolates both by restriction fragment length polymorphism of kinetoplast DNA (kDNA) and genomic DNA. The genomic DNA was probed with a cDNA probe B2a1. Using a kDNA restriction pattern technique, different isolates of Leishmania donovani could be differentiated from the UR6 strain of L. tropica, but it was not possible to differentiate between newer local isolates of L. donovani with most of the restriction enzymes except AluI. However, the B2a1 cDNA probe was able to differentiate these isolates effectively. Both of these techniques could differentiate newer local isolates of L. donovani from the older isolates of L. donovani from India, i.e., DD8, RMRI and SS. The Indian isolates of L. donovani could also be differentiated from isolates of L. donovani from Jeddah and Germany using both techniques. The present study indicates that the cDNA probe B2a1 can be used as an important adjunct to kDNA restriction analysis for the characterization of Leishmania species.

Animals↗

PubMed1987

The aryl hydrocarbon hydroxylase (Ah) locus and a novel restriction-fragment length polymorphism (RFLP) are located on mouse chromosome 12.

The aryl hydrocarbon hydroxylase (Ah) locus that controls the induction of chemical carcinogen-metabolizing enzymes in mice has been found to be linked to a new restriction-fragment length polymorphism (RFLP). Only C57BL/6 and closely related inbred strains displayed a 7.6-kb HindIII restriction fragment, while all other inbred strains tested displayed an 11.2-kb HindIII restriction fragment when using plasmid pRC2.3 as the hybridization probe. Polymorphisms in this region can also be detected with two other restriction enzymes: SacI and EcoRV. Linkage of Ah and the restriction-fragment length polymorphism was first detected using the BXD (C57BL/6 x DBA/2) recombinant inbred strains and was confirmed by a backcross. Both the restriction-fragment length polymorphism and Ah were not linked to the standard genetic markers Hba, Hbb, b, d, C-3, and W. However, comparison of the RFLP strain distribution pattern in the BXD recombinant inbred set with the strain distribution pattern of another RFLP, known to be located on chromosome 12, shows complete concordance in 24 of 24 strains, thereby locating Ah on chromosome 12.

Animals↗

PubMed1986

An overview of the restriction fragment length polymorphism of the HLA-D region: its application to individual D-, DR- typing by computerized analyses.

HLA-D/DR alleles as defined by cellular and serological typing can also be identified by biochemical methods. The Southern blot technique provides an additional typing facility which can be applied to DNA obtained from any source of nucleated cells. The polymorphism revealed by Southern blot analyses, the so-called restriction fragment length polymorphism (RFLP), depends upon the restriction enzyme and cDNA probes used. To identify HLA-DR specificities a protocol was developed based on the use of the results of southern blot analyses with several restriction enzymes and cDNA probes within a panel of HLA-D/DR homozygous cells representing the DR1 to DRw8 alleles. First, hybridizations with the 3' untranslated sequence of the DR beta cDNA probe, after digestion of the DNA with PvuII (PvuII-DR beta 3') allows the selective identification of DR1, DR2 and DRw8; DR3, DR5 and DRw6 are found as one group as well as DR4 and DR7 as another. Second, TaqI-DQ alpha hybridization allows the splitting of DRw6-Dw18, DRw6-Dw19 and DRw6-Dw9 from the DR3, DR5 and DRw6 group. The other alleles DR3, DR4, DR5, DRw6-Dw16 and DR7 are revealed by dehybridization and rehybridization of the blot with a DR beta cDNA probe. This protocol was used to test whether in a panel of 30 randomly chosen individuals the HLA-DR typing could be performed. The results were highly concordant to the serotyping. Furthermore by adding the Pst-DR beta and TaqI-DQ alpha RFLPs, most of the MLC defined Dw specificities could also be identified. An overview of the specific fragments described here has been summarized in matrices which can be used as references for DNA-typing in computerized analyses.

Collodion↗

PubMed1994

Dde I restriction fragment length polymorphism of the alpha 2-adrenoceptor gene does not correlate with blood pressure in the F2 generation obtained from crossing stroke-prone spontaneously hypertensive rats and Wistar-Kyoto rats.

OBJECTIVE: A pathogenetic role of altered alpha 2-adrenoceptors in essential hypertension has been suggested, based on studies in humans and animals. To examine the role of the alpha 2-adrenoceptor in genetically hypertensive rats, we compared the alpha 2-adrenoceptor genes of stroke-prone spontaneously hypertensive rats (SHRSP) and Wistar-Kyoto (WKY) rats by restriction fragment length polymorphism analysis using human alpha 2-adrenoceptor probes (alpha 2-C10) and Dde I restriction endonuclease, and conducted a genetic cosegregation study. METHOD: Five female WKY rats were bred with five male SHRSP. Eight pairs of F1 rats were mated in brother-sister pairs to yield an F2 population of 84 rats. Systolic blood pressure was determined by tail-cuff sphygmomanometry. Direct arterial blood pressure was taken under ether anaesthesia just before the rats were killed. Southern blots were performed using alpha 2C10 as a probe and the DNA from the F2 generation. RESULTS: A restriction fragment length polymorphism of the SHRSP allele of a 1.6-kb fragment and a WKY rat allele of a 0.9-kb fragment with a common band of 1.3 kb in SHRSP and WKY rats was found, as reported previously. The distribution of the genotype based on restriction fragment length polymorphism conformed to a 1:2:1 ratio in F2 rats, as expected for a Mendelian trait. There was no significant difference in the blood pressure of F2 rats with respect to alpha 2-adrenoceptor genotype. CONCLUSION: This study demonstrated that the alpha 2-adrenoceptor gene restriction fragment length polymorphism distribution is a Mendelian trait in the F2 rats of crossed SHRSP and WKY rats, but failed to show genetic cosegregation of this restriction fragment length polymorphism with blood pressure in this generation.

Alleles↗

PubMed2004

PrP polymorphisms in Basque sheep breeds determined by PCR-restriction fragment length polymorphism and real-time PCR.

Two new PCR-based methods were developed to decode prion protein (PrP) gene polymorphisms at codons 136, 154 and 171: a PCR-restriction fragment length polymorphism (RFLP) analysis consisting of two PCR reactions followed by three enzymatic digestions, and a real-time PCR consisting of four reactions with seven fluorogenic probes. Both methods were used to study the distribution of PrP gene polymorphisms in a representative sample (1297 animals) of the populations of the two native breeds of sheep of the Spanish Basque Country, Latxa and Carranzana. Fourteen genotypes were found in the Latxa breed, in which ARQ/ARQ was the genotype most frequently observed (49.3 per cent), followed by ARR/ARQ (32.6 per cent) and ARQ/ARH (5.8 per cent). The genotype associated with the highest resistance to scrapie (ARR/ARR) was present in 5 per cent of the animals analysed. Similar results were observed in the Carranzana sheep.

Animals↗

PubMed2001

Identification of 54 mycobacterial species by PCR-restriction fragment length polymorphism analysis of the hsp65 gene.

A total of 121 reference and clinical strains of both slowly and rapidly growing mycobacteria belonging to 54 species were studied for restriction fragment length polymorphism of a PCR-amplified 439-bp segment of the gene encoding the 65-kDa heat shock protein. Restriction digests were separated by 10% polyacrylamide gel electrophoresis (PAGE). By including a size standard in each sample, the restriction fragment profile was calculated using a computer-aided comparison program. An algorithm describing these 54 species (including 22 species not previously described) is proposed. We found that this assay based on 10% PAGE provided a more precise estimate than that based on agarose gel electrophoresis of the real size of restriction fragments as deduced from the sequence analysis and allowed identification of mycobacteria whose PCR-restriction fragment length polymorphism analysis patterns were unequivocally identified by fragments shorter than 60 bp.

Algorithms↗

PubMed1989

CR1 polymorphism in hydralazine-induced systemic lupus erythematosus: DNA restriction fragment length polymorphism.

The contribution of genetic factors in the reduction in erythrocyte CR1 levels observed in hydralazine (Hz) induced systemic lupus erythematosus (SLE) was investigated by determining the frequency of a HindIII restriction fragment length polymorphism (RFLP) in the CR1 gene. This RFLP is associated with quantitative erythrocyte CR1 expression. Individuals who have developed SLE as a reaction to Hz therapy, consanguinous relatives of the Hz-SLE patients, controls who had been treated with Hz without any adverse reaction, and the consanguinous relatives of these controls were included in this study. No difference was found in the frequency of occurrence of the alleles associated with CR1 expression between the Hz-SLE patients and the control groups (P greater than 0.2). Individuals from the Hz-SLE group who were homozygous for the 7.4 kb 'high expressor' allele had lower mean levels of erythrocytes CR1 (564 +/- 65) than the corresponding homozygous subgroups within the Hz-SLE relative group (774 +/- 46), the Hz control group (756 +/- 80) and the Hz control relatives group (825 +/- 66). In addition, 50% of the Hz-SLE patients in the 'high expressor' subgroup who had less than 500 CR1 per erythrocyte had elevated levels of circulating immune complexes. This study suggests that individuals who are genetically low expressors of erythrocyte CR1 are not predisposed to developing SLE in response to Hz therapy, and that in a subgroup of genetically 'high expressors', low CR1 levels are associated with elevated levels of circulatory immune complexes.

Alleles↗

PubMed1998

Restriction fragment length polymorphism species-specific patterns in the identification of white truffles.

A molecular method for the identification of ectomycorrhizae belonging to five species of white truffle is described. The polymerase chain reaction (PCR) and universal primers were used to amplify internal transcribed spacers and 5.8S rDNA, target sequences present in a high number of copies. The amplified products were digested with restriction enzymes in order to detect interspecific polymorphisms. Species-specific restriction fragment length polymorphism patterns were determined for all five species. The use of PCR in conjunction with restriction enzymes provides a sensitive and efficient tool for use in distinguishing ectomycorrhizal species and monitoring inoculated seedlings or field mycorrhizal populations.

Ascomycota↗

PubMed1999

Identification of Bartonella species directly in clinical specimens by PCR-restriction fragment length polymorphism analysis of a 16S rRNA gene fragment.

It is now established that two species of Bartonella, namely, Bartonella henselae and B. quintana, cause bacillary angiomatosis in human immunodeficiency virus-infected patients. In addition, B. henselae causes cat scratch disease and B. quintana, B. henselae, and B. elizabethae can cause bacteremia and endocarditis in immunocompetent persons. We have developed a PCR-restriction fragment length polymorphism-based assay for direct detection and identification to species level of Bartonella in clinical specimens. This is accomplished by PCR amplification of Bartonella DNA using primers derived from conserved regions of the gene carrying the 16S ribosomal DNA, followed by restriction analysis using DdeI and MseI restriction endonucleases. We amplified a Bartonella genus-specific 296-bp fragment from 25 clinical samples obtained from 25 different individuals. Restriction analysis of amplicons showed that identical patterns were seen from digestion of B. henselae and B. quintana amplicons with DdeI, whereas a different unique pattern was seen by using the same enzyme with B. vinsonii and B. elizabethae. With MseI digestion, B. henselae and B. vinsonii gave nearly identical patterns while B. quintana and B. elizabethae gave a different pattern. By combining the restriction analysis data generated with MseI and DdeI, unique "signature" restriction patterns characteristic for each species were obtained. These patterns were useful in identifying the Bartonella species associated with each tissue specimen.

Angiomatosis, Bacillary↗

PubMed2006

Trypanosoma evansi: genetic variability detected using amplified restriction fragment length polymorphism (AFLP) and random amplified polymorphic DNA (RAPD) analysis of Kenyan isolates.

We compared two methods to generate polymorphic markers to investigate the population genetics of Trypanosoma evansi; random amplified polymorphic DNA (RAPD) and amplified restriction fragment length polymorphism (AFLP) analyses. AFLP accessed many more polymorphisms than RAPD. Cluster analysis of the AFLP data showed that 12 T.evansi isolates were very similar ('type A') whereas 2 isolates differed substantially ('type B'). Type A isolates have been generally regarded as genetically identical but AFLP analysis was able to identify multiple differences between them and split the type A T. evansi isolates into two distinct clades.

Animals↗

PubMed2000

DNA fingerprints of Trichinella as revealed by restriction fragment length polymorphism and single-strand conformational polymorphism (RFLP-SSCP).

A method was developed for gene fingerprinting, combining the principles of restriction fragment length polymorphism and single-strand conformational polymorphism (PCR-RFLP-SSCP). Taking advantage of this method, we analysed the genotypes of 20 isolates from five species of Trichinella (Trichinella spiralis, Trichinella britovi, Trichinella nativa, Trichinella murrelli and Trichinella pseudospiralis) and two uncertain genotypes (Trichinella T6 and Trichinella T8). Target genes for the analysis included three kinds of random amplified polymorphic DNA (RAPD), the gene encoding 43 kDa excretory-secretory protein, the gene encoding mitochondrial cytochrome c -oxidase subunit I and the gene of 18 S rRNA. The genotype revealed by this method was in good concordance with the taxonomy of Trichinella (six species and two uncertain genotypes which is currently accepted based on morphology, isozyme pattern and reproductive isolation). More important, this method revealed intraspecies polymorphism among geographical isolates of T. spiralis and T. britovi.

Animals↗

PubMed2006

Rapid screening of fluoroquinolone resistance determinants in Streptococcus pneumoniae by PCR-restriction fragment length polymorphism and single-strand conformational polymorphism.

A rapid method, using PCR-restriction fragment length and single-strand conformation polymorphism (SSCP), was applied to screen for mutations of the fluoroquinolone resistance determinants in Streptococcus pneumoniae. One hundred nonduplicate Streptococcus pneumoniae isolates with ciprofloxacin MICs of > or = 4.0 microg/ml from the Prince of Wales Hospital, Hong Kong, years 2000 to 2003, were examined. For each isolate, PCR amplicons of quinolone resistance-determining regions (QRDRs) of gyrA, gyrB, parC, and parE genes were digested with AluI, HinfI, Sau3AI, and MspI, respectively, and analyzed by SSCP. Each SSCP pattern was given a number, and each isolate obtained a four-digit code, e.g., 1111, that represented the SSCP profile. The SSCP patterns were correlated to mutations characterized from sequence analyses of PCR amplicons. The most common SSCP profile obtained was no. 5232 (40%), which included strains with two amino acid substitutions in the ParC (Lys-137-Asn) and ParE (Ile-460-Val) genes, followed by the SSCP profile 5223 (17%), which included strains with amino acid substitutions in the ParE (Ile-460-Val) gene only. Ten isolates (10%) with amino acid substitutions at GyrA and ParE (+/-ParC) genes were resistant to levofloxacin with a MIC of > or = 16 microg/ml. Other SSCP profiles were unique in distinguishing the common amino acid substitutions in GyrA (Ser-81-Phe) and ParC (Lys-137-Asn, Ser-79-Phe plus Lys-137-Asn, Asp-83-Asn plus Lys-137-Asn, Ser-79-Phe, and Glu-96-Asp). SSCP analysis of restricted fragments generated patterns that were highly discriminative for mutations present in the QRDRs of gyrA, gyrB, parC, and parE. This method provides a database of high resolution profiles on these mutations and allows rapid screening for new mutations of the fluoroquinolone resistance genes.

Amino Acid Substitution↗

PubMed1988

Restriction-fragment-length polymorphism in insulin-receptor gene and insulin resistance in NIDDM.

Restriction-enzyme analysis of genomic DNA from 52 White and Hispanic nondiabetic subjects and 51 subjects with non-insulin-dependent diabetes (NIDDM) was carried out with insulin-receptor cDNA probes. A polymorphic 5.8-kilobase SstI fragment was found in 12 (23.5%) of 51 NIDDM subjects but only in 4 (7.7%) of 52 nondiabetic control subjects. This association is significant by chi 2-analysis (P less than .05). Furthermore, the nondiabetic subjects with the polymorphism were found to have hyperinsulinemia and/or nondiagnostic glucose tolerance. The polymorphism is a genetic marker for a phenotype that is neither necessary nor, by itself, sufficient for NIDDM. Nevertheless, it may indicate that insulin resistance functionally related to an insulin-receptor gene polymorphism is the proximal cause of NIDDM in at least one subset of the population.

Aged↗

PubMed1987

Globin gene-associated restriction-fragment-length polymorphisms in southern African peoples.

The combination of polymorphic restriction-enzyme sites in the 3' region of the beta-globin gene cluster shows very little variation in southern-African Bantu-speaking black and Kalahari !Kung San populations. The sites of the 5' region, on the other hand, show marked variation, and two common haplotypes are present--the "Negro" type (- - - - +) and the "San" type (- + - - +)--in frequencies of .404 and .106, respectively, in the Bantu-speakers and .262 and .405, respectively, in the San. Twenty of 23 beta s-associated haplotypes in southern-African Bantu-speaking black subjects were the same as that found commonly in the Central African Republic (CAR)--i.e., the "Bantu" type--a finding providing the first convincing biological evidence for the common ancestry of geographically widely separated speakers of languages belonging to the Bantu family. The (-alpha) haplotype has a frequency of .21 in the Venda, .07 in both the Sotho-Tswana and the Nguni, and .06 among the !Kung San. These data are interpreted in the light of Plasmodium falciparum malaria selection and population movements in the African subcontinent.

Africa, Southern↗

PubMed1991

Techniques used to define human MHC antigens: restriction fragment length polymorphisms.

Polymorphisms within the HLA-DRB1, -DRB3, -DQB1 and -DQ A1 genes are detectable using restriction fragment length polymorphism (RFLP) analysis. DNA is isolated from EDTA-treated blood or from spleen or lymph nodes. The DNA is digested to completion with the restriction endonuclease TaqI and resolved using agarose gel electrophoresis. The DNA after denaturation is then transferred to a nylon membrane (Southern blotting) and hybridised with radiolabelled cDNA probes: HLA-DR beta pRTV1, HLA-DQ beta pII-beta-1 and HLA-DQ alpha pDCH1. After autoradiography the membrane is dehybridised prior to rehybridisation. This system is very useful in those situations where serological assignment is difficult due to poor quality or low numbers of circulating B cells and where there is a lack of reliable antisera for certain specificities. The RFLP techniques can also define subtypes of DR and DQ serological specificities. However, certain alleles have the same RFLP. In some instances by identifying the DQ allele the DR allele can be determined by association due to linkage disequilibrium (e.g., DRw17-Dw25-DQw2 and DRw13-Dw25-DQw6). In other instances (e.g., DR1 and DRBr), the problem can be resolved using serology. In addition the RFLP system cannot be applied prospectively to the cadaver donor situation because of time restrictions. Thus the RFLP system complements existing serological techniques. However, it can be very useful as a quality control for the serological methods especially in the assessment of the quality of antisera and in the determination of discrepancies between centres.(ABSTRACT TRUNCATED AT 250 WORDS)

Alleles↗