Polymorphic Restriction Fragment Length Polymorphism Research Articles

Genotyping of Malassezia pachydermatis disclosed genetic variation in isolates from dogs in Colombia

ABSTRACT: Malassezia pachydermatis is a lipophilic and lipid-dependent yeast mostly isolated from animals’ skin; hence, it is regarded as a zoophilic species causing otitis externa in dogs. Aspects associated with its epidemiology and pathogenicity is a matter of interest. This study aimed to conduct a molecular characterization of 43 isolates of M. pachydermatis obtained from dogs with otitis externa. For this purpose, the 5.8S internal transcribed spacer 2 (ITS2) and D1/D2 26S rRNA regions were amplified, sequenced and analyzed using restriction fragment length polymorphism (RFLP) with AluI, CfoI, and BstF5I endonucleases. Phylogenetic analyses revealed that these isolates grouped with the sequence types I, IV and V, previously proposed for M. pachydermatis. Interestingly, we found a new polymorphic RFLP pattern using BstF5I, these isolates were associated with the sequence types IV and V, nevertheless an association between polymorphic RFLP patterns, and fosfolipase activity or canine population data was not observed. These findings underline the genetic diversity of M. pachydermatis and provide new insights about the epidemiology of this species in the analyzed population.

P0194 Association between abdominal obesity and type 2 diabetes and HSP70-2 (+1267) gene mutation in colorectal cancer

<h3>Background</h3> The number of patients with colorectal cancer (CRC) is increasing, as is mortality for patients with CRC. Previous reports have shown that heat-shock protein 70-2 (+1267) may be associated with CRC. However, genetic analysis has not been done for effect of the <i>HSP70-2</i> (+1267) gene in association with obesity and type 2 diabetes in CRC. The aim of this study is to investigate whether <i>HSP70–2</i> (+1267) gene polymorphism is involved in susceptibility to CRC in Iranian patients with obesity and type 2 diabetes and whether it correlates with CRC progression in these patients. <h3>Methods</h3> 50 genetically unrelated patients with CRC and 50 healthy controls were typed for the <i>HSP70-2</i> (+1267) PstI polymorphism with use of restriction fragment length polymorphism (RFLP) analysis (alleles A and B). Patients with CRC were classified into two types: those with obesity and type 2 diabetes and those without obesity and type 2 diabetes. <h3>Findings</h3> Our RFLP PCR analysis and polymorphism analysis revealed that <i>HSP70</i>-<i>2</i> has three type of allele, and that the BB allele (1267 A/G) was significantly more common in patients with obesity and type 2 diabetes than in those in the control group (<i>p</i>=0.0001). <h3>Interpretation</h3> Our data suggest that the BB allele of <i>HSP70-2</i> (+1267 A/G) in CRC patients is associated with hereditary obesity, and in lower efficiency, with type 2 diabetes.

Genotypic Characterization of Phage-Typed Indigenous Soybean Bradyrhizobia and Their Host Range Symbiotic Effectiveness

Analysis of genetic diversity among indigenous rhizobia and its symbiotic effectiveness with soybean cultivar is important for development of knowledge about rhizobial ecology. In India, little is known about the genetic resources and diversity of rhizobia nodulating soybean. Indigenous bradyrhizobia isolated from root nodules of soybean plants, collected from traditional cultivating regions of two states (Madhya Pradesh and Uttar Pradesh) of India, were screened for bacteriophage sensitivity to identify successful broad host range symbiotic effectivity. Of 172 rhizobial isolates, 91 showed sensitivities to eight lytic phages and form ten groups on the basis of sensitivity patterns. The genetic diversity of 23 isolates belonging to different phage groups was assessed along with that of strains USDA123 and USDA94 by the restriction fragment length polymorphism (RFLP) analysis of 16S rDNA, intergenic spacer (IGS) (16S-23S rDNA), and DnaK regions. RFLP analysis of 16S rDNA formed 5 groups, whereas 19 and 9 groups were revealed by IGS and the DnaK genes, respectively. The IGS regions showed many amplified polymorphic bands. Nine isolates which revealed high RFLP polymorphism in the abovementioned regions (16S rRNA, IGS, DnaK) were used for 16S rRNA sequence analyses. The results indicate that taxonomically, all isolates were related to Rhizobium etli, Bradyrhizobium spp., and Bradyrhizobium yuanmingense. The doubling time of isolates varied from 9h (MPSR155) to 16.2h (MPSR068) in YM broth. Five isolates which did not show cross infectivity with isolated phage strains were studied for symbiotic efficiency. All isolates showed broad host range symbiotic effectiveness forming effective nodules on Vigna mungo, Vigna radiata, Vigna unguiculata, and Cajanus cajan. The present study provides information on genetic diversity and host range symbiosis of indigenous soybean rhizobia typed by different phages.

Molecular evidence for the interspecific hybrid origin of Ilex × wandoensis

The hybrid origin of Ilex × wandoensis C. F. Mill. & M. Kim was studied by polymerase chain reaction (PCR) using taxa-specific primers, restriction fragment length polymorphism (RFLP) analysis of the internal transcribed spacer 2 (ITS2) regions, and sequence analysis of PCR amplicons of the ITS region. Five accessions of I. integra Thunb., three accessions of I. cornuta Lindl. & Paxton and 22 accessions of I. × wandoensis, tentatively characterized based on morphological characters, were selected and subjected to PCR analysis. Amplified products of the selected accessions were sequenced. Significant variations were observed in leaf morphology of I. × wandoensis accessions based on the presence of single or multiple teeth and cuneate or rounded base of the leaf blade. Based on the PCR amplifications, sequence and RFLP analysis using HhaI and HaeIII restriction enzymes, the hybrid origin of I. × wandoensis was confirmed. Some accessions appeared to be introgressant and either crossed back to I. integra or to I. cornuta based on the RFLP polymorphisms. Analysis of the ITS2 region of the nuclear ribosomal DNA (nrDNA) using primers specific to certain taxon or taxa, and RFLP analysis of PCR amplicons proved to be useful tools to study the nature of interspecific hybridization and introgressions in complement with morphological characters.

RFLP analysis of mitochondrial DNA in two cytoplasmic male sterility systems (CMS-D2 and CMS-D8) of cotton

Cytoplasmic male sterility (CMS) in higher plants is a maternally inherited trait and CMS-associated genes are known to be located in the mitochondrial genome. However, CMS-inducing genes in CMS-D2 and CMS-D8 of Upland cotton (Gossypium hirsutum L., AD1) are currently unknown. The objective of this study was to identify potential candidate DNA or gene sequences for CMS-D2 and CMS-D8 through restriction fragment length polymorphism (RFLP) analysis. Seven mtDNA gene probes and five restriction enzymes were first used to compare D2 (from G. harknessii Brandegee) and AD1 cytoplasms. With cox1, cox2, and atp1 as probes, RFLP polymorphisms were detected with one or more restriction enzyme digestions. The most notable difference was an additional fragment in the normal AD1 cytoplasm detected by cox2 in digests of three enzymes, and by cox1 and atp1 in digests with PstI. The RFLP analysis was then conducted among CMS-D2, CMS-D8 (from G. trilobum (DC.) Skovst.), and AD1 cytoplasms. Two probes from maize, atp1 and atp6, detected polymorphism among the different cytoplasmic lines. However, no difference in RFLP patterns was noted between male sterile (A) and restorer (R) lines with the D2 or D8 cytoplasm, indicating that the presence of the D2 or D8 restorer gene does not affect mtDNA organization in Upland cotton. The results demonstrate that RFLP using atp1 and atp6 as probes can distinguish the three cytoplasms. The atp1 and atp6 in CMS-D8 and these two genes together with cox1 and cox2 in CMS-D2 could be the candidates of CMS-associated genes in the mitochondrial genome, providing information for further molecular studies and developing PCR-based markers for the CMS cytoplasms in breeding. This research represents the first work using RFLP to analyze the genetic basis of CMS in cotton.

Genetic Diversity of Heat-Labile Toxin Expressed by Enterotoxigenic Escherichia coli Strains Isolated from Humans

The natural diversity of the elt operons, encoding the heat-labile toxin LT-I (LT), carried by enterotoxigenic Escherichia coli (ETEC) strains isolated from humans was investigated. For many years, LT was supposed to be represented by a rather conserved toxin, and one derivative, produced by the reference H10407 strain, was intensively studied either as a virulence factor or as a vaccine adjuvant. Amplicons encompassing the two LT-encoding genes (eltA and eltB) of 51 human-derived ETEC strains, either LT(+) (25 strains) only or LT(+)/ST(+) (26 strains), isolated from asymptomatic (24 strains) or diarrheic (27 strains) subjects, were subjected to restriction fragment length polymorphism (RFLP) analysis and DNA sequencing. Seven polymorphic RFLP types of the H10407 strain were detected with six (BsaI, DdeI, HhaI, HincII, HphI, and MspI) restriction enzymes. Additionally, the single-nucleotide polymorphic analysis revealed 50 base changes in the elt operon, including 21 polymorphic sites at eltA and 9 at eltB. Based on the deduced amino acid sequences, 16 LT types were identified, including LT1, expressed by the H10407 strain and 23 other strains belonging to seven different serotypes, and LT2, expressed by 11 strains of six different serotypes. In vitro experiments carried out with purified toxins indicated that no significant differences in GM1-binding affinity could be detected among LT1, LT2, and LT4. However, LT4, but not other toxin types, showed reduced toxic activities measured either in vitro with cultured cells (Y-1 cells) or in vivo in rabbit ligated ileal loops. Collectively, these results indicate that the natural diversity of LTs produced by wild-type ETEC strains isolated from human hosts is considerably larger than previously assumed and may impact the pathogeneses of the strains and the epidemiology of the disease.

Fungal colonization of the roots of selected halophytes from Sečovlje salterns

To reveal their mycotrophic status, we have here analysed the fungal colonisation of the roots, and the identity of these fungi, of the most abundant halophytic plant species from Sečovlje salterns (Slovenia): Aster tripolium, Limonium angustifolium and Salicornia europaea. The highest frequency of fungal colonisation was seen for the roots of A. tripolium, followed by those of L. angustifolium and S. europaea. Hyphae and occasional microsclerotia, the assumed structures of dark septate endophytes, were seen in the roots of all three species, whereas arbuscules, as typical structures of arbuscular mycorrhiza, were seen in the roots of A. tripolium and in one specimen of L. angustifolium. Fungal partial small subunit ribosomal DNA (SSU-rDNA) fragments were amplifi ed from total root DNA extracts for further restriction fragment length polymorphism (RFLP) analyses of the structures of the fungal root communities. Fifteen different RFLP profi les were obtained that grouped into two major clusters. Two RFLP profi les strongly dominated in samples of all three of the plant species. Sequencing revealed that one of these profiles corresponds to the species Cylindrobasidium laeve (Basidiomycota), and the second to Capnobotryella sp. / Phaeotheca fi ssurella, putative dark septate endophytes from the class Dothideomycetes (Ascomycota). Intraspecific RFLP polymorphisms were demonstrated for both species. To our knowledge, this is the first report on dark septate endophyte fungi occurrence in roots of A. tripolium, L. angustifolium and S. europaea.

CpDNA‐RFLP in Ceramium (Rhodophyta): intraspecific polymorphism and species‐level phylogeny

Restriction fragment length polymorphism (RFLP) analysis of chloroplast (cp) DNA is a powerful tool for the study of microevolutionary processes in land plants, yet has not previously been applied to seaweed populations. We used cpDNA-RFLP, detected on Southern blots using labeled total plastid DNA, to search for intraspecific and intrapopulational cpDNA RFLP polymorphism in two species of the common red algal genus Ceramium in Ireland and Britain. In C. botryocarpum one polymorphism was detected in one individual among 18 from two populations. Twenty-six individuals of C. virgatum from five populations at three locations exhibited a total of four haplotypes. One was frequent (80.8% of individuals); the others were rare (7.7, 7.7, and 4.2%) and were private to particular populations. Polymorphism was observed in two populations. The corrected mean was 2.26 ± 0.36 haplotypes per population, which was within the typical range determined for higher plants using similar techniques. The spatial distribution of haplotypes was heterogeneous, with highly significant population differentiation (P = 0.00018; Fisher's exact test). Intraspecific polymorphism in C. virgatum had no impact on species-level phylogenetic reconstruction. This is the first unequivocal report of both intraspecific and intrapopulational cpDNA-RFLP polymorphism in algae.

Investigation of infection with Campylobacter jejuni in a man with hypogammaglobulinaemia using PCR-single-strandedconformational polymorphism (PCR-SSCP) typing

Abstract This study investigated several episodes of infection of Campylobacter jejuni in an immunocompromised male with hypogammaglobulinaemia, presenting with diarrhoea and bacteraemia over a 16-month period, by employing three phenotyping and four genotyping schemes, including the single-stranded conformational polymorphism (SSCP) technique to establish if infection was re-infection or persistent infection. Four isolates from blood culture and two faecal isolates of Campylobacter jejuni were obtained from the patient by direct selective plating on Skirrow Selective agar. Isolates were characterised at the sub-species level by Penner serology, Preston biotyping, Preston phage-typing, as well as E3CJC2 restriction fragment length polymorphism (RFLP), 16S ribotyping, flagellin ( flaA) RFLP and single-stranded conformational polymorphism (SSCP) analyses. Phenotyping and genotyping sub-species analyses demonstrated that the patient was infected with at least two different strains of Campylobacter jejuni, i.e. one strain that persisted throughout the 16-month period and another strain that was transient suggesting reinfection from a different source. SSCP analysis was the most discriminatory of all the typing schemes examined and demonstrated an altered genotype of the persistent strain, whereby there were subtle modifications to the hypervariable regions of the flaA gene. Overall, as SSCP examines the hypervariable region of the flaA gene and as this technique can detect point mutations, differences between SSCP banding patterns may represent markers and thus examine mutations that occur under immune selection, thereby permitting the C. jejuni to evade the host immune response. In conclusion, this study describes the novel use of SSCP genotyping of C. jejuni and demonstrated that this method is a highly discriminatory technique which may be beneficial in outbreak characterisation, but which is not suitable to examine the clonal patterns of C. jejuni over a long period of time.

Physical location of homoeologous groups 5 and 6 molecular markers mapped in Triticum aestivum L.

In situ hybridization was used to map 21 restriction fragment length polymorphism (RFLP) probes to linkage groups 5 and 6 of hexaploid wheat (Triticum aestivum L. em Thell.) in order to compare physical distances and genetic distances between adjacent markers. All 21 probes hybridized to the corresponding homoeologous chromosome arms. The linear order and linkage relationships among the DNA probes on the in situ-based physical maps were generally the same as those on the RFLP-based genetic maps. However, significant differences were observed between the centiMorgan distances on a linkage map and the physical distances of the probes using in situ-based techniques. The results indicated a clustering of polymorphic RFLP markers in the middle of all of the homoeologous group 5 and 6 chromosome arms. This suggests that the available linkage maps do not completely cover the physical length of the chromosomes. As with the genetic maps, the physical map clearly showed the presence of nonhomoeologous rearrangements in the terminal regions of chromosome arms 5AL and 6BS. However, the physical mapping gave an indication of the physical size of the rearrangements as well as their arm location.

Polymorphisms in the Vitamin D Receptor Gene and the Androgen Receptor Gene and the Risk of Benign Prostatic Hyperplasia

Objective: Little is known about risk factors for the development of benign prostatic hyperplasia (BPH). Recently, associations were observed between prostate cancer (CaP) risk and polymorphisms in the vitamin D receptor (VDR) gene and the androgen receptor (AR) gene. Since both receptors are relevant for prostate growth, the VDR and AR are also expected to be involved in the development of BPH. The objective of this study is to establish the relationship between the risk of BPH and a polymorphism in the number of CAG repeats in the AR gene and a TaqI restriction enzyme polymorphism in the VDR gene.Methods: For this study, 98 patients who had been treated for BPH–related complaints and 61 convenience controls (predominantly bladder cancer patients) were recruited from the outpatient clinic. DNA was isolated from peripheral blood, and genotyping was performed with PCR–based methods. Means as well as odds ratios (ORs) with 95% confidence intervals (CI) were calculated using SPSS software.Results: The mean number of CAG repeats in the AR gene in patients and controls was found to be similar: 21.8 (SD = 2.8) and 21.9 (SD = 2.9), respectively. In the subgroup of patients with a prostate volume of at least 50 cm³, the mean number of repeats was 21.5 (SD = 2.6). The OR for BPH for individuals with homozygous presence of the VDR TaqI restriction fragment length polymorphism (RFLP) (tt) versus individuals with homozygous absence (TT) or heterozygotes (Tt) was found to be 1.0 (95% CI 0.4–2.4). For individuals with a prostate volume of at least 50 cm³, the OR was 1.2 (95% CI 0.5–3.2).Conclusion: Unlike earlier observations in prostate cancer, we did not find an association between the CAG repeat polymorphism in the AR gene and the TaqI RFLP polymorphism in the VDR gene and the risk of BPH.

Improved Polymerase Chain Reaction-Based Mitochondrial Genotype Assay for Identification of the Africanized Honey Bee (Hymenoptera: Apidae)

A polymerase chain reaction (PCR)-based restriction fragment-length polymorphism (RFLP) assay was developed that discriminates among the 4 mitotypes found in North, Central, and South American honey bee racial groups—eastern European (Apis mellifera ligustica Spinola, caucasica Gorbachev, and carnica Pollman), western European (A. m. mellifera Linnaeus), Egyptian (A. m. lamarckii Cockerell Lepeletier), and sub-Saharan African (A. m. scutellata). Before the development of this assay, 13% of southern Californian feral bees collected before the arrival of the Apis mellifera scutellata (Africanized) race were found to contain a non-European mitochondrial genotype that could not be distinguished from that of A. m. scutellata. DNA sequence analysis suggests the unusual mitotype to be that of A. m. lamarckii. An RFLP polymorphism was identified that distinguished this subspecies from all others present in North America. This polymorphism was not found in any of 96 bees collected primarily in Mexico and Central America. Thus, the Egyptian mitochondrial type is either absent or extremely rare in these regions. The PCR assay also distin- guishes A. m. lamarckii from 2 other north African racial types, A. m. intermissa Buttel-Reepen and A. m. sahariensis Baldensperger.

Comparisons of RFLP and PCR-based markers to detect polymorphism between wheat cultivars.

Previously chromosome 3A of wheat (Triticum aestivum L.) was reported to carry genes influencing yield, yield components, plant height, and anthesis date. The objective of current study was to survey various molecular marker systems for their ability to detect polymorphism between wheat cultivars Cheyenne(CNN) and Wichita (WI), particularly for chromosome3A. Seventy-seven `sequence tagged site' (STS), 10simple sequence repeat (SSR), 40 randomly amplified polymorphic DNA (RAPD) markers, and 52 restriction fragment length polymorphism (RFLP) probes for wheat homoeologous group 3 chromosomes, were investigated. Three (3.9%) STS-PCR primer sets amplified polymorphic fragments for the two cultivars, of which one was polymorphic for chromosome 3A. Sixty percent of SSR markers detected polymorphism between CNN and WI of which 50% were polymorphic for chromosome 3A. Twenty percent of RAPD markers detected polymorphism between CNN and WI in general, but none of these detected polymorphism for chromosome 3A. Of the fifty-two RFLP probes, 78.8% detected polymorphism between CNN and WI for group 3 chromosomes with one or more of seven restriction enzymes and 42% of the polymorphic fragements were for chromosome 3A. These high levels of RFLP and SSR polymorphisms between two related wheat cultivars could be used to map and tag genes influencing important agronomic traits. It may also be important to reconsider RFLP as the most suitable marker system at least for anchor maps of closely related wheat cultivars.

RFLP markers associated with major genes controlling heading date evaluated in a barley germ plasm pool

Adjustment of crop phenology to resources and constraints of the production environment is crucial for barley adaptation to dry-land environments. The aims of this study were to characterize (under field and under controlled conditions) the diverse heading responses of barley cultivars grown in Spain, and to identify phenotypic trait-molecular marker associations with restriction fragment length polymorphism (RFLP) probes which are linked to major loci controlling vernalization (Sh and Sh2) and photoperiod (Ppd-H1 and Ppd-H2) sensitivities in barley. Thirty-two cultivars were sown in field trials for 3 years in four locations in northern Spain (10 autumn and nine winter sowings), and for 1 year in southern Spain (autumn sowing), and 2 years in Scotland (spring sowings); they were also studied under four glasshouse treatments, combining presence/absence of vernalization and short/long photoperiod. These cultivars were examined for the presence of RFLP polymorphisms with four probes and three restriction enzymes. Analyses of variance detected a number of associations which were quite consistent with expectations, suggesting that marker-loci associations found in mapping populations are, to some extent, maintained in a germ plasm pool, and may be useful for germ plasm characterization and marker-assisted selection in breeding programmes. Two possible epistatic interactions among the probes were detected, one of them possibly related to a short-day vernalization response.

A T1083C polymorphism in the human adenosine A2a receptor gene is not associated with essential hypertension

The adenosine A2a receptor (A2aAR) gene is thought to be involved in essential hypertension because adenosine elicits vasodilation and decreases arterial blood pressure via this receptor, and because disruption of the A2aAR gene increases blood pressure in mice. Therefore, using a restriction fragment length polymorphism (RFLP) of the A2aAR gene, we performed an association study in patients with essential hypertension. One hundred forty-two patients with essential hypertension and 142 age-matched subjects with normal blood pressure were studied. Polymerase chain reaction (PCR) was applied to amplify the T1083C polymorphic site in the A2aAR gene, and restriction analysis of the PCR product was employed to score the T and C alleles. Overall distributions of allele frequencies in the two groups were not significantly different. Thus, the alleles detected by this RFLP polymorphism in the A2aAR gene are not associated with essential hypertension.

Molecular epidemiology of penicillin-resistant Streptococcus pneumoniae isolates recovered in Italy from 1993 to 1996.

Thirty-nine penicillin-resistant Streptococcus pneumoniae isolates recovered among the approximately 700 pneumococcal strains collected from 1993 to 1996 in central and northern Italy were analyzed for several characteristics, including serotype, antibiotic susceptibility profile, chromosomal relatedness (by using pulsed-field gel electrophoresis [PFGE]), restriction fragment length polymorphism (RFLP) of the penicillin-binding protein (PBP) genes 1A, 2X, and 2B, and the presence of a variety of antibiotic resistance genes (determined by hybridization with appropriate DNA probes). The MICs of penicillin for most of the isolates (30 of 39) were high, in the range of 1 microgram/ml or higher, and these 30 isolates carried additional resistance traits to two or more drugs (erythromycin, chloramphenicol, co-trimoxazole, and tetracycline) and expressed serotypes 9, 19, and 23 and three distinct PFGE patterns. More than half (22 of 30) of the isolates for which MICs were high were identified as representatives of two widespread international epidemic clones of S. pneumoniae. The first one of these clones (seven isolates) expressed serotype 23F and possessed all properties characteristic of the widespread Spanish/USA international clone. Seven additional strains with serotype 19 also had the same PFGE pattern, PBP gene, and RFLP polymorphisms, and other properties typical of the serotype 23 Spanish/USA clone, suggesting that these strains were the products of a capsular transformation event (from serotype 23F to serotype 19) in which the Spanish/USA clone was the recipient. The second international clone was represented by eight serotype 9 isolates which were resistant to penicillin and trimethoprim-sulfamethoxazole and had the molecular properties of the French/Spanish epidemic clone. The remaining eight isolates for which penicillin MICs were high appeared to represent a hitherto-undescribed "Italian" clone; they had a novel PFGE type, unique RFLPs for the PBP genes, and resistance to tetracycline, trimethoprim-sulfamethoxazole, and erythromycin, and the penicillin MICs for these isolates were 2 to 4 microgram/ml.

Comparison of three restriction endonucleases in IS 1245-based RFLP typing of Mycobacterium avium

IS1245-based restriction fragment length polymorphism (RFLP) analysis has been proposed recently for molecular typing of Mycobacterium avium isolates. As there is no standardised method with respect to the optimal restriction enzyme, three restriction endonucleases were tested for analysis of 17 human isolates. The restriction endonucleases, selected on the basis of the physical maps of IS1245 and of the highly homologous IS1311, were BsaAI, that cleaves IS1245, PvuII, that cleaves IS1311, and NruI, that cleaves both IS1245 and IS1311. All the restriction endonucleases yielded polymorphic and complex RFLP patterns. However, BsaAI- and NruI-generated bands were more evenly distributed and easier to detect than PvuII-generated bands, most of which clustered in a narrow zone of the fingerprint. In some cases, DNA digestion with BsaAI or NruI yielded probe-specific restriction fragments of molecular size lower than expected. Moreover, digestion with NruI, which was expected to generate the highest numbers of bands in all the isolates, yielded fewer bands than were obtained with BsaAI or PvuII in 14 and 5 isolates, respectively. These findings might suggest the existence of unidentified IS1245-related insertion element(s) in M. avium isolates. Computer analysis of the IS1245-based RFLP patterns of M. avium isolates showed that the restriction endonucleases were capable, although with minor differences, of defining distinct banding patterns and clusters of identical or highly related isolates, thus confirming IS1245-based RFLP analysis as a useful technique for epidemiological studies.

New chicken Rfp-Y haplotypes on the basis of MHC class II RFLP and MLC analyses.

New chicken Rfp-Y haplotypes were determined by the use of restriction fragment length polymorphism (RFLP) and mixed lymphocyte culture (MLC) in four different chicken haplotypes, B15, B19, B21, B201. The RFLP polymorphism was mapped to the Rfp-Y system by the use of a subclone (18.1) which maps near a polymorphic lectin gene located in the Rfp-Y system and DNA from families with known segregation of the implicated RFLP polymorphism. For the first time it is shown that major histocompatibility complex class II genes in the Rfp-Y system have functional implications. Sequence information of the B1 domain of the proposed Rfp-Y haplotypes was obtained which supported the functional data.

Rsa I polymorphism at the cytochrome P4502E1 locus is not related to the risk of alcohol-related severe liver disease.

Ethanol-inducible cytochrome P4502E1 is the main pathway in the non-alcohol dehydrogenase oxidation of ethanol. Its coding gene, CYP2E1, is polymorphic at the Rsa I restriction site in the 5'-flanking region. The mutant genotype c2c2 has a higher transcriptional activity than the genotype c1c1 or c1c2. Heavy drinkers carrying the c2 allele might be at a higher risk of alcoholic cirrhosis since they might synthesize greater amounts of acetaldehyde, the compound believed responsible for hepatotoxicity of ethanol. With the aim of establishing if the c2 allele increases the risk of cirrhosis in heavy drinkers, we studied 58 (6 female) chronic heavy drinkers with liver cirrhosis and 137 healthy normal controls of the same ethnic (white Spaniards) origin. After extraction of DNA from white blood cells, alleles c1 and c2 of CYP2E1 were identified by restriction fragment length polymorphism (RFLP) with endonuclease Rsa I. Fifty-six patients and 130 controls were classified as homozygous c1c1 and two and seven, respectively, as heterozygous c1c2. No homozygous c2c2 were detected. The c2 allele frequencies were 0.017 in patients and 0.026 in controls (non-significant differences). We conclude that the Rsa I RFLP polymorphism is probably not related to the risk of cirrhosis in Spanish heavy drinkers.

A comparative analysis of the genetic relationships between rye cultivars using RFLP and RAPD markers

The genetic similarities of eight closely related rye cultivars were estimated using two molecular marking techniques: restriction fragment length polymorphism (RFLP) and random amplified polymorphic DNA (RAPD). Cultivars were evaluated for variation by 11 random cDNA and genomic clones used in combination with four restriction enzymes and 40 decamer primers. A total of 53 polymorphic RFLP fragments and 94 polymorphic RAPD fragments were observed. Based on the presence/absence of fragments, two genetic similarity matrices were calculated which were then used in cluster analysis. Differences between pair of cultivars were observed in RFLP and RAPD dendrograms. RFLP analysis produced estimates of genetic relationships more in accordance with the partially known pedigree of the cultivars than did RAPD analysis. The use of bulk samples of DNA in these analyses affected the sensitivity of RAPD assays more strongly. Dendrograms which took into account all fragments produced, either by RFLP or RAPD, reflected better the relationships between cultivars than did dendrograms based on only one type of marker. This reflects the importance of the number of markers used in determining the genetic relationships between genotypes.