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Genotyping human and bovine isolates of Cryptosporidium parvum by polymerase chain reaction-restriction fragment length polymorphism analysis of a repetitive DNA sequence.

In order to define transmission routes of cryptosporidiosis and develop markers that distinguish Cryptosporidium parvum isolates, we have identified 2 polymorphic restriction enzyme sites in a C. parvum repetitive DNA sequence. The target sequence was amplified by polymerase chain reaction from 100 to 500 oocysts and the amplified product was subjected to restriction enzyme digestion. Typing of 23 isolates showed that 10/10 calf isolates had the same profile. In contrast, 2 patterns were observed among human isolates: 7/13 displayed the calf profile, and 6/13 presented another pattern. The PCR-RFLP assay described here is a sensitive tool to distinguish C. parvum isolates.

Animals↗

Segregation of restriction fragment length polymorphism in an interspecies cross of laboratory and wild mice indicates tight linkage of the murine IFN-beta gene to the murine IFN-alpha genes.

Southern blot analysis with murine (Mu) interferon (IFN)-alpha cDNA of restricted genomic DNA of three inbred strains of mice belonging to the species Mus musculus domesticus (BALB/c, C57BL/6, and DBA/2) revealed only a limited degree of polymorphism. For example, with HindIII there were only two polymorphic bands out of 14 hybridizing fragments. With Mu IFN-beta cDNA there was no polymorphism at all between BALB/c and C57BL/6 in DNA restricted with seven different enzymes. In contrast, HindIII-restricted DNA of an inbred strain of wild mice (M. spretus Lataste) hybridized with the IFN-alpha probe displayed a high degree of polymorphism compared with the three strains of laboratory mice and was also polymorphic when probed with IFN-beta cDNA. Although M. musculus domesticus and M. spretus Lataste represent different species, certain interspecies crosses are possible in the laboratory. This enabled us to follow segregation of restriction fragment length polymorphism in HindIII-restricted DNA obtained from 18 backcross progeny of a (DBA/2 X M. spretus)F1 X DBA/2 interspecies cross. There was complete coincidence between the segregation of parental (DBA/2) and (DBA/2 X M. spretus)F1-type IFN-beta and IFN-alpha restriction fragment length polymorphism, indicating tight linkage of the IFN-beta and IFN-alpha genes. In addition, in 15 of 18 progeny the segregation coincided with that of the brown locus on chromosome 4, in accord with previous results obtained with the IFN-alpha probe in strains derived from crosses between BALB/c and C57BL/6 mice. Thus, the Mu IFN-beta gene is tightly linked to the Mu IFN-alpha gene cluster on chromosome 4 near the brown locus.

Animals↗

Identification of restriction fragment length polymorphism and random amplified polymorphic DNA markers linked to downy mildew resistance genes in lettuce, using near-isogenic lines.

Near-isogenic lines were used to identify restriction fragment length polymorphism (RFLP) and random amplified polymorphic DNA (RAPD) markers linked to genes for resistance to downy mildew (Dm) in lettuce. Two pairs of near-isogenic lines that differed for Dm1 plus Dm3 and one pair of near-isogenic lines that differed for Dm11 were used as sources of DNA. Over 500 cDNAs and 212 arbitrary 10-mer oligonucleotide primers were screened for their ability to detect polymorphism between the near-isogenic lines. Four RFLP markers and four RAPD markers were identified as linked to the Dm1 and Dm3 region. Dm1 and Dm3 are members of a cluster of seven Dm genes. Marker CL922 was absolutely linked to Dm15 and Dm16, which are part of this cluster. Six RAPD markers were identified as linked to the Dm11 region. The use of RAPD markers allowed us to increase the density of markers in the two Dm regions in a short time. These regions were previously only sparsely populated with RFLP markers. The rapid screening and identification of tightly linked markers to the target genes demonstrated the potential of RAPD markers for saturating genetic maps.

Base Sequence↗

HLA class II genotyping by polymerase chain reaction-restriction fragment length polymorphism method (PCR-RFLP) in systemic lupus erythematosus patients.

Recently, genotyping of HLA class II alleles by digestion of polymerase chain reaction (PCR) amplified polymorphic DNA region with restriction endonucleases (PCR-RFLP) has been reported and claimed to be a simple and reliable technique. We tested the use of PCR-RFLP for genotyping of DQA 1, DQB 1, DPB 1 and DRB 1 alleles of 40 normal individuals and 19 patients with systemic lupus erythematosus (SLE). In agreement with previous reports, we observed an association of SLE with the DRB 1*1501-DQA 1*0102-DQB 1*0602 haplotype and noted an unreported association with DPB 1*0501. Although these observations must be confirmed with larger sample sizes, we believe that the PCR-RFLP is simpler and more practical than the sequence-specific oligonucleotides (PCR-SSO) for genotyping and can be invaluable to the more widespread molecular determination of HLA-class II specificities, including in non-specialized laboratories.

Genotype↗