A PstI restriction fragment length polymorphism in sheep detected with a human myelin basic protein (MBP) probe.

Source and Description of Probe. The probe was obtained from a pUC8 clone, pHBP-1, that carried the complete coding sequence of the human MBP gene (Kamholz et al., 1986; American Type Culture Collection, Rockville, MD). A 1.2-kb cDNA insert was excised from pHBP-1 using the enzyme EcoRI. Method of Detection. Sheep genomic DNA was extracted from whole blood. The DNA digests were resolved in 1% agarose gels and Southern blotted onto nylon membranes. The blots were prehybridized at 60°C in 5x SSPE ( . 9 M NaCl, 50 mM sodium phosphate, 5 mM EDTA, pH 7.4), 5x Denhardt’s reagent, 5% SDS, and .1 mg/mL sheared denatured herring sperm DNA. Hybridization was done at 60°C in the same solution, but without the Denhardt’s reagent. Final washin was in l x SSC and .l% SDS a t 60°C. The MBP probe was [f2P]-labeled using the random priming method. Description of Polymorphism. Twenty-four unrelated animals were screened for RFLP with enzymes EcoRI, MspI, PstI, and TuqI. Polymorphisms were detected with PstI and TuqI; only the PstI RFLP was further characterized. In the PstI polymorphism, two sets of allelic bands were observed, designated set A and set B: the set A alleles were A1 (4.6 kb ) and A2 (3.3 kb), and the set B alleles were B1 (1.5 k b ) and B2 ( . 8 kb) (Figure 1). Besides the variant bands, there was a monomorphic band of 1.2 kb. All four possible haplotypes (AlB1, AlB2, A2B1, and A2B2) were discernible in the animal samples used in this work. Inheritance Pattern. Five two-generation families, with two offspring each, were checked for Mendelian inheritance. Codominant segregation of the A and B alleles was confirmed in four and two families, respectively. Transmission of A1B2 and A2B2 haplotypes was observed in four of the families. Frequency. Frequencies of the A and B alleles, estimated from 33 unrelated individuals, were 5 2 ( A l ) , .48 (A2), .2 ( B l ) , and .8 (B2) . Haplotype frequencies, calculated from 29 of the animals, were .05 ( A l B l ) , .47 (AlB2) , .1 (A2B1), and .38 (A2B2). The sheep breeds included Coopworth, Merino, Perendale, Romney, and their crosses. The PIC values (Botstein et al., 1980) were generally low for the individual sets of alleles: .374 (A alleles) and .269 ( B alleles). When haplotyping was used, the MBP PstI RFLP became a more informative marker, as indicated by its increased PIC value of 549. Chromosomal Location. Unknown.