Radiation hybrid mapping of the two highly homologous human-variant pMCHL genes by PCR-SSCP.
When gene loci are very similar in sequence, as in gene families or multiple pseudogenes, it is difficult to determine the specific location of the individual genes. We show here that applying PCR-SSCP to a radiation hybrid panel allowed mapping and specific sequencing of two genes with only a few sequence differences. Human-variant forms of the promelanin-concentrating hormone (pMCH) gene are found in two locations in the genome, previously localized by FISH to 5p14 and 5q12-q13. Without prior knowledge of sequence variation between the loci, we observed a difference in migration pattern in PCR-SSCP, indicating the presence of at least one point of sequence divergence. PCR-SSCP of 93 samples from a human-hamster radiation hybrid panel revealed the location of the genes to be between markers WI-4804 and AFM225YC5 on chromosome 5p, and between markers WI-3133 and WI-4225 on chromosome 5q. Sequencing of the two 680-bp PCR products from the hybrid panel demonstrated 3 bases of sequence difference between the 5p and 5q locations.
Mental Health Research Institute, University of Michigan, Ann Arbor, Michigan 48109 USA.
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Miller
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Thompson
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Burmeister
D000483:Alleles; D000818:Animals; D002874:Chromosome Mapping / Q000379:methods*; D002895:Chromosomes, Human, Pair 5 / Q000235:genetics; D006224:Cricetinae; D006801:Humans; D006822:Hybrid Cells / Q000378:metabolism* / Q000528:radiation effects; D007028:Hypothalamic Hormones / Q000235:genetics*; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods*; D018807:Polymorphism, Single-Stranded Conformational; D011498:Protein Precursors / Q000235:genetics*; D017422:Sequence Analysis, DNA; D012689:Sequence Homology, Nucleic Acid
D007028:Hypothalamic Hormones; D011498:Protein Precursors; C059991:melanin-concentrating hormone precursors