Identification of the homologous beige and Chediak-Higashi syndrome genes - PubMed (original) (raw)

FIG. 2

Intragenic deletion of Lyst in bg11J DNA. a–c, Southern blot identification of an intragenic Lyst deletion in bg11J DNA. A Southern blot was sequentially hybridized with 3 Lyst probes: a, the probe (nucleotides 1,262–3,433 of Lyst cDNA) extends upstream from the bg11J deletion; b, the probe (nucleotides 2,835–3,433 of Lyst cDNA) is completely deleted; c, the probe (nucleotides 3,594–4,237 of Lyst cDNA) extends downstream from the bg11J deletion. Restriction endonucleases are indicated at the bottom of each panel, and molecular size standards (in kb) are shown to the left. Similar results were obtained with 3 additional restriction endonucleases (data not shown). The bg11J mutation was discovered in 1992 at The Jackson Laboratory in a C57BL/6J-jb mouse at generation N4 after transfer from B6C3Fe-a/a. The mutation jb had, in turn, been discovered 14 generations earlier in B6C3Fe-a/a-hyh mice at generation N3 after transfer form C57BL/10J. The hyh mutation arose in C57BL710J mice, and was maintained in that strain until transfer at F15. Thus the possible contributors of genetic information to bg11J include C57BL/6J, C3HeB/FeJ and C57BL/10J. Southern blots were prepared from genomic DNA of all potential progenitor mouse strains, but only C57B/V10J, C57BL/6J and C57BI/6J-bg11J are shown, d–f, PCR analysis of the bg11J deletion. C57BL/10J, C3HeB/FeJ, C57BL/6J and C57BL/6J-bg11J genomic DNA and Lyst cDNA were used as templates in the PCR reactions. Amplicons illustrated correspond to: d, Lyst cDNA nucleotides 1,337–1,837, which represent exon β and are upstream from the deletion; e, nucleotides 2,670–3,210, which represent exon γ, which is deleted in bg11J DNA; and f, nucleotides 4,913–5,433, which represents an exon downstream from the deletion. No amplicon was observed in control PCR reactions performed without template. More than 30 other STSs that had been localized within the bg non-recombinant interval PCR amplified normally from bg11J DNA. g, Genomic structure of Lyst in the vicinity of the bg11J deletion. Lyst exons (α, β, γ, δ, ε and φ) are depicted by black boxes, and intervening introns by a solid line. Nucleotides of the mouse Lyst cDNA that correspond to exonic boundaries are indicated above the boxes. The 3′ end of exon β, and all of exons γ and δ, are deleted in bg11J DNA. The region of Lyst protein that is deleted in bg11J contains a pair of helices with N-terminal phosphorylation sites. Genomic structure and intronic sequences were ascertained by sequence analysis of nested PCR products, performed with exonic primers and P1 clone DNA as template. Boundaries of the bg11J deletion were determined by PCR of genomic DNA.