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Results: 4

FIG. 4

FIG. 4. From: Identification of the homologous beige and Chediak-Higashi syndrome genes.

Northern blot analysis of mouse and human Lyst. a, b, Northern blots of 2 µg poly(A)+ RNA from various mouse tissues (Clontech) hybridized with probes that correspond to a, nucleotides 4,423–4,631, and b, nucleotides 1,430–2,457 (exon β) of mouse Lyst cDNA. c, d, Northern blot of 2µg poly(A)+ RNA from c, various human lymphoid tissues, or d, human cancer cell lines, hybridized with a probe that corresponds to nucleotides 357–800 of human LYST cDNA. Molecular size standards (in kb) are shown to the left. Hybridization of mouse mRNA with probes from mouse Lyst exons α and γ gave identical results to those shown with exon β (b), whereas probes from exons δ, ε, and ϕ gave results identical to those shown in a.

Maria D. F. S. Barbosa, et al. Nature. ;382(6588):262-265.
FIG. 1

FIG. 1. From: Identification of the homologous beige and Chediak-Higashi syndrome genes.

Genetic and physical map of the bg non-recombinant interval on mouse chromosome 13 showing the location of Lyst. Mouse chromosome 13 is shown by a horizontal line with the centromere on the left. The bg critical region is delineated by chromosome crossovers (denoted with an X) in animals 134 and 137 of an intersub-specific mouse backcross12 [C57BL/6J – bgJ × (C57BL/6J – bgJ × CAST/EiJ)F1]. Microsatellite markers D13MM172 and D13Mit239 flank bg proximally; D13Mitl62 and D13Mit305 lie distal to bg (indicated by turquoise circles). YAC and P1 clones identified by PCR screening25,26 with oligo-nucleotides corresponding to Nid or D13Sfkl3 are shown above the chromosome. Novel sequence-tagged sites (STS, indicated by dark blue circles), generated by inverse repetitive element PCR or direct cDNA selection13, were used to order clones within the contiguous array. Novel mouse chromosome 13 STSs are numbered 1–18, corresponding to D13Sfkl to D13Sfkl8, respectively13. Lyst was isolated from YAC 195A8, a 650-kb clone, by using direct cDNA selection13. The physical location of Lyst-associated STSs on YAC and P1 clones are shown in red (MGD accession number MGD-PMEX-13).

Maria D. F. S. Barbosa, et al. Nature. ;382(6588):262-265.
FIG. 3

FIG. 3. From: Identification of the homologous beige and Chediak-Higashi syndrome genes.

Amino-acid sequence and predicted peptide structure of mouse and human Lyst cDNAs. a, Predicted amino-acid sequence of the mouse Lyst cDNA. The region (residues 463–536) with sequence similarity to stathmin is underlined, as is a C-terminal prenylation motif, b, Predicted secondary structure27–30 and motifs of mouse Lyst protein. The solid line denotes the protein sequence. Above the line are the stathmin-like region and areas rich in acid (A; Asp, Glu), basic (B; His, Arg, Lys), and turn (T; Pro, Gly, Ser, Thr, Asp) residues, the latter, especially at regions 600–625 and 875–895, are too long to be a simple turn between secondary structural elements, and are either very flexible or very rigid (in a manner reminiscent of collagen, although they do not fit the collagen consensus motifs). Helices predicted with >80% confidence are shown as solid lines below the protein sequence, and asterisks identify helices with N-terminal phosphorylation motifs. A tyrosine phosphorylation motif (residue 1,214) is designated Y. c, Nuceiotide and predicted amino-acid sequence of the 5′ end of the human LYST coding domain showing a frame-shift mutation in a patient with CHS. Line A illustrates the predicted normal human amino-acid sequence; line B shows the normal human nucleotide sequence; line C shows the nucleotide sequence from a patient with CHS; line D shows the predicted amino-acid sequence from the CHS patient. The CHS nucleotide sequence contains an insertional mutation at nucleotide 117–118 that results in a frame shift and premature termination (asterisk).

Maria D. F. S. Barbosa, et al. Nature. ;382(6588):262-265.
FIG. 2

FIG. 2. From: Identification of the homologous beige and Chediak-Higashi syndrome genes.

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 hybridized11 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 template14. Boundaries of the bg11J deletion were determined by PCR of genomic DNA.

Maria D. F. S. Barbosa, et al. Nature. ;382(6588):262-265.

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