Rao_Miller_1997

Rao_Miller_1997 - REPORTS 15. sternum, and lower jaw...

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sternum, and lower jaw aplasia were also commonly seen. 15. W. F. Lever and G. S. Lever, Histopathology of the Skin (Lippincott, Philadelphia, 1990); S. Miller, J. Am. Acad. Dermatol. 24 , 1 (1991). 16. A. C. Markey, E. B. Lane, D. M. MacDonald, I. M. Leigh, Br. J. Dermatol. 126 , 154 (1992). 17. A. Stoler, R. Kopan, M. Duvic, E. Fuchs, J. Cell. Biol. 107 , 427 (1988). 18. J. Stanley, J. Beckwith, R. Fuller, S. Katz, Cancer 50 , 1486 (1982); P. Savoia, L. Trusolino, E. Pepino, O. Cremona, P. Marchisio, J. Invest. Dermatol. 101 , 352 (1993); Z. Lazarova, N. Domloge-Hultsch, K. Yancey, Exp. Dermatol. 4 , 121 (1995); J. Fairley, P. Heintz, M. Neuburg, L. Diaz, G. Giudice, Br. J. Der- matol. 133 , 385 (1995). 19. M. Cooper and H. Pinkus, Cancer Res. 37 , 2544 (1977); R. E. Grimwood et al. , Cancer 56 , 519 (1985); G. Stamp, A. Quaba, A. Braithwaite, N. A. Wright, J. Pathol. 156 , 213 (1988); S. Hales, G. Stamp, M. Evans, K. Fleming, Br. J. Dermatol. 120 , 351 (1989). 20. We performed mouse skin grafts as in ( 27 ), except that we used transgenic or wild-type dorsal trunk skin from B6CBF2 embryos that had been dissected away from underlying muscle and grafted it onto 8- to 12-week-old male scid recipient mice. Dressings were removed after 3 weeks. Each animal was pho- tographed weekly. 21. Genomic sequences containing SHH were isolated from a bacterial artificial chromosome library ob- tained from Research Genetics. Primers used to screen this library from exon 2 [ACC GAG GGC TGG GAC GAA GAT GGC and GCG AGC CAG CAT GCC GTA CTT GCT G ( 28 )] identified BAC 270A17, which was digested with restriction enzymes and ligated with vectorette linkers ( 29 ). Exon-intron boundaries for the three exons were determined by sequencing polymerase chain reaction (PCR) products amplified using the universal vectorette primer and SHH cDNA primers selected from published sequences. Since we were unable to obtain sequences from the exon 2–intron 3’ boundary, a primer from the 3 9 end of exon 2 was used. Primers used to amplify genomic SHH were as follows: exon 1, CCG CCG CGC GCA CTC G and AAG GAG CGG GTG AAA TCA CC; exon 2, TAA CGT GTC CGT CGG TGG G and TGC TTT CAC CGA GCA GTG G; and exon 3, CCT CCT CCC CGA GAC GC and GGC CCC CTC CCG CGC C. Mutations were identified by single-strand confor- mation polymorphism (SSCP) analysis of PCR prod- ucts amplified from genomic DNA. The PCR prod- ucts were sequenced on both strands directly from the PCR-produced templates and after cloning into Bluescript. Subsequent to our completion of this work, another group has published intron sequenc- es and primers useful for amplifying SHH exons from genomic DNA ( 26 ). 22. DNA from one other BCC had a methionine to iso- leucine change at position 115, but this change was also present in DNA from the patient’s blood. This unusually large BCC was diagnosed at age 40 in a patient with no other phenotypic abnormalities sug- gestive of BCNS. 23. K. F. Liem, G. Tremmi, H. Roelink, T. M. Jessell,
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Rao_Miller_1997 - REPORTS 15. sternum, and lower jaw...

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