High-resolution FISH analysis.

Map order, orientation, and gap or overlap distance of closely linked DNA probes may be determined using fluorescent hybridization to decondensed DNA. The linear arrangement of released chromatin fibers not only simplifies the task of gene ordering, but also provides higher resolution with probes separated by greater distances than can be achieved in FISH with intact interphase nuclei. The Basic Protocol 1 of this unit describes an alkaline lysis procedure for generating free chromatin from cultured cells for FISH analysis. A support protocol describes an empirical approach to optimize conditions for preparation of free chromatin. An Alternate Protocol 1 provides a method for producing free chromatin from cultured lymphocytes with drug treatment. The Basic Protocol 2, high-resolution FISH mapping with free chromatin, is a modification of the method used for FISH mapping of interphase nuclei.link_to_subscribed_fulltex

Genomic Organization and FISH Mapping of Human Pmel 17, the Putative Silver Locus

The Pmel 17 gene is expressed preferentially in pigment cells. It has been mapped to human chromosome 12 pter-q21 and mouse chromosome 10, near the silver locus. The Pmel 17 gene contains an insertional mutation at its carboxyl terminus in the silver mouse, suggesting that the silver locus might correspond to the gene. In the current studies, we have isolated and characterized human Pmel 17 genomic clones and employed FISH mapping for a precise localization of this gene in the human chromosome. The FISH mapping placed the Pmel 17 gene at human chromosome 12 q12-q13. The human gene consists of nine exons and eight introns, and the entire coding region of the gene spans approximately 7.9 kb of the human chromosome 12. The putative functional domains, such as the signal sequence, histidine-rich, 26-amino acid repeats, cysteine-rich, transmembrane and cytoplasmic domains, were encoded by separate exons. Cis-transcription elements such as a TATA, a CAT and other potential elements for pigment cell-specific gene expression were found within 1100 base pairs of the 5'flanking region

Genomic organization and FISH mapping of human Pmel 17, the putative silver locus

The Pmel 17 gene is expressed preferentially in pigment cells. It has been mapped to human chromosome 12 pter-q21 and mouse chromosome 10, near the silver locus. The Pmel 17 gene contains an insertional mutation at its carboxyl terminus in the silver mouse, suggesting that the silver locus might correspond to the gene. In the current studies, we have isolated and characterized human Pmel 17 genomic clones and employed FISH mapping for a precise localization of this gene in the human chromosome. The FISH mapping placed the Pmel 17 gene at human chromosome 12 q12-q13. The human gene consists of nine exons and eight introns, and the entire coding region of the gene spans approximately 7.9 kb of the human chromosome 12. The putative functional domains, such as the signal sequence, histidine-rich, 26-amino acid repeats, cysteine-rich, transmembrane and cytoplasmic domains, were encoded by separate exons. Cis-transcription elements such as a TATA, a CAT and other potential elements for pigment cell-specific gene expression were found within 1100 base pairs of the 5' flanking region.This work was supported by NIH Grant R01 AR-40248 (BSK). Kack-Kyun Kim is supported by a fund from the Cancer Research Center, SNU, Korea (KOSEF-SRC-56-crc-21).