High-resolution mapping of mammalian genes by in situ hybridization to free chromatin.

Abstract

Fluorescence in situ hybridization to metaphase chromosomes or chromatin fibers In Interphase nuclei is a powerful technique in mapping genes and DNA segments to specific chromosome region. We have been able to release the chromatin fibers from cells arrested at GI and G2 phases using different drugs and a simple alkailne lysis procedure. We have also demonstrated specific hybridization of fluorescencelabeled probes to single-copy genoic DNA sequences on the free chromatins. Fluorescence in situ hybridztion signals have been detected for sequences separated as close as 21 klobase pairs and as far as 350 kilobase pairs, with excellent correspondence between the observed and expetd distances. The resolution of this technique should approach 10 kilobase pairs and its coverage should span millions of base pairs. Therefore, free chromatin mapping can be generally used to study the structure and organization of mammalian genomes.

The adenovirus E1A binding protein BS69 is a corepressor of transcription through recruitment of N-CoR

“…The procedure for FISH detection on human lymphocytes was performed according to (Heng et al, 1992). FISH signals and the DAPI banding pattern was recorded separately by taking photographs, and the assignment of the FISH mapping data with chromosomal bands was achieved by superimposing FISH signals with DAPI banded chromosomes (Heng and Tsui, 1993).…”

Human fibroblast growth factor-18 stimulates fibroblast cell proliferation and is mapped to chromosome 14p11

“…Using the¯uorescence in situ hybridization (FISH) technique (Heng et al, 1992;Heng and Tsui, 1993), the biotinylated FGF-18 cDNA probe was used to map the human chromosome. A speci®c region of one chromosome showed the FISH positive with the FGF-18 probe ( Figure 6a).…”

“…The procedure for FISH detection was performed as previously described (Heng et al, 1992;Heng and Tsui, 1993). Brie¯y, the cell slides were baked at 558C for 1 h. After RNase treatment, the slides were denatured in 70% formamide in 26SSC for 2 min at 708C followed by dehydration with ethanol.…”

“…Probes were denatured at 758C for 5 min in a hybridization bu er containing 50% formamide and 10% dextran sulfate, and loaded onto the denatured chromosomal slides. After 16 ± 20 h hybridization, the slides were washed and incubated with¯uorescein isothiocyanate (FITC)-conjugated avidin (Vector Laboratories), and the signal was ampli®ed as described (Heng et al, 1992). FISH signals and the 4',6'-diamidino-2-phenylindole (DAPI) banding patterns were recorded separately by taking photographs, and the assignment of the FISH mapping data with chromosomal bands was achieved by superimposing FISH signals with the DAPI banded chromosomes (Heng and Tsui, 1993).…”

Multicolor fluorescence in situ hybridization on metaphase chromosomes and interphase Halo-preparations using cosmid and YAC clones for the simultaneous high resolution mapping of deletions in the dystrophin gene

“…Multiple haptenization and detection protocols, and combinatorial labeling approaches allow for the simultaneous visualization of several target regions in metaphase chromosomes and interphase nuclei (Nederlof et al 1990;Ried et al 1992a, b,c;Lenganer et al 1993;Wiegant et al 1993). The high spatial resolution of fluorescent signals is improved considerably when extended chromatin preparations are used for FISH analysis (Heng et al 1992;Wiegant et al 1992). Moreover, with the progress of the Human Genome Project, an increasing number of DNA clones has become available (see e.g., Bellann6-Chantelot et al 1992) that can be applied to specifically delineate chromosomal aberrations.…”