Region-specific cosmids and STRPs identified by chromosome microdissection and FISH.

A strategy for identifying short tandem repeat (STR)-containing cosmid clones from a specific chromosomal region is described. The approach is based on the use of uncloned, PCR-amplified DNA derived from chromosome microdissection and pooled groups of STR sequences as hybridization probes to screen a cosmid library. Cosmid clones that display a positive signal common to both hybridizations are then characterized for repeat length polymorphisms. This method has been applied to chromosome bands 17q12-q21, a region that includes a gene (BRCA1) involved in early onset familial breast and ovarian cancer. Of 1536 chromosome 17-specific cosmid clones tested, 38 were identified by the dual screening procedure. Fluorescence in situ hybridization revealed that 19 cosmids originated from the microdissected target region. Thirteen of the 19 cosmids were mapped between markers flanking the BRCA1 region and selected for further characterization. Tetranucleotide repeats were identified in 10 of these 13 cosmids. Primers designed for each marker were tested on a panel of 80 CEPH parents for allele sizes, frequencies, and observed heterozygosities. From these studies six polymorphic and one nonpolymorphic STRs were identified. A similar approach should be applicable for screening whole genomic or chromosome-specific cosmid libraries in efforts to isolate new polymorphic markers from any chromosomal region of interest.

Localization of the human HTF4 transcription factors 4 gene (TCF12) to chromosome 15q21.

Identification and localization of genes that encode regulators of transcription could provide landmarks for functional analysis of the human genome. Toward this goal, we examined a panel of somatic cell hybrids and assigned the gene (TCF12) encoding the helix-loop-helix transcription factors 4 (HTF4) to chromosome 15. Fluorescence in situ hybridization further localized TCF12 to chromosome 15q21. Northern analysis revealed that the relative abundance of HTF4 gene transcripts is not constant but varies depending on the human cell-line or tissue examined.

Multicolor FISH mapping of YAC clones in 3p14 and identification of a YAC spanning both FRA3B and the t(3;8) associated with hereditary renal cell carcinoma.

Human chromosome band 3p14 contains two tightly linked cytogenetic markers of broad interest, FRA3B and the t(3;8) breakpoint associated with hereditary renal cell carcinoma (RCC). The common fragile site at 3p14.2 (FRA3B) is the most sensitive site on normal human chromosomes to breakage when DNA replication is perturbed by aphidicolin or folate stress. The t(3;8)(p14.2;q24.1) translocation segregates with RCC in a large family and could mark the location of a tumor suppressor gene involved in renal cancers. In studies aimed at positional cloning of FRA3B and the t(3;8) breakpoint, we have used multicolor fluorescence in situ hybridization analysis (FISH) on metaphase spreads and interphase nuclei to order 14 yeast artificial chromosomes (YACs) in 3p14. The YACs used in this study were identified by a group of unordered lambda clones that had been previously localized to the 3p14 region and mapped proximal or distal to the t(3;8) breakpoint. FISH analysis was used to order the YACs and to map them in relation both to the t(3;8) translocation breakpoint and to FRA3B induced on normal chromosomes by treatment with aphidicolin. YACs that closely flanked both the t(3;8) translocation breakpoint and the fragile site were identified. A YAC walk from the closest distal YAC allowed the identification of a 1.3-Mb YAC derived from the CEPH large insert YAC library that spans both the FRA3B and the t(3;8) breakpoint. The order of the YACs and cytogenetic landmarks in 3p14 is cen-(126E1/230B9)-181H6-B15-D20F4-258B7-++ +280D2-70E12-168A8- 403B2-143C5-413C6-468B10-[850A6/t(3;8)/ FRA3B]-74B2.(ABSTRACT TRUNCATED AT 250 WORDS)

Mapping of the gene for interferon-inducible dsRNA-dependent protein kinase to chromosome region 2p21-22: a site of rearrangements in myeloproliferative disorders.

Recent evidence suggests that the human interferon-inducible double-stranded RNA-dependent protein kinase may function as a tumor suppressor. Here we describe the mapping of the gene for this kinase to chromosome region 2p21-22 by fluorescence in situ hybridization. A combined analysis of cytogenetic data from a series of 341 patients with hematologic disorders that exhibited cytogenetic abnormalities and from published reports indicates that abnormalities involving 2p21-22 occur nonrandomly and are observed among patients with acute myelogenous leukemia, raising the possibility of a role for this protein kinase in leukemogenesis.

Multicolor FISH mapping with Alu-PCR-amplified YAC clone DNA determines the order of markers in the BRCA1 region on chromosome 17q12-q21.

A gene designated BRCA1, implicated in the susceptibility to early-onset familial breast cancer, has recently been localized to chromosome 17q12-q21. To date, the order of DNA markers mapped within this region has been based on genetic linkage analysis. We report the use of multicolor fluorescence in situ hybridization to establish a physically based map of five polymorphic DNA markers and 10 cloned genes spanning this region. Three cosmid clones and Alu-PCR-generated products derived from 12 yeast artificial chromosome clones representing each of these markers were used in two-color mapping experiments to determine an initial proximity of markers relative to each other on metaphase chromosomes. Interphase mapping was then employed to determine the order and orientation of closely spaced loci by direct visualization of fluorescent signals following hybridization of three probes, each detected in a different color. Statistical analysis of the combined data suggests that the order of markers in the BRCA1 region is cen-THRA1-TOP2-GAS-OF2-17HSD-248yg9-RNU 2-OF3-PPY/p131-EPB3-Mfd188- WNT3-HOX2-GP3A-tel. This map is consistent with that determined by radiation-reduced hybrid mapping and will facilitate positional cloning strategies in efforts to isolate and characterize the BRCA1 gene.