Fluorescence in situ hybridization (FISH) is now widely used for the localization of genomic DNA fragments, and the identification of chromosomes by painting. We now show that half of the chromosomal complement can be painted in twelve different colors by using human chromosome specific libraries carrying three distinct labels mixed in multiple ratios. The photographs are in 'real' color rather than 'colorized'. The painting technique described here can be used for the identification of small or complex chromosomal rearrangements and marker chromosomes in humans or in any other species for which well defined chromosome specific libraries exist in a laboratory equipped with a conventional fluorescence microscope. The versatility of this novel cytogenetic technology may well constitute an advancement comparable to the introduction of chromosome banding and high resolution analysis of chromosomes in prometaphase.

The pericentric inversion of chromosome 16 and the t(16;16) are two recurrent aberrations in bone marrow of patients with acute nonlymphocytic leukemia subtype M4 Eo, characterized by abnormal eosinophilic granulation. We describe here the precise localization of the breakpoints using fluorescence in situ hybridization (FISH) with cosmids spread over the short arm of chromosome 16 and the detection, isolation and characterization of a 14Kb EcoRI fragment containing a cluster of breakpoints. First, cosmids were mapped to intervals defined by constitutional 16p rearrangements, second, the inv(16) and t(16;16) breakpoints were mapped to one of the intervals using FISH with the mapped cosmids and third, cosmids within this interval were ordered using two color interphase FISH. An STS of the cosmid closest to the breakpoints was then used to isolate five YACs, which did span all of the 16 inv(16) breakpoints and one t(16;16) breakpoint analysed. In the DNA of one inv(16) patient we detected an additional submicroscopic deletion immediately proximal to the 16p breakpoint. Since this patient has the same phenotype, the 16p sequences proximal to the breakpoint seem non-essential to M4 Eo. This implies that the pathologic event is the juxtaposition of sequences distal to the 16p breakpoint with sequences proximal to the 16q breakpoint. While four of the five YACs showed instability of the region around the inv(16) breakpoint, DNA halo analysis allowed us to identify one YAC which was co-linear with normal genomic DNA and has yielded the actual breakpoint sequences which could be subcloned into cosmids and fosmids.(ABSTRACT TRUNCATED AT 250 WORDS)

Summary Loss of heterozygosity (LOH) on chromosome arm 16q occurs in 48-65% of breast tumours. One small region of overlap is located at 1 6q24.3. Two genes located in this region, the cellular adhesion regulatory molecule (CMAR) and the breast basic conserved gene (BBC1), are plausible candidate tumour-suppressor genes. Mutational analysis of the retained copy of these genes has been performed by direct sequencing in a selected set of breast tumours that show LOH at 1 6q24.3 but not at other regions on chromosome arm 1 6q. In CMAR no other alterations than the previously described 4-bp insertion of CACA at nucleotide 241 could be detected, which was also present in constitutional DNA of the same patients. This polymorphism occurs homozygously in germline DNA of normal individuals and breast cancer patients. LOH analysis at this locus shows no preferential loss of a particular variant of the 241 polymorphism. In the BBC1 gene, three different alterations were found, but only one resulted in an amino acid substitution. This is a known polymorphism, however, also appearing in germline DNA. The absence of tumour-specific mutations in CMAR and BBC1 in this selected series of breast tumours implies that another gene at 1 6q24.3 must be the tumour-suppressor gene that is the target for LOH in breast cancer.Keywords: breast cancer; tumour-suppressor gene; chromosome 16; breast basic conserved gene 1; cellular adhesion regulatory molecule Loss of heterozygosity (LOH) on the long arm of chromosome 16 occurs frequently in breast cancer and other tumours. Percentages of LOH in breast cancer vary from 48% to 65% in different regions of 16q in different studies (Cleton-Jansen et al, 1994;Tsuda et al, 1994;Dorion-Bonnet et al, 1995). This suggests the presence of tumour-suppressor genes on this chromosome arm. One of two identified small regions of overlap is located at 16q24.3 between the markers APRT and D16S303 (Cleton-Jansen et al, 1994).Two genes, the cellular adhesion regulatory molecule (CMAR) and the breast basic conserved gene (BBCJ), located in this region are probable candidates for the gene targeted by LOH (Adams et al, 1992;Pullman and Bodmer, 1992;Cleton-Jansen et al, 1995a). The CMAR gene product enhances binding of integrins to extracellular matrix components (Pullman and Bodmer, 1992). A 4-bp insertion of CACA at nucleotide 241 occurs in 38% of Caucasians and in 30% of Japanese (Koyama et al, 1992;Durbin et al, 1994). This 4-bp insertion probably results in an alternative start site for the gene (Durbin et al, 1994).The BBC] gene was identified by differential screening of cDNA libraries of a primary breast carcinoma and of a benign fibroadenoma (Adams et al, 1992). Expression of this gene is higher in fibroadenomas than in carcinomas. The gene is expressed in a great variety of tissues and it is also highly conserved in other species (Bertauche et al, 1994). At the genetic level 85% homology is found with the gene coding for the rat ribosomal protein L13, at the protein level 97% homology is found (Olvera et...

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