Analysis of large and small colony L5178Y tk-/- mouse lymphoma mutants by loss of heterozygosity (LOH) and by whole chromosome 11 painting: detection of recombination.

Analysis of 122 spontaneous large and small colony mutants derived from L5178Y tk +/- mouse lymphoma cells at 28 heteromorphic microsatellite loci on chromosome 11 showed that extensive loss of heterozygosity (LOH) is common in both large colony and small colony mutants, eliminating most chromosome 11 loci as candidates for a putative growth control locus. These results, in conjunction with historical cytogenetic data, suggest that a putative growth control locus lies distal to the thymidine kinase (Tk1) gene, near the telomere. Thirty seven mutants were hybridized with a chromosome 11-specific whole chromosome painting probe for analysis of rearrangements. Generally, painting confirmed earlier observations that large colony mutants are karyotypically normal, whereas small colony mutants frequently have detectable rearrangements. A point probe distal to Tk1 revealed no evidence of chromosome breakage in small colony mutants that appeared normal on whole 11 painting and had no LOH. Therefore, the molecular difference between large and small colony mutants remains unknown. Models to explain large and small colony mutants consistent with our findings are presented, including loss of a putative growth control gene, differential mechanisms of chromosome breakage/repair and second site mutations as explanations for small colony mutants. Painting revealed translocations and aneuploidy and showed that non-disjunction was not a common explanation for complete LOH. The most common finding was that large regions of LOH do not result from deletions, demonstrating that these cells can detect recombination events as well as previously observed chromosomal rearrangements, deletions and point mutations.

Comparative genome mapping: mouse and rat homologies revealed by fluorescence in situ hybridization.

Mouse and rat genome studies are vital to the use of rodents as models of biology and human genetic disease. In this study, comparative cytogenetic maps of individual homologous mouse (Mus musculus) and rat (Rattus norvegicus) chromosomal regions are presented as defined by cross-species fluorescence in situ hybridization. Such "Zoo-FISH" methods permit direct visual observation of the location of DNA segments from one species on mitotic chromosomes of evolutionarily diverged species. Mouse whole chromosome paint (WCP) probes generated from microdissection and degenerate oliogonucleotide primed (DOP) PCR were hybridized on slides containing a mixture of both mouse (the reference species) and rat (the diverged/ comparative species) metaphase chromosomes. Using six different mouse WCPs, eight regions on seven rat chromosomes were shown to be evolutionarily conserved between these species. The specific chromosomal sites of homology delineated in this study between mouse (MMU) and rat (RNO) genomes include the following: MMU 1 to RNO 9q21-q36 and to RNO 13 from bands q11 to the telomere, MMU 4 to all of RNO 5, MMU 11 to all of RNO 10 and the distal region of RNO 14 (14q21-q22), MMU 7 and MMU 19 both to RNO 1, from bands 1q21 to 41 (MMU 7) and 1q42 to the telomere (MMU 19), and MMU X to all of RNO X. Additionally, several new mouse and rat map assignments have been predicted based on the observed cross-species hybridization patterns in conjunction with known mapping data for mouse or rat genes.

Differential destabilization of repetitive sequence hybrids in fluorescence in situ hybridization.

A method for painting a chromosome or chromosome region by fluorescence in situ hybridization (FISH) without blocking DNA is described. Both unique sequence and repetitive sequence components of a fluorescently labeled probe are hybridized under low-stringency conditions, but the chromosomes are washed in such a manner that repetitive sequences are differentially removed, while region-specific unique sequence fragments remain bound to the target chromosomes. We refer to this differential retention and removal of probe components as differential stability FISH.

Selection of hybrids by affinity capture (SHAC): a method for the generation of cDNAs enriched in sequences from a specific chromosome region.

We have established a method for preparing cDNA sublibraries enriched in sequences from specific chromosome regions, called selection of hybrids by affinity capture (SHAC). This procedure can be described in two stages. In the first stage, a particular chromosome region, in this study mouse chromosome 11, was microdissected, followed by PCR amplification with a universal degenerate primer. This material is referred to as the "target" DNA. In the second stage, a mouse liver cDNA library with unique linker-adapter ends, referred to as the "source" cDNA, was hybridized to the biotin-labeled target DNA prepared during the first stage. The resulting DNA duplexes were captured by streptavidin-coated magnetic beads. The cDNAs were released from their biotin-labeled target homologs by alkaline denaturation and recovered by PCR amplification. These cDNAs were referred to as the SHACcDNAs. Specificity of the SHACcDNA to chromosome 11 was verified by FISH analysis. To examine representation of the SHACcDNA, we confirmed the presence of seven genes or single-copy DNA segments known to be localized on mouse chromosome 11, using a dot blot assay. In addition, a second round of SHAC was performed to achieve even higher specificity for the resulting chromosome 11 SHACcDNA. The SHAC technology should facilitate construction of cytogenetically defined cDNA libraries and should assist in the fields of gene discovery and genome mapping.

Mouse chromosome-specific painting probes generated from microdissected chromosomes.

Using degenerate primer amplification of chromosomes microdissected from banded cytogenetic preparations, we constructed both whole chromosome painting probes for mouse Chromosomes (Chrs) 1, 2, 3, and 11 and a centromere probe that strongly paints most mouse centromeres. We also amplified a Robertsonian translocation chromosome microdissected from unstained preparations to construct a painting probe for Chrs 9 and 19. The chromosome probes uniformly painted the respective chromosomes of origin. We demonstrated the utility of the Chr 11 probe in aberration analysis by staining mutants that we had previously identified as containing a Chr 11 translocation, and in some mutant cell lines we observed chromosome rearrangements not previously detected in stained cytogenetic preparations. The technology of microdissection and amplification applies to all mouse chromosomes or to specific subchromosomal regions and will be useful in mouse genetics, in aberration analysis, and for chromosome identification.