Physical mapping of genes in somatic cell radiation hybrids by comparative genomic hybridization to cDNA microarrays
- Johann Y Lin†1,
- Jonathan R Pollack†2, 3,
- Fan-Li Chou1,
- Christian A Rees4,
- Allen T Christian6,
- Joel S Bedford6,
- Patrick O Brown3, 5 and
- Mark H Ginsberg1Email author
© Lin et al., licensee BioMed Central Ltd 2002
Received: 6 February 2002
Accepted: 18 April 2002
Published: 14 May 2002
Somatic cell mutants can be informative in the analysis of a wide variety of cellular processes. The use of map-based positional cloning strategies in somatic cell hybrids to analyze genes responsible for recessive mutant phenotypes is often tedious, however, and remains a major obstacle in somatic cell genetics. To fulfill the need for more efficient gene mapping in somatic cell mutants, we have developed a new DNA microarray comparative genomic hybridization (array-CGH) method that can rapidly and efficiently map the physical location of genes complementing somatic cell mutants to a small candidate genomic region. Here we report experiments that establish the validity and efficacy of the methodology.
CHO cells deficient for hypoxanthine:guanine phosphoribosyl transferase (HPRT) were fused with irradiated normal human fibroblasts and subjected to HAT selection. Cy5-labeled genomic DNA from the surviving hybrids containing the HPRT gene was mixed with Cy3-labeled genomic DNA from normal CHO cells and hybridized to a microarray containing 40,185 cDNAs, representing 29,399 genes (UniGene clusters). The DNA spots with the highest Cy5:Cy3 fluorescence ratios corresponded to a group of genes mapping within a 1 Mb interval centered near position 142.7 Mb on the X chromosome, the genomic location of HPRT.
The results indicate that our physical mapping method based on radiation hybrids and array-CGH should significantly enhance the speed and efficiency of positional cloning in somatic cell genetics.
Systematic forward genetics using somatic cell mutants is a powerful tool in elucidating biochemical pathways . However, it is often difficult to identify the genetic loci responsible for a particular somatic cell mutant phenotype. Map-based positional cloning strategies can be used in somatic cell hybrids to analyze mutants with recessive phenotypes. By tracking DNA sequence variants that co-segregate with heritable traits, the genes accounting for these traits can be localized to specific chromosomal locations . Recent advances in genome sequencing have facilitated the assembly of physical maps of candidate regions, but fine mapping and narrowing of candidate regions is still often tedious and remains a major obstacle in somatic cell genetics. Consequently, there is a need to efficiently map the positions of genes in somatic cell mutants.
We previously established a method to enrich for human genes that complement rodent somatic cell mutants . The mutant rodent cells are fused with wild-type human fibroblasts and the resulting hybrids are irradiated and selected for absence (that is, complementation) of the mutant phenotype. Retention of the complementing gene(s) during selection leads to enrichment, in the selected population of cells, of DNA copy number of the genomic region containing the complementing gene. Hybridization of genomic DNA from the selected hybrids to human metaphase chromosomes can be analyzed by fluorescence in situ hybridization (FISH), to locate the complementing genes by the elevated fluorescence ratios at the corresponding chromosomal loci. However, metaphase-CGH (a map of DNA copy number as a function of chromosomal location on metaphase spreads) requires specialized facilities, has a limited resolution and it is difficult to detect changes in small regions of the genome.
Recent developments in microarray technology have been applied in comparative genomic hybridization (array-CGH) by replacing metaphase chromosomes with arrayed DNAs such as bacterial artificial chromosomes (BACs), P1 artificial chromosomes and cDNAs representing specific genetic loci [4,5,6]. DNA microarrays have shown reliable quantitative capability in expression monitoring as well as greater sensitivity and resolution in array-CGH. We therefore defined and tested a radiation-hybrid method for mapping the complementing genes in somatic cell hybrids using high-density array-CGH. Using this method, we were able to map the human gene that complemented a recessive mutation in a CHO line to a region spanning less than 1 million bases (Mb) or 0.1% of the genome.
Results and discussion
We believe that the procedure we describe here can substantially increase the speed and efficiency of complementation cloning in somatic cell genetics. Approaches employing expression cloning using cDNA-based libraries have had success in identifying complementing genes [7,8]. The present method offers potential advantages over expression cloning. All human genes are represented in the donor-cell genome; consequently, limitations associated with quality and representation in cDNA libraries are overcome. Moreover, for mapping dominant mutations in human cells, the procedure obviates the requirement for a cDNA library from the cells of interest.
Materials and methods
Cell lines and mutant selection
Genomic DNA from a monochromosomal human-hamster hybrid containing the human X chromosome was obtained from NIGMS Human Genetic Mutant Cell repository (number GM0631).
CHO cell lines defective in the hprt gene were obtained as described . Briefly, CHO cells were chemically mutagenized by the point mutagen EMS or the frameshift mutagen ICR-191. After initial mutagenesis, the cells were cultured in the presence of the toxic base analog 6-thioguanine (6-TG), which selects for those cells that lack HPRT and are therefore resistant to killing by 6-TG. Before cell-fusion experiments, Hprt mutants were cultured for several passages in 6-TG to ensure stability of the mutant phenotype.
Generation of radiation hybrids
Radiation hybrids were generated by polyethylene-glycol-mediated cell fusion between lethally gamma-irradiated (150 Gy) human male AG1522 fibroblasts and the CHO mutants. The hybrids were selected for the presence of the HPRT gene by culturing in the presence of HAT medium. The complemented hybrids were analyzed as the test population after three weeks of HAT selection.
Labelling and hybridizations
DNA from the test and reference populations was harvested using Qiagen Genomic Tips 500 according to the manufacturer's protocol. For each labeling, we DpnII-digested (New England Biolabs) genomic DNA (4 μg), which was then purified (Qiaquick PCR kit) and random-primer labeled using a Bioprime Labeling kit (Gibco BRL), modified to include in a 100 μl reaction, dATP, dGTP and dTTP (120 μM each), dCTP (60 μM) and Cy5-dCTP or Cy3-dCTP (60 μM). We purified labeled products using a Microcon 30 filter (Amicon) and Cy5- and Cy3-labeled probes were combined with 50 μg human Cot-1 DNA (BRL), 20 μg poly(dA)-poly(dT) (Sigma), and 100 μg yeast tRNA (Gibco BRL). A Microcon 30 filter (Amicon) concentrated the hybridization mixture, which was then adjusted to contain 3.4 × SSC and 0.3% SDS in a 40 μl final volume. Following denaturation (100°C, 2 min) and a 30 min Cot-1 pre-annealing step (37°C), the probe was hybridized to the array under a glass coverslip at 65°C for 16-20 h, which was then washed in 2× SSC, 0.03% SDS (65°C, 5 min), followed by 5 min each at room temperature in 1× SSC and 0.2× SSC. cDNA microarrays were fabricated as described . In brief, we PCR-amplified IMAGE human cDNA (ESTs) clones in a 96-well format from DNA minipreps (Qiagen) using modified M13 universal primers. Most PCR products were 0.5-2 kb. The purified PCR products in 3× SSC were robotically arrayed (175 μm spacing between spots) onto polylysine-coated glass microscope slides. The cDNA microarrays described here contained 40,185 different sequence-validated human cDNAs, representing 29,399 different human UniGene clusters, of which 13,267 were named genes and the remaining 16,132 were ESTs.
Imaging and data analysis
Hybridized arrays were imaged using a GenePix scanner (Axon Instruments). Fluorescence ratios were calculated after background subtraction (background calculated as the median fluorescence signal of non-target pixels) using the GenePix software package. To correct for differences in DNA-labeling efficiency between samples, fluorescence ratios were normalized across each array to achieve an average log ratio of 0 (average ratio of 1; that is, no DNA copy-number change) for all cDNA elements on the array. Poorly quantified data (fluorescence signal < 40% above background for the wild-type CHO genomic DNA sample) were excluded from analysis (approximately 10-20% of array spots). Mean fluorescence ratios were calculated in log space to weight DNA gains (fluorescence ratios > 1) and losses (fluorescence ratios < 1) equally. When indicated, data are expressed as a 'moving average' of fluorescence ratios, calculated for sets of five adjacent genes along the chromosome. A moving-average ratio serves to average across multiple elements any imprecision in measurement, along with inaccuracies due to mapping misassignments. Genomic map positions of the arrayed cDNAs were determined using the 'Golden Path' assembly of the human genome draft sequences . We were able to assign genomic map positions for approximately 85% of all arrayed genes/ESTs. Fluorescence ratios were visualized by chromosomal nucleotide position using the CaryoScope website ( and C.A.R., unpublished data).
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