Precise gene localization by phenotypic assay of radiation hybrid cells

Abstract

A high resolution map of the human genome previously has been constructed by using the G3 panel of human/hamster radiation hybrid cell lines and >15,000 unique human genetic markers. By determining whether human DNA sequences are present or absent in each of the hybrids, localization of single genes may routinely be achieved at approximately 250-kb resolution. In this paper we have tested whether similarly precise localization might be achieved by phenotypic screening of the hybrids to facilitate positional cloning of unknown genes. We assayed the susceptibility of each of the hybrid cell lines to transduction by retroviral vectors bearing different retroviral envelope proteins that recognize receptors present on human but not on hamster cells. The results for each of the retroviral vectors were informative and allowed precise localization of the receptor genes for the RD114 cat endogenous retrovirus, xenotropic murine leukemia virus, and type C feline leukemia virus. After cloning of the receptors for these retroviruses, we found that standard genotypic mapping by PCR gave results that were nearly identical to those from phenotypic mapping. These experiments show that precise gene localization by phenotypic assay of radiation hybrids is practical and was not appreciably impacted by the known instability of such hybrid cells. This technique should be applicable to many other human genes having discernible phenotypes in hamster cells and, with completion of the human genome project, will allow rapid identification of unknown genes on the basis of phenotype.

Figure 1

Results of screening radiation hybrids for the presence of retrovirus receptors by phenotype (Transduction) and genotype (Receptor PCR) as compared with results from the PCR analysis of closely linked markers. The radiation hybrid clones are arrayed horizontally with numbering equivalent to the standard used for hybrid DNA analysis. A “1” on a black background indicates that the radiation hybrid was transduced at a titer of ≥10⁴ FFU/ml in the case of transduction analysis or that a PCR product was detected in the case of DNA analysis. A “0” indicates that the hybrid was transduced at a titer of <10 FFU/ml in the case of transduction analysis or that a PCR product was not detected in the case of DNA analysis. The shaded boxes indicate unavailable or ambiguous data. Note that the radiation hybrid cell lines numbers 48, 76, and 78 were not available for transduction analysis. The third through fifth rows of data for each receptor represent the PCR results for the closest SHGC-ordered markers for each receptor.

Figure 2

Two-point distances between retrovirus receptors and ordered markers (x axis) in comparison to SHGC-ordered marker map distance (y axis). The location and identification numbers of the SHGC-ordered markers are shown below the diagrams with the distances (cR) shown between markers. All distances are drawn to the same scale.

Figure 3

Multipoint analysis of the likelihood of linkage (lod) between ordered markers and the retrovirus receptors by phenotypic and genotypic analysis. SHGC-ordered marker numbers are shown below the curves. Map distance is plotted on the x axis. lod scores below 0 are not shown. Multipoint lod scores were computed as described (20) with the computer program radmap written by L.K.