Construction of a genetic map of human chromosome 17 by use of chromosome-mediated gene transfer.

Abstract

We used somatic-cell hybrids, containing as their only human genetic contribution part or all of chromosome 17, as donors for chromosome-mediated gene transfer. A total of 54 independent transfectant clones were isolated and analyzed by use of probes or isoenzymes for >20 loci located on chromosome 17. By combining the data from this chromosomemediated gene transfer transfectant panel, conventional somatic-cell hybrids containing well-defined breaks on chromosome 17, and in situ hybridization, we propose the following order for these loci: pter- (i) although substantial lengths of DNA may be transferred intact (8, 9), interstitial deletions do frequently occur; (ii) multiple fragments of transfected chromatin can be found in the same clone; and (iii) there is also selection for centromeric sequences (8,9).In addition, during the course of this study we obtained some transfectants that contained only the small regions of chromosome 17 particularly relevant to the study of APL and von Recklinghausen neurofibromatosis. MATERIALS AND METHODSCulture Conditions. The cells were cultured either in RPMI 1640 or in Dulbecco's minimal essential medium supplemented with 10% fetal bovine serum. PCTBA1.8, PJT2/A1, and all CMGT transfectants, PLT, KLT, and TLT were maintained in HAT medium (100 ,uM hypoxanthine/10 ,M methotrexate/10 ,M thymidine), which selects for thymidine kinase. The hybrids GPT17.3.2K41 and TRID62 were maintained in MX medium [mycophenolic acid (25 ,ug/ml)/xanthine (250 ,g/ml)]. Back selection of CMGT transfectants with 5-bromo-2-deoxyuridine was done by the addition of 5-bromo-2-deoxyuridine (50 ,g/ml) to the medium, thus selecting for those CMGT transfectants that have lost the TKI locus. This medium was replenished every 3 days for 2-3 weeks. Resulting colonies were maintained in RPMI 1640/ 10% fetal bovine serum and were denoted by the letter B after the name of the original transfectant.Cytogenetic Analysis. Chromosomes from human lymphocytes, hybrids, and CMGT transfectants were all prepared as described (10). Additional

Genetic analysis of transgenome structure and size of chromosome-mediated gene transfer lines

“…A series of 9 transfectants generated by chromosome-mediated gene transfer [2] were surveyed. The more detailed analysis of these transfectants and other transfectants can be found in reference 2.…”

“…Previously, we reported the generation of transfectants for chromosome 17 by CMGT for linking and ordering genes on chromosome 17 over the whole chromosome [2]. This produced the longer distance than the high resolution mapping techniques of DNA-mediated gene transfer or cosmid cloning and at a more sensitive level than can be achieved by somatic cell hybrids or in situ hybridization.…”

Genetic and physical map of the von Recklinghausen neurofibromatosis (NF1) region on chromosome 17.

“…A series of somatic cell hybrids containing fragments of chromosome 17 was produced by chromosome-mediated gene transfer (10). One of these, KLT8, was used to construct a cosmid library, as it was known to contain about 10% of chromosome 17, from dot-blot analysis, and to contain the NFI region.…”

“…KLT8, PLT6B, and PLT8 are CMGTs containing fragments of chromosome 17 (10 (21). Cosmid DNA was linearized by digesting with BamHI, and 60 ng was used in the random-primer labeling method as described by Feinberg and Vogelstein (22).…”

“…Probes that map close to the translocations have been used to make long-range maps of this region (8,9). By using a chromosome-mediated gene transfectant (CMGT), KLT8, containing about 10% of chromosome 17, including the NFl region (10), we have isolated cosmids flanking the NFI translocations. Restriction fragment length polymorphisms (RFLPs) in these cosmids have allowed us to map them by genetic linkage.…”