Nucleic Acids Research: VOLUME 40 WEB SERVER ISSUE JULY 1, 2012

The clinical association of Wilms tumour with anlridia, genitourinary abnormalities and mental retardation (WAGR syndrome) is characterised cytogenetically by variable length, constitutional deletion of the short arm of chromosome 11, which always includes at least part of band Ilpl3. HRAS1selected chromosome mediated gene transfer (CMGT) generated a transformant, E65-6, in which the only human genes retained map either to band Ilpl3 or, with HRASl, in the region Ilpl5.4-pter. Human recomblnants isolated from E65-6 were mapped to a panel of five WAGR deletion hybrids and two clinically related translocations. We show that E65-6 is enriched ^400-fold for HpI5.4pter markers and -200-fold for Ilpl3 markers. 'Hitch-hiking' from HRASl with CMGT markers has allowed us to define seven discrete intervals which subtend band Ilpl3. Both associated translocations co-locate within the smallest region of overlap for the WAGR locus, which has been redefined by identifying a new interval closer than FSHB. INTRODUCTION In an earlier study (1), we described the establishment and cytogenetics of lymphoblastoid cell lines derived from several WAGR patients, the segregation of deleted from normal chromosomes 11 In somatic cell hybrids and their characterisation with defined gene markers. We showed that CAT and the cell surface markers MIC4 and MICH all map to band Hpl3 and are frequently but not ubiquitously deleted in WAGR patients. In a parallel study (2), we described the development of the chromosome mediated gene transfer (CMGT) technique with selection for expression of the activated HRASl oncogene, mapping at Hpl5.4-pter. This procedure facilitates the stable isolation of human chromosome 11 fragments in mouse C127 cell3. However, lntrachromosomal rearrangement accompanies the CMGT process such that markers centromere proximal to HRASl can be co-transferred while intervening markers are lost. Most recently (3), we described the isolation and sub-chromosomal localisation of forty-four human DNA recombinants from one HRAS1-CMG transformant, E67-1. This transformant contains =50Mbp of human DNA inserted interstitially © IR L Press Limited, Oxford, England. 5 1 Nucleic Acids Research at a single site on a mouse chromosome (4). Several chromosome 11 markers had been co-transferred, including ones known to flank either side of the WAGR locus (2). Mapping of the E67-1 derived recombinants to our panel of WAGR deletions (now extended to five patients, three of which retain part of band Ilpl3), together with two clinically associated Ilpl3 translocations, allowed us to define ten discrete intervals on the short arm of chromosome 11 (3). These included a centromere distal interval previously defined only by FSHB (5,6) and a centromere proximal interval previously defined by CAT and MICH (1,6). Thus, the CMGT approach rapidly generated a valuable and extensive set of anonymous DNA markers for fine-structure mapping of the short arm of chromosome 11. The enrichment for Hpl3 markers was substantial and significantly higher than would be expected from a chromosome 11 only hybrid or from a flow sorted, chromosome 11 enriched library. However, although three new intervals on the short arm of chromosome 11 were defined, none were closer to the WAGR locus than the FSHB or the CAT/MICH intervals. The success of the clone isolation and mapping exercise with E67-1 encouraged us to test other HRAS1selected CMGT's, in particular those for which we had evidence for complex but potentially adventitious intrachromosomal rearrangement. The HRASl-selected CMG transformant E65-6 contains -lOMbp of human DNA (2). LI "fingerprinting" provides evidence for complex but stable amplification and rearrangement of the isolated human DNA sequences (2). In situ hybridisation analysis demonstrates the presence of three discrete blocks of human chromatin, each carrying a copy of the HRAS1 oncogene (4,7). Of particular relevance to the present study, only LDHA, FSHB and MICH, of all the chromosome 11 genes tested, have been co-transferred along with HRAS1 (Table 1). Furthermore, several of the E67-1 derived recombinants which map to band Ilpl3 cross-hybridise to E65-6 (Porteous et al., in prep.). E65-6 should therefore provide an ideal further test of the CMGT approach, not only for isolating chromosomal segments which directly flank the selected locus (8,9), but also for cloning syntenic sequences originally at a distance (CMGT "hitch-hiking"). HATERIALS AND METHODS Chromosome 11 somatic cell hybrids The human-mouse cell hybrids 1W1.LA4.9 and IB.8.1.6 retain chromosome 11 as their sole human component (1,6). We have tested over 150 gene specific and anonymous DNA markers on chromosome 11 and all are present in 1W1LA4.9, whereas IB.8.1.6 appears to have a terminal deletion of short arm material,