Ah Vanderhout

A straightforward approach to isolate DNA sequences with potential linkage to the retinoblastoma locus

SummaryFrom a human-Chinese hamster somatic cell hybrid a clone was derived containing chromosome 13 in duplicate as its only human material. This clone was used to construct a human chromosome 13-specific recombinant DNA-library. Overlapping Sau3AI DNA sequences (11.9–17.2 kb) from the cell hybrid were inserted into the lambda phage vector EMBL4. From eleven recombinants having a human insert thirteen putative unique DNA sequences were isolated and cloned into the plasmid vector pBR329. A human-mouse hybrid containing a human chromosome 13 with a deletion of 13q14 and lacking its undeleted homologue was constructed to be used in a selection procedure for DNA sequences belonging to band q14. Three probes originating from two different phages were assigned to 13q14 because they did not hybridise to DNA from this cell hybrid. One of these 13q14 probes detects a low frequency (2/44) Msp I restriction fragment length polymorphism. The probes are now being used for screening a cosmid library to find adjacent polymorphic sequences with a RFLP information content suitable for application in the diagnosis of hereditary retinoblastoma.

LOCALIZATION BY INSITU HYBRIDIZATION OF DNA PROBES WITH TIGHT LINKAGE TO THE LOCUS FOR THE CYSTIC-FIBROSIS MUTATION

Probes 7C22 and pJ3.11 known to be closely linked to the cystic fibrosis locus were made available to us by Prof. Williamson, London. In situ hybridizations were carried out on metaphase preparations of lymphocytes from a patient having an apparent deletion in band q22 of one of the chromosomes 7, but no CF. The site and number of grains were scored for the normal and the deleted homologue. The majority of the grains resulting from hybridization with 7C22 were found in the region q22-qter, with a clustering at the distal part of band 31 and at band 32. For pJ3.11 most of the grains were in the region q31-qter. The normal homologue was more frequently labelled than the deleted one. This difference might be due to random variation, although alternatively structural chromosome aberrations other than deletion might be involved in this patient. In summary, our preliminary results suggest the distal half of the long arm of chromosome 7 to be the location of these probes. Our studies are now being extended to some other probes linked to the cystic fibrosis locus.