Isolation of mouse X‐chromosome specific DNA from an X‐enriched lambda phage library derived from flow sorted chromosomes

Abstract

A lambda phage library enriched in X(7) chromosomal material has been constructed from flow sorted chromosomes isolated from mice carrying the Cattanach translocation T(X;7)1Ct. The flow sorted fraction that was cloned contained 40% X(7) chromosomes, so that the resulting lambda phage library should be more than 10-fold enriched for X chromosomal DNA. Approximately 100,000 lambda phage clones were obtained; of these, at least 80% were recombinant. Three quarters of recombinants were positive for mouse repetitive DNA as detected either by phage plaque filter hybridization or by Southern blotting. Recombinant DNA inserts were prepared from some of the remaining nonrepetitive phage fraction. The X-chromosome specificity of cloned DNA inserts was tested by hybridization to DNA from mouse-hamster somatic cell hybrids that had retained all or most of the mouse X as the only mouse chromosome and by comparison of the extent of hybridization to DNA from male and female mice. Out of nine cloned unique sequence segments successfully examined thus far, two were presumably derived from the X. Possession of phage library highly enriched for mouse X DNA should facilitate molecular studies of the control of X chromosome gene expression.

Historical development of analysing large-scale changes in the human genome

“…The DOE laboratories at Los Alamos and Lawrence Livermore developed especially dedicated high-speed cell sorters and were gradually making chromosome-specific libraries available to the mapping community. However, several groups including those of Sam Latt (Disteche et al, 1982), Malcom FergusonSmith (Harris et al, 1985) and Bryan Young (Davies et al, 1981) successfully sorted human metaphase chromosomes and made chromosome-specific libraries by cloning inphage vectors using much slower, commercially available FACS machines than those at the DOE laboratories. It was subsequently discovered that the libraries could be used for painting human chromosomes (Lichter et al, 1988;Telenius et al, 1992) and, by adjusting the stringency of the hybridisation, also in cross-species hybridisation even between species which are evolutionarily very distant from each other.…”