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The direct screening of cosmid libraries with YAC clones.

The yeast artificial chromosome (YAC) cloning system has permitted the cloning of large fragments of mammalian DNA (1). However, YAC clones can be difficult to analyse and manipulate and conversion of a YAC into cosmids provides a useful step in the characterisation of the cloned region. Here we describe a method for screening cosmid libraries directly with the human artificial chromosome. We have used two overlapping YAC clones, YGA5 (410 kb) and YGA10 (440 kb), that were isolated from a YAC library constructed from a 48XXXX human cell line (2), and which lie within the Huntington's disease gene candidate region (Bates unpublished) on 4pl6.3. The yeast chromosomes were separated by pulsed field gel electrophoresis in LMP agarose (SeaPlaque GTG) and after staining with ethidium bromide, the human artificial chromosome was excised from the gel with the aid of UV irradiation (360 nm). After treatment with Geneclean (BIO 101), the DNA was resuspended in TE to S ng//tl. Approximately 50 ng of YAC DNA was labelled by random oligonucleotide priming (3) with 40 AtCi each of [a-32P]dGTP and [a-32P]dATP. Repetitive sequences were removed from probes by prehybridisation with 1.5 mg/ml of sheared human placental DNA (Sigma) in 0.12 M Na2HPO4 pH 6.8 for 3 hours at 65°C. A flow sorted human chromosome 4 cosmid library, contained in 263 microtitre dishes, has been spotted in high density arrays onto Nylon membranes (Hybond N +) using a robotic device as described in (4). Hybridisations were performed in 50% formamide at 42°C (5). Filters were prehybridised with 100 tsg/ml of denatured sonicated human placental DNA (Sigma) for 24 hours and then hybridised with probes at a concentration of 106 cpm per ml. The filters were washed to 0.1 xSSC, 0.1% SDS and autoradiography was for 2-3 days. The hybridisation of YGA5 and YGA1O to a cosmid filter is illustrated in Figure 1. A subset of the positive clones have been verified by the hybridisation to the filters of single copy probes and cosmids known to map to the region. The number of clones found to date has been consistent with the expected coverage of the library and the size of each YAC insert. This technique allows immediate access to chromosome 4 DNA contained within chimaeric YACs and clones containing 2 artificial chromosomes. It may also simplify the analysis of unstable YACs. In such cases, hybridisation with the largest artificial chromosome may circumvent complications arising from deletion products by utilising a cloned source of the region that has not been subject to rearrangements originating from propagation in yeast. Comparison of the hybridisation patterns of a number of overlapping YACs will also permit the grouping of these cosmids into defined regions within each YAC.