Worm Breeder's Gazette 8(2): 37
These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author.
During the next few years, a major thrust of C. elegans research will be the cloning pf developmentally important genes. The absence of known products means that such genes must be isolated either by the direct insertion of a transposon or by walking along the genome from some genetically identified starting point. Transposition may not be applicable to all loci, and walking is tedious. In the hope of speeding up the cloning process, and of providing information about the genome as a whole, we have begun the task of generating a physical map of the genome - that is to say, of creating an ordered library of cloned DNA fragments which will parallel the genetic map. At the least, such a library will facilitate access to any given region; ultimately, as we identify increasing numbers of transcription units, it may allow immediate isolation of any genetic locus. In order to build up the library we have cloned nematode DNA ( partially digested with Sau3a1) into the cosmid vector pJB8, and have set out some 6000 individual colonies in microtitre wells. In this form, the collection can conveniently be transferred to nitrocellulose (384 clones per 130mm filter) and screened by hybridization. We are now systematically 'fingerprinting' the clones, by the following procedure. The cosmid DNA is cut with a restriction enzyme that recognizes a six base pair sequence, end labelled, and then cut again with a restriction enzyme that recognizes a four base pair sequence. The resulting fragments are separated on a thin polyacrylamide gel, and the positions of the radioactive bands are recorded relative to a set of standards. The pattern derived from each clone is compared by computer with all the others, and significant overlaps are noted. So far we have looked at 1500 clones, and have identified 325 multiple contigs (i.e. groups of overlapping clones); we estimate that about 40% of the genome is now covered. We are continuing at a rate of about 100 clones per week, and expect to finish this phase of the project in 1-2 years. At that time random cloning will have become unproductive, and we shall resort to hybridization to fill the remaining gaps, of which there will be at least several hundred. If there are many sequences in C. elegans that clone poorly or not at all in E. coli, we shall then be in difficulties, (although our contigs will already be useful to genome walkers). In that event, we would hope to continue mapping by a combination of in situ hybridization (Albertson, this newsletter) and genetic markers. It is clear from the outset that the physical map will only become a reality as a communal project. In its final stages it will have to be completed opportunistically, and in any case numerous markers will be required to align it with the genetic map. We therefore invite anyone who has genetically positioned DNA that is >10 Kb in length and available for distribution to collaborate with us, by sending a sample for fingerprinting and comparison with the database. In return, we can send you any flanking cosmids that we find, as well as any others that you need.