A SCHIZOSACCHAROMYCES-POMBE ARTIFICIAL CHROMOSOME LARGE DNA CLONING SYSTEM

The feasibility of using the fission yeast, Schizosaccharomyces pombe, as a host for the propagation of cloned large fragments of human DNA has been investigated. Two acentric vector arms were utilized; these c arry autonomously replicating sequences (ars elements), selectable mar kers (ura4(+) or LEU2) and 250 bp of S,pombe terminal telomeric repeat s. All cloning was performed between the unique sites in both vector a rms for the restriction endonuclease Notl, Initially the system was te sted by converting six previously characterized cosmids from human chr omosome 11p13 into a form that could be propagated in S.pombe as linea r episomal elements of 50-60 kb in length. In all transformants analys ed these cosmids were maintained intact, To test if larger fragments o f human DNA could also be propagated total human DNA was digested with Notl and size fractionated by pulsed field gel electrophoresis (PFGE) , Fractions of 100-1000 kb were ligated to Notl-digested vector arms a nd transformed into S.pombe protoplasts in the presence of lipofectin, Prototrophic ura(+) leu(+) transformants were obtained which upon exa mination by PFGE were found to contain additional linear chromosomes m igrating at between 100 and 500 kb with a copy number of 5-10 copies/c ell. Hybridization analyses revealed that these additional bands conta ined human DNA, Fluorescent in situ hybridization (FISH) analyses of s everal independent clones indicated that the inserts were derived from single loci within the human genome, These analyses clearly demonstra te that it is possible to clone large fragments of heterologous DNA in fission yeast using this S.pombe artificial chromosome system which w e have called SPARC, This vector-host system will complement the vario us other systems for cloning large DNA fragments.