Nucleic Acids Research, Vol 26, Issue 22 5052-5060, Copyright © 1998 by Oxford University Press
DJ Young, ER Nimmo and RC Allshire
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 carry
autonomously replicating sequences ( ars elements), selectable markers (
ura4(+) or LEU2 ) and 250 bp of S. pombe terminal telomeric repeats. All
cloning was performed between the unique sites in both vector arms for the
restriction endonuclease Not I. Initially the system was tested by
converting six previously characterized cosmids from human chromosome 11p13
into a form that could be propagated in S.pombe as linear episomal elements
of 50-60 kb in length. In all transformants analysed these cosmids were
maintained intact. To test if larger fragments of human DNA could also be
propagated total human DNA was digested with Not I and size fractionated by
pulsed field gel electrophoresis (PFGE). Fractions of 100-1000 kb were
ligated to Not I- digested vector arms and transformed into S.pombe
protoplasts in the presence of lipofectin. Prototrophic
ura+leu+transformants were obtained which upon examination by PFGE were
found to contain additional linear chromosomes migrating at between 100 and
500 kb with a copy number of 5-10 copies/cell. Hybridization analyses
revealed that these additional bands contained human DNA. Fluorescent in
situ hybridization (FISH) analyses of several independent clones indicated
that the inserts were derived from single loci within the human genome.
These analyses clearly demonstrate that it is possible to clone large
fragments of heterologous DNA in fission yeast using this S.p ombe
artificial chromosome system which we have called SPARC. This vector- host
system will complement the various other systems for cloning large DNA
fragments.
ARTICLES
A Schizosaccharomyces pombe artificial chromosome large DNA cloning system
MRC Human Genetics Unit, Western General Hospital, Crewe Road, Edinburgh EH4 2XU, UK.
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