Investigation of Schizosaccharomyces pombe as a cloning host for human telomere and alphoid DNA.
The fission yeast Schizosaccharomyces pombe (Sch. pombe) has been proposed as a possible cloning host for both mammalian artificial chromosomes (MACs) and mammalian genomic libraries, due to the large size of its chromosomes and its similarity to higher eukaryotic cells. Here, it was investigated for its ability to form telomeres from human telomere sequence and to stably maintain long stretches of alphoid DNA. Using linear constructs terminating in the telomere repeat, T2AG3, human telomere DNA was shown to efficiently seed telomere formation in Sch. pombe. Much of the human telomeric sequence was removed on addition of Sch. pombe telomeric sequence, a process similar to that described in S. cerevisiae. To investigate the stability of alphoid DNA in fission yeast, bacterial artificial chromosomes (BACs) containing 130 and 173 kb of alphoid DNA were retrofitted with the Sch. pombe ars1 element and ura4+ marker using Cre-lox recombination. These alphoid BACs were found to be highly unstable in Sch. pombe deleting down to less than 40 kb, whilst control BACs of 96 and 202 kb, containing non-repetitive DNA, were unrearranged. Alphoid DNA has been shown to be sufficient for human centromere function, and this marked instability excludes Sch. pombe as a useful cloning host for mammalian artificial chromosomes. In addition, regions containing repetitive DNA from mammalian genomes may not be truly represented in libraries constructed in Sch. pombe.
Mann
K L
KL
Section of Molecular Genetics, Division of Biomedical Sciences, Imperial College School of Medicine, London, UK.
Huxley
C
C
D001483:Base Sequence; D003001:Cloning, Molecular / Q000379:methods*; D004247:DNA; D004276:DNA, Satellite; D006801:Humans; D019426:Integrases; D008969:Molecular Sequence Data; D012568:Schizosaccharomyces / Q000235:genetics*; D016615:Telomere; D014764:Viral Proteins
D004276:DNA, Satellite; D014764:Viral Proteins; D004247:DNA; C045073:Cre recombinase; D019426:Integrases