Introduction of YACs into intact yeast cells by a procedure which shows low levels of recombinagenicity and co-transformation.
Yeast artificial chromosomes (YACs) enable the cloning and analysis of large segments of genomic DNA and permit the isolation of sequences which are impossible to maintain in Escherichia coli. However, the construction of genome libraries in YAC vectors is beset by a number of technical problems, not least of which is the creation of cloned fragments which are not true representatives of the donor genome. These artefactual clones arise mainly due to intra-fragment rearrangements or inter-fragment chimaera formation, both phenomena resulting from the activity of the host yeast's mitotic recombination system. We demonstrate that this system is significantly stimulated by the spheroplasting step of the standard YAC transformation system. In contrast, the transformation of intact yeast cells by either the lithium method or a new lithium-free protocol is much less recombinagenic. It is not possible to introduce high molecular weight YACs into yeast using the lithium protocol, but we find that such molecules may be introduced into pde2-mutants using the lithium-free approach. Since intact cells are transformed by this method, automation of post-transformation steps in the construction of YAC libraries is facilitated. Moreover, the frequency of cotransformation (and, therefore, chimera formation) is significantly reduced. However, these advantages do incur a penalty. Yields of YAC transformants by this simplified intact cell approach are reduced some 25- to 30-fold compared to those obtained by the spheroplast transformation route. Nevertheless, the considerable advantages of the new system recommend it for a number of applications.
Heale
S M
SM
Department of Biochemistry and Applied Molecular Biology, UMIST, Manchester, UK.
Stateva
L I
LI
Oliver
S G
SG
D018244:Chromosomes, Artificial, Yeast; D003001:Cloning, Molecular / Q000379:methods*; D005822:Genetic Vectors; D015698:Genomic Library; D011995:Recombination, Genetic; D013104:Spheroplasts; D014170:Transformation, Genetic; D015003:Yeasts / Q000235:genetics