Polyamines eliminate an extreme size bias against transformation of large yeast artificial chromosome DNA.
The recent development of vectors and methods for cloning large linear DNA as yeast artificial chromosomes (YACs) has enormous potential in facilitating genome analysis, particularly because of the large cloning capacity of the YAC cloning system. However, the construction of comprehensive libraries with very large DNA segments (400-500 kb average insert size) has been technically very difficult to achieve. We have examined the possibility that this difficulty is due, at least in part, to preferential transformation of the smaller DNA molecules in the yeast transformation mixture. Our data indicate that the transformation efficiency of a 330-kb linear YAC DNA molecule is 40-fold lower, on a molar basis, than that of a 110-kb molecule. This extreme size bias in transformation efficiency is dramatically reduced (to less than 3-fold) by treating the DNA with millimolar concentrations of polyamines prior to and during transformation into yeast spheroplasts. This effect is accounted for by a stimulation in transformation efficiency of the 330-kb YAC molecule; the transformation efficiency of the 110-kb YAC molecule is not affected by the inclusion of polyamines. Application of this finding to the cloning of large exogenous DNA as artificial chromosomes in yeast will facilitate the construction of genomic libraries with significantly increased average insert sizes. In addition, the methods described allow efficient transfer of YACs to yeast strain backgrounds suitable for subsequent manipulations of the large insert DNA.
Connelly
C
C
Department of Molecular Biology and Genetics, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205.
McCormick
M K
MK
Shero
J
J
Hieter
P
P
D015825:Chromosomes, Fungal; D004247:DNA / Q000187:drug effects*; D015723:Gene Library; D015894:Genome, Human; D006801:Humans; D011073:Polyamines / Q000494:pharmacology*; D014170:Transformation, Genetic
D011073:Polyamines; D004247:DNA