Recovery of YAC-end sequences through complementation of an Escherichia colipyrF mutation
Recovery of YAC-end sequences through complementation of an Escherichia coli pyrF mutationDavid A. Wright, Sei-Kyoung Park1, Dongying Wu2, Gregory J. Phillips1, Steven R. Rodermel2 and Daniel F. Voytas*
Department of Zoology and Genetics, 1Department of Microbiology, Immunology and Preventive Medicine and 2Department of Botany, Iowa State University, Ames, IA 50011, USA
Received March 10, 1997;Revised and Accepted May 13, 1997
ABSTRACT
We have developed a genetic means to recover sequences from YAC-ends near the yeast selectable marker URA3.This strategy is based on the ability of URA3 to complement mutations in pyrF, an Escherichia coli gene required for pyrimidinebiosynthesis.We have developed an E.coli strain with a non-reverting allele of pyrF that is also suitable for cloning (recA-, hsdR-).We demonstrate the utility of this complementation strategy to obtain right-end clones from three YACs containing Arabidopsis thaliana DNA.
Yeast artificial chromosomes (YACs) are widely used for cloning genes by chromosome walking. This strategy typically requires generating a YAC contig that encompasses the gene of interest. To assemble a contig, a YAC near the desired locus is used as a starting point for the walk. DNA sequences are isolated from the ends of this YAC and used as hybridization probes to identify additional overlapping YACs. A difficulty with this procedure is the ability to quickly and reliably obtain YAC-end clones. One YAC-end (the left-end) carries a bacterial origin of replication and selectable marker. Left-ends can be recovered by digesting the YAC with restriction enzymes that release these sequences and part of the insert DNA; the product of self-ligation can be recovered in Escherichia coli as an autonomous plasmid (plasmid rescue). The isolation of the other YAC-end (the right-end), however, is more difficult. A variety of methods have been employed, including direct cloning strategies, which are often labor intensive, and variations of the polymerase chain reaction (PCR) (for examples see 1 -4 ). PCR methods are often unreliable, because the sequence of the insert DNA is unknown and cannot be used to make primers for traditional amplification strategies. In addition, PCR is limited by the size of DNA fragments that can be amplified, and amplification products often have to be cloned for detailed analysis.
We have developed a novel genetic method to clone YAC right-ends that utilizes the URA3 gene found at the right end of most YACs, including those constructed from the popular vector pYAC4 (5 ). The URA3 gene encodes orotidine-5'-phosphate decarboxylase, an enzyme involved in uracil biosynthesis. In E.coli, this enzyme is encoded by pyrF, and the yeast URA3 gene can complement pyrF mutations (6 ). We reasoned that the URA3 gene and adjacent YAC insert DNA could readily be recovered by utilizing the strong genetic selection afforded by pyrF complementation. In initial experiments, we tested this strategy with the pyrF strain MH1066 ([Delta]lacX74, hsr-, rpsL, pyrF::Tn5, leuB600, trpC9830, galE, galK). We found, however, that the pyrF allele in MH1066 reverts at a high enough frequency to make recovery of rare URA3-containing plasmids difficult. We have developed a pyrF- strain that overcomes this problem.
REFERENCES
1 Ausubel,F.M., Brent,R., Kingston,R.E., Moore,D.D., Seidman,J.G., Smith,J.A. and Struhl,K. (1987) Current Protocols in Molecular Biology. Greene/Wiley Interscience, New York, NY.
