Recombination walking: genetic selection of clones from pooled libraries of yeast artificial chromosomes by homologous recombination.

Recombination walking is based on the genetic selection of specific human clones from a yeast artificial chromosome (YAC) library by homologous recombination. The desired clone is selected from a pooled (unordered) YAC library, eliminating labor-intensive steps typically used in organizing and maintaining ordered YAC libraries. Recombination walking represents an efficient approach to library screening and is well suited for chromosome-walking approaches to the isolation of genes associated with common diseases.

Comparison of Coxiella burnetii plasmids to homologous chromosomal sequences present in a plasmidless endocarditis-causing isolate.

Coxiella burnetii is the etiological agent of human Q fever and chronic endocarditis. Different plasmids have been found in C. burnetii isolates and a correlation between disease state and plasmid type has been established. The plasmid QpRS was found in all but four of the endocarditis-causing isolates examined. These four isolates did not contain a detectable plasmid. However, when DNA from the plasmidless isolates is hybridized with 32P-labeled QpRS, homologous sequences are detected. It was hypothesized that plasmid sequences had inserted into the chromosomal DNA of the plasmidless isolate. A cosmid chromosomal gene bank was constructed from one of the plasmidless isolates and a number of clones were obtained. One clone, pEAS137, contained all of the EcoR I fragments with homology to the C. burnetii plasmids plus several non-homologous fragments. The EcoR I fragments in pEAS137 were in the same linear order as present in the chromosome of the plasmidless isolate and were shown to exist as a single contiguous sequence. This information supports the hypothesis that plasmid sequences have inserted into the chromosomal DNA and makes pEAS137 a good candidate for studying the relationships between the plasmids. Initial studies comparing pEAS137 to QpRS and QpH1 suggest that pEAS137 is more closely related to QpRS than to QpH1.