Cowdria ruminantium DNA is unstable in a SuperCos1 library.
A Cowdria ruminantium genomic library was constructed in a cosmid vector to serve as a source of easily accessible and pure C. ruminantium DNA for molecular genetic studies. The cosmid library contained 846 clones which were arrayed into microtitre plates. Restriction enzyme digestion patterns indicated that these clones had an average insert size of 35 kb. Probing of the arrays did not detect any bovine clones and only one of the known C. ruminantium genes, pCS20, was detected. Due to the high AT content and the fact that C. ruminantium genes are active in the Escherichia coli host, the C. ruminantium clones were unstable in the SuperCos1 vector and most clones did not grow reproducibly. The library was contaminated with E. coli clones and these clones were maintained with greater fidelity than the C. ruminantium clones, resulting in a skewed representation over time. We have isolated seven C. ruminantium clones which we were able to serially culture reproducibly; two of these clones overlap. These clones constitute the first large regions of C. ruminantium DNA to be cloned and represent almost 10% of the C. ruminantium genome.
Brayton
K A
KA
Molecular Biology Section, Onderstepoort Veterinary Institute, South Africa.
De Villiers
E P
EP
Fehrsen
J
J
Nxomani
C
C
Collins
N E
NE
Allsopp
B A
BA
D000818:Animals; D002417:Cattle; D003001:Cloning, Molecular / Q000379:methods; D003360:Cosmids / Q000235:genetics*; D016995:Ehrlichia ruminantium / Q000235:genetics*; D005821:Genetic Techniques / Q000592:standards; D005820:Genetic Testing; D016680:Genome, Bacterial; D015698:Genomic Library; D006357:Heartwater Disease / Q000209:etiology; D009693:Nucleic Acid Hybridization