John J. Dunn, Laura-Li Butler-Loffredo, Jan Kieleczawa and F. William Studier
Biology Department, Brookhaven National Laboratory, Upton, New York 11973
Borrelia burgdorferi is a spirochete that causes Lyme disease. It has a linear chromosome of about 935 kbp that contains about 70% AT base pairs. Although other workers have reported difficulty in cloning and maintaining long pieces of Borrelia DNA in E. coli, we readily obtained a library in our fesmid vector, presumably because the cloned DNA is propagated at very low copy number under control of the replication and partitioning functions of the F factor. Using a Sau3A partial digest of DNA from a very early passage strain, we picked 250 clones with inserts averaging about 35 kbp, an estimated 9-10 fold coverage of the Borrelia chromosome. We are beginning to sequence this chromosome as a test system in which to develop methods for sequencing DNA by primer walking using a hexamer library.
We have completed the sequence of one fesmid insert by generating both random and specific subclones in pGEM-based plasmid vectors, sequencing both ends of each subclone using standard vector primers, and filling the gaps and merging the entire sequence by primer walking with hexamer strings or by cycle sequencing with 18-mers formed by ligation of hexamers on hexamer templates. Many of the subclones were obtained by partial digestion with EcoRI under relaxed "star" conditions or with Tsp509 I, since both enzymes leave AATT overhangs that can be directly ligated into the EcoRI site of the pGEM vectors. BglII, HindII and PstI sites were also used in generating the subclones. A gap not covered by the subclones was filled by primer walking directly on the fesmid DNA.
Both strands of this fesmid insert were completely sequenced, a total of 34,820 base pairs. Sequences determined by priming with hexamer strings or ligated hexamers cover more than 80% of the insert. This part of Borrelia DNA contains a high density of coding sequence (97%), with 36 complete open reading frames plus incomplete coding sequences at each end, all transcribed in the same direction. Similarities with genes from other bacteria indicate that operons for septum formation and biosynthesis of the basal body and flagellum are contained in this region of the Borrelia genome.
Supported by the Office of Health and Environmental Research of the U. S. Department of Energy and by a grant from the National Institute of Allergy and Infectious Diseases.