Construction of bacterial artificial chromosome libraries from the parasitic nematode Brugia malayi and physical mapping of the genome of its Wolbachia endosymbiont.
The parasitic nematode, Brugia malayi, causes lymphatic filariasis in humans, which in severe cases leads to the condition known as elephantiasis. The parasite contains an endosymbiotic alpha-proteobacterium of the genus Wolbachia that is required for normal worm development and fecundity and is also implicated in the pathology associated with infections by these filarial nematodes. Bacterial artificial chromosome libraries were constructed from B. malayi DNA and provide over 11-fold coverage of the nematode genome. Wolbachia genomic fragments were simultaneously cloned into the libraries giving over 5-fold coverage of the 1.1 Mb bacterial genome. A physical framework for the Wolbachia genome was developed by construction of a plasmid library enriched for Wolbachia DNA as a source of sequences to hybridise to high-density bacterial artificial chromosome colony filters. Bacterial artificial chromosome end sequencing provided additional Wolbachia probe sequences to facilitate assembly of a contig that spanned the entire genome. The Wolbachia sequences provided a marker approximately every 10 kb. Four rare-cutting restriction endonucleases were used to restriction map the genome to a resolution of approximately 60 kb and demonstrate concordance between the bacterial artificial chromosome clones and native Wolbachia genomic DNA. Comparison of Wolbachia sequences to public databases using BLAST algorithms under stringent conditions allowed confident prediction of 69 Wolbachia peptide functions and two rRNA genes. Comparison to closely related complete genomes revealed that while most sequences had orthologs in the genome of the Wolbachia endosymbiont from Drosophila melanogaster, there was no evidence for long-range synteny. Rather, there were a few cases of short-range conservation of gene order extending over regions of less than 10 kb. The molecular scaffold produced for the genome of the Wolbachia from B. malayi forms the basis of a genomic sequencing effort for this bacterium, circumventing the difficult challenge of purifying sufficient endosymbiont DNA from a tropical parasite for a whole genome shotgun sequencing strategy.
Foster
Jeremy M
JM
New England Biolabs, 32 Tozer Rd, Beverly, MA 01915, USA. foster@neb.com
Kumar
Sanjay
S
Ganatra
Mehul B
MB
Kamal
Ibrahim H
IH
Ware
Jennifer
J
Ingram
Jessica
J
Pope-Chappell
Jesse
J
Guiliano
David
D
Whitton
Claire
C
Daub
Jennifer
J
Blaxter
Mark L
ML
Slatko
Barton E
BE
D000818:Animals; D001483:Base Sequence; D017178:Brugia malayi / Q000235:genetics*; D002874:Chromosome Mapping / Q000379:methods*; D022202:Chromosomes, Artificial, Bacterial / Q000235:genetics*; D020451:Contig Mapping / Q000379:methods; D004269:DNA, Bacterial / Q000235:genetics; D016054:DNA, Protozoan / Q000235:genetics; D016680:Genome, Bacterial; D018503:Genome, Protozoan; D015698:Genomic Library; D008970:Molecular Weight; D010957:Plasmids; D012329:RNA, Bacterial / Q000235:genetics; D012335:RNA, Ribosomal / Q000235:genetics; D015183:Restriction Mapping / Q000379:methods; D017422:Sequence Analysis, DNA / Q000379:methods; D013559:Symbiosis / Q000235:genetics; D020577:Wolbachia / Q000235:genetics*
D004269:DNA, Bacterial; D016054:DNA, Protozoan; D012329:RNA, Bacterial; D012335:RNA, Ribosomal