Characterization of yeast artificial chromosomes from Plasmodium falciparum: construction of a stable, representative library and cloning of telomeric DNA fragments.
Molecular genetic studies of the human malaria parasite Plasmodium falciparum have been hampered in part due to difficulties in stably cloning and propagating parasite genomic DNA in bacteria. This is thought to be a result of the unusual A+T bias (>80%) in the parasite's DNA. Pulsed-field gel electrophoretic separation of P. falciparum chromosomes has shown that large chromosomal polymorphisms, resulting from the deletion of DNA from chromosome ends, frequently occur. Understanding the biological implications of this chromosomal polymorphism will require the analysis of large regions of genomic, and in particular telomeric, DNA. To overcome the limitations of cloning parasite DNA in bacteria, we have cloned genomic DNA from the P. falciparum strain FCR3 in yeast as artificial chromosomes. A pYAC4 library with an average insert size of approximately 100 kb was established and found to have a three to fourfold redundancy for single-copy genes. Unlike bacterial hosts, yeast stably maintain and propagate large tracts of parasite DNA. Long-range restriction enzyme mapping of YAC clones demonstrates that the cloned DNA is contiguous and identical to the native parasite genomic DNA. Since the telomeric ends of chromosomes are underrepresented in YAC libraries, we have enriched for these sequences by cloning P. falciparum telomeric DNA fragments (from 40 to 130 kb) as YACs by complementation in yeast.
de Bruin
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D
Department of Molecular Biology, Cornell University Graduate School of Medical Sciences, New York, New York 10021.
Lanzer
M
M
Ravetch
J V
JV
D000818:Animals; D001483:Base Sequence; D015139:Blotting, Southern; D015825:Chromosomes, Fungal; D003001:Cloning, Molecular; D016054:DNA, Protozoan / Q000235:genetics*; D016521:Electrophoresis, Gel, Pulsed-Field; D015723:Gene Library; D017125:Genes, Protozoan; D008969:Molecular Sequence Data; D010963:Plasmodium falciparum / Q000235:genetics*; D016615:Telomere
D016054:DNA, Protozoan