Insertional tagging of at least two loci associated with resistance to adenine arabinoside in Toxoplasma gondii, and cloning of the adenosine kinase locus.
A genetic approach has been exploited to investigate adenylate salvage pathways in the protozoan parasite Toxoplasma gondii, a purine auxotroph. Using a new insertional mutagenesis vector designed to facilitate the rescue of tagged loci even when multiple plasmids integrate as a tandem array, 15 independent clonal lines resistant to the toxic nucleoside analog adenine arabinoside (AraA) were generated. Approximately two-thirds of these clones lack adenosine kinase (AK) activity. Parallel studies identified an expressed sequence tag (EST) exhibiting a small region of weak similarity to human AK, and this locus was tagged in several AK-deficient insertional mutants. Library screening yielded full-length cDNA and genomic clones. The T. gondii AK gene contains five exons spanning a approximately 3 kb locus, and the predicted coding sequence was employed to identify additional AK genes and cDNAs in the GenBank and dbEST databases. A genomic construct lacking essential coding sequence was used to create defined genetic knock-outs at the T. gondii AK locus, and AK activity was restored using a cDNA-derived minigene. Hybridization analysis of DNA from 13 AraA-resistant insertional mutants reveals three distinct classes: (i) AK-mutants tagged at the AK locus; (ii) AK- mutants not tagged at the AK locus, suggesting the possibility that another locus may be involved in regulating AK expression; and (iii) mutants with normal AK activity (potential transport mutants).
Sullivan
W J
WJ
Department of Biology, University of Pennsylvania, Philadelphia 19104-6018, USA.
Chiang
C W
CW
Wilson
C M
CM
Naguib
F N
FN
el Kouni
M H
MH
Donald
R G
RG
Roos
D S
DS
D000248:Adenosine Kinase / Q000235:genetics*; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D017931:DNA Primers / Q000235:genetics; D018076:DNA, Complementary / Q000235:genetics; D016054:DNA, Protozoan / Q000235:genetics; D004351:Drug Resistance / Q000235:genetics; D017125:Genes, Protozoan; D006801:Humans; D008954:Models, Biological; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D011687:Purines / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D016324:Sequence Tagged Sites; D014122:Toxoplasma / Q000187:drug effects / Q000201:enzymology* / Q000235:genetics*; D014740:Vidarabine / Q000494:pharmacology*
D017931:DNA Primers; D018076:DNA, Complementary; D016054:DNA, Protozoan; D011687:Purines; D000248:Adenosine Kinase; D014740:Vidarabine