TY - JOUR
T1 - Insertional tagging, cloning, and expression of the Toxoplasma gondii hypoxanthine-xanthine-guanine phosphoribosyltransferase gene
T2 - Use as a selectable marker for stable transformation
AU - Donald, Robert G.K.
AU - Carter, Darrick
AU - Ullman, Buddy
AU - Roos, David S.
PY - 1996
Y1 - 1996
N2 - A nonhomologous integration vector was used to identify the Toxoplasma gondii hypoxanthine-xanthine-guanine phosphoribosyl transferase (HXGPRT) gene by insertional mutagenesis. Parasite mutants resistant to 6-thioxanthine arose at a frequency of ∼3 × 10-7. Genomic DNA flanking the insertion sites was retrieved by marker rescue and used to identify molecular clones exhibiting unambiguous homology to H(X)GPRT genes from other species. Sequence analysis of vector/genome junction sites reveals that integration of the linearized vector occurred with minimal rearrangement of either vector or target sequences, although the addition of filler DNA and small duplications or deletions of genomic sequences at the transgene termini was observed. Two differentially spliced classes of cDNA clones were identified, both of which complement hpt and gpt mutations in Escherichia coli. Kinetic analysis of purified recombinant enzyme revealed no significant differences between the two isoforms. Internally deleted clones spanning the genomic locus were used to create "knock-out" parasites, which lack all detectable HXGPRT activity. Complete activity could be restored to these knock-out mutants by transient transformation with either genomic DNA or cDNA-derived minigenes encoding both enzyme isoforms. Stable HXGPRT+ transformants were isolated under selection with mycophenolic acid, demonstrating the feasibility of HXGPRT as both a positive and negative selectable marker for stable transformation of T. gondii.
AB - A nonhomologous integration vector was used to identify the Toxoplasma gondii hypoxanthine-xanthine-guanine phosphoribosyl transferase (HXGPRT) gene by insertional mutagenesis. Parasite mutants resistant to 6-thioxanthine arose at a frequency of ∼3 × 10-7. Genomic DNA flanking the insertion sites was retrieved by marker rescue and used to identify molecular clones exhibiting unambiguous homology to H(X)GPRT genes from other species. Sequence analysis of vector/genome junction sites reveals that integration of the linearized vector occurred with minimal rearrangement of either vector or target sequences, although the addition of filler DNA and small duplications or deletions of genomic sequences at the transgene termini was observed. Two differentially spliced classes of cDNA clones were identified, both of which complement hpt and gpt mutations in Escherichia coli. Kinetic analysis of purified recombinant enzyme revealed no significant differences between the two isoforms. Internally deleted clones spanning the genomic locus were used to create "knock-out" parasites, which lack all detectable HXGPRT activity. Complete activity could be restored to these knock-out mutants by transient transformation with either genomic DNA or cDNA-derived minigenes encoding both enzyme isoforms. Stable HXGPRT+ transformants were isolated under selection with mycophenolic acid, demonstrating the feasibility of HXGPRT as both a positive and negative selectable marker for stable transformation of T. gondii.
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U2 - 10.1074/jbc.271.24.14010
DO - 10.1074/jbc.271.24.14010
M3 - Article
C2 - 8662859
AN - SCOPUS:0029895115
SN - 0021-9258
VL - 271
SP - 14010
EP - 14019
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
IS - 24
ER -