INSERTIONAL MUTAGENESIS AND MARKER RESCUE IN A PROTOZOAN PARASITE - CLONING OF THE URACIL PHOSPHORIBOSYLTRANSFERASE LOCUS FROM TOXOPLASMA-GONDII
INSERTIONAL MUTAGENESIS AND MARKER RESCUE IN A PROTOZOAN PARASITE - CLONING OF THE URACIL PHOSPHORIBOSYLTRANSFERASE LOCUS FROM TOXOPLASMA-GONDII
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DOI：
10.1073/pnas.92.12.5749
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发表时间：
1995-06-06
影响因子：
11.1
通讯作者：
ROOS, DS
中科院分区：
文献类型：
--
作者：
DONALD, RGK;ROOS, DS
非同源整合载体已被用来证明插入诱变的可能性在单倍体速殖子的原生动物寄生虫弓形虫。以约10（-6）的频率（约2 × 10（-5）的稳定转化体）鉴定对5-氟尿嘧啶具有抗性的突变克隆。分离到4个独立的突变体，它们均显示缺乏尿嘧啶磷酸核糖基转移酶（UPRT）活性，并且含有整合在紧密连锁的基因座处的转基因，表明UPRT编码基因失活。（沿着整合的载体）通过用限制性酶切的、自连接的总基因组DNA进行细菌转化而容易地回收，用回收的片段筛选基因组文库，鉴定出与来自其他物种的已知UPRT编码基因具有高度同源性的序列，并分离出含有预测编码244个氨基酸的酶的单个开放阅读框的cDNA克隆。同源重组载体被利用来创建基因敲除在UPRT基因座，这是缺乏酶活性，但可以补充的瞬时转化与野生型序列正式确认鉴定的功能UPRT基因。转基因插入点的映射表明，在UPRT基因内的不同位点的整合产生多个独立的突变体，这表明非同源整合是足够随机的，允许在一个单一的转化标记整个寄生虫基因组。
Nonhomologous integration vectors have been used to demonstrate the feasibility of insertional mutagenesis in haploid tachyzoites of the protozoan parasite Toxoplasma gondii. Mutant clones resistant to 5-fluorouracil were identified at a frequency of approximate to 10(-6) (approximate to 2 x 10(-5) of the stable transformants). Four independent mutants were isolated, all of which were shown to lack uracil phosphoribosyltransferase (UPRT) activity and harbor transgenes integrated at closely Linked loci, suggesting inactivation of the UPRT-encoding gene, Genomic DNA flanking the insertion point (along with the integrated vector) was readily recovered by bacterial transformation with restriction-digested, self-ligated total genomic DNA, Screening of genomic libraries with the recovered fragment identified sequences exhibiting high homology to known UPRT-encoding genes from other species, and cDNA clones were isolated that contain a single open reading frame predicted to encode the 244-amino acid enzyme. Homologous recombination vectors were exploited to create genetic knock-outs at the UPRT locus, which are deficient in enzyme activity but can be complemented by transient transformation with wild-type sequences-formally confirming identification of the functional UPRT gene. Mapping of transgene insertion points indicates that multiple independent mutants arose from integration at distinct sites within the UPRT gene, suggesting that nonhomologous integration is sufficiently random to permit tagging of the entire parasite genome in a single transformation.