Molecular biological research for correlation between isozyme type of Toxoplasma specific enzyme and virulence

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We have previously reported the presence of a novel nucleoside triphosphate hydrolase (NTPase) in the rapidly multiplying tachyzoite form of Toxoplasma gondii. On further examination, it was found that tachyzoites contain two forms of NTPase (NTPase-I and NTPase-II). The nucleotide sequences of these genes have also been determined. With kinetic studies for these isozymes of many Toxoplasma strains, it has been presumed that NTPase-II is essentially present in all the strains and NTPase-I is only present in virulent strains. In the present study, it was found that the presence of isozymes in many strains correlated to the presence of genes which corresponded to these isozyme, that is to say, the gene encoding NTPase-I was not present in avirulent strains. It has also been presumed that all the strains have a pseudogene which dose not produce mRNA.However, an avirulent strain without the pseudogene was also recognized in the present study. We have cloned the gene which contains a promoter area for NTPase gene from a cosmid library of Toxoplasma. Subsequently, we studied a condition for reconstruction of functional gene by using some PCR products, and the constructed gene was inserted to a Phlomycin resistant plasmid. This plasmid was then transfected to avirulent strain of Toxoplasma. Unfortunately, we did not succeed to produce NTPase-I in avirulent strain. Probably, the pseudogene may play a critical role for expression of NTPase-I gene. An avirulent strain without the pseudogene recognized in the present study may be useful for next study.