DETERMINATION OF COPY NUMBER OF RIBOSOMAL-RNA GENES IN PNEUMOCYSTIS-CARINII F SP. HOMINIS

Differential PCR was performed to determine the copy number of rRNA ge nes in Pneumocystis carinii f, sp, hominis. Two different reference ge nes, thymidylate synthase (TS) and beta-tubulin (BTU) genes, were used . Primers for the internal transcribed spacer (ITS) region of nuclear rRNA genes and either the TS or BTU gene were mixed together to perfor m PCR on seven different bronchoalveolar lavage specimens from patient s with P, carinii pneumonia, The radioactivity derived from the incorp orated radioactive nucleotides of each PCR product band was then used to calculate the copy number of the ITS relative to that of the TS or BTU gene. The copy number ratio between the ITS and the TS gene was de termined to be 0.8, and that between the ITS and the BTU gene was also 0.8, These results suggest that the ITS has the same copy number as t he TS or BTU gene, Since the copy number of the TS or BTU gene is pres umed to be 1, the results also suggest that P, carinii f, sp. hominis has only one copy of the ITS and thus one copy of the nuclear rRNA gen es. Therefore, two types of ITS sequences derived from a specimen woul d indicate that the patient is infected by two types of P, carinii f. sp, hominis.