Wright T W, Bissoondial T Y, Haidaris C G, Gigliotti F, Haidaris P J
Department of Microbiology and Immunology, University of Rochester School of Medicine and Dentistry, NY 14642, USA.
DNA Res. 1995;2(2):77-88. doi: 10.1093/dnares/2.2.77.
Two ferret P. carinii gpA cDNA clones were identified that reacted identically with a panel of anti-gpA monoclonal antibodies, although their nucleotide sequences were 22% divergent. Each clone hybridized to a single mRNA species of 3,600 nucleotides only in P. carinii-infected lung mRNA, but RT-PCR analysis demonstrated that these cDNA clones were derived from two distinct gpA mRNA transcripts. Further PCR analysis demonstrated that the ferret P. carinii genome contains at least two gpA genes lying in tandem on a single chromosome separated by a 329-bp intergenic region. Based on the terminal gene sequences of this tandem repeat and the cDNA clones, a composite full-length ferret P. carinii gpA coding sequence was constructed. The intergenic region immediately downstream of the stop codon of the first gpA gene contains three putative polyadenylation signals, and constitutes the 3' untranslated region (UTR) of the gpA mRNA. Primer extension of the gpA mRNA resulted in products extending 74 and 244 nucleotides into the 5' UTR. However, the intergenic region lying greater than 25 nucleotides upstream of the first methionine of the second gpA gene was found to be absent from the 5' UTR.
鉴定出两个雪貂卡氏肺孢子虫gpA cDNA克隆，它们与一组抗gpA单克隆抗体的反应完全相同，尽管它们的核苷酸序列有22%的差异。每个克隆仅在感染卡氏肺孢子虫的肺mRNA中与一种3600个核苷酸的单一mRNA物种杂交，但逆转录聚合酶链反应（RT-PCR）分析表明，这些cDNA克隆源自两种不同的gpA mRNA转录本。进一步的PCR分析表明，雪貂卡氏肺孢子虫基因组在一条单染色体上至少包含两个串联的gpA基因，它们被一个329 bp的基因间隔区隔开。基于这种串联重复序列的末端基因序列和cDNA克隆，构建了一个复合的全长雪貂卡氏肺孢子虫gpA编码序列。第一个gpA基因终止密码子紧邻下游的基因间隔区包含三个假定的聚腺苷酸化信号，并构成gpA mRNA的3'非翻译区（UTR）。gpA mRNA的引物延伸产生了延伸到5' UTR中74和244个核苷酸的产物。然而，在第二个gpA基因第一个甲硫氨酸上游大于25个核苷酸的基因间隔区在5' UTR中未被发现。
