Nelson D M, Foresman M D, Ronnei B J, McIvor R S
Institute of Human Genetics, University of Minnesota, Minneapolis 55455.
Gene. 1992 Apr 15;113(2):215-21. doi: 10.1016/0378-1119(92)90398-9.
To isolate murine purine nucleoside phosphorylase-encoding cDNA sequences (PNP), a murine BALB/c liver cDNA library in lambda gt10 was screened for recombinants hybridizing to a human PNP cDNA probe. Two of three clones recovered included inserts large enough to contain the full-length coding sequence. Sequence analysis of the largest clone revealed an 867-nucleotide open reading frame encoding 289 amino acids with 84% residue identity to that encoded by human PNP and 351 bp of 3'-untranslated region. The 5' end of the murine PNP message was specifically amplified by PCR using the RACE (rapid amplification of cDNA ends) protocol, revealing a 5'-untranslated region of 78 bp. Northern hybridization using the murine PNP cDNA sequence as a probe identified a message of approx. 1.6 kb in mouse NIH3T3 cells which was slightly smaller than the human message observed in HeLa cells. The cloned murine PNP cDNA coding sequence was inserted into a mammalian expression vector under transcriptional regulation of the Moloney murine leukemia virus long terminal repeat. Transfection of this plasmid into human 293 cells resulted in the expression of PNP activity which co-focused with murine PNP activity extracted from NIH3T3 cells, verifying that the isolated murine PNP cDNA clone encoded catalytically active PNP protein.
为了分离小鼠嘌呤核苷磷酸化酶编码cDNA序列（PNP），用λgt10载体构建的小鼠BALB/c肝脏cDNA文库筛选与人类PNP cDNA探针杂交的重组体。回收的三个克隆中有两个包含足够大的插入片段，足以包含全长编码序列。对最大的克隆进行序列分析，发现一个867个核苷酸的开放阅读框，编码289个氨基酸，与人类PNP编码的氨基酸序列有84%的残基同一性，以及351bp的3'非翻译区。使用RACE（cDNA末端快速扩增）方法通过PCR特异性扩增小鼠PNP信使RNA的5'端，揭示了一个78bp的5'非翻译区。以小鼠PNP cDNA序列为探针进行Northern杂交，在小鼠NIH3T3细胞中鉴定出一条约1.6kb的信使RNA，略小于在HeLa细胞中观察到的人类信使RNA。将克隆的小鼠PNP cDNA编码序列插入到受莫洛尼小鼠白血病病毒长末端重复序列转录调控的哺乳动物表达载体中。将该质粒转染到人293细胞中，导致PNP活性表达，该活性与从NIH3T3细胞中提取的小鼠PNP活性共聚焦，证实分离的小鼠PNP cDNA克隆编码具有催化活性的PNP蛋白。
