Monaco L, Vicini E, Conti M
Institute of Histology and General Embryology, University of Rome, Italy.
J Biol Chem. 1994 Jan 7;269(1):347-57.
The products of two phosphodiesterase (PDE) genes (ratPDE3/IVd and ratPDE4/IVb) are present in the rat Sertoli cell in culture, and their expression is under the control of the gonadotropin follicle-stimulating hormone (Swinnen, J.V., Tsikalas, K.E., and Conti, M. (1991) J. Biol. Chem. 266, 18370-18377). To understand the basis of the sequence heterogeneity found in the 5'-region of the different cDNAs thus far characterized, the structure of the coding region of these two cAMP PDE genes was investigated. Analysis of five ratPDE3/IVd and ratPDE4/IVb genomic clones showed that the coding region of these genes expressed in the Sertoli cell is divided into 11 exons distributed over 35-45 kilobases of genomic DNA. The intron/exon boundaries agreed, with some exceptions, with the established consensus sequences and were located in the same position in the coding region of the two genes. Also present were similarities to the exon composition of the Drosophila melanogaster "dunce" gene, the ancestor of these mammalian cAMP PDEs. Multiple AUG codons and short open reading frames were present at the 5'-untranslated end of the ratPDE4/IVb mRNA, but not in the ratPDE3 mRNA. By using polymerase chain reaction amplification or Northern analysis, it was determined that at least two forms of ratPDE3/IVd mRNA are present in rat Sertoli and FRTL-5 thyroid cells, but not in the brain. These mRNA variants are generated by inclusion or removal of an intron sequence that produces a frameshift affecting the position of the initiation AUG codon. Both mRNA species were efficiently translated into cAMP PDE proteins with different molecular masses in a transient transfection assay in COS cells. Polymerase chain reaction amplification demonstrated that heterogeneity of ratPDE4/IVb mRNAs was present in the same location as in the ratPDE3/IVd mRNA. Two ratPDE4/IVb mRNAs with different 5'-ends were expressed in Sertoli and FRTL-5 cells and in the brain. This heterogeneity is caused by the presence of an intron promoter that controls the transcription of this mRNA in Sertoli and FRTL-5 cells, but not in the brain. Upstream exons and additional promoters are probably present and necessary to generate the brain-specific mRNAs. These findings demonstrate that the cAMP-specific PDE genes have complex structure and that cAMP PDE proteins with different amino termini are derived from these genes.
两种磷酸二酯酶（PDE）基因（大鼠PDE3/IVd和大鼠PDE4/IVb）的产物存在于培养的大鼠支持细胞中，其表达受促性腺激素卵泡刺激素的调控（斯温嫩，J.V.，齐卡拉斯，K.E.，和孔蒂，M.（1991年）《生物化学杂志》266卷，18370 - 18377页）。为了解迄今为止所鉴定的不同cDNA 5'区域中序列异质性的基础，对这两个环磷酸腺苷（cAMP）PDE基因编码区的结构进行了研究。对五个大鼠PDE3/IVd和大鼠PDE4/IVb基因组克隆的分析表明，在支持细胞中表达的这些基因的编码区被分为11个外显子，分布在35 - 45千碱基的基因组DNA上。内含子/外显子边界除了一些例外情况，与既定的共有序列一致，并且位于这两个基因编码区的相同位置。还存在与果蝇“迟钝”基因外显子组成的相似性，该基因是这些哺乳动物cAMP PDE的祖先。在大鼠PDE4/IVb mRNA的5'非翻译末端存在多个AUG密码子和短开放阅读框，但在大鼠PDE3 mRNA中不存在。通过聚合酶链反应扩增或Northern分析确定，至少两种形式的大鼠PDE3/IVd mRNA存在于大鼠支持细胞和FRTL - 5甲状腺细胞中，但不存在于脑中。这些mRNA变体是通过包含或去除一个内含子序列产生的，该内含子序列会导致移码，影响起始AUG密码子的位置。在COS细胞的瞬时转染实验中，这两种mRNA都能有效地翻译成具有不同分子量的cAMP PDE蛋白。聚合酶链反应扩增表明，大鼠PDE4/IVb mRNA的异质性与大鼠PDE3/IVd mRNA位于相同位置。两种具有不同5'末端的大鼠PDE4/IVb mRNA在支持细胞、FRTL - 5细胞和脑中表达。这种异质性是由一个内含子启动子的存在引起的，该启动子控制该mRNA在支持细胞和FRTL - 5细胞中的转录，但在脑中不控制。可能存在上游外显子和其他启动子，它们对于产生脑特异性mRNA是必要的。这些发现表明，cAMP特异性PDE基因具有复杂的结构，并且具有不同氨基末端的cAMP PDE蛋白源自这些基因。
