GENE EXPRESSION/KIDNEY SPECIFIC VACUOLAR PROTON ATPASE

肾集合管是质子调节的最终部位 维持氢离子平衡所需的分泌物。氢离子 该片段中的运输由嵌入的细胞载体执行 空泡型H+-ATPase在这一段的离子外转运 它被极大地放大并以极化分布的形式分布在 质膜。申请者最近证明了一个 液泡H+ATPase 56kD亚基亚基的异构体 在肾间质细胞中扩增表达，但存在于 在其他组织中含量很低或检测不到。这就是我们的目的 研究基因表达调控机制的项目 编码空泡H+ATPase 56kD亚基的“肾”亚基，在 以便深入了解如何放大和专门使用 发生在质子运输细胞中的液泡H+-ATPase。这个 具体目标是： 1)克隆基因并测定其5‘侧翼的基因结构 区域和大鼠“肾”MR 56,000的第一内含子和外显子 液泡H+ATPase的亚基。 2)确定编码肾脏的高稳定水平的mRNA是否“ 空泡H+ATPase MR 56,000亚基的亚型可用 单独的高水平转录，或额外的转录后 机制，如信使核糖核酸稳定性。 3)确定基因组调控元件的程度 有助于“肾”的组织特异性和扩增表达 液泡H+ATPase亚基MR 56000亚基的异构体。 4)确定mRNA稳定性对组织的贡献程度 空泡“肾”异构体的特异扩增表达 H+ATPase MR 56,000亚基。 5)确定空泡的“肾”亚型的表达是否 H+ATPase MR 56,000亚基受体内酸摄入量增加的调节 老鼠， 6)建立永生化嵌合细胞系，通过使小鼠 “肾”亚型控制下的SV40大T抗原转基因 推动者。

The kidney collecting duct is the final site for the regulation of proton secretion required to maintain hydrogen ion balance. Hydrogen ion transport in this segment is carried out by The intercalated cells carry out ion transport in this segment by means of a vacuolar-type H+ATPase that is greatly amplified and distributed in a polarized distribution on the plasma membrane. The applicant recently demonstrated that one isoform of the 56 kD subunit of the vacuolar H+ATPase exhibits greatly amplified expression in the renal intercalated cells, but is present at low or undetectable levels in other tissues. It is the aim of this project to examine the mechanisms regulating expression of the gene encoding the "kidney" isoform of the vacuolar H+ATPase 56 kD subunit, in order to attain insights into the amplification and specialized use of the vacuolar H+ATPase that occurs in proton transporting cells. The specific aims are: 1) Clone the gene and determine the gene structure of the 5' flanking region and the first intron and exon of the rat "kidney" Mr 56,000 subunit of the vacuolar H+ATPase. 2) Determine if the high steady state level of mRNA encoding the kidney" isoform of vacuolar H+ATPase Mr 56,000 subunit can be accounted for by high levels of transcription alone, or additional posttranscriptional mechanisms, such as mRNA stability. 3) Determine the extent to which the genomic regulatory elements contribute to tissue specific and amplified expression of the "kidney" isoform of the vacuolar H+ATPase Mr 56,000 subunit. 4) Determine the extent to which mRNA stability contributes to tissue specific and amplified expression of the "kidney' isoform of the vacuolar H+ATPase Mr 56,000 subunit. 5) Determine if the expression of the "kidney' isoform of the vacuolar H+ATPase Mr 56,000 subunit is modulated by increased acid intake in the rat, 6) Develop an immortalized intercalated cell line by making mice transgenic for SV40 large T antigen under control of the "kidney isoform promoter.