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。 氢离子 这一节段的运输是由闰细胞进行的。 通过液泡型H + ATP酶在此节中向外离子运输 它被极大地放大并以极化分布的方式分布在 质膜。 申请人最近证明， 液泡膜H~+ ATP酶56 kD亚基的一种同工型， 在肾间质细胞中表达增强，但在 在其他组织中水平低或检测不到。 这是我们的目的 研究基因表达调控机制的项目 编码液泡H + ATP酶56 kD亚基的"肾"亚型， 为了获得对放大和专门使用的见解， 质子运输细胞中的液泡H + ATP酶。 的 具体目标是： 1)克隆该基因并确定其5 '侧翼的基因结构 区域和第一内含子和外显子的大鼠"肾"先生56，000 液泡H + ATP酶亚基。 2)确定是否高稳态水平的mRNA编码的肾脏" 液泡H + ATP酶Mr56，000亚基亚型可通过以下方法解释： 高水平的单独转录，或额外的转录后 例如mRNA的稳定性。 3)确定基因组调控元件 有助于"肾"的组织特异性和扩增表达 液泡H + ATP酶Mr 56，000亚基的同种型。 4)确定mRNA稳定性在多大程度上有助于组织 特异性和扩增表达的"肾"亚型的空泡 H + ATP酶Mr 56，000亚基。 5)确定空泡细胞的"肾"亚型的表达是否 H + ATP酶Mr56，000亚基通过增加酸摄入调节， 老鼠， 6)用小鼠建立永生化闰细胞系 在"肾同种型"控制下的SV40大T抗原转基因 启动子

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.