DIETARY RESTRICTION EFFECT ON GENE EXPRESSION

饮食限制（DR）是唯一已知的实验操作， 延缓哺乳动物的衰老;然而， DR的延长作用目前尚不清楚。一个有吸引力 一种解释是，DR的影响至少部分地通过以下方式产生： 基因表达的变化。以热休克基因hsp 70为模型， 系统阐明衰老和DR改变基因的机制 转录，我们已经发现hsp 70转录的变化， 随着年龄的增长而发生，DR与特定 转录因子：HSF（热休克转录因子）。高等 在真核生物中，HSF存在于非应激细胞中的非活性（即，不表示 DNA结合）在细胞质中的单体形式。温度升高， 或其它应激，导致HSF活化成寡聚体， 结合hsp 70基因启动子中的热休克元件（HSE）。我们 已经表明年龄和DR改变HSF的激活。的目标 本提案中描述的研究旨在阐明 衰老和DR改变HSF的激活机制。 具体目标： 1.确定HSF激活的变化是否是由于 HSF表达。HSF的蛋白质和mRNA水平将被测量为 年龄和DR的函数。 2.为了确定HSF激活的变化是否是由于 HSF的寡聚化。寡聚化、磷酸化和 将在从大鼠获得的细胞提取物中测量HSF的易位 不同年龄段的动物自由进食和DR饮食。 3.为了确定HSF激活的变化是否与 HSF理化性质的改变。水动力 HSF的性质，HSF对HSE的亲和力，以及HSF与HSE的相互作用。 将研究具有hsp 70启动子的HSF作为年龄和DR的函数。 4.为了确定HSF的激活是否随年龄和DR的变化而变化， 产生于抑制细胞增殖的因素的水平/活动的变化。 激活HSF。设计了一种新的系统，用于筛选 从老年大鼠肝脏表达cDNA文库中检测是否存在 编码HSF活化抑制剂的cDNA。 5.为了证明HSF是诱导变化的原因， hsp 70的转录。重组HSF逆转HSF的能力， 热休克蛋白70启动子/报告基因体外转录的变化 将研究模板，并将使用转基因小鼠来确定是否 HSF的过表达可逆转hsp 70的衰老性下降 转录。

Dietary restriction (DR) is the only experimental manipulation known to retard aging in mammals; however, the mechanism responsible for the life prolonging action of DR is unknown at the present time. One attractive explanation is that the effect of DR arises at least partially through changes in gene expression. Using the heat shock gene hsp70 as a model system to elucidate the mechanism by which aging and DR alter gene transcription, we have found that the changes in hsp70 transcription that occur with age and DR are correlated to changes in a specific transcription factor: HSF (heat shock transcription factor). In higher eukaryotes, HSF is found in non-stressed cells in an inactive (i.e., non- DNA binding) monomeric form in the cytoplasm. An increase in temperature, or other stress, results in the activation of HSF to an oligomer that binds the heat shock element (HSE) in the promoter of the hsp70 gene. We have shown that age and DR alter the activation of HSF. The objective of the research described in this proposal is to elucidate the molecular mechanism whereby aging and DR alter the activation of HSF. Specific Aims: 1. To determine if the changes in HSF activation are due to an alteration in HSF expression. The protein and mRNA levels of HSF will be measured as a function of age and DR. 2. To determine if the changes in HSF activation arise from alterations in the oligomerization of HSF. The oligomerization, phosphorylation, and translocation of HSF will be measured in cell extracts obtained from rats of various ages fed ad libitum and a DR diet. 3. To determine if the changes in HSF activation can be correlated to alterations in the physiochemical properties of HSF. The hydrodynamic properties of HSF, the affinity of HSF for the HSE, and the interaction of HSF with the hsp70 promoter will be studied as a function of age and DR. 4. To determine if the changes in the activation of HSF with age and DR arise from changes in the levels/activities of factors that inhibit the activation of HSF. A novel system has been designed for screening an expression cDNA library from the liver of old rats for the presence of cDNAs coding for inhibitors of HSF activation. 5. To demonstrate that HSF is responsible for the changes in the induction of hsp7o transcription. The ability of recombinant HSF to reverse the changes in the in vitro transcription of an hsp70-promoter/reporter template will be studied, and transgenic mice will be used to determine if the overexpression of HSF can reverse the age-related decline in hsp 70 transcription.