Roles of stress protein hsp105 during mouse embryo development.

1. 105 kda热休克蛋白（HSP105）是高分子量热休克蛋白家族的一员。为了阐明小鼠HSP105的基因组结构并研究其基因的表达调控，我们分离并表征了小鼠HSP105基因，其中包括约1.2kb的5'侧区。(1)小鼠HSP105基因全长约22 kb，由18个外显子和17个内含子组成。(2) Southern blotting分析显示HSP105存在单拷贝。(3)引物延伸分析表明，转录起始位点位于ATG翻译起始密码子上游165 bp处。(4) HSP105基因5′-启动子区包含1个TATA盒、1个CAAT盒、1个倒CAAT盒和2个GC盒。在nt -64和nt -128处发现两个热休克元件（HSE）序列为4个nGAAn重复序列。(5)利用缺失衍生物进行启动子分析发现，一个包含两个一致的HSE序列的最小区域对热休克有反应，也对基因的组成表达有活性。探讨HSP105在细胞分化和凋亡中的作用。将pcDNA3载体构建的小鼠HSP105alpha cDNA表达质粒导入小鼠畸胎瘤F9细胞，分离出2个HSP105alpha过表达细胞，其HSP105表达水平比亲本细胞高2-3倍。(2) hsp105α过表达细胞对热休克、放线菌素D、依托泊苷、过氧化氢等多种应激反应的敏感性均高于亲本细胞或转染pcDNA3载体的细胞。(3)由于对这些应激的敏感性增加是由于凋亡细胞死亡的增加，因此HSP105alpha可能促进细胞凋亡。阐明HSP105alpha细胞凋亡的确切机制的实验正在进行中。

1. The 105-kDa heat shock protein (HSP105) is a member of the high molecular mass heat shock protein family. To elucidate the genomic structure of mouse HSP105 and to examine the regulation of expression of its gene, we have isolated and characterized the mouse HSP105 gene including about 1.2kb of the 5'-flanking region. (1) The mouse HSP105 gene spans about 22 kb, consisting of 18 exons separated by 17 introns. (2) Southern blotting analysis revealed the existence of a single copy of HSP105. (3) Primer extension analysis revealed that the transcription initiation site was located 165 bp upstream of the ATG translation initiation codon. (4) The 5'-promoter region of the HSP105 gene contained a TATA box, a CAAT box, an inverted CAAT box and two GC boxes. Two heat shock element (HSE) sequences were found as four nGAAn repeats at nt -64 and nt -128. (5) Promoter analysis using deletion derivatives revealed that a minimal region which contained the two consensus HSE sequences was active in response to heat shock and also for constitutive expression of the gene.2. To examine the role of HSP105 for differentiation and apoptosis.of mouse teratocarcinoma F9 cells, mouse HSP105alpha cDNA expression plasmid constructed with pcDNA3 vector was introduced into F9 cells, and isolated two HSP105alpha-overexpressing cells which expressed HSP105 at 2-3-fold higher levels than parent cells. (2) In response to various stresses such as heat shock, actinomycin D, etoposide and hydrogen peroxide, the HSP105alpha-overexpressing cells were more sensitive than parent cells or the cells transfected with pcDNA3 vector. (3) Since the increased sensitivity to these stresses was due to an increase of apoptotic cell death, HSP105alpha was suggested to enhance apoptosis. Experiments to elucidate precise mechanisms of HSP105alpha for apoptosis are now in progress.