Roles of stress protein hsp105 during mouse embryo development.

1.105 kDa热休克蛋白(HSP105)是大分子质量热休克蛋白家族的成员。为了阐明小鼠HSP105的基因组结构并研究其基因表达的调控，我们分离并鉴定了小鼠HSP105基因，其5‘侧翼区长约1.2kb。(1)小鼠HSP105基因全长约22kb，由18个外显子和17个内含子组成。(2)Southern印迹分析显示HSP105只有一个拷贝。(3)引物延伸分析表明，转录起始点位于ATG翻译起始密码子上游165bp。(4)HSP105基因5‘端启动子区含有一个TATA盒、一个CAAT盒、一个倒置CAAT盒和两个GC盒。在NT-和NT-128处发现了两个热休克元件(HSE)序列，分别为4个NAGAN重复序列。(5)利用缺失导数进行启动子分析表明，包含两个共同的HSE序列的最小区域在热激反应中是活跃的，并且对于该基因的结构性表达也是活跃的。为探讨热休克蛋白105(HSP105)在小鼠畸胎癌F9细胞分化和凋亡中的作用，将构建的小鼠HSP105基因表达载体导入F9细胞，分离出两个高表达HSP105的细胞，其表达水平是亲本细胞的2~3倍。(2)HSP105α过表达细胞对热休克、放线菌素D、依托泊苷、过氧化氢等应激反应比亲本细胞和pcDNA3载体转染组敏感。(3)由于HSP105α对这些应激反应的敏感性增加是由于凋亡细胞死亡增加所致，因此建议HSP105α促进细胞凋亡。阐明HSP105α诱导细胞凋亡的确切机制的实验目前正在进行中。

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.