DNA Methylation and Human Endogenous Retroviruses Linking Genetic and Environmental Factors in the Pathogenesis of Psychiatric Disorders

(1)全面筛选具有脑内转录潜力的人内源性逆转录病毒（HERV） EST数据库，并对其活性进行实验确认。在460万条EST中，我们从各种脑相关组织和细胞系中选取了44万条EST，从中提取了12个可能具有转录活性的HERV基因座。最后，我们通过RT-PCR和随后的克隆和测序分析鉴定了12个herv中的6个。在之前的一项研究中，我们鉴定了属于HERV- h和HERV- k家族的两个HERV基因座。HERV-H（22q12）位于SYN3基因下游约4kb的反向位置。为了研究反义RNA对HERV-H读透产物SYN3的影响，我们构建了三个表达载体，其中包含从HERV-H的3'LTR到SYN3的最后一个外显子的基因组序列，除了最后一个外显子被反转之外的基因组序列相同，以及没有外显子的序列。正在研究实验条件以获得最佳结果。(3)对HERV-K家族的甲基化状态进行了初步研究，采用甲基化敏感酶进行Southern blot杂交检测。材料为来自三个不同个体的外周白细胞、胎脑、胎肝和胎盘。虽然几乎所有的位点都被重度甲基化，但甲基化状态在组织和个体之间存在差异。甲基化状态依次为：外周血白细胞>脑>肝>胎盘。当个体之间的差异在胎盘中为A bb0 B≧C时，这个顺序在肝脏中也保持不变。

(1)Comprehensive screening of EST database for human endogenous retroviruses(HERVs) with transcriptional potential in the brain and experimental confirmation of their activitiesAmong 4.6-million ESTs, we selected 440 thousands that were prepared from various brain-related tissues and cell lines From the 440-thousand ESTs, we extracted 12 HERV loci that may have transcriptional activities. Finally we identified six out of the 12 HERVs by RT-PCR and subsequent cloning & sequencing analysis.(2)Investigation of an anti-sense RNA effect on nearby genes by HERV-read-through transcriptsIn a previous study, we identified two HERV loci belonging to HERV-H and HERV-K families. That HERV-H(22q12) was located about 4 kb downstream of the SYN3 gene in the reverse orientation. To investigate an anti-sense RNA effect on SYN3 of the HERV-H read-through product, we constructed three expression vectors containing a genomic sequence from the 3'LTR of the HERV-H to the last exon of SYN3, the same genomic sequence except the last exon being reversed, and no sequence of the exon. Experimental conditions are being examined to obtain the best result.(3)A preliminary study on HERV-K methylationMethylation states of the HERV-K family were examined by Southern blot hybridization using a methylation sensitive enzyme. Materials were peripheral leukocytes, fetal brain, fetal liver, and placenta from three different individuals. Although almost loci were heavily methylated, difference of methylation states was observed between tissues and between individuals. The methylation states were higher in the following order : peripheral leukocytes> brain> liver> placenta. When the difference between individuals was A> B≧C in the placentas, this order was also kept in the livers.,