In vitro methylation inhibits the promotor activity of a cloned intracisternal A-particle LTR.

We studied the relation between LTR methylation and expression of the family of endogenous retrovirus-like elements related to mouse intracisternal A-particles (IAP). Comparative HpaII/MspI and HhaI restriction analysis of genomic DNA's showed that in cells and tissues with a low level of IAP gene expression, HpaII and HhaI sites within the 5' LTR were heavily methylated, while in cells abundantly expressing IAP's 20 to 30% of the 5' LTRs were demethylated at these sites. The effects of methylation on the promoter activity of a cloned IAP 5' LTR was studied directly, using the plasmid pMIA5' L-cat in which this LTR was linked to the chloramphenicol acetyl transferase (CAT) gene. In vitro methylation of three HhaI sites located between -137 and -205 bp from the RNA start site of this LTR completely inactivated the promoter activity of pMIA5' L-cat transfected into COS7 cells. Methylation of a HpaII site located 94 bp downstream from the RNA start site reduced the promoter activity by 75%. The results show that methylation at sites both upstream and downstream from the RNA start site profoundly effects the promoter activity of this LTR and suggest that methylation within the 5' LTR can serve to regulate IAP gene expression in vivo.     

柞蚕核型多角体病毒(ApNPV)转移载体质粒pAp M2614的组建

自从美国科学家G.Smith等首次建立苜蓿尺蠖核型多角体病毒(AcNPV)转移载体表达系统以来,已被广泛用于外源基因的表达,成为世界上一新的具有巨大潜力的载体表达系统。为了进一步提高表达产量,降低成本,日本科学家前田进建立了家蚕核型多角体病毒(BmNPV)载体表达系统,并获得了高效表达。柞蚕是我国特产,以蛹滞育越冬,保存时间长,个体大,可工厂化生产。因此,组建柞蚕NPV转移载体,进而建立该载体表达系统,是目前利用昆虫活体为宿主进行外源基因表达较理想的昆虫杆状病毒载体表达系统。