KasI vs. SspDI: Comparative analysis of enzyme activity for restriction digestion

Type II restriction enzymes are routinely used by molecular biologists in designing and implementation of cloning experiments without referring to the literature on enzymes in use, and at times, face some unforeseeable problems. In our laboratory too, we encountered one such problem while working with KasI restriction enzyme which recognizes GGCGCC sequence, and we further analyzed the issue. Our observations corroborate the fact that KasI acts as monomer and cleaves double stranded DNA through nicking mechanism. It introduces breaks in two strands of DNA after substantial time gap which can be owed to two independent nickase activities in the opposite strands. Moreover, this time gap between two nickase activities results in formation of different topological forms of DNA. Since molecular biologists working with common restriction enzymes are not familiar with such nickase activity, they may misinterpret their restriction digestion results. However, no such problem was observed with the use of SspDI restriction enzyme which also recognizes the same sequence (GGCGCC) and produces the identical overhangs as by KasI. Hence, SspDI suits better for routine cloning and genetic modification purposes over KasI while using GGCGCC as cloning site.

Cloning of an ovule specific promoter from Arabidopsis thaliana and expression of beta-glucuronidase.

Tissue specific expression of transgenes in plant species has several advantages over constitutive expression. Identification of ovule specific promoters would be useful in genetic engineering of plants with a variety of desirable traits such as genetically engineered parthenocarpy, female sterile plants or seedless fruits. Relative inaccessibility and difficulty in harvesting adequate amounts of tissue at known developmental stages has impeded the progress in cloning of promoters involved in ovule development. In the present study an ovule specific promoter was cloned from Arabidopsis AGL11 gene and used to express GUS (beta-glucuronidase) gene in transgenic Arabidopsis. Histochemical staining of GUS appeared in the center of young ovary (ovules), but no detectable GUS activity was observed in vegetative plant tissues, sepals, petals and androecium. AGL11 gene promoter can be useful to modify the developmental path of plants by expressing either plant hormones or lethal genes for agronomic purpose.