Cloning of human ADAMTS-2 Promoter: Strategies for cloning extremely GC rich promoters

Abstract

ADAMTS (A Disintegrin and Metalloproteinase with Thrombo-spondin Motifs) are zinc dependent proteases have a part in impor-tant physiologic processes such as development, homeostasis andfertility. To date, 19 different ADAMTS proteases have been iden-tified. ADAMTS-2 is a member of ADAMTS family. Togetherwith ADAMTS-3 and ADAMTS-14, ADAMTS-2 has procollagenN-proteinase activity. It mainly processes type I, II, III, and V col-lagen precursors that have a key role for all humans. This processis important for the correct fibril and fiber conformation of con-nective tissue. Therefore, ADAMTS-2 has been implicated in somehuman diseases like Ehler-Danlos syndrome type VIIC and derma-tosparaxis. Recently, It also has been postulated it’s anti-angio-genic and anti-tumoral functions. ADAMTS-2 gene expressionwas determined in some tissues like aorta, bone, skin, tendon,bladder, retina, lung, kidney, liver and skeletal muscle.There isn’tany study about transcriptional regulation of this gene. Therefore,this study is focused on transcriptional regulation of ADAMTS-2gene. Different strategies for the amplication of ADAMTS-2 pro-motor that has extremely secondary structures and 80% GC richsequences have been used without any success. These strategies arethe use of different thermostable enzymes, some additives andenhancers, different primers, PCR techniques such as touch-downPCR and genome walker strategies. Putative 760bp of ADAMTS-2 promoter region was able to amplify from human genomic DNAusing some additives and cloned in pGEM-T-Easy. These GC-richamplications strategies will be discussed in detail.