Identification and application of novel autonomously replicating sequences (ARSs) for promoter-cloning and
Shigehito Ikushima1, Toshiko Minato, Keiji Kondo
1Central Laboratories for Frontier Technology, KIRIN Holdings Co., Ltd. Shigehito_Ikushima@kirin.co.jp
Abstract:
In order to develop practical recombinant DNA techniques in the industrially important yeast Candida utilis, at least six plasmids harboring autonomously replicating sequences (ARSs) were isolated from a C. utilis genomic library. Two ARSs were subjected to detailed analysis. Sequences of 1.9 and 1.8 kb were found to be necessary to exert ARS activity in a plasmid as assessed by transformation efficiency and mitotic stability. Both fragments were found to be rich in AT content (69.5% and 70.8% respectively), and to contain an 11-bp ARS consensus sequences (10 and 13 motifs with one base difference respectively). Using the ARS-containing plasmid as a promoter-cloning vector, several DNA fragments having promoter activities were cloned and characterized. Co-transformation of C. utilis with an integrating DNA fragment and a replicating plasmid yielded plasmid-free transformants harboring the fragment integrated into the C. utilis genome.
