|Budget Amount *help
¥1,700,000 (Direct Cost : ¥1,700,000)
Fiscal Year 1994 : ¥600,000 (Direct Cost : ¥600,000)
Fiscal Year 1993 : ¥500,000 (Direct Cost : ¥500,000)
Fiscal Year 1992 : ¥600,000 (Direct Cost : ¥600,000)
There are many kinds of restriction enzymes which are produced in microorganisms to protect themselves from viral infection. Among them, type II enzyme recognize and digest palindromic sequences of dsDNA,hence they have been utilized in recombinant DNA technology.
Our first purpose is to produce new kinds of restriction enzymes through protein engineering. New restriction enzymes which hydrolyze ssDNA,dsRNA,DNA-RNA hybrid, non-palindromic sequence, etc., will be helpful in further progress in molecular biology, gene therapy, and many fields handling nucleic acids. We started working on EcoRI : site-directed mutagenesis EcoRI gene. Unfortunately, this attempt was failed. We could not obtain mutants which generate altered EcoRI.Then, we tried to perform random mutation on EcoRI gene. We failed again. In every case, EcoRI methylase gene was co-transfected with mutagenized EcoRI gene. However, this enzyme did not methylate the sequence which the altered EcoRI recognized. This problem could
not be dissolved in ant system we employed.
During these works, Haemophilus influenzae Rd which produces restriction enzyme HindIII,was presented from Nippon Gene. We tried to purify HindIII in order to prepare its gene and the adjacent HindIII methylase gene. These genes will be used for mutagenesis. In the purification of HindIII,we found there are two forms (P1 and P2) of HindIII.The P2 which was eluted with high concentration of KCI from phosphocellulose column, was stable and showed higher activity than P1 (eluate with lower concentration of KCI), with high affinity to the substrate sequence. Addition of high amount of phage T7 to the bacterial culture resulted in disappearance of P2 fraction. The P2 was rich when lower amount of T7 was added. These results indicate that there would be a factor which is bound to HindIII to form P2 and is influenced by phage attack.
Addition of disaccharides such as maltose and trehalose to HindIII extract showed increase of P2 content. The factor mentioned above could be easily bound to HindIII in presence of disaccharide. Addition of urea to the extract resulted in interesting phenomena. When 2 M urea was added, a broad peak of HindIII was observed in phosphocellulose chromatography. Addition of 4 M urea, on the contrary, yielded a single peak. HindIII could be purified from this fraction by subsequent DEAE-cellulose chromatography. We consider that there is only one molecular speceis in HindIII,and this enzyme shows active form upon interaction with the factor.
Finally, as to alteration of restriction enzymes EcoRI and HindIII will be continued foa couple of years. We are going to examine a system using Baculovirus and insect cells. Some researchers are now thinking about collaboration with us. Less