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Site‐directed mutagenesis of hamster complement C1S: Characterization with an active form‐specific antibody and possible involvement of C1S in tumorigenicity
Author(s) -
Sakiyama Hisako,
Nishida Munehiro,
Sakai Norie,
Nagino Ken,
Miyatake Shoichiro,
Saito Takashi,
ImajohOhmi Shinobu
Publication year - 1996
Publication title -
international journal of cancer
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 2.475
H-Index - 234
eISSN - 1097-0215
pISSN - 0020-7136
DOI - 10.1002/(sici)1097-0215(19960611)66:6<768::aid-ijc10>3.0.co;2-#
Subject(s) - complementary dna , transfection , microbiology and biotechnology , hamster , mutagenesis , biology , site directed mutagenesis , antibody , cdna library , plasmid , cell culture , mutation , dna , biochemistry , gene , genetics , mutant
Abstract We have previously shown that non‐transformed mouse A31 cells became tumorigenic when they were transfected with hamster CIs cDNA expression plasmid BCMGSNeoCS. In the present study, mutations were introduced into the cDNA at the activation cleavage site, Arg423(AGG) and the active center Ser617(AGC). These amino‐acids were replaced by His423 (CAC) and Thr617(ACC), respectively. The mutated cDNAs were inserted into BCMGSNeo and transfected to A31 and its polyoma‐virus‐transformed SEA7 cells. CIs produced from these transfectants lost their enzyme activity. Transfectants of these mutated CIs cDNA did not form tumors in nude mice. To distinguish between active and inactive CIs in situ , we have developed novel antibodies, one directed to the NH 2 ‐terminal neoepitope of the L chain and the other specific for uncleaved inactive CIs. These antibodies were used to characterize CIs produced by transfectants, so as to determine whether or not it was cleaved at the right position. © 1996 Wiley‐Liss, Inc.