was digested with 3 units of psti in 10 u03bcl of h buffer for an hour the above two reaction mixtures were combined and incubated in 0 5 units of t4 dna ligase 0 5 mm atp and 10 mm dtt at 4 u00b0 c for 20 hours the ligated dna thus obtained was used to transform escherichia coli c600r m atcc 33525 according to the method described in molecular cloning page 250 cold spring harbor laboratory 1982 the resulting transformed cells were plated on lb agar containing 5 u03bcg ml of tetracycline and incubated at 37 u00b0 c overnight the tetracycline resistant colonies were transferred to lb agar containing 20 u03bcg ml of chloramphenicol to lb agar containing 50 u03bcg ml of ampicillin and to mm agar containing 50 u03bcg ml threonine lacking leucine respectively and cultured at 37 u00b0 c overnight the ap s tc r cm r leu colonies were obtained and the plasmid dna was named pbr leu the pbr leu dna was isolated according to the method described in advanced bacterial genetics page 116 cold spring harbor laboratory 1980 sterile ac medium 100 ml was inoculated with mycelium from an agar slant of a chrysogenum atcc 11550 and cultured for 3 days at 25 u00b0 c 250 rpm in 500 ml shaking flask cells were harvested and washed with kp buffer the cells were suspended in 20 ml of kp buffer containing 10 mm dtt and incubated for 30 minutes at 37 u00b0 c then 20 mg of zymolyase 60 000 seikagaku kogyo co was added to the suspension and which was shaken for 2 hours at 37 u00b0 c the cells were centrifuged at 1 000 u00d7 g for 10.