ecori or from psvcow7 supra to produce fragment 11 fragment 11 is cloned between the ecori and smai cleavage sites of puc9 obtained from pharmacia pl or atcc to give pcowt1 pcowt1 is cut with sali the ends made blunt with t4 dna polymerase ecori linkers are added the dna is cut with ecori and the 0 6 kb fragment fragment 12 is isolated this is the same as fragment 11 except that it has two ecori ends and a polylinker sequence at one end plasmid pdie50 is cut with ecori and fragment 12 is cloned into it to produce plasmid pdie50pa digestion with bamhi and pvuii produces a fragment of 1 1 kb in the case where the polyadenylation signal is in the proper orientation the plasmid can also be constructed by cloning in the polyadenylation sequence before the promoter plasmid pdie50pa is used to transfect cho dhfr u2212 cells dxb 11 g urlaub and l a chasin proc natl acad sci usa 77 pp 4216 20 1980 by calcium phosphate co precipitation with salmon sperm carrier dna f l graham and a j van der eb virol 52 pp 456 67 1973 the dihydrofolate reductase positive dhfr transfected cells are selected in dulbecco 39 s modified eagle 39 s medium plus eagle 39 s non essential amino acids plus 10 fetal calf serum selected dhfr cho cells produce gp50 as detected by immunofluorescence with anti gp50 monoclonal antibody 3a 4 or by labelling with 14 c glucosamine and immunoprecipitation with 3a 4 monoclonal antibody 3a 4 is produced as described in copending u s patent application ser no 817 429.