The single transmembrane segment of gp210 is sufficient for sorting to the pore membrane domain of the nuclear envelope

Synthesis of the 5' fragment was complicated by the lack of a complete template encoding this region. To circumvent this problem, inserts from three overlapping, partial cDNA clones, which together span this region, 50-17, 45-A1, and 47-10 07), were used to synthesize a hybrid cDNA of sufficient length to act as a template for amplification. Synthesis of this template was performed as follows. Two overlapping eDNA clones, 50-17 and 45-A1 ('~50 rig each), were cleaved with EcoRI and XhoI, respectively, to liberate two overlapping inserts. The resulting DNA fragments were combined and heat denatured at 95~ for 10 min in lx Taq polymerase buffer (Perkin-Elmer Cetus Instruments, Norwalk, CT). The denatured DNA was allowed to reanneai at 65~ for 30 rain and dATP, dCTP, dGTP, and dTTP were added to a final concentration of 0.2 mM each. The reaction temperature was raised to 72~ and 2.5 U ofTaq polymerase (Perkin Elmer Cetus Instruments) was added to fill in the fraction of DNA molecules formed by the hybridization of overlapping single-stranded inserts from 50-17 and 45-A1. The polymerization was allowed to proceed for 8 rain at 72~ The DNA from this reaction, containing the double-stranded 50-17/45-A1 hybrid, was combined with ~50 ng of EcoRI/AatlI cut 47-10 eDNA, and the denaturation, annealing, and polymerization steps repeated as above to form a 50-17/45-A1/47-10 hybrid eDNA. Although this species likely represents only a fraction of the total eDNA present in the reaction, it proved sufficient for amplification. With this template, a PCR was performed using a sense oligonucleotide encoding a 5' SalI site followed by nucleotides -12-33 (sense primer) and an antisense oligonucleotide encoding nucleotides 1,967-1,997 (antisense primer).