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

Deletion of the TM and the CT of gp210 was achieved by inserting a stop codon after nucleotide 5,424 into the gp210:HA cDNA. This was accomplished by inserting a PCR product into a unique BgllI site at nucleotide 5,139. This PCR product is identical to the gp210 eDNA from the BglH site downstream to the codon for amino acid residue 1,808, after which a stop eodon is inserted. It was synthesized using the eDNA clone 49-10 as a template and two oligonucleotide primers, a sense primer encoding nucleotides 5,125-5,141 and an antisense primer encoding nncleotides 5,409-5,424 followed by an in-frame stop codon and a 5' BglII linker. After cleavage with BglII, the PCR product was introduced into the gp210:HA reading frame at the BgllI site. The orientation and the reading frame of the insert were determined by DNA sequencing. This gp210-(TM + CT):HA mutant (see Fig. 1 ) was inserted into the pSVL plasmid for expression studies.