The first membrane spanning region of the lamin B receptor is sufficient for sorting to the inner nuclear membrane

The specific mRNA encoding p58 was prepared by T3 RNA polymerase in vitro transcription of p58cDNA linearized with XhoI. mRNA encoding bovine preprolactin was prepared by T7 RNA polymerase in vitro transcription of pGEMBPl (Connolly and Gilmore, 1986 ) linearized with Ecorl. The mRNAs were used to program in vitro translation reactions using the reticulocyte lysate system (Promega, Madison, WI) as described previously (Nicchitta and Blobei, 1989) . Before analysis of translation products, reactions were fractionated by ammonium sulfate precipitation according to Nicchitta and Blobel (1990) . The pellets were dissolved in sample buffer containing 0.5 M Tris base, 5% SDS, and 50 mM DTT, incubated for 30 rain at 55~ followed by bath sonication for 60 s. Samples were fractionated on 12.5% SDS-polyacrylamide gels according to the procedure of Laemmli (1970) , enhanced with Enlightning (Dupont New England Nuclear, Boston, MA), and autoradiographed.