Primary sequence domains required for the retention of rotavirus VP7 in the endoplasmic reticulum

pGEM3 DNA constructs containing wild-type VP-/, the VP7 deletions (Al-14, A47-61, A47-61/dhl, A51-61 and A51-61/dhl), wild-type amylase, or the VP763-amylase chimera, were obtained by standard bacterial lysis procedures and purified by precipitation or by cesium chloride-ethidium bromide ultracentrifugation followed by precipitation and resuspension in water. Before use as a template for in vitro run off transcription, plasmid DNA was linearized at a suitable restriction site downstream of the 3' end of the DNA coding sequence of interest. After being linearized, the DNA was extracted one time each with phenol, phenol/chloroform (1:1), and then chloroform and precipitated according to usual cloning procedures (22) . The transcription reactions were performed using the Promega Riboprobe Gemini System (Promega Biotec), according to the recommendations and procedures of the manufacturer. Briefly, for a 50-0.1 transcription mix, a 5 × transcription buffer consisting of 200 mM Tris-HC1 pH 7.5, 30 mM MgCI2, 10 mM spermidine and 50 mM NaCI, was used and dithiothreitol (DTT) added to a final concentration of 10 mM, rNTPs were each added to a final concentration of 0.5 mM, 25-50 U of ribonuclease inhibitor RNasin (Promega Biotec) were added, 2-5 0`g of linearized DNA, and 10-50 U of either Riboprobe SP6 or T7 RNA polymerase, and the reaction mixture incubated at 40°C for 90 rain. For wild-type amylase the cap mTG(5')ppp(5')G (Pharmacia Fine Chemicals) was also added to a final concentration of 0.5 mM. At the end of the incubation period, 3 vol of water and 15 lag of yeast tRNA were added as carrier before the sample was phenol and chloroform extracted as above, and precipitated with ethanol and 1 M sodium acetate, pH 8.0. The pellet retrieved was washed in 70% ethanol, dried, and then resuspended in 15-40 I11 of water.