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

The dideoxy method of DNA sequencing (34) was used to confirm the amino terminal VP7 coding sequence and the VP7-amylase junctions for the constructs A51-6163/dhl/Am, Al-14m/Am and A47-61m/dhl/Am. The coding sequences of the chimeric VP7-amylase molecules were each cloned into either pTZ18R or pTZ19R (Pharmacia Fine Chemicals, Piscataway, NJ). For A51-61jdhl/Am, this was achieved by cutting the plasmids with Barn HI and cloning the 1,700-hp fragment into the Barn HI site in pTZ18R. Xho I/Barn HI fragments of Al-14m/Am and A47-61m/dhl/Am were cloned into the Barn HI/Sal I site of pTZ19R. JM101 or JMI09 strains of bacteria were transformed with the ligation mixtures and colonies were screened for inserts, and in the case of A51-6163/dhl/Am in pTZ18R, orientation monitored by restriction enzyme analysis. The transformed bacteria containing the construct in the desired orientation were infected with helper phage to generate single stranded DNA. Sequencing reactions used the T7 primer or a synthesized primer, 5' TATAATTTATCd3ATTTCT 3' which annealed within the 5' terminal coding sequence of VP7, thus allowing an increased range of sequencing of the chimeric coding regions. Reaction mixtures incorporating 35S-ATP were run on acrylamide-urea gels for analysis.