Differential Evolution of Antiretroviral Restriction Factors in Pteropid Bats as Revealed by APOBEC3 Gene Complexity

Hayward, Joshua A; Tachedjian, Mary; Cui, Jie; Cheng, Adam Z; Johnson, Adam; Baker, Michelle L; Harris, Reuben S; Wang, Lin-Fa; Tachedjian, Gilda

Bat A3 cDNAs were PCR amplified from parental vectors and subcloned into pET24(þ) vectors (EMD Biosciences). Plasmids were transformed into calcium competent C43 (DE3) strain E. coli (gift from Dr. Do-Hyung Kim) and grown in LB plates with kanamycin. Single colonies were selected and grown in LB with kanamycin for 28 h to ensure stationary phase growth. Thereafter, cultures were spread on either rifampicin plates or serially diluted in M9 salt media and spread on kanamycin plates. Colony counts were obtained after 18 h. rpoB mutation frequency was obtained by dividing the number of viable colonies on rifampicin plates by colonies on kanamycin plates and correcting for the dilution factor. A portion of the rpoB gene was PCR amplified using primers: forward 5 0 -TTGGCGAAATGGCGGAAAACC and reverse 5 0 -CACCGACGGATACCACCTGCTG. PCR products were enzymatically purified using Exonuclease I and rSAP (NEB). Purified fragments were sequenced using the forward primer (GeneWiz, NJ).