Bacterial Artificial Chromosomes: A Functional Genomics Tool for the Study of Positive-strand RNA Viruses

Yun, Sang-Im; Song, Byung-Hak; Kim, Jin-Kyoung; Lee, Young-Min

transcription reaction, both the m 7 G(5')ppp(5')A and m 7 G(5')ppp(5')G cap analog can be used equally well, although the latter places an unrelated extra G nucleotide upstream of the viral 5'-end, but that addition does not alter the infectivity or replication of synthetic RNA. 27 Moreover, removal of the cDNA template from the RNA transcripts by DNase I digestion is not necessary for RNA infectivity tests, because the cDNA template itself is not infectious. 27 The BAC technology has now been applied to constructing infectious cDNA clones for a handful of positive-strand RNA viruses, namely, two JEVs, CNU/LP2 27 and SA 14 -14-2 28 (genome size, ~11 kb); two dengue viruses, BR/90 26 and NGC 29 (~11 kb); the bovine viral diarrhea construction is the high genetic stability of the large, 1-or 2-copy BAC plasmids; however, the intrinsic nature of its extremely low-copy number is also a great disadvantage, because of very low yields of BAC DNA and the consequent reduction in the purity of the BAC DNA with respect to host chromosomal DNA. In the current protocol, the yield of BAC DNA is maximized by growing E. coli DH10B transformed with the infectious BAC pBAC/SA 14 -14-2 in a nutrient-rich medium, 2xYT. Despite this effort, the average yield is only ~15 µg of BAC DNA from 500 ml of 2xYT broth. Also, the purity of the BAC DNA is best achieved by using CsCl-EtBr density gradient centrifugation for purification, rather than the commonly used column-based plasmid isolation. However, it is important to keep in mind that the BAC-transformed E. coli should not overgrow because it might jeopardize the genetic stability of the cloned cDNA, and higher growth does not necessarily lead to greater yields or higherpurity BAC DNA.