FIG. 1.
Insertion of BAC vector pKSO-gpt into the RRV genome. (A) The RRV genome is shown with the region of BAC vector insertion enlarged to show the relative location and orientation of ORFs. The genome of RRV17577 consists of a long unique region of 131,364 bp with flanking terminal repeat sequences. The BamHI and HindIII digestion patterns for the enlarged region are noted, with the predicted size (in kb) of restriction fragments listed above each fragment. The BAC vector pKSO-gpt was inserted into the HindIII site at nt 79010 (*), which is located in the intergenic region between ORF57 and R6, by performing homologous recombination with a 1,600-bp RRV genomic fragment (nt 78376 to 80000) engineered to contain pKSO-gpt. pKSO-gpt contains a chloramphenicol resistance gene (CAMr) and the selection marker guanosine phosphoribosyl transferase (gpt) and is flanked by loxP sites. (B) 3′ RACE analysis of RNA from wild-type RRV-infected primary rhesus fibroblasts results in single discrete products for R6 and ORF57. (C) Sequencing of 3′ RACE products identified the poly(A) signal utilized by ORF57 and R6 and the location of the poly(A) tail addition for ORF57 at nt 79001 (>) and for R6 at nt 79194 (<). ORFs are denoted by boldface type, with arrows indicating the direction of transcription. The HindIII site utilized for pKSO-gpt insertion (*) is located after the transcript termination site for both ORFs. Three clones of each RACE product were sequenced.