Figure 4

Splicing junction between SD⁶¹⁴⁰ and SA⁸⁵¹⁴ sites occurs in CAEV-infected cells. A, Proviral organization and splicing pattern of CAEV genome. The nucleotide numbers of SD sites (open triangles) and SA sites (solid triangles) are shown. All splice sites were identified by cDNA sequencing. Exons are represented by solid lines. Alternative exons which are present in only some of the mRNAs are shown in parenthesis. The putative structure of rtm transcript generated by splicing between SD⁶¹⁴⁰ and SA⁸⁵¹⁴ sites is shown. The arrows represent PCR primers used for cDNA amplification. B, Southern blot analysis of cDNAs from either transfected or infected cells. Cytoplasmic RNAs extracted from either 293T cells transfected with plasmids pKRB1 (lane 1) and pKRmB1 (lane 2) or CAEV-infected (lane 3) and non-infected (lane 4) GSM cells were submitted to RT/PCR. Primer pairs PK5/M3b and Mar52/M3b were used to amplify in a first-round PCR the cDNAs from transfected and infected cells, respectively. Primer pair MarN/M3b was used in the second-round PCR. PCR-amplified cDNA fragments were electrophoresed through an 2.5% agarose gel, blotted to nylon, and hybridized to either probe MarN2 (left panel) or probe MarS (right panel). Size of PCR-amplified fragments corresponding to the splice junction 6140–8514 is indicated.