TABLE 1.
Characterization of cDNA clones representing recombinant and parental (nonrecombined substrate) BMV RNA3 (−) sequences within the sgp region after in vitro copying of RW1 (−) RNA3 and RW2 (−) RNA3 templates either with BMV replicase or with protein 2a
Enzyme prepn and type of RNA producta | Clone no. | Crossover regionb | Crossover typec | Length (nt) (SD) of poly(A) tractd |
---|---|---|---|---|
BMV replicase | ||||
Recombinant sequence | 1 | II | 1 | 18 (0.43) |
2 | II | 1 | 18 (0.43) | |
3 | II | 2 | 19 (0.43) | |
4 | II | 2 | 19 (0.43) | |
5 | II | 1 | 18 (0.43) | |
6 | II | 1 | 18 (0.43) | |
7 | II | 2 | 17 (0.43) | |
Parental sequence | RW1 (−) RNA3 | NA | NA | 18 (0.25) |
RW1 (−) RNA3 | NA | NA | 18 (0.25) | |
RW2 (−) RNA3 | NA | NA | 18 (0.25) | |
RW2 (−) RNA3 | NA | NA | 19 (0.25) | |
2a | ||||
Recombinant sequence | 1 | I | 1 | 16 (2) |
2 | I | 2 | 20 (2) | |
3 | I | 2 | 18 (2) | |
4 | I | 2 | 19 (2) | |
5 | II | 2 | 19 (2) | |
6 | II | 2 | 17 (2) | |
7 | II | 1 | 19 (2) | |
8 | II | 1 | 19 (2) | |
9 | II | 1 | 22 (2) | |
10 | II | 2 | 16 (2) | |
11 | II | 2 | 17 (2) | |
12 | II | 1 | 21 (2) | |
13 | II | 1 | 25 (2) | |
Parental sequence | RW1 (−) RNA3 | NA | NA | 17 (1.8) |
RW1 (−) RNA3 | NA | NA | 20 (1.8) | |
RW1 (−) RNA3 | NA | NA | 16 (1.8) | |
RW2 (−) RNA3 | NA | NA | 15 (1.8) | |
RW2 (−) RNA3 | NA | NA | 19 (1.8) |
Types of RNA products were determined by sequencing of RT-PCR-generated cDNAs from copying reaction products of a 1:1 mixture of RW1 (−) and RW2(−) RNA3 templates (reaction time, 90 min). The sequenced sgp regions carried flanking markers derived from either parental [RW1 (−) or RW2 (−)] or recombined (between two RNA templates) (−) RNA3 sequences (see Fig. 1 for more details).
Region of crossovers within the tested sgp sequence (see Fig. 1), as follows: region I, 116-nt sequence between C-4/5 and XhoI-1320; and region II, 34-nt sequence between EcoRI-1169 and C-4/5. NA, not applicable.
Direction of crossovers, as follows: type 1, RW1 (−) RNA3 (donor)→RW2 (−) RNA3 (acceptor); and type 2, RW2 (−) RNA3 (donor)→RW1 (−) RNA3 (acceptor).
Standard deviations for the lengths of poly(A) tracts were calculated based on the 18-adenine wild-type length.