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. 2018 Feb 12;8:2851. doi: 10.1038/s41598-018-21317-w

Figure 3.

Figure 3

Definition of messenger RNAs transcribed from CMI1.252. (a) Linear representation of the CMI1.252 genome including ORFs larger than 40 amino acids for the sense strand. The position of the RNA probes for northern blot analyses (Fig. 3e) are indicated. For 5′-RACE, nested PCR was performed using primers located within the Rep-ORF (indicated as red arrows) and within the ORF-2 (blue arrows). Each reproducibly detected transcription start site for each primer is plotted against the location within the genome. For 3′-RACE, nested PCR was performed using the indicated primers (black and grey arrows). Each reproducibly detected polyadenylation site is plotted against the location within the genome. Transcripts arising from the combination of the 5′- and 3′-RACE results, whose existence was supported by northern blots, as well as their coding potentials are shown. (b) Agarose gel image of the CMI1.252-specific 5′-RACE products obtained by PCR using the different primers indicated in panel (a). cDNA of mock-transfected cells was used as a negative control. Specific start sites are indicated by letters according to panel (a). (c) Agarose gel image of the respective 3′-RACE products obtained by nested PCR using the primers indicated in panel (a). Specific polyadenylation sites are indicated as letters according to panel (a). (d) Validation of continuous transcripts using RT-PCR. Total RNA of CMI1.252-transfected cells was reverse transcribed using oligo-dT primers (+RT) and the resulting cDNA was subjected to PCR reactions using the indicated primer combinations. RT reactions lacking reverse transcriptase was used as negative controls (−RT) and PCR reactions using linearized CMI1.252 DNA as a template served as a positive control. (e) Northern blot validation of CMI1.252 transcripts in HEK293TT cells. Antisense RNA probes complementary to nt 883–1051 (probe 1) or nt 2074–2264 (probe 2) of CMI1.252 were used to detect transcripts in CMI1.252-transfected HEK293TT cells. Mock-transfected cells served as a negative control and circular CMI1.252 genome served as a positive control.