Quantitation and Structural Analysis of SV40 RNAs

The 5243-bp genome of SV40 contains two transcriptional units (
see
Fig. 1 ); the early one, which is first expressed early in the lytic cycle of infection, and the late unit, which is expressed at a significant level only after the onset of viral
DNA
replication (
1
). The early genes encode the viral regulatory proteins, small, and large T-antigens. These proteins play roles in replication of the viral genome and transcriptional transactivation of the late genes (
seerefs.2
–
4
and references therein). The late genes encode the capsid proteins that encapsulate the viral DNA into new virions. The
cis
-acting transcriptional elements that regulate the synthesis of both the early and late viral RNAs (
seerefs.5
,
6
and references therein), the mechanisms that regulate the temporal expression of the SV40 genome (
seeref.4
and references therein) and the patterns by which the viral transcripts are spliced (
see refs. 7
,
8
and references therein) are well understood. In this chapter, we describe methods for quantifying and mapping the 5 ends of the viral RNAs synthesized in SV40-transfected cells. Minor variations of these methods can be used to map the 3 ends as well (
9
) and the splice sites (
8
) of the SV40 RNAs synthesized, not only in SV40-transfected cells, but also in SV40-infected and transformed cells. Fig. 1.
Schematic of the SV40 genome. (
A
) Map of wild-type SV40 DNA. (
B
) Schematic of the origin-promoter region of SV40. This bidirectional promoter controls both early and late transcription. The thick and thin arrows indicate the positions at which transcription initiates and the direction transcription proceeds. The objects indicate the cis-acting sequences and the transcription factors that bind in trans. The nucleotide numbering system is that of Buchman et al. (
21
).