In vitro transcription of adenovirus

Abstract

A series of recombinants of adenovirus DNA fragments and pBR322 was used to test the transcriptional activity of the nine known adenovirus promoters in a cell-free extract. Specific initiation was seen at all five early promoters as well as at the major late promotor and at the intermediate promoter for polypeptide IX. The system failed to recognize the two other adenovirus promoters, which were prominent in vivo only at intermediate and late stages in infection. Microheterogeneity of 5' termini at several adenovirus promoters, previously shown in vivo, was reproduced in the in vitro reaction and indeed appeared to result from heterogeneous initiation rather than 5' processing. To test for the presence of soluble factors involved in regulation of nRNA synthesis, the activity of extracts prepared from early and late stages of infection was compared on an assortment of viral promoter sites. Although mock and early extracts showed identical transcription patterns, extracts prepared from late stages gave 5- to 10-fold relative enhancement of the late and polypeptide IX promoters as compared with early promoters.

Accurate transcription initiation by RNA polymerase II in a soluble extract from isolated mammalian nuclei

“…18 and Figure 7), a substantial and readily detectable level of accurate initiation is observed with a template containing only the EIIA promoter (D. H. Huang and R. G. Roeder, unpublished observation). This may be of significance in view of the fact that the major EILA-early promoter does not contain a canonical TATA box and that this promoter is recognized only at a very low level in the other polymerase II transcription systems (20,21). Thus, the nuclear system could be more active in recognizing a broader group of class II promoters with differing requirements for polymerase II factors, although this important point remains to be further investigated.…”

Cycloheximide stimulates early adenovirus transcription if early gene expression is allowed before treatment

“…Fire et al (8) showed that at least some extracts made from Ad2-infected cells late in infection have different in vitro transcription activities than do extracts from early-infected or mock-infected cells when early versus late transcription units are assayed. Although they found no difference between mock-and early-infected cell extracts, it would be interesting to know whether a difference would be seen between early-infected extracts and extracts from cells which are given CH after the establishment of early infection.…”

Unusual heterogeneity of the 5′-termini of human adenovirus type 2 early region E2 mRNA

“…These sequences appear to be transcription initiation sites. Subsequently, Fire et al (18) and Mathis et al (19) transcribed E2 RNA using an in vitro transcription system. Analysis of the RNA runoff products indicated that the 5'-termini were heterogeneous.…”