Selective transcription of a cloned cauliflower mosaic virus DNA fragment in vitro by soybean RNA polymerase II in the presence of dinucleotide primers

Abstract

Transcription of a cloned cauliflower mosaic virus (CaMV) DNA fragment (plasmid pCa8) was studied at a low enzyme : DNA ratio. Preincubation with purine nucleoside triphosphates leads to essentially random transcription, while in the presence of a dinucleoside monophosphate and a purine nucleoside triphosphate in the preincubation medium certain combinations prime preferential transcription of the eucaryotic moiety of the chimeric plasmid.Characterisation of transcription primed by the most efficient combination, ApG + ATP, shows that a low enzyme :DNA ratio is absolutely essential for selective initiation. Interestingly the presence of the eucaryotic insertion is essential for the transcription of vector sequences. Analysis of RNA primed by ApG + ATP and of short chains synthesised in the presence of the GTP analogue 3'-OMeGTP shows a high degree of selectivity of transcription initiation sites, Hybridisation of primed RNA to restriction fragments of pCa8 shows that initiation occurs within a limited region of the inserted CaMV fragment.The cauliflower mosaic virus (CaMV) genome provides an excellent material for studies of plant gene expression. The complete sequence of several isolates is now known [I, 21, the transcription pattern in vivo has been partially established [3] and transcription in vitro from probable in vivo promoters has been studied in a heterologous cell-free system [4].In previous studies we have used CaMV DNA as a template to investigate the mode of action of wheat germ RNA polymerases [5 -71. We have shown that RNA polymerase I1 transcribes CaMV DNA even in the presence of heparin, but that this transcription is initiated principally at the level of the three single-stranded interruptions, which are present on the CaMV genome [8], initiation being more efficient at one of these interruptions [6]. Subsequent electron microscope studies using the entire CaMV genome cloned in the vector pATl53 [7], and thus lacking the single-strand interruptions, showed that RNA polymerase I1 forms stable, binary complexes at several sites on this template, certain of which are relatively resistant to the action of heparin.Most previous studies on transcription in vitro, including our own, have been carried out at relatively high enzyme: DNA ratios and have led to the general conclusion of non-specificity on purified templates. However, several recent reports have indicated that, under certain conditions, yeast RNA polymerase B is capable of selective initiation on the eucaryotic moiety of supercoiled cloned yeast templates at low enzyme:DNA ratios and in the presence [9, 101 or absence [I1 -131 of dinucleotide primers. No similar studies have been carried out for plant or animal RNA polymerases. In fact, no selective or specific transcription by a plant RNA polymerase I1 hdS yet been obtained, even in factor-supplemented systems. We therefore felt it of interest to investigate the effect of low enzyme : DNA ratio and dinucleotide priming in the transcription by soybean RNA polymerase I1 of a supercoiled tem...

Enzymatic properties of plant RNA polymerases

“…As described above, the kinetic analysis on denaturing polyacrylamide gels of (ApG + ATP) primed RNA synthesized on cloned CaMV fragments shows that transcription is not only initiated but also terminated at a limited number of discrete sites. This termination is most probably due to a pausing phenomenom at the low nucleoside triphosphate concentrations used (10). Similar observations have been reported for yeast RNA polymerase II (42) and E.coli RNA polymerase (39).…”

“…This could be due either to the presence on the insertion of sequences necessary for enzyme-DNA interactions or to teleactivation effect of the insertion on the vector as described by Carnevali et aL (4) Hybridisation of primed RNA to restriction fragments of the chimaeric plasmid shows that initiation occured within a limited region of the inserted CaMV fragment. Up to the present, these observations constitute unique examples showing that pure plant enzymes can selectively initiate RNA transcription on homologous templates (10).…”

Accurate in vitro transcription of plant promoters with nuclear extracts prepared from cultured plant cells

“…These observations indicate that the apparent constitutive expression of the 35S promoter is due to number of regulatory elements acting alone, or in combination, to augment transcription in a wide variety of tissues [5]. While the 35S promoter maybe ideal for expression of genes in transformed plant cells, the complex transcriptional controls on it make it less than ideal for use as a model promoter in the development and characterization of plant in vitro transcription systems [1,6,7,12,48].…”

Isolation and characterization of an unusual repeated sequence from the ribosomal intergenic spacer of the crucifer Sisymbrium irio

“…Digests were fractionated by agarose gel electrophoresis on horizontal gels run in 0.04 M Tris-acetate, 2 m M EDTA (pH 7) buffer. Size markers were Hind III fragments from lambda phage D N A of Hind II fragments of a recombinant, pCa8 [4].…”