Properties of an isolated transcription stimulating sequence derived from the cauliflower mosaic virus 35S promoter

Abstract

As a highly active plant viral promoter that is able to function in a wide variety of cell types, the cauliflower mosaic virus (CaMV) 35S promoter has the potential for harboring a plant enhancer element. We tested this possibility and demonstrated that a 338 base pair fragment isolated from the region upstream of the 35S TATA box can increase the expression of a low-activity heterologous promoter up to the level observed for the intact 35S promoter. This fragment is fully active in both orientations when placed 150 base pairs upstream of the transcription start site. However, the activity of this fragment is sensitive to location, demonstrating a reduction in activity and loss of orientation-independent function when the distance from the transcription start site is increased. By assaying fragments of different sizes, we have also characterized regions that are functional in directing the stimulation of the heterologous promoter.

Multiple cis regulatory elements for maximal expression of the cauliflower mosaic virus 35S promoter in transgenic plants.

“…Ow et al (1987) reported that, in carrot protoplasts, the -89 5' deletion mutant has 23% WT activity, whereas the -68 5' deletion mutant has only 0.8%. Odell et al (1988) found that deletion of the sequence between -90 and -55 reduces transient expression levels of the -392 to -90 fragment by twofold. We have confirmed and extended these results in transgenic tobacco plants.…”

“…Thus, CaMV fragments containing 400 to 1000 base pairs of 35S upstream sequences have been shown to be active when integrated into the nuclear genome of transgenic tobacco (Odell et al, , 1987Nagy et al, 1987;Kay et al, 1987;Jefferson et al, 1987) and petunia (Sanders et al, 1987). Moreover, the promoter can also be expressed transiently in protoplasts of several dicots and monocots (On-Lee et al, 1986;Fromm et al, 1985;Ow et al, 1987;Nagata et al, 1987;Odell et al, 1988). Quantitative measurements of relative transcript levels in transformed tobacco cells Nagy et al, 1985) or transgenic petunia plants (Sanders et al, 1987) showed that the 35S promoter is at least 30 times stronger than the nopaline synthase promoter.…”

“…Ow et al (1987) placed the 5' boundary of the 35S promoter at -148 and showed that deletion of the distal region (-148 to -89) of the promoter decreases activity by 80%. Odell et al (1988) reported that an upstream fragment, from -393 to -90, can increase the expression level of the nopaline synthase promoter by about threefold. However, increasing the copy number of this activating fragment does not increase the expression level further.…”

Analysis of the enhancer-blocking function of the TBS element from Petunia hybrida in transgenic Arabidopsis thaliana and Nicotiana tabacum

“…However, this strategy is often problematic due to the positionand orientation-independent ability of enhancers to trigger enhancer-promoter interference and disturb the expression of the transgene of interest. This phenomenon is particularly prevalent in transgenic plants harboring constructs in which the enhancer from the CaMV 35S promoter (Odell et al 1988) is in relatively close proximity to the promoter of another transgene, and results in both a loss of specificity and an increase in the level of expression induced by the transgenic promoter Zheng et al 2007). Interestingly, this enhancer-mediated activation of proximal promoters appears to be a fairly common property of a wide range of enhancers, and as a result, has incited interest in the development of strategies with which to prevent such interactions (Gudynaite-Savitch et al 2009;Singer et al 2010b).…”

New chimeric promoter useful for expression of selectable marker genes in rice transformation

“…The enhancer-like element (Ϫ351 to Ϫ84) of the CaMV35S promoter (Odell et al 1988) was prepared by PCR from pBI221 with primers 35S-Sal (5Ј-CGTCGACAGGGCAATTGAGAC-3Ј) and 35S-Hind (5Ј-TCAAGCTTTGGAGATATCACATC-3Ј). The PCR cycle conditions were 30 s at 98°C, 30 s at 55°C, and 30 s at 72°C, for 25 cycles.…”