Functional regions of the cauliflower mosaic virus 35S RNA promoter determined by use of the firefly luciferase gene as a reporter of promoter activity

Abstract

The cauliflower mosaic virus (CaMV) 35S RNA promoter has been dissected and examined in a transient expression system using the firefly luciferase gene as a reporter of promoter activity. Deletion analysis has shown that the 35S RNA promoter is composed of at least three regions-distal, medial, and proximal-which are essential for activity. The distal region contains three smaller elements homologous to the simian virus 40 "core" enhancer element, the medial region possesses a CCAAT-like box, and the proximal region contains a TATA box. A DNA segment encompassing the distal region is capable of activating the CaMV 35S core promoter in an orientation-independent, but not position-independent, fashion. The distal region can also activate a heterologous weak promoter, the CaMV 19S RNA promoter, albeit not to the high levels of the 35S RNA promoter. Multimers of the distal region are able to activate the 35S RNA promoter core to even greater levels of expression than the native 35S promoter. These experiments demonstrate that elements outside the boundaries of the core promoter (composed of proximal and medial elements) are recognized in a plant cell transient expression system. Eukaryotic promoters that have been examined in detail are composed of multiple cis-acting elements, which are required for promoter function. These elements have been recognized in DNA manipulation experiments (deletions, mutagenesis, linker scanning analysis, element relocation, etc.) and bind trans-acting factors, which activate transcription (reviewed in refs. 1 and 2). Many, but not all, plant and animal promoters are composed of a TATA box, 25-30 bases upstream from the start of transcription, often a CCAAT box at about -80, and other elements that may confer greater or regulated activity on a promoter. The TATA box establishes polarity to the eukaryotic promoter; binds protein factors (3, 4); and alterations in sequence, orientation, or position dramatically reduce transcription altogether or from the normal start site (5). The CCAAT box, in promoters, in which it i,s found, is affected by alterations in sequence (5), appear,s in one instance (HV thymidine kinase promoter) in an inverted orientation with respect to the start of transcription (6), and binds distinct protein factors (7). Other upstream elements may differ from one promoter to another and presumably bind different trans-acting protein factors that may confer specialized functions, such as high level constitutive expression, tissue-specific expression, or response to environmental cues (reviewed in ref.2). The elements that bind the transcriptional factor SP1 are well characterized and are found in a number of animal promoters, such as those in the 21-base-pair (bp) repeats of the simian virus 40 (SV40) early promoter (8, 9). Other upstream elements can act as enhancers, such as those in the 72-bp repeats of the SV40 promoter-that is, they can function in a position-and orientation-independent fashion and are able to empower or regulate heterologous core promoters...

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

“…The underlined sequences, GGTAATAC, GTGGAAAAG, GTGGAAAAAG, and GTGGATTG, resemble the enhancer core sequence of SV40 (Serfling et al, 1985). The putative CCAAT sequences are CCACT, CACAAT, and CCACT (Ow et al, 1987). The TATA box sequence and the TGACG repeats are boxed.…”

“…In several animal genes the activity of certain enhancer elements has been shown to require the "CCAAT" box (Dierks et al, 1983;Bienz and Pelham, 1986). There are three CCAAT-like boxes in the 35S promoter: CCACT (-85 to -81), CACAAT (-64 to -59), and CCACT (-57 to -53) (Ow et al, 1987). Since all three boxes are contained within -90 to -46, we have examined the effect of this region on transcription.…”

“…Recently, Ow et al (1987) reported a detailed 5' deletion analysis of the 35S promoter using a protoplast transient expression system. They found little change in activity when the upstream sequence is deleted from -1600 to -148 and, accordingly, they fixed the 5' boundary of the 35S promoter at -148.…”

“…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.…”

“…DNA sequences for 35S promoter activity have also been studied by transient expression assays in protoplast cultures. 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.…”