|Morey, Kevin - COLORADO STATE UNIVERSITY|
Submitted to: Journal of Virology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: February 24, 2005
Publication Date: February 24, 2005
Citation: Velten, J.P., Morey, K.J., Cazzonelli, C.I. 2005. Viral intergenic dna sequence repeats with transcription enhancing activity. Virology Journal. 2(16). Interpretive Summary: As part of a project intended to generate new synthetic plant promoters we have tested the transcriptional enhancing activity of a set of short, repeated DNA sequence elements found within geminivirus and nanovirus intergenic regions. A number of the elements tested were originally identified as sequences that are repeated within geminivirus and intergenic segments, but have never been directly tested for transcription enhancing function. The enhancer screen was successful in that we identified several novel elements (approximately 13% of 105 repeats tested were active). Although the survey is not fully comprehensive, it represents the first extensive functional (transcription-wise) screen of small repeated geninivirus intergenic sequence elements and would provide a valuable resource to scientists interested in plant viral gene regulation during infection.
Technical Abstract: The geminivirus and nanovirus families of DNA plant viruses have proved to be a fertile source of viral genomic sequences, clearly demonstrated by the large number of sequence entries within public DNA sequence databases. Due to considerable conservation in genome organization, these viruses contain easily identifiable intergenic regions that have been found to harbor multiple DNA sequence elements important to viral replication and gene regulation. As a first step in a broad screen of geminivirus and nanovirus intergenic sequences for DNA segments important in controlling viral gene expression, we have 'mined' a large set of viral intergenic regions for transcriptional enhancers. Viral sequences that are found to act as enhancers of transcription in plants are likely to contribute to viral gene activity during infection. DNA sequences from the intergenic regions of 29 geminiviruses or nanoviruses were scanned for repeated sequence elements to be tested for transcription enhancing activity. 105 elements were identified and placed immediately upstream from a minimal plant-functional promoter fused to an intron-containing luciferase reporter gene. Transient luciferase activity was measured within Agrobacteria-infused Nicotiana tobaccum leaf tissue. Of the 105 elements tested, 14 were found to reproducibly elevate reporter gene activity (>25% increase over that from the minimal promoter-reporter construct, p < 0.05), while 91 elements failed to increase luciferase activity. A previously described "conserved late element" (CLE) was found to have intrinsic enhancer activity in the absence of viral gene products and was identified within 5 different viral species. The remaining 9 active elements have not been previously demonstrated to act as functional promoter components. Biological significance for the active DNA elements identified is supported by repeated isolation of a previously defined viral element (CLE), and the finding that two of three viral enhancer elements examined were markedly enriched within both geminivirus sequences and within Arabidopsis promoter regions. These data provide a useful starting point for virologists interested in undertaking more detailed analysis of geminiviral promoter function.