Viral and chloroplastic signals essential for initiation and efficiency of translation in Agrobacterium tumefaciens

Ahmad T, Venkataraman S, Hefferon K, AbouHaidar MG

Biochem. Biophys. Res. Commun. 2014 Sep;452(1):14-20

PMID: 25117444

Abstract

The construction of high-level protein expression vectors using the CaMV 35S promoter in concert with highly efficient translation initiation signals for Agrobacterium tumefaciens is a relatively less explored field compared to that of Escherichia coli. In the current study, we experimentally investigated the capacity of the CaMV 35S promoter to direct GFP gene expression in A. tumefaciens in the context of different viral and chloroplastic translation initiation signals. GFP expression and concomitant translational efficiency was monitored by confocal microscopy and Western blot analysis. Among all of the constructs, the highest level of translation was observed for the construct containing the phage T7 translation initiation region followed by the chloroplastic Rubisco Large Subunit (rbcL) 58-nucleotide 5′ leader region including its SD-like sequence (GGGAGGG). Replacing the SD-like (GGGAGGG) with non SD-like (TTTATTT) or replacing the remaining 52 nucleotides of rbcL with nonspecific sequence completely abolished translation. In addition, this 58 nucleotide region of rbcL serves as a translational enhancer in plants when located within the 5′ UTR of mRNA corresponding to GFP. Other constructs, including those containing sequences upstream of the coat proteins of Alfalfa Mosaic Virus, or the GAGG sequence of T4 phage or the chloroplastic atpI and/or PsbA 5′ UTR sequence, supported low levels of GFP expression or none at all. From these studies, we propose that we have created high expression vectors in A. tumefaciens and/or plants which contain the CaMV 35S promoter, followed by the translationally strong T7 SD plus RBS translation initiation region or the rbcL 58-nucleotide 5′ leader region upstream of the gene of interest.

An international bioinformatics infrastructure to underpin the Arabidopsis community

Plant Cell 2010 Aug;22(8):2530-6

PMID: 20807877

Abstract

The future bioinformatics needs of the Arabidopsis community as well as those of other scientific communities that depend on Arabidopsis resources were discussed at a pair of recent meetings held by the Multinational Arabidopsis Steering Committee and the North American Arabidopsis Steering Committee. There are extensive tools and resources for information storage, curation, and retrieval of Arabidopsis data that have been developed over recent years primarily through the activities of The Arabidopsis Information Resource, the Nottingham Arabidopsis Stock Centre, and the Arabidopsis Biological Resource Center, among others. However, the rapid expansion in many data types, the international basis of the Arabidopsis community, and changing priorities of the funding agencies all suggest the need for changes in the way informatics infrastructure is developed and maintained. We propose that there is a need for a single core resource that is integrated into a larger international consortium of investigators. We envision this to consist of a distributed system of data, tools, and resources, accessed via a single information portal and funded by a variety of sources, under shared international management of an International Arabidopsis Informatics Consortium (IAIC). This article outlines the proposal for the development, management, operations, and continued funding for the IAIC.

Taking the next step: building an Arabidopsis information portal

Plant Cell 2012 Jun;24(6):2248-56

PMID: 22751211

Abstract

The Arabidopsis information portal (AIP), a resource expected to provide access to all community data and combine outputs into a single user-friendly interface, has emerged from community discussions over the last 23 months. These discussions began during two closely linked workshops in early 2010 that established the International Arabidopsis Informatics Consortium (IAIC). The design of the AIP will provide core functionality while remaining flexible to encourage multiple contributors and constant innovation. An IAIC-hosted Design Workshop in December 2011 proposed a structure for the AIP to provide a framework for the minimal components of a functional community portal while retaining flexibility to rapidly extend the resource to other species. We now invite broader participation in the AIP development process so that the resource can be implemented in a timely manner.