Alternative splicing of the Vupur3 transcript in cowpea produces multiple mRNA species with a single protein product that is present in both plastids and mitochondria
John D. Bussell A B , Doug J. Hall A , Anthea J. Mann A , Danica E. Goggin A , Craig A. Atkins A and Penelope M. C. Smith A CA School of Plant Biology, The University of Western Australia, 35 Stirling Highway, Nedlands, WA 6009, Australia.
B Current address: Umeå Plant Sciences Center, Department of Forest Genetics and Plant Physiology, Swedish University of Agricultural Sciences (SLU), 901 83 Umeå, Sweden.
C Corresponding author. Email: pmsmith@plants.uwa.edu.au
Functional Plant Biology 32(8) 683-693 https://doi.org/10.1071/FP05044
Submitted: 4 March 2005 Accepted: 28 April 2005 Published: 3 August 2005
Abstract
A heterogeneous population of cDNAs (designated Vupur3) encoding phosphoribosylglycinamide formyltransferase (GART; EC 2.1.2.2) was isolated from a cowpea (Vigna unguiculata L. Walp.) nodule library. Three classes of cDNA with the same ORF, but differing in their 3′-UTRs, were identified. Southern analysis and sequencing of genomic DNA confirmed that these differences result from alternative splicing of the primary transcript of a single Vupur3 gene. Alternative splicing does not appear to play a role in the production of soybean (Glycine max Merrill.) pur3 transcripts. The presence of the protein product of the Vupur3 gene, GART, in plastids and mitochondria was confirmed by immunoblotting with antibodies raised against the recombinant protein. The antibodies recognised two proteins with apparent molecular masses of 27 and 27.5 kDa in both mitochondria and plastids. All Vupur3 transcripts have two in-frame start codons that are active in wheatgerm in vitro transcription / translation experiments suggesting a mechanism by which the gene product could be targeted to two organelles. Like other genes encoding enzymes for purine synthesis, Vupur3 is expressed in nodules before nitrogen fixation begins but in contrast to these genes its expression does not increase markedly after nitrogen fixation begins.
Keywords: alternative splicing, cowpea, N assimilation, nodules, purine biosynthesis, soybean, 3′-UTR.
Acknowledgments
This research was supported by grants (to CAA and PMCS) from the Australian Research Council and by an Australian Postgraduate Award (to DEG). The authors are grateful to Mr S Mole for plant culture, Assoc Prof J Whelan for the soy cDNA library and p35S : GUS construct, and to Dr K Schnorr for providing the Atpur3 cDNA.
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