This Article Full Text Full Text (PDF) All Versions of this Article: 138/3/1746    most recent pp.105.063040v1 Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in ISI Web of Science Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via ISI Web of Science (25) Citing Articles via Google Scholar Google Scholar Articles by Quesada-Vargas, T. Articles by Daniell, H. Search for Related Content PubMed PubMed Citation Articles by Quesada-Vargas, T. Articles by Daniell, H. Agricola Articles by Quesada-Vargas, T. Articles by Daniell, H.

GENETICS, GENOMICS, AND MOLECULAR EVOLUTION

Characterization of Heterologous Multigene Operons in Transgenic Chloroplasts. Transcription, Processing, and Translation¹

Department of Molecular Biology and Microbiology, University of Central Florida, Orlando, Florida 32816–2364

The first characterization of transcriptional, posttranscriptional, and translational processes of heterologous operons expressed via the tobacco (Nicotiana tabacum) chloroplast genome is reported here. Northern-blot analyses performed on chloroplast transgenic lines harboring seven different heterologous operons revealed that polycistronic mRNA was the predominant transcript produced. Despite the lack of processing of such polycistrons, large amounts of foreign protein accumulation was observed in these transgenic lines, indicating abundant translation of polycistrons. This is supported by polysome fractionation assays, which allowed detection of polycistronic RNA in lower fractions of the sucrose gradients. These results show that the chloroplast posttranscriptional machinery can indeed detect and translate multigenic sequences that are not of chloroplast origin. In contrast to native transcripts, processed and unprocessed heterologous polycistrons were stable, even in the absence of 3' untranslated regions (UTRs). Unlike native 5'UTRs, heterologous secondary structures or 5'UTRs showed efficient translational enhancement independent of cellular control. Abundant read-through transcripts were observed in the presence of chloroplast 3'UTRs but they were efficiently processed at introns present within the native operon. Heterologous genes regulated by the psbA (the photosystem II polypeptide D1) promoter, 5' and 3'UTRs have greater abundance of transcripts than the endogenous psbA gene because transgenes were integrated into the inverted repeat region. Addressing questions about polycistrons, and the sequences required for their processing and transcript stability, are essential in chloroplast metabolic engineering. Knowledge of such factors would enable engineering of foreign pathways independent of the chloroplast complex posttranscriptional regulatory machinery.

² Present address: Midwest Research Institute, 1470 Treeland Blvd., S.E. Palm Bay, FL 32909–2211.

Article, publication date, and citation information can be found at www.plantphysiol.org/cgi/doi/10.1104/pp.105.063040.

^* Corresponding author; e-mail daniell{at}mail.ucf.edu; fax 407–823–0956.

Received March 17, 2005; returned for revision May 16, 2005; accepted May 17, 2005.

This article has been cited by other articles:

				D. Verma and H. Daniell Chloroplast Vector Systems for Biotechnology Applications Plant Physiology, December 1, 2007; 145(4): 1129 - 1143. [Full Text] [PDF]

				A. Limaye, V. Koya, M. Samsam, and H. Daniell Receptor-mediated oral delivery of a bioencapsulated green fluorescent protein expressed in transgenic chloroplasts into the mouse circulatory system FASEB J, May 1, 2006; 20(7): 959 - 961. [Abstract] [Full Text] [PDF]

				V. Koya, M. Moayeri, S. H. Leppla, and H. Daniell Plant-Based Vaccine: Mice Immunized with Chloroplast-Derived Anthrax Protective Antigen Survive Anthrax Lethal Toxin Challenge Infect. Immun., December 1, 2005; 73(12): 8266 - 8274. [Abstract] [Full Text] [PDF]