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Paper 2 - Engineering the Provitamin A-Carotene...

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DOI: 10.1126/science.287.5451.303 , 303 (2000); 287 Science , et al. Xudong Ye (Carotenoid-Free) Rice Endosperm -Carotene) Biosynthetic Pathway into β Engineering the Provitamin A ( This copy is for your personal, non-commercial use only. clicking here. colleagues, clients, or customers by , you can order high-quality copies for your If you wish to distribute this article to others here. following the guidelines can be obtained by Permission to republish or repurpose articles or portions of articles ): March 3, 2011 www.sciencemag.org (this infomation is current as of The following resources related to this article are available online at http://www.sciencemag.org/content/287/5451/303.full.html version of this article at: including high-resolution figures, can be found in the online Updated information and services, http://www.sciencemag.org/content/287/5451/303.full.html#ref-list-1 , 1 of which can be accessed free: cites 16 articles This article 543 article(s) on the ISI Web of Science cited by This article has been http://www.sciencemag.org/content/287/5451/303.full.html#related-urls 92 articles hosted by HighWire Press; see: cited by This article has been http://www.sciencemag.org/cgi/collection/botany Botany subject collections: This article appears in the following registered trademark of AAAS. is a Science 2000 by the American Association for the Advancement of Science; all rights reserved. The title Copyright American Association for the Advancement of Science, 1200 New York Avenue NW, Washington, DC 20005. (print ISSN 0036-8075; online ISSN 1095-9203) is published weekly, except the last week in December, by the Science on March 3, 2011 www.sciencemag.org Downloaded from
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13. The AAPK coding sequence was amplified by PCR from the AAPK cDNA clone with the primers 5 9 -GA- ATCTCCACTACGACGCCGTTTACTTCCG-3 9 and 5 9 - CCGTGCAACCATGGATATGGCATATACAAT-3 9 . The pAAPK-GFP construct was created by inserting (via Nco I digestion) the amplified AAPK coding sequence down- stream of the 35 S promoter and upstream of, and in frame with, the GFP coding sequence in the GFP expression vector (pGFP) described in ( 17 ). To create pAAPK(K43A)-GFP, the lysine at position 43 in the AAPK coding sequence was substituted with an alanine by site-directed mutagenesis {overlapping PCR method; [S. N. Ho, H. D. Hunt, R. M. Horton, J. K. Pullen, L. R. Pease, Gene 77 , 51 (1989)]}. Constructs were se- quenced to confirm correct junction, orientation, and/or site mutation. 14. Fifteen million V. faba guard cell protoplasts were transfected with pGFP, pAAPK-GFP, or pAAPK (K43A)-GFP by polyethylene glycol–mediated DNA transfer ( 17 ). Protoplasts were lysed and recombi- nant protein was immunoprecipitated with GFP pep- tide antibodies (Clontech, Palo Alto, CA) and protein A–Sepharose CL-4B (Amersham Pharmacia Biotech, Picataway, NJ). Immunoprecipitated proteins were assayed for kinase activity using histone III-S (Sigma) as substrate ( 4 ). No histone phosphorylation by the pGFP control immunoprecipitate was observed. Anal- ogous to native AAPK ( 4 ), histone phosphorylation by the pAAPK-GFP immunoprecipitate was enhanced when the immunoprecipitate was isolated from ABA- treated guard cells. When the same experiment was
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