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C. B. Yohn, A. Cohen, A. Danon, S. P. Mayfield, in preparation
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C. B. Yohn, A. Cohen, A. Danon, S. P. Mayfield, in preparation.
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16
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2642602476
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note
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The amino acid sequences of tryptic peptides of the RB60 protein were determined by J. Leszyk, Worcester Foundation for Biomedical Research, Worcester, MA, USA.
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23
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2642699257
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note
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An immunoblot analysis of isolated chloroplasts from C. reinhardtii cells was performed with both antisera to tubulin and C. reinhardtii RB60. Although a specific 60-kD band was detected in isolated C. reinhardtii chloroplasts, it was not possible to obtain pure chloroplasts free of cytoplasmic proteins by density gradient centrifugation because of the similar density of whole cells and isolated chloroplasts. We therefore used pea chloroplasts for this assay, which can be readily obtained in pure form by Percoll gradient centrifugation.
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24
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2642686015
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note
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Full-length r-RB47 is 69 kD and contains a COOH-terminal domain that is cleaved in vivo to yield the native 47-kD protein (8). To generate the endogenous form of RB47, we mapped the location of the 47-kD polypeptide on the full-length recombinant protein by comparing mass spectrometric data of tryptic digests of the C. reinhardtii 47-kD protein with the full-length recombinant protein. These data showed that the 47-kD protein encompasses the four RNA recognition motif domains found in all polyadenylate [poly(A)]-binding proteins, but lacked the COOH-terminal portion of the protein.
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25
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2642655675
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To examine whether auto-phosphorylation of r-RB60 could affect the binding of r-RB47 to the 5′-UTR of psbA mRNA, we incubated r-RB47 and r-RB60 in the presence or absence of 3 mM ATP followed by gel mobility-shift assay. The RNA binding activity of r-RB47 was not altered by the addition of ATP, showing that auto-phosphorylation of r-RB47 or r-RB60 is not responsible for reducing r-RB47 RNA binding.
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S. P. Mayfield, C. B. Yohn, A. Cohen, A. Danon, Annu. Rev. Plant Physiol. Plant Mol. Biol. 46, 147 (1995).
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0011904098
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N. Harris and K. J. Oparka, Eds. Oxford Univ. Press, Oxford
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C. Robinson, in Plant Cell Biology, A Practical Approach, N. Harris and K. J. Oparka, Eds. (Oxford Univ. Press, Oxford, 1994), pp. 273-276.
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Plant Cell Biology, a Practical Approach
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Robinson, C.1
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2642686014
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note
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We thank R. Beachy, A. Cohen, and R. Bruick for critical reading of the manuscript; A. Cohen for help with fast protein liquid chromatography; and B. Huang for anti-tubulin. Supported by grants from the NIH (GM54659) and the U.S. Department of Energy (DE-FG03-93ER70116) to S.P.M. J.K is supported by a Skaggs Institute postdoctoral fellowship.
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