Cloning and expression of a nuclear encoded plastid specific 33 kDa ribonucleoprotein gene (33RNP) from pea that is light stimulated

Citation
Mk. Reddy et al., Cloning and expression of a nuclear encoded plastid specific 33 kDa ribonucleoprotein gene (33RNP) from pea that is light stimulated, GENE, 263(1-2), 2001, pp. 179-187
Citations number
25
Language
INGLESE
art.tipo
Article
Categorie Soggetti
Molecular Biology & Genetics
Journal title
GENE
ISSN journal
0378-1119 → ACNP
Volume
263
Issue
1-2
Year of publication
2001
Pages
179 - 187
Database
ISI
SICI code
0378-1119(20010124)263:1-2<179:CAEOAN>2.0.ZU;2-K
Abstract
We report the cloning and sequencing of both cDNA and genomic DNA of a 33 k Da chloroplast ribonucleoprotein (33RNP) from pea. The analysis of the pred icted amino acid sequence of the cDNA clone revealed that the encoded prote in contains two RNA binding domains, including the conserved consensus ribo nucleoprotein sequences CS-RNP1 and CS-RNP2, on the C-terminus half and the presence of a putative transit peptide sequence in the N-terminus region. The phylogenetic and multiple sequence alignment analysis of pea chloroplas t RNP along with RNPs reported from the other plant sources revealed that t he pea 33RNP is very closely related to Nicotiana sylvestris 31RNP and 28RN P and also to 31RNP and 28RNP of Arabidopsis and spinach, respectively. The pea 33RNP was expressed in Escherichia coli and purified to homogeneity. T he in vitro import of precursor protein into chloroplasts confirmed that th e N-terminus putative transit peptide is a bona fide transit peptide and 33 RNP is localized in the chloroplast. The nucleic acid-binding properties of the recombinant protein, as revealed by South-Western analysis, showed tha t 33RNP has higher binding affinity for poly (U) and oligo dT than for ssDN A and dsDNA. The steady state transcript level was higher in leaves than in roots and the expression of this gene is light stimulated. Sequence analys is of the genomic clone revealed that the gene contains four exons and thre e introns. We have also isolated and analyzed the 5' flanking region of the pea 33RNP gene. (C) 2001 Elsevier Science B.V. All rights reserved.