Cloning and expression of a nuclear encoded plastid specific 33 kDa ribonucleoprotein gene (33RNP) from pea that is light stimulated.
We report the cloning and sequencing of both cDNA and genomic DNA of a 33 kDa chloroplast ribonucleoprotein (33RNP) from pea. The analysis of the predicted amino acid sequence of the cDNA clone revealed that the encoded protein contains two RNA binding domains, including the conserved consensus ribonucleoprotein 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 chloroplast RNP along with RNPs reported from the other plant sources revealed that the pea 33RNP is very closely related to Nicotiana sylvestris 31RNP and 28RNP and also to 31RNP and 28RNP of Arabidopsis and spinach, respectively. The pea 33RNP was expressed in Escherichia coli and purified to homogeneity. The in vitro import of precursor protein into chloroplasts confirmed that the N-terminus putative transit peptide is a bona fide transit peptide and 33RNP is localized in the chloroplast. The nucleic acid-binding properties of the recombinant protein, as revealed by South-Western analysis, showed that 33RNP has higher binding affinity for poly (U) and oligo dT than for ssDNA and dsDNA. The steady state transcript level was higher in leaves than in roots and the expression of this gene is light stimulated. Sequence analysis of the genomic clone revealed that the gene contains four exons and three introns. We have also isolated and analyzed the 5' flanking region of the pea 33RNP gene.
Reddy
M K
MK
International Centre for Genetic Engineering and Biotechnology (ICGEB), Aruna Asaf Ali Marg, New Delhi 110 067, India. reddy@icgeb.res.in
Nair
S
S
Singh
B N
BN
Mudgil
Y
Y
Tewari
K K
KK
Sopory
S K
SK
D000595:Amino Acid Sequence; D001483:Base Sequence; D015152:Blotting, Northern; D002467:Cell Nucleus / Q000235:genetics*; D002736:Chloroplasts / Q000378:metabolism; D003001:Cloning, Molecular; D004247:DNA / Q000235:genetics / Q000378:metabolism; D018076:DNA, Complementary / Q000737:chemistry / Q000235:genetics; D018744:DNA, Plant / Q000737:chemistry / Q000235:genetics; D004274:DNA, Recombinant; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D018506:Gene Expression Regulation, Plant / Q000528:radiation effects; D005796:Genes / Q000235:genetics; D008027:Light; D008969:Molecular Sequence Data; D008970:Molecular Weight; D018532:Pisum sativum / Q000235:genetics* / Q000528:radiation effects; D010957:Plasmids / Q000235:genetics; D018087:Plastids / Q000378:metabolism*; D016133:Polymerase Chain Reaction; D011485:Protein Binding; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism / Q000528:radiation effects; D012261:Ribonucleoproteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D017422:Sequence Analysis, DNA; D014018:Tissue Distribution; D014158:Transcription, Genetic
D018076:DNA, Complementary; D018744:DNA, Plant; D004274:DNA, Recombinant; D012333:RNA, Messenger; D012261:Ribonucleoproteins; D004247:DNA