Cloning and expression of a nuclear encoded plastid speci®c 33 kDa ribonucleoprotein gene (33RNP) from pea that is light stimulated q M.K. Reddy * , Suresh Nair, B.N. Singh, Yashwanti Mudgil, Krishna K. Tewari 1 , S.K. Sopory International Centre for Genetic Engineering and Biotechnology (ICGEB), Aruna Asaf Ali Marg, New Delhi 110 067, India Received 4 July 2000; received in revised form 12 October 2000; accepted 24 November 2000 Received by W. Martin Abstract 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 puri®ed to homogeneity. The in vitro import of precursor protein into chloroplasts con®rmed that the N-terminus putative transit peptide is a bona ®de 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 af®nity 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 0 ¯anking region of the pea 33RNP gene. q 2001 Elsevier Science B.V. All rights reserved. Keywords: In vitro transcription and translation; Pisum sativum; Plant promoter; Primer extension; Rapid ampli®cation of cDNA ends-polymerase chain reaction; Transcript analysis 1. Introduction The chloroplast genome expression exhibits both prokar- yotic and eukaryotic features (Sugiura, 1989). The psbB operon is transcribed as a tetracistronic precursor, like a prokaryotic system, (Tanaka et al., 1987; Westhoff and Herrmann, 1988). The coding information of plastid speci®c rps12 gene in barley and psaA gene in Chlamydomonas are interrupted by introns, like a eukaryotic system, (Hubsch- mann et al., 1996; Hahn et al., 1998). Most of plastid mRNA precursors in higher plants undergo a series of complex maturation events that include cleavage of poly cistronic RNAs (Westhoff and Herrmann, 1988), splicing of introns (Tanaka et al., 1987) and 3 0 end cleavage and processing (Schuster and Gruissem, 1991; Hayes et al., 1996). Each of these steps are essential for gene expression and/or for its proper regulation. However, relatively little is known about RNA processing and the factors involved in its regulation in plants when compared to animal and yeast systems (Krai- ner, 1997). A class of proteins were identi®ed in the plastids of tobacco (Li and Sugiura, 1990; Ye et al., 1991; Mieszc- zak et al., 1992.), Arabidopsis (Ohta et al., 1995) and spinach (Schuster and Gruissem, 1991) which bound, in vitro, to a number of RNA substrates with varying degree of af®nity. The amino acid analysis of these proteins derived from their cDNA sequence suggested that they are RNA- binding proteins (RNPs).The chloroplast RNPs show several structural similarities to hnRNPs and is speculated to be involved in splicing and/or processing of chloroplast mRNAs (Li and Sugiura, 1990; Ye et al., 1991; Mieszczak et al.,1992; Ohta et al., 1995). Later in spinach many of the chloroplast RNPs have shown to be involved in in vitro 3 0 end processing complex of petD mRNA. In the presence of Gene 263 (2001) 179±187 0378-1119/01/$ - see front matter q 2001 Elsevier Science B.V. All rights reserved. PII: S0378-1119(00)00574-6 www.elsevier.com/locate/gene Abbreviations: cDNA, complementary DNA; CTP, C-terminus primer; NTP, N-terminus primer; PCR, polymerase chain reaction; RACE, rapid ampli®cation of cDNA ends; RNP, ribonucleoprotein q The nucleotide sequence data reported here appears in the EMBL, GenBank and DDBJ Nucleotide Sequence Databases under the accession numbers Y14557, AF255058, AF255059. * Corresponding author. Tel.: 191-11-618-1242; fax: 191-11-616-2316. E-mail address: reddy@icgeb.res.in (M.K. Reddy). 1 Present address: Department of Molecular Biology and Biochemistry, University of California, Irvine, CA 92717, USA.