Introduction Directory UMM :Data Elmu:jurnal:P:PlantScience:PlantScience_Elsevier:Vol158.Issue1-2.Sept2000:

Plant Science 158 2000 97 – 105 Identification and characterization of the trnSpseudo-tRNAnad 3 rps 12 gene cluster from Coix lacryma-jobi L: organization, transcription and RNA editing Sandra Martha G. Dias a , Susely F. Siqueira a , Bernard Lejeune b , Anete P. de Souza a,c, a Centro de Biologia Molecular e Engenharia Gene´tica CBMEG , Uni6ersidade Estadual de Campinas UNICAMP , Cidade Uni6ersita´ria ‘ Zeferino Vaz ’ , 13083 - 970 C.P. 6010 Campinas SP, Brazil b Institut de Biotechnologie des Plantes, Uni6ersite´ de Paris Sud, Baˆtaille 630 , 91405 Orsay Cedex, France c Departamento de Gene´tica e E6oluc¸a˜o, Instituto de Biologia IB , Uni6ersidade Estadual de Campinas UNICAMP , Cidade Uni6ersita´ria ‘ Zeferino Vaz ’ , 13083 - 970 C.P. 6109 Campinas SP, Brazil Received 13 April 2000; received in revised form 31 May 2000; accepted 2 June 2000 Abstract During a study of mitochondrial sequence conservation between the liverwort Marchantia polymorpha and several Angiosperm species, as revealed by heterologous hybridization experiments, the trnSpseudo-tRNAnad 3 rps 12 gene cluster in Coix lacryma- jobi L., an Asian grass species from the Andropogoneae, was identified using the mitochondrial probe orf 167 from M. polymorpha. The Coix gene cluster was cloned and sequenced, and its expression analyzed. The gene sequence and gene locus organization were found to be similar to the corresponding cluster in wheat and maize. Northern hybridization and reverse transcription-polymerase chain reaction analyses indicated that nad 3 and rps 12 genes were co-transcribed as a 1.25 kb RNA molecule. The transcript displayed 20 and six RNA edition sites, in the nad 3 and rps 12 genes, respectively, that changed the codon identities to amino acids, which are better conserved in different organisms. Twenty-three cDNA clones were analysed for the edition process and revealed different partial editing patterns without apparent sequential processing. © 2000 Elsevier Science Ireland Ltd. All rights reserved. Keywords : Mitochondrial DNA; NADH dehydrogenase subunit 3; Ribosomal S12; RNA editing; Coix lacryma-jobi L. www.elsevier.comlocateplantsci

1. Introduction

The mitochondrial genomes of higher plants are much larger than those of non-plant organ- isms, from which they differ in general structure, variable gene arrangement, encoding capacity, and gene expression [1,2]. Almost all of the genes for protein complexes in the respiratory chain, encoded by animal mitochondrial genomes, have also been identified in higher plant mitochondria. The mitochondrial genome of Arabidopsis thaliana contains 57 genes with at least 42 putative open reading frames [3]. RNA editing occurs widely in higher plant mitochon- dria and involves C to U and, less frequently, U to C alterations [4 – 6]. General understanding of the information con- tent of plant mitochondrial genomes has been greatly advanced by the sequencing of the entire mitochondrial genome of the liverwort Marchan- tia polymorpha [7] and of the higher plant A. thaliana [3]. In the M. polymorpha mitochondrial genome, 28 open reading frames orf were pre- dicted as being possible genes. Five of these orf 228 , 509 , 169 , 322 and 277 are homologous to Corresponding author. Present address: Centro de Biologia Molecular e Engenharia Gene´tica CBMEG, Universidade Estadual de Campinas UNICAMP, Cidade Universita´ria ‘Zeferino Vaz’, 13083-970 C.P. 6010 Campinas SP, Brazil. Tel.: + 55-19-7881132; fax: + 55-19-7881089. E-mail address : aneteunicamp.br A.P. de Souza. 0168-945200 - see front matter © 2000 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 1 6 8 - 9 4 5 2 0 0 0 0 3 0 8 - 3 genes required for cytochrome c biogenesis in Rhodobacter capsulatus, a photosynthetic bac- terium. Homologous genes to those already men- tioned have also been found in higher plant species [8 – 16]. Genes are widely dispersed in the mitochondrial genome of higher plants and their disposition varies considerably among plant species. This vari- ability is the result of frequent rearrangements within mitochondrial genomes [2]. Co-transcrip- tion of adjacent genes has been found to occur in the mtDNA of several plant species. Polycistronic transcripts such as the rrn 18 rrn 5 gene in wheat [17], and rps 3 rpl 16 [18] and atpAatp 9 genes in maize [19] may contain sequence coding for re- lated products. However, co-transcribed genes such as atp 9 rps 13 in tobacco [20], nad 3 rps 12 in wheat and maize [21], and orf 25 cox 3 in rice [22] are involved in different metabolic pathways. The co-transcription of genes encoding proteins, acting in different metabolic pathways, indicates that post-transcriptional andor translational regula- tion is important for the control of gene-product abundance [16]. Ribosomal protein genes are generally scattered throughout the mitochondrial genome of An- giosperms and, in some cases, may be linked to non-ribosomal protein genes [8,9,16,21]. Some of these associations have been conserved over large evolutionary distances. For example, the nad 3 gene encoding mitochondrial NADH-ubiquinone- oxidoreductase subunit 3 and the rps 12 gene small subunit ribosomal protein 12 are closely linked and co-transcribed in the mtDNA of An- giosperm families as distant as the monocot grasses Sorghum [23], rice [24], maize and wheat [21] and the dicot Brassicaceae Arabidopsis [25], radish [26], rapeseed and other Brassica species [16] and also in the mtDNA from Pinus syl6estris and other Gymnosperms [27]. In maize and wheat, the trnS and a pseudo-tRNA gene are located upstream to nad 3 [21]. In this report, the analysis of a 1.4 kb BglII fragment from the mtDNA of Coix lacryma-jobi L. a south-east Asia grass species from the An- dropogoneae tribe identified by heterologous hy- bridization with DNA sequence of the mitochondrial orf 167 of M. polymorpha, was de- scribed. This orf I 67 was found to hybridize with C. lacryma-jobi nad 3 gene, located on a gene cluster that includes trnS, pseudo-tRNA and rps 12 genes. The organization, sequence analysis, tran- scription and editing pattern of this gene cluster are described.

2. Materials and methods