Structure and expression of an asparaginyl-tRNA synthetase gene located on chromosome IV of Arabidopsis thaliana and adjacent to a novel gene of 15 exons

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Abstract

The gene AtNS1 coding for an asparaginyl-tRNA synthetase and located on chromosome IV of Arabidopsis thaliana has been characterized. AtNS1 is the first asparaginyl-tRNA synthetase gene described in higher plants. The genomic environment of AtNS1 has been studied, as well as a partial cDNA of a second homologous asparaginyl-tRNA synthetase gene, AtNS2. Both AtNS1 and AtNS2 exhibit the highest similarity with prokaryotic homologues. A large novel gene of 15 exons, named AtG2484-1, is located adjacent to AtNS1. AtG2484-1 shows features rarely described in plants including large exons and one 3′ non-coding exon. PCR and Northern analyses were carried out to obtain information about the expression of these genes in various A. thaliana tissues.

Introduction

The aminoacyl-tRNA synthetases are a large family of house-keeping enzymes essential for the translation of genetic information into protein gene products, and are found in the cytosol and in all protein-synthesizing organelles. Eukaryotic cells have a complement of 20 enzymes, one for each amino acid (glutaminyl-tRNA synthetase is lacking from some bacteria and most mitochondria and chloroplasts).

A few aminoacyl-tRNA synthetase sequences from Arabidopsis thaliana are available in the databases, but in most cases they are partial (ESTs). In this paper, the first higher plant gene coding for asparaginyl-tRNA synthetase is described together with its cognate cDNA and a homologous cDNA. Part of the gene is located on a 9263-bp fragment sequenced by the laboratory in the framework of the European Scientists Sequencing Arabidopsis (ESSA) programme. This fragment comes from the cosmid G2484 (YAC EW4G11) located on the bottom arm of chromosome IV. With the release of the 1.9-Mb sequence by the ESSA consortium [1], a detailed and complete analysis of the genes present in this region was possible. The work presented in this article integrates biological and complementary computer approaches, and shows that the automatic annotations associated with systematic sequencing must be checked and reinforced.

As in two previous studies [2, 3], PCR has been extensively used to elucidate the structure and the expression of the analysed genes with the objective of applying this method to numerous genes released by the Arabidopsis Genome Initiative (AGI). Finally, sequence comparisons and computer analyses give information about the putative targeting of the asparaginyl-tRNA synthetase and gene duplications which occurred during the evolution of the plant genome.

Section snippets

Sequencing strategy

The nucleotide sequence of the 9263-bp-long fragment was determinated by the shotgun method [4]. Plasmid DNA was prepared using the Qiawell+ method (Qiagen) and sequencing was carried out using the dideoxy chain termination reaction. The sequence was completed using a primer-walking approach.

Computer analyses and gene characterisation

The search of similarities in databases was carried out using BLAST software [5] and the putative splicing sites in the genes were predicted by the NETPLANTGENE software [6] especially realized for A.

Results and discussion

The 9263-bp fragment sequenced is a part of the 207 674-bp genomic sequence AtFCA8 (accession no. Z97343). Fig. 1a shows the organisation and the position of the genomic fragment analysed. Because the putative gene (with a single intron) predicted between the two studied genes had no similarity with database entries and no expression was detected by PCR (Table 1), its study has not been pursued.

Conclusion

Two homologous A. thaliana genes, AtNS1 and AtNS2, coding for asparaginyl-tRNA synthetases have been characterized. An analysis of their translation products suggest that they are not located in the cytosol. The AtNS1 protein contains an amino-terminal presequence which might target the protein to mitochondria. The expression patterns of AtNS1 and AtNS2 are clearly different, indicating that the two genes are not redundant. The two genes probably encode proteins with different subcellular

Acknowledgements

We thank M. Bevan for coordinating the ESSA programme and I. Bancroft for coordinating the DNA production and distribution. We are grateful to the Arabidopsis Biological Resource Center and the stock donor (Ohio State University, USA) for providing various ESTs. We are grateful to I. Small for his critical review of the manuscript and M. Dornelas for the ASKα gene expression. The technical help of I. Gy and the photographic art work of R. Boyer are acknowledged. This work was carried out in the

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