NOTES
Cloning, sequencing and structural analysis of a pea cDNA encoding EF-la CONG Xiangyu , LING Shiyun & ZHU Yuxian National Laboratory of Protein Engineering and Plant Genetic Engineering, College of Life Science, Peking University, Beijing 100871, China Correspondence should be addressed to Zhu Yuxian (e-mail:
[email protected])
Abstract A cDNA library with the capacity of 2.0X 10' was constructed using mRNA extracted, from gibberellic acid (GA)-treated G2 pea seedlings and a 1.6 kb cDNA with 100 nucleotides of 5 ' non-coding region and 223 nucleotides of 3' non-coding region was obtained by random screening. The DNA fragment contains an open reading frame of 1 344 nucleotides and encodes 447 amino acids. Sequence analysis of DNA and deduced polypeptide demonstrated that it encoded for a pea elongation factor 1-alpha (EF-la). The functional domains of EF-la is well conserved and EF-1a can be a good candidate for studying molecular evolution. Keywrds: pea cDNA library, EF-la, sequence analysis.
The function of EF-la in polypeptide chain elongation in eucaryotes has been basically expounded. It binds GTP and aminoacyl-tRNA and leads to the codon-dependent positioning of this aminoacyl-tRNA at the A site on the ribosome[']. EF- la may be involved in the other functions besides protein synthesis. It was found that EF-la promoted degradation of microtubules in animal cell mitotic cycle. This degradation is important not only in microtubule reorganization during the transition EF-la was also from interphase to mitosis, but also in detachment of microtubules from centros~mes'~~. found associated with plant cytoskeletal network. Through regulations of protein-body formation in endosperm cells, it can influence the lysine content of storage proteins of cereal grain[3'41.To date, many genes encoding EF-la have been cloned and sequenced. These EF-la genes all encode proteins the which are highly homologous to each other. Since EF-la genes may form a large gene
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similarities and differences among EF-la genes are used for phylogenetic analysis of different The interaction between EF-la and cytoskeleton may aslo be regulated by ~a~'/calmodulin and In order to further study the function of EF-la in plant growth and development, to elucidate its possible roles in microtubule depolymerization, and to further expound the interaction between GA and EF-la, we cloned the EF-la cDNA from GA-treated G2 pea which is a mutant line with a tightly regulated senescence phenotype. We found that the pea EF-la cDNA not only contains highly conserved functional domains specific for protein synthesis, but also potentially different sites for other purposes. 1 Materials and methods ( i ) Materials. G2 pea ( Pisum sativum L.) was provided by Prof. P. J. Davies of Cornell University. Ten grams of seeds were soaked for three days and germinated in a mixture of peat and vermiculite (1:1, v/v). Ten-day-old plants were harvested for RNA extraction 2 h after being spread with 10 mmol/L GA3. ( i i ) RNA isolation and cDNA library construction. Total RNA was isolated according to RNeasy Plant Total RNA kit (Qiagen). High-quality mRNA was obtained by using Promega's PolyATtract kit. A cDNA library with titer of 2.0 X lo6 was constructed by using ZAP-cDNA synthesis kit (Stratagene). The resultant cDNAs were inserted into the Uni-ZAP XR vector in a sense orientation ( EcoR 1 -Xho 1 ) with respect to the lac2 promoter. Uni-ZAP XR clones growing in E.coli strain XL-blue MRF were converted to the pBluescript phagemids by superinfecting with the filamentous helper phage ExAssist. Clones containing recombinant plasmids were screened by a-complementation. Using blue-white selection, we determined that the ratio of recombinant to nonrecombinant was greater than 20 : 1. (iii) Screening and cloning of pea E F - l a cDNA. One hundred recombinant clones (white plaques) were picked and pBluescript SK' phagmids were purified using alkaline lysis method. Nine clones with insert longer than 800 nucleotides were sequenced using the dideoxy-mediated chain termination method. The 26th clone was found to possess sequence homologous to barley and tobacco EF- 1a gene. (iv) DNA sequence analysis. DNA and derived amino acid sequences were analyzed using the DNAsis program. ( v ) Alignment of amino acid sequences and construction of the molecular phylogenetic tree. CLUASTA ~ 1 . 7 ' " ' program was used for the alignment of amino acid sequences and PHYLIP''~] program was used to construct the molecular phylogenetic tree.
2 Results ( i ) Subclone construction and sequence analysis. The entire cDNA insert was sequenced by using ExoIII-Sl nuclease nested deletion method since there exists no restriction endonuclease site in about 800 nucleotides in the middle of our putative EF-la gene. The dideoxy method was used to sequence the deletion series. It contains 1 667 nucleotides in length with a deduced coding region of 447 amino acids (fig. 1). The sequence of EF-la gene has been accepted by EMBL database with the accession number X96555. ( ii ) Amino acid sequence analysis. Amino acid sequence alignment of pea EF-la with tobacco, barley, yeast and human EF-la sequences obtained from EMBL and SWISSPROT databases was performed using CLUASTA W 1.7 program. It reveals that all the major functional domains specific for protein synthesis were highly conserved among those available sequences. Through sequence analysis, we found that EF- la contains potentially different sites for other purposes as well (fig. 2). (iii) Construction of molecular phylogenetic tree. In order to observe the conservation of EF-la during evolution, we analyzed EF-la in SWISSPROT database using PHYLIP program with sequences obtained from 25 different species. Since these species covered most categories of the phylum, it is considered that the molecular phylogenetic tree comes from a solid background and
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Tobacco
WCKEKFHI N I VVICHVDSGKSmCHLIYKLCOIDKRVIERFEKEMENNKRSFKYAWVL
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Fig. 2. Comparisons of EF- la amino acid sequences of tobacco, pea, barley, yeast and human. See legend of fig. 1 for sign designation. The black letters represent the amino acids involved in homologous functional domains. The EMBL accession numbers of sequences above are U04632, X96555.223130, J04617 and M29934, respectively.
reflects to a certain extent the genetic flow from ancient to modem times (fig. 3). 3 Discussion The presence of 2 in-frame stop codons in the 5' untranslated region of pea EF-la gene is a common phenomenon among all known EF-la genes (fig. 1, and ref. [7]). In pea EF-la gene, an adenine residue is found at -3 position and a guanine residue at +4 position relative to the initiation codon. The nucleic acid sequence surrounding the initiation codon represents a consensus signal for Chinese Science Bulletin
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-
B, mori D. mehnogaster
A. salina
-
I
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Tobacco Tomato Wheat Barley Soybean Carrort
I Pea D. disuoideum B. hominis S. acidocaldarius
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Fig. 3. Molecular phylogenetic tree of EF-la. The MI (M-sub-L) method in CONSENSE program of PHYLIP was used for data analysis. DRAWGRAM program of PHYLIP was used to construct the EF- lo: molecular phylogenetic tree. The distances between branches represent the extent of affinity.
efficient translation it~itiation'~]. This coincides with the important function of EF-la in protein synthesis. Comparisons between the pea EF-la amino acid sequence with that of tobacco, barley, yeast and human sequences from the EMBL and SWISSPROT databases c o n f i i e d the existence of GTP-binding sites and tRNA binding sites (fig. 2). This also indicated the importance of EF-la in protein synthesis. EF-la has many other functions, too[24*9~'01. Previous work reported that PVNl (plant vitronectin-like 1) from potato may be a member of the EF-la ~ u ~ e r f a m i PVNl l ~ [ ~ ~which . contains RYD motif as well has very high sequence identity with E F - l a of plant origin. The RYD (Arg-'Qr-Asp) motif is believed to be associated with the cell joining function of PVN1. The homology between PVNl and EF-la indicates that the two proteins may be structurally similar even though they are located in different places and the former may have nothing to do with protein synthesis['31. G2 pea is a mutant line which grows unlimitedly in short-day conditions and displays nonnal apical senescence after flowering in long-day conditions. This phenotype was reverted by GA Since the treatment and the pattern of gene expression was different with or without GA treatn~ent['~.'~]. tissue- and developmental-stage-specific expression and regulation of EF-la may influence the plant growth and development, genetic studies of EF-la may help elucidating the mechanism of G2 mutation. In general, the molecular phylogenic tree of EF-la is consistent with the principle of evolution in living organisms which is supposed to develop' from lower to higher and from simple to complicated. The consistency between projected molecular evolution process and conventional 342
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evolution process may reflect the important function of EF-lain living organisms and the universal mechanism of protein synthesis in biosphere. Acknowledgements This work was supported by a grant from the Excellent Young Scientist Program of the National Nahlral Science Foundation of China (Grant No. 39725002). a Rockefeller Biotechnology Career Fellowship grant to Y-X Zhu.
References 1. Riis, B., Rattan, S. L. S., Clark, B. F. C. et al., Eukaryotic protein elongation factors, TIBS, 1990.15: 420. 2. Shiina, N., Gotoh, Y., Kubomura, N. et al., Microtubule severing by elongation factor-la, Science, 1994,266: 282. 3. Clore, A. M., Dannenhoffer, J. M., Larkins, B. A., EF-la is associated with a cytoskeletal network surrounding protein bodies in maize endosperm cells, The Pant Cell, 1996. 8: 2003. 4. Habben. J. E., Moro, G. L., Hunter, B. G. et al., Elongation factor-la concentration is highly correlated with the lysine content of maize endosperm, Roc. Natl. Acad. Sci. USA, 1995,92: 8640. 5. Axelos, M., Bardet, C., Liboz, T. et al., The gene family encoding the Arabidopsis thaliana translation elongation factor EF-la: Molecular cloning, characterization and expression, Mol. Gen. Genet., 1989,219: 106. 6. Bonafonte, M. T., Preist, J. W., Garmon, D. et al.. Isolation of the gene coding for elongation factor-la in Cryptosporidium pawum, Biochim. et. Biophys. Acta, 1997, 1351: 256. 7. Pokalsky, A. R., Hiatt, W. R., Ridge, N. et al.. Structure and expression of elongation factor - l a in tomato, Nucl. Acids Res., 1989, 17: 4661. 8. Qing, Y. L.. Baldauf, S. L. Reith, M. E., Elongation factor-la genes of the red alga Porphyra purpurra include a novel, developmentally specialized variant, Plant. Molecular Biology, 1996.31: 77. 9. Durso, N. A., Cyr, R. J., A calmodulin-sensitive interaction between microtubules and a higher plant homolog of elongation factor-la, The Plant Cell, 1994.6: 893. 10. Kurasawa, Y., Hanyu, K., Watanabe, Y. et al., F-actin bundling activity of Tetrahymena elongation factor la is regulated by ~a~+/calmodulin, J. Biochem., 1996, 119: 791. 11. Thompson, J. D., Higgins, D. G., Gibson, T. I., CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, positions-specific gap penalties and weight matrixchoice, Nucleic Acids Research, 1994.22: 4673. 12. Baum, B. R., PHYLIP: Phylogeny Inference Package, Version 3.2. (Software review), Quarterly Review of Biology, 1989, 64: 539. 13. Zhu, J. K., Damsz, B., Andrzej, K. et al., A higher plant extracellular vitronectin-like adhesion protein is related to the translational elongation factor-la, The Plant Cell, 1994.6: 393. 14. Zhu, J. K., Damsz, B., Andrzej, K. et al.. A higher plant extracellular vitronectin-like adhesion protein is related to the translational elongatio P. J., The control of apical bud growth and senescence by auxin and gibberellin in genetic lines of peas. Plant Physiol., 1997, 113: 631. 15. Zhu, Y. X., Zhang, Y. E,Luo, J. et al., PPF-1, a post-floral-specific gene expressed in short-day grown G2 pea may be important for the maintenance of vigorous growth, Gene, 1998,208: 1. (Received March 30,1999)
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