Expression and deletion analysis of an Arabidopsis SUPERMAN-like zinc finger gene
Introduction
In animal systems, the zinc finger protein gene family is the largest group of transcription factors, and its members play a wide and important role in growth and development. Similarly, plants also contain a large array of zinc finger proteins, with many being unique to plants (for reviews see [1], [2]). The canonical C2H2 zinc finger motif is one of the best characterized due to numerous studies of the TFIIIA protein from Xenopus laevis and ZIF268 protein from mice [3], [4]. Crystal structures of these proteins bound with DNA have resulted in a clear understanding of the role of the zinc fingers in DNA binding [4], [5] and has resulted in the “designing” of zinc finger genes that bind to specific DNA sequences [6]. Animal zinc finger proteins generally contain multiple fingers separated by short spacers, generally 6–8 amino acids, where the coordination of fingers with the DNA appears to be an essential feature of these zinc finger proteins [7], [8]. Plant C2H2 zinc finger proteins, on the other hand, contain only one to four zinc fingers and have long and variable spacer regions of 19–65 amino acids [2], [9]. In contrast to the positioning and DNA binding of animal C2H2 zinc finger proteins, the DNA–protein interaction of plant zinc finger proteins are only beginning to be characterized [10], [11].
Comparisons of the Arabidopsis C2H2 zinc finger proteins reveal that most contain the canonical CX2CX3FX8HX3H zinc finger domain. The best characterized zinc finger family in plants is the EPF type, so named as it was first characterized as a component binding to the promoter of the 5-enolpyruvulshikimate-3-phosphate synthase gene [12]. Subsequently, additional EPF zinc finger genes have been identified that are expressed temporally during flowering in petunia [9], [13]. The only other C2H2 EPF-type zinc finger proteins documented to date are those that have been found in Arabidopsis. One class of the Arabidopsis zinc finger genes, exemplified by the SUPERMAN and ZFP1-8 genes, contain a single zinc finger domain [14], [15]. It has not been determined if or how single zinc finger proteins bind DNA, but it suggests that they should interact with other proteins, or dimerize, if they are to function in DNA binding. A leucine rich region in the carboxyl terminus of most of the single zinc finger proteins was speculated to be involved in protein–protein interactions [14]. This region is similar to the ERF transcriptional repression domain that has been shown to be required for SUPERMAN and other zinc finger proteins to function as a transcriptional repressor [16], [17]. With the exception of the SUPERMAN gene, no known phenotype has been associated with any of the other single C2H2 zinc finger genes. In this study we have analyzed expression of one of these genes, AtZFP11, in Arabidopsis and demonstrated that ectopic expression in Arabidopsis and tobacco disrupts normal development. Overexpression of the AtZFP11 gene lacking the coding region for the leucine rich region, the ERF motif, in Arabidopsis and tobacco resulted in normal transgenic plants suggesting the necessity of this region for normal protein function. We also delimit the nuclear localization region of the AtZFP11 protein using N-terminal deletion fusion protein constructs with the GFP protein.
Section snippets
DNA manipulations
Oligonucleotide primers were synthesized based on sequences found on BAC MHK10 on chromosome 2 in the Arabidopsis genome database that encodes the putative protein NP_181770 in GenBank and At2g42410 in the Arabidopsis database. For expression, primers were synthesized to include a start and stop codon (in bold) with the addition of a restriction endonuclease site (underlined) to aid in cloning in the plant expression vectors described below (AtZFP11For: TTG TTG GA AAA TAT GAA GAG AAC
Arabidopsis single zinc finger genes
Putative SUPERMAN-like C2H2 zinc finger genes were identified in the Arabidopsis protein database using SUPERMAN (SUP) as a starting point. As putative single zinc finger genes were identified, they were then confirmed and used to search for additional zinc finger genes. Numerous C2H2 zinc finger genes were identified, many containing two or more zinc fingers with homology in the zinc finger domain. Twenty-eight putative genes that had only a single C2H2 zinc finger domain and that were similar
Discussion
There are 28 putative single EPF C2H2 zinc finger genes in the Arabidopsis genome. One of these, the SUPERMAN (SUP) gene, is required in flower development [15], [24]. A second member of the EPF single C2H2 zinc finger protein family in Arabidopsis, AtZFP1, has been shown to be developmentally regulated and is expressed in the shoot apex of developing seedlings following exposure to light [25]. The expression profile of eight other single C2H2 proteins have been documented [14], [26], [27], but
Acknowledgements
The authors wish to thank Whitney Greer, Curtis Meurer, Carl Redmond and Ray Stevens for technical assistance in the laboratory and greenhouse. This research was supported by KAES Hatch funds to G.B. Collins. Part of this research was conducted by C. Pflipsen as part of the Independent Honors Project (ABT 395) for the Agricultural Biotechnology BS degree in the Agronomy Department. This paper (Number 02-06-53) is published with the approval of the Director of the Kentucky Agricultural
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