Partial Sequencing of an Arabidopsis thaliana cDNA encoding a novel zinc finger DNA binding protein

Emily Gray, Soumit Ryo, and Charles McMurrough

University of Texas at Austin

 Abstract

            We isolated a cDNA fragment from a pZL1 plasmid containing an Arabidopsis thaliana insert. A partial sequence of the cDNA fragment was obtained using dideoxy chain termination sequencing reactions. Searches in BLAST databases identified homologous regions between our translated protein sequence and DNA binding proteins of six different plant species.

             A section of 32 amino acids appears to be a conserved sequence found in plants. The sequence has been analyzed in sweet potato SPF-1, wild oat ABF, cucumber SPF-1 type, and arabidopsis Zap1. This conserved sequence, C1-X4- C1-X23-H1-X1- H1, is believed to encode a novel cysteine/histidine zinc finger motif. The genomic DNA of sweet potato, cucumber, and Arabidopsis contain single copies of the gene and increased expression is seen in different plant parts. The cDNA fragment characterized in this study appears to be part of a small multigene family and is expressed ubiquitously in arabidopsis. The protein also shows increased expression consequent to wounding which may indicated a functional role of the protein in repair mechanisms of arabidopsis. Further characterization of the entire protein would be necessary to elucidate possible connection between this and the various other characterized zinc dependent DNA binding proteins of plants.

 

Introduction

            Transcription factors play an active role in the regulation of gene expression7. Binding of these factors to site-specific DNA sequences can influence the activation or inactivation of a particular transcriptional event resulting in increased or decreased production of RNA3. There are several structural binding motifs that are common among these DNA-binding proteins. These structural motifs include the helix-loop-helix, leucine zipper, and zinc finger3.

            Several zinc finger binding proteins have been isolated from plants with the characteristic histidine, cysteine, and zinc ion interactions7. Recently a unique class of zinc finger like binding motifs7 with an unusual spacing of cysteine and histidine residues6 has been characterized in Ipomoea batatas (sweet potato)2, Avena fatua (wild oat)5, Cucumis sativus (cucumber)6, and Arabidopsis thalaian (arabidopsis)7. The cDNA from these plants contain one or more homologous sequences for a supposed zinc finger binding motif, C1-X4- C1-X23-H1-X1- H1(figure 3).

            In this study, we isolated and partially sequenced an Arabidopsis thalaian cDNA fragment that encodes one C2H2 zinc finger like motif. An open reading frame of 324 nucleotides was determined in Mac Vector and entered into Blast search programs to search the DNA/protein sequence databases for homologies. The search produced several high scoring segment pairs (range 151-179) of our partial protein sequence (108 amino acids) and DNA binding proteins of sweet potato SPF1 (p = 6.5X10-16), wild oat ABF (p = 5.8X10-16), cucumber SPF1-type (p= 2.8X10-18), and arabidopsis ZAP1 (p = 2.1X10-14).

            The C2H2 motif is duplicated in sweet potato SPF1, wild oatABF, cucumber SPF1-type, and arabidopsis ZAP17 (figure 3). The gene coding for the sweet potato SPF1 was found to be present in one or a few copies and RNA was abundant in leaves, stems, and tuberous root2. Cucumber cDNA contains a few genes of SPF1-type that have increased expression in cotyledons6. Arabidopsis contains a single copy of the ZAP1 gene expressed in roots and floral tissue7. Wild oat ABF is suggested to belong to a multigene family and only contains one C2H2 motif5.

            Southern and Northern analysis of the cDNA clone sequence indicates the gene may belong to a multigene family (figure 4) and is expressed throughout the whole plant and upon wounding shows an RNA production spike at 5hrs (figure 5)which may indicated activity of the protein in delayed repair mechanisms.

            The characterization of the Arabidopsis thalaian cDNA fragment indicates a possible zinc dependent DNA binding protein. The homology of this 32 amino acid sequence between the 6 plant species contributes to the theory that this conserved region plays a role in binding of these transcription factors7. Further characterization of the entire gene will be necessary to determine if this protein is functionally homologous to any of the various plant proteins6.

 

Methods1

Plasmid Isolation and Subcloning

            An Escherichia coli stock containing pZL1 plasmid DNA with an unidentified Arabidopsis thaliana cDNA insert was obtained from the Arabidopsis Biological Resource Center. A modified alkaline lysis method was used to extract the plasmid DNA from E. coli. The isolated plasmid DNA was treated with EcoR1 and HindIII, restriction endonucleases that cut at particular bp sites, to excise the cDNA insert and determine a partial restriction map of the plasmid (figure 1). The insert was subsequently subcloned into a pBluescript plasmid and transformed into XL1-blue competent cells for blue/white color screening.

PCR and DNA sequencing

            A Qiagen miniprep kit was used to isolate plasmids from transformed colonies. The polymerase chain reaction, using T7 and T3 primers, was performed to determine if the insert was present in the transformed colonies. Dideoxy chain termination reactions (Sanger et al 1977) were performed using a T7 primer for manual sequencing of the pBluescript plasmid subclone and for automated sequencing of the original pZL1 plasmid clone. The automated DNA sequencing provided a partial sequence for the Arabidopsis thaliana cDNA insert that was used to search the gene databases. MacVector and Blast software were used to analyze the sequence.

Genomic DNA Isolation and Southern Transfer

            Genomic DNA was isolated from frozen Arabidopsis thaliana plant tissue by grinding in liquid N2, treating with SDS/NaOH and SDS/phenol/chloroform followed by PEG precipitation. The DNA was digested with EcoRI, BamHI, and HindIII, fractionated in an 0.8% agarose gel and transferred to a nylon membrane by capillary transfer. Alpha-32P-labeled DNA probes were prepared by a random primer method using the PCR amplified insert product (see PCR methods). The primers were hybridized to the Southern blot overnight and then washed with a low stringency solution of 5% SDS, 40nMNA2HPO4pH 7.4, and 1mM EDTA at 60 degrees Celsius. (The melting temperature of the probe duplex allowed for a 5% mismatch pairing and a 95% homology hybridization between probe and fragmented genomic DNA4.)

Total RNA Isolation and Northern Transfer

            Total RNA was isolated from frozen plant tissue of wounded seedlings at times 0, 1hr, 5hrs, 8hrs, and from stems, floral tissue, leaves and whole plant. The frozen tissue was ground in liquid N2, RNA was extracted with phenol/chloroform in SDS/EDTA and precipitated under salt conditions. For Northern hybridization the total RNA was sized fractionated in an 1.2% formaldehyde agarose gel. RNA was transferred to a nylon membrane by capillary transfer. The same Southern hybridization probe and preparatory conditions were used for Northern analysis.

Results

            The pZL1 recombinant plasmid was isolated and cut with EcoR1 and HindIII to excise the insert and determine a partial restriction map of the plasmid (figure 1). The restriction digestion fragments were run on a gel using gel electrophoresis and the insert size was determined to be approximately 750bp.

            The cDNA clone was sequenced and analyzed in MacVector to determine the best open reading frame (ORF) of 324 nucleotides (figure 2). The translated ORF coded for a 108 amino acid protein that was compared with BLAST search databases. High scoring segment pairs ranged from 151 to 179. The probability of randomness (desired p<.01) ranged form 10-14 to 10-18 indicating partial homology matches between proteins. The matches pulled up DNA binding proteins of sweet potato SPF1 (p = 6.5X10-16), wild oat ABF (p = 5.8X10-16), cucumber SPF1-type (p= 2.8X10-18), and arabidopsis ZAP1 (p = 2.1X10-14). The homologous sequence of significance consists of amino acid 451to 481, C1-X4- C1-X23-H1-X1- H1 (figure 3).

            Southern blot analysis was performed to determine the number of genes homologous to the cDNA fragment. The PCR amplified probe hybridized to a series of EcoR1, BamHI, and HindIII fragments suggesting a small multigene family of DNA binding proteins with the same novel binding motif. The strongest hybridization bands were 6.6kb and 1kb EcoR1 fragments and a 3kb HindIII fragment. Resolution of bands and the elimination of weak hybridization bands could be accomplished with a high stringency wash. A clearer picture of the genes belonging to the family could then be determined. Northern hybridization of total RNA from wounded seedlings, stems, floral tissue, leaves, and whole plants demonstrated the presence of the protein mRNA throughout the plant in approximately equal quantities. The probe hybridized to a 0.6kb length RNA. The insert isolated contained 0.75kb and is therefore may contain the complete copy of the gene. The Northern analysis of wounded seedlings showed a decrease of RNA production after 1 hr and a spike in production at 5hrs.

           

Discussion

            In this study, we partially sequenced and characterized an Arabidopsis thaliana cDNA fragment that appears to encode a DNA binding protein with a novel zinc finger like binding motif. The translated sequence of the cDNA clone coded for a protein composed of 108 amino acids. 32 amino acids of the translated sequences have high homology with the DNA binding protein sequences of 6 different plant species. This conserved sequence, C1-X4- C1-X23-H1-X1- H1, is believed to form a distinct class of zinc dependent DNA binding proteins.

            Southern and Northern analysis of the arabidopsis cDNA demonstrated the existence of a multigene family that is expressed throughout the whole plant in approximately equal quantities. This evidence deviates slightly from gene copy number and gene expression in sweet potato, cucumber, and arabidopsis. These plant species contained single gene copies and increased expression in roots, coytledons, and stems/floral tissue, respectively. Continued characterization of the entire protein could clarify more of the structural composition of the gene. Once the entire sequence is available a larger probe could be used to isolate and determine key genes in the multigene family.

            Northern analysis of wounded seedlings demonstrated a decrease of gene expression after the initial wounding and then a spike in expression after 5hrs. This may indicate the functional role of this protein in delayed repair mechanisms of the plant but further studies would need to be conducted on functions of the protein before conclusions could be made.

            The proteins SPF-1 of sweet potato, ABF of wild oat, SPF-1 type of cucumber, and ZAP1 and unknown cDNA fragment of arabidopsis show homologies in binding motifs but they all may function in various biological processes. Besides the 32 amino acid binding motif little homology outside of short flanking regions is seen.

            The data collected in this study contributes to the evidence of a distinct and unusual class of zinc finger motif found in a conserved sequence of some plant DNA binding proteins. It would be interesting to continue comparison studies of these unique binding proteins.

 

Acknowledgments

            We thank Dr. Sathasivan, Ryo, and Wednesday Group 2 for their guidance and contributions to this study.

 

References

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 5. Rushton, P.J., Macdonald, H., Huttly, A.K., Lazarus, C.M., and Hooley, R.: Members of a new family of DNA-binding proteins to a conserved cis-element in the promoters of alpha-Amy 2 genes. Plant Mol. Bio. 29:691-702. (1995)

 6. Kim, D-J, Smith, S.M., and Leaver, C.J.: A cDNA encoding a putative SPF1-type DNA binding protein from cucumber. Gene 185:265-269. (1997)

 7. Pater, S.de, Greco, V., Pham, K., Memelink, J. and Kijne,J.: Characterization of a zinc-dependent transcriptional activator from Arabidopsis. Nucleic Acid Research 24:4624-4631. (1996)