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Comparative Study
. 1996 Nov 12;93(23):13371-6.
doi: 10.1073/pnas.93.23.13371.

A cdc5+ homolog of a higher plant, Arabidopsis thaliana

T Hirayama  1 , K Shinozaki
Affiliations
Comparative Study

A cdc5+ homolog of a higher plant, Arabidopsis thaliana

T Hirayama et al. Proc Natl Acad Sci U S A. .

Abstract

We cloned and characterized a cDNA corresponding to a cdc5+ homolog of the higher plant, Arabidopsis thaliana. The cDNA, named AtCDC5 cDNA, encodes a polypeptide of 844 amino acid residues. The amino acid sequence of N-terminal one-fourth region of the predicted protein bears significant similarity to that of Schizosaccharomyces pombe Cdc5 and Myb-related proteins. Overexpression of the AtCDC5 cDNA in S. pombe cells is able to complement the growth defective phenotype of a cdc5 temperature-sensitive mutant. These results indicate that the AtCDC5 gene is a plant counterpart of S. pombe cdc5+. This is the first report of a cdc5(+)-like gene in a multicellular organism. We also demonstrated that a recombinant AtCDC5 protein possesses a sequence specific DNA binding activity (CTCAGCG) and the AtCDC5 gene is expressed extensively in shoot and root meristems. In addition, we cloned a PCR fragment corresponding to the DNA binding domain of human Cdc5-like protein. These results strongly suggest that Cdc5-like protein exists in all eukaryotes and may function in cell cycle regulation.

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Figures

Figure 5
Figure 5
The expression pattern of AtCDC5. (A) In situ analysis of AtCDC5. Using antisense and sense RNA probes, whole mount in situ hybridization was performed. Microscopic views of root tips and a vegetative shoot apex are shown. Note that high background signals in cotyledons was due to their overlapping as indicated. (B) Northern blot analysis of AtCDC5 and CDC2a. RNAs from leaves, developing siliques, shoots, roots, and flowers were hybridized with the AtCDC5 cDNA probe or the CDC2a cDNA probe (3).
Figure 5
Figure 5
The expression pattern of AtCDC5. (A) In situ analysis of AtCDC5. Using antisense and sense RNA probes, whole mount in situ hybridization was performed. Microscopic views of root tips and a vegetative shoot apex are shown. Note that high background signals in cotyledons was due to their overlapping as indicated. (B) Northern blot analysis of AtCDC5 and CDC2a. RNAs from leaves, developing siliques, shoots, roots, and flowers were hybridized with the AtCDC5 cDNA probe or the CDC2a cDNA probe (3).
Figure 1
Figure 1
Deduced amino acid sequence of AtCDC5 cDNA. The putative Myb-like DNA binding domain is underlined.
Figure 2
Figure 2
The structure of AtCDC5 compared with other Myb-related proteins. (A) Comparison of the amino acid sequence of the N-terminal region of AtCDC5 with that of S. pombe Cdc5. Identical amino acid residues are indicated with asterisks and conservative amino acid substitutions are indicated with dots. (B) The structure of the threonine-proline rich regions of AtCDC5 and Cdc5 proteins. Thr-Pro sequences are indicated with reversed characters. (C) The predicted amino acid sequence of the Myb-like repeats of AtCDC5 is compared with S. pombe Cdc5 (SpCdc5), putative human Cdc5 protein (HsCdc5), Arabidopsis ATMYB2 (42), Arabidopsis GL1 (AtGL1; ref. 44), Zea mays C1 (ZmC1; ref. 45), human c-MYB (HCMYB; ref. 46), Drosophila MYB (DmMYB; ref. 47). The amino acid residues that are identical in AtCDC5 and another Myb-related protein are indicated with reversed characters. Closed circles and open circles indicate conserved tryptophan residues. Asterisks represent the amino acid residues that are presumed to be involved in the target recognition of human c-Myb protein. Note that the first five amino acid sequences of human Cdc5 protein corresponds to primer 1.
Figure 3
Figure 3
Complementation of the cdc5-120 temperature-sensitive mutation by AtCDC5 cDNA. KGY372 (cdc5-120) was transformed with recombinant plasmid DNAs, streaked on EMM-agar (3 mM thiamin + or −) plates and incubated at the permissive temperature (26°C) or nonpermissive temperature (36°C). “pIRT2+cdc5+” indicates that the cells were transformed with the chimeric plasmids DNA that consist of pIRT2 and the cdc5+ gene. “pREP1+AtCDC5,” “pREP1+AtCDC5 (inverted characters),” and “pREP1” indicates the cells transformed with the chimeric pREP1 plasmid carrying the AtCDC5 cDNA in appropriate direction, the cells transformed with the chimeric pREP1 plasmid carrying the AtCDC5 cDNA in reverse direction and the cells transformed with pREP1 vector only, respectively.
Figure 4
Figure 4
The GST-AtCDC5 fused protein possessed a sequence specific DNA binding activity. (A) Summary of the nucleotide sequence of DNA fragments recognized by the GST-AtCDC5 fused protein in random binding site selection experiments. The base constitution around the consensus sequence was indicated in percent. (B) Gel mobility shift assay. Five nanograms per lane of the GST-AtCDC5 protein was used. C, A DNA fragment with the consensus sequence (68-mer left arm-ACCAACGGGCGCTGAGCTGATGTCG-68-mer right arm); N, a DNA fragment without the consensus sequence (68-mer left arm-ACACCAAGTCTTACGCGCTGTCTCGC-68-mer right arm).
Figure 6
Figure 6
Zoo blot analysis. Genomic DNAs from various organisms such as human, mouse, Xenopus, Drosophila, Caenorhabditis elegans, Arabidopsis, and S. pombe were subjected to Southern blotting analysis. (Upper) The autoradiogram of the Southern hybridization using a cDNA fragment of AtCDC5 as a probe; (Lower) the photograph of the electrophoresed agarose gel stained with ethidium bromide.
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