Young Ju CHOI and Gynheung AN
Institute of Biological Chemistry, Washington State University, Pullman, Washington 99164, USA
The calcium-binding protein calmodulin (CaM) is a ubiquitous protein that is thought to be a primary transducer of intracellular calcium signals (Means et al. 1982; Poovaiah et al. 1987). CaM is a niember of the calcium-modulated protein family and has been identified in all eukaryotes examined. The cDNA clones coding for plant CaM were isolated from potato (Jena et al. 1989), barley (Ling and Zielinski 1989), alfalfa (Barnett and Long 1990) and Arabidopsis thaliana (Ling et al. 1991). All the cDNA clones code for 148 amino acid peptides which are highly conserved in all eukaryotes (Means et al. 1985). Between the monocot and dicot species, the homology was higher than 90%. These results suggest that biological function of CaM is conserved throughout living organisms. Using the potato clone, it was found that CaM mRNA is present in various tissues. However, a high level of CaM was detected in stolon tips (Jena et al. 1989). In addition, it was reported that auxin treatment of detached strawberry fruits increased the CaM mRNA level. Role of CaM in phytochrome-regulated gene expression was also proposed (Jena et al. 1989; Lam et al. 1989). In response to water spray, wind, touch, wounding, or darkness, Arabidopsis thaliana accumulated CaM and CaM like proteins (Bream 1990). These observations indicate that the CaM activity is regulated by a variety of environmental or developmental signals in plants. In an attempt to further understand the molecular mechanisms involved in regulation of the CaM gene expression, we have isolated and characterized the structure of two rice CaM genomic clones.
Southern analysis of rice genomic DNA using potato cDNA clone as a probe indicated that there are at least four copies of CaM or CaM-related genes. Using the potato probe, we have obtained several phage clones containing a putative CaM gene from a rice genomic library. In order to group identical clones, restriction enzyme mapping (Fig. 1A) and Southern blot analysis (Fig. 1B) were carried out. The lambda phage clones 9, 11, 13 and 20 appeared to carry overlapping fragments which contained an identical CaM gene. The restriction enzyme digestion patterns were similar to each other and all four clones contained a 1.2 Kbp BamHI fragment which hybridized with the cDNA probe. The length of the EcoRI fragment that contains the CaM gene was different from each other since one of the EcoRI site was derived from the multiple linker sequence located in the cloning vector. The CaM gene located in this group was called CaM-1. Clones 14, 18, and 22 share a similar map and contain the same 4.5 Kb EcoRI fragment that hybridized with the cDNA probe. The gene located in the second group was named CaM-2. Clone 15 seems different from the remaining.
Fig. 1. Analysis of lambda phage clones. (A). Lambda phage DNA was digested with either EcoRI or BamHI and separated on 0.7% agarose gel. Ethidium bromide stained DNA bands were visualized by taking a picture with a Polaroid Land camera. H, the size markers (Kbp) generated by digestion of lambda phage DNA with HindIII; L. 123 ladder.
(B). Hybridization of the gel with potato CaM cDNA probe.
To facilitate characterization of the genes, the EcoRI fragments and HindIII fragments of these phages were subcloned into a phagemid pBluscript II KS+ and position of the CaM gene in the plasmid clones was identified by hybridizing various restriction fragments with the potato cDNA probe. Both CaM-1 and CaM-2 genes were sequenced and compared with the sequence of cDNA clones of other plant species. It was revealed that an intron disrupts the 25th codon of the CaM genes.
Amino acid sequence was deduced from the DNA sequence of the rice CaM genes (Fig. 2). Between two rice CaMs there is only one difference at the tenth amino acid from the COOH terminus. The sequence of rice CaM-1 is identical to a barley CaM and only one amino acid different from wheat and two from Arabidopsis or alfalfa. Potato CaM is the most diverged from the other plant CaM. Unlike the high conservation of the amino acid sequences, however, DNA sequence homology was much less. The rice CaM is 84% homologous to a barley clone and 78% to a potato gene. Much of the mismatch occurred in the third position of each codon due to different codon usage.
Fig. 2. Comparison of CaM sequences. Asterisks indicate Ca++ binding amino acid and underlined are is Ca++ binding domain.
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