BERGAMO, ITALY
Istituto Sperimentale per la Cerealicoltura
Phylogenetic analysis reveals that a maize member of the MSI/RbAp sub-family of WD-repeat proteins clusters in an evolutionary separate group --Lanzanova, C, Locatelli, S, Hartings, H, Rossi, V Members of the MSI/RbAp sub-family of WD-repeat proteins are widespread in eukaryotes and are part of a variety of multiprotein complexes involved in different biological pathways, including chromatin assembly, regulation of gene transcription, and cell division (reviewed in Verreault, A, Genes Dev 14: 1430-1438, 2000). Recently, we have identified and characterized a cDNA sequence from Zea mays encoding a homologue of the Retinoblastoma associated protein (ZmRbAp1). This gene shows structural and functional features common to the MSI/RbAp proteins, including the ability to bind acetylated histones H3 and H4, and to negatively regulate the Ras/cAMP pathway in yeast. During the molecular characterization of ZmRbAp1 we have identified two additional partial cDNAs (ZmRbAp2 and ZmRbAp3) that exhibit 81% and 96% nucleotide identity with ZmRbAp1, respectively. This finding, together with Southern analysis, which revealed a complex hybridization pattern, suggests that maize RbAp genes belong to a gene family. Because MSI/RbAp sub-family members have been found in different eukaryotes and because many organisms possess multiple copies of these genes, we performed a phylogenetic analysis to compare the MSI/RbAp amino acid sequences available in databank. We used BLASTP (scores > 70, p values < 0.05 Altschul, SF et al., J Mol Biol 215: 403-410, 1990) to search in the non-redundant peptide sequence database at the National Center for Biotechnology Information for proteins similar to human RbAp48, yeast MSI1 and ZmRbAp1. Eighteen amino acid sequences, among the most representative for different species, were aligned using ClustalW software (Thompson, JD et al., Nucl Acid Res 22: 4673-4680, 1994) and a tree construction was performed (neighbor joining method using MEGA software v.1.0; Kumar, S et al., Pennsylvania State University). Interestingly, two separate groups were identified (see Fig. 1). ZmRbAp1 clustered together with AtMSI4 and SlY1, suggesting a common origin for these proteins. The two additional ZmRbAp clones we have identified also belong to this group. A second major cluster contained MSI/RbAp members of Homo sapiens, Drosophila melanogaster and Saccharomyces cerevisiae; for these proteins a role in chromatin modification, histone assembly and binding to retinoblastoma protein has been reported. Particularly, it has been shown that mammalian MSI/RbAp components possess a partially distinct activity (reviewed in Verreault, A, Genes Dev 14:1430-1438, 2000).

Our analysis indicates that at least three copies of functionally related MSI/RbAp genes exist in maize and that these genes have evolved differently with respect to the best characterized members of the MSI/RbAp sub-family of WD-repeat proteins. Peptide microsequencing of a recently described acetyltransferase HATB-associated RbAp protein (Lusser, A et al., Nucl Acid Res 27: 4427-4435, 1999) revealed a low degree of similarity with ZmRbAp1. Altogether these findings suggest the presence in the maize genome of different MSI/RbAp members performing specific tasks, while maintaining other functions common to all members of this sub-family.

Figure 1. Phylogenetic tree based on the alignment of 18 members of the MSI/RbAp sub-family of WD-repeat proteins. ZmRbAp1 from Z. mays was aligned with 17 MSI/RbAp-like proteins from H. sapiens (HsRbAp46 and HsRbAp48), G. gallus (GgCAF-p48), X. laevis (XlRbAp48), D. melanogaster (Dmp55), C. elegans (Celin53; CeRBA1), L. esculentum (LeMSI1), A. thaliana (AtMSI1; AtMSI2; AtMSI3 and AtMSI4), S. latifolia (SlY1), S. pombe (SpCAF and SpWD-repeats), S. cerevisiae (ScMSI1 and ScHAT2). The lengths of the tree branches are proportional to the genetic distance. Bootstrap values based on 500 replicates supporting the branches at 75% cut-off value are indicated..
 
 


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