Evolution and human tissue expression of the Cres/Testatin subgroup genes, a reproductive tissue specific subgroup of the type 2 cystatins.

The cystatin family comprises a group of generally broadly expressed protease inhibitors. The Cres/Testatin subgroup (CTES) genes within the type 2 cystatins differs from the classical type 2 cystatins in having a strikingly reproductive tissue-specific expression, and putative functions in reproduction have therefore been discussed. We have performed evolutionary studies of the CTES genes based on gene searches in genomes from 11 species. Ancestors of the cystatin family can be traced back to plants. We have localized the evolutionary origin of the CTES genes to the split of marsupial and placental mammals. A model for the evolution of these genes illustrates that they constitute a dynamic group of genes, which has undergone several gene expansions and we find indications of a high degree of positive selection, in striking contrast to what is seen for the classical cystatin C. We show with phylogenetic relations that the CTES genes are clustered into three original groups, a testatin, a Cres, and a CstL1 group. We have further characterized the expression patterns of all human members of the subfamily. Of a total of nine identified human genes, four express putative functional transcripts with a predominant expression in the male reproductive system. Our results are compatible with a function of this gene family in reproduction.

MAP20, a microtubule-associated protein in the secondary cell walls of hybrid aspen, is a target of the cellulose synthesis inhibitor 2,6-dichlorobenzonitrile.

We have identified a gene, denoted PttMAP20, which is strongly up-regulated during secondary cell wall synthesis and tightly coregulated with the secondary wall-associated CESA genes in hybrid aspen (Populus tremula x tremuloides). Immunolocalization studies with affinity-purified antibodies specific for PttMAP20 revealed that the protein is found in all cell types in developing xylem and that it is most abundant in cells forming secondary cell walls. This PttMAP20 protein sequence contains a highly conserved TPX2 domain first identified in a microtubule-associated protein (MAP) in Xenopus laevis. Overexpression of PttMAP20 in Arabidopsis (Arabidopsis thaliana) leads to helical twisting of epidermal cells, frequently associated with MAPs. In addition, a PttMAP20-yellow fluorescent protein fusion protein expressed in tobacco (Nicotiana tabacum) leaves localizes to microtubules in leaf epidermal pavement cells. Recombinant PttMAP20 expressed in Escherichia coli also binds specifically to in vitro-assembled, taxol-stabilized bovine microtubules. Finally, the herbicide 2,6-dichlorobenzonitrile, which inhibits cellulose synthesis in plants, was found to bind specifically to PttMAP20. Together with the known function of cortical microtubules in orienting cellulose microfibrils, these observations suggest that PttMAP20 has a role in cellulose biosynthesis.