Genes upregulated during flagellar regeneration in the scaly green alga Scherffelia dubia: identification of the first scale-associated protein, a novel protein.

Abstract

The flagella of the unicellular green alga Scherffelia dubia are entirely covered with layers consisting of thousands of discrete structures called scales. This complex covering represent a primitive form of extra cellular matrix which was developed before the land plants evolved from the green algae. Recent studies indicated that 2-keto sugar acid-containing scaly algae might be the ancestors of land plants. The flagellar scales of S. dubia are mainly composed of rare 2-keto sugar acids to which about twenty glycoproteins associate. They are produced in the Golgi apparatus and synthesized when new flagella are growing. The ultrastructure and carbohydrate composition of S. dubia's flagellar scales have been studied in great detail but not the scale associated proteins (SAP). In the present thesis SAPs were characterised and the isolation of a 2,4 kb cDNA corresponding to the C-terminal end of a SAP of 98 kDa was performed successfully. The sequence of the SAP98 cDNA indicated that this protein is not similar to any other known protein. It was also observed that the SAP98 gene was upregulated during experimentally-induced flagellar regeneration. Since the procedure used to isolate the SAP98 cDNA could not be successfully repeated for other SAPs, we isolated genes which are upregulated during flagellar regeneration in Scherffelia dubia by constructing a subtracted cDNA library (flagellar regenerating cells � interphase cells) using suppression subtraction hybridization (SSH). The flagellar regeneration-specific cDNA library suffered from significant redundancy and rRNA contamination. A significant proportion (55%) of the contigs could not be identified by similarity searches. It was estimated than 5% of the contigs of the library corresponded to upregulated genes and 35 contigs were identified as potential candidate for flagellar regeneration-upregulated genes, of which 24 might represent novel genes. The SAPs were not found in this library, but two genes coding for proteins involved in the scale polysaccharides biosynthesis, GDP-mannose-3,5-epimerase and UDP-glucose dehydrogenase, were present