Suppressive Subtractive Hybridization of and Differences in Gene Expression Content of Calcifying and Noncalcifying Cultures ofEmiliania huxleyiStrain 1516

Abstract

The marine coccolithophorid Emiliania huxleyi is a cosmopolitan alga intensely studied in relation to global carbon cycling, biogeochemistry, marine ecology, and biomineralization processes. The biomineralization capabilities of coccolithophorids have attracted the attention of scientists interested in exploiting this ability for the development of materials science and biomedical and biotechnological applications. Although it has been well documented that biomineralization in E. huxleyi is promoted by growth under phosphate-limited conditions, the genes and proteins that govern the processes of calcification and coccolithogenesis remain unknown. Suppressive subtractive hybridization (SSH) libraries were constructed from cultures grown in phosphate-limited and phosphate-replete media as tester and driver populations for reciprocal SSH procedures. Positive clones from each of the two libraries were randomly selected, and dot blotting was performed for the analysis of expression patterns. A total of 513 clones from the phosphate-replete library and 423 clones from the phosphate-limited library were sequenced, assembled, and compared to sequences in GenBank using BLASTX. Of the 103 differentially expressed gene fragments from the phosphate-replete library, 34% showed significant homology to other known proteins, while only 23% of the 65 differentially expressed gene fragments from the phosphate-limited library showed homology to other proteins. To further assess mRNA expression, real-time RT-PCR analysis was employed and expression profiles were generated over a 14-day time course for three clones from the phosphate-replete library and five clones from the phosphate-limited library. The fragments isolated provide the basis for future cloning of full-length genes and functional analysis.Coccolithophorids are unicellular marine algae that produce a wide variety of highly sculpted calcium carbonate cell coverings. Calcification in some coccolithophorids occurs intracellularly and is thought to begin with the conversion of calcium and bicarbonate ions into crystals of calcium carbonate that are deposited in a defined array in association with an organic matrix (35). A coccolith vesicle developing from the Golgi apparatus provides the microenvironment that promotes the formation of the crystallographically intricate coccolith structures, which are subsequently extruded from the cell to form the coccosphere (7,20). The coccolith vesicle fuses with the plasma membrane to release the individual coccoliths. While calcification and coccolithogenesis are presumed to occur in a genetically controlled manner, the functional significance and genetic control of these complex structures and the variation in crystal shape, orientation, order, and connectivity that occurs across species are poorly understood The size and morphology of the calcite structures are as diverse as the coccolithophorids that produce them. Some of the calcite elements are simple rhombohedral units, while others appear as ornate oval platelets, extended spic...

Long Serial Analysis of Gene Expression for Gene Discovery and Transcriptome Profiling in the Widespread Marine Coccolithophore Emiliania huxleyi

“…The high number of unique and up-regulated tags in the ϪP library suggests that we may be observing the up-regulation of genes involved in calcification or calcium homeostasis (e.g., tag 11952 [calmodulin]) in addition to those involved in P scavenging. Substantial efforts have been made to identify the genes involved in calcification, and the most recent approaches have compared E. huxleyi EST libraries (40) or used suppressive subtractive hybridization with calcifying and noncalcifying cells (22). Of the five tags most highly expressed in our long SAGE ϪP library (Table 4), none clearly map to the most prevalent ESTs in the calcifying library analyzed by Nguyen et al (22).…”

“…Substantial efforts have been made to identify the genes involved in calcification, and the most recent approaches have compared E. huxleyi EST libraries (40) or used suppressive subtractive hybridization with calcifying and noncalcifying cells (22). Of the five tags most highly expressed in our long SAGE ϪP library (Table 4), none clearly map to the most prevalent ESTs in the calcifying library analyzed by Nguyen et al (22). Moreover, of the tags up-regulated or unique to the ϪP library, over 60 are orphan tags, which do not map to any of the genes identified to date in calcifying cells (22,40).…”

“…While genomic research with marine cyanobacteria is rapidly advancing our understanding of their role in the sea (12,27), there are few genome sequences (3), differential gene expression studies (2,22,37), and transcriptome analyses with eukaryotic marine algae. In the case of coccolithophores, fundamental gaps in our molecular-level understanding of calcification and even basic N and P scavenging mechanisms remain.…”

Gene expression profiles of the heterotrophic microalga Chlorella pyrenoidosa F-9

“…For the green alga Acetabularia acetabulum, expressed sequence tags were used to compare the adult phase and the juvenile phase with a technology that was similar to SSH (Henry et al, 2004). The SSH technique was used in the analysis of gene expression in calcifying and noncalcifying cultures of the marine microalga Emiliania huxleyi strain 1516 (Nguyen et al, 2005).…”

A coccolithophore concept for constraining the Cenozoic carbon cycle

“…Marsh, 2003;Nguyen et al, 2005), or the detailed photosynthetic mechanism of coccolithophores. Coupling of calcification with species-specific Rubisco specificity provides a tangible means to preserve the CO 2 /O 2 composition at the time of origin of photosynthetic phyla (Giordano et al, 2005;Tcherkez et al, 2006).…”