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CLONING, SEQUENCING AND EXPRESSION OF A HISTONE‐LIKE PROTEIN FROM THE PHOTOSYNTHETIC DINOFLAGELLATE GONYAULAX POLYEDRA
Author(s) -
Fagan F. T.,
Li J.F.,
Chudnovsky J.,
Rizzo P.J.,
Hastings J.W.
Publication year - 2000
Publication title -
journal of phycology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.85
H-Index - 127
eISSN - 1529-8817
pISSN - 0022-3646
DOI - 10.1046/j.1529-8817.1999.00001-64.x
Subject(s) - biology , dinoflagellate , biochemistry , histone , isoelectric point , gene , microbiology and biotechnology , botany , enzyme
The presence of small basic DNA‐binding proteins, or histone‐like proteins (HLPs), in dinoflagellate nuclei has been well documented. Genes for HLPs have been cloned from Crypthecodinium cohnii ( HCc 1 and 2) and Alexandrium fundyense ( HAf ) but their predicted protein sequences show no homology to histones from other eukaryotes or to bacterial HLPs. The precise role of these HLPs in dinoflagellate nuclei is uncertain; the HLP:DNA ratio is too low to facilitate packaging of the entire (very large) genome. In order to investigate the role of these proteins we have cloned a gene for a HLP from Gonyaulax polyedra ( HGp ). Unlike the genes from C. cohnii , the G. polyedra gene contains no introns. It codes for a protein with a predicted molecular mass of 10,985 Da, and an isoelectric point of 10.5. We over‐expressed this protein in Escherichia coli using a construct that expresses the full‐length protein with an N‐terminal histidine tag, and purified it by Nickel‐affinity and cation‐exchange chromatography. While the predicted molecular mass of the fusion protein is 12,973 Da, similar to sizes observed for acid‐soluble G. polyedra nuclear proteins, the relative mobility on denaturing SDS‐polyacrylamide gel electrophoresis is 20,000 Da. We attribute the slower mobility in denaturing page to the basicity of the protein and predict that the relatively faster mobility exhibited by native HGp is due in part to post‐translational modification of basic residues. Such modifications have been observed in histones and play an important role in the regulation of transcription in eukaryotes, a function that HLPs may assume in dinoflagellates. This possibility is being investigated.