The intertidal copepod Tigriopus japonicus small heat shock protein 20 gene (Hsp20) enhances thermotolerance of transformed Escherichia coli.
To understand the role of the Tigriopus japonicus Hsp20 gene, we isolated this gene from a whole body cDNA library and found two heat shock factor elements at the 5'-UTR. The transformed bacteria containing Tigriopus Hsp20 showed thermotolerance against heat shock (54 degrees C) with different ranges of time. The Tigriopus Hsp20 gene is comprised of 174 amino acid residues and shows similarity to Caenorhabditis elegans (27% identity), silkworm (24.1% identity), moth (24.1% identity), Mexican tetra (19.5% identity), zebrafish (19.5% identity), and spiny dogfish (17.2% identity) genes, but shows more similarity in the C-terminal region that contains an alpha-crystallin domain. Protein motifs such as an N-glycosylation site (67-70 NKSE) and a casein kinase II phosphorylation site were found in Tigriopus Hsp20. The genomic structure of the Tigriopus Hsp20 gene did not contain introns. To characterize the biochemical characteristics of the Tigriopus Hsp20 protein, we expressed Tigriopus Hsp20 in Escherichia coli and purified the soluble protein via 6x His-tag chromatography. To analyze the gene expression of Tigriopus Hsp20 against environmental stresses (e.g., water temperature and salinity), we performed a semi-quantitative reverse transcriptase-polymerase chain reaction (RT-PCR). On exposure to different salinities, significant change in the expression of Tigriopus Hsp20 was not observed. However, upon heat shock (30 degrees C), Tigriopus Hsp20 expression was significantly increased, but in the case of cold shock (4 or 10 degrees C), expression was likely downregulated. These findings provide a better understanding of cellular protection mechanisms against environmental stress such as heat shock.
Seo
Jung Soo
JS
Department of Molecular and Environmental Bioscience, Graduate School, Hanyang University, Seoul 133-791, Republic of Korea.
Lee
Young-Mi
YM
Park
Heum Gi
HG
Lee
Jae-Seong
JS
D020121:5' Untranslated Regions; D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015153:Blotting, Western; D012831:Bombyx; D017173:Caenorhabditis elegans; D047390:Casein Kinase II / Q000378:metabolism; D003001:Cloning, Molecular; D033342:Copepoda / Q000378:metabolism*; D017931:DNA Primers / Q000737:chemistry; D018076:DNA, Complementary / Q000378:metabolism; D004284:Dogfish; D004591:Electrophoresis, Polyacrylamide Gel; D004777:Environment; D004926:Escherichia coli / Q000378:metabolism*; D020224:Expressed Sequence Tags; D005786:Gene Expression Regulation; D015723:Gene Library; D006031:Glycosylation; D050886:HSP20 Heat-Shock Proteins / Q000737:chemistry / Q000502:physiology*; D006360:Heat-Shock Proteins / Q000737:chemistry; D006358:Hot Temperature; D008969:Molecular Sequence Data; D009036:Moths; D016366:Open Reading Frames; D010766:Phosphorylation; D010802:Phylogeny; D017434:Protein Structure, Tertiary; D012333:RNA, Messenger / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017386:Sequence Homology, Amino Acid; D013696:Temperature; D013997:Time Factors; D015027:Zebrafish
D020121:5' Untranslated Regions; D017931:DNA Primers; D018076:DNA, Complementary; D050886:HSP20 Heat-Shock Proteins; D006360:Heat-Shock Proteins; D012333:RNA, Messenger; D011994:Recombinant Proteins; D047390:Casein Kinase II