Heat-Shock Response in a Molluscan Cell Line: Characterization of the Response and Cloning of an Inducible HSP70 cDNA

• Published in: Journal of invertebrate pathology Vol. 70; no. 3; pp. 226 - 233
• Main Authors: Laursen, Jeffrey R., di Liu, Hong, Wu, Xiao-Jun, Yoshino, Timothy P.
• Format: Journal Article
• Language: English
• Published: Amsterdam Elsevier Inc 01.11.1997; Elsevier
• Subjects: Amino Acid Sequence; Animals; Base Sequence; Bge cell line; Biochemistry. Physiology. Immunology; Biological and medical sciences; Biomphalaria - cytology; Biomphalaria - genetics; Biomphalaria glabrata; cDNA; Cell Line; cloning; DNA, Complementary - genetics; embryonic cell line; Freshwater; Fundamental and applied biological sciences. Psychology; Gene Library; heat-inducible HSP70 gene; Heat-Shock Proteins - biosynthesis; Heat-Shock Response; HSP70 Heat-Shock Proteins - biosynthesis; HSP70 Heat-Shock Proteins - genetics; Invertebrates; Molecular Sequence Data; Mollusca; Physiology. Development; Sequence Analysis, DNA; Sequence Homology, Amino Acid; Biomphalaria glabrata; heat-shock response; embryonic cell line; heat-inducible HSP70 gene; cDNA; Bge cell line; cloning; Cell line; Pulmonata; Complementary DNA; Nucleotide sequence; Heat shock protein; Gastropoda; Invertebrata; Mollusca; Molecular cloning; Gene expression; Hyperthermia
• ISSN: 0022-2011; 1096-0805
• DOI: 10.1006/jipa.1997.4686

Sublethal heat-shock of cells of the Bge (Biomphalaria glabrataembryonic) snail cell line resulted in increased or new expression of metabolically labeled polypeptides of approximately 21.5, 41, 70, and 74 kDa molecular mass. Regulation of this response appeared to be at the transcriptional level since a similar protein banding pattern was seen upon SDS–PAGE/fluorographic analysis of polypeptides produced byin vitrotranslation of total RNA from cells subjected to heat shock. Using a yeast (Saccharomyces cerevisiae) 70-kDa heat-shock protein (HSP70) probe to screen a cDNA library from heat-treated Bge cells, we isolated a full-length cDNA clone encoding a putative Bge HSP70. The cDNA was 2453 bp in length and contained an open reading frame of 1908 bp encoding a 636-amino-acid polypeptide with calculated molecular mass of 70,740 Da. Comparison of a conserved region of 209 amino acid residues revealed >80% identity between the deduced amino acid sequence of Bge HSP70 and that of yeast (81%), the human blood flukeSchistosoma mansoni(for whichB. glabrataserves as intermediate host) (81%),Drosophila(81%), human (84%), and the marine gastropodAplysia californica(88%, 90%). In addition to the extensive sharing of sequence homology, the identification of several eukaryotic HSP70 signature sequences and an N-linked glycosylation site characteristic of cytoplasmic HSPs strongly support the identity of the Bge cDNA as encoding an authentic HSP70. Results of a Northern blot analysis, using Bge HSP70 clone-specific probes, indicated that gene expression was heat inducible and not constitutively expressed. This is the first reported sequence of an inducible HSP70 from cells originating from a freshwater gastropod and provides a first step in the development of a genetic transformation system for molluscs of medical importance.