Qin-Bao Li, James V. Anderson, and Charles 1. Guy*. Program for ... The 5' and 3' oligonucleotides were. GGT ATT ... 2220-nucleotide sequence with a 3' poly(A) tail. It contains ... Neven LG, Haskell DW, Guy CL, Denslow N, Kliein PA, Green.
Plant Physiol. (1994) 105: 457-458
Plant Gene Register
A cDNA Clone Encoding a Spinach 70-Kilodalton Heat-Shock Cognate' Qin-Bao Li, JamesV. Anderson, and Charles 1. Guy*
Program for Plant Molecular and Cellular Biology, Department of Environmental Horticulture, lnstitute of Food and Agricultural Science, University of Florida, Gainesville, Florida 3261 1-051 2
The genes for the Hsp70s of plants, in addition to being under developmental control, are activated by a variety of diverse stresses in addition to that of a heat shock (Neumann et al., 1989). For example, wounding, water deficits, ABA, arsenite, or cold can induce the synthesis of the Hsp70s (Heikkila et al., 1984; Lin et al., 1984; Neven et al., 1992). Some members of this gene family are expressed under normal growth conditions as well as being stress inducible. Hsp70s have been identified in a number of cell compartments, including the cytosol, ER, mitochondria, and chloroplasts. The induction by various apparently unrelated stresses and chemical agents suggests that Hsp70 could play a general role in stress adaptation (Vierling, 1991). Members of the 70-kD heat-shock gene family are highly conserved across a wide range of organisms. As such, the Hsp70s have been implicated in a variety of essential cellular processes, ranging from DNA replication to protein folding and transport. In an effort to learn more about the evolution and possible functions of higher plant Hsp70s, we isolated a cDNA clone encoding a putative cytosolic member (Hscl) of this family of proteins. This clone was obtained from a cDNA library prepared from poly(A)+ RNA isolated from coldacclimated spinach leaf tissue. The library was screened by in situ plaque hybridization using a 597-bp hybridization probe prepared from total cDNA by PCR using primers to highly conserved regions in the N-terminal ATPase domain (Flaherty et al., 1990). The 5' and 3' oligonucleotides were GGT ATT GA(CT) CTC GGT ACC AC(CT) TAC (CG24) and TCC ACC ACC AAG GTC GAA GAC (CG14), respectively. The characteristic features of the cDNA clone are described in Table I. Double-stranded dideoxy sequencing resulted in a 2220-nucleotide sequence with a 3' poly(A) tail. It contains an open reading frame of 1941 bp. It can encode a polypeptide of 647 amino acid residues with a predicted molecular m a s of 70,876 D. The 647 amino acid residues exhibit a 97.8% identity with SCE70 of spinach (Ko et al., 1992) and a 94.3% identity with the tomato HSC-2 Hsp70 (Lin et al., 1991). SCE70 and Hscl may represent sequences for the same gene, but the sequence for Hscl conforms more closely to that of typical Hsp70 gene sequences. The spinach Hscl
Table 1. Characteristics of a spinach cDNA clone (HSCI)
Organism: Spinacia oleracea L. cv Bloomsdale. Gene Product: Hsp70. Clone Type: cDNA clone, 2220-bp Pstl and Xhol fragment in pBluescript SK
+I-. Cenome Location: Nuclear genome; chromosome location unknown. Cene Copy Number: A member of Hsp70 gene family. Gene Function: Encodes for cytosolic Hsc70, which functions as a molecular chaperone. Source:
cDNA library in XUni-Zapll constructed from poly(A)+R N A
from cold-acclimated spinach leaf tissue. Sequencing Strategy: Dideoxy sequencing of both strands using synthetic primers with Applied Biosystems automated sequencer. Method of Identification: Screening with 597-bp PCR product; cDNA library DNA was amplified using Taq polymerase for PCR; CG24 and CG14 as primers. Amino acid sequence comparison with CenBank, EMBL, PIR, and SwissProt sequence data bases using GCG. Expression Characteristics: The Hscl m R N A is expressed at normal growth temperatures and is slightly upregulated during heat shock and at low temperatures. Structural Features of the Protein: An open reading frame of 1941 bp encodes a 647-amino acid protein with a predicted molecular m a s of 70,876 D and an isoelectric point of 5.061, Antibodies: Monoclonal 5B7 is avaiiable.
exhibits the most similarity with the ssal-4 family of Hsp70s in yeast (Craig et al., 1993). Hsc70s and Hsp70s in plants and other systems are known to bind to ATP and to use the energy of ATP hydrolysis for their functions. The ATP-binding domain of the bovine heatshock cognate protein has been characterized by x-ray crys-
Financia1 support for this work was provided by the National Science Foundation (DCB-9017625).This is Florida Agricultural Experiment Station Joumal Series No. R-03512. * Corresponding author; fax 1-904-392-3870.
Abbreviations: Hsc70, 70-kD heat-shock cognate; Hsp70, 70-kD heat-shock orotein. 457
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tallography (Flaherty et al., 1990). A calmodulin-binding motif KRAVRRLRTACERAKRTLSSS has been noted for mouse, rat, and human Hsp70s (Stevenson and Calderwood, 1990). The Hscl protein contains a related sequence at positions 263 to 283. Received November 23, 1993; accepted December 20, 1993. Copyright Clearance Center: 0032-0889/94/105/0457/02. The GenBank accession number for the sequence reported in this article is L26243. LITERATURE CITED
Craig EA, Gambill BD, Nelson RJ (1993) Heat shock proteins: molecular chaperones of protein biogenesis. Microbiol Rev 57: 402-414 Flaherty KM, Deluca-Flaherty C, McKay DB (1990) Three-dimensional structure of the ATPase fragment of a 70 K heat-shock cognate protein. Nature 346 623-628 Heikkila JJ, Papp JET, Schultz GA, Bewley JD (1984) Induction of
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