Plant dnaj homologue: molecular cloning, bacterial expression, and expression analysis in tissues of cucumber...

Proteins homologous to the bacterial dnaJ protein have been implicated as molecular chaperones in different compartments of eukaryotes. A plant equivalent is now described in various cucumber tissues. Using a cDNA library constructed from poly(A)-rich RNA of cotyledons of etiolated seedlings and dnaJ-specific probes, we isolated clones encoding full-length cDNAs: clone CSDNAJ-1 contained a 1.7-kb insert with a single open reading frame of 413 amino acid residues encoding a protein of M(r) 46, 012; pCSDNAJ-2 differed from pCSDNAJ-1 by an additional nucleotide and predicted a protein of M(r) 26, 104. Peptides of similar sizes were observed when the cDNA information was expressed by in vitro transcription and translation or expressed in vivo in bacteria. Using mRNA isolated by hybrid selection for in vitro translation we observed the formation of CSDNAJ-1 protein. Comparisons revealed that the similarity in primary structure between CSDNAJ-1 protein and dnaJ homologues in eukaryotes is most pronounced in the N-terminal region. The high degree of identity (39%) of CSDNAJ-1 protein with the yeast dnaJ homologue YDJ1 protein suggests that csdnaJ proteins assist intracellular protein transfer. The predicted CSDNAJ-1 protein is characterized by a fourfold repetition of the motif CXXCXGX(G) and by a C-terminus -QCAQQ. Analysis of gene expression at the level of mRNA showed that the putative dnaJ protein is expressed in cucumber seedlings in all tissues, but exceedingly high in hypocotyledons and roots. The level of dnaJ mRNA was transiently increased by a factor of 1.5-2.0 when heat shock was applied to the seedlings.