The University of Western Ontario

Characterization and expression of a gene encoding a third member of the 18kDa HSP family in an inbred
--Manish Raizada, David B. Walden and Burr G. Atkinson

All organisms possess genes whose expression is up-regulated by heat shock/stress and code for a group of proteins referred to as the heat shock proteins (HSPs). Unlike animal HSPs, the majority of HSPs synthesized in plants appear to consist of a complex group of low molecular weight proteins with Mrs ranging from 15,000 to 30,000. Although the size and complexity of the low molecular weight HSPs vary among plant species, proteins of approximately 18kDa comprise the most prominent HSP class in maize, and, much like their counterparts in other organisms, are considered to be encoded from a complex, multigene family. In previous reports (Goping et al., MNL64:79-80, 1990; Plant Mol. Biol. 16:699-711, 1991), we described the genetic information from cDNAs encoding two different members of this family (cMHSP18-9 and cMHSP18-3). In this communication, we report the isolation and characterization of cDNA and genomic clones encoding information for a third member of this HSP family (g/c MHSP18-1).

A comparison of the sequence in the genomic clone with that obtained from the cDNA clone establishes that no introns are present in the gene encoding this 18kDa HSP. The 5' untranscribed region of gMHSP18-1 contains three, paired (decanucleotide), exact heat shock element (HSE) consensus sequences (all within an area of 62 nucleotides), and two putative TATA boxes. One TATA box is located 44 nucleotides upstream from the most 5' HSE and the other is located 18 nucleotides downstream from the most 3' HSE. The cDNA clone (cMHSP18-1) is 780 nucleotides long and contains a 465 nucleotide open reading frame (ORF) terminated by a TGA codon, flanked by a 5' untranslated region (UTR) of 58 nucleotides and a 3' UTR of 257 nucleotides containing a complete, animal-like polyadenylation signal sequence, AATAAA, 21 nucleotides upstream from the polyadenylated site.

A comparison of cMHSP18-1 with the two other 18kDa HSPs in this same maize inbred reveals that although the 3' and 5' UTRs share little identity (34-54%), the ORF of cMHSP18-1 shares 94.6% identity with the ORF in the other 18kDa HSP mRNAs. Analyses of the amino acid sequence derived from the putative ORF of cMHSP18-1 predict that its protein product contains 154 amino acids, and has an Mr of 17,027 and an isoelectric point (pI) of 8.4. Although the Mr and pI predicted for the protein of cMHSP18-1 result in a protein slightly smaller and much more basic than the other characterized 18kDa HSPs in this inbred, the derived amino acid sequence of cMHSP18-1 shares 93% identity with the one derived from cMHSP18-3 and 89% identity with the one derived from cMHSP18-9.

Northern and dot blot hybridization analyses, using a DNA fragment specific (as shown by Southern hybridization analyses) for cMHSP18-1 (see Atkinson et al., this volume) with RNAs isolated from plumules and radicles of control (25 C) and heat-shocked (41 C) 5-day-old maize seedlings, substantiate that the expression of mRNA transcripts encoded from this gene are up-regulated during heat shock. Two-dimensional (IEF/SDS) polyacrylamide gel electrophoretic separation of the protein product transcribed and translated from cMHSP18-1, and of the protein product translated from hybrid-selected mRNAs, demonstrate that the nucleotide sequence in the ORF of g/cMHSP18-1 represents a gene which encodes another member of the 18kDa HSP multigene family in this inbred line. 

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