In previous reports (Goping et al., Plant Mol. Biol. 19:679-711, 1991; Atkinson et al., Develop. Genetics, in press; Raizada et al., this volume), we described the genetic information from genomic and cDNA clones encoding three different members of this family (designated as g/cMHSP18-1, g/cMHSP18-3 and cMHSP18-9) in an inbred line (Oh43). In this communication, we describe (1) the preparation of DNA fragments from these clones which contain nucleotide sequences which are either common to all of the characterized members of the 18kDa HSP family or specific for each of the 18kDa HSPs we characterized in this family, and (2) the use of these DNA fragments as probes to assess the expression of these genes during microsporogenesis and gametophyte development in Oh43.
We excised and subcloned, into pBluescript II SK-, a 0.342kb Pst/SpeI DNA fragment (scMHSP19-9-2) from the open reading frame (ORF) of cMHSP18-9 to use as a probe for a sequence common to all of the 18kDa HSP sequences we characterized in Oh43; it shares 90% identity with a sequence in both g/cMHSP18-1 and g/cMHSP18-3. We also excised and subcloned, into pBluescript II SK-, DNA fragments from the 3' untranslated regions (UTRs) of each of the 18kDa HSP sequences we characterized in Oh43, and used them as probes specific for each of these 18kDa HSP sequences. These specific probes include a 0.242kb BstXI/XhoI DNA fragment (scMHSP18-1-1) from the 3' UTR of cMHSP18-1, a 0.139kb HindIII/EcoRI DNA fragment (scMHSP18-3-3) from the 3' UTR of cMHSP18-3, and a 0.235kb SalI/EcoRI DNA fragment (scMHSP18-9-3) from the 3' UTR of cMHSP18-9. Southern blot hybridization analyses confirmed the commonality of the scMHSP18-9-2 probe and the specificity of each of the other probes.
Northern and dot blot hybridization analyses with these 18kDa HSP probes, using RNAs isolated from plumules and radicles of control (25 C) and heat-shocked (42 C) 5-day-old maize seedlings, confirmed their ability to recognize mRNAs encoding the 18kDa HSPs in this inbred. Since studies in our laboratories (Frappier et al., in Stress Protein and the Heat Shock Response, Cold Spring Harbor Press, p.53, 1991; Atkinson et al., Develop. Genetics, in press) and other laboratories (Dietrich et al., Plant Physiol. 96:1268-1271, 1991) have reported an enhanced expression and/or accumulation of the 18kDa HSP mRNAs in the male gametophyte of maize in the absence of heat stress, we elected to assess the levels of mRNAs encoding specific members of this 18kDa HSP family during microsporogenesis and gametophyte development in Oh43. Our results, from Northern and dot blot hybridization analyses, demonstrate that mRNA transcripts encoding different members of this 18kDa family are expressed and/or accumulate independently, in a stage-specific manner during microsporogenesis and male gametophyte development. For example, mRNA transcripts recognized by g/cMHSP18-1 are predominant early in microsporogenesis (mid-prophase I through meiosis II), those recognized by cMHSP18-9 are most abundant in the binucleate stage of the gametophyte, and those detected by g/cMHSP18-3 do not appear to accumulate significantly at any stage of microsporogenesis or gametophyte development. These observations imply that the stage-specific expression of genes encoding particular members of this family results from gene-specific regulation during these phases of male gametophyte development, rather than from an overall activation of the heat shock or stress response. Moreover, these results suggest that particular members of this 18kDa HSP family may serve specific functions in the normal development of maize meiotic cells and/or the male gametophytes they produce.
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