Polymerase chain reaction as a tool for developing stress protein probes

Because of the high degree of evolutionary conservation of stress proteins, potential exists for the development of nucleic acid probes from particular species that could be used to monitor stress related changes in mRNA abundance The polymerase chain reaction (PCR) is a powerful tool that can be applied to the generation of these probes, provided that primer sequences can be identified that specifically amplify sequences of interest from a wide variety of organisms We identified such sequences from multiple alignments of published chaperomn and stress‐70 sequences, and tested their ability to amplify appro priately sized fragments from genomic DNA from a variety of vertebrates and invertebrates Although no primer pair could be used successfully with all species, we were able to derive specific products from most species by testing different pairs One primer pair for chaperomn proved particularly useful Products were obtained from all tested species, and with a single exception (human), these primers appeared to amplify a single copy sequence We determined the nucleotide sequence of the product obtained from the rotifer Brachionus plicatilis and determined by phylogenetic analysis of the inferred protein product that the product obtained is most likely derived from a rotifer DNA template Finally, we show that this product can be used to detect changes in abundance of homologous mRNA in heat stressed rotifers We suggest that this approach may prove useful not only in the context of development of hybridization probes for stress proteins, but also for designing peptides to be used for generation of specific antibodies, as well as for obtaining probes for other stress regulated genes that are less conserved than the classical stress proteins