POLYMERASE CHAIN-REACTION AS A TOOL FOR DEVELOPING STRESS PROTEIN PROBES

Because of the high degree of evolutionary conservation of stress prot eins, potential exists for the development of nucleic acid probes from particular species that could be used to monitor stress-related chang es in mRNA abundance. The polymerase chain reaction (PCR) is a powerfu l tool that can be applied to the generation of these probes, provided that primer sequences can be identified that specifically amplify seq uences of interest from a wide variety of organisms. We identified suc h sequences from multiple alignments of published chaperonin and stres s-70 sequences, and tested their ability to amplify appropriately size d fragments from genomic DNA from a variety of vertebrates and inverte brates. Although no primer pair could be used successfully with all sp ecies, we were able to derive specific products from most species by t esting different pairs. One primer pair for chaperonin proved particul arly 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 o btained from the rotifer Brachionus plicatilis and determined by phylo genetic analysis of the inferred protein product that the product obta ined is most likely derived from a rotifer DNA template. Finally, we s how that this product can be used to detect changes in abundance of ho mologous mRNA in heat-stressed rotifers. We suggest that this approach may prove useful not only in the context of development of hybridizat ion probes for stress proteins, but also for designing peptides to be used for generation of specific antibodies, as well as for obtaining p robes for other stress-regulated genes that are less conserved than th e classical stress proteins.