Worm Breeder's Gazette 10(2): 135
These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author.
Our studies of the genes encoding 16 kilodalton heat shock proteins ( hsp16's) in C. elegans have resulted in the characterization of two hsp16 loci. One locus, hsp16-1/48, encodes a pair of divergently transcribed genes, hsp16-1 and hsp16-48, which are exactly duplicated in an inverted orientation, forming a 4 kb inverted repeat containing four hsp16 genes. This locus has been assigned to a 368 kb contig which maps to chromosome V, by John Sulston and Alan Coulson. The other locus, which has not yet been mapped, codes for another pair of divergently arranged genes, hsp16-2 and hsp16-41. All four genes are under tight heat shock control. However, Northern blot analysis had shown that there is approximately 15-fold more mRNA from locus 16-2/41 than from 16-1/48, on a per gene basis (Jones et al., 1986). To explore the molecular basis for this difference in expression levels, we have used locus-specific hybridization probes to measure the mRNA content of embryos under a variety of heat shock conditions. Nuclear run-on assays demonstrate that the difference in expression of the hsp16 loci is not primarily due to transcription rates - the rate of hsp16-2 transcription in vitro is at most two-fold higher than that of hsp16-1. In contrast, the two loci differ dramatically in the duration of their active phase and in the kinetics of inactivation during a prolonged heat shock. When embryos are placed abruptly at 33 C, the induction of both hsp16-1 and hsp16-2 message is detectable within minutes, and both transcripts accumulate at similar rates. However, hsp16-1 transcript levels begin to decline in less than one hour, while those of hsp16-2 continue to increase rapidly for an additional 40 min. before beginning to decrease; under these conditions, the peak mRNA levels of the two genes differ by four fold. When embryos are heated gradually (1 C per 15 min.) to 33 C, the difference between loci is even more marked - the level of hsp16-2 message continues to increase at a high rate for more than 30 min. after hsp16-1 mRNA reaches a plateau, and the ratio of 16-2:16-1 message reaches 7:1, or 14:1 on a per gene basis. Although we cannot measure mRNA half lives directly due to the impermeability of the embryos to inhibitors of transcription, the levels of hsp16-1 and hsp16-2 message drop in parallel during recovery from heat shock, suggesting that the two messages have similar stabilities. Thus the major reason for the differences in expression of the two loci seems to be due to a difference in the shutdown phase of the heat shock response: although both loci are transiently activated by heat shock, locus 16-1/48 is more rapidly inactivated than locus 16-2/41. The hsp16 genes are inducible at all stages of development, but the induced levels of message are highest in L1 and lowest in adults. The difference in expression between the two loci is also greatest at L1. The thermotolerance of embryos was examined - abrupt 30 min. heat shocks had little effect on survival until 36 C was reached, and at 38 C the survival was only 30%. If embryos were raised gradually to 38 C, and kept at that temperature for 30 min., survival jumped to approximately 90%. Likewise, if embryos were pre-treated at 32 C for 30 min., then subjected to 38 C for 30 min., a greater than two-fold increase in survival was seen, relative to the abrupt heat shock. Pre- induction of the heat shock response thus greatly enhances survival in C. elegans as in other organisms, and the effect is most significant for a gradual temperature increase, a situation which is undoubtedly much more frequently seen by worms living outside in the real world.