A DROSOPHILA MELANOGASTER Strain From Sub-Equatorial Africa Has Exceptional Thermotolerance But Decreased Hsp70 Expression
Olga G. Zatsepina1,
Vera V. Velikodvorskaia1,
Vasilii B. Molodtsov1,
David Garbuz1,
Daniel N. Lerman2,
Brian R. Bettencourt3,
Martin E. Feder2,3,* and
Michael B. Evgenev1
1 Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, Vavilov Street 32, 117984 Moscow, Russia,
2 The Committee on Evolutionary Biology and
3 Department of Organismal Biology and Anatomy, The University of Chicago, 1027 East 57th Street, Chicago, IL 60637, USA
View larger version (25K):
[in a new window]
|
Fig. 1. Basal and inducible thermotolerance in the Oregon R (OR25), T25 and T32 strains of Drosophila melanogaster. Batches of 4-day-old adults were exposed to the indicated temperatures for 30min either with or without a heat pretreatment (PT) as indicated. Means are plotted ±1 S.E.M. (N=2–5), which the plotted points often obscure. All axes use identical scales to facilitate comparison. (A) Thermotolerance in flies without pretreatment. (B–D) Thermotolerance of the three strains with and without a pretreatment. In B–D, without-pretreatment data are replotted from A.
|
|
View larger version (26K):
[in a new window]
|
Fig. 2. Effects of heat-shock temperature and time after heat shock on normalized relative Hsp70 concentration in lysates of whole 4-day-old flies of the Oregon R (OR25), T25 and T32 strains of Drosophila melanogaster. Inset (top right): effects of heat-shock temperature on Hsp70 levels 1h after a 30min heat shock. Results are expressed relative to the signal obtained with a standard derived from heat-shocked D. melanogaster tissue culture cells (see Materials and methods). Each point is the mean of 5–9 independendent measurements (except for OR25 at 34°C, for which N=3). Means are plotted ±1 S.E.M., which the plotted points often obscure. The arrow in the inset indicates a mean for T32 strain flies reared at 25°C. All axes use identical scales to facilitate comparison.
|
|
View larger version (25K):
[in a new window]
|
Fig. 3. Immunoblots of Hsp70 (detected with antibody 7FB) and all Hsp70 family members (detected with antibody 7.10) in salivary glands of third-instar larvae of the Oregon R (OR25), T25 and T32 strains of Drosophila melanogaster undergoing various temperature treatments. The blots were stripped and reprobed for actin as a standard for loading. DAB, detection by 3,3'-diaminobenzidine; ECL, detection by chemiluminescence.
|
|
View larger version (19K):
[in a new window]
|
Fig. 4. Effects of strain, culture temperature and heat shock on hsp70 mRNA levels.
|
|
View larger version (75K):
[in a new window]
|
Fig. 5. Proteins synthesized in control conditions and during the hour after a 20min 39°C heat shock in salivary glands of OR25 and T25 strain larvae, as indicated by L-[35S]methionine labeling, two-dimensional gel electrophoresis and autoradiography. Arrows indicate the positions of actin (a) and of identified or presumptive heat-shock proteins of interest. 27, Hsp27; 68, Hsp68; 70, Hsp70; 83, Hsp83; ‘70’ indicates the position on the gel where Hsp70 should be detected. b, d, e, presumptive small Hsps; c, unknown presumptive Hsp.
|
|
View larger version (91K):
[in a new window]
|
Fig. 6. Proteins present in control conditions and 1 and 3h after a 30min 37.5°C heat shock in salivary glands of OR25 larvae and of T32 strain larvae acclimated for 3 days at 25°C, as detected by silver staining and immunoblotting of two-dimensional electrophoresis gels. (A,B) Silver staining of the Hsp70 region in OR25 and T32 strains, respectively. 70c, Hsp70 cognates. (C,E) Hsp70 family members, as detected by antibody 7.10, in OR25 and T32 strains, respectively. Note the difference in Hsp68 (68) accumulation in the two strains and in the protein positioned immediately underneath the major Hsp70 spot (low MW). (D,F) Actin, as detected by silver staining, in OR25 and T32 strains, respectively.
|
|
View larger version (46K):
[in a new window]
|
Fig. 7. Restriction digests of genomic DNA of the OR25, T25 and T32 strains of Drosophila melanogaster probed with the ClaI–BamHI fragment of hsp70. Note the differences among strains.
|
|
View larger version (36K):
[in a new window]
|
Fig. 8. Polymorphism at the 87A7 and 87C1 hsp70 gene clusters. (A) PCR products from individual flies differing at the 87A7 and 87C1 hsp70 gene clusters. Left-hand lane, DNA size markers; other lanes, PCR products from single flies of each genotype as indicated. (B) A 265bp H.M.S. Beagle long-terminal repeat (LTR) is present in the 87A7 intergenic region of the T strains. The insertion lies 391bp upstream of the 56H8/122 insertion/deletion polymorphism (triangle, 142bp; see text). The nucleotide sequence begins 161bp upstream of the H.M.S. Beagle insertion. The remainder of the 87A7 intergenic region was not sequenced: its size ranges from 1.6kb in 122-type clusters to 3.6kb in 56H8-type clusters (Bettencourt, 2001; Ish-Horowicz et al., 1979; Mason et al., 1982). (C) Site of insertion of H.M.S. Beagle element showing duplicated host DNA (bold). (D) A 1.4kb fragment corresponding to the 3' end of the Jockey element is inserted 107bp upstream of the hsp70Ba transcription start site in the T strains. The orientation of Jockey is inverted with respect to the hsp70Ba gene, with the oligo(dT) at the distal end of the fragment. The insertion intervenes between HSE 2 and HSE 3, displacing HSE 3 and HSE 4 as well as three GAGA elements (hatched boxes; defined as the pentamer consensus sequence GAGAG; Omichinski et al., 1997; Wilkins and Lis, 1997). (E) Site of insertion of Jockey. Duplicated host DNA (bold) contains one of the putative GAGA elements. (t)n, reverse complement of poly(A) tail.
|
|
View larger version (24K):
[in a new window]
|
Fig. 9. Analysis of heat-shock element (HSE) binding activity in OR25 and T32 flies reared at 25°C. The latter strain yielded the same results as for T25 and T32 flies reared at 32°C; data not shown. (A) Effect of heat shock and identification of specific HSE complexes (A–C) in the OR25 strain. Lane 1, control (25°C); lane 2, after a 30min 37.5°C heat shock; lane 3, a 30min 37.5°C heat shock plus anti-heat-shock-factor (HSF) antibody, with supershift implicating HSF as a component of complex 1; lane 4, a 30min 37.5°C heat shock plus anti-Ku autoantigen antibody, with supershift implicating Ku autoantigen as a component of complex 2; lane 5, a 30min 37.5°C heat shock plus 200-fold molar excess of unlabeled HSE. This image is a composite of several gels. (B) Effect of heat-shock intensity and recovery time on HSE complexes in the two strains.
|
|
View larger version (17K):
[in a new window]
|
Fig. 10. Hypothesized effects of evolution at moderately high temperatures on the Hsp70 versus temperature norm of reaction. The solid line represents an idealized norm of reaction for wild-type Drosophila melanogaster reared at typical culture temperatures, 25°C or below, and approximates diverse data for Hsp70 expression and heat-shock factor (HSF) activation in this species (Bettencourt et al., 1999; Dahlgaard et al., 1998; Feder et al., 1997; Lerman and Feder, 2001; Lindquist, 1980). Given this norm of reaction, D. melanogaster cultured at a constant 28°C (Cavicchi 28°C lines; Bettencourt et al., 1999) or 31–32°C (T strain; T32) would constitutively undergo low levels of Hsp70 expression, incurring the deleterious consequences of Hsp70 (see Discussion) but none of the benefits realized only at higher temperatures. It is suggested that evolution at these moderately high temperatures acts to modify the norm of reaction (broken line) so that heat shocks at temperatures above 35°C result in lower concentrations of Hsp70 than in the wild type.
|
|
© The Company of Biologists Ltd 2001
