Fig 1.
A. General arrangement of the HSPA1 cluster in mammals. B. The level of transcription activity of individual hsp70 gene promoters comprising the cluster based on the measurement of transient luciferase luminescence driven by constructs carrying different human and camel hspA1 promoters. (i)—HSPA1A, (ii)—HSPA1B and (iii)—HSPA1L. The signal levels are shown as the ratio of the intensity of the luminescence of firefly (pFF) and renilla (pRL) luciferase. C. EMSA experiments with human and camel HSPA1L promoters fragments: 1—H. sapiens promoter without 112 bp fragment specific for primates only, 2—isolated H. sapiens 112 bp fragment, 3—C. dromedarius promoter. *** and ### p ≤ 0.001, ** p ≤ 0.01, * p ≤ 0.05.
Fig 2.
A. Various hsp70 promoter strengths based on the luciferase luminescence levels in S2 cells and the effects of experimental GAGA element insertion into the hsp70S3 promoter. B. The level of S. singularior and D. melanogaster hsp83 promoter activity in D. melanogaster S2 cells. *** p ≤ 0.001, ## p ≤ 0.01.
Fig 3.
A. EMSA experiments with protein extracts from S2 cells and adult D. melanogaster flies exploring labeled fragments of the D. melanogaster hsp70Aa and S. singularior hsp70S3 and S4 genes. 1—Control (25°C), 2—heat shock (37°C), 3—heat shock + preimmune serum, 4—super shift with anti-HSF. The arrow indicates the position of the HSF-HSE complex. B. Recombinant D. melanogaster GAF protein effectively binds to the D. melanogaster hsp70 and S. singularior hsp70S3 promoters with the experimental insertion of three GAGA elements (lanes 1 and 4) but not with the wild-type hsp70S3 and hsp70S4 promoters (lanes 2 and 3). The arrow indicates the position of the GAF-GAGA complexes.
Fig 4.
Left panel: General arrangement of constructs used in the transformation experiments.
The thick green arrows in I(S-GAGA) indicate the position of the inserted GAGA elements. HSE, GAGA elements and TATA boxes are indicated by square boxes of different color. Right panel: in situ hybridization of heat-shocked salivary gland chromosomes with the white gene fragment included in the constructs. The sites of the inserts are shown by arrows with puffs formed only in the strains (the bottom panel) containing constructs with D. melanogaster hsp70 promoters. In all panels, the heat shock puffs formed in the locations of major D. melanogaster hsp genes (i.e., 63B, 61C and 95D) that represent an internal control are indicated. 3B is the white locus that hybridizes with the labeled probe and represents an internal control for hybridization efficiency. In each strain, at least ten larvae were used for puff detection after HS.
Table 1.
List of transgenic strains used in the investigation.
Fig 5.
The results of RT-PCR experiments using RNA isolated from adult flies (A) or the third instar larvae (B) of D. melanogaster strains transformed with constructs containing different hsp70 promoters.
A significant dichotomy in the expression of the I(S-GAGA) construct in adults and larvae is evident, while all other constructs exhibit similar strengths at both stages. *** p ≤ 0.001.