Part:BBa_K4890014
MRE-Hsp70-Hid
This part is responsible to the expression of Hid gene driven by MRE in Drosophila. It consists of MRE sequence (BBa_K4890002), Hsp70 sequence (BBa_K4890004) and Hid gene (BBa_K4890003). Metal response element (MRE) is derived from Drosophila melanogaster. In the cell nucleus, activated MTF-1 binds to MRE. The MRE is typically located in the promoter regions associated with genes involved in the response to heavy metals. Binding of MTF-1 to MRE activates the transcription of the corresponding genes, thereby promoting heavy metal-related gene expression. Head involution defective (Hid) gene is derived from Drosophila melanogaster. Hid induces cell apoptosis in Drosophila. Hsp70 is derived from pUAST plasmid. Hsp70 is a promoter that can bind to RNA polymerase and start transcription.
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal EcoRI site found at 360
Illegal XbaI site found at 1616
Illegal PstI site found at 222
Illegal PstI site found at 964
Illegal PstI site found at 1193
Illegal PstI site found at 1337 - 12INCOMPATIBLE WITH RFC[12]Illegal EcoRI site found at 360
Illegal PstI site found at 222
Illegal PstI site found at 964
Illegal PstI site found at 1193
Illegal PstI site found at 1337
Illegal NotI site found at 378 - 21INCOMPATIBLE WITH RFC[21]Illegal EcoRI site found at 360
Illegal BglII site found at 372
Illegal XhoI site found at 487
Illegal XhoI site found at 1393 - 23INCOMPATIBLE WITH RFC[23]Illegal EcoRI site found at 360
Illegal XbaI site found at 1616
Illegal PstI site found at 222
Illegal PstI site found at 964
Illegal PstI site found at 1193
Illegal PstI site found at 1337 - 25INCOMPATIBLE WITH RFC[25]Illegal EcoRI site found at 360
Illegal XbaI site found at 1616
Illegal PstI site found at 222
Illegal PstI site found at 964
Illegal PstI site found at 1193
Illegal PstI site found at 1337
Illegal NgoMIV site found at 1322 - 1000COMPATIBLE WITH RFC[1000]
Results
1 Construction of pMRE-Hid plasmid
We also reformed the pUAST plasmid into pMRE plasmid by restrictive endonuclease (Pst1) digestion to obtain a linearized pUAST vector and then substitute UAS sequence for MRE sequence. MRE was obtained by DNA synthesis and T4 ligase was used to combine the linearized vector into complete plasmid. pMRE plasmid was transformed into E. coli DH5α strain. Colony PCR and DNA electrophoresis (600 bp) was performed to confirm the positive colonies. These colonies were transferred and expanded. Plasmid extracted from the colonies was confirmed to be pMRE by gene sequencing.
Start from pMRE as template, we used restrictive endonuclease (NotI and XbaI) digestion to obtain a linearized pMRE vector. Hid gene fragment was amplified from the cDNA of wildtype Drosophila melanogaster by PCR. DNA electrophoresis confirmed the length of the PCR product (1233bp). Hid gene fragment was ligated with pMRE linearized vector by T4 ligase. pMRE-Hid plasmid was transformed into E. coli DH5α strain. Colony PCR and DNA electrophoresis (1233bp) was performed to confirm the positive colonies. These colonies were transferred and expanded. Plasmid extracted from the colonies was confirmed to be pMRE-Hid by gene sequencing.
2 Transient transfection of Drosophila S2 cells with pUAST-MTF-1, pMRE-Hid, and pAc-GAL4 plasmids
We cultured Drosophila S2 cells on plates for 24h, and then transiently co-transfected pUAST-MTF-1 (refer to BBa_K4890013), pMRE-Hid and pAc-GAL4 (which contains actin-GAL4) plasmids into the S2 cells. pAc-GAL4 plasmid was previous constructed by Genetic Lab, School of Life Science and Technology, Tongji University. The transfected S2 cells were notated as Drosophila UAS-MTF-1/MRE-Hid/Ac-GAL4 cells.Drosophila UAS-MTF-1/MRE-Hid/Ac-GAL4 cells were cultured for 48h and then divided into 5 groups. The control group received no treatment, and the other 4 groups were treated with 10μM ZnCl2, 100μM ZnCl2, 10μM CdCl2, and 100μM CdCl2, respectively, for 4h.
Real-time PCR and Western Blot results confirmed that both mRNAs and proteins of MTF-1 and Hid were expressed in Drosophila UAS-MTF-1/MRE-Hid/Ac-GAL4 cells treated with 10μM and 100μM ZnCl2 or CdCl2 (Figure 5-6). The mRNA and protein levels of Hid were concentration-dependent (P<0.05). The mRNA level of MTF-1 was not changed with the addition of metal ions (P>0.05).
(Note: MTF-1 was detected by antibody of HA tag, which was co-expressed at the N-terminal of MTF-1. Hid was detected by antibody of Myc tag, which was co-expressed at the C-terminal of Hid.)
3 Generation of Drosophila lines with genotype of UAS-MTF-1; MRE-Hid/GMR-GAL4 , UAS-MTF-1；MRE-Hid/ Vg-GAL4 and UAS-MTF-1；MRE-Hid/ ptc-GAL4
pUAST-MTF-1 and pMRE-Hid were micro-injection into embryos of Drosophila W1118 respectively to obtain Drosophila UAS-MTF-1 and Drosophila MRE-Hid (Micro-injection was performed by Core Facility of Drosophila Resource and Technology, CEMCS, CAS). Drosophila UAS-MTF-1 was crossed with Drosophila MRE-Hid to obtain the offspring with genotype of UAS-MTF-1;MRE-Hid. This progeny was crossed with Drosophila GMR-GAL4 or Drosophila Vg-GAL4 or Drosophila ptc-GAL4 to obtain the progeny with genotype UAS-MTF-1;MRE-Hid/GMR-GAL4 or UAS-MTF-1;MRE-Hid/Vg-GAL4 or UAS-MTF-1;MRE-Hid/ptc-GAL4.
Each cell line of Drosophila UAS-MTF-1;MRE-Hid/GMR-GAL4 and Drosophila UAS-MTF-1;MRE-Hid/Vg-GAL4 was divided into 5 groups for larvae Acridine Orange (AO) staining and adult phenotype. The control group received no treatment, and the other 4 groups were treated with 10μM ZnCl2, 100μM ZnCl2, 10μM CdCl2, and 100μM CdCl2, respectively. Each cell line of Drosophila UAS-MTF-1;MRE-Hid/GMR-GAL4 and Drosophila UAS-MTF-1;MRE-Hid/ptc-GAL4 were divided into 3 groups for larvae Death Caspase-1 (Dcp-1) staining. The control group received no treatment, and the other 2 groups were treated with 10μM CdCl2, and 100μM CdCl2, respectively.
3.1 Heavy metal response of Drosophila larvae: cell death
The third instar larvae were collected in about 5 days and imaginal discs were dissected.
In Drosophila UAS-MTF-1;MRE-Hid/GMR-GAL4, AO staining detected increased cell death in the eye imaginal discs when cultured with ZnCl2 (100μM) or CdCl2 (10μM and 100μM) (P<0.01) (Figure 7). The level of cell death was correlated with the concentration of CdCl2 (P<0.05). More cell death was observed in Drosophila grown under 10μM and 100μM CdCl2 than in those under the same concentration of ZnCl2 (P<0.05).
In Drosophila UAS-MTF-1;MRE-Hid/Vg-GAL4, AO staining detected increased cell death in the wing imaginal discs when cultured with 10μM and 100μM ZnCl2 or CdCl2 (P<0.05) (Figure 8). The level of cell death was correlated with the concentration of metal ions (P<0.05). More cell death was observed in Drosophila grown under 10μM and 100μM CdCl2 than in those under the same concentration of ZnCl2(P<0.05).
3.2 Heavy metal response of Drosophila larvae: cell apoptosis
Dcp-1 staining detected enhanced cell apoptosis in the eye or wing imaginal discs of Drosophila UAS-MTF-1;MRE-Hid/GMR-GAL4 and Drosophila UAS-MTF-1;MRE-Hid/ptc-GAL4 cultured under 100μM CdCl2 (P<0.05)(Figure 9-10). Cultured under 10μM CdCl2, their discs also showed increased trend of cell apoptosis, but the difference was not statistically significant compared with the control (P>0.05).
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