Part:BBa_K4890013

UAS-Hsp70-MTF1

This part is responsible to the expression of MTF-1 gene driven by UAS in Drosophila. It consists of UAS sequence (BBa_K3281012), Hsp70 sequence (BBa_K4890004) and MTF-1 gene (BBa_K4890001). UAS and Hsp70 are derived from pUAST plasmid. Upstream activating sequence (UAS) is a cis-acting regulatory sequence. It increases the expression of a neighbouring gene when binds to GAL4. Hsp70 is a promoter that can bind to RNA polymerase and start transcription. MTF-1 gene is derived from Drosophila melanogaster. It encodes MTF-1 (metal-responsive transcription factor-1) which is a transcription factor. MTF-1 protein can be activated by heavy metals. In the cell nucleus of Drosophila cells, MTF-1 binds to MRE to recruit RNA polymerase, in turn increasing the expression of Mto/Mtn to detoxicate metals.

Sequence and Features

Assembly Compatibility:

10
INCOMPATIBLE WITH RFC[10]
Illegal EcoRI site found at 382
Illegal EcoRI site found at 2296
Illegal PstI site found at 244
Illegal PstI site found at 2054
Illegal PstI site found at 2232
12
INCOMPATIBLE WITH RFC[12]
Illegal EcoRI site found at 382
Illegal EcoRI site found at 2296
Illegal NheI site found at 509
Illegal PstI site found at 244
Illegal PstI site found at 2054
Illegal PstI site found at 2232
21
INCOMPATIBLE WITH RFC[21]
Illegal EcoRI site found at 382
Illegal EcoRI site found at 2296
Illegal BglII site found at 527
Illegal BamHI site found at 479
Illegal BamHI site found at 1468
Illegal BamHI site found at 1864
Illegal BamHI site found at 1891
23
INCOMPATIBLE WITH RFC[23]
Illegal EcoRI site found at 382
Illegal EcoRI site found at 2296
Illegal PstI site found at 244
Illegal PstI site found at 2054
Illegal PstI site found at 2232
25
INCOMPATIBLE WITH RFC[25]
Illegal EcoRI site found at 382
Illegal EcoRI site found at 2296
Illegal PstI site found at 244
Illegal PstI site found at 2054
Illegal PstI site found at 2232
Illegal NgoMIV site found at 1730
1000
COMPATIBLE WITH RFC[1000]

Results

1 Construction of pUAST-MTF-1 plasmid

We took a commercialized recombinant plasmid pUAST as template, and used restrictive endonuclease (BglII and XhoI) digestion to obtain a linearized pUAST vector. MTF-1 gene fragment was amplified from the cDNA of wildtype Drosophila melanogaster by PCR. DNA electrophoresis confirmed the length of the PCR product (2376bp). MTF-1 gene fragment was ligated with the pUAST linearized vector by T4 ligase. pUAST-MTF-1 was transformed into E. coli DH5α strain. Colony PCR and DNA electrophoresis (2376bp) was performed to confirm the positive colonies. These colonies were transferred and expanded. Plasmid extracted from the colonies was confirmed to be pUAST-MTF-1 plasmid by gene sequencing (Figure 1-2).

Figure 1 Gel electrophoresis of MTF-1

Figure 2 Gel electrophoresis of pUAST-MTF-1 plasmid
(From left to right: marker, pUAST-MTF-1, pMRE-Hid and pMRE-GFP)

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, pMRE-Hid (refer to BBa_K4890014) 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 3-4). 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).

Figure 3 mRNA levels of MTF-1 and Hid in Drosophila UAS-MTF-1/MRE-Hid/Ac-GAL4 cells treated with different concentrations of ZnCl₂ or CdCl₂
(unpaired t-test: ***P<0.001, ****P<0.0001, ns: P>0.05)

Figure 4 Protein levels of MTF-1 and Hid in Drosophila UAS-MTF-1/MRE-Hid/Ac-GAL4 cells treated with different concentrations of ZnCl_{2_{or CdCl₂
(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 3rd 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 5). 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 6). 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).

Figure 5 Cell death in the eye imaginal discs of Drosophila UAS-MTF-1;MRE-Hid/GMR-GAL4 cultured under ZnCl₂ and CdCl₂ for 5 days detected by AO staining (unpaired t-test (vs control): **P<0.01, ***P<0.001, ****P<0.0001, ns: P>0.05)

Figure 6 Cell death in the wing imaginal discs of Drosophila UAS-MTF-1;MRE-Hid/Vg-GAL4 cultured under ZnCl₂ and CdCl₂ for 5 days detected by AO staining (unpaired t-test (vs control): *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001)

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 7-8). 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).

Figure 7 Cell apoptosis in the eye imaginal discs of Drosophila UAS-MTF-1;MRE-Hid/GMR-GAL4 cultured under CdCl₂ for 5 days detected by Dcp-1 staining (unpaired t-test (vs control): ***P<0.001, ns: P>0.05)

Figure 8 Cell apoptosis in the wing imaginal discs of Drosophila UAS-MTF-1;MRE-Hid/ptc-GAL4 cultured under CdCl₂ for 5 days detected by Dcp-1 staining (unpaired t-test (vs control): ****P<0.0001, ns: P>0.05)