Analysis of the structure and activity of the promoter regions of the metallothionein genes of the freshwater pearl mussel Hyriopsis schlegelii

Figures & data

Table 1. Primers used in this study.

Definition	Sequence (5′-3′)	Application
HsMT1-GSP1	GTCTCAAGGCAGTTGCATGCGTCAG	Cloning HsMT1 promoter region
HsMT1-GSP2	GTCTTAGTATGCCGTGTGCTACGTG
HsMT1-GSP3	CCGTGTTGTAACTGCCTTGTGATGC
HsMT2-GSP1	CTCCAAACACTGGCATGATTCCGAC	Cloning HsMT2 promoter region
HsMT2-GSP2	GAGTCGTGTGCAAAACGAAGTGCGT
M13R(-48)	AGCGGATAACAATTTCACACAGGA	Sequencing and identification
M13F(-47)	CGCCAGGGTTTTCCCAGTCACGAC
pMT1-1121 forward primer	CTTGGTACCCTGAGGGACCACGGGTCTTT	Constructing deletion mutants for HsMT1 5′-upstream region
pMT1-587 forward primer	TCTGGTACCGTGTCCACCTTGTTTGCATGAAT
pMT1 reverse primer	TCGAAGCTTAATCTTCGGTGCTTCAAAATCTG
pMT2-1121 forward primer	CTTGGTACCAGCAAGCAGATAATCGTTGTGTC	Constructing deletion mutants for HsMT2 5′-upstream region
pMT2-541 forward primer	TCTGGTACCACATATGGTCTTAGACGGAAACTC
pMT2-136 forward primer	TTAGGTACCACGACTCTTTGCAATACTGTTCGC
pMT2 reverse primer	GTCGCTAGCCCGCTCTTTATGTATCTGCAATCG
RVprimer3	CTAGCAAAATAGGCTGTCCC	Sequencing and identification
GLprimer2	CTTTATGTTTTTGGCGTCTTCCA
HsMTF-like-F	CTAGCTAGCATGGAAAACACAGCAACGTTCCGTGAGGC	Cloning of HsMTF-1 whole ORF cDNA
HsMTF-like-R	CCCAAGCTTTCAGGATTTGAGCTTATAACTGACATCTGT
HsMT1-MREmutF1	GACACGGTACCCTGAGGGACCACGGGTC	Mutagenesis of the MRE in HsMT1 promoter
HsMT1-MREmutR1	CAAGGTGGACACGTGCACAGAAAATTACCGTATATC
HsMT1-MREmutF2	GATATACGGTAATTTTCTGTGCACGTGTCCACCTTG
HsMT1-MREmutR2	GACACaAGCTTAATCTTCGGTGCTTCAAAATC
HsMT2-MREmutF1	GAGAGAGGTACCAACAGTTTTGTGCGCACATATG	Mutagenesis of MREs in HsMT2 promoter
HsMT2-MREmutR1	GAGAGAGCTAGCAAGAGTCGTGTGCAAAACGAAG
HsMT2-MREmutF2	GAGAGAGGTACCAACAGTTTTGTatGCACATATG
HsMT2-MREmutR2	GAGAGAGCTAGCAAGAGTCGTGTatAAAACGAAG

Kpn I (in bold), Hind Ш (underlined), and Nhe I (in italic and bold) restriction enzyme were adopted to the 5′ and 3′ primers, respectively.

Figure 1. Nucleotide sequences of the 5′-upstream regions of the H. schlegelii MT1 (A) (GenBank Accession No. KX279831) and MT2 (B) (GenBank Accession No. KX279832) genes. MREs and other putative transcription factor binding sites are shown.

Figure 2. Transcriptional activity of the HsMT1 5′-promoter regions (A) and HsMT2 5′- promoter regions (B) after induction using Zn²⁺ (50, 100, and 150 μM), Cu²⁺ (50, 100, and 150 μM), and Cd²⁺ (0.5, 1.0, and 1.5 μM). The background activity of the pGL3-Basic vector were set as 1.0 and all the other activities were adjusted accordingly. The number on the bars indicates the induction ratio compared with uninduced basal activity. Significant differences across control are indicated with ** at p < 0.05.

Figure 3. Transcription activities of each deletion mutants of the HsMT1 and HsMT2 promoters in the presence of 100 μM Zn²⁺, 100 μM Cu²⁺, and 1.0 μM Cd²⁺, respectively. (A) Schematic diagram of the deletion mutants, the main cis-acting elements are marked. (B) Induction ratio of the five deletion mutants in the presence of 100 μM Zn²⁺, 100 μM Cu²⁺, and 1.0 μM Cd²⁺ , calculated by the ratio of the induced promoter activity to the uninduced basal promoter activity. The background activity of the pGL3-Basic vector were set as 1.0 and all the other activities were adjusted accordingly. Significant differences across control are indicated with ** at p < 0.05.

Figure 4. Alignment of the deduced amino acid sequences of HsMTF-like and MTF-1s of other species in NCBI. Their GenBank accession numbers are as follows: hMTF: Homo sapiens X78710; zMTF: Danio rerio AF458116; fMTF: Fugu rubripes AJ131393; mMTF: Mus musculus X71327. F1-F6 represent six zinc fingers regions of HsMTF-like. Zinc fingers 1 to 6 are indicated by numbered brackets above the corresponding sequences.

Figure 5. Effects of HsMTF-like on the promoter activity of HsMT1 in HepG2 cells. (A). Schematic diagram of site-directed mutagenesis of the MRE motif in the HsMT1 and HsMT2 promoters. (B). The cells were co-transfected with promoter reporter plasmids containing a MRE (pMT1-1121) or a site-mutated MRE motif (pMT1-1121-MREmut) together with the HsMTF-like expression vector (pcDNA-HsMTF-like). The cells co-transfected with pGL3-Basic and pcDNA3.1 were used as control and its relative luciferase activities was arbitrarily set to 1.0 and all the other activities were adjusted accordingly. (C & D). HepG2 cells were treated with 100 μM Zn²⁺ in growth medium when cotransfection of expression vectors (pcDNA3.1 or pcDNA-HsMTF-like) and reporter genes (pMT1-1121, pMT1-1121-MREmut or pMT2-1121). The relative luciferase activity of the samples cotransfected with the pMT1-1121 and pcDNA-HsMTF-like (C) or pcDNA3.1 (D) was arbitrarily set to 1.0 and all the other activities were adjusted accordingly. Each bar represented the mean ± S.D of triplicates. Significant differences across control are indicated with ** at p < 0.05. (E). HepG2 cells were treated with 100 μM Zn²⁺ in growth medium when cotransfection of expression vectors (pcDNA3.1 or pcDNA-HsMTF-like) and reporter genes (pMT2-1121, pMT2-1121-MREabmut, pMT2-1121-MREamut or pMT2-1121-MREbmut). The relative luciferase activity of the samples cotransfected with the pMT2-1121 and pcDNA3.1 was arbitrarily set to 1.0 and all the other activities were adjusted accordingly. Each bar represented the mean ± S.D of triplicates. Significant differences across control are indicated with ** at p < 0.05.