Metallothionein promoter

Although not as popular as the baculovirus-system, stable transformations of insect cells can be used to circumvent the problems mentioned in the last paragraph. Common hosts include the fruitfly and mosquito. The expressed genes are often under the control of the Drosophila metallothionein promoter. Genes coding for resistance to antibiotics such as hygromycin and neomycin are used as selection markers. 

The mechanism by which MTF-1 facilitates zinc-induction of metallothionein promoter through the MREs is not known, but several models have been proposed. First, zinc may act as a coinducer by binding to MTF-1 and creating an allosteric change, allowing MTF-1 to bind to the MREs. The model proposed for mammalian MTF-l/MRE interaction has already been proven for yeast copper metallothionein systems (Furst et al., 1988). Another possibility may be that, under normal conditions, an inhibitor binds MTF-1. When an influx of zinc occurs, MTF-1 binds the zinc, undergoes a conformational change and is released from the inhibitor. The protein would then have the ability to bind to the MREs. Finally, upon an increase in intracellular zinc concentration, a specific coactivator may bind zinc and interact with MTF-1 to maximally induce transcription. 

Figure 6.30. Rat Growth Hormone-Metallothionein Gene Construct. The gene for rat growth hormone (shown in yellow) was inserted into a plasmid next to the metallothionein promoter, which is activated by the addition of heavy metals, such as cadmium ion.

Dalton TP, Paria BC, Fernando LP, Huet-Hudson YM, Dey SK and Andrews GK (1997) Activation of the chicken metallothionein promoter by metals and oxidative stress in cultured cells and transgenic mice. Comp Biochem Physiol B Biochem Mol Biol 116 75 -86. 

Bunch TA, Grinblat Y, Goldstein LS (1988) Characterization and use of the Drosophila metallothionein promoter in cultured Drosophila melanogaster cells. Nucleic Acids Res 16 1043-1061... 

Ract-Hadh (Swevers et al. 1996) was made by substituting an actinSC promoter fragment for the metallothionein promoter of pRmHa-1 pRmHa-1 contains a metallothionein promoter followed by a polylinker and a polyadenylation region from Adh (Bunch et al. 1988). 

To detoxify heavy metals, the liver contains metallothioneins, a group of cysteine-rich proteins with a high af nity for divalent metal ions such as Cd Cu Hg, and Zn T These metal ions also induce the formation of metallothioneins via a special metal-regulating element (MRE) in the gene s promoter (see p. 244). 

Radtke, F., Heuchel, R., Georgiev, O., Hergersberg, M., Gariglio, M., Dembic, Z. and Schaffner, W. (1993) Cloned transcription factor MTF-1 activates the mouse metallothionein I promoter. EMBO J., 12,1355-1362. 

Stuart, G.W., Searl, P.F., Chen, H.Y., Brinster, R.L. and Palmiter, R.D. (1984) A 12-base-pair DNA motif that is repeated several times in metallothionein gene promoters confers metal regulation to a heterologous gene. Proc. Natl. Acad. Sci. USA, 81, 7318-7322. 

Figure 21.9. Human MT promoter. (Adapted from Murphy, B. J., Andrews, G. K. Activation of metallothionein gene expression by hypoxia involves metal response elements and metal transcription factor-1. Cancer Res. 59,1315-1322, 1999.)...

Cu-induced transcriptional activation of metallothionein is known in Neurospora crassa, hut the factor that mediates Cu activation is unknown (Munger el a/., 1987). The MT gene does not contain an Acel-hinding site in its 5 promoter sequences. 

Dalton TP, Li Q, Bittel D, Liang L, Andrews GK (1996) Oxidative stress activates metal-responsive transcription factor-1 binding activity. Occupancy in vivo of metal response elements in the metallothionein-I gene promoter. J Biol Chem 271 26233-26241 Danscher G, Howell G, Perez-Clausell J, Hertel N (1985) The dithizone, Timm s sulphide silver and the selenium methods demonstrate a chelatable pool of zinc in CNS. A proton activation (PIXE) analysis of carbon tetrachloride extracts from rat brains and spinal cords intravitally treated with dithizone. Histochemistry 83 419 22 Danscher G, Jensen KB, Frederickson CJ, Kemp K, Andreasen A, Juhl S, Stoltenberg M, Ravid R (1997) Increased amount of zinc in the hippocampus and amygdala of Alzheimer s diseased brains a proton-induced X-ray emission spectroscopic analysis of cryostat sections from autopsy material. J Neurosci Methods 76 53-59... 

Evidence suggests that the regulation of metallothionein levels by metal ions results from the binding of zinc (or other metal ions) to a special transcription factor (molecular weight = 105 kDa, with the subsequent binding of the zinc/transcription factor complex to d promoter that resides near the metallothionein gene. The zinc/transcription factor complex actually binds to the metal response element that resides in the promoter The sequence of the metal response element is ... 

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