Isolation and Characterization ofthe Metallothionein Promoterin Artemia

Background metallothionein = MT metal-binding protein four isoforms identified in mammals: MT-I, MT-II, MT-III and MT-IV diverse functions -metal ion homeostasis -heavy metal sequestration -protection from oxidative damage -metal reservoir

Metallothioneindumbbell shape, cysteine-rich domain and domainclusters of metal ions www.unizh.ch/~mtpage/into.html

Metallothionein

Fischer et al., Proc.Natl.Acad.Sci. 1998Maret, W. Am.Soc.Nutr.Sci 2000Zinc-thiolate clustersEo very low

Metallothionein Regulationregulation transcriptional levelinduction metals, oxidants,cytokines, hormonesmetal induction- metal responsive elements (MREs)MREs- conserved core sequence TGC(G/A)CNCmetal transcription factor, MTF-1 regulates expression - binding to MRE - activity induced by metals but needs zinc to bind to MRE

Metallothionein Regulation

Artemiabrine shrimp Artemia salinahttp://www.captain.at/artemia/ http://www.acuariolasmercedes.com/Guia-de-cuidado/artemia-salina-brine-shrimps-introducion-1.htm AdultNauplius

MetallothioneinWhy Artemia?small, easy to grow, and relatively cheap to purchase embryonic development extensively studiedbiomarker for heavy metal contamination in aquatic ecosystems-phthalate ester embryotoxicityfour Artemia MT isoforms identifiedcoding region of one of the isoforms sequenced

Metallothionein

Goal: isolate and sequence the promoter region, ID position and number of MREs, and determine extent of promoter activity when induced by heavy metals - experiment design will determine if four isoforms regulated by one promoter, or if each isoform is regulated by its own promoter

Metallothionein

5ATGGACTGCTGCAAGAACGGTTGCACCTGTGCCCCAAATTGCAAATGTGCCStart asp cys cys lys asn gly cys thr cys ala pro asn cys lys cys alaAAAGACTGCAAATGCTGCAAAGGTTGTGAGTGCAAAAGCAACCCAGAATGC lys asp cys lys cys cys lys gly cys glu cys lys ser asp pro glu cysAAATGTGAGAAGAACTGTTCATGCAACTCATGTGGTTGTCACTGA3 lys cys glu lys asn cys ser cys asn ser cys gly cys his Stop

coding sequence of Artemia MT isoform

Methods and MaterialsGenomic DNA digested with restriction enzymesDNA walking method- amplification of an unknown sequence adjacent to a known sequence -

touchdown PCR annealing/extension temperature is higher than Tm of the primers

Methods and Materials

DNA walking Clone into vector clone into GFP reporter geneSend for sequencing generate MT promoter- ID MREs GFP constructs transfect into COS cells treat with nM metal concentrations for 24h incubation measure fluorescence

Methods and MaterialsMT-GFP vectorGFPMT-GFP constructs showing deletions

Expected Resultsinduction by Zn greater than Cdextent of induction decreased as MREs removed

Chart2

0.10.50.5

0.2510.5

0.532

143

1.55.54.5

1.565

Basal Activity

Zn

Cd

MT Deletion Contructs

GFP Fluorescence

Functional Analysis Of Artemia MT Promoter Deletions

Sheet1

0.10.50.5

0.2510.5

0.532

143

1.55.54.5

1.565

Sheet1

Basal Activity

Zn

Cd

MT Deletion Contructs

GFP Fluorescence

Functional Analysis Of Artemia MT Promoter Deletions

Sheet2

Sheet3

Future Perspectives4 promoters for 4 isoforms or 1 promoter for 4 isoforms - expression is tissue-specific - differential expression due to environmental factorscooperative interaction among MREs promoter sequenced other regulatory elements can be identified and studied -ARE/USF oxidative stress

AcknowledgementsDr Acey (Research Mentor)HHMIDr Mason (Instructor)