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Isolation and Characterization of the Metallothionein Promoter in 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 - PowerPoint PPT Presentation
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HHMI Spring 2004 Gwen Jordaan
Isolation and Characterization ofthe Metallothionein Promoter
in Artemia
HHMI Spring 2004 Gwen Jordaan
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
HHMI Spring 2004 Gwen Jordaan
Metallothionein dumbbell shape,
cysteine-rich domain and
domain clusters of metal
ions
www.unizh.ch/~mtpage/into.html
HHMI Spring 2004 Gwen Jordaan
Metallothionein
Fischer et al., Proc.Natl.Acad.Sci. 1998Maret, W. Am.Soc.Nutr.Sci 2000
Zinc-thiolate clustersEo very low
HHMI Spring 2004 Gwen Jordaan
Metallothionein Regulation
regulation – 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
HHMI Spring 2004 Gwen Jordaan
Metallothionein Regulation
HHMI Spring 2004 Gwen Jordaan
Artemiabrine shrimp – Artemia salina
http://www.captain.at/artemia/ http://www.acuariolasmercedes.com/Guia-de-cuidado/artemia-salina-brine-shrimps-introducion
-1.htm
Adult Nauplius
HHMI Spring 2004 Gwen Jordaan
Metallothionein
Why 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
HHMI Spring 2004 Gwen Jordaan
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
HHMI Spring 2004 Gwen Jordaan
Metallothionein
5’ATGGACTGCTGCAAGAACGGTTGCACCTGTGCCCCAAATTGCAAATGTGCCStart 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
HHMI Spring 2004 Gwen Jordaan
Methods and Materials
Genomic 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
HHMI Spring 2004 Gwen Jordaan
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
HHMI Spring 2004 Gwen Jordaan
Methods and Materials
MT-GFP vector
GFP
MT-GFP constructs showing deletions
HHMI Spring 2004 Gwen Jordaan
Expected Results
Functional Analysis Of Artemia MT Promoter Deletions
0 2 4 6 8
1
3
5
MT De
letion
Contr
ucts
GFP Fluorescence
Cd
Zn
Basal Activity
induction by Zn greater than Cdextent of induction decreased as MREs removed
HHMI Spring 2004 Gwen Jordaan
Future Perspectives
4 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
HHMI Spring 2004 Gwen Jordaan
Acknowledgements
Dr Acey (Research Mentor)HHMIDr Mason (Instructor)
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