isolation and characterization of the metallothionein promoter in artemia

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HHMI Spring 2004 Gwen Jordaan lation and Characterization the Metallothionein Promoter in Artemia

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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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Page 1: Isolation and Characterization of the Metallothionein Promoter in  Artemia

HHMI Spring 2004 Gwen Jordaan

Isolation and Characterization ofthe Metallothionein Promoter

in Artemia

Page 2: Isolation and Characterization of the 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

Page 3: Isolation and Characterization of the Metallothionein Promoter in  Artemia

HHMI Spring 2004 Gwen Jordaan

Metallothionein dumbbell shape,

cysteine-rich domain and

domain clusters of metal

ions

www.unizh.ch/~mtpage/into.html

Page 4: Isolation and Characterization of the Metallothionein Promoter in  Artemia

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

Page 5: Isolation and Characterization of the Metallothionein Promoter in  Artemia

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

Page 6: Isolation and Characterization of the Metallothionein Promoter in  Artemia

HHMI Spring 2004 Gwen Jordaan

Metallothionein Regulation

Page 7: Isolation and Characterization of the Metallothionein Promoter in  Artemia

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

Page 8: Isolation and Characterization of the Metallothionein Promoter in  Artemia

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

Page 9: Isolation and Characterization of the Metallothionein Promoter in  Artemia

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

Page 10: Isolation and Characterization of the Metallothionein Promoter in  Artemia

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

Page 11: Isolation and Characterization of the Metallothionein Promoter in  Artemia

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

Page 12: Isolation and Characterization of the Metallothionein Promoter in  Artemia

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

Page 13: Isolation and Characterization of the Metallothionein Promoter in  Artemia

HHMI Spring 2004 Gwen Jordaan

Methods and Materials

MT-GFP vector

GFP

MT-GFP constructs showing deletions

Page 14: Isolation and Characterization of the Metallothionein Promoter in  Artemia

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

Page 15: Isolation and Characterization of the Metallothionein Promoter in  Artemia

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

Page 16: Isolation and Characterization of the Metallothionein Promoter in  Artemia

HHMI Spring 2004 Gwen Jordaan

Acknowledgements

Dr Acey (Research Mentor)HHMIDr Mason (Instructor)