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Cloning and Characterization of the MaxiK Ion Channel
Promoter of Xenopus
Vanessa ProvencioDr. Elba Serrano
Biology Department
- In the United States, about 28 million people or one in every ten individuals have some sort of hearing loss.
- Dysfunction of ion channels, through genetic mutations, can cause hearing loss.
- Ion channels are important molecules that help transduce sound into electrical activity in the nervous system.
Hearing loss is becoming an epidemic
Hearing, the perception of sound, begins in the inner ear.
Organ of Corti
Hair cell stereocilia
Bear, Connors, Paradiso, Neuroscience: Exploring the Brain (2002)
Hair cells of the inner ear are the mechanoreceptors for sound
• Cilia movement opens ion channels within hair cells.• Ion channels, found in the membrane of cells, are important
for electrical signaling to the brain
A. Hair cell B. Cross section of cell membrane C. Ion channel Bear, Connors, Paradiso, Neuroscience: Exploring the Brain
Ion channels like MaxiK are essential for hearing
Mammalian high conductance, calcium-activated potassium (MaxiK) channels are composed of two dissimilar subunits, alpha and beta.
Objective
To isolate and characterize the function of the MaxiK channel β-subunit promoter from amphibians.
Promoters regulate gene expression
• Promoters are regions of DNA that control gene expression
• Give us a better understanding of the mechanisms involved in development and maintenance of tissues in organisms
hnRNA
Exon 1 Exon 2
Transcription Factor
Promoter
TFBS
ATG
Xenopus tropicalis and Xenopus laevis will be used in the investigations of the
this promoter.
Xenopus is our model organism for understanding the mechanisms of hearing
Serrano Lab Photo
These amphibians can regenerate damaged hair cells
X. laevis
http://www.iacuc.arizona.edu/training/xenopus/images/xenopus.jpg
1. Purify X. tropicalis genomic DNA
2. Obtain and analyze sequence– Primer design
3. Isolate 2,000 bp of genomic DNA upstream of protein coding sequence using PCR
4. Clone promoter
5. Analyze promoter function– Bioinformatics
– Transfections
Project Goals
High integrity X. tropicalis genomic DNA was isolated
• DNA extraction protocol from Sambrook et al. 1989
• 1 gram of liver & muscle tissue
• Quantification by spectrophotometer– >1 µg/µl– 260nm/280nm = 1.6
• Visual inspection of DNA stained with EtBr on agarose gel
Hi M
W
Lo M
WX
. tro
p G
DN
AH
. sap
iens
G D
NA
50kb
3kb
Bioinformatic tools were used to identify and analyze sequence of interest
• Identified region of DNA sharing homology with mammalian beta1 subunit– BLAST X. tropicalis genome sequencing project
Putative promoter region
X. tropicalis genomic DNA
Human MaxiK beta protein sequence
Bioinformatic tools were used to identify and analyze sequence of interest
• Primer Design – Primer 3 (http://frodo.wi.mit.edu/cgi-bin/primer3/primer3_www.cgi)
– Upstream of ATG start codon from MaxiK gene using Primer3 program
– Short & Long PCR Products:• Yellow = short (877bp) product
• Blue= long (2500bp) product
• Fuchsia= overlap of primers
X. tropicalis Genomic DNAShort PCR ProductLong PCR Product
Bioinformatic tools were used to identify and analyze sequence of interest
X. tropicalis Genomic DNAShort PCR ProductLong PCR Product
Genomic PCR how it works
Components for PCR include:
– DNA template
– Forward and reverse primers
– Taq polymerase
– Deoxynucleotides-triphosphate
– Buffer
Currently optimizing PCR to isolate promoter
1. Varied concentration of DNA• Concentrated (1 µg) and diluted DNA (100ng)• Positive control to test the Picomaxx kit components• No product observed
2. Change TAQ polymerase
(Catalyzes DNA synthesis)• Newly isolated plasmid (xslo α6)
(10ng,20ng,227ng) used as a control• Finally, some product!
1kb
ladd
er
Lo M
W
10ng
10kb
3kb
20ng
227n
g
PCR Performance OptimizationPicomaxx Stratagene Manual
Conclusions
• Isolated high integrity X. tropicalis genomic DNA
• Found a putative MaxiK beta subunit gene in X. tropicalis genome
• Developed primers for PCR
• Currently optimizing PCR
• Future studies include cloning promoter region and analyzing promoter function
Promoter functional analysis
• Construct fusion protein downstream of promoter• EGFP as a reporter protein
– Used in protein localization studies with increase in sensitivity
• BK alpha as a reporter• Take dividing eggs and cultured cells• Transfect with lipofectamine and plasmid
The BK alpha, hearing, and sex connection…
• Males and females differ in their hearing abilities.• To what extent does estrogen play a role in preserving
hearing?• The β-subunit of the Maxi-K channel is thought to be
estrogen regulated. Estrogen is present in both males and females (highly expressed in females).
Acknowledgements
• Dr. Elba Serrano
• Dr. TuShun Powers
• David Sultemeier
• Members of the Serrano Lab
• NMSU Alliance for Minority Participation Program• NSF CREST
• Funded by NIH RO1 DC 003292 to EES
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