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