1. Cloning and Characterization of the MaxiK Ion Channel Promoter of Xenopus
Vanessa Provencio
Dr. Elba Serrano
Biology Department

2. Hearing loss is becoming an epidemic
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.

3. Hearing, the perception of sound, begins in the inner ear.
Organ of Corti
Hair cell
stereocilia
Bear, Connors, Paradiso, Neuroscience: Exploring the Brain (2002)

4. 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

5. 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.

6. Objective
To isolate and characterize the function of the MaxiK channel β-subunit promoter from amphibians.

7. 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

8. regenerate damaged hair cells
Xenopus is our model organism for understanding the mechanisms of hearing
Xenopus tropicalis and Xenopus laevis will be used in the investigations of the this promoter.
Serrano Lab Photo
X. laevis
These amphibians can
regenerate damaged hair cells

9. Project Goals Purify X. tropicalis genomic DNA
Obtain and analyze sequence
Primer design
Isolate 2,000 bp of genomic DNA upstream of protein coding sequence using PCR
Clone promoter
Analyze promoter function
Bioinformatics
Transfections

10. High integrity X. tropicalis genomic DNA was isolated
Hi MW
Lo MW
X. trop G DNA
H. sapiens G DNA
50kb
3kb
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
Fig. 5. X. tropicalis genomic DNA isolated from liver and muscle tissue. Lane 1. High molecular weight DNA ladder. Lane 2. kB DNA ladder. Lane 3. X. tropicalis genomic DNA. Lane 4. High integrity H. sapien genomic DNA.

11. 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

12. Bioinformatic tools were used to identify and analyze sequence of interest
Primer Design
Primer 3 (
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 DNA
Short PCR Product
Long PCR Product

13. Bioinformatic tools were used to identify and analyze sequence of interest
X. tropicalis Genomic DNA
Short PCR Product
Long PCR Product

14. Genomic PCR how it works
Components for PCR include:
DNA template
Forward and reverse primers
Taq polymerase
Deoxynucleotides-triphosphate
Buffer

15. Currently optimizing PCR to isolate promoter
Varied concentration of DNA
Concentrated (1 µg) and diluted DNA (100ng)
Positive control to test the Picomaxx kit components
No product observed
Change TAQ polymerase
(Catalyzes DNA synthesis)
Newly isolated plasmid (xslo α6)
(10ng,20ng,227ng) used as a control
Finally, some product!
1kb ladder
Lo MW
10ng
10kb
3kb
20ng
227ng

16. PCR Performance Optimization
Picomaxx Stratagene Manual

17. 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

18. 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

19. 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).

20. 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 to EES