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

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.

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

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 http://www.iacuc.arizona.edu/training/xenopus/images/xenopus.jpg These amphibians can regenerate damaged hair cells

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

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.

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 DNA Short PCR Product Long PCR Product

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

PCR Performance Optimization Picomaxx 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