Outline for a Scientific Research Talk and Poster

• Introduction-– Briefly provide background relevant to the hypothesis, and state your hypothesis. “We

are investigating how arsenic is taken up into cells” Our hypothesis is that AQP3 mediates iAs uptake in KF gill and that increased salinity reduces AQP3 gene expression and iAs uptake” We report PCR studies demonstrating that AQP3 mRNA XXXXX when fish are exposed to increased salinity and that iAs XXXX AQP3 mRNA” Describe what AQP3 is and why you think it changes in SW and why you think it is an iAs transporter and why you think iAs may regulate it’s expression.

• Methods– Briefly summarize the methods (i.e. isolated RNA by…, made first strand cDNA by kit,

Q-PCR using AQP3 primers kin a Stratagene XXXX.• Results

– This should be the largest section of the talk poster (see poster). Transition to each result slide by telling the audience why you did the experiment, how you did it (briefly (I.e., Q-PCR) and what you found.

• Conclusions– Brief- 4-5 bullet points

• References Cited – If any. Cite source of any images not generated by you.

• Acknowledgements– Acknowledge grant funding and people who provided substantial assistance

• Note: As good poster has very little text and has the first name (not initial) of all presenters.

Molecular cloning of p38 MAP kinase cDNA from killifish (Fundulus heteroclitus)

Erin E. Flynn1,2, M. Christine Chapline1, Jordan A. Francke1,3, Cecily J. Swinburne1,4, Joseph R. Shaw1,5, Bruce A. Stanton1,6, J. Denry Sato1

1MDI Biological Laboratory, Salsbury Cove, ME; 2John Bapst HS, Bangor, ME; 3Presque Isle HS, Presque Isle, ME; 4College of the Atlantic, Bar Harbor, ME; 5University of Indiana, Bloomington, IN; 6Dartmouth Medical School, Hanover, NH

INTRODUCTION

Serum- and glucocorticoid-inducible kinase (SGK) regulates the cystic fibrosis transmembrane regulator (CFTR) chloride ion channel in the adaptation of killifish, a euryhaline teleost, to increased environmental salinity1,2. SGK function is regulated at the levels of transcription, kinase activation, and protein degradation3.

Acknowledgments. We thank Christine Smith of the MDIBL Marine DNA Sequencing Center for DNA sequencing. This research was supported by INBRE grant P20-RR016463 from NCRR, grant RO1-DK45881 from NIDDK to BAS, a Cystic Fibrosis Foundation research development program grant to BAS, and by NIEHS Center grant P30-ES03828. CJS was supported by INBRE grant P20-RR016463 from the National Center for Research Resources. EEF and JAF were STEER Scholars.

Based on p38 MAPK cDNA sequences from salmon, carp and zebrafish we designed forward and reverse synthetic p38 MAPK oligonucleotide primers for polymerase chain reaction (PCR) experiments (Fig. 2). Using pairs of primers we amplified several p38 MAPK cDNA fragments; the longest of which was approximately 1 kb in length (Fig 3). The DNA sequence of this fragment corresponded to approximately 90% of the coding region of p38 MAPK in carp and zebrafish (Fig. 4).

LITERATURE CITED

1. Sato, J.D., M.C. Chapline, R. Thibodeau, R.A. Frizzell, and B.A. Stanton (2007) Regulation of human cystic fibrosis transmembrane conductance regulator (CFTR) by serum- and glucocorticoid-inducible kinase (SGK1). Cell. Physiol. Biochem. 20: 91-98.

2. Shaw, J.R., J.D. Sato, J. VanderHeide, T. LaCasse, C.R. Stanton A. Lankowski, S.E. Stanton, C. Chapline, B. Coutermarsh, R. Barnaby, K. Karlson, and B.A. Stanton

(2008) The role of SGK and CFTR in acute adaptation to seawater in Fundulus heteroclitus. Cell. Physiol. Biochem., 22: 69-78.

3. Firestone, G.L., J.R. Giampaolo and B.A. O’Keeffe (2003) Stimulus-dependent regulation of serum and glucocorticoid-inducible protein kinase (SGK) transcription, subcellular localization and enzymatic activity. Cell. Physiol. Biochem. 13: 1-12.

CONCLUSIONS

Three nested fragments of killifish p38 MAPK cDNA have been cloned and sequenced.

The largest fragment (~1 kb) encodes approximately 90% of the kinase.

We have detected putative allelic polymorphisms in p38 MAPK

(~ 13% of aa residues).

5’ RACE experiments will be necessary to obtain the nucleotide sequence spanning the 5’-end of the p38 MAPK coding region.

Figure 1. Hypothesis: up-regulation of SGK function by p38 MAPK stimulates chloride secretion by CFTR from killifish gill epithelia.

.

RESULTSABSTRACT To draw a connection, whether direct or indirect, between the activities of the stress-responsive p38 MAP kinase (MAPK 14) and serum- and glucocorticoid-inducible kinase (SGK), separate gene “knockdowns” of the two kinases will be performed on killifish to compare the effects. Cloning of p38 MAPK cDNA was necessary to design an anti-sense DNA morpholino for the knockdown. First strand cDNA was synthesized from killifish liver total RNA, and putative p38 MAPK cDNA fragments were amplified with primers based on areas of homology between carp, salmon, and zebrafish p38 MAPK sequences. Several cDNA fragments were cloned, purified and sequenced. Nested p38 MAPK cDNA fragments of 400 bp, 700 bp and 1 kb were identified; the 1 kb fragment encodes approximately 90% of the killifish p38 MAPK protein.

The stress-responsive kinase p38 MAP kinase (p38 MAPK/MAPK 14) regulates mammalian SGK gene transcription. Our working hypothesis is that in response to increased salinity p38 MAPK increases CFTR activity in killifish gill tissue through its stimulation of SGK activity (Fig. 1). We are cloning killifish p38 MAPK cDNA to design anti-sense morpholino oligonucleotides.The morpholinos will be used to test our hypothesis in killifish transferred from fresh water to sea water by knocking down p38 MAPK expression and measuring survival and SGK levels in the gills of treated fish.

Figure 2. Locations of synthetic oligonucleotide primers used to amplify killifish p38 MAP kinase cDNA fragments.

Figure 3. Photograph of a 1.2% agarose gel in which 1 kb killifish p38 MAPK cDNA fragments from two cDNA preparations were resolved.

Figure 4. Alignment of the deduced amino acid sequences of p38 MAPK from carp, zebrafish, human and killifish. Asterisks denote identical residues.