1. RACE-Amplification and Cloning of Winter Flounder Psuedopleuronectes americanus Cytochrome P450 1A. Abstract: Winter flounder are found all along the US Atlantic coastline. CYP1A gene expression has been useful biomarker of environmental contaminants and exposure to these contaminants (in Winter Flounder) is known to alter CYP expression. Therefore, the goal of this research was to identify the full CYP1A coding gene by using a partial clone Cyp1A (OU99) that was identified previously from Winter Flounder, along with CYP1A Forward and Reverse Primers and the use of GeneRacer™ Kit and TOPO TA Cloning® Kit. Our results indicated that a PCR product at 329bp does confirm that the 3’ and 5’ RACE products span theS full length CYP1A gene. » Nicole Castle-Madensky and Dr. Peter F. Straub 1 1 Richard Stockton College, Pomona, NJ 08240-0195 Methods Young of the year Winter Flounder were collected from Newark Bay, NJ in the Hudson- Raritan estuary and from offshore Great Bay, NJ (Fig. 1.). Total RNA was isolated from Winter Flounder and ran on Experion RNA Analyzer to make sure RNA was still viable after being stored at -70 ⁰ C. Primers were then designed using known cytochrome P450 (CYP1A) for use with the GeneRacer™Kit to amplify the 5’ and 3’ end of the gene. The steps involved included Dephosphorylating RNA- where we treated the total RNA with calf intestinal phosphatase (CIP) to dephosphorylate non-mRNA. After, dephosphorylating and precipitating the RNA, we removed the 5’ cap structure from full-length mRNA. Next, we ligated the GeneRacer™ RNA Oligo to the 5’end of the mRNA. (The GeneRacer™ oligo is specifically designed to optimize ligation to decapped mRNA.) Then, we reversed transcribed the mRNA into cDNA using the SuperScript™III RT Reaction. (The primer contains a dT tail of 24 nucleotides.) Later, we moved on to amplifying the cDNA ends by Touchdown PCR setup with Taq DNA Polymerase High Fidelity. (Touchdown PCR increases specificity and reduces background amplification.) Finally, we ended the GeneRacer™Kit by performing a nested PCR, to increase the specificity and sensitivity of RACE products for the 5’ and 3’ ends of our gene. Once product was confirmed in our gels, then we used Wizard® SVGel and PCR Clean Up to extract and purify DNA fragments. TOPO TA Cloning®Kit was used on the 3’(CYP 1A Forward) end. (To provide an efficient one step cloning strategy “TOPO®Cloning” for the direct insertion of Taq polymerase amplified PCR products into a plasmid vector for sequencing.) Support: NSF DBI- #0619611 and NJ Sea Grant # NA16RG1047 and NOAA Fisheries for samples. l Control site Impacted site Stockton Philadelphia Fig. 1. Study sites-NJ-NY coast. The Newark Bay,is a highly impacted urban environment, The South Jersey coast, offshore of the Mullica estuary, has a low urban impact Figure 3. Polychorobiphenyl (PCB’s) concentrations (ng/g) in young of the Winter Flounder. Compared to DDTs levels found in the fish. Maroon color= Newark Bay, Light Blue = Gravelling Point (Mullica Estuary). From Dr. Straub unpublished. Fig 8. PCR amplification of the 3’ Race product, 5’ Race product, and the control partial clone Cyp1A (OU99) with gene specific cyp1A primers Fig 2. Winter Flounder samples used. RNA was extracted from these Winter Flounder on August 8, 2007. Results Fig 4. shows that the first Touchdown PCR only amplified the 3’ flounder RACE end. New PCR parameters were used to amplify the 5’ flounder RACE. We started at 55°C and allowed it drop 0.2°C every cycle for 25 cycles ending at 50°C. These conditions amplified the flounder 5’ RACE (Fig 5). Then we cut the bands from the 3’ and 5’ gels at approximately 1300bp mark and purified the RACE products (Fig 6. and Fig 7) These size bands correspond with a full length cyp1A from Pleuronectes yokohamae (a similar Japanese flounder) from GenBank. To confirm the identity of the RACE products, Cyp1A gene specific primers were used to amplify the RACE products and a partial flounder cyp1A clone (OU 99). Fig 8. Shows PCR amplification in both RACE products and the control and allowed us to confirm that indeed they are parts of the full CYP1A coding gene. Fig 6. The gel purified 3’flounder forward RACE end. Background Winter flounder are vulnerable to pollution. The cytochrome P450 (CYP) is a superfamily of heme-containing enzymes of endogenous and exogenous compounds. Because activity of these enzymes is often the determining factor in susceptibility of organisms to toxicity, knowledge of substrate specificity and regulation of CYPs has been useful in predicting toxicity across species. M 3’ 329bp Fig 7. The gel purified 5’ flounder RACE end 1300bp M5’ Plasmid M 3’ 5’ OU99 Fig 9. Is a visual diagram of the methods used in the GeneRacer™ Kit. Figure taken from GeneRacer™ manual. Fig 5. The 5’ flounder Reverse before Wizard® SV Gel Cleanup. M 5’ 1300bp M 5C 5’ 3C 3’ 1300bp Fig 4. First Touchdown PCR. (5C)The 5’ control, (5) the 5’ flounder RACE sample, (3C) the 3’ control, and (3’) the 3’ flounder RACE sample. <<<point reached Conclusion The goal of this research project was achieved, since we were able to amplify both ends of the full CYP1A coding gene by using sequence and primers from the partial clone Cyp1A (OU99). For me, the next step is to sequence the RACE products and analyze the sequence.