RACE-Amplification and Cloning of Bluefish Pomatomus saltatrix Cytochrome P450 1A.

1. RACE-Amplification and Cloning of Bluefish Pomatomus saltatrix Cytochrome P450 1A. Rachel Sura and Dr. Peter F. Straub Richard Stockton College, Pomona, NJ 08240-0195 • Abstract: • Our study attempted to clone the entire bluefish Cytochrome P 450 1A gene using a RT-PCR process. To do this, a GeneRacer (Invitrogen) kit, RNA ligase-mediated rapid amplication of 5’ and 3’ of cDNA ends, was used with total RNA of bluefish from clean waters of Gravelling Point in South Jersey and bluefish from polluted Newark Bay in the Bayonne area of North Jersey. Primers were ligated onto the RNA, a 5’ primer to the 5’ end and a 3’ poly T primer to the 3’ end. Transcription led to formation of cDNA, which was amplified with cytochrome P450 1A gene specific primers (from previous studies) to obtain a 5’ and 3’ PCR product to overlap the entire gene (to clone and sequence). The results were a 3’ product not well resolved on the gel electrophoresis and two distinct 5’ products, both long and short shown on the gel. The two 5’ products were cut out of the gel and purified using a SV Gel Purification and PCR Clean-Up System (Promega). The products were checked by a standard PRC with GSP forward and reverse primers. Both 5’samples and the control showed diagnostic PCR product. In conclusion the RACE amplified 5’ products are both part of the bluefish CYP1A gene. • Conclusion: • This research project achieved the RACE amplification of the CYP1A 5’ end and obtained an unresolved 3’ product. Both 5’ samples and the control showed diagnostic PCR product. Therefore, the RACE amplified 5’ products are both part of the bluefish CYP1A gene which used the using CYP1A forward and reverse primers. Future research would be to clone and sequence the 5’ end and resolve the 3’ end by another PCR reaction. Results Our gel results show that a short band at 800 bp and long band at 1200 bp was seen in the 5’ RACE while the 3’ product was a smear (Fig. 3). The 800 and 1200 bp bands were cut out and purified. On PCR analysis the result was that both the 5’ 800 and 1200 bp and control produced CYP1A a specific PRC product of 300bp (Fig. 4). Introduction: Bluefish are migratory marine fish that is distributed all over the world. They are especially important in the Northeast for recreational and commercial fishing. Local bays and rivers are nurseries for juvenile bluefish, called snappers. However, in the urban areas’ bays and rivers have high levels of pollution, mainly hydrocarbons from oil and PCBs. To see how pollution affected the bluefish, the enzyme Cytochrome P 450 1A, important in liver detoxifying pollutants within the body was examined. Our study attempted to clone the entire bluefish Cytochrome P 450 1A gene using a RT-PCR process. l Methods Bluefish RNA was extracted from fish that came from two different geographic locations (Fig. 2) within New Jersey; Bayonne-Newark Bay (North Jersey) and Gravelling Point (South Jersey). The Bayonne Bluefish represents a specimen from polluted waters, where as Gravelling Point has cleaner water. Total RNA was isolated from Bluefish and run on a BioRad Experion RNA Analyzer to make sure RNA was still intact after being stored at -70ºC for four years. Primers for cytochrome P450 (CYP1A) sequence were used with a GeneRacer™Kit (Invitrogen) to amplify the 5’ and 3’ end of the gene (Fig. 1). The total RNA from each sample was treated with calf intestinal phosphates (CIP) to remove the 5’ phosphates, this dephosphorylated the RNA. With the RNA dephosphorylated, tobacco acid pyrophosphate (TAP) was applied to remove the 5’ cap structure that led to an open 5’ phosphate which is necessary for ligation. The GeneRacer RNA Oligo was ligated to the 5’ end of the mRNA, providing a known priming site for the GeneRacer RNA Oligo primer. This ligated mRNA was reverse transcribed with SuperScript III RT and the GeneRacer Oligo dT primer. The transcription led to the first strand of cDNA, with known priming sites on the 5’ and 3’ ends. The 5’ and 3’ ends of the CYP1A were obtained by RACE amplication of the cDNA using CYP1A gene specific primer (GSP) reverse CYP1A GSP and GeneRacer 5’ Primer for the 5’ end and forward CYP1A GSP and GeneRacer 3’ Primer at the 3’ end. Agarose gel electrophoresis was used to analyze the PCR products and showed Bayonne 5’ end to have discrete products at 800bp and 1200bp. The 3’ product was represented by a smear on the gel. The 5’ products at 800bp and 1200bp were cut out of the gel and purified by SV Gel Purification and PCR Clean-Up System. The purified gel products were used in a PCR reaction with CYP1A gene specific reverse and forward primers. The control for the PCR reaction was a plasmid UP019 which contained a partial CYP1A clone from bluefish, isolated previously. M L S C hh 300 bp 3’ M 5’ Figure 4: Shows PCR amplification of a short diagnostic product of the long (L0 and short (S) 5’ products and the control (C) bluefish CYP1A UP019 plasmid (partial clone). M is 100 bp ladder. Figure 3: The 5’ and 3’ RACE product. M is 100 bp ladder. Figure 2: New Jersey Locations of Bluefish: Stared area is Bayonne Bay (highly polluted) Starfish area is Gravel Point (low pollution) Figure 1. Outline of procedures for GeneRacer™Kit (Invitrogen) Support: Richard Stockton College, The National Research Council, Research Associate Program; NSF DBI-#0619611 and NJ Sea Grant # NA16RG1047.