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Strategy for Identifying and Sequencing the Polycomb Gene in Danio rerio (Zebrafish)

Strategy for Identifying and Sequencing the Polycomb Gene in Danio rerio (Zebrafish). Celeste Nadal, YW Peter Lin, Gerhild Packert and Teresa Petrino. Barry University, Miami Shores, FL. Introduction. Polycomb gene ( Pc ) Regulates gene expression by repressing other regulatory genes

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Strategy for Identifying and Sequencing the Polycomb Gene in Danio rerio (Zebrafish)

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  1. Strategy for Identifying and Sequencing the Polycomb Gene in Danio rerio (Zebrafish) Celeste Nadal, YW Peter Lin, Gerhild Packert and Teresa Petrino Barry University, Miami Shores, FL

  2. Introduction • Polycomb gene (Pc) • Regulates gene expression by repressing other regulatory genes • Mechanism of gene repression is unknown • Related to some human cancers

  3. Long-term Goal • Evaluate the role of the Pc gene in regulating gene expression using zebrafish as a vertebrate model

  4. Why zebrafish? • Extra-uterine development • Easily bred and maintained

  5. Objective of Research • Clone and sequence the Polycomb gene

  6. Methods • Multisequence Alignment • Design primers • Polymerase Chain Reaction (PCR) • Gel electrophoresis • Subclone PCR products • DNA sequence analysis

  7. Polycomb Sequence Alignment MELPAVG---------------EHVFAVESIEKKRIRKGRVEYLVKWRGW 35 Homo sapiens MELPAVG---------------EHVFAVESIEKKRIRKGRVEYLVKWRGW 35 Mus musculus MELPAVG---------------EHVFAVESIEKKRIRKGRVEYLVKWRGW 35 Gallus gallus MELPAVG---------------EHVFAVESIEKKRIRKGRVEYLVKWRGW 35 Xenopus laevis MTGRGKGSKGKLGRDNATDDPVDLVYAAEKIIQKRVKKGVVEYRVKWKGW 50 Drosophila melanogaster * . * : *:*.*.* :**::** *** ***:** SPKYNTWEPEENILDPRLLIAFQNRERQEQLMGYRKRGPKPKPLVVQVPT 85 SPKYNTWEPEENILDPRLLIAFQNRERQEQLMGYRKRGPKPNPLVVQVPT 85 SPKYNTWEPEENILDPRLLIAFQNRERQEQLMGYRKRGPKPKPLVVQLPS 85 SSKYNTWEPEENILDPRLLVAFQNRERQEQIMGYRKRGPKPKNNLVSMPS 85 NQRYNTWEPEVNILDRRLIDIYEQTNKSSGTPSKRGIKKKEKE---PDPE 97 . :******* **** **: ::: ::.. . * * : * FARRSNVLTGLQDSSTDNRAKLDLGAQGKGQGHQYELNSKKHHQYQPHSK 135 FARRSNVLTGLQDSSADNRAKLELGTQGKGQGHQYELNSKKHHQYQPHSK 135 FARRSNILTGLQDPTVDTRPKLDLGSSGKSQQHQYELNSKKHHQYQPNGK 135 FARRSNVLSGLHDSSGENRTKLDLGPIPKS--HQYQLNSKKHHQYQPSGK 133 PESEEDEYTFTENDVDTHQATTSSATHDKE--SKKEKKHHHHHHHHHHIK 145 ..: : .: :.. . .. * : : : ::**::: * E--GKPRPPGKSGKYYYQLNSKKHHPYQPDPKMYDLQYQGGHKEAPSPTC 183 ERSGKPPPARKSGKYYYQLNSKKHHPYQPDPKMYDLQYQGGHKEAPSPTC 185 E-SGVKHQSHSKGKYYYQLNSKKHHHYQPDPKMYEPHYHPSGKEPQGQAC 184 D-HNEKHHSSNKKKYYYQLNSKKHHHYQPDPKLYE-QYTIEKESQISTDV 181 S-------ERNSGRRSESPLTHHHHHHHHESKRQR-------IDHSSS–- 179

  8. Conserved Amino Acid Sequence SPKYNTWEPEENILDPRLLIAFQNRE SPKYNTWEPEENILDPRLLIAFQNRE SPKYNTWEPEENILDPRLLIAFQNRE SSKYNTWEPEENILDPRLLVAFQNRE NQRYNTWEPEVNILDRRLIDIYEQTN . :******* **** **: :::

  9. Conserved Nucleotide Sequence

  10. cDNA Library (Dr. Bruce Appel, Univ. Oregon) • 15-19 hour embyros • PolyA+ RNA • Uni-ZAP XR lambda vector • Vector primers • T3 Forward • T7 Reverse

  11. Amplifying thePc Gene T7 Poly R2 Poly F1 Poly R1 Polycomb Gene Poly F Poly R T3

  12. PCR Products • Possible amplified portions of Pc gene PolyF & T7 (>1.6) PolyF1 & T7 (>0.9) PolyF & PolyR1 (~0.7) PolyR & T3 (>0.2) ~1.6 kb ~1.5 kb ~2.2 kb ~1.5 kb ~0.7 kb ~1.5 kb ~1.3 kb ~0.8 kb ~0.7 kb PolyR1 & T3 (>0.9)

  13. Subcloning • Millipore DNA extraction • Ligation • pGem T Vector • Transformation • JM109 Competent Cells • Blue and white screening

  14. TA Plasmid Analysis Empty plasmids

  15. DNA Sequencing Analysis • Blast Comparison • Sequence similarity search tool • Nucleotide databases • Protein databases • Design specific oligonucleotide primers

  16. Conclusion • Promising PCR Results • Successful ligation of PCR products • Successful cloning of transformed cells

  17. Acknowledgements • Dr. Bruce Appel • University of Oregon • Saioa Torrealday • Flona Redway, PhD • Sister John Karen Frei, OP, PhD • MARC U*STAR/MBRS SCORE/ Barry University Research Scholarship Grants

  18. Questions

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