Yu-Wai P. Lin, Gerhild Packert, Teresa Petrino, Celeste Nadal, Saioa Torrealday

1. Cloning and Sequencing of Danio rerio (zebrafish) Polycomb 2 and Polycomb 3 Genes Pc3 Protein Alignment Pc2 Protein Alignment Fig. 2 Total RNA isolated from zebrafish embryos Fig. 3 Results of 5’ and 3’ RACE PCR b a 1kb M 1 2 3 4 5 67 0.5 kb M 1 2 3 4 2 kb DNA Fig.3a Lanes 1-4 show results of the Outer PCR using 5’RACE Outer primer with gene specific primers Pc3R1, R2, R3, R4. Cycle as follows: 3min-94ºC; 35 cycles: 94ºC-30sec, 57 ºC-30sec, 72ºC-2min; 10min: 72ºC. No visible product was detected. These Outer PCRs were then used as template for the nested PCR using 5’ RACE Inner primer with gene specific Pc3R5 and R6. Cycle as follows: 3min-94ºC; 35 cycles: 94ºC-30sec, 60ºC-30sec, 72ºC-1min; 10min: 72ºC. Gel electrophoresis: 10µl of the PCR products were loaded into a 1.2% agarose gel, run at 120V for 1hr, stained with Ethidium Bromide. M lane contains markers. Lane 5 is a negative control. In the nested PCR, only one product >0.5kb was obtained (lane 6) with primer Pc3R5 using template Pc3R1(Outer PCR, lane 1). This product was cloned and sequenced and contains the 5’end of ZPc3. Fig.3b PCR using 3’RACE Outer and ZfF10 primers. Cycle as follows:3min-94ºC; 35 cycles: 94ºC-30sec, 55ºC-30sec, 72ºC-2min; 10min: 72ºC. Gel electrophoresis: 25µl of the PCR reaction was loaded into a 1.2% agarose gel, run at 120V for 1hr, stained with Ethidium Bromide. M lane contains markers. All other lanes show PCR products. Expected fragment size was around 2kb. The 2kb fragment was isolated from the gel, cloned into the pGEM-T vector and sequenced (contains 3’end of ZPc2 and ZPc3). 28S 18S Fig. 2 The overall quality of the RNA preparation was determined by evaluating the intensity and sharpness of the 28S and 18S ribosomal RNA in a 1% agarose gel in TBE buffer electrophoresed at 90V for 1hr 30min. Lane 1 through 4 each contained approximately 0.5µg of the total RNA. These RNA preparations were used to obtain cDNA by reverse transcription for the subsequent 5’ and 3’ RACE PCRs. Fig. 1 Marble Breading 24 Hr Embryos Program No. 337.1 ABSTRACT: The Polycomb (Pc) gene has been implicated in the repression of gene activity during development in fruit fly, mouse, and human. The molecular mechanism underlying the role of the Pc gene in the stable transmission of gene activity has yet to be elucidated. In order to evaluate the role of Pc gene in the regulation of homeotic gene expression, the isolation and cloning of the zebrafish Pc gene was carried out in this study. For this, degenerate primers were designed based on regions of Polycomb Pc2-­like proteins and genes that show conserved amino acid and nucleotide sequences. These primers were then used in PCR to amplify specifically a region of the zebrafish Pc gene. cDNA obtained by reverse transcription of zebrafish embryonic total RNA was used as the template for the PCR reaction. A PCR product of 403 base pair was isolated, cloned, and sequenced. BLAST analysis indicated that the above zebrafish clone sequence encodes the predicted 134 amino acids of the amino terminal (chromodomain) of the Pc protein and shows 85% homology to Pc proteins of human, mouse, chicken, and frog. Based on this result, specific primers were designed to perform 5’ and 3’ RACE. The complete coding sequences for two zebrafish Pc genes: Polycomb 2 (477 amino acids) and Polycomb 3 (342 amino acids) were then determined by sequence analysis of overlaping clones [Supported by NIH-MBRS SCORE (S06 GM 45455), MARC (T34 GM 08021), NSF (DBI-0116080) and Barry Research Scholarship Grants] Yu-Wai P. Lin, Gerhild Packert, Teresa Petrino, Celeste Nadal, Saioa Torrealday School of Natural and Health Sciences, Barry University, Miami Shores, Florida 33161 RESULTS INTRODUCTION RNA isolation: Total RNA (~61 g) was isolated after homogenization of the embryos in a Lysis/binding Solution using the RNAqueous kit (Ambion) (Fig. 2). This RNA preparation was used for the reverse transcription reactions (RT) and rapid amplification of 3' and 5' cDNA ends (RACE).(Fig. 3). Isolation of the zebrafish Pc (ZPc) genes: Based on the amino acid sequences conserved in homologous protein of other species, we designed several degenerate oligonucleotide primers (Table 1). With Polycomb F2 (forward primer) and Zebra R6 (reverse primer), a partial cDNA fragment was obtained by RT-PCR (Polymerase Chain Reaction). Additional gene specific primers were synthesized based on this sequence and used in conjunction with the 5' and 3' RACE primer adapters (Ambion) to amplify the fragments corresponding to the 5' and 3' ends of zebrafish mRNA. Subcloning of PCR products: PCR products were isolated and purified from agarose gels using the MinElute gel extraction kit (Qiagen), ligated into a pGEM-T plasmid vector (Promega), and transformed into JM109 High Efficiency Competent Cells (Promega). Transformants were isolated based on blue/ white color selection, insert size confirmed by PCR and sequenced (Automated Sequencing, University of Miami, FL). Polycomb (Pc) is a member of the Polycomb group (PcG) proteins that serve as factors regulating homeotic gene expression (Lewis, 1978; Duncan and Lewis, 1982). Homeotic genes (Hox) determine the fate of cells, like those involved in the antero-posterior body segmentation, during development. These homeotic genes are expressed in precise temporal and spatially-restricted patterns. Mutation in PcG genes lead to abnormal expression of the homeotic genes in body segments where they are supposed to be inactive (Denell and Frederick, 1983). Thus Pc and PcG gene products are transcriptional repressors that silence (repress) and maintain the homeotic genes in a stable and heritable manner throughout the embryonic development. Although Pc gene was first identified in fruit fly (Drosophila) (Paro and Hogness, 1991), several homologs of Pc have been identified in vertebrates (human, Gecz et al. 1995; mouse, Pearce etal. 1992; frog, Reijnen et al. 1995), suggesting that the Pc repression of gene activity is evolutionary conserved. The molecular mechanism underlying the role of the Pc gene in the stable transmission of gene activity has yet to be elucidated. All Pc proteins had a highly conserved sequence motif called chromodomain (chromatin organization modifier) at the amino-terminal. The observation that Pc chromodomain shows a high homology to the heterochromatin-associated protein HP1 suggests that Pc activity may be mediated through its effect on chromatin structure (Paro and Hogness, 1991). In addition, a conserved carboxy-terminal COOH box (C-box) was also observed in all Pc proteins (Bardos et.al. 2000). While in Drosophila only one Pc gene is present, multiple members have been identified in more complex organisms including frog, chicken, mouse, and human. Here we describe our attempts to identify Pc genesin the zebrafish, Danio rerio, using degenerate oligonucleotide primed-polymerase chain reaction (DOP-PCR). Degenerate primers were designed based on regions of Pc chromodomain that show conserved amino acid and nucleotide sequences. These primers were then used in DOP-PCR to amplify specifically a region of the zebrafish Pc gene. So far, three Pc genes have been described for zebrafish: Pc1 (Kawamura et al., 2002 Accession No. AB074154), Pc2 and Pc3. The latter two have been identified in our lab, termed ZPc2 (Lin et al., 2003 Accession No. AY186581 ), and ZPc3 (Packert et al., 2002 Accession No. AY179330). Data on the cloning and sequencing of zebrafish ZPc2 and ZPc3 are presented. Zebrafish Polycombs Protein Alignment MATERIAL AND METHODS Animals and collection of embryos: Zebrafish (Danio rerio) were obtained from a vendor in Miami. Fish were maintained in a 25 gal. aquarium on a 14/10 hr light/ dark cycle, at 28.5C, and fed three times a day with flake food (TetraMin). Embryos were collected from fish (8 females: 4 males) producing eggs over marble as described (Westerfield,1995) (Fig. 1 ). 24 hr embryos were staged according to age in hours postfertilization and morphology, manually dechorionated with forceps (the yolk was also removed), and placed (150 embryos/3 ml/tube) in RNAlater (Ambion) at 4C for one week. CONCLUSIONS • The mature proteins for ZPc2 and Zpc3 are predicted (using conceptual translation) to be 477 and 342 amino acids(aa) long respectively. Both zebrafish Pc proteins contain the highly conserved chromodomain and COOH-Box sequence motif. • A multiple sequence alignment comparison of the ZPc2 protein sequence with human (gi|2317723), mouse (gi|2781392), chicken (gi|3649787), and Xenopus (gi|1086556) Pc2 shows: 91% (69/76) homology from aa 1 to aa 76, a region that spans the chromodomain (aa 10 to aa 50); 90% (37/41) homology in the chromodomain; and 81% (13/16) homology in the COOH-Box (aa 458 to aa 473). Several other very conserved regions (aa 121-134, aa 150-170, aa 244-281) were also observed in ZPc2 proteins. • A multiple sequence alignment comparison of the ZPc3 protein sequence with mouse (gi|6707290), rat (gi|27690850), and human (gi|9957028) Pc3 shows: 87% (82/94) homology from amino aa 1 to aa 94, a region that spans the chromodomain (aa 10 to aa 50); 90% (37/41) homology in the chromodomain (with only 4 conserved amino acids change); and 84% (26/31) homology with respect to the ~31 amino acids (aa 312-342) conserved at the COOH-Box region (aa 315-330); and 94% (15/16) homology in the COOH-Box. • A comparison between the three zebrafish Pc proteins (Pc1, ZPc2, ZPc3) shows: 58% (24/41) homology in the chromodomain and 56% (9/16) homology in the COOH-Box. REFERENCES ACKNOWLEDGEMENTS Bardos, J.I., Saurin, A.J., Tissot, C., Duprez, E., and Freemont, P.S. 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