1. Template provided by: “posters4research.com” CONCLUSIONS AND FUTURE WORKS REFERENCE Cytochrome P450 (CYPs) are known to play a central role in insecticide resistance, typically through up regulation in resistant individuals allowing them to detoxify insecticides at a higher rate. Cytochrome P450 (CYPs) are known to play a central role in insecticide resistance, typically through up regulation in resistant individuals allowing them to detoxify insecticides at a higher rate. Among them, members of CYP6M2 gene have been repeatedly implicated in resistance to insecticides. Among them, members of CYP6M2 gene have been repeatedly implicated in resistance to insecticides. To our knowledge, there have been no studies into understanding the complex mechanisms regulating CYP6M2 gene expression in insecticide resistance in Anopheles gambiae. To our knowledge, there have been no studies into understanding the complex mechanisms regulating CYP6M2 gene expression in insecticide resistance in Anopheles gambiae. Understanding these mechanisms will potentially have implication in the control of malaria. Understanding these mechanisms will potentially have implication in the control of malaria. To isolate and characterise 5’ 896 bp region upstream of CYP6M2 hypothesised to contain the promoter in insecticide resistant Tiassalé and susceptible Kisumu strains of Anopheles gambiae. To isolate and characterise 5’ 896 bp region upstream of CYP6M2 hypothesised to contain the promoter in insecticide resistant Tiassalé and susceptible Kisumu strains of Anopheles gambiae. Cytochrome P450 (CYPs) are known to play a central role in insecticide resistance, typically through up regulation in resistant individuals allowing them to detoxify insecticides at a higher rate. Cytochrome P450 (CYPs) are known to play a central role in insecticide resistance, typically through up regulation in resistant individuals allowing them to detoxify insecticides at a higher rate. Among them, members of CYP6M2 gene have been repeatedly implicated in resistance to insecticides. Among them, members of CYP6M2 gene have been repeatedly implicated in resistance to insecticides. To our knowledge, there have been no studies into understanding the complex mechanisms regulating CYP6M2 gene expression in insecticide resistance in Anopheles gambiae. To our knowledge, there have been no studies into understanding the complex mechanisms regulating CYP6M2 gene expression in insecticide resistance in Anopheles gambiae. Understanding these mechanisms will potentially have implication in the control of malaria. Understanding these mechanisms will potentially have implication in the control of malaria. To isolate and characterise 5’ 896 bp region upstream of CYP6M2 hypothesised to contain the promoter in insecticide resistant Tiassalé and susceptible Kisumu strains of Anopheles gambiae. To isolate and characterise 5’ 896 bp region upstream of CYP6M2 hypothesised to contain the promoter in insecticide resistant Tiassalé and susceptible Kisumu strains of Anopheles gambiae. METHODS RESULTS [1].Misra, R. Jyoti., Lam, G., Thummel, S. Carl. Constitutive activation of the Nrf2/Keap1 pathway in insecticide- resistant strains of Drosophila, Insect Biochemistry and Molecular Biology, 43(12): 111 -1124, 2013. We thank Tertiary Education Trust Fund (TETFUND) and University of Abuja, Nigeria for sponsoring this research Correspondence– balarabemohammed161@yahoo.co.uk Phylogenetic Tree of the Anopheles gambiae CYP6M2 gene sequence Phylogenetic Tree of the Anopheles gambiae CYP6M2 gene sequence Genomic DNA and mRNA sequence retrieval Genomic DNA and mRNA sequence retrieval Analysis of CYP6M2 gene sequence and putative promoter region for CYP6M2 Analysis of CYP6M2 gene sequence and putative promoter region for CYP6M2 Putative transcription binding sites found along Anopheles gambiae at 70% cut off score Figure 1.3: TFBS analysis on 896 bp CYP6M2 promoter. Reverse compliment (http://www.Bioinformatics.rg/sms/ndex.html)was performed on the sequenced CYP6M2 Figure 1.3: TFBS analysis on 896 bp CYP6M2 promoter. Reverse compliment (http://www.Bioinformatics.rg/sms/ndex.html)was performed on the sequenced CYP6M2.http://www.Bioinformatics.rg/sms/ndex.html Figure 1.1: Phylogenetic tree of CYP6M2 gene sequence (Ensembl Genome Browser). The following tree was generated by Ensembl and takes gene orthologs and paralologs into account. The phylogenic tree highlights “CYP6M2, Anopheles gambiae” in red font. Paralogues have been shown in blue. Figure 1.2: Shows image of the retrieved CYP6M2 promoter gene indicating the CYP6M2 protein coding (Red boxes) entire forward and reverse strands of 896 bp (Black arrows). Multiple amino acid sequence alignments of CnCC from Drosophila melanogaster, Nf2e1 from Anopheles gambiae and Nrf2 from Homo sapiens Figure 1.4B: Amino acid alignment of FBgn003513 (Ss) sequences from D. melanogaster (Dm), with AGAP010259 from Anopheles gambiae (Ag) and ENST000242057 (AhR) in Homo sapiens (Hs) species. Multiple amino sequence alignments of Spineless from D. melanogaster, AGAP010259 from Anopheles gambiae and AhR from Homo sapiens Multiple amino sequence alignments of Spineless from D. melanogaster, AGAP010259 from Anopheles gambiae and AhR from Homo sapiens Figure 1.4A : Amino acid alignment of FBgn0262975 (CnCC) sequences from Drosophila melanogaster (Dm), with AGAP005300 Anopheles gambiae (Ag) and ENSG00000116044.11 (Nrf2) Homo sapiens (Hs) species. Mammalian Drosophila melanogaster Anopheles gambiae AhR Ss AGAP010259 ARNT Tgo AGAP009748 Nrf2 CnCC Nf2e1 Keap 1 dKeap 1 AGAP003645 Key: AhR (Aryl hydrocarbon receptor), ARNT (Aryl hydrocarbon nuclear translocator), Ss (Spineless), Tgo (Tango), Nrf2 (Nuclear factor erythroid 2 – related factor 2), CnCC (Cap ‘n’ collar isoform C), Nf2e1 (Nuclear factor erythroid 2 invertebrate). Key: AhR (Aryl hydrocarbon receptor), ARNT (Aryl hydrocarbon nuclear translocator), Ss (Spineless), Tgo (Tango), Nrf2 (Nuclear factor erythroid 2 – related factor 2), CnCC (Cap ‘n’ collar isoform C), Nf2e1 (Nuclear factor erythroid 2 invertebrate). Table 1.1: The orthologs to AhR /ARNT and Nrf2 /ARE Table 1.1: The orthologs to AhR /ARNT and Nrf2 /ARE Gene IDAAEL009496AGAP008212Fbgn0000473CPIJ016809 ENSG000000 59377 AAEL009496100.0044.9937.2635.2735.18 AGAP00821244.99100.0036.1837.7736.79 Fbgn000047337.2636.18100.0042.4040.75 CPIJ01680935.2737.7742.40100.0043.55 ENSG000000 59377 35.1836.7940.7543.55100.00 Table 1.2: Percent Identity Matrix - created by Clustal2.1 Table 1.3: The prediction of TSSs with upstream region in the cloned CYP6M2_Kis and CYP6M2_Tias Promoter Length (bp) position TATA BOX (TSS)cPG count Ratio of O/E (%GC) CYP6M2_Kis-1915-824-97420.7550.50-53.00 CYP6M2_Kis-2896-818-108310.9950.50-52.50 CYP6M2_Tias-1929-838-50560.7850.50-53.00 CYP6M2_Tias-2929-838-50560.7850.50-53.00 Figure 1.5A: Sequence alignment and TFBS analysis of Anopheles gambiae gene (CYP6M2) promoter using conservation cut off score of 30% and TF score threshold of 80% Figure 1.5B: Sequence alignment and TFBS analysis of Drosophila melanogaster (CYP6G1) gene promoter using conservation cut off Score of 30% and TF score threshold of 80%. The present insilico study established the presence of putative AhR / ARNT (AGAP010259 / AGAP009748) and / or Nrf2 / Keap 1 (AGAP005300/ AGAP003645) binding sites in the region upstream of CYP6M2 in Anopheles gambiae. The present insilico study established the presence of putative AhR / ARNT (AGAP010259 / AGAP009748) and / or Nrf2 / Keap 1 (AGAP005300/ AGAP003645) binding sites in the region upstream of CYP6M2 in Anopheles gambiae. This potentially suggests that CYP6M2 is under the control of Nrf2 and or AhR through in the 5’–flanking region. This potentially suggests that CYP6M2 is under the control of Nrf2 and or AhR through in the 5’–flanking region. It is therefore proposed that AhR / ARNT or Nrf2 / Keap 1 pathways are involved in the cellular network that maintains redox homeostasis in order to protect cells from oxidative stress induced by their respective ligands. It is therefore proposed that AhR / ARNT or Nrf2 / Keap 1 pathways are involved in the cellular network that maintains redox homeostasis in order to protect cells from oxidative stress induced by their respective ligands. In additional experiments, we have used the dual luciferase assay to investigate the promoter activity of the region upstream of CYP6M2 (See Mohammed et al., 2014 a, b and c ) In additional experiments, we have used the dual luciferase assay to investigate the promoter activity of the region upstream of CYP6M2 (See Mohammed et al., 2014 a, b and c for details ) ACKNOWLEDGEMENTS Prediction of putative promoter elements within CYP6M2 gene Figure 1.6 A, B, C & D: McPromoter analysis of the 896 bp region upstream CYP6M2_Kis-1, Kis-2,Tias-1 and Tias-2 promoter. Grey lines indicate the boundaries of CpG dinucleotides identified using the UCSC browser (http://tools. genome.duke.edu/gene regulation/McPromoter/). http://tools. genome.duke.edu/gene regulation/McPromoter/http://tools. genome.duke.edu/gene regulation/McPromoter/ A B C D CYP6M2_Kis-1 CYP6M2_Kis-2 CYP6M2_Tias-1 CYP6M2_Tias-1 CYP6M2_Tias-2 CYP6M2_Tias-2 Insilico Analysis of Selected Regulatory Elements within the Promoter Region of the Cytochrome P450 Gene, CYP6M2 in Anopheles gambiae 1 Balarabe R. Mohammed, 2 Craig, S. Wilding, 1 Collier J. Phillip and 1 Deeni Y. Yusif 1 School of Science, Engineering and Technology, Abertay University, Dundee, United Kingdom 2 School of Natural Science and Psychology, Liverpool John Moores University, Liverpool, United Kingdom INTRODUCTION AIM RESULTS CONT. - -885-890 bp - -349-359 bp -32- 42 bp 6-11bp - 6-11bp RESULTS CONT.. YOUNG LIFE SCIENTISTS’ SYMPOSIUM (YLS), 2015 REDBLUEMAGENTAGREEN Small+ hydrophobic AcidicBasic Hydoroxyl + Amin + Basic REDBLUEMAGENTAGREEN Small+ hydrophobic AcidicBasic Hydoroxyl + Amin + Basic CYP6M2 promoter genomic sequence retrieval from the genomic database Phylogenetic tree of CYP6M2 gene sequence