Comparative Genomics: Conservation of DNA in New World Primates
Isolation of the Chorionic Gonadotropin Gene Promoter in the Owl Monkey ( Aotus nancymaae )
LH and CG: Two Glycoprotein Hormones that Regulate Reproduction CG LH Luteinizing hormone Chorionic gonadotropin Anterior pituitary Placenta Reproductive functions Maintains progesterone of males and females secretion in the corpus luteum
Expression of Luteinizing Hormone and Chorionic Gonadotropin Old World Primates CG LH (human, rhesus monkey) New World Primates (common marmoset, squirrel monkey) CG LH CG Pituitary Placenta Maston and Ruvolo 2002 Mol Biol Evol 19:320 M űller et. al. 2004 J. Mol Endo 32:115
Expression of Luteinizing Hormone and Chorionic Gonadotropin New World Primates CG LH (common marmoset, squirrel monkey) CG Pituitary Placenta How is CG expressed in the pituitary? Isolation of the promoter region of the CG gene M űller et. al. 2004 J. Mol Endo 32:115 Vasauskas, Hubler et al. 2011 Gen. And Comp. Endocrinol. 170:509, 170:514
Expression of Luteinizing Hormone and Chorionic Gonadotropin New World Primate Aotus nancymaae (owl monkey) CG LH CG Pituitary Placenta How is CG expressed in the pituitary? Isolation of the promoter region of the CG gene
The owl monkey CG (omCG) gene and promoter Transcription start site Promoter 5’ 3’ DNA Exon Intron CG gene
Isolation of owl monkey CG (omCG) gene promoter Transcription start site Promoter 5’ 3’ DNA Exon Intron CG gene
Polymerase chain reaction (PCR) amplification of omCG promoter from genomic DNA 1. Isolated genomic DNA from owl monkey lymphocytes (OML) PCR primers designed from squirrel monkey CG gene sequence 2. ATG Forward Reverse Size primer primer std 10 kb 3 3. Predicted 0.8 kb product 2 0.8 kb 1 4. Excised and sequenced 0.5 0.25
Results Promoter 5’ 3’ ATG Exon Intron 5’ 3’ …CCCTGAGG TATAAA GCCAGGTACACGAGGCAGGGGAGGCACCAAGGA … TATA box
Comparison of omCG and smCG promoters 97% identity omCGp Query 1 CGGGGTCTCGACCGAGCCTGGACCACACTCACCGAGTCTATCGGCAATGTGCGGGCGGGA 60 smCGp ||||||||||||||||||||| ||||||||| ||||||||||||||||||||||||||| Sbjct 1 CGGGGTCTCGACCGAGCCTGGGTCACACTCACTGAGTCTATCGGCAATGTGCGGGCGGGA 60 Query 61 CTAGCCCAGACACGCCCCGCGGCTTCGCGGGGAGGGGACTAAGTCCAGAAAATATTGATG 120 ||||||||||||||||||||||||||| ||||||||| ||||||||||||||| || ||| Sbjct 61 CTAGCCCAGACACGCCCCGCGGCTTCGTGGGGAGGGGGCTAAGTCCAGAAAATGTTCATG 120 Query 121 CAGGCTGTGGCCCCGCGGGCAGGACACGCCCCCTGCGGGTGTTTTAAATAATGAGTTAAA 180 ||||||||||||||| ||||||| |||||||||||||||||||||||||||||||||||| Sbjct 121 CAGGCTGTGGCCCCGAGGGCAGG-CACGCCCCCTGCGGGTGTTTTAAATAATGAGTTAAA 179 http://blast.ncbi.nlm. Query 181 TAACCTGAACCACACTCATTTCTGGGGACTTCTCCGGGCATCCTGGCTTGAGAGTGGTGT 240 |||| |||||||| ||||||||||| |||| ||| |||| ||||||| |||||||||||| Sbjct 180 TAACGTGAACCACGCTCATTTCTGGAGACTGCTCTGGGCTTCCTGGC-TGAGAGTGGTGT 238 nih.gov/Blast.cgi Query 241 GGGAGAAAGTTCCTAAGGGAGAGGTGGGGCTCCGGCTGAATCCCTGTTTGGTGAGTATCT 300 ||||||| |||||||||||||||||||||||||||||||||||||||||||||||||||| Sbjct 239 GGGAGAAGGTTCCTAAGGGAGAGGTGGGGCTCCGGCTGAATCCCTGTTTGGTGAGTATCT 298 Query 301 GAGTCCAGCGGCTCCCCTCGTAGTAGTCTCGCGGAGGCCCTCTCGGACTGGGCAGAAGCT 360 | |||||||||||||||||| || |||||||||||||||||||||||||||||||||||| Sbjct 299 GGGTCCAGCGGCTCCCCTCGCAGCAGTCTCGCGGAGGCCCTCTCGGACTGGGCAGAAGCT 358 Query 361 AACACCGAAGCCGCGGCGCTCCTGGGAATCTGGACTGTGGTGCAGGAATGGCTCTGGTGG 420 |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| Sbjct 359 AACACCGAAGCCGCGGCGCTCCTGGGAATCTGGACTGTGGTGCAGGAATGGCTCTGGTGG 418 Query 421 AGGTGAGTTGACGCTTCAGTCTAGTGCCTTTCTTGGGTCACGGCCTCCTCCTGGCTCCCC 480 ||||||||||||||||||||||||| |||||||||||||||||||||||||||||||||| Sbjct 419 AGGTGAGTTGACGCTTCAGTCTAGTACCTTTCTTGGGTCACGGCCTCCTCCTGGCTCCCC 478 Query 481 CGCCTCAGGAAGGGACAGGGCGTGGCCTTTCTGTACCCTACTTCCTGTGCCTCCAACCCC 540 |||||||||||| ||||||||| ||||||||||||||||||||||||||||||||||||| Sbjct 479 CGCCTCAGGAAGAGACAGGGCGAGGCCTTTCTGTACCCTACTTCCTGTGCCTCCAACCCC 538 Query 541 GACTCAGCTGAGTCTCTGAGGTCACTTCATCGTGGTCTCCGGGAGAGGGCTGGAGTGCTC 600 |||||| |||||||||||||||||||||||||||||||||||||||||||||||| |||| Sbjct 539 GACTCAACTGAGTCTCTGAGGTCACTTCATCGTGGTCTCCGGGAGAGGGCTGGAGCGCTC 598 Query 601 CACTGAGCCCCTGCTGCGTCTCCCTGGCCTTGTCCGCCTCCAGCCCCCGGGGGGATTAGT 660 ||||||||||||||||||||||||||||||||||||||||| |||||||||||||||||| Sbjct 599 CACTGAGCCCCTGCTGCGTCTCCCTGGCCTTGTCCGCCTCCCGCCCCCGGGGGGATTAGT 658 Query 661 GTCCAGGTTACCCCAGGATGCTAACACCACCTGGTGGCCTTGTCGCCCCCACAACCCTGA 720 TATA box ||||||||||||||||||||||||||||||||||||||||||||||||||||||||| || Sbjct 659 GTCCAGGTTACCCCAGGATGCTAACACCACCTGGTGGCCTTGTCGCCCCCACAACCCCGA 718 in red Query 721 GGTATAAAGCCAGGTACACGAGGCAGGGGAGGCACCAAGGA 761 ||||||||||||||||||||||||||||||||||||||||| Sbjct 719 GGTATAAAGCCAGGTACACGAGGCAGGGGAGGCACCAAGGA 759
Expression of Luteinizing Hormone and Chorionic Gonadotropin Old World Primates CG LH (human, rhesus monkey) New World Primates (common marmoset, squirrel monkey) CG LH CG Pituitary Placenta Maston and Ruvolo 2002 Mol Biol Evol 19:320 M űller et. al. 2004 J. Mol Endo 32:115
Cell specific gene expression Pituitary-specific transcription factors TATAAA Egr-1 IN HUMAN PITUITARY LH gene Placenta-specific transcription factors TATAAA SF-1 CG gene IN HUMAN PLACENTA Pituitary-specific transcription factors TATAAA Egr-1 CG gene IN SM PITUITARY Jameson and Hollenberg 1993 Endo Rev 14:2 Vasauskas, Hubler et al. Gen. And Comp. Endocrinol. 170:514
Conservation of pituitary-specific transcription factor binding sites in the omCG promoter omp Query 541 GACTCAGCTGAGTCTCTGAGGTCACTTCATCGTGGTCTCCGGGAGAGGGCTGGAGTGCTC 600 |||||| |||||||||||||||||||||||||||||||||||||||||||||||| |||| smp Sbjct 539 GACTCAACTGAGTCTCTGAGGTCACTTCATCGTGGTCTCCGGGAGAGGGCTGGAGCGCTC 598 Egr-1 Query 601 CACTGAGCCCCTGCTGCGTCTCCCTGGCCTTGTCCGCCTCC AGCCCCCGG GGGGATTAGT 660 ||||||||||||||||||||||||||||||||||||||||| |||||||| |||||||||| Sbjct 599 CACTGAGCCCCTGCTGCGTCTCCCTGGCCTTGTCCGCCTCC CGCCCCCGG GGGGATTAGT 658 Egr-1 Query 661 GTCCAGGTTACCCCAGGATGCTAACACCACCTGGTGGCCTTGT CGCCCCCAC AACCCTGA 720 ||||||||||||||||||||||||||||||||||||||||||| ||||||||| ||||| || Sbjct 659 GTCCAGGTTACCCCAGGATGCTAACACCACCTGGTGGCCTTGT CGCCCCCAC AACCCCGA 718 +1 Query 721 GGTATAAAGCCAGGTACACGAGGCAGGGGAG G CACCAAGGA 761 ||||||||||||||||||||||||||||||| | ||||||||| Sbjct 719 GGTATAAAGCCAGGTACACGAGGCAGGGGAG G CACCAAGGA 759 1. Transcription start site indicated in blue 2. One base pair difference in distal Egr1 (in red) 3. Suggests similar tissue expression of CG in pituitary of om and sm
Summary CG LH New World Primates (common marmoset, squirrel monkey) CG Pituitary Placenta How is CG expressed in the pituitary? CG gene Promoter 5’ 3’ Pituitary-specific transcription factors M űller et. al. 2004 J. Mol Endo 32:115 Vasauskas, Hubler et al. 2011 Gen. And Comp. Endocrinol. 170:509, 170:514
Summary CG LH New World Primates (common marmoset, squirrel monkey) CG owl monkey ? Pituitary Placenta How is CG expressed in the pituitary? CG gene Promoter 5’ 3’ Pituitary-specific transcription factors
Summary CG LH New World Primates (common marmoset, squirrel monkey) CG owl monkey ? Pituitary Placenta How is CG expressed in the pituitary? 1. Functional assays of promoter 2. Analysis of mRNA
Acknowledgments Dr. Tina Hubler University of North Alabama Department of Biology University of North Alabama Intramural Research Grants Dr. Jonathan Scammell, University of South Alabama
The human LH-CG b gene cluster on chromosome 19 CG CG CG CG CG CG LH 52 kb Jameson and Lindell Mol & Cell Biol 1988 8:5100 Policastro et al. J. Biol Chem 1986 261:5907
New World and Old World Primates New World Old World North Eurasia America Africa South America Squirrel monkey Rhesus Owl monkey Cynamolgous Common marmoset Orangutan Dusky titi monkey Human
LH and CG: Two Glycoprotein Hormones that Regulate Reproduction Heterodimers a b = LH a b = CG Species-specific a subunit Hormone-specific b subunits
Cell specific gene expression Pituitary-specific transcription factors TATAAA IN HUMAN PITUITARY LH gene Placenta-specific transcription factors TATAAA CG gene IN HUMAN PLACENTA Placenta-specific Pituitary-specific transcription factors transcription factors TATAAA CG gene IN NWP PITUITARY AND PLACENTA Jameson and Hollenberg 1993 Endo Rev 14:2
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