Conservation of DNA in New World Primates Isolation of the - - PowerPoint PPT Presentation

Comparative Genomics: Conservation of DNA in New World Primates

Isolation of the Chorionic Gonadotropin Gene Promoter in the Owl Monkey (Aotus nancymaae)

Reproductive functions

• f males and females

LH and CG: Two Glycoprotein Hormones that Regulate Reproduction

CG

Chorionic gonadotropin

Anterior pituitary

LH

Luteinizing hormone

Placenta Maintains progesterone secretion in the corpus luteum

Old World Primates (human, rhesus monkey) New World Primates (common marmoset, squirrel monkey)

CG LH

Műller et. al. 2004 J. Mol Endo 32:115 Maston and Ruvolo 2002 Mol Biol Evol 19:320

Expression of Luteinizing Hormone and Chorionic Gonadotropin

Pituitary Placenta

CG CG LH

New World Primates (common marmoset, squirrel monkey)

Pituitary Placenta

CG CG LH

How is CG expressed in the pituitary?

Isolation of the promoter region of the CG gene

Expression of Luteinizing Hormone and Chorionic Gonadotropin

Műller et. al. 2004 J. Mol Endo 32:115 Vasauskas, Hubler et al. 2011 Gen. And Comp. Endocrinol. 170:509, 170:514

New World Primate Aotus nancymaae (owl monkey)

Pituitary Placenta

CG CG LH

Expression of Luteinizing Hormone and Chorionic Gonadotropin

How is CG expressed in the pituitary?

Isolation of the promoter region of the CG gene

The owl monkey CG (omCG) gene and promoter

CG gene 3’ 5’ Promoter Exon Intron

DNA Transcription start site

Isolation of owl monkey CG (omCG) gene promoter

3’ 5’ Promoter Exon Intron

DNA Transcription start site

CG gene

Polymerase chain reaction (PCR) amplification of omCG promoter from genomic DNA

1. Isolated genomic DNA from owl monkey lymphocytes (OML) 3. Predicted 0.8 kb product 0.8 kb 4. Excised and sequenced 10 kb 3 2 1 0.5 0.25 Size std 2. PCR primers designed from squirrel monkey CG gene sequence

Forward primer Reverse primer ATG

…CCCTGAGGTATAAAGCCAGGTACACGAGGCAGGGGAGGCACCAAGGA …

5’ 3’ TATA box

3’ 5’ Promoter Exon Intron

ATG

Results

Comparison of omCG and smCG promoters 97% identity TATA box in red

• mCGp

smCGp

Query 1 CGGGGTCTCGACCGAGCCTGGACCACACTCACCGAGTCTATCGGCAATGTGCGGGCGGGA 60 ||||||||||||||||||||| ||||||||| ||||||||||||||||||||||||||| Sbjct 1 CGGGGTCTCGACCGAGCCTGGGTCACACTCACTGAGTCTATCGGCAATGTGCGGGCGGGA 60 Query 61 CTAGCCCAGACACGCCCCGCGGCTTCGCGGGGAGGGGACTAAGTCCAGAAAATATTGATG 120 ||||||||||||||||||||||||||| ||||||||| ||||||||||||||| || ||| Sbjct 61 CTAGCCCAGACACGCCCCGCGGCTTCGTGGGGAGGGGGCTAAGTCCAGAAAATGTTCATG 120 Query 121 CAGGCTGTGGCCCCGCGGGCAGGACACGCCCCCTGCGGGTGTTTTAAATAATGAGTTAAA 180 ||||||||||||||| ||||||| |||||||||||||||||||||||||||||||||||| Sbjct 121 CAGGCTGTGGCCCCGAGGGCAGG-CACGCCCCCTGCGGGTGTTTTAAATAATGAGTTAAA 179 Query 181 TAACCTGAACCACACTCATTTCTGGGGACTTCTCCGGGCATCCTGGCTTGAGAGTGGTGT 240 |||| |||||||| ||||||||||| |||| ||| |||| ||||||| |||||||||||| Sbjct 180 TAACGTGAACCACGCTCATTTCTGGAGACTGCTCTGGGCTTCCTGGC-TGAGAGTGGTGT 238 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 ||||||||||||||||||||||||||||||||||||||||||||||||||||||||| || Sbjct 659 GTCCAGGTTACCCCAGGATGCTAACACCACCTGGTGGCCTTGTCGCCCCCACAACCCCGA 718 Query 721 GGTATAAAGCCAGGTACACGAGGCAGGGGAGGCACCAAGGA 761 ||||||||||||||||||||||||||||||||||||||||| Sbjct 719 GGTATAAAGCCAGGTACACGAGGCAGGGGAGGCACCAAGGA 759

http://blast.ncbi.nlm. nih.gov/Blast.cgi

Old World Primates (human, rhesus monkey) New World Primates (common marmoset, squirrel monkey)

CG LH

Műller et. al. 2004 J. Mol Endo 32:115 Maston and Ruvolo 2002 Mol Biol Evol 19:320

Expression of Luteinizing Hormone and Chorionic Gonadotropin

Pituitary Placenta

CG CG LH

Cell specific gene expression

TATAAA

LH gene

IN HUMAN PITUITARY TATAAA

CG gene

IN HUMAN PLACENTA

Jameson and Hollenberg 1993 Endo Rev 14:2

Pituitary-specific transcription factors Placenta-specific transcription factors

TATAAA IN SM PITUITARY

CG gene

Vasauskas, Hubler et al. Gen. And Comp. Endocrinol. 170:514

Egr-1

Pituitary-specific transcription factors

Egr-1 SF-1

Conservation of pituitary-specific transcription factor binding sites in the omCG promoter

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

• mp

smp

Query 541 GACTCAGCTGAGTCTCTGAGGTCACTTCATCGTGGTCTCCGGGAGAGGGCTGGAGTGCTC 600 |||||| |||||||||||||||||||||||||||||||||||||||||||||||| |||| Sbjct 539 GACTCAACTGAGTCTCTGAGGTCACTTCATCGTGGTCTCCGGGAGAGGGCTGGAGCGCTC 598 Egr-1 Query 601 CACTGAGCCCCTGCTGCGTCTCCCTGGCCTTGTCCGCCTCCAGCCCCCGGGGGGATTAGT 660 ||||||||||||||||||||||||||||||||||||||||| |||||||||||||||||| Sbjct 599 CACTGAGCCCCTGCTGCGTCTCCCTGGCCTTGTCCGCCTCCCGCCCCCGGGGGGATTAGT 658 Egr-1 Query 661 GTCCAGGTTACCCCAGGATGCTAACACCACCTGGTGGCCTTGTCGCCCCCACAACCCTGA 720 ||||||||||||||||||||||||||||||||||||||||||||||||||||||||| || Sbjct 659 GTCCAGGTTACCCCAGGATGCTAACACCACCTGGTGGCCTTGTCGCCCCCACAACCCCGA 718 +1 Query 721 GGTATAAAGCCAGGTACACGAGGCAGGGGAGGCACCAAGGA 761 ||||||||||||||||||||||||||||||||||||||||| Sbjct 719 GGTATAAAGCCAGGTACACGAGGCAGGGGAGGCACCAAGGA 759

Summary New World Primates

(common marmoset, squirrel monkey)

Pituitary Placenta

CG CG LH

How is CG expressed in the pituitary?

CG gene 3’ 5’ Promoter

Pituitary-specific transcription factors

Műller et. al. 2004 J. Mol Endo 32:115 Vasauskas, Hubler et al. 2011 Gen. And Comp. Endocrinol. 170:509, 170:514

Summary New World Primates

(common marmoset, squirrel monkey)

• wl monkey ?

Pituitary Placenta

CG CG LH

How is CG expressed in the pituitary?

CG gene 3’ 5’ Promoter

Pituitary-specific transcription factors

Summary New World Primates

(common marmoset, squirrel monkey)

• wl monkey ?

Pituitary Placenta

CG CG LH

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-CGb gene cluster on chromosome 19

CG CG CG CG CG CG LH

Jameson and Lindell Mol & Cell Biol 1988 8:5100 Policastro et al. J. Biol Chem 1986 261:5907

52 kb

New World and Old World Primates

North America South America Eurasia Africa

New World Old World

Rhesus Cynamolgous Orangutan Human Squirrel monkey Owl monkey Common marmoset Dusky titi monkey

Heterodimers

Species-specific a subunit

Hormone-specific b subunits

= LH = CG

a b a b

LH and CG: Two Glycoprotein Hormones that Regulate Reproduction

Cell specific gene expression

TATAAA

LH gene

IN HUMAN PITUITARY TATAAA

CG gene

IN HUMAN PLACENTA

Jameson and Hollenberg 1993 Endo Rev 14:2

Pituitary-specific transcription factors Placenta-specific transcription factors

TATAAA IN NWP PITUITARY AND PLACENTA

CG gene

Pituitary-specific transcription factors Placenta-specific transcription factors