Gonadotropin Beta Subunit Gene in the Squirrel Monkey ( Saimiri - PowerPoint PPT Presentation

Organization of the Chorionic Gonadotropin Beta Subunit Gene in the Squirrel Monkey ( Saimiri boliviensis )

Glycoprotein Hormones Regulating Gonad Function CG FSH LH Follicle stimulating hormone Luteinizing hormone Chorionic gonadotropin Anterior pituitary Anterior pituitary Placenta Maintains progesterone Reproductive functions secretion in the of males and females corpus luteum

Glycoprotein Hormones Regulating Gonad Function Heterodimers a b = LH a b = CG Species-specific a subunit Hormone-specific b subunits

Expression of Luteinizing Hormone and Chorionic Gonadotropin Old World Primates CG β LH β (humans,rhesus monkey) New World Primates CG β LH β (common marmoset, squirrel monkey) CG β 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 β LH β (common marmoset, squirrel monkey) CG β Pituitary Placenta Does CG assume LH function? How is CG expressed in the pituitary? 1. Isolate CG b gene 2. Isolate promoter region of the CG b gene

Isolation of squirrel monkey CG b (smCG b ) gene Transcription start site Promoter 5’ 3’ DNA Exon Intron CG β gene

Polymerase chain reaction (PCR) amplification of smCG b from genomic DNA 1. Isolated genomic DNA from squirrel monkey lymphocytes (SML) Start Stop codon codon 2. PCR primers ATG TAA human CG b gene Forward Reverse primer primer 3. Predicted 1.1 kb product 1.1 kb 4. Excised and sequenced

smCG b gene sequence Start codon ATGGAGATGC TCCAGGTAAG ACTGCAGGGC CCCTGGGTAC CTTCCACCGC Exon 1 CCTCCAGGCC ATCACTGGCA TGAAGAGGGG CAGAGTCGTG TGAGCTGGGG AAGGAGGCCT TTTTCTGGAG GGGTGTGACT CTGCAGTAAG CTTCAGGTGG AGAAGTCCCT GAGGGTGGAG AACTGAAATG TTGGGCTGGG GGTGGGCTCT GAAAGGCAGG TGTCTGGGTG GCAGGTCCTG AATAAGACAT GCCAGGCAGG Intron A GTCCCTGGGT CCTTGAGGGT GGTATACCCC TGGGGATGGG CCAGGGCTCA GGGCTTCAGT CTCAGGCTCG GCTGAAGCAC CGGTCTTGTC CCAGGGACTG CTGCTGTGTC TGCTGCTGAG CACAGGTGGG GCATGGGCAT CCAAGGAGCC ACTTCGGCCG CCGTGCCGCC CCACCAATGT CATCCTGGCT GTTGAGAAGG Exon 2 AGGGCTGCCC TGTTTGCGTT CCCTTCAACA CCACCATCTG CGCCGGCTAC TGCTCCAGCA TGGTGAGCTG CCCGGGACCG GGGGCAGGTG CTGCCACCTC AGGGCGGGGC CCACAGAGGC ACTGGGGAAG GGTGTCTGGC TCTCTGGGCA GGGGCTGGGA AATGGGGCTG GAGGGCAGGA ACAGATGGCT TCCTGGACAT Intron B GAGTCTGGGA CCTGTGGAGG GGGCTGGGGT GCTCAGCTGA GGTGCTGGCC CCCAGACACA TGCCCACTCT CCCACCCACA TGGCCTTAGG TACGAGTGAT GCAGACCTTG CCGCCCTTAC CCCAGACGGT GTGCAACTAC CACGAGCTGC GCTTCACCTC CGTCCGGCTC CCTGGCTGTC GGCGCGGCGT GGATCCCGTG Exon 3 GTCTACATGC CCATGGCTGT CAGCTGTCGC TGTGCACTCT GCCGCCGAAG CTATTCTGAC TGTGGGAGTT TCAGGAACGA GTCCCTGGGC TGTGACTACG CCACCTCCCA GGACTCTTCC TCTAATGTCC CTCCCAGCAA CCTTACAAGT Stop CCATCCCAAC TCCTGGAGCC AGCAGTCACT CCATTAGTCC CACAATAA codon

smCG b gene sequence ATGGAGATGC TCCAGGTAAG ACTGCAGGGC CCCTGGGTAC CTTCCACCGC CCTCCAGGCC ATCACTGGCA TGAAGAGGGG CAGAGTCGTG TGAGCTGGGG AAGGAGGCCT TTTTCTGGAG GGGTGTGACT CTGCAGTAAG CTTCAGGTGG AGAAGTCCCT GAGGGTGGAG AACTGAAATG TTGGGCTGGG GGTGGGCTCT GAAAGGCAGG TGTCTGGGTG GCAGGTCCTG AATAAGACAT GCCAGGCAGG GTCCCTGGGT CCTTGAGGGT GGTATACCCC TGGGGATGGG CCAGGGCTCA GGGCTTCAGT CTCAGGCTCG GCTGAAGCAC CGGTCTTGTC CCAGGGACTG CTGCTGTGTC TGCTGCTGAG CACAGGTGGG GCATGGGCAT CCAAGGAGCC ACTTCGGCCG CCGTGCCGCC CCACCAATGT CATCCTGGCT GTTGAGAAGG AGGGCTGCCC TGTTTGCGTT CCCTTCAACA CCACCATCTG CGCCGGCTAC TGCTCCAGCA TGGTGAGCTG CCCGGGACCG GGGGCAGGTG CTGCCACCTC AGGGCGGGGC CCACAGAGGC ACTGGGGAAG GGTGTCTGGC TCTCTGGGCA GGGGCTGGGA AATGGGGCTG GAGGGCAGGA ACAGATGGCT TCCTGGACAT GAGTCTGGGA CCTGTGGAGG GGGCTGGGGT GCTCAGCTGA GGTGCTGGCC CCCAGACACA TGCCCACTCT CCCACCCACA TGGCCTTAGG TACGAGTGAT GCAGACCTTG CCGCCCTTAC CCCAGACGGT GTGCAACTAC CACGAGCTGC GCTTCACCTC CGTCCGGCTC CCTGGCTGTC GGCGCGGCGT GGATCCCGTG GTCTACATGC CCATGGCTGT CAGCTGTCGC TGTGCACTCT GCCGCCGAAG CTATTCTGAC TGTGGGAGTT TCAGGAACGA GTCCCTGGGC TGTGACTACG CCACCTCCCA GGACTCTTCC TCTAATGTCC CTCCCAGCAA CCTTACAAGT CCATCCCAAC TCCTGGAGCC AGCAGTCACT CCATTAGTCC CACAATAA GT- AG rule for introns : GT……AG

sm CG b ( smCG b) protein -20 +1 SmCGβ MEMLQGLLLC LLLSTGGAWA SKEPLRPPCR PTNVILAVEK EGCPVCVPFN TTICAGYCSS 40 H CGβ ---F-----L ----M--T-- -------R-- -I-AT----- ------ITV- --------PT 40 SmCGβ MVRVMQT*LP PLPQTVCNYH ELRFTSVRLP GCRRGVDPVV YMPMAVSCRC ALCRRSYSDC 99 H CGβ -T--L-GV-- A---V----R DV--E-I--- --P---N--- SYAV-L--Q- ------TT-- 100 SmCGβ GSFRNESLGC DYATSQD*SS SNVPPS NLTS PSQLLEPAVT PLVPQ 143 H CGβ -GPKDHP-T- -DPRF--S-- -KA--PS-P- --R-PG-SD- -IL-- 145

Homology to human CG b ( hCG b) protein 64 % identity 76 % similarity -20 +1 Sm CGβ MEMLQGLLLC LLLSTGGAWA SKEPLRPPCR PTNVILAVEK EGCPVCVPF N TTICAGYCSS 40 H CGβ ---F-----L ----M--T-- -------R-- -I-AT----- ------ITV- --------PT 40 Sm CGβ MVRVMQT*LP PLPQTVCNYH ELRFTSVRLP GCRRGVDPVV YMPMAVSCRC ALCRRSYSDC 99 H CGβ -T--L-GV-- A---V----R DV--E-I--- --P---N--- SYAV-L--Q- ------TT-- 100 Sm CGβ GSFRNESLGC DYATSQD*SS SNVPPS NLTS PSQLLEPAVT PLVPQ 143 H CGβ -GPKDHP-T- -DPRF--S-- -KA--PS-P- --R-PG-SD- -IL-- 145

Homology to human CG b ( hCG b) protein 64 % identity 76 % similarity -20 +1 Sm CGβ MEMLQGLLLC LLLSTGGAWA SKEPLRPPCR PTNVILAVEK EGCPVCVPF N TTICAGYCSS 40 H CGβ ---F-----L ----M--T-- -------R-- -I-AT----- ------ITV- --------PT 40 Sm CGβ MVRVMQT*LP PLPQTVCNYH ELRFTSVRLP GCRRGVDPVV YMPMAVSCRC ALCRRSYSDC 99 H CGβ -T--L-GV-- A---V----R DV--E-I--- --P---N--- SYAV-L--Q- ------TT-- 100 Sm CGβ GSFRNESLGC DYATSQD*SS SNVPPS NLTS PSQLLEPAVT PLVPQ 143 H CGβ -GPKDHP-T- -DPRF--S-- -KA--PS-P- --R-PG-SD- -IL-- 145 Predicted N-linked Glycosylation N-X-S/T O-linked glycosylation

Mammalian Expression of Chorionic Gonadotropin and Luteinizing Hormone New World Primates CG β LH β (common marmoset, squirrel monkey) CG β Pituitary Placenta How is CG expressed in the pituitary? 1. Isolate CG b gene 2. Isolate promoter region of the CG b

Isolation of squirrel monkey CG b gene promoter Promoter 5’ 3’ ATG TAA Exon Intron CG β gene

Genome Walking Genomic DNA from SML DNA Restriction enzyme Library Adaptor PCR amplification Adaptor primer Gene specific primer exon 2 smCG b Products analyzed by gel electrophoresis CG b ?

Results M Pvu II Dra I EcoR V 1.0 kb 0.25 kb Promoter 5’ 3’ Exon Intron 0.25 kb 1 kb

Results Promoter 5’ 3’ ATG Exon Intron 5’ 3’ …CCCCGAGG TATAAA GCCAGGTACACGAGGCAGGGGAGGCACCAAGG ATG … TATA box Start codon

Summary How is CG b expressed in the squirrel monkey pituitary? Promoter 5’ 3’ Exon Intron CG β gene  Obtained genomic sequence for smCG b gene  Confirmed coding sequence for smCG b protein  Obtained promoter sequence for smCG b gene  Currently analyzing transcription factor binding sites in promoter sequence

Acknowledgments Dr. Jonathan Scammell Dr. Tina Hubler Audrey Vasauskas Donna Willis NSF-REU

Summary CG β LH β Squirrel Monkey CG β Pituitary Placenta How is CG expressed in the pituitary? CG β gene Promoter 5’ 3’ Pituitary-specific transcription factors

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

Cell specific gene expression HUMAN PITUITARY TATAAA LH b promoter LH b gene HUMAN PLACENTA TATAAA CG b gene CG b promoter Pituitary-specific transcription factors SM PITUITARY TATAAA CG b gene CG b promoter Jameson and Hollenberg 1993 Endo Rev 14:2

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

Homology to human CG b ( hCG b) protein 64 % identity 76 % similarity -20 +1 SqM CGβ MEMLQGLLLC LLLSTGGAWA SKEPLRPPCR PTNVILAVEK EGCPVCVPF N TTICAGYCSS 40 Hum CGβ ---F-----L ----M--T-- -------R-- -I-AT----- ------ITV- --------PT 40 SqM CGβ MVRVMQT*LP PLPQTVCNYH ELRFTSVRLP GCRRGVDPVV YMPMAVSCRC ALCRRSYSDC 99 Hum CGβ -T--L-GV-- A---V----R DV--E-I--- --P---N--- SYAV-L--Q- ------TT-- 100 SqM CGβ GSFRNESLGC DYATSQD*SS SNVPPS NLTS PSQLLEPAVT PLVPQ 143 Hum CGβ -GPKDHP-T- -DPRF--S-- -KA--PS-P- --R-PG-SD- -IL-- 145 Predicted N-linked Glycosylation N-X-S/T O-linked glycosylation