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)

Anterior pituitary Reproductive functions

• f males and females

Glycoprotein Hormones

Regulating Gonad Function

CG

Chorionic gonadotropin

Placenta Maintains progesterone secretion in the corpus luteum Anterior pituitary

FSH

Follicle stimulating hormone

LH

Luteinizing hormone

Heterodimers

Species-specific a subunit

Hormone-specific b subunits

= LH = CG

a b a b

Glycoprotein Hormones

Regulating Gonad Function

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

CGβ LHβ

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

CGβ CGβ LHβ

New World Primates (common marmoset, squirrel monkey)

Pituitary Placenta

CGβ CGβ LHβ

Does CG assume LH function? How is CG expressed in the pituitary?

• 1. Isolate CGb gene
• 2. Isolate promoter region of the CGb gene

Expression of Luteinizing Hormone and Chorionic Gonadotropin

Isolation of squirrel monkey CGb (smCGb) gene

CGβ gene 3’ 5’ Promoter Exon Intron

DNA Transcription start site

Polymerase chain reaction (PCR) amplification of smCGb from genomic DNA

1. Isolated genomic DNA from squirrel monkey lymphocytes (SML) human CGb gene 2. PCR primers 3. Predicted 1.1 kb product 1.1 kb 4. Excised and sequenced Start codon

ATG

Forward primer

Stop codon

TAA

Reverse primer

smCGb 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 Start codon Stop codon Exon 2 Intron A Exon 3 Exon 1 Intron B

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

smCGb gene sequence

GT-AG rule for introns : GT……AG

• 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 SNVPPSNLTS PSQLLEPAVT PLVPQ 143 H CGβ -GPKDHP-T- -DPRF--S-- -KA--PS-P- --R-PG-SD- -IL-- 145

sm CGb (smCGb) protein

• 20 +1

Sm CGβ MEMLQGLLLC LLLSTGGAWA SKEPLRPPCR PTNVILAVEK EGCPVCVPFN 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 SNVPPSNLTS PSQLLEPAVT PLVPQ 143 H CGβ -GPKDHP-T- -DPRF--S-- -KA--PS-P- --R-PG-SD- -IL-- 145

Homology to human CGb (hCGb) protein

64 % identity 76 % similarity

• 20 +1

Sm CGβ MEMLQGLLLC LLLSTGGAWA SKEPLRPPCR PTNVILAVEK EGCPVCVPFN 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 SNVPPSNLTS PSQLLEPAVT PLVPQ 143 H CGβ -GPKDHP-T- -DPRF--S-- -KA--PS-P- --R-PG-SD- -IL-- 145

64 % identity 76 % similarity

Predicted N-linked Glycosylation N-X-S/T O-linked glycosylation

Homology to human CGb (hCGb) protein

New World Primates (common marmoset, squirrel monkey)

Mammalian Expression of Chorionic Gonadotropin and Luteinizing Hormone

Pituitary Placenta

CGβ CGβ LHβ

How is CG expressed in the pituitary?

• 1. Isolate CGb gene
• 2. Isolate promoter region of the CGb

Isolation of squirrel monkey CGb gene promoter

CGβ gene 3’ 5’ Promoter Exon Intron

ATG TAA

Genome Walking

Genomic DNA from SML

Restriction enzyme

Library

Adaptor

PCR amplification

Adaptor primer Gene specific primer exon 2 smCGb DNA

Products analyzed by gel electrophoresis

? CGb

Results

1.0 kb 0.25 kb

M Pvu II Dra I EcoR V

3’ 5’ Promoter Exon Intron

0.25 kb 1 kb

…CCCCGAGGTATAAAGCCAGGTACACGAGGCAGGGGAGGCACCAAGGATG …

5’ 3’ TATA box Start codon

3’ 5’ Promoter Exon Intron

ATG

Results

Summary How is CGb expressed in the squirrel monkey pituitary?

CGβ gene 3’ 5’ Promoter Exon Intron

 Obtained genomic sequence for smCGb gene  Confirmed coding sequence for smCGb protein  Obtained promoter sequence for smCGb gene  Currently analyzing transcription factor binding sites in promoter sequence

Acknowledgments

• Dr. Jonathan Scammell
• Dr. Tina Hubler

Audrey Vasauskas Donna Willis NSF-REU

Summary Squirrel Monkey

Pituitary Placenta

CGβ CGβ LHβ CGβ gene 3’ 5’ Promoter

How is CG expressed in the pituitary?

Pituitary-specific transcription factors

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

Cell specific gene expression

TATAAA

LHb gene

TATAAA

CGb gene

Pituitary-specific transcription factors

HUMAN PITUITARY HUMAN PLACENTA

LHb promoter CGb promoter

TATAAA SM PITUITARY

CGb gene CGb promoter

Jameson and Hollenberg 1993 Endo Rev 14:2

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

• 20 +1

SqM CGβ MEMLQGLLLC LLLSTGGAWA SKEPLRPPCR PTNVILAVEK EGCPVCVPFN 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 SNVPPSNLTS PSQLLEPAVT PLVPQ 143 Hum CGβ -GPKDHP-T- -DPRF--S-- -KA--PS-P- --R-PG-SD- -IL-- 145

64 % identity 76 % similarity

Predicted N-linked Glycosylation N-X-S/T O-linked glycosylation

Homology to human CGb (hCGb) protein