A Single Gene Encodes the ,8Subunits of Equine Luteinizing Hormone and Chorionic Gonadotropin

G. B. Sherman*, D. S. Threadgillt,

M. W. Wolfe*, D. C. Sharp,

T. A. Farmerie, and J. H. Nilson

C. M. Clay,

Department of Pharmacology Case Western Reserve University School of Medicine (G.B.S., M.W.W., T.A.F., C.M.C., D.S.T.,J.H.N.) Cleveland, Ohio 44106 Department of Veterinary Biosciences University of Illinois (G.B.S.) Urbana, Illinois 61801 Department of Animal Science University of Florida (D.C.S.) Gainesville, Florida 32611

Equine (e) CG and LH P-subunits have identical amino acid sequences, including an extended carboxyl-terminal peptide (CTP). This suggests that unlike the corresponding human genes, the P-subunits of eCG and eLH may be encoded by a single gene and share a common proximal promotor region. To explore this, we isolated and characterized the eLH/CG@ gene(s). Data from Southern analyses suggest that the eCGP and eLH@ subunits are products of the same single copy gene (eLH/CGB). Overlapping fragments of the eLH/CGB gene and cDNA were amplified from equine genomic DNA and pituitary gland mRNA by the polymerase chain reaction, cloned, and sequenced. The eLH/CG@ gene spans less than 1.2 kilobasepairs and has three exons that translate a CTP-containing polypeptide identical in sequence to that previously reported for the mature equine protein. There is, however, little amino acid homology shown between the CTP of human or equine CG/3 subunit. In addition, unlike the human genes, the same TATAA-like element appears to be involved in directing initiation of transcription of the eLH/CG@ gene in placenta and anterior pituitary. Based upon these differences, we suggest that the CGB genes evolved independently in humans and equids and that different mechanisms are involved in their patterns of placenta-specific expression. (Molecular Endocrinology 6: 951-959, 1992)

in regulating gonadal function, whereas CG is essential for maternalrecognitionand maintenanceof pregnancy (1). Both gonadotropins are heterodimeric glycoproteins composed of two noncovalently associated subunits, designated 01 and p. The a-subunit is identical for LH, CG, and two other heterodimeric glycoprotein hormones found in the pituitary (FSH and TSH). The common a-subunit is encoded by a single copy gene (2), whereas the P-subunits are encoded by separate genes (3, 4). All mammals exhibit pituitary-specific expression of LH, FSH, and TSH (1) whereas placental expression of CG has been clearly demonstrated only in primates (5) and equids (6). In humans, CGP and LHB subunits share extensive, but incomplete, amino acid sequence homology (7, 8) contain different carboxyl-termini due to changes in the reading frame of their coding sequences (6), and are encoded by a distinct gene(s) within a cluster of structurally linked genes (3, 4). Despite the high degree of nucleotide homology between human (h) LHP and CGP genes, they do not share common promotor-proximal regions. The hCG@ mRNA contains a 5’-untranslated region of 350 nucleotides (4) whereas the LH@ 5’-untranslated region is only a few nucleotides in length in most mammals studied to date (9-12). The start site of transcription in the hCGP gene is specified by a non-TATAA promoter, whereas a classical TATAA element appears to direct the start of transcription in the LHB gene. In striking contrast to hLH@ and hCGfl subunits, recent analysis of the primary structures of equine (e) LHfi and CG/3 subunits (13, 14) has revealed that their amino acid sequences are identical. Both have extended carboxyl-termini usually associated with hCG@

INTRODUCTION In primates, LH and CG are synthesized in the anterior pituitary and placenta, respectively. LH has a major role oeee-8809/92/0951-0959$03 00/o Molecular Endocmology CopyrIght 0 1992 by The Endocme

Srmety

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MOL ENDO. 1992 952

Vol6 No. 6

(15). This unexpected finding presents the intriguing possibilitythat the P-subunitsof eCG and eLH may be encodedby the samegene. Previously, we presentedevidence suggestingthat placenta-specificexpression of the equine a-subunit evolved via a pathway distinct from that of the human a-subunit (16). In this report we begin to addressacquisition of placenta-specificexpression of the eCG/3 subunit. Our results indicate that the eCGp gene is distinct from the hCG/3 gene with regard to certain aspectsof structure and function, and that evolution of CG expression in the equid lineage presumably occurred through disparate mechanismscompared to thosein the primatelineage.In horses,this involved the genesisof a singlecopy gene capable of expressing the sameP-subunitin pituitary and placenta.Since the P-subunithaspropertiesof both LHPand CGP,we refer to the encoded product as eLH/CGP.

RESULTS For initial characterization of the eLHP and eCG0 gene(s),a fragment correspondingto a portion of the putative third exon of the equinep gene(s)was amplified from genomic DNA by the polymerasechain reaction (PCR).The primersusedto generatethis fragmentwere basedon the aminoacid sequenceof eCGP and eLHp (13, 14), and the nucleotide sequence of the related bovine LHP gene (12). Because the amino acid sequencesof eCGPand eLH/3are identical(13,14), probe derived from this fragment would be expected to efficiently hybridize with any eLH@or eCGPgenespresent. Southern blot analysis(Fig. 1) resulted in singlebands when equine genomicDNA was digestedwith several different restriction enzymes, strongly suggestingthat a single gene encodes the P-subunitsof pituitary LH and placentalCG. PCR methodologieswere then used to isolate and characterize the equine p gene. Several overlapping fragments, which together spanned the entire gene, were amplified, cloned, and sequenced(Fig. 2). We have determinedapproximately 1700 basepairs(bp) of DNA sequence,includingthe entire coding portion of the eLH/CG@gene, approximately450 bp of 5’-flanking sequence, and 150 bp of 3’-untranslated sequence. Sequencediscrepanciesin overlapping regions of the gene were not observed, a finding consistent with the presenceof a singlegene. To further establishthe likelihoodof a single gene and validate the sequenceobtained by genomic PCR, pituitary RNA was amplifiedby PCR, cloned, and sequenced (Fig. 2). There was perfect agreement between the sequenceof the overlappingcDNA fragments and genomicsequence.Moreover, this sequencetranslated a polypeptidewith a primary structure identicalto the previously reported aminoacid sequencefor eLHp and eCGP(13, 14). Comparisonof cDNA and genomic sequencespermitted us to establishthe positions of

Fig. 1. Southern Blot Analysis of Equine Genomic DNA DNA was incubated with the indicated restriction enzymes and hybridized using a homologous third exon probe (see Materials and Methods). Kb, Kilobases.

introns and exons (Fig. 28). In addition, the sequence we report confirms that the C-terminal amino acid of eLH/CGp is Ser14’,as was suggestedfrom the peptide analysis(13). Interestingly,the coding sequencereads through to a TGA stop codon located 8 bp beyond the polyadenylationsignal.This is due to a frameshiftcompared to that of the hCG@gene and accounts for the lack of amino acid homology between the carboxyterminalpeptide (CTP) regionof humanand horse. The serine codon residing at the N-terminusof the secretedform of eLH/CGP is located within the second exon. The sequence5’ to this codon correspondsto the primary amino acid sequence for the previously uncharacterizedsignalpeptideof the eLH/CGPsubunit. The nucleotidesequenceshown in Fig. 28 predicts a 20-amino acid signal sequence correspondingto the entire first exon (five aminoacids)and the first 15 amino acids encodedby the secondexon. Initiationof transcriptionin most LHPgenesis TATAA associated(9-l 2) whereasinitiationof transcriptionin hCG@occurs 350 bp upstreamfrom the corresponding hLHP start site and is not spatially associatedwith a

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Equine

LH/CGP

Subunit

A

953

Gene

eLHlCGl3 gene

T3L

-

-

eLH/CGO

--

-

-

--‘I,

---

mRNA

B 5’-------------ct~~gg~~gg~ctttggc~c8gg~~tggctttc~gttca~gctg~ggagcgtg~acctg~~gcttc~gccc~tctc c~tttgagggtcgtcgg~ggggcg~gcat~cctt~cttc~tgtgtcccc8gc~cg~cctttctagcccctgcgg~ctgagtctgcgaagttgg tctc~cctgggtgtctcctgctt~aggcgcct~gc~ctcagcgagtgggggg~ggggttgctcc~gggagccactcctgc~cctccctg~cct tgtccgcctctcgcccgggggcactt~gtgtccaggtt~ccctgcc~tgcggc~tgccc~gtggccttgcctcccccacagcctgcctgcgg g~accagtPg~g*cc*ggg~MGtCC*GCG*~*GT*GTGTMG*~~*G*GT~~~G~*G*GG*GG~~~G*GG -20 Met ATG

Glu Thr Le" GAG ACG CTC

Gln CA0

gtaaggctgt~gggccctgggccttgc~~ctcc~tcc~gggtcc~gttggcgcg~~g~gggagcgtcttatga

cctggc~~~ggtgtggacegcc~gcgttcctgcgttcctgccaggagggccgcgggtagggc~gggcctg~gtgggt~tctg~ggtgttggggtccct ggggt~ttgggatgggggatc~aagtggagggtccctg~~tgga~gatgtc~ggcag~gg~gctgggtccctcaatgtgt~tctggg~cggga Gly Le" GGG CTG

gggtgagggcccagggctgaggtctcaggctggccctgaggcactggccttgtccc~g Ser ACT

Val OTT

Gly CCC

Ala Ala Glu GCT GCT GAG

Gly Val Trp GGG OTC TOG +20 Lys MC

Glu GAG

Ala GCA

Ala Cys CCC TGC

+1 Ser KC

Arg

Pro CCC

AGO

Gly GGG

110

Cys

ATTC

TGC

Leu Arg CTG COG

Pro CCA

+10 Lo" Cys Arg Pro Ile CTG TGC COG CCC ATC

110

Thr

Phe

+30 Thr

Thr

Sar

ATC

AC!C

TTC

ACC

ACC

AGC

Pro CCA

Val Arg Val ".t GTG CCC GTG ATG

gagggcccaecccaggcagetgctcetgctgctgcaccccctctctccag Pro Gln CCC CAG

Pro CCA

Val Cys Thr GTG TGC ACC

Val Asp GTG GAC

Pro CCC

Met ATG

Tyr TAC

+60 Arg Glu Lau Arg Phe COT GAG CTG CCC TTT

Pro CCA

Pro 110 CCA ATA

Lys AAG

Thr ACT

+50 Pro Ala 11s CCC GCC ATT

110 ATC

Lys MC

Thr ACT

SQI: Smr KC TCT

LyS MC

+90

Pro CCC

+110 Le" Ala Cy‘ Ala TTG CCC TGT WC

Pro Gln CCC CAD

Ala Ser CCC TCC

Ser KT

Thr

+130 Ser KC

Thr

Pro

Thr

ACC

CCA

ACT

Gly Ala Ser GGG CCC AGC

Arg

Asp

AGA

GAC

Leu Thr CTC ACA

SW KC

+149 S-r TCT

Pro CCC

Gly GOT

Gin CA0

Gin Pro CAA CCT

Tyr Cys TAC TGC

Pro Pro CCC CCT

Cys Gln TGC CA0

Ser TCC

Gly CCC

Gly Cys GGC TGC

Gly Pro GGG CCC

Phe T-K

Leu CTG

Ala Ala Lau GCT GCC CTG

CCA

WC

Pro Val Ala Lou Ser Cys Eis Cys CCC GTG CCC CTC ACT TOT CAC TGC

Gly Val GGG OTT

Ala Thr CCC ACT

Pro

Ala

Leu Lmu CTG CTG

Asn MC

Cys TGT

Val Ser Phe GTC TCC TTC

+100 Cye TGC

-10 Trp Met TOG ATG

110 ATC

+70 Ala Ser 110 Arg Leu Pro GCT TCC ATC CCC CTC CCC

+so

Asp GAC

Le" Leu CTG CTG

Tar TGA ACTAC

ACA

Pro CCT

Thr ACC

+120

Arg AGA

+140 Arg Ser Ser CGT TCC TCT

PI0 CAT CCC

Asp

His Pro CAT CCC

Leu CTC

ecactgtggggttgtttttgggctcaggggtgcgtgtgcacgtgtatacacacacacagtgg

Fig. 2. Cloning Strategy and Nucleotide Sequence of eLH/CG@ A, Cloning and sequencing scheme. Boxed regions correspond to exons. Filled boxes represent 5’- and 3’-untranslated regions. Thick lines indicate intervening or flanking sequence. Dashed lines represent vector sequence. Triangles represent primers used for PCR, sequencing, or both. Lines extending from these triangles depict sequence obtained using a given primer (above gene and mRNA diagrams) or PCR-amplified fragments generated from pairs of primers (below gene and mRNA diagrams). A,, Poly(A) tail of cDNA; M, Mae1 restriction site used for inverse PCR, T3 and T7 represent primers directed against the BSK- vector. B, Nucleotide sequence of the eLH/CG gene. Flanking and intervening sequences are in lowercase letters. The cDNA sequence is in capital letters, with mRNA cap sites indicated by asterisks. Amino acids are numbered, with the first amino acid of the mature protein as +l The consensus TATAA promoter (31) and AATAAA polyadenylation (32) sequences are underlined. The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 07 April 2015. at 11:09 For personal use only. No other uses without permission. . All rights reserved.

MOL ENDO. 1992 954

Vol6 No. 6

TATAA element (4, 11) (see Discussion). Therefore, it was important to determine whether similar differences in initiation sites for transcription were associated with expression of the single eLH/CGP gene in pituitary and placenta. Analysis of initiation sites for transcription by primer extension (Fig. 3) revealed that the same three principal extension products were observed for mRNA from both pituitary and placenta. Furthermore, all mRNA CAP sites were located 20-30 bp downstream of one of two tandemly repeated TATAA elements (see Fig. 2B). The minor bands 5’ to the three primary initiation sites in the equine pituitary lane probably represent products resulting from priming of nontissue-specific sequences present in the total RNA preparations used as template. After longer exposure, minor bands were also observed in the same location in equine liver and placenta (data

GATCLC

PE -1353

hCG8 + (-350) - 194

- 118 TATAA

C

TATAA

C

-3

-

72

t

Fig. 3. Mapping of the Transcription lnltiatlon Sites of eLH/CG in Pituitary and Placenta (Endometrial Cups) by Primer Extension Lane identification: G,A,T,C, DNA sequencing ladder; L, equine liver RNA; 0, oligodeoxynucleotide primer only; P, equine pituitary RNA; E, equine endometrial cup RNA. Arrows indicate the three predominant primer extension products. TATAA elements are delineated by small brackets. The approximate location of hCG transcriptional initiation, relative to the eLH/CG 5’-flanking region, is indicated for comparison. The locations of DNA standards (basepairs) are shown on the right.

not shown). These results suggest that the same start sites direct initiation of eLH/CG/I transcription in both pituitary gland and endometrial cups (equine placental tissue that expresses CG). There was no evidence for transcriptional initiation in the region further upstream in the eLH/CGP gene corresponding to the site associated with initiation of transcription in hCGP. Northern analyses were performed to further characterize expression of the eLH/CGP gene. Analysis of RNA prepared from equine pituitaries revealed a transcript of the predicted size, the levels of which peaked in the summer and were lowest in winter (Fig. 4A). This change in eLHP RNA with season of the year is consistent with the seasonal profile of LH secretion in the horse (17). Expression of the eLH/CGP gene was also examined in endometrial cups isolated from fetuses collected at various stages of development. Northern analysis revealed that the size of the eCGP RNA (placental origin) was similar to that of the eLH@ RNA (pituitary origin). Levels of eCGP RNA in endometrial cups were high during the early stages of gestation (2.5 and 5-cm fetuses), but were considerably lower during later stages of gestation (Fig. 4B), thus paralleling the profile of eCG secretion during gestation (17). While the predominant RNA species identified by the probe was of the predicted size in each case, some variation in the length of P-subunit RNAs was noted (Fig. 4). Because similar heterogeneity was also observed with the bovine pituitary RNA, these variations in length may be attributed to partial degradation of RNA and differences in the length of polyadenylated tails.

DISCUSSION

Acquisition of CG synthesis in primates and equids requires expression of both the a-subunit and CGP subunit genes in placenta. We previously reported that humans and horses use different cis-acting elements and presumably trans-acting factors to direct expression of the a-subunit gene to placenta (16). We now extend this observation to include placental expression of the gene encoding the eCGp subunit. Based upon the data presented, evolution of expression of eCGp appears to have occurred through a mechanism independent and distinct from that of the hCG/I gene cluster. The single copy eLH/CGP gene appears to be quite similar to LHP and CGP genes of other species (Fig. 5A). Positions of intron/exon boundaries within the equine gene correspond precisely to highly conserved mRNA splice sites reported for hCG/3 and LHP genes (Fig. 5A) (4, 10, 12, 18, 19). Differences between the eLH/CGP gene and others are primarily centered around two areas, the promoter and C-terminus. In primates, the LHP gene is expressed exclusively in pituitary, whereas CGP genes are expressed only in placenta. This divergent pattern of LHP and CG/3 expression in primates is associated with different sites

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Equine LH/CGP Subunit Gene

A.

Equine Pituitaries .y

Julian Da”

,qa 3

$

@

3 Q .p .$ ($8 &

&

1.77 1.52 1.28 0.78 0.53 0.40 0.28 0.16

B.

Equine Endo. Cups

Crown-rump Length 2.5

5

23

25

36

1.77 1.52 1.28 0.78 0.53 0.40 0.28 0.16 Fig. 4. Blot Analysis of eLH/CGP mRNA A, Anterior pituitary. Numbers at the top of lanes indicate the day of the year on which pituitary tissue was harvested (day 1 being January 1). B, Endometrial cups. Numbers at the top of lanes represent crown-rump length (centimeters) of the equine fetus at the time of endometrial cup collection. Estimated days of gestation based on fetal crown-rump lengths of 2.5, 5, 23, 25, and 36 cm are 40, 53, 112, 118, and 147 days, respectively (17). The locations of RNA standards (kilobasepairs) are shown on the leff of each blot.

of transcriptional initiation, that reflects different promoters used to achieve tissue-specificexpression (4, 11). Our data indicate that unlike primates, the same transcription start sites are used when the eLH/CGP gene is expressed in both pituitary and placenta. This suggests that the same promoter is active in both pituitary and placenta. Comparisonof the 5’-flanking and -untranslatedregions of LH& CGP, and LH/CGP genes (Fig. 58) indicates the presence of a 52-bp

insertion that is unique to eLH/CG@ This insertion appearsto have resultedfrom replicationof the 12-bp sequenceGTATAAGACCAG and gives rise to tandem TATAA elements.Whilethis causesthe 5’-untranslated sequenceto be elongated relative to other LHP genes, transcriptional initiation remains linked to the TATAA elements.In contrast, the start site for transcription in hCGP is located greater than 350 bp 5’ to the TATAA sequence.Thesedata suggestthat the promoterregion responsiblefor initiation of transcription of the eLH/ CGPgene in both pituitary and placenta is more similar to LHP (TATAA-associated) than hCGP (non-TATAAassociated).This finding provides support for different evolutionary pathways leadingto expressionof human and equineCGP. A comparisonof the C-terminalregion of characterized LHP and CGPgenesrevealsthat similarevolutionary events led to acquisitionof a CTP domainin primatesand equids(Fig. 5C). In primates,emergenceof a CTP appearsto result from a singlebasepairdeletion in a replicatedcopy of the ancestralLHPgene (4). This deletion results in readthroughto a distal translational stop codon locatedwithin the polyadenylationsignal.In horses,a deletionof 10 hp occurred in the samevicinity as the single base deletion observed in the coding region of hCGP. The lo-bp deletion creates a reading frameshiftthat resultsin readthroughof the stop codon used by human and bovine LHP. In contrast to hCG& other insertionsand deletions further downstream altered the readingframe of the eLH/CG@gene, resulting in the use of a stop codon 8 bp beyond the polyadenylation signal.Together these insertionsand deletions account for the differences in length of the eLH/CG& hCGP(4) and donkey CGP(/LHP?)(19) CTPs (Fig. 5, A and C). Furthermore, these differences result in little amino acid sequencehomology between equine and human CTPs. Interestingly, the donkey CTP initially is in-frame with that of the horse, but then shifts to the reading frame of hCTP (Fig. 5C) (19). Presumably, differences between the CTPs of horse and donkey occurred after radiation of these two species. Thus, these three CTP-containing species display marked differencesin aminoacid sequence,suggestingthat a strict primary structure is not a critical determinant of CTP function or, alternatively, the CTP has an insignificant role in CG function. A model summarizingthe independentevolution of ,quine and primate CGP genes is illustrated in Fig. 6. While other evolutionary pathways are possible, the model presented requires the fewest recombinatorial events to establishthe extant structures of the equine and primate LHP-CGPloci. We proposethat the ancestral gene was singlecopy, expressedonly in pituitary, and encoded a LHP subunit lacking a CTP domain. In primates, the LHP gene duplicated with one of the copies acquiring a CTP domain and a novel promoter that directed expressionto placenta.Presumably,these events inactivated the pituitary-specificpromoter. Subsequentduplicationevents of the new CGPgeneled to a cluster of six CGPgenesin additionto the LHP gene

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MOL 956

ENDO.

Vol6

1992

No. 6

TATA

A

Donkey W

(LHP)

q

Nontranscribed

0

Untranslated

n

Conserved

regions regions protein

coding

regions

0

Human-like

LB

Horse-like

CTP CTP

* 4 7 Frameshift

mutations

B.

hLm hcGB

gtggccttgccgcccccacaaccccgaggt~aagccag..................... GTGGCCTTGCCGCCCCCACACCCCGAGG~~GCCAG.-..*.'

eLfl/CG6

gtggccttgcctcccccacagcctgcgggtataaaaccaq ccggccttgccgcccccacagcctgcagg~gaccag~~~~~~~~~~~~~~~~~~~~~

bLUB

mfi

hCGB eJX/CG~ hub

"...

......‘.

.

taatataaaaccaqggTAAGC

............................ ~~atacacgaggcaggggaTGCACCAAGG ............................. .GTACACCAGGCAGGGGACGCACCAAGG ACCAGCAAAGATCAGTAGTGTAAGACCAGACCAGAGTAAACACGGCAGAGGAGGCACCGAGG ............................. .gtaaacacagcaggggAGGCACCAAGG

ATQ ATQ ATQ

ATQ

C.

huB hCGB eLH/CGE

CDHPQLSG C D D P C A . ' ....

dCGB bLUB

C

hLIlB hCGB eur/cce dCGB bl2lB

hCGB

dJI/CGE dCGfl

hl.RB hCGB

eL3I/CGB dCGB bLJB

A

.

.R FQ . ..P Q

. . . . . . ..P

Q

L L F L Z DS S S S KA. P P P S,.L A S S S S K D P P S Q P T S S S C K DPPSQPLTFH

P T

L

.s

P

.S

T

CDHPPLPDILFLZ n TGTGACCACCCCCAACTCTCAGGCCTCCTCTTCCTC~aga.ccctccccgcagc' .cttccaa.gtcc TGTQATGACCCCC.GCTTCCAGGACTCCTCTTCCTCAAAOOC,CCCTCCCCCCAGC,.CTTCCM.GCCC TGTQCCC' . . . * * * . "CCCAQQCCTCCTCTTCCTCTMGQATCCCCCATCCCMCCTCTCACAT'CCAC TQTQCCC.......... CCCAGACCTCCTCTTCCTGTMGGATCCCCCATCCCMCCTCTCACATTCCAC TGTGACCACCCCCCGCTCCCAGACATCCTCTTCCTC~ggatgccccacttcaac. .ctcccat'gccc

. . . .S R L P G P S D .S T P T P G A S RR IPPQLLGPADVPLIPSQZ

. ..T S

P S

I H

L P

P L

Q P

Z I

K

T

S

Z

at..... cccgactcctggagccctgaca~~~ccccgatcctcccac~ataa~ggcttctcaatccgc AT'.. "CCCGACTCCGGQGGCCCTCGGACA... CCCCGATCCTCCCACAA~aggcttctcaatccgc AT~CCACCCC~CTCCTGGGGCCAGCAGAcGTTCCTCTCATCCccTccc~T~GACTTCT~actac ATTCCACCCCMCTCCTGGGGCCAGCAGACGTTCCTCTCATCCCCTCCCM~acacttcttgaaccac at... '.cctaactctggaaaccagcagacactcttcccctcccttccca~gacttctcaaactgc

Fig. 5. Comparison of eLH/CGp with hCG (4) LH (4, 10, 12) and Donkey (d) CG(/LH) (19) Genes A, Schematic representation of LH and CGP genes. Translational termination codons are underlined. B, Comparison of promoter-proximal 5’-flanking and untranslated regions. Uppercase text designates sequence present in the mature mRNA. boldface ATG represents the initiation codon. The consensus TATAA sequence is brackefed, functional TATAA elements double undefined, and the 12-bp repeat as well as similar sequences in eLH/CGP gene are underlined. C, Comparison of

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the The are the

Equine

LH/CGP

Evolution

Subunit

957

Gene

of Placental

Gonadotropins

animals. This should reveal critical regulatory regions that confer pituitary- and placenta-specific properties to the proximal TATAA-containing promoter of the eLH/ CGP gene.

p - Subunit II Equids

/LHP/

MATERIALS

Other mammals

AND METHODS

Materials

i

c

/LHP Ancestral Fig. 6. Model

0

Pituitary

& placenta

mammal

for the Evolution

of CG@ and LH/3 Genes

found in humans. Most other mammals have retained the ancestral form of the single copy LHP gene, with the exception of equids. In horses, the ancestral-like LH/I gene remained single copy, retained the property of pituitary-specific expression, and acquired both a CTP domain and the additional property of placentaspecific expression. These events bolster the conclusion that the appearance of placental gonadotropins in primates and horses has occurred through independent evolutionary pathways involving several genes (a-subunit, LHP, CG& and LH/CGP) and promoter elements. Although our recent evidence indicates that placentaspecific expression of the a-subunit gene in primates and equids has involved selection of different &-acting elements and trans-acting factors, the mechanisms responsible for this same property in CGP genes remain vague. With characterization of the equine LH/CGP gene and in comparison with the primate CGP genes, it again appears that completely different mechanisms have been used to achieve placenta-specific expression. For the primate CGP genes, pursuit of this mechanism has been difficult due in part to the dearth of information regarding functional requirements of promoters that lack TATAA elements. Because expression of the eLH/CGP gene in pituitary and placenta occurs from the same transcription start sites, it is likely that the same promoter confers both properties by using a spectrum of regulatory elements to form unique combinatorial codes. Thus, we now have a target promoter region that can be explored through the use of deletion mutagenesis and subsequent expression in transgenic

Restriction enzymes and other enzymes for molecular biology were obtained from Boehringer Mannheim Biochemicals (Indianapolis, IN) and Gibco-Bethesda Research Laboratories (Gaithersburg, MD). Nylon membranes were obtained from CUNO, Inc. (Meriden, CT), and nitrocellulose membranes from Schleicher and Schuell (Keene, NH). Oligodeoxynucleotide primers were obtained from the Case Western Reserve University Molecular Biology Core Laboratory (Cleveland, OH). PCR was performed with Taq DNA polymerase obtained from Perkin-Elmer Cetus (Norwalk, CT) and Gibco-Bethesda Research Laboratories using a Perkin-Elmer Cetus DNA thermal cycler. Bluescript plasmid vector (pBSK-) used for DNA cloning was obtained from Stratagene (La Jolla, CA). All radionuclides were purchased from New England Nuclear (Boston, MA). The random prime labeling kit was purchased from Boehringer Mannheim Biochemicals. Sequencing was conducted using Sequenase purchased from U.S. Biochemical Corp. (Cleveland, OH). Reverse transcription of RNA was conducted using Superscript reverse transcriptase and buffer obtained from Gibco-Bethesda Research Laboratories. All other chemicals were obtained from Pharmacia (Piscataway, NJ), Mallinckrodt, Inc. (Paris, KY), Sigma (St. Louis, MO), and Gibco-Bethesda Research Laboratories. Genomic

Blot

Genomic DNA was prepared from equine sperm as previously described (20). Equine genomic DNA (10 pg) was digested with restriction endonucleases, analyzed by electrophoresis through a 1% agarose gel, and blotted to a nylon membrane

(21). The probe used for Southern hybridization was a PCRamplified DNA fragment corresponding to amino acids 42-l 10 of the eLH/CGP gene. Consensus primers for this fragment were based upon a comparison of the previously reported eLHP and eCGp amino acid sequence (13,14) and the nucleotide sequence of the bovine LHP gene (12). After confirming that the fragment encoded bona fide eLH/CGfl by direct sequencing (22, 23) the fragment was cloned into a Bluescript vector (pBSK-). The fragment was labeled with [n-32P]deoxyCTP 13000 Ci/mM) to soecific activities of 1 08-1 O9 dom/ua bv the random primer method (24) and was purified from’&incorporated nucleotide using a Sephadex G-50 spun column (25). Prehybridization and hybridization buffers containing 50% formamide were prepared as previously described (26). Blots were prehybridized at 37 C for 2 h, followed by hybridization with labeled probe (1 O6 cpm/ml) at 37 C for 18 h. To remove the unhybridized probe, the membranes were washed sequentially in 1 x SSC (1 x SSC = 0.15 M NaCI, 0.015 M sodium citrate, pH 7.0)-0.1% sodium dodecyl sulfate (room temperature) and 0.5 x SSC-0.1% sodium dodecyl sulfate (65 C). The

CTPs and 3’-flanking regions. Boldface uppercase text designates sequence present in the mature mRNA. Lowercase text designates 3’-flanking sequence. The hLHfi and bovine (b) LHP stop codons (and similar regions in the CGB genes) and AATAAA polyadenylation sequences are bracketed. Translation stop codons are underlined. Amino acid sequences are shown above the nucleotide sequences: A, Ala; C, Cys; D, Asp; E, Glu; F, Phe; G, Gly; H, His; I, Ile; K, Lys; L, Leu; M, Met; N, Asn; P, Pro; Q, Gln; S, Ser; T, Thr; V, Val; W, Trp; Y, Tyr; Z, termination codon.

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MOL 958

ENDO.

Vol6

1992

No. 6

washed filter was placed against Kodak XAR-5 film (Eastman Kodak, Rochester, NY) with a DuPont Cronex Lightning Plus intensifying screen (DuPont, Wilmington, DE) at -70 C for l7 days.

was isolated and analyzed by blot transfer-hybridization niques, as previously described (30).

Genomic

The authors wish to express their appreciation to Dr. Deborah Hamernik, Dr. Ruth Keri, Ms. Robin Pittman, and Ms. Joan N. Clay for their advice and technical assistance at various stages of these investigations. The authors are grateful to Dr. Lisa Lund for her help in preparing several of the figures included in this work. We also thank Dr. E. L. Squires for providing horse sperm for DNA analysis.

PCR

Fragments of the eLH/CGp gene were amplified using consensus primers derived from sequence comparison of other mammalian LHB and CGfi genes as well as specific primers based on the nucleotide sequence obtained from the third exon probe used for Southern blots. Overlapping fragments were cloned using restriction endonuclease sites engineered into primers or oligodeoxynucleotide linker ligation and subsequently sequenced using the Sanger dideoxynucleotide procedure (27). Products of several different PCR reactions were sequenced to reduce the possibility of obtaining sequence errors resulting from nucleotide misincorporation during PCR generation of the clones. Sequence corresponding to the 3’-flanking region of the eLH/CG gene was obtained using inverse PCR methodologies (28, 29). Equine genomic DNA was digested with the enzyme Mael, diluted to a concentration of 3 ng/pl, and circularized using T4 DNA ligase. This circularized DNA served as template for PCR, and the product generated was cloned and sequenced. Complementary

DNA PCR

tech-

Acknowledgments

Received March 3, 1992. Revision received April 2, 1992. Accepted April 2, 1992. Address requests for reprints to: Dr. John H. Nilson, Department of Pharmacology, Case Western Reserve University, 2109 Adelbert, Cleveland, Ohio 44106. This work was supported by the NIH (Grant DK-43039; to J.H.N.), the Grayson-Jockey Club Research Foundation, Inc. (to J.H.N. and D.C.S.), a Cancer Research Grant (PBO-CA43730; to J.H.N.), NIH Training Program in Developmental Genetics (HD-07138; to M.W.W.), NIH Training Program in Metabolism (DK-0731; to T.A.F. and D.S.T.), an American Heart Association Award, Northeast Ohio Affiliate (F195; to C.M.C.), and the USDA (Grant 9001872; to D.C.S.). The first two authors have contributed equally to this work. T Current address: Department of Molecular Biology and Microbiology, Case Western Reserve University School of Medicine, Cleveland, Ohio 44106. l

Equine pituitary RNA served as template for PCR-mediated amplification of the eLH/CGp cDNA. The first strand of the cDNA was synthesized using reverse transcriptase (following conditions recommended by the manufacturer) and a primer composed of polythymidine sequence with additional 5’ anchor sequence (restriction endonuclease sites for cloning) or specific primers derived from the sequence of genomic clones. The second strand was synthesized, and the resulting cDNA was amplified using PCR with specific upstream primers and conditions previously used to amplify the genomic clones. The cDNA fragments generated were cloned and sequenced. Transcription

Start

Site Mapping

A 20-bp oligodeoxynucleotide primer complimentary to 5’untranslated sequence was end labeled using T4 polynucleotide kinase and [gamma-32P]ATP (3000 Ci/mM) to an approximate specific activity of 10’ dpm/pg. Labeled primer was combined with 100 pg equine pituitary RNA, 50 Kg equine endometrial cup RNA (plus 50 Fg yeast transfer RNA), or 100 Fg equine liver RNA and precipitated with ethanol. The primerRNA mixtures were resuspended in 9 ~1 water and 10 ~1 Superscript reverse transcriptase buffer. After heating to denature nonspecific associations (95 C; 5 min). the temperature was lowered to 50 C, and 20 U RNAase inhibitor were added. The final mixture was then incubated for 4 h at 50 C. After hybridization of the primer to the RNA, the reactions were incubated at 54 C for 5 min, and the following were added to the reaction mixtures to attain the final indicated concentrations: 6 mM dithiothreitol, 0.5 mM deoxy-NTPs, and 400 U reverse transcriptase. The reaction mixture was incubated for 1 h at 54 C. RNAase-A (10 Kg) was then added, and the reaction tube was incubated at 37 C for 30 min. Single stranded DNA products were isolated from the reaction mixture by phenol-chloroform extraction, followed by ethanol precipitation The resultant DNA was separated by electrophoresis through an 8% denaturing polyacrylamide gel. Kodak XAR5 film and a DuPont Cronex Lightning Plus intensifying screen were used for autoradiography (l-7 days). Northern

Analyses

Tissues for RNA analysis were collected at an abattoir, immediately frozen in liquid nitrogen, and stored at -80 C. RNA

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Gene

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A single gene encodes the beta-subunits of equine luteinizing hormone and chorionic gonadotropin.

Equine (e) CG and LH beta-subunits have identical amino acid sequences, including an extended carboxyl-terminal peptide (CTP). This suggests that unli...
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