The nucleotide sequences of baboon chorionic gonadotropin β-subunit genes have diverged from the human

161

Gene, 46 (1986) 161-169 Elsevier GENE

1732

The nucleotide sequences of baboon chorionic gonadotropin j-subunit human

genes have diverged from the

(Recombinant DNA; multigene family; placenta; placental gene expression; gene conversion; antipregnancy vaccine; A page vectors)

Robert J. Crawford*, Geoffrey W. Tregear and Hugh D. Niall** Howard Florey Institute of Experimental Physiology and Medicine, Universityof Melbourne, Parkville, Victoria 3052 (Australia) Tel. (03) 344-5654 (Received

February

(Revision

received

(Accepted

lOth, 1986) June 20th, 1986)

July 19th, 1986)

SUMMARY

The placental glycopeptide hormone chorionic gonadotropin (CG) is involved in establishing and maintaining pregnancy. The hormone consists of two different non-covalently associated subunits termed a and /I. In man there are seven closely linked genes coding for /ICG-like peptides, but only three of these appear capable of expression in the placenta. The organization of BCG-like genes in man and baboon appears to be similar. We demonstrate here that the baboon genome contains multiple copies (at least five) of /3CG-related genes, and that these genes are closely linked in the genome. Nucleotide sequence data from several BCG cDNA clones indicates that at least two of these BCG-related genes are expressed in the baboon placenta. Analysis of /?CG sequences from baboons and human subjects demonstrates that these genes have diverged markedly between species. In contrast, these sequences are remarkably homogeneous within their respective genomes. Gene conversion events may be responsible for retaining the high degree of identity among the various /?CG gene family members. Knowledge of /?CG sequences from baboon may lead to development of a long-term antipregnancy vaccine. The ability of CG antibodies to interfere with the maintenance of pregnancy can now be investigated within a homologous system.

* To whom

correspondence

and

reprint

requests

should

be

INTRODUCTION

addressed. ** Present

address:

Boulevard,

South

Genentech, San

Inc.,

Francisco,

460 Point CA

San

94080

Bruno

(U.S.A.)

Tel. (415)952-1000. Abbreviations: complementary

aa, amino acid(s); bp, base pair(s); cDNA, to mRNA;

follicle-stimulating

hormone;

mone; nt, nucleotide(s)

0378-l 119/86/$03.50

0

CG, chorionic

gonadotropin;

DNA FSH,

kb, 1000 bp; LH, luteinizing

TSH, thyroid-stimulating

1986 Elsevier

Science Publishers

hor-

hormone.

B.V. (Biomedical

A glycoprotein hormone from the placenta, CG, plays a vital role in early pregnancy by rescuing the corpus luteum and maintaining the synthesis of progesterone (Canfield et al., 1978; Pierce and Parsons, 1981). FSH, LH and TSH, each synthesized in the anterior pituitary, are the other members of the glycoprotein hormone family. Each hormone is composed Division)

162

of two different subunits (termed cc and p) linked non-covalently. The CIsubunits of each hormone are

MATERIALS

identical,

(a) Analysis of baboon DNA

while

responsible

the B subunits

for the physiological

are unique,

and

AND METHODS

specificity (Canlield

et al., 1978). In man, the /3CG subunit

shares about

85 % aa sequence identity with the BLH subunit,

and

Baboon

DNA

was isolated

digested

with restriction

30 % to 40 % identity with the /I subunits of FSH and TSH, respectively. However, it is longer than the /l

Hind111

and

subunits

of the other glycoprotein

an extension C terminus

described

PstI

(Pharmacia)

by Maniatis

hormones,

due to

was

at the

phage 3, Hind111 markers,

(Morgan

et al.,

1975).

The

human

lulose (Southern,

BamHI, using

on

1%

agarose

and

EcoRI,

procedures

et al. (1982). Restricted

of from 25 to 30 aa residues

electrophoresed

from placenta

enzymes

DNA

gels using

transferred

to nitrocel-

1975) and hybridized

with appro-

genome contains a single gene encoding the tl subunit (Fiddes and Goodman, 198 1; Boothby et al., 198 1). In contrast seven BCG-related genes and one BLH gene have been cloned from the human genome (Boorstein et al., 1982; Policastro et al., 1983; Talmadge et al., 1984a).

priate 32P-labelled human /ICG cDNA probes (Maniatis et al., 1982). The human cDNA clone (Fiddes and Goodman, 1980) was kindly provided Dr. J. Fiddes (California Biotechnology, by

We report here an analysis of the BCG sequences in the baboon, (Papio anubis and Papio cyanocephalus) which demonstrates that the baboon genome also contains several /3CG-related genes. Furthermore, nucleotide sequence analysis of several different BCG cDNA clones indicates that at least two of these genes are expressed in the baboon placenta. Comparison of the predicted peptide sequences from baboon with that of man (Fiddes and Goodman, 1980) indicates that a large number of aa differences, many of them nonconservative, have occurred during the relatively short evolutionary period between baboons and human beings. Knowledge of the baboon j3CG sequence has relevance to the development of a long-term antipregnancy vaccine. One approach to contraception relies on the ability to specifically inhibit CG activity.

(b) Preparation

The unique C-terminal extension of the /I subunit provides a specific target for antibody action that should not interfere with the biological activity of the other glycoprotein hormones. Indeed, studies reported by Stevens et al. (198 1) have clearly demonstrated antifertility effects in female baboons immunized with C-terminal peptides of the p subunit of human CG. Data presented here enable the synthesis of peptide fragments corresponding in sequence to the C-terminal extension of baboon BCG. The ability of these peptides to induce antibody production against pregnancy can now be investigated in an homologous system.

Mountain

View, CA). of cDNA library and screening

Baboon (Papio cyanocephalus and Papio anubis) placentae were obtained from Dr. T. Kuehle (San Antonio, TX) and Dr. V. Stevens (Columbus, OH), respectively. Poly(A)+ RNA was isolated from 27-day baboon placentae using guanidinium thiocyanate (Chirgwin et al., 1979). Double-stranded cDNA was synthesized (Gubler and Hoffman, 1983) and cloned into @lo vector (Huynh et al., 1984). The library was probed with human BCG cDNA probes (1977).

RESULTS

as described

by Benton

and Davis

AND DISCUSSION

(a) The number of /KG-related genome

genes in the baboon

Two introns interrupt the human j3CG gene at nt positions 40 and 211 of the corresponding cloned cDNA sequence (Talmadge et al., 1984a) (Fig. 1). A single PslI site, spanning nt 220 through 225, therefore divides the cloned cDNA into one fragment that exclusively encompasses the 3’ exon, and another that contains the entire middle exon (as well as small fragments of the 5’ and 3’ exons). Two separate 32P probes, each containing essentially one of the two human j?CG exons (Fig. 1) were hybridized to genomic baboon DNA previously

163

lntron B

lntron A

23.1 9.4 6.6 4.4

f

2.3 2.03

0.66

-

J

ub

.

m 1234

1234

Fig. 1. Hybridization

of baboon

DNA with 5’ exon and 3’ exon

human BCG cDNA probes. cDNA fragments /KG

exons

acrylamide

were isolated gel electrophoresis

and Goodman, (Maniatis (Taylor

of human

1980). The separate

et al., et al.,

1982),

1976), and

PstI fragments

32P-labelled

using

hybridized

(Southern,

1975). The filters were exposed

probe,

from hybridization respectively.

different 3, HindHI; fragments

baboon

The left-hand

restriction 4, Pstl.

(Papio

enzymes.

baboon Lanes:

film for

autoradiographs

rectangles

DNA digested 1, BamHI;

represent

used as 5’ and 3’ exon probes.

nitrocellulose

with

2, EcoRI;

separate

Triangles

the intron positions

to the PstI site within the human

onto

filters

anubis) DNA

to X-ray

PstI

(labelled

with respect

BCG gene.

(Southern,

(b) The nt and predicted aa sequences placental /ICC cDNAs

of baboon

primers

with the 5’ exon probe and 3’ exon

intron A and intron B) indicate

transferred

(Fiddes

were eluted

random

and right-hand

Lanes contain Striped

separate 8% poly-

to nitrocellulose

restriction

resulted

digested

and

PCG cDNA

containing

7 days at -80°C.

encoding

after PstI digestion

izing gene, then this result further suggests there are more than five baboon flCG-related genes. In support of this interpretation, no PstI sites were observed in the corresponding 3’ exon of several baboon j?CG cDNA clones (Fig. 2 and Table I). EcoRI digestions provided evidence that the BCG genes are closely linked. Both the 5’ and 3’ probes hybridized to a single EcoRI fragment larger than 23 kb (Fig. 1). Results with BarnHI- or HindIIIdigested DNA are consistent with the conclusion that the baboon genome contains multiple copies of closely linked /?CG related genes (Fig. 1). The human genome also contains multiple /?CGrelated genes (seven BCG-like genes and one BLH gene) that are probably all linked within single Sal1 and EcoRI digestion fragments (Boorstein et al., 1982; Talmadge et al., 1984a). Hence the number and linkage of genes hybridizing to 32P-labelled /KG probes are similar in both the baboon and human genomes.

1975).

We

reasoned that the use of single exon probes would result in a close correspondence between the number of hybridizing bands and the number of BCG-related genes in the baboon genome. The 3’ exon probe hybridizes to five PstI fragments of different size, with varying intensity (Fig. 1). This result indicates the presence of multiple /3CG related genes in the baboon genome. If each hybridizing PstI fragment contains a complete 3’ exon, and the darker bands (e.g., the 2.4-kb and 4.4-kb bands, Fig. 1) contain more than one hybrid-

Poly(A) + RNA isolated from a 27-day-old baboon (Papio anubis) placenta was cloned using the RNase H approach to generate second-strand synthesis (Gubler and Hoffman, 1983) and JgtlO vector (Huynh et al., 1984). About 30000 independent clones were screened with 32P-labelled human /KG probes (Fiddes and Goodman, 1980). About one clone in 500 was positive indicating that approx. 0.2% of 27-day placental baboon poly(A)+ RNA contains fiCG sequences. Analysis of several /KG inserts by enzyme digestion and blot hybridization (Southern, 1975) showed that these clones contained inserts ranging in size from 400 bp to 700 bp (not shown). Inserts from these clones were subcloned into M13mp8 or M13mpl0, and the nt sequence in four of the I clones was determined by the dideoxy method (Sanger et al., 1980). The longest clone (Cl) was 642 nt in length, and the complete sequence of this clone is shown in Fig. 2. This clone encodes a peptide similar in structure to human /ICG. The complete translation product is 165 aa which includes a hydrophobic leader sequence characteristic of secretory proteins (Blobel and Dobberstein, 1975; Habener et al., 1978). Furthermore the predicted baboon peptide bears close

1G1u Thr

Met

AA~:~I~~I~A~C;TATAAAC;CC:AC~GTA~~A~I~A~~~~~~A~G~~~~AI::~~~~A~~:I~AA~~~ AT13 GAG

Arg L4u

Trp

ICTG

TOG

Lcu CTG

Lcu ICTG

Lcu CTrj

Cys

Arg

Pro

114

Asn

Ala

TIX

Cl:iC CCC: ATE

AAT

lIil:c

Gly

IIily

:34r M4t A1312 ATI~

-1

Ala 01n C;I;A ~:Arj I;

ICKil: l3;G

)

Lru

lliln

Al::Ci CTC:

Gly

Lcu Leu C:TG CTO

C:ACi 13Xi

1

Ala lX:A

Scr TI,I:

Arg Al:ifi

13111 F’ro L4u Arg I~AI:; I::I:I~ I::TI~ I:I:I:

I+,:, cl::A

L4u I:TI>

Lys

Glu

Ala

lCys

C:ys

Val

Thr

AAG

IIiAlIi I~I~lZ Tl:iT 1~121~ GTlIi I::

Tl3lz

GTIZ

AIZI)

I;ln

Ala

Val GTC

a

Val Asn r-jTt; ,+.,I;

Lcu l;TCi

Thr At;C

Lcu I,TC

Arg lzoc

Pro F’ro Val 1:1;13 CC’C GTI;

Thr ACC

Gly

IXT

13lIiC TGC

Cys TOT

Pro

Gln

I:IX:

C:AG I~TG

Val

Val

Asn

Cys TIX

GTI;

Met ATEi

Arg 011~ I:C;C: I:AI~

TyrAAC: TAI:

PrIII Val

Met ATCi

Arg Cl33

Val CiTG

CTG

Val

Arg

Phc

Glu

I;TG

I::I:~I:: TTI::

I~AI~ TC:l: ATE

Ar-g Cl;fi

Val

Pro

Ala

Cys

Lru

C:AG CilX

C;cr

114

T

Pro

Cys TOT

Lcu Ala Ala lrilu AlI:lz C:TG C;lI:T 1~1~~ GAG

1:~s Pro Thr I14 Cys Ala rIily Tyr A-f11 TI:~T I-J::C: I~I>C: TAt:: TI:~I:: c:l::C ACC

Thr &;I;

A

Thr

Cys

Ala l3lzA

Asp GAT

Pro

Lcu IITC

Asp GAC

Pro

1G1y

Val

Asp

I”C:C; IXT

133:

GTI;

C;AC I,IX

Cys TIX

At-g Cl2c

Pro

Asn His Asn

Val Val Met

Pro

Scr

Val

AT13 13 I3

-‘TC: TIX: IJ

!f;etTIZT

Asp l3AlI

Cys TQT

Ssr 1313: Tl::C

Scr TCT

Arg lzl3c

S4r Al3C

ThrAIZC

Lcu CTC

Gln

Ala

GTT I::IX 1:

Vel

Leu

!Scr

GTC; l:iC:T ICTI:

AI;C: TGT

T

l>GCi GIliT

b-a Lys CCC: AAG

l>AC

His IZAC

b-o IZIZT

Ser TIX

!34r TCA

Lys AAG

Asp I~AC

Pro IXT

Fro 1332

Pro 1332

!jcr AIX

Ala

131~ Thr

Pro

F’he

L~IJ Pro

145 I:;ln

nwr

WA

133C

TTC:

l:TC:

CAA

TAA

131~ IIily

Asp

0

0

Cl:12 AAC c:

CAG

L4u TTlIi

Pro IXT

A

0 PVU WA

Sar AGT

Pro CCA

Ser TCC

Arg CGA

Leu CTC

L4u CTG

Glu Pro GAG CCA A G

AC:C I::I,~

r;CG

Al;GCTTC

TCAATCTGCAAAAAAAA Fig. 2. The nt and predicted into M13mp

8 or Ml3mp

aa sequences

10 (Messing

1980) using either a universal by the solid-phase

The complete

sequence.

Only nt (written

C2 and C3. Asterisks marks

serine

sequence:

Melbourne). sequence

residues

mature

/ICG cDNAs.

or specific baboon

(McBride

using computer

homologous cleavage

to O-linked

to human

glycosylation

primers.

1983) and purified by preparative by Staden

glycosylated

point is shown by a downward

arrow

(Sanger

et al.,

were synthesized

gel electrophoresis.

Sequence

by T. Kyne (Walter

(Papio anubL~) placenta

were

and extend to the poly(A)

above Cl then C2) that differ from Cl are shown for clones

sites (Asn-X-Thr sites within

method Primers

(1977; 1978) and modified

of cDNA derived from a single baboon (written

BCG probe were subcloned

by the dideoxynucleotide

BCG oligodeoxyribonucleotide

developed

nt sequences

from I hybridizing

Clones C2 and C3 begin at nt 135 and 354, respectively,

below Cl then C2) and aa positions N-linked

Inserts

were determined

and Carruthers,

programmes

Three separate

of Cl is shown.

identify consensus

peptide

primer

procedure

and analysed

and Eliza Hall Institute, aligned.

sequencing

phosphoramidite

data were assembled

of baboon

and Vieira, 1982). Sequences

or Asn-X-Ser)

human between

at aa residues

/JCG (Canfield aa -1 and

33 and 50. The letter 0

et al., 1978). The proposed

+ 1.

leader

165

TABLE

I

Nucleotide

differences

in baboon Position

cDNA

clone b

/KG

cDNA

clones

of nt difference a

184

257

A

N

N

B

N

N

341

344

423

434

528

596

634

N

N

N

N

A

G

C

N

N

G

C

A

A

T

Cl

A

T

C

G

A

T

A

G

T

c2

G

C

A

T

G

C

C

A

T

c3

N

N

N

N

G

T

A

G

T

a Positions prepared

of nt differences and isolated

are numbered

as described

according

in MATERIALS

to Fig. 2. N indicate AND METHODS,

undetermined

nt in short cDNA

and nt sequences

clones. cDNA

clones were

as described

in the legend

were determined

to Fig. 2. b Clones, A and B originate

from separate

Pupio cyanocephalus placentae.

Clones Cl, C2 and C3 were derived from a single Pupio anubis

placenta.

similarity to the human /?CG peptide (Fiddes and Goodman, 1980). We therefore conclude that this cDNA clone codes for a baboon flCG peptide. Each of the other cDNA clones sequenced are shorter than clone Cl. One is 382 nt in length, and is identical in sequence to clone Cl. The other two clones contain sequence differences when compared to Cl. Clone C2 contains a sequence that encodes a complete /KG peptide, but lacked the two 5’-terminal codons of the leader peptide, and clone C3 extends from nt 354 to 642 when compared to Cl (Fig. 2). The nt differences are apparent at eight positions when the sequences of the three separate Papio anubis PCG clones are compared (Table I; Fig. 2). Three of the eight nt differences result in aa changes. The single nt difference between Cl and C3 (at nt 423, Fig. 2) alters the Met residue in Cl to Val. This change, as well as two additional aa differences are apparent when Cl is compared to C2 (Fig. 2). His replaces the Asn residue of Cl (nt change at 528, Fig. 2) and within the leader sequence the Cl Gln residue is changed to Arg (nt position 184, Fig. 2). Clones Cl and C2 encode complete /?CG peptides. Within the hormone-coding region these clones differ at 7 nt positions (1.6%) and 2 aa positions (1.4%) (Table II). A different baboon species (Papio cyanocephalus) was used to prepare two additional cDNA clone banks, and one /KG clone was analysed from each.

The nt sequences of these two clones differ from each of the Papio anubis clones (Table I). However, the peptide sequences predicted from the Papio cyanocephalus clones are identical with the correTABLE

II

Baboon

and human

Comparison

HPCG Hj?CG

a

:BabBCG-Cl :BabBCG-C2

Babj3CG-Cl: HBCG

)?CG comparison

BabBCG-C2

: HfiLH

nt changes”

aa changes’

(%)

(%)

43/438 (9.8)

27/145 (18.6)

40/438 (9.1)

26/145 (17.9)

7/438 (1.6)

2/145 ( 1.4)

26/334 (7.8)

17/115 (14.8)

a BCG comparisons

were made over the hormone

coding region,

from

start

1 up

(Figr3).

The

HBLH

: HBCG comparison

was made over the homologous

hor-

codon

mone-coding

to

region from codon

nt deletion

codon

number

ratio

146

1 up to codon 94, where a single

in the BCG gene accounts

results in the )?CG C-terminal b The

stop

for the frameshift

extension

of different

(Talmadge

nt

was

that

et al., 1984a)

determined

from

total nt BabfiCG-Cl H/KG

and BabfiCG-C2

and HBLH comparisons

of Fiddes

and Goodman

cDNA sequences

shown in Fig. 2.

were determined

from the data

(1980) and Talmadge

et al. (1984a),

respectively. c The ratio

number

of aa differences

was determined

from pep-

total aa tide sequences

predicted

from the cDNAs

from the data

of Fiddes

and Goodman

et al. (1984a).

shown in Fig. 2 and (1980) and Talmadge

166

sponding region of peptide C3 from Papio anubis. Knowledge of baboon /?CG sequences will allow antipregnancy vaccine studies (Stevens et al., 1981) to proceed in an homologous system. It is particularly relevant to these studies that the predicted BCG C-terminal peptide sequence is identical for each of five different cDNA clones from two species of baboon. (c) At least two /KC

genes are expressed

in the

baboon placenta

Three different /?CG cDNA clones (Cl, C2 and C3) were isolated from a library derived from a single baboon placenta. Two of these clones (Cl and C2) showed differences at 8 nt positions, while the third clone (C3) was different to clone Cl at only 1 of 5 nt positions compared (Table I). It is possible that differences between any two of the clones are polymorphic and that these clones are products of separate alleles. It follows that the third clone originates from a non-allelic gene. It is also possible that the three clones result from transcription of three separate /?CG genes. Regardless of these alternatives, it is clear that more than one PCG related gene is expressed in the baboon placenta. These data are in agreement with that from human subjects where two different /?CG mRNAs from separate genes were also detected in the lirsttrimester placenta. Additional evidence suggests that no more than three of the seven human /?CG-like genes are active in the placenta (Talmadge et al., the additional genes are 1984b). Whether pseudogenes, or expressed in other tissues remains to be determined. Simultaneous expression of more than one /?CG-like gene in the placenta suggests that the peptides might exert separate hormonal effects. Hence it may be worthwhile to investigate whether differences in the biological activities of the respective peptides can be detected. Such differences may be difficult to demonstrate considering their nearidentical peptide sequence. It is also possible that separate PCG-like genes are expressed within the placenta at varying stages of pregnancy. This would explain, at least to some extent, why the baboon and human genomes contain multiple BCG-related genes. This possibility could be investigated by determining the nt sequence of PCG mRNAs within placenta both older and younger than the 25 to 30 day tissue used here.

(d) Comparison

of baboon

and human

/XC

se-

quences

A total of 49 nt differences are apparent when the coding region of baboon /?CG clone Cl is compared to that of man (Fig. 3). There are 43 (9.8%) nt differences in the hormone region, resulting in 27 aa (18.6%) differences (Fig. 3; Table II). Similarly the extent of nt and aa variation between baboon clone C2 and human is 9.1% and 17.9x, respectively (Table II). This level of difference contrasts with the closer similarity between some other baboon and human peptides. For example about 4% and 7% diversity is observed when B globin (Nute and Mahoney, 1980; Braunitzer et al., 1961) and a-1-antitrypsin (Kurachi et al., 1981; Bollen et al., 1983) peptides are aligned, respectively. Many of these differences are non-conservative in nature. For example aa residues 8 and 83 are Leu and Pro in baboon and Arg and Ala in man (Fig. 3). Despite the many aa differences, the hydropathy profiles (Kyte and Doolittle, 1982) of the baboon and human BCG peptides are virtually identical (Fig. 4). These profiles illustrate that the two most hydrophobic regions of the hormone (apart from the leader sequence) are localised approximately between aa residues 23 and 57 and residues 75 and 93. The most hydrophilic region covers the C-terminal portion of the peptide from about Cys residue 93 to about Gly 139. One particular aa difference between the peptides is noteworthy. It has been suggested that an Ala codon instead of Asp at aa position 117 in the human peptide, correlates with nonexpression in the human placenta (Talmadge et al., 1984b). This is clearly not the case in baboon because each of the baboon BCG cDNAs sequenced contain an Ala codon at this position. (e) Evolution of /KG genes

The pituitary glycoprotein hormones are synthesized in a wide range of species, whereas placental CG synthesis appears restricted to certain mammals (Canfield et al., 1978; Pierce and Parsons, 1981). Hence PCG is believed to be the most recently evolved member of the glycoprotein hormone /? subunit family. At least in man CG most closely resembles LH in structure (Dayhoff, 1976) and

VP1

36

Ila

Asn

Ala

Thr

Leu

Ala

Ala

I;I:I:

ATC

AAT

oc:c

ACI:

I:TF

C;IX

Ile

Cys

Ala

Gly

Tyr

TI>T

rjtX

1:~s At-g Fra TljC

C132

Val

Asn

Thr

Thr

ijTC

AAC

ACC

AI::I: ATI:

Lys

131~ Ala

I;C:I: I;AI~ TO

AAG

l>Al2 l?l~l~: Tl:iT l::l~:l: l>Tl> 1: G

Cys

Pro

Thr

Met

IXC

TAC: TOI:

l::lX

AN:

ATl_7 ATI> lZl3E l3TG t: t: 1:

Val

Val

Asn

Tyr

Arg

ASP Olu

Val

Arg

Pha

Glu

GTC

t3Trj

l:y!tt TtX

AAC

TAC

IZuC

l:AG

OTt2

IZcic

TTC:

GAG

Tyr Val

Ala Pro

Val

Ala

*

Ala Leu Lcu Pro Fro Val Pro rjln tzTt3 CCC CCC GTCi Cl~l~ CAG t: T 12

Lcu

Pro

C;ly

Cys

Pro

CTI=

CCT

GOC

TGC

CtX

I3 1 n Arg Cys

Ala

Leu

t:fiC

t;CA

CTC

TI>T

Arg Fro

Thr Mat

Cys

Ar’g

Pro

Val

Val

Lcu

Asn Asp

cily

Val

CCT t;c

tXtc.

GTG

GAI: A

Cys

Arg

Arg

Scr

TGC.

t;GC

CTjl:

AtX

Thr AtX

Fro t:tX

Val Met

Val

ATIS t:

GTC

!Ser Tt:T

Ser TtX

Asp

t::ys tily

t;Ar;

TOT

Arg Phe Asp Asp Pru Asn Lcu t3AT GAC CCC AAC: CTr, CG T

Leu

CGA

CTtZ

I>TI~ A

01n

Thr AIX

Gl y Ala Val l3tX t>

CiTtZ

:Ser

Ile

Arg

TCC

ATC

Ct>G

Leu

Ser

ASP Gin Ala CAG GtX A

tGTtIi GC:T t::TC AGC

01~

Fro

Lys

Asp

His

CCC

AAG

t3At:

CAC: XT

tGt37 tXiT

Fro Lru

Oly Glu

Cl73 1:

GAG G

Ala

0 !&r

Ser

Lys

Tt::C Tt:T

TW

TCA

AAG

Pra

11s Phe

Pro

Ser Ala

Asp rjly

Thr

CCA

WA

t:t3C

AtX

CTG

Fro

A

TOT

LWJ

TTtz t;

Set-

!&r

Cys

rjTT CCC TAC: Ci

Asp GAC 1:

Pro CCT

F’ro CCC

Fro CtX

Leu t;C:o TTC: CTI: A

Pro

14.3 rjln

)ew

lXt3

CAA

TAA

0 Arg

Tl>ll

t:

A

Pro Ssr Fro,Ser CCA At3T CCA TCC

Val

1Z:TG IZAG

Thr

0

Cys

T

AA

Thr CYS ACC TGT

IlC

GlY

Glu.

0

Let1

!%r AGC

Fr-a CCT T

AiX:TTC

A

Fig. 3. The nt and predicted aa sequences of baboon and human jCG subunits. The sequences of baboon jCG clone Cl (see Fig. 2) are aligned with those of human fiCG (Fiddes and Goodman, 1980). Only those positions which differ from baboon BCG are shown for the human sequence (nt differences below and aa replacement substitutions above the baboon sequence). Asterisks and letters 0 identify N-linked and O-linked glycosylation sites, respectively. The Ser residue at aa 158 is O-linked glycosylated in man (Canfield et al., 1978). The aa residue at this position in the baboon is Ala.

biological activity (Pierce and Parsons, 1981). This clone similarity is also apparent in the gene sequences of the respective fi subunits (Talmadge et al., 1984a). It is therefore likely that the jS33 gene originated by duplication of an ancestral BLH-like and gene, and that subsequent duplications rearrangements of the PCG gene were responsible

for the /?CG multigene family detected in humans and baboons. The evolutionary events from the point of fiCG$LH divergence are not clear. These events have resulted in a marked divergence of /KG-related genes between baboon and human. (In fact human PCG more closely resembles human /?LH (92.2% nt

168

It is significant

that a high proportion

of replace-

ment changes has occurred between human and baboon /?CG-genes; of 43 nt differences in the hormone

coding

region,

30 are replacement.

more many of the aa changes suggesting the

Hydropathy

B

Fig. 4. Hydropathy Doolittle

Sequences

were

analysed

(1982) using the computer

(A) Baboon

BCG; (B) human

human

and baboon

according programme

to Kyte

/XC and

of Dr. T. Kyne.

BCG.

and 85.2% aa homology) than baboon /?CG (90-91x nt and 82% aa homology; Table II). In contrast the sequences of /?CG-related genes within species (baboon or human) are very similar. These observations suggest that recombinational events, equal crossing-over or gene conversion events which may have resulted in the present BCG gene family, occurred much later than the primary /?CG//?LH duplication, and after the evolutionary divergence of baboon and man. However, this evolutionary course is unlikely because the j?CG gene duplication probably occurred soon after the separation of /3CG and /?LH genes, since the divergence rate of the introns from human PCG genes 5 and 6 is similar to that of PLH and fiCG introns (Talmadge et al., 1984a). Hence it is not clear how divergence of /?CG genes proceeded between species while maintaining sequence conservation within species. It is possible that gene conversion, which can operate to maintain homogeneity within a gene family (Slightom et al., 1980) has been involved in conserving j?CG sequences within species. However, such a conversion process has excluded the PLH gene, despite the close physical linkage of /?LH and PCG-like genes.

sequence

of BCG

explanations

First,

CG

may be under less selective pressure

than

hormonal

possible.

species.

Several

other

are

between

functions most

plot of aligned

that there is not a strict need to conserve

peptide

receptor

peptides.

Further-

are nonconservative,

activities.

may be capable

Second,

of responding

the CG to hetero-

geneous CG-related peptides, thus allowing the evolution of diverse CG-like peptides. The likelihood of this suggestion is supported by the observation that LH is capable of exerting CG-like activities (Pierce and Parsons, 1981) despite the high degree of sequence divergence between human /?LH and j?CG peptides (Table II). Alternatively selective pressure may act on the interaction between the CG peptide and its receptor rather than the flCG sequence alone. Divergence of BCG sequences between species would then be permitted if the BCG sequence and its receptor evolved congruently.

ACKNOWLEDGEMENTS

We thank Dr. T. Kuehl (San Antonio, TX) and Dr. V. Stevens (Ohio State University, Columbus, OH) for providing baboon placentae, and Dr. J. Fiddes for allowing the use of the human /?CG cDNA clone. We are grateful for the excellent technical assistance of Vicki Hammond and Lucy Duncan, and to Dr. Tony Kyne for use of computer programmes. This work was supported in part by the Special Programme for Research in Human Reproduction, World Health Organisation, and by the National Health and Medical Research Council of Australia.

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