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0013.7227/96/$03.00/O Endocrinology Copyright 0 1996 by The Endocrine Society Vol. 137, No. 11 Printed in U.S.A. Prolactin-Like Protein-C Variant: Complementary Deoxyribonucleic Acid, Unique Six Exon Gene Structure, and Trophoblast Cell-Specific Expression* GUOLI DAI, BING LIU, CLAUDE SZPIRER, GORAN LEVAN, SIMON C. M. WOK, AND MICHAElL J. SOARES Department of Physiology (G.D., B.L., M.J.S.), University of Kansas Medical Center, Kansas City, Kansas 66160; Department de Biologie Moleculaire (C.S.), Universite Libre de Bruxelles, B-1640 Rhode St. Genese, Belgium; Department of Genetics (G.L.), University of Gothenburg, S-400 33 Gothenburg, Sweden; Department of Obstetrics and Gynecology (S.C.M.K.), Albert Einstein Medical Center, Philadelphia, Pennsylvania 19141-3025 ABSTRACT The rat placental PRL family consists of proteins structurally re- lated to pituitary PRL. As a consequence of attempting to characterize the gene for one of the members of the family, PRL-like protein-C (PLP-C), we identified a related gene that we have termed PLP-C variant (PLP-Cv). In this study, we present information on the PLP-Cv gene and its pattern of expression. Screening of a rat genomic library with a PLP-C cDNA resulted in the isolation of four phage clones. Nucleotide sequence analysis of the clones revealed a gene, PLP-Cv, closely related but distinct from PLP-C. The PLP-Cv gene possessed a six exon/five intron organization, unique among members of the PRL family, and was localized to chromosome 17 of the rat genome, similar to other PRL family members. A PCR strategy in- volving primers based on the PLP-Cv gene was used to isolate a placental PLP-Cv cDNA. PLP-Cv showed 90 and 78% sequence iden- tity with PLP-C at nucleotide and amino acid levels, respectively. Expression of PLP-Cv was restricted to the trophoblast lineage and was coordinately activated with PLP-C beginning at day 11 of ges- tation and continuing until term. Primer extension analysis revealed multiple putative transcription start sites. A 2.1-kilobase pair PLP-Cv promoter-luciferase reporter construct was specifically acti- vated in differentiating rat trophoblast cells but not in other cell types. In conclusion, we have identified a new member of the PRL family possessing considerable homology to PLP-C, a unique gene structure, and displaying a trophoblast-specific pattern of transcriptional acti- vation. (Endocrinology 137: 5009-5019, 1996) T HE RAT PRL family represents a cluster of several genes located on chromosome 17 (l-3). Members of the fam- ily include: PRL (l), placental lactogen-I (PL-I; 4, 5), PL-I variant (PL-Iv; 6,7), PL-II (g-lo), PRL-like protein-A (PLP-A; 9,11,12), PRL-like protein-B (PLP-B; 13), PRL-like protein-C (PLP-C; 14, 15), and decidual/ trophoblast PRL-related pro- tein (d/ tPRP, 16). These genes are expressed by the anterior pituitary, uterus, or placenta during specific phases of preg- nancy and participate in the coordination of maternal and fetal tissue responses (2, 3). Some members of the family (PL-I, PL-Iv, and PL-II) are functional agonists of PRL (17-19) and represent the principal ligands for the PRL receptor, especially during the second half of gestation (20). PLP-A and PLP-C do not utilize the PRL receptor signaling pathway and appear to possess unique actions (21, 22). Thus far, only the gene structure for PRL has been reported (23-25). During the course of analyzing the PLP-C gene, we iden- tified a closely related gene that we have termed, PLP-C variant (PLP-Cv). In this report, we present data on the Received June 10, 1996. Address all correspondence and requests for reprints to: Michael J. Soares, Department of Physiology, University of Kansas Medical Center, Kansas City, Kansas 66160-7401. * This work was supported by grants from the National Institute of Child Health and Human Development (HD-29036 and HD-29797), by the Fund for Scientific Medical Research (FRSM, Belgium), and by the Belgian Programme on interuniversity attraction poles initiated by the Belgian State. structure of the PLP-Cv gene and cDNA and demonstrate the trophoblast cell-specific characteristics of its expression. Materials and Methods Reagents FBS and donor horse serum (HS) were purchased from JRH Bio- science (Lenexa, KS). Reagents for PAGE were purchased from Bio-Rad (Hercules, CA). All restriction enzymes, polymerases, and DNA ligase were purchased from New England Biolabs (Beverly, MA). The GH, pituitary tumor and L929 cell lines and a Rous sarcoma virus promoter- P-galactosidase (RSV-P-GAL) reporter plasmid were obtained from the American Type Culture Collection (Rockville, MD). Transformation competent Sure bacterial cells, flash nonradioactive gene mapping kit, and a rat genomic library were acquired from Stratagene (La Jolla, CA). DNA extraction kits were purchased from Qiagen (Chatsworth, CA). Nitrocellulose and nylon membranes were obtained from Schleicher and Schuell (Keene, NH). The pGL-2 basic vector and an RSV promoter- luciferase reporter plasmid were purchased from Promega (Madison, WI). T7 DNA sequencing kits were acquired from United States Bio- chemical (Cleveland, OH). PCR cloning kits were obtained from In- vitrogen (San Diego, CA) and CLONTECH (Palo Alto, CA). Radio- labeled nucleotides were purchased from DuPont-NEN (Boston, MA). TRIzol reagent for RNA extraction, RT-PCR kits, and LipofectAmine reagent for transfection were obtained from Life Technologies (Gaith- ersburg, MD). Kits for monitoring P-GAL activities were acquired from Tropix (Bedford, MA). Unless otherwise noted, all other chemicals and reagents were purchased from Sigma Chemical Co. (St. Louis, MO). Animals and tissue preparation Holtzman rats were obtained from Harlan Sprague-Dawley (Indianapolis, IN). The animals were housed in an environmentally controlled facility, with lights on from 0600-2000 h, and allowed free 5009 on March 9, 2006 endo.endojournals.org Downloaded from

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0013.7227/96/$03.00/O Endocrinology Copyright 0 1996 by The Endocrine Society

Vol. 137, No. 11 Printed in U.S.A.

Prolactin-Like Protein-C Variant: Complementary Deoxyribonucleic Acid, Unique Six Exon Gene Structure, and Trophoblast Cell-Specific Expression*

GUOLI DAI, BING LIU, CLAUDE SZPIRER, GORAN LEVAN, SIMON C. M. WOK, AND MICHAElL J. SOARES

Department of Physiology (G.D., B.L., M.J.S.), University of Kansas Medical Center, Kansas City, Kansas 66160; Department de Biologie Moleculaire (C.S.), Universite Libre de Bruxelles, B-1640 Rhode St. Genese, Belgium; Department of Genetics (G.L.), University of Gothenburg, S-400 33 Gothenburg, Sweden; Department of Obstetrics and Gynecology (S.C.M.K.), Albert Einstein Medical Center, Philadelphia, Pennsylvania 19141-3025

ABSTRACT The rat placental PRL family consists of proteins structurally re-

lated to pituitary PRL. As a consequence of attempting to characterize the gene for one of the members of the family, PRL-like protein-C (PLP-C), we identified a related gene that we have termed PLP-C variant (PLP-Cv). In this study, we present information on the PLP-Cv gene and its pattern of expression. Screening of a rat genomic library with a PLP-C cDNA resulted in the isolation of four phage clones. Nucleotide sequence analysis of the clones revealed a gene, PLP-Cv, closely related but distinct from PLP-C. The PLP-Cv gene possessed a six exon/five intron organization, unique among members of the PRL family, and was localized to chromosome 17 of the rat genome, similar to other PRL family members. A PCR strategy in-

volving primers based on the PLP-Cv gene was used to isolate a placental PLP-Cv cDNA. PLP-Cv showed 90 and 78% sequence iden- tity with PLP-C at nucleotide and amino acid levels, respectively. Expression of PLP-Cv was restricted to the trophoblast lineage and was coordinately activated with PLP-C beginning at day 11 of ges- tation and continuing until term. Primer extension analysis revealed multiple putative transcription start sites. A 2.1-kilobase pair PLP-Cv promoter-luciferase reporter construct was specifically acti- vated in differentiating rat trophoblast cells but not in other cell types. In conclusion, we have identified a new member of the PRL family possessing considerable homology to PLP-C, a unique gene structure, and displaying a trophoblast-specific pattern of transcriptional acti- vation. (Endocrinology 137: 5009-5019, 1996)

T HE RAT PRL family represents a cluster of several genes located on chromosome 17 (l-3). Members of the fam-

ily include: PRL (l), placental lactogen-I (PL-I; 4, 5), PL-I variant (PL-Iv; 6,7), PL-II (g-lo), PRL-like protein-A (PLP-A; 9,11,12), PRL-like protein-B (PLP-B; 13), PRL-like protein-C (PLP-C; 14, 15), and decidual/ trophoblast PRL-related pro- tein (d/ tPRP, 16). These genes are expressed by the anterior pituitary, uterus, or placenta during specific phases of preg- nancy and participate in the coordination of maternal and fetal tissue responses (2, 3). Some members of the family (PL-I, PL-Iv, and PL-II) are functional agonists of PRL (17-19) and represent the principal ligands for the PRL receptor, especially during the second half of gestation (20). PLP-A and PLP-C do not utilize the PRL receptor signaling pathway and appear to possess unique actions (21, 22). Thus far, only the gene structure for PRL has been reported (23-25).

During the course of analyzing the PLP-C gene, we iden- tified a closely related gene that we have termed, PLP-C variant (PLP-Cv). In this report, we present data on the

Received June 10, 1996. Address all correspondence and requests for reprints to: Michael J.

Soares, Department of Physiology, University of Kansas Medical Center, Kansas City, Kansas 66160-7401.

* This work was supported by grants from the National Institute of Child Health and Human Development (HD-29036 and HD-29797), by the Fund for Scientific Medical Research (FRSM, Belgium), and by the Belgian Programme on interuniversity attraction poles initiated by the Belgian State.

structure of the PLP-Cv gene and cDNA and demonstrate the trophoblast cell-specific characteristics of its expression.

Materials and Methods

Reagents

FBS and donor horse serum (HS) were purchased from JRH Bio- science (Lenexa, KS). Reagents for PAGE were purchased from Bio-Rad (Hercules, CA). All restriction enzymes, polymerases, and DNA ligase were purchased from New England Biolabs (Beverly, MA). The GH, pituitary tumor and L929 cell lines and a Rous sarcoma virus promoter- P-galactosidase (RSV-P-GAL) reporter plasmid were obtained from the American Type Culture Collection (Rockville, MD). Transformation competent Sure bacterial cells, flash nonradioactive gene mapping kit, and a rat genomic library were acquired from Stratagene (La Jolla, CA). DNA extraction kits were purchased from Qiagen (Chatsworth, CA). Nitrocellulose and nylon membranes were obtained from Schleicher and Schuell (Keene, NH). The pGL-2 basic vector and an RSV promoter- luciferase reporter plasmid were purchased from Promega (Madison, WI). T7 DNA sequencing kits were acquired from United States Bio- chemical (Cleveland, OH). PCR cloning kits were obtained from In- vitrogen (San Diego, CA) and CLONTECH (Palo Alto, CA). Radio- labeled nucleotides were purchased from DuPont-NEN (Boston, MA). TRIzol reagent for RNA extraction, RT-PCR kits, and LipofectAmine reagent for transfection were obtained from Life Technologies (Gaith- ersburg, MD). Kits for monitoring P-GAL activities were acquired from Tropix (Bedford, MA). Unless otherwise noted, all other chemicals and reagents were purchased from Sigma Chemical Co. (St. Louis, MO).

Animals and tissue preparation

Holtzman rats were obtained from Harlan Sprague-Dawley (Indianapolis, IN). The animals were housed in an environmentally controlled facility, with lights on from 0600-2000 h, and allowed free

5009

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PRL-LIKE PROTEIN-C VARIANT Endo. 1996 Vol 137 . No 11

access to food and water. Timed pregnancies and tissue dissections were (32) using oligonucleotide primers corresponding to regions of the I’LP-C performed as previously described (26). The presence of a copulatory cDNA to sequence all exons and exon/intron boundaries. Sequencing plug or sperm in the vaginal smear was designated day 0 of pregnancy. reactions used [35S] dATI’ and were performed with T7 sequencing kits. Timed pregnant Wistar Kyoto outbred rats were obtained from Taconic Reaction products were resolved in 6% polyacrylamide urea gels, dried, Farms (Germantown, NY). and exposed to Kodak XAR film.

Protocols for the care and use of animals were approved by the University of Kansas Animal Care and Use Committee. Chromosomal assignment

Cell lines

A series of trophoblast and nontrophoblast cell lines were examined for their ability to express PLP-Cv. The Rcho-1 trophoblast cell line was derived from a rat choriocarcinoma and is capable of differentiating along the trophoblast giant cell lineage (27). Rcho-1 trophoblast cells were routinely maintained in subconfluent conditions with NCTC-135 culture medium supplemented with 20% FBS, 50 PM 2-mercaptoethanol, 1 mrvr sodium pyruvate, 100 U/ml of penicillin, and 100 Kg/ml of streptomycin (27). Differentiation was induced by growing the cells to confluence in FBS supplemented culture medium and then replacing the serum supplemen- tation with 10% HS (28, 29). HIV-1 trophoendodermal stem cell line rep- resents a cell population with both trophoblast and yolk sac attributes (30). HRP-1 trophoendodermal cells were routinely maintained in RPMI-1640 culture medium containing 10% FBS and the above supplements. GH, cells were derived from a rat pituitary tumor (31) and were maintained in DMEM culture medium supplemented with 10% FBS and antibiotics. L929 cells represent a mouse fibroblast cell line and were maintained in RPMl culture medium supplemented with 10% FBS and antibiotics.

Cell hybrids used in the chromosomal localization of the PLP-Cv gene were derived from the fusion of mouse hepatoma cells (BWTG3) with adult rat hepatocytes and have been used in the chromosomal assign- ment of several rat genes (33). Chromosomal preparations were gener- ated as previously reported (34, 35). DNA was extracted and analyzed by PCR using primers specific for PLP-Cv. The PCR reaction was per- formed for 35 cycles (denature, 94 C for 30 set; annealing and extension, 68 C for 6 min). Reaction products were electrophoretically separated in 1% agarose gels, transferred to nylon membranes, and probed with a [32P]-labeled PLP-Cv cDNA (36).

Zsolation and characterization of the PLP-Cv cDNA

Zsolation and characterization of the PLP-Cv gene

A genomic DNA library generated from lZweek-old male Wistar Kyoto outbred rat liver and packaged in the Lambda DASH II vector was obtained from Stratagene. The library was screened with a rat PLP-C cDNA as previously described (15). Positive plaques were amplified and used to inoculate LE392 Escherechiu coli. Phage DNA from four positive clones was isolated, spotted on to nylon membranes and probed with oligonucleotides representing either the 5’ or 3’ end of the PLP-C cDNA. The oligonucle- otides were end-labeled with T4 polynucleotide kinase and [r3 I’] ATP. Th e genomic DNA was excised with Not1 and then partially digested with HindIII, BamHl, and Xbal to generate a restriction map. Restriction frag- ments containing exons were obtained by complete digestion with these restriction enzymes and Southern blotting with a radiolabeled PLP-C cDNA probe. HindIIl restriction fragments hybridizing to the PLP-C cDNA were subcloned into pBluescript sk+ vector (Stratagene) for sequencing. DNA sequencing was performed by the dideoxy chain termination method

Specific primers for the amplification of the PLP-Cv cDNA were designed based on sequences from the 5’ and 3’ untranslated regions of the PLP-C cDNA and the corresponding sequences predicted for the PLP-Cv cDNA from the sequence of the PLP-Cv gene. Total RNA was extracted with TRIzol Reagent (Life Technologies) from the junctional zone of the rat chorioallantoic placenta on day 19 pregnancy. RT-PCR was performed according to the manufacturer’s instructions (Super- Script preamplification system for first strand cDNA Synthesis Kit, Life Technologies). Five pg of total placental RNA and 0.5 PLg of oligo(de- oxythymidine) were used for the RT reaction. The PCR reaction was performed for 30 cycles (denature, 94 C for 1 min; annealing, 60 C for 2 min; extension, 72 C for 2 min). The amplified products were subcloned into the pCR II vector flanked by Sp6 and T7 promoters (Invitrogen) and sequenced as described above. Oligonucleotides complementary to the Sp6 and T7 promoters and to two internal segments of the PLP-Cv cDNA were used as primers for the sequencing reactions.

Tissue distribution of PLP-C and PLP-Cv expression

Total RNA was extracted from a range of different rat tissues in- cluding, spleen, liver, thymus, hypothalamus, anterior pituitary, ovary, uterus, decidua, and the labyrinth and junctional zones of the rat cho- rioallantoic placenta and from the Rcho-1 trophoblast, L929 fibroblast, GH, pituitary, and HRP-1 trophoendodermal cell lines, with the TRIzol

- 1 kb

FIG. 1. Schematic representation of the PLP-Cv genomic clone (RGLC-31). Restriction map of the 15.4-kb RGLC- Blgenomic clone. Complete digestion with Not1 and Hind111 resulted in the generation of six fragments, two of which hybridized with the PLP-C probe in a Southern blot (2.8 and 3.6 kb). A third fragment (3.0 kb) generated from the Not1 and Hind111 digestion and lo- cated immediately 5’ to the hybridizing fragments subsequently was found to contain the first exon. Please note that shaded boxes in the top panel corre- spond to genomic fragments hybridiz- ing with the PLP-C cDNA probe. Shaded boxes in the lower panel corre- spond with actual exon sizes. H, Hin- dII1; X, Xba 1; B, BamHl; E, exon; I, intron.

Not I Not I

ATG Poly A Signal

4 4’ 1 IA 1 IS 1 IC 1 ID 1 IE m

5’ I II 111 3’

El E2 E3 E4 E5 E6

PLP-Cv Genomic Clone (154kb)

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PRL-LIKE PROTEIN-C VARIANT 5011

Reagent as described above. RT-PCR was performed with primers spe- cific for PLP-C or PLP-Cv as outlined above. Control reactions were performed with primers to rat p-actin (upstream primer: 5’ ATCGT- GGGCCGCCCTAGGCA 3’; downstream primer: 5’ TGGCCTTAGGGT- TCAGAGGGG 3’). Reaction products were electrophoretically sepa- rated in 1% agarose gels, transferred to nylon membranes, and probed with [32P]-labeled PLP-C or PLP-Cv cDNAs (36).

Identification of the PLP-C and PLP-Cv transcription start sites

The transcription start sites for PLP-C and PLP-Cv were determined by primer extension analysis. Specific oligonucleotides complementary to regions in the proximity of the respective start codon (ATG) for PLP-C or PLP-Cv were synthesized and end labeled with T4 polynucleotide

TTGCACAAGGATCAACATGTAGGAACAACCAAAGA GJUAACATGCAGACAGATATTAAGTATGATGTATGATGT

AACAAAACAGTTAACATCCCCTAAAGATATATATGGTTTCTTGTTGTC ***

GCATGGCTGTGCGTGCCTGAGGGTTATCTACTA TCAGGAAACTAACTGAATGTACAATTCCAGTGA il **

4 --an ACTAGATCTTCAGAGACAGAACTCATCCTGCTTAGGAACTCCTCAGTG ATG GAG CTG GCA TTG ACT CAA

M E L A L T Q

CCT CCC TTC T gtaagtaaatcattccacacctgtgctctcttagtaaaagtatttaattcaggggtttcataaa P P F

tttattttaatggaattctttatttgcaatctttgatgttctatctggcatgaagagatatttgttcatggatttcca

ggggtagattactgcatagactttatattacctttaaaattattttttcaaagaaactgtacagggttacaggagaca

attggattagtagttttcaatatcattt....INTRON A (-2.OKb)....aaaaaaaaatctaaatactaaaatc

ctttggccagatacaacagtctggtctgaggagaactcagattaatacaccatctctaagttgggttttgtttctatg

-20 -10 tag TT GGC ACA CTC CTG ATG CTG GTG GTT TCA AAC TTG CTC TTG TGG GAG AAA GCT GCA

FGTLLMLVVSNLLLWEKAA

-1 v +1 +10 TCA ATT CCT GCA TGT ATG GTG GAA GAC GGT GGC TGC TGG GAC CCT CTC GAG GAA ACA GT

SIPACMVEDGGCWDPLEETV

+20 +30 A AAC AGT GCT ATC CAG AAA GCT GAA AAC CTC CGT AAT CTT GCT CAC CAC TTA TAT GTA

NSAIQKAENLRNLAHHLYV

+40 GAA TTC gtaagtacctaaacctccatactaatacttgaactatatgagaatgagaaacgcaaaacatcatgataaa

E F

aattatttgagaatacagcttatagttaaaagatgcccaaactactgaaacaaggacagggccaattggacagtg...

. . . INTRON B (-l.OKb) . . . . aaaagaatgtgtctctgttaggaataaacctgttgtcatattttttattgacta

+50 gatgttatttctctctccttaacattactcag TAC CAG AAC CAA TTC TCA TCT AGA CAG TTT GCA G

YQNQFSSRQFA

AT CTT gtaagtaccttgctttcttctagccgcttccctataagaacctcatgtcaatgattgtgctttagaaaaga D L

aagcattgtaaagcatttgtatttcctgttcttaatattcagtgtttcaacttcttaaccttcatgattattcatgaa

ttagctgattaattgaaataaagataagtaaatattaattgttaaa....INTRON C (-1.2Kb)....gctctac

tcttcagctaaatccccaacccctacatgaaagtttcttaattcctcaatatctgtattttgtgaattacttatgtta

aaatttaaagatccaacaaaatgcattaatattctgttcatatgtttatttatgatttctgaactctgtttttttgtt

FIG. 2. PLP-Cv gene (nucleotide sequence and exonhntron boundaries) and PLP-Cv cDNA structure. Nucleotide sequence of the PLP-Cv structural gene is shown. The locations of six exons are indicated by capital letters and flanking and intronic regions are in lower case. Three prime untranslated region of the PLP-Cv cDNA is also presented in capital letters. The location of the complementary oligonucleotide used for primer extension analysis is denoted by the horizontal arrow (see Fig. 7). Putative transcription start sites are denoted by asterisks and TATA boxes are indicated by solid boxes. Encoded amino acids are indicated by single letter designations beneath their respective codons. A putative N-linked glycosylation site is denoted by shading and the location of the presumed polyadenylation site is underlined.

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5012 PRL-LIKE PROTEIN-C VARIANT Endo. 1996 01 137 l No 11

+60 +70 tcatttgcag AAT TCA CAA CTG ATG AGG TGG GAT GAG ACT GTT GTC AAA ACT GGA ACT TAC

NSQLMRWDETVVKTGTY

+so

TGC CAT TCA ACT CTT GCA AGA CCC CAA AAT AAA GGA ATA AAT ATT GAA gtgagtttccttac CHSTLARPQNKGINIE

tgggtttttcacagaactccagaagcattacattgtgtctgtggtgctgtcagttattacaggcattttacaataata

aggtatccagaaaaaa....INTRON D (-l.lti) . . . . tacagctttacatatatattataggagtttaagctgc

+90 taaactgaaaccatttaatgcaattgctcaaatgtgtttcacag ATT GAA GTG TAT TTG CAA ACG TTG A

I E V Y L Q T L

+100 +110 TC AAT TTT GTG GGT TCA TGG ATC AGC CCT CTC CTC CAT TTA GTA ATT GAA CTG AGT GCC I' NFVGSWISPLLHLVIELSA

+120 +130 ATG GAA GGT GTC CCT GAA ACT ATC CTC TCT AAA GCT AAG GAT CTG GAA GAG AAC AAC AG

MEGVPETILSKAKDLEENNR

+140 A CAA CTT CTG GAT GAC CTT AGG TGG ATA CTC ACC AAG gtgagcccttcccaggagtttattttc

QLLDDLRWILTK

actcataaaataggagagtgtggaaattaacaagaataacaaatatcaattctaaaataccatatatctccaggaaga

aatatacatattt.... INTRON E (--1 .3Kb) . . . . tttctgaatgtatttctctgatcgggcacagaaggaataa

+150 gaagaaaggaaactctaacaatgtaatcaacaagtaactgattatcattttgatttttag GTC TCT CCT ACA G

V S P T

+160 +170 CA GAG ATG AAG GAA GAA TTT CCC AGC TGG GGA TAT CTT TCA TTC TTA AAA TCA AGT AGC AEMKEEFPSWGYLSFLKSSS

+1so +190 AAA AAT CAC AAA TTT TTG GCA GTG TTT AAC CTT TCC AAC TGT CTA GAA GAT GAT ACA AA

KNHKFLAVF-NCLEDDTK

+200 G TTC ACT CTA TAT CAT CTC AGA ATA TTG AAA TGT CTC ATA ACT GGG AAA GAT TGC TAA

F T L Y H L R I L K C L I T G K D C -

TTGTACATTTATTATGTCTGCTTGGGCTATGCTCTAAGACTT

TCAAATGCATGTGTAGGTACAATGTGTCTCTTTCAAAAAATAAAAA GATTCTCTTTCAgcatgcatttcgtggcatca

gcaatattttctgcctgggtttggtgtttgtatgtacatgggctggatcaccaggtatggcgggctctgaatagcaag

tcattcattcattctttgctcaaaactttgtctccatatctcctcctatgaatat

FIG. 2. CONTINUED.

kinase and [-r-32P] ATP (PLP-C: 5’ CAGTTCCATCACTGGGGAGCC 3’; PLP-Cv: 5’ CAGCTCCATCACTGAGGAGTT 3’). The labeled primer (final concentration 10 PM) was extracted with phenol/chloroform, pre- cipitated with ethanol, and hybridized with 5 fig of total RNA from the junctional zone of the chorioallantoic placenta or from the spleen. RT was performed using Superscript preamplification system for first strand cDNA synthesis kit (Life Technologies). Reactions were termi- nated by the addition of sample running buffer (98% formamide, 2.5 rnr.4 EDTA, 0.1% bromophenol blue, 0.1% xylene cyanol) and then separated on a 6% polyacrylamide/7 M urea sequencing gel. A known DNA sequence was run on the same gel to indicate the size of the primer extended products.

PLP-Cv promoter analysis in trophoblast and nontrophoblast cell types

A 2,106-bp fragment flanking the 5’ end of the PLP-Cv gene was excised with KpnI and BglII and subcloned into KpnI and BglII cloning sites upstream of the luciferase reporter gene within the pGL-2 basic vector. Promoter-reporter constructs were transiently transfected into the Rcho-1 trophoblast, HIV-1 trophoendodermal, GH, pituitary tumor, and L929 fibroblast cell lines using a liposome-mediated delivery sys- tem. Cells were seeded in 35-mm tissue culture dishes (3 x 105), grown to approximately 70-80% confluence, and then transfected with 2 pg of the 2.1-kilobase pair (kb) I’LP-Cv promoter-luciferase construct, Rous

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PRL-LIKE PROTEIN-C VARIANT 5013

TABLE 1. Cosegregation of the rat PLP-Cv gene and chromosome 17 in mouse x rat cell hybrids

Hybrids Rat Rat chromosomes*

PLP-cv genea x 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20

LB20 + + - (+) (+) - - (-) + - - - - + + - - + (+) + + - LB150-1 + + - - + + - - +- + (+) + + + - - (+) (+) + + - LB161 + + - + + + + + +- + + - (+) + + + + + + + (+) LBSlO-I - +------------++---++-

LB251 + + + + - + - (+) + - - + - + + - - - + - + - LB330 + +- + ++- +--- +-+---- f---

LB360B + f--+++-++-+++++++++++ LB510-6 + +-+++--+----+++++++-- LB600 - + + + + + + (+) + - (-) + + + + + + + - + + - LB630 - + (-) - + + (+) + + - + - + + + (+) + + - + + (-) LB780 + +-++++-+--++-+---++-+ LB810 + +-+++-+++-++++++++- + (+) LB860 + +-+++--+-+-+++-++++ - C-t) LB1040 - + - - + + (-) + + - - + + + - - + + - + - + Independent 4 10 3 4 4 9 8 4 8 8 5 8 4 4 9 8 6 o 7 6 6

discordant clones’

a + and - = presence or absence of the rat gene, respectively. b + = rat chromosome present in more than 55% of the metaphases; (+) = rat chromosome present in 25-55% of the metaphases; (-) =

rat chromosome present in less than 25% of the metaphases; - = rat chromosome absent. ’ When a chromosome was present in less than 25% of the metaphases (- in parentheses), the hybrid in question was not taken in account

to establish the number of discordancies for that particular chromosome.

sarcoma virus promoter-luciferase (RSV-Luc; positive control), or pGL-2 basic vector (negative control). An RSV promoter-P-GAL construct (RSV-P-GAL; 0.5 ~g) was cotransfected and used to evaluate transfec- tion efficiency. Forty-eight hours following transfection, cells were col- lected, and lysates prepared via three cycles of freezing and thawing. Luciferase activity was measured with a luminometer according to the procedure of Brasier et al. (37). P-GAL activities and total protein con- centrations in the lysates were determined with a Galacto-Light kit (Tropix) and the protein-dye binding method (38), respectively.

Results

Isolation and characterization of PLP-Cu genomic clones

Approximately 1 X lo6 pfu of phage were screened re- sulting in the identification of four positive phage clones: RGLC-31 (15.4 kb), RGLC-32 (16 kb), RGLC-43 (18.4 kb), and RGLC-44 (17 kb). Dot blot analysis with oligonucleotide probes corresponding to 5’ and 3’ regions of the PLP-C cDNA demonstrated that RGLC-31 and RGLC-32 clones con- tained the entire coding sequence, whereas RGLC-43 and RGLC-44 were incomplete lacking 5’ and 3’ regions, respec- tively. The restriction and Southern blot analyses of the RGLC-31 clone are summarized in Fig. 1. Complete digestion with Not1 and Hind111 resulted in the generation of six frag- ments, two of which hybridized with the PLP-C probe in a Southern blot (2.8 and 3.6 kb), and were subsequently sub- cloned into pBluescript sk+ for sequencing. A third fragment (3.0 kb) generated from the Not I and Hind111 digestion and located immediately 5’ to the hybridizing fragments was also subcloned into pBluescript sk+ for sequencing.

All exons and exon/intron boundaries were sequenced (Fig. 2). Exonic regions showed extensive nucleotide homol- ogy to the PLP-C cDNA; however, significant differences were evident. The genomic clone was determined not to contain the PLP-C gene but instead a related gene that we termed PLP-C variant (PLP-Cv). Sequences from the three additional genomic clones (RGLC-32, RGLC-43, and RGLC-

44) were analyzed. Each sequence was identical to RGLC31 corresponding to the PLP-Cv sequence. We initially hypoth- esized that PLP-Cv may represent an allele for PLP-C that was solely expressed in the Wistar Kyoto outbred rat (source of the genomic library). Placental RNA was analyzed from Wistar Kyoto outbred and Holtzman rats for PLP-C and PLP-Cv expression by RT-PCR. PLP-C and PLP-Cv were expressed in both strains (data not shown). The PLP-C gene has subsequently been isolated from the Wistar Kyoto out- bred genomic library using a PCR-based strategy (data not shown). The organization of the PLP-Cv gene was deter- mined by aligning the PLP-Cv cDNA (see below) with the corresponding regions of the RGLC-31 clone. The complete PLP-Cv cDNA sequence was identified in this clone, con- firming that it contained the PLP-Cv gene. Consensus GT and AG splicing junctions were evident in each intron. The PLP-Cv gene was shown to exhibit a six exon and five intron arrangement, unique among members of the PRL family. The existence of the additional exonic and intronic sequences was further verified by PCR analysis using primers correspond- ing to exons 2, 3, and 4 of PLP-Cv. Comparison of the pro- totypical PRL family gene structure with the PLP-Cv gene indicated the existence of a unique exon 3 encoding for 13 amino acids in the PLP-Cv gene. Amino acids encoded by exon 3 of PLP-Cv are highly homologous to corresponding regions in the rat PLP-C, rat decidual/ trophoblast PRL-re- lated protein (d / tPRP), and mouse proliferin related protein (PRP) sequences (15,16, 39). Genes encoding these proteins have not been cloned but may also possess the unique six exon and five intron structure exhibited by the PLP-Cv gene.

Chromosomal assignment

The PLP-Cv gene was localized using somatic cell hybrids that segregate rat chromosomes. PCR-based identification of

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5014 PRL-LIKE PROTEIN-C VARIANT

FIG. 3. Structural comparisons of PLP- Cv, PLP-C, decidualkrophoblast PRL- related protein (d/tPRP), and proliferin related protein (PRP; top panel) and hy- drophobicity profiles of PLP-Cv and PLP-C (bottompanel). Toppanel, Align- ment of rat PLP-Cv, PLP-C, d/tPRP, and PRP amino acid sequences. The predicted amino acid sequences of PLP-Cv is compared with amino acid sequences of PLP-C (15), d/tPRP (161, and PRP (39). Shaded areas denote identity with the PLP-Cv sequence. Bottompanel, Hydrophobic character of rat PLP-Cv (broken line) and PLP-C (solid line) as determined by the Pustell Sequence Analysis Program (Interna- tional Biotechnologies, Inc.). Hydropho- bic regions are shown as an upward ex- cursion from the x-axis, whereas hydrophilic regions are shown as a downward excursion from the x-axis. Analyses are based on published se- quences of PLP-Cv (present study) and PLP-c (15).

Endo. 1996 Vol 137 . No 11

L

51 100 PLP-cv PLP-c d/tPRP PRP

101 150 PLP-cv PLP-c d/tPRP PRP TVEDVEETSF

151 200 PLP-cv PLP-c

d/ tPRP PRP

201 PLP-cv PLP-c d/ tPRP PRP

....... 238

....... 238

....... 239 VCYSEF. -244

PLP-c -

PLP-cv - - - -

I , I / I

40 80 120 160 200

Residue Number

the PLP-Cv gene in the cell hybrids co-segregated with rat other rat chromosomes are shown in Table 1. The data is chromosome 17. Discordant clones were not observed for consistent with a localization of the PLP-Cv gene on chro- this chromosome. Independent discordant clones for the mosome 17.

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PRL-LIKE PROTEIN-C VARIANT 5015

FIG. 4. Validation of the PCR analysis of PLP-C and PLP-Cv. Specific primers for the amplification of PLP-C and PLP-Cv cDNAs were designed based on sequences from the 5’ and 3’ untrans- lated regions of the PLP-C and PLP-Cv cDNAs (Top panel). The PCR reaction was performed for 30 cycles (denature, 94 C for 1 min; annealing, 60 C for 2 min; extension, 72 C for 2 min) using the PLP-C and PLP-Cv cDNAs as tem- plates for the reactions. Reaction prod- ucts were electrophoretically separated in 1% agarose gels and stained with ethidium bromide (lower panel). Please note each set of primers was specific for either PLP-C or PLP-Cv and generated amplified products of 780 bp.

ATG TAA

1 1

5’ AGAGACAGAACTCATCCTGCTTAGGAACTCCTCAGTG

PLP-Cv 5’ PCR Primer

TTGTACATTTATTATGTCTGCTTGGGCTATGCTCTAAG 3’

A TACAGACGAACCCGATACG

PLP-Cv 3’ PCR Primer

ATG

1

5’ AGAGACAGAACTCAGCCTCCTTAGG CTCCCCAGTG

PLP-C 5’ PCR Primer

TAA

1

GTGCACA~ACCGTGTCTGCTTGGGAGATGCTCTAAG 3’ G CACAGACGAACCCTCTACG

PLP-C 3’ PCR Primer

PLP-Cv PCR PLP-C PCR

Primers Primers

PLP!E--L!C PLp!i-z!!

M cDNA cDNA cDNA cDNA

Isolation and characterization of PLP-Cv cDNA clones

Two PLP-Cv cDNA clones (PLP-Cv9 and PLP-CvlO) have been sequenced. Their sequences were identical to each other and to the coding sequence of the PLP-Cv gene (Figs. 2 and 3). An error was discovered in the published PLP-C sequence (15) between amino acids +131 to +136 (K. Shiverick, per- sonal communication), which has been corrected (see Fig. 3). PLP-Cv showed 90 and 78% sequence identity with PLP-C at nucleotide and amino acid levels, respectively. The predicted PLP-Cv protein contains 238 amino acids and was calculated to possess a mol wt of 27,199 and a p1 of 5.9, virtually identical to PLP-C. Hydropathy plots for PLP-Cv and PLP-C were also very similar (Fig. 3). PLP-Cv showed progressively less amino acid sequence identity with decidual / trophoblast PRL-related protein (d / tPRP, 58%), and proliferin related protein (PRP, 30%); however, the amino acid region associ- ated with the unique exon 3 (Tyr4’ to Leu53) is present in each of these PRL family members but not in other PRL family members (2, 16, 39). Unlike PLP-C, which has two putative N-linked glycosylation sites at Asn3’ and Asn18’ (15), PLP-Cv possesses only a single site at Asn18’ (see Fig. 2).

Tissue distribution of PLP-C and PLP-Cv

The sequence similarities between PLP-C and PLP-Cv dic- tated that we establish an assay that could distinquish be- tween these two close relatives. A PCR-based strategy shown in Fig. 4, used specific primers that resulted in the specific amplification of either PLP-C or PLP-Cv. The size of the amplified products was 780 bp. A survey of various tissues indicated that PLP-Cv and PLP-C were coordinately ex-

PLP-cv

PLP-c

780bp

+ 780bp

FIG. 5. RT-PCR analysis of the tissue distribution of PLP-C and PLP- Cv. Total RNA was extracted from a range of different rat tissues including, spleen, liver, thymus, hypothalamus, anterior pituitary, ovary, uterus, decidua, and the labyrinth and junctional zones of the rat chorioallantoic placenta and from the Rcho-1 trophoblast cell line. RT-PCR was performed with primers specific for PLP-C or PLP-Cv (see Fig. 4). The sizes of the amplified PLP-C and PLP-Cv products were 780 bp. Reaction products were electrophoretically separated in 1% agarose gels, stained with ethidium bromide (upper panel of each set.), transferred to nylon membranes, and probed with a [32P]-labeled PLP-C or PLP-Cv cDNAs (lower panel of each set). Please note the coordinate expression of PLP-C and PLP-Cv and the restriction of their expression to trophoblast.

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5016 PRL-LIKE PROTEIN-C VARIANT Endo l 1996 Vol137. No 11

Day of Pregnancy

M Dll Dl3 D15 Dl7 D19 021

Day of Pregnancy

Dll D13 D15 D17 D19 021 M

FIG. 6. Gestational pattern of placental PLP-C and PLP-Cv messen- ger RNA and protein expression. Total RNA was extracted from the junctional zone ofthe rat placenta on days 11 through 21 of pregnancy. RT-PCR was performed with primers specific for PLP-C or PLP-Cv (see Fig. 4). The sizes of the amplified PLP-C and PLP-Cv products were 780 bp. Reaction products were electrophoretically separated in 1% agarose gels, stained with ethidium bromide (upper panel of each set), transferred to nylon membranes, and probed with a [32Pl-labeled PLP-C or PLP-Cv cDNAs (lower panel of each set). Please note the coordinate expression of PLP-C and PLP-Cv.

pressed in trophoblast tissues and not detectable in nontro- phoblast tissues (Figs. 5 and 6).

Analysis of the PLP-C and PLP-Cv transcription start sites

Primer extension analysis was used to identify transcrip- tion start sites for PLP-C and PLP-Cv in junctional zone placenta. Several putative transcription start sites were iden- tified for both PLP-C and PLP-Cv (Fig. 7). These were located in two general regions relative to their respective translation start site (ATG), -43, and -60 nucleotides (Figs. 2 and 7). Sequence analysis of 5’ flanking DNA corresponding to the PLP-Cv gene indicated the presence of consensus TATA boxes located -48 to -42 bp (ATATATA) and -234 to -229 (TATAAT) upstream of the most proximal transcription start site (see Fig. 2). Primer extended products were not gener- ated from spleen RNA (Fig. 7).

Trophoblast cell-specific activation of the PLP-Cv promoter

Trophoblast and nontrophoblast cells were examined for their abilities to express PLP-Cv and a 2.1 kb PLP-Cv pro- moter-luciferase reporter construct. PLP-Cv was detectable by RT-PCR analysis in differentiated Rcho-1 trophoblast cells (day 9) but not in undifferentiated Rcho-1 trophoblast cells (dav 1). HRP-1 troDhoendoderma1 stem cells. GH, &uitarv \ , ,I I

FIG. 7. Primer extension analysis of PLP-C and PLP-Cv transcrip- tion start site in the junctional zone placenta. Several putative tran- scription start sites were identified for both PLP-C (top panel) and PLP-Cv (bottom panel). These were located in two general regions relative to their respective translation start site (ATG), -43 and -60 nucleotides (see also Fig. 2). Primer extended products were not gen- erated from spleen RNA. DNA of known sequence was used to de- . . -.

~~, -~~~J =~~.~~~.~~, termine the size of the primer extenaed proaucts.

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PRL-LIKE PROTEIN-C VARIANT 5017

+ pActin

6000

5000

N i 4000 a a

6 2 3000

8

iit 2000

1000

0

-- -7

GH3 HRP-1 L929 Rcho-1

FIG. 8. Trophoblast cell-specific activation of the PLP-Cv promoter. Top panel, RT-PCR analysis of PLP-Cv expression in trophoblast and nontronhoblast cell lines. Total RNAwas extracted from GHsX, HRP-1. L929, and Rchol trophoblast cell lines. RT-PCR was performed with primers specific for PLP-Cv and 6-actin (see Fig. 4). The sizes of the amplified PLP-Cv and 6-actin products were 780 and 244 bp, respec- tively. Reaction products were electrophoretically separated in 1% agarose gels, transferred to nylon membranes, and probed with a [32P1-labeled PLP-Cv cDNA. Please note that PLP-Cv expression was restricted to differentiated Rcho-1 trophoblast cells (Rcho-1 D9). Bot- tom panel, PLP-Cv promoter analysis in trophoblast and nontropho- blast cell lines. GH,, HRP-1, L929, and Rchol trophoblast cell lines were transiently cotransfected with promoter-luciferase reporter con- structs (2.1 kb PLP-Cv promoter or a promoterless, pGL-2 construct) and RSV-p-Gal plasmid. Forty-eight hours after transfection cells were harvested, lysates prepared, and analyzed for luciferase or p-Gal activities. Promoter activities are shown relative to promoterless- luciferase reporter activities (pGL-2). Luciferase activities in GH,, HRP-1, L929, and Rcho-1 trophoblast cell lines transfected with the pGL-2 promoterless construct were 0.86 ? 0.25,0.40 t 0.10,175.48 2 21.20, and 21.77 ? 3.16 light unitsl6-gal unitslpg protein, respec- tively. Each value is the mean + SEM of triplicate measurements. Please note the activation of the PLP-Cv promoter was restricted to trophoblast cells.

tumor cells, or L929 fibroblasts (Fig. 8). Similarly, the 2.1 kb PLP-Cv promoter-luciferase construct was active in the Rcho-1 trophoblast cells but not in the other cell lines (Fig. 8). The promoterless construct (pGL-2) showed minimal activity in each of the cell lines (Fig. 8). An RSV-Luc construct was maximally active in each of the cell lines, exhibiting luciferase activities greater than 1000 times activities noted for the promoterless construct (data not shown).

Discussion

In this report, we have identified and characterized a new member of the PRL gene family possessing considerable homology to PLP-C, a unique gene structure, and a tropho- blast cell-specific pattern of transcriptional activation. This new member is referred to as PLP-C variant (PLP-Cv).

Members of the PRL family typically possess a five exon/ four intron gene organization that has been extremely well conserved across species (23-25,40-42). Partial 5’ genomic clones for PLP-A and PLP-B are consistent with the five exon/four intron gene structure of PRL (13,23-25,43). The PLP-Cv gene represents only the, second member of the rat PRL family to be completely characterized and the first shown to exhibit a six exon/ five intron arrangement (present study). Five of the exons are entirely homologous with exons from the PRL gene (Fig. 9). The additional exon is situated between exons two and three of the prototypical PRL gene structure (Fig. 9). Exon three of PLP-Cv is unique, relatively short, and encodes for a region rich in aromatic amino acids that also appears to be present in a subgroup of the PRL family, including PLP-C, d / tPRP, and mouse proliferin re- lated protein (15,16,39). Constituents of this subgroup do not appear to signal through the PRL receptor signaling pathway (22,44). Mouse proliferin related protein has been shown to antagonize blood vessel development (44). It remains to be determined whether this subgroup shares similar biological actions and whether their actions are influenced by inclusion of an additional exon.

Each of the currently identified members of the rat PRL gene family (PRL, PL-I, PL-Iv, PL-II, PLP-A, PLP-8, PLP-C, PLP-Cv, and d/ tPRP) have been assigned to chromosome 17 of the rat genome (1,2,15,18,19); present study). The specific arrangement of these genes on chromosome 17 has not been reported. Members of the human and mouse PRL gene fam- ilies have been assigned to human chromosome 6 and mouse chromosome 13, respectively (45, 46). Only one member of the PRL gene family, PRL, has been identified in the human, whereas five members have been identified in the mouse. These include mouse homologs for PRL, PL-I, and PL-II plus two novel mouse genes, proliferin and proliferin related protein (45). The arrangement of the mouse PRL family of genes on chromosome 13 has also not been reported. Physical relationships of the PRL family genes along their respective chromosomes may provide insights into the nature of the coordinated tissue- and cell-specific expression patterns for several of the PRL family genes.

Each of the members of the rat PRL family except PRL are expressed by trophoblast cells in a lineage- and temporal- specific pattern. The trophoblast ceil types involved are tro- phoblast giant cells and spongiotrophoblast cells. Tropho-

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5018 PRL-LIKE PROTEIN-C VARIANT Endo . 1996 Vol 137. No 11

FIG. 9. Schematic representations of the rat PRL (rPRL), mouse placental lactogen-II (mPL-II), and rat PLP-Cv (rPLP-Cv) genes. Exons are depicted by boxes. Numbers within the boxes refer to the size of the exons. Please note that the rPLP-Cv gene contains an extra exon situated between second and third exons of the prototypical PRL exonic or- ganization seen in both rPRL and mPL- II. Derived from present results (rPLP- Cv) and previously published data for rPRL (23-25, 39) and mPL-II (52).

rPRL 108 180 313

mPL-II 108 180 311

rPLP-Cv 179 180 311

t ATG

blast giant cells are the exclusive sources of PL-I between days 6-11 of gestation (4, 5) and PL-II from midgestation until parturition (5, 9). During the latter part of gestation, trophoblast giant cells are minor contributors to the produc- tion of PLP-A, PLP-C, and PL-Iv (5, 6, 9, 14, 15, 47). Spon- giotrophoblast cells are the major sources of PLP-A, PLP-B, PLP-C, and PL-Iv, the exclusive trophoblast source of PLP-B, and are first expressed between between days 13 and 15 of gestation and continue until term (6, 7, 9, 14, 15, 47-49). d / tPRP and PLP-B are also expressed during the first half of pregnancy in uterine decidual tissue (16, 50, 51). PLP-Cv exhibits a lineage- and temporal-specific expression pattern similar to PLP-C (present study), which also closely resem- bles the expression patterns for PLP-A and PL-Iv. Thus, cell-specific factors regulating trophoblast giant cells and spongiotrophoblast cells and temporal-specific factors active during the second half of gestation likely participate in the control of PLP-Cv gene activation.

Some significant progress has been made in understand- ing the control of PRL family gene expression in trophoblast cells. Most of the data has been generated with either the Rcho-1 trophoblast cell line or through the use of transgenic approaches (43, 52). Promoter constructs representing 5’ flanking DNA associated with each of the PL-I, PLP-A, and PLP-Cv genes can direct reporter gene activities in Rcho-1 trophoblast cells (43, 52, 53); present study). More specifi- cally, AP-1 and GATA elements and fos, jun, GATA-2, GATA3, and possibly the basic helix-loop-helix transcrip- tion factor, Hxt / Thing-l / eHAND, have been implicated in the control of PL-I promoter activity (43, 53-55). GATA fac- tors have also been implicated in the transcriptional control of the a-subunit gene of CG in human trophoblast cells (56). A consensus GATA element is located within the first 300 bp upstream of the PLP-Cv gene proximal transcriptional start site; however, whether the element is functionally important remains to be determined. Rcho-1 trophoblast cells are re- stricted to the trophoblast giant cell lineage (27) and thus the above factors may contribute to the control of trophoblast giant cell PLP-Cv gene activation. As indicated above, PLP-Cv is also likely expressed in spongiotrophoblast cells. Research directed toward understanding regulatory factors governing spongiotrophoblast cell gene expression has been hindered by a paucity of in vitro models. Nonetheless, gene targeting strategies have implicated Mash-2, a basic helix- loop-helix transcription factor (27, 57), and the epidermal growth factor receptor in the control of spongiotrophoblast

POLY A SiTE (AATAAA)

cell development (58, 59). Consequently, Mash-2 and the epidermal growth factor receptor, either directly or indi- rectly, may also participate in the regulation of spongiotro- phoblast cell PLP-Cv gene activation.

It is not yet entirely apparent why the rodent placenta expresses an array of genes related to PRL. Specificity con- cerning functions, expression patterns, and accessibility are certainly reasonable hypotheses. PLP-Cv is a newly identi- fied member of the PRL family with structural features and expression patterns indicative of a role in the maintenance of pregnancy. The availability of the PLP-Cv cDNA and gene are necessary prerequisites for the implementation of addi- tional molecular and transgenic strategies directed toward understanding the role of PLP-Cv in the physiology of preg- nancy in the rat.

Acknowledgments

We acknowledge the assistance and advice of Belinda M. Chapman, Christopher 8. Cohick, and Drs. Michael Wolfe and Kyle E. Orwig. We also thank Dr. Kathleen Shiverick for sharing information on the PLP-C sequence.

References

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2. Duckworth ML, Robertson MC, Schroedter IC, Szpirer C, Friesen H G 1993 Molecular genetics and biology of the rat placental prolactin family. In: Soares MJ, Handwerger S, Talamantes F (eds) Trophoblast Cells: Pathways for Ma- ternal-Embryonic Communication. Springer-Verlag, New York, pp 169-190

3. Soares MJ, Faria TN, Roby KF, Deb S 1991 Pregnancy and the prolactin family of hormones: coordination of anterior pituitary, uterine, and placental expres- sion. Endocr Rev 12402-423

4. Robertson MC, Craze F, Schroedter IC, Friesen HG 1990 Molecular cloning and expression of rat placental lactogen-I complementary deoxyribonucleic acid. Endocrinology 127:702-710

5. Faria TN, Deb S, Kwok SCM, Talamantes F, Soares MJ 1990 Ontogeny of placental lactogen-I and placental lactogen-II expression in the developing rat placenta. Dev Biol 141:279-291

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7. Robertson MC, Schroedter IC, Friesen HG 1991 Molecular cloning and ex- pression of rat placental lactogen-Iv, a variant of rPL-I present in late pregnant rat placenta. Endocrinology 1292746-2756

8. Duckworth ML, Kirk KL, Friesen HG 1986 Isolation and identification of a cDNA clone of rat placental lactogen II. J Biol Chem 261:10871-10878

9. Campbell WJ, Deb S, Kwok SCM, Joslin JA, Soares MJ 1989 Differential expression of placental lactogen-II and prolactin-like protein-A in the rat chorioallantoic placenta. Endocrinology 125:1565-1574

10. Deb S, Hashizume K, Boone K, Southard JN, Talamantes F, Rawitch A, Soares MJ 1989 Antipeptide antibodies reveal structural and functional char- acteristics of rat placental lactogen-II. Mol Cell Endocrinol 63:45-56

11. Duckworth ML, Peden LM, Friesen HG 1986 Isolation of a novel prolactin-like cDNA clone from developing rat placenta. J Biol Chem 261:10879-10884

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12.

13.

14.

15.

16.

17.

18.

19.

20.

21.

22.

23.

24.

25.

26.

27.

28.

29.

30.

31.

32.

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