Expression of new members of the prolactin growth hormone gene family in bovine placenta. Isolation and characterization of two prolactin-like cDNA clones.

Two prolactin-like proteins (bPLP-I and bPLP-II) were deduced from the nucleotide sequence analyses of the cDNA clones derived from a bovine (Bos taurus) term placenta. These proteins resembled bovine prolactin but were different from the reported bovine placental lactogens or prolactin-related proteins. The predicted amino acid sequences of these clones showed 45-51% identity with bovine prolactin and 23-24% with bovine growth hormone. The two new clones show 62 and 39% overall homology with each other at the levels of nucleotide and amino acid sequences, respectively. bPLP-I, bPLP-II, placental lactogens, prolactins (PRLs), and other prolactin-like proteins isolated from cow, mouse, and rat share 7 common amino acid residues. Five of the 7 residues are conserved by other members of the family such as growth hormones, suggesting that they may be essential for the common structural features of the gene family. The other 2 residues are uniquely conserved in bovine, mouse, and rat placental lactogens, PRLs, and PRL-like proteins, predicting their indispensable roles in binding to the specific receptors. bPLP-I and bPLP-II, as well as bPLP-III, are shown to be expressed stage specifically and predominantly in full-term bovine placentas.

Two prolactin-like proteins (bPLP-I and bPLP-II) were deduced from the nucleotide sequence analyses of the cDNA clones derived from a bovine (Bos taurus) term placenta. These proteins resembled bovine prolactin but were different from the reported bovine placental lactogens or prolactin-related proteins. The predicted amino acid sequences of these clones showed 45-51% identity with bovine prolactin and 23-24% with bovine growth hormone.
The two new clones show 62 and 39% overall homology with each other at the levels of nucleotide and amino acid sequences, respectively.
Five of the 7 residues are conserved by other members of the family such as growth hormones, suggesting that they may be essential for the common structural features of the gene family. The other 2 residues are uniquely conserved in bovine, mouse, and rat placental lactogens, PRLs, and PRL-like proteins, predicting their indispensable roles in binding to the specific receptors. bPLP-I and bPLP-II, as well as bPLP-III, are shown to be expressed stage specifically and predominantly in full-term bovine placentas.
the placenta (9,(14)(15)(16)(17). Recently, Schuler and Hurley (18) have reported the isolation of a new PRL-related clone, bPRC1, expressed in bovine fetal placenta from 6 months of gestation. They have suggested that multiple PRL-related genes expressed in the placenta may be a general phenomenon in nonprimates.
During the course of our study on the characterization and regulation of the PRL-GH gene family, we have analyzed the structure-function relationship of the PRLs and related proteins from different vertebrate species (19). Elucidation of the primary structures of avian, mammalian, and teleost PRLs enabled the extended analysis of the specific and conserved amino acid residues and domains on the PRL molecules. In order to extend our study further in this respect, we have investigated a bovine placental cDNA library and isolated multiple clones hybridizing to bovine PRL cDNA probes. In this paper, we describe the isolation and characterization of two cDNA clones from bovine placenta, bPLP-1 and bPLP-11, which have not been reported previously.
The nucleotide and amino acid sequences of these clones indicate that they belong to the PRL-GH gene family and are related more closely to PRL (20) than to GH (21) or PLs (22, 23).

EXPERIMENTAL PROCEDURES
Several peptide hormones originating from the placenta are thought to play important roles in fetal development during pregnancy (1, 2). One of the dominant hormones of the placenta is placental lactogen (PL),' which is a memher of the prolactin (PRL)-growth hormone (GH) gene family (1,(3)(4)(5)(6)(7)(8). In addition to the PLs, other members of this family have been identified in the placentas from mouse (9-12) and rat (13,14). The expression of the genes of these new members during gestation has also been investigated, and the differently programmed appearances have suggested their important roles in the growth and development of the fetus and/or  Bovine GH (bGH) cDNA clone, phGH95, which had heen established in this laboratory, was digested with PL,uII. The resulting 494.base pair fragment was also radiolabeled as described above to use as another DNA prohe. The prehyhridization was performed for 5 h at 42 "C in a solution containing 5 x Denhardt's solution (31), 5 X SSPE (Z9), 0.1 mg/ml salmon iperm DNA, and 35% formamide.
The filters were then hybridized with the probes for 16 h at the same temperature.
The filters were washed three times with 2 x SSC (29) before exposure to x-ray films. The signal-positive clones were confirmed hy t.he second colony hybridization screening under the same conditions as described above.

Plasmid Isolation and &stric&r~
Mapping-Plasmid DNA was isolated from each of the signal-positive clones by the alkaline-SDS method (32)

RESULTS
Isolation and Nucleotide Sequence of bPLP-I and bPLP-II Clones-The bovine placental cDNA library was first screened with the bGH cDNA probe. However, under the conditions employed, no signal-positive clones were obtained with that cDNA probe. Then, the library was hybridized with the bPRL cDNA probes. From about 5000 colonies, 66 clones were detected to give positive signals. The positive clones were subjected further to the secondary screening, and 24 clones were confirmed to show strong positive signals. Restriction mapping analysis revealed that these positive clones could be classified into three major groups. These three groups of cDNA clones were found to be distinct from those of bPRL (20) or bGH (21). We designated these three clones as bPLP-I, bPLP-II, and bPLP-III. In this study, bPLP-I and bPLP-II have been sequenced and analyzed. The restriction maps and sequencing strategies are shown in Fig. 1. The sequencing analyses were performed as described under "Experimental Procedures." The complete nucleotide sequences of bPLP-I and bPLP-II are presented in Fig. 2. Each cDNA clone possesses a single large open reading frame, and their deduced amino acid sequences are also shown in Fig. 2. These sequence data indicate that bPLP-I and bPLP-II are similar to bPRL but not identical with bovine PLs (bPLs), whose amino acid compositions (22) and other properties (23) have been reported previously.
The proteins encoded by bPLP-I and bPLP-II are composed of 236 and 238 amino acid residues, respectively. The amino-terminal regions of both proteins are rich in hydrophobic amino acid residues, which are characteristic of signal peptides (39,40). Schuler and her group have previously isolated a bovine placental PRL-related clone, bPRC I (18), and recently a bPL (41) and two other PRLrelated clones, bPRC II and bPRC III (42). bPLP-I of this study was almost identical with the bPL clone except that the amino-terminal residue of bPL was alanine instead of valine found for bPLP-I. bPLP-II was not identical with any protein or clone ever isolated. bPLP-I had a concensus sequence for N-glycosylation, Asn-X-Ser/Thr (43) at the position of 89-91. On the other hand, bPLP-II contained four N-glycosylation sites at positions 70-72, 92-94, 146-148, and 160-162. Codon Usage in bPLP-I and bPLP-II-As is the case for other eukaryotes (44)(45)(46)(47), the codon usage in bPLP-I and bPLP-II cDNAs is apparently nonrandom.
G or C in the third position of the codon is preferentially used (55% in bPLP-I and 58% in bPLP-II) over A or T, as has been observed in other members of the PRL-GH gene family (44,48,49). Hydropathy Profile of bPLP-I and bPLP-II- Fig.   3 shows the hydropathy profiles of bPLP-I and bPLP-II. Highly hydrophobic regions were found in the predicted signal peptide sequences of bPLP-I and bPLP-II. The profiles of bPLP-I and bPLP-II resemble those of bPRL and bPRC-I, indicating the structural similarity of these proteins.
Stage  Placenta-Another novel PRL-Like cDNA clone, bPLP-III, has also been isolated from bovine placenta in this laboratory (50). Stage-specific expressions of bPLP-I, bPLP-II, and bPLP-III mRNAs were investigated in bovine placenta. As shown in Fig. 4, bPLP-I and bPLP-II were expressed dominantly in full-term placentas and slightly in placentas of 2-month or 3-month gestation. bPLP-III was also expressed in full-term placentas, but this species was not expressed in the early stages of gestation such as 2 or 3 months. Each of bPLP-I, bPLP-II, and bPLP-III showed a mRNA size around 1 kilobase, consistent with that of those cDNAs (Fig. 2 and Ref. 50). Another larger species of RNA of approximately 4 kilobases was also detected clearly in full-term placenta for bPLP-III and, to less extents, for bPLP-I and bPLP-II.
On the other hand, no mRNA expression was observed for bPLP-I, bPLP-II, and bPLP-III in the pituitary of nonpregnant cow (Fig. 4). Using cDNA of bPRL or bGH as the probe, no RNA was detected in the placenta of those gestation stages. DISCUSSION We report here the isolation and characterization of two novel prolactin-like cDNA clones, bPLP-I and bPLP-II, from bovine (B. taurus) term placenta. The third clone we have obtained recently, bPLP-III, is also shown to be a member of the gene family (50). Schuler and her group have recently isolated bovine placental PRL-related clones, bPRC-I (X3), bPRC-II and bPRC-III (42), and a bPL (41) clone. The three clones we obtained were different from any of these four clones, but they were closely related to each other. In Fig. 5, the deduced amino acid sequences of bPLP-I and bPLP-II are compared with the preforms of bPLP-III, PLs, PRLs, and other PRL-like proteins from cow, mouse, and rat. bPLP-I of the present study is very similar to bPL reported previously (4l), except for the predicted amino-terminal residues of the two mature forms. The nucleotide sequences for these residues are GTG and GCG, respectively, showing a single nucleotide difference. We analyzed seven individual clones isolated as bPLP-I for the corresponding sequences, finding that four of the seven clones had GTG, and the other three clones had GCG. Schuler and her group (41) have reported previously the presence of 2 amino-terminal residues, valine and alanine, in equal molar amounts for the isolated mature bPL hormones, consistent with our results. We consider that these two clones (or proteins) are derived from the different genes. We propose to designate bPLP-I and bPL as bPL(Va1) and bPL(Ala), respectively. bPLP-II, on the other hand, shows a close relationship to bPRC-II reported by Kessler et al. (42). Only 1 residue in the predicted mature portion of bPLP-II, Asp-189, is replaced by asparagine in mature bPRC-II. However, most of the predicted signal peptide of bPRC-II is quite different from those of bPLP-I, bPLP-II, bPLP-III, bPL, or bPRC-I, which are highly conserved (Fig. 5). The nucleotide sequence for the mature portion of bPLP-II is identical to that for bPRC-II (42), except that GAC (Asp-189) in bPLP-II is replaced by AAC (asparagine) in bPRC-II. However, the 5'-noncoding and signal-coding sequences of these two clones are quite different, and several nucleotide replacements are observed in the 3'-noncoding regions. In Table I, percent identities in amino acid and nucleotide sequences among members of bovine GH-PRL gene family are shown. bPLP-I, bPLP-II, and bPLP-III are more homologous to PRL than to GH. The order of homology to PRL among the preforms of members is PRL > bPLP-I > bPL > bPLP-II > bPRC-III > bPRC-I > bPLP-III > bPRC-II > bGH (Table I and Ref. 42). bPLP-I, bPLP-II, and bPLP-III are expressed in a stagespecific manner, being predominant in the term placenta. Similar gestation stage-specific expressions of PLs and PRLlike proteins have been observed in the developing rodent placentas (9,(13)(14)(15)(16)(17). Protein of bPLP-I, or bPL(Val), has been identified by Schuler and her group (41); however, those for bPLP-II and bPLP-III remain to be isolated. We have also obtained three clones for bPL(Ala), but no clone of bPRC-I, bPRC-II, or bPRC-III was detected in our cDNA library constructed from full-term placenta. These clones have been obtained with the cDNA library constructed from fetal cotyledons of 6 months of gestation (18,42). This difference of the source may have affected the species of isolated clones. Together with the stage-specific expressions and the structures of the mature hormones for these clones, the physiological roles of bovine PLs and other placental PRL-like proteins should be clarified hereafter.
When the primary structures of bovine PLs, PRL-like proteins, and PRL are compared with those for mouse and rat (Fig. 5), 7 amino acid residues are completely conserved in the 16 hormones. These 7 residues are distributed to four domains (LDl-LD4) of relatively high conservation. Recently, we have shown that the PRL molecules from various vertebrate species contain four highly conserved domains (PDl-PD4) (19), whereas the GH molecules are shown to possess five conserved domains (GDl-GD5) (51). LDl falls on the PD2 or GD2 region, and LD2 fits PD3 or GD3. LD3 overlaps PD4, and LD4 falls within the PD4 or GD5 region. The four domains from LDl to LD4 are considered to have important roles in the biological activities of those lactogenic proteins. Five of the 7 conserved amino acid residues, Cys-98 in LDl and Cys-214, Asp-218, Cys-231, and Cys-236 in LD4, are shared not only by these 16 lactogenic hormone but also by all other known members of the gene family including the GHs. This may suggest that the LDl and LD4 domains are essential for the common structural features of the PRL-GH gene family and that LD2 and LD3 are important for the specific activities of the PLs and PRL-like proteins.