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Defining Regulatory Regions in the Rat Prolactin Gene Family Locus Using a Large P1 Genomic Clone

Departments of Physiology (A.Ö., A.F., A.S., M.L.D.) and Chemistry (H.W.D.), University of Manitoba, Winnipeg, Manitoba, Canada R3E 3J7

Address all correspondence and requests for reprints to: Dr. Mary Lynn Duckworth, Department of Physiology, University of Manitoba, Room 421, Basic Medical Sciences Building, 730 Bannatyne Avenue, Winnipeg, Manitoba, Canada R3E 3J7. E-mail: mdckwth{at}cc.umanitoba.ca.

	Abstract

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Members of the large rat prolactin gene family located on chromosome 17 are expressed in one or more placental trophoblast cell types and maternal decidua at specific times during pregnancy. Studies to identify the factors involved in these highly specific developmental expression patterns, using limited amounts of 5'-flanking DNA, have met with only partial success. Here we report the isolation and characterization of an 80-kb rat genomic clone, P1 12830, containing linked rat placental lactogen II, rat prolactin-like protein-I, and rat prolactin-like protein-B genes with substantial amounts of 5'- and 3'-flanking DNA as well as a rat placental lactogen II-related pseudogene, the first to be described in this gene family. This clone was used to create F0 transgenic mice, and the levels of expression of the three rat genes were compared with those of the endogenous mouse genes, using RT-PCR. Each rat gene was expressed differently in the same placenta, confirming the importance of sufficient flanking sequences in the expression of the individual genes. These studies emphasize the need for large genomic clones in defining the complete complement of factors that regulate the developmental expression of the rat prolactin gene locus.

	Introduction

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IN RODENTS, MATERNAL PITUITARY prolactin (PRL) plays an essential role in establishing and maintaining the early stages of pregnancy (1, 2). Seminal experiments in the 1930s demonstrated, however, that once the fetal placenta had been established, pregnancy would continue even after maternal hypophysectomy. This key observation led to the hypothesis that a hormone or hormones expressed by the placenta could replace the function(s) of PRL during mid- to late pregnancy. Placental extracts were subsequently demonstrated to have both luteotropic and mammotropic activities (reviewed in Ref. 3). We now know that in rats, mice, and several other nonprimate species, the developing placenta and the maternal decidua are the sources of a large number of proteins that are highly related to PRL in primary sequence, gene structure, and chromosomal location. In rodents, two of these proteins, placental lactogens (PLs) I and II bind to the PRL receptor and may be responsible for maintaining the corpus luteum and establishing pregnancy-related changes in mammary gland structure. Although no specific receptors have as yet been identified for the remaining proteins, these have been classified as cytokines because of their structural similarity to PRL and GH. The roles of these proteins are largely unknown, but it has been speculated that they function in maternal adaptations to pregnancy and potentially in fetal development itself (4). Some of these proteins may have a role in placental angiogenesis and antiangiogenesis, in preventing rejection of the fetus by the mother’s immune system and in the growth and differentiation of hematopoietic cells, in particular megakaryocytes and erythrocytes (reviewed in Refs. 3 and 5).

The novel PRL-related genes expressing these proteins have been identified by hybridization to cDNA clones for PRL or other known family members, or by searches of expressed sequence tag (EST) databases for related sequences. Most recently new members have been identified through homology searches of the mouse genome database. PRL and another 25 related genes have now been identified in the mouse and shown to be arranged in a cluster on chromosome 13 (6). In the rat, PRL gene family members have been localized to chromosome 17 (7, 8, 9, 10, 11, 12). In general the rat genes are orthologs of the mouse genes, several having been initially identified in the rat, but fine structure mapping and sequence confirmation of the gene order has been largely lacking. The recent availability of the first draft version of the rat genome sequence has now provided an essential tool for the comprehensive comparison of the rodent PRL loci.

Although much is known about the tissue-specific regulation of PRL gene expression in the pituitary (13), we have a more limited understanding of the regulation of those PRL family members that are expressed only during pregnancy. Expression of the individual genes in the locus follows very specific developmental programs, suggesting that transcriptional regulation may be complex. Some genes, like PLI and PLII, have unique expression patterns, but others share similar trophoblast cell-specific and temporal expression patterns (reviewed in Ref. 5). Most of the rat and mouse orthologs are similarly expressed, although slight differences have been noted for a few of the genes (14, 15). Unlike the human placental chorionic somatomammotropin genes, which are highly related in both their coding and flanking regions to the human (h)GH gene expressed in pituitary (16), the individual rodent PRL-related genes appear to have distinct flanking sequences (17, 18, 19, 20, 21, 22). Regulation of the hGH/human placental chorionic somatomammotropin locus is dependent on a locus control region (LCR) that is marked by a set of Dnase I hypersensitive sites between 15 and 32 kb upstream of the most 5' gene in the locus, hGHN. Without these sites, expression of the hGH family of genes in transgenic mice is low and sporadic (23). Whether the rodent PRL locus is similarly regulated by an LCR remains to be investigated. It has been shown, however, that as little as 3 kb of rat PRL 5'-flanking DNA is able to target high-level reporter gene expression strictly to pituitary lactotrophs in transgenic mice (24).

We have been investigating the factors that regulate the developmental expression of the rPLII gene in the rat. We have previously demonstrated that 3 kb of the rat PLII 5'-flanking region is sufficient to target luciferase expression to the placenta in F0 transgenic mice (12) and have identified an enhancer element within this region that is important for expression in the rat choriocarcinoma cell line, Rcho (21). Highly variable placental expression, and ectopic expression in some fetuses, suggests that there are elements outside this 3-kb region that are required for the complete developmental expression of the rat (r)PLII gene. Whether these elements flank the gene itself, as appears to be the case for the rat PRL gene, or whether, as in the hGH locus, far distal elements are required has yet to be rigorously examined. The arrangement of the genes in the rodent PRL locus is more complex than that in the hGH locus. Genes appear to be arranged largely according to the relatedness of sequence and gene structure rather than cell type or temporal expression patterns (Ref. 6 and our data).

To further our understanding of the regulation of genes in the rat PRL locus, we have now isolated and extensively characterized an 80-kb P1 rat genomic clone. This clone contains the entire rPLII, rat PRL-like protein (rPLP)-I and rPLP-B genes with varying amounts of 5'- and 3'-flanking sequence as well as a previously undescribed rPLII-related pseudogene. We used this clone to create F0 transgenic mice and examined, by RT-PCR, the expression of the three rat genes in placenta at d 14 of pregnancy. Each of the three rat genes is expressed differently, suggesting that expression is dependent on sequences that are proximal to or within the genes. These experiments demonstrate the feasibility of using large genomic clones to study the regulation of the rat PRL family members in transgenic mice and their potential for identifying the factors that are necessary for the complete developmental expression patterns of these genes.

	Materials and Methods

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Clones
The rPLP-C, rPLP-Cv, and decidual/trophoblast PRL-related protein (d/t PRP) cDNA clones were generously provided by Dr. Michael Soares (University of Kansas) (8, 9, 10). The cDNA clones for rPLI, rPLII, and rPLP-A and rPLP-B were previously isolated in our laboratory (25, 26, 27, 28). The rPLP-I cDNA (29) was cloned from d 18 rat placental RNA using RT-PCR with the primer pair RIH-cDNAF and RIH-cDNAR (Table 1). The PCR fragment was cloned into the vector pCR2.1 (Invitrogen Canada, Burlington, Ontario, Canada) by the TA cloning method. The rPLP-I cDNA was confirmed by sequencing.

Animals
CD1 mice for the production of transgenic embryos were supplied by the University of Manitoba Mouse Breeding Facility (Winnipeg, Manitoba, Canada) or Charles River Laboratories (St. Constant, Québec, Canada). Protocols for the production and use of these mice were approved by the University of Manitoba Protocol Management and Review Committee.

Screening a rat genomic P1 library for rat prolactin family genes
A rat genomic P1 library in the vector, pAD10SacBII (31), was initially screened by Genome Systems (St. Louis, MO) with primer pair, IIF-1262 and IIR-1549 (Table 1), corresponding to sequences in the 5'-flanking region of the rPLII gene. The clone P1 12830 was isolated. P1 DNA was purified using a protocol supplied by Genome Systems and the clone was analyzed to identify additional family members that were present on P1 12830 and test for potential rearrangements of the genomic DNA. Hybridization to cDNA clones was used as the detection method for rPRL (32), rPLI (26), rPLP-A (27), rPLP-B (28), rPLP-C (8), rPLP-Cv (10), and d/t PRP (9). PCR was used to screen for genes for rPLP-D; primers DF1 and DR1 (33); rPLP-F; primers FF1 and FR1 (34); rPLP-H; primers HF1 and HR1 (35); rPLP-I; primers RIHF and RIHR (29); rPLP-J; primers JF1 and JR1; rPLP-K; primers KF1 and KR1 (15, 29); rPLP-L; primers LF1 and LR1 (37); rPLP-M; primers MF1 and MR1 (15, 37); rat proliferin-related protein (rPRP); and primers PRPF1 and PRPF2 (37). Accession numbers for these sequences are shown in Table 2. Custom primers, supplied by Sigma-Aldrich Canada (Oakville, Ontario, Canada) or QIAGEN Inc. (Mississauga, Ontario, Canada) (Table 1), were selected to amplify within coding sequence only. Amino acid sequences were compared with that of rPRL and intron/exon boundaries predicted from the known locations of introns in the rPRL gene. HotStarTaq (QIAGEN) was used for PCRs. The program for all PCRs consisted of an initial 15-min enzyme activation step at 94 C, followed by 30 cycles of a 1-min denaturation step at 94 C, 1-min annealing step at 55 C, a 1-min extension step at 72 C, and a final 10-min extension step at 72 C. All primer pairs gave the predicted fragment sizes when tested with rat genomic DNA, confirming that only exon sequences were amplified.

An internal 9.5-kb SacI fragment from P1 12830 was subcloned into the SacI site of the plasmid vector pBluescript SK (Stratagene, La Jolla, CA) for further mapping. The ends of the SacI subclone were sequenced using the T7 and T3 primer sites in the vector. Restriction enzyme mapping of this clone was carried out, and further BamHI subfragments were also cloned into pBluescript for sequencing using the T3 and T7 primers. Primers IIvX4 and IIvX5 were designed for sequence analysis of an internal region of the SacI clone.

RNA expression analysis
BLASTX analysis of the DNA sequence from the 9.5-kb SacI fragment showed limited homology to exons 4 and 5 of rat, mouse, and hamster PLII genes. To determine whether this region of the clone contained a transcribed gene for an unidentified member of the rat prolactin gene family, primers were developed for RT-PCR using sequences from these homologous regions. Single-stranded cDNA was synthesized from 1 µg of d 12, 14, and 16 rat placental RNA using mouse mammary tumor virus reverse transcriptase (Invitrogen Canada). One microliter of this reaction was amplified using PCR primer pair X5F and X5R. The primer pair, IIF1-RNA/IIR2-RNA, that amplified rPLII cDNA served as a positive control for the reverse transcriptase (RT) reaction. The PCR program used was as described for the analysis of P1 12830. When no RT-PCR product was obtained, the X5F/X5R primers were tested for the ability to amplify the SacI DNA fragment. Extension times were increased from 1 min to 5 min because the priming sites were separated by approximately 6 kb in this clone, and 35 cycles of amplification were carried out. Small amounts of a high-molecular-weight fragment of the predicted fragment size were produced. To verify that this was the expected fragment, 2 µl of PCR product was reamplified with the nested primer set X6F and X6R. Amplification was for 35 cycles. PCR product was electrophoresed on a 2% agarose gel, blotted, and hybridized to a 2-kb BamHI subfragment of the SacI clone, which contained the region amplified by the X6F and X6R primers.

To examine the expression of rPLP-I in placenta, the primer pair RIH-BF/RIH-BR was used for RT-PCR with d 11, 12, 14, 16, 18, and 21 rat placental RNA and RNA from differentiated Rcho trophoblast cells collected at various days after plating. The rPLII primer pair, IIF1-RNA/IIR2-RNA, was used as a positive control for the RT reactions.

Generation and analysis of P1 12830 F0 transgenic mice
P1 12830 was grown using the protocol provided by Genome Systems. DNA was isolated from a 75-ml culture using a NucleoBond BAC maxikit with a BAC 100 column (BD Biosciences/Clontech, Mississauga, Ontario, Canada), according to the supplier’s instructions. In all subsequent steps the DNA was dissolved in polyamine buffer containing 10 mM Tris (pH 7.5), 0.1 mM EDTA, 30 µM spermine, 70 µM spermidine, and 100 mM NaCl. DNA was linearized at the unique NotI vector site, isolated in 1% low melt agarose by pulse field gel electrophoresis, as described, and purified from gel slices using ß-agarase (New England Biolabs Canada) according to the supplier’s protocol. DNA was concentrated by a Centricon YM-30 filtering device, dialyzed against polyamine buffer, and adjusted to 3 ng/µl in polyamine buffer without NaCl for injection into the male pronucleus of CD1 fertilized mouse eggs according to standard procedures. Pregnant female recipients were killed at d 14 of pregnancy, and the extraembryonic membranes, placenta, head, trunk, and abdomen were collected separately for each of 17 fetuses.

To identify transgenic conceptuses, DNA was isolated from fetal membranes and analyzed for the presence of the transgene by PCR, using primer pairs for three regions of P1 12830: 5' (IIF-1262, IIR-1549), middle (X6F, X6R; RIH-BF, RIH-BR), and 3' (B-intron DF, B-intron DR). Amplification was carried out using the standard PCR program for 30 cycles. For further assessment of transgene integrity, 10 µg of genomic DNA from each conceptus was digested with either SacI or BamHI, blotted, and hybridized to ³²P-labeled rPLII and rPLP-B cDNA clones, respectively.

For copy number estimation, 10 µg of genomic DNA from each of the transgenic and representative nontransgenic fetuses was digested with PstI. P1 12830 DNA was mixed with control nontransgenic genomic DNA to give the equivalent of 1, 2, 5, and 10 copies per genome based on a transgene size of 100 kb and a haploid genome size of 3 x 10⁹ kb and also digested with PstI. All samples were electrophoresed on the same 0.8% gel, blotted, and hybridized to a P³²-labeled rPLII cDNA probe. Autoradiograms were exposed to XAR film (Kodak, Rochester, NY) at -70 C with an intensifying screen and analyzed by densitometry.

Transgene expression analysis
Expression of rPLII, rPLP-B, and rPLP-I mRNA in the placentas of transgenic fetuses was determined using RT-PCR. One-microgram samples of total RNA from transgenic and nontransgenic mouse placentas and d 18 rat placenta was transcribed using mouse mammary tumor virus reverse transcriptase. One microliter of each reaction was used for PCR. Primer pairs that would amplify both the rat and mouse cDNAs were selected, producing PCR products that could be distinguished by restriction enzyme digestions. In all cases the primers represent the rat sequence. PLII cDNA was amplified using the primers IIF1-RNA (nucleotides 26–49) and IIR2-RNA (nucleotides 431–408). The rat-specific fragment contained a unique PvuII site, the mouse-specific fragment a unique ClaI site. The PLP-B cDNA was amplified using the primers BF1-RNA (nucleotides 304–327) and BR9-RNA (nucleotides 829–807); the rat-specific fragment contained a unique Sph I site, the mouse-specific fragment a unique PstI site. The rPLP-I cDNA was amplified using the primer pair RIH-RNAF (nucleotides 382–405) and RIH-RNAR (nucleotides 609–633). The rat-specific PCR fragment contained a unique Taq I site, the mouse-specific fragment a unique BclI site. Primer pairs are shown in Table 1. Lowercase, underlined nucleotides indicate differences and dashed lines extra bases in the mouse sequence. PCR conditions were the same as those for assessing genomic DNA, and 25 or 30 cycles were used. Fragments were separated on 2% agarose gels.

Total RNA was also isolated separately from the head, trunk, and abdomen of the transgenic fetuses and nontransgenic littermates. Fetal RNA was analyzed by PCR for 30 cycles using the same primers and conditions as for the placental RNA.

To estimate the relative amounts of the rat and mouse PLII and PLP-B mRNAs in each transgenic, PCRs were carried out in which one primer of each primer pair was end labeled using T4 polynucleotide kinase and P³²ATP. For assessment of PLII mRNA levels, the forward primer was end labeled; the reverse primer was labeled for assessment of PLP-B expression. After 25 or 30 cycles, the PLII reactions were digested with PvuII, generating labeled rat and mouse fragments of 225 and 404 bp, respectively. The PLP-B PCRs were digested with PstI, generating labeled rat and mouse fragments of 526 and 373 bp, respectively. Samples were electrophoresed on 2% agarose gels, dried, autoradiographed, and analyzed by densitometry.

	Results

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Isolation and mapping of rat genomic clone, P1 12830
A P1 rat genomic library was screened by PCR using a primer pair specific for sequences from -2514 to -2534 (primer IIF-1262) and -2248 to -2223 (primer IIR-1549) in the 5'-flanking region of the rPLII gene. A single positive clone, P1 12830, was isolated and shown to hybridize to both the 5' and 3' regions of an rPLII cDNA clone (25). P1 12830 was further screened for the presence of 15 other known members of the rat PRL gene family (see Materials and Methods), either by hybridization to cDNA clones or PCR, using primer pairs that would amplify within a single exon. Only rPLP-B and rPLP-I (28, 29) were positive (data not shown). The rPLP-I gene on P1 12830 is the rat ortholog of the mouse gene that was previously designated 1600016E11Rik on the mouse genome map and has recently been identified as the mouse PLP-I gene (6).

To determine that no deletions or rearrangements had occurred within the P1 clone, Southern blot analysis was carried out on a series of restriction enzyme digests of P1 12830 and rat genomic DNA. Hybridization patterns for the rPLII and rPLP-B genes were identical in both DNAs for all digests (Fig. 1).

View larger version (89K): [in this window] [in a new window]   FIG. 1. Southern blots of P1 12830 and rat genomic DNA. P1 12830 and rat genomic DNA were digested with restriction enzymes as indicated and hybridized to either an rPLII or rPLP-B cDNA clone. P1 12830 (A) and rat DNA (B) hybridized to rPLII; P1 12830 (C) and rat genomic DNA (D) hybridized to rPLP-B. All rat genomic DNA fragments are present in P1 12830 for both rPLII and rPLP-B, confirming that no rearrangements of these genes had taken place in the clone.

View larger version (94K): [in this window] [in a new window]   FIG. 2. Pulse field electrophoresis of the rat genomic clone, P1 12830. Rat genomic DNA was cloned into a BamHI site between unique SalI and NotI sites in the vector, pAD10SacBII. To establish the size of the cloned insert and locations of the rPLII, rPLP-B, and rPLP-I genes, DNA was digested with SalI, NotI, and SmaI, electrophoresed on a pulse field gel, blotted, and hybridized to cDNA clones. The NotI digest shows a fragment of approximately 97 kb, which contains the insert and vector. A 23-kb SalI fragment and a 43-kb SmaI fragment, marked by asterisks contain the rPLII gene. The 57-kb SalI/NotI fragment and the 56-kb SmaI fragment, marked by arrows, contain both the rPLP-B and rPLP-I genes. Both SmaI fragments also contain vector sequences. The 16-kb fragment in the SalI/NotI digest is the vector. Sizes were determined using HindIII cut lambda DNA and midrange I pulse field gel markers.

View larger version (15K): [in this window] [in a new window]   FIG. 3. A detailed restriction enzyme map of P1 12830. The entire 80-kb P1 12830 genomic insert is illustrated, cloned between a SalI and NotI site in the vector, pAD10SacBII. The locations of internal SalI and SmaI sites, which each divide the clone into two large fragments are indicated. Further restriction enzyme sites that were used in characterizing the clone are also shown. Exons for the rPLII, rPLP-I, and rPLP-B genes are illustrated by filled boxes. Locations of exons within the rPLII gene were previously established (12 ). The positions of the exons within the rPLP-B clone were determined by hybridization of restriction enzyme digested P1 12830 DNA to subclones of the rPLP-B cDNA clone and by DNA sequence. These locations have been confirmed by data from the rat genome sequence. The location of the rPLP-I gene was established by hybridization to an rPLP-I cDNA clone. Exons were located using the rat genome sequence database. Boxed regions on an internal SacI fragment represent sequences that are highly related to exons 4 and 5 of rPLII. Arrows indicate the positions of primers X5F and X5R that were used for RT-PCR to investigate whether these putative exons were transcribed.

To map the location of the rPLP-I gene within P1 12830, we first cloned the cDNA by RT-PCR using the primer pair RIH-cDNAF/RIH-cDNAR to amplify RNA from d 18 rat placenta. As well as the predicted fragment of 775 bp, a second smaller PCR product was also amplified. Each fragment was cloned into pCR2.1 and sequenced. Both fragments contained rPLP-I sequence, except that the smaller fragment was missing 177 internal nucleotides in a region that, by comparison with the rat genomic sequence, appears to be in the second exon, suggesting the possibility of alternative splicing of rPLP-I transcripts. The longer clone, which represents the published cDNA sequence (29), was used for hybridizations. The rPLP-I gene was localized to the central region of P1 12830, using a series of XhoI, BamHI, and PstI digests (Fig. 3) and therefore contains complete 5'- and 3'-flanking sequences.

The distance between the rPLII and rPLP-B genes suggested that there could be further genes present on the P1 clone. When Southern blots of P1 12830 restriction enzyme digests were probed with ³²P-labeled cDNA synthesized from d 17 rat placental mRNA, a number of fragments hybridized that had not been previously associated with the identified genes, supporting this possibility. We subcloned one of these, a 9.5-kb SacI fragment, for further analysis by restriction enzyme mapping and sequencing. This clone contained a SalI site, placing it precisely within the central region of P1 12830 (Fig. 3). The orientation of the SacI fragment within P1 12830 was established by combinations of XhoI, SmaI, and SalI digests of P1 12830 hybridized to a 1.3-kb BamHI/SacI fragment from one end of the SacI subclone. Further characterization, as outlined below, suggests that this fragment represents a portion of an rPLII-related pseudogene.

Evidence for an rPLII-related pseudogene
To investigate whether the 9.5-kb SacI fragment contained a further unidentified member of the rat PRL family, nucleotide sequence was obtained from both ends of this subclone and compared with the mammalian genomic and the rodent EST databases using the BLASTN and BLASTX programs. The BLASTN search produced a very short region of homology between the rPLII cDNA sequence and sequence from the 5' end of the 9.5-kb SacI subclone. The BLASTX search indicated two regions of homology with the rat, mouse, and hamster PLII proteins. One region near the 5' end of the SacI subclone showed homology to exon 4 and the other region, approximately 6 kb more 3', showed homology to exon 5. A detailed map of this region is shown in Fig. 3.

When translated, both the PLII exon 4 and exon 5-related sequences were incomplete and contained frame shifts and stop codons. DNA sequence was verified using the internal primers, IIvX4 and IIvX5, which were designed for sequencing on both strands in the region of the putative exon 5. Figure 4 shows a comparison of the deduced PLII-related amino acid sequence with that of the rat, mouse, and hamster PLII protein sequences as identified by the BLASTX program. At a similar location to the exon 4/intron D boundary in the rat and mouse PLII genes, a GT nucleotide doublet is also present in the novel sequence and may represent a splice donor site. Sequence immediately 3' of this dinucleotide shows no homology to translated PLII sequences. The putative exon 5 sequence potentially extends 16 amino acids more 5' than is shown, but these do not represent conserved residues in PLII and were not identified by BLASTX analysis. An AG doublet is located immediately 5' of a leucine codon as indicated in Fig. 4 and may represent the remnant of a splice acceptor site.

View larger version (28K): [in this window] [in a new window]   FIG. 4. Amino acid sequence comparison of the rPLII-related pseudogene fragment with rat, mouse, and hamster PLIIs. Nucleotide sequence from the 5' and 3' regions of the 9.5-kb SacI fragment were compared by the BLASTX program with the mammalian nucleotide database. Two regions as illustrated in Fig. 3 showed homology to translated exons 4 and 5 of the rat, mouse, and hamster PLII cDNAs. Identical amino acids that are conserved between the pseudogene and at least one of the PLII genes are shaded. No homology was detected in the putative pseudogene exon 4 beyond the region shown. Conserved cysteine residues in the PLII sequences are marked by an asterisk, and conserved tryptophans are marked by an inverted V. The BLASTX program inserted two Xs, marked in bold, in the sequence in which a shift in reading frame is required to maintain amino acid sequence homologies. Asterisks within the pseudogene sequence indicate stop codons; one insertion as indicated by a hyphen was introduced by the program to produce a better fit. The locations of introns in the rPLII sequence are indicated by arrows. There is a GT dinucleotide in the pseudogene sequence at the exon 4/intron D boundary and translated amino acid sequence similarity with the PLIIs is lost after this point. The putative exon 5 is approximately 6 kb more 3'. Pseudogene homology with PLII exon 5 as determined by the BLASTX program sequence ends at an asparagine. Immediately 5' of the leucine residue, shown in bold there is an AG dinucleotide.

To test the X5F/X5R primers, PCRs were carried out on the 9.5-kb SacI subclone itself, using longer extension times. A small amount of an approximately 6-kb product was synthesized, corresponding to the predicted fragment size. Using the nested primers set X6F/X6R, this PCR fragment could be amplified to produce the expected 211-bp product, which hybridized to a BamHI/SacI fragment from the 3' end of the SacI clone (data not shown). These PCR results support the conclusion that the SacI fragment contains a portion of a pseudogene rather than a transcribed member of the rat prolactin family. A pseudogene has not been previously described in the rodent prolactin family, probably because most members have been identified using hybridization to cDNA clones or EST analysis, both of which are dependent on gene transcription. A more thorough analysis of the now-complete mouse and rat genome sequences may reveal further examples.

Expression of the rPLP-I gene in placenta
The original rPLP-I clone was identified as an EST and isolated from a rat spleen cDNA library (29). No information was available on its expression pattern in placenta or decidua, in which all other novel members of the rodent PRL family are expressed. Hybridization of the rPLP-I cDNA clone to an RNA blot containing 30 µg of total rat placental RNA from d 11, 12, 14, 16, 18, and 21 of pregnancy showed the presence of rPLP-I RNA only on d 18 and 21 (data not shown). RT-PCR using the RIH-B primer set and 30 cycles of amplification showed very low levels of PCR product until d 18 and 21 when there was a large increase (Fig. 5). Similar results were obtained with the primers used for cloning the rPLP-I cDNA (data not shown). When RNA from differentiated Rcho cultures collected at 4, 10, and 18 d post plating were similarly amplified, only very low levels of PCR product were detected. RT-PCR using the rPLII primer pair, IIF1-RNA/IIR2-RNA, with the same Rcho RT reactions, amplified product at all times after plating as expected (Fig. 5). These results suggest that giant cells are not likely to be the cell type expressing rPLP-I in rat placenta. It has recently been reported that in mouse placenta only spongiotrophoblasts express mPLP-I (6).

View larger version (39K): [in this window] [in a new window]   FIG. 5. RT-PCR determination of rPLP-I expression in rat placenta and Rcho cells. RT-PCRs were carried out using 1 µg of total RNA isolated from rat placenta at the indicated days of pregnancy and from differentiated Rcho cell cultures at 4, 10, and 18 d after plating. One microliter of the RT reactions was amplified using the rPLP-I primer pair, RIH-BF and RIH-BR. After 30 amplification cycles, the expected fragment size of 135 bp was clearly visible in d 18 and d 21 rat placental RNA but was barely detectable in d 11–16 placental RNA. RT-PCR of the Rcho RNA gave essentially no product. A 382-bp PCR product was clearly visible when the rPLII-specific primer pair, IIF1-RNA and IIR2-RNA, were used to amplify the same Rcho RT reactions. This result confirms the presence of rPLII-expressing trophoblast giant cells in these cultures and suggests that these cells are not the source of the rPLP-I mRNA in placenta. Control samples without RT (C) showed no PCR product.

P1 12830 was linearized by NotI digestion and gel purified as described in Materials and Methods. Whole linearized P1 clone was microinjected into fertilized CD1 mouse zygotes and implanted into pseudopregnant recipients. F0 placentas, embryos, and extraembryonic membranes were collected for each conceptus at d 14 of gestation, which is approximately equivalent to d 16 in the rat. Both the rat and mouse PLII and PLP-B genes are normally highly expressed at this time (14, 25, 28, 39). Genomic DNA was prepared from the extraembryonic membranes and analyzed for the presence of the transgene using PCR. Three of 17 normally developing conceptuses (715.3, 715.5, 715.6) tested positive using the rPLII 5'-flanking primer pair IIF-1262 and IIR-1549. Transgene integrity in these animals was established using primers from different regions of P1 12830 for PCR (Fig. 6) and by Southern blot analysis (data not shown).

View larger version (79K): [in this window] [in a new window]   FIG. 6. The complete P1 12830 is present in all transgenic animals. DNA was isolated from the extraembryonic membranes of the fetuses and tested by PCR for presence of all known genes on the P1 clone. The primer pairs IIF1262 /IIR1549 from the 5'-flanking region of the rPLII gene, B-intron DF/B-intron DR from the 3' end of the rPLP-B gene, X6F/X6R from the pseudogene, and RIK-BF/RIK-BR from the rPLP-I gene were used. DNA from the three transgenic animals, 715–3, 715–4, and 715–6, gave the expected sized fragments with each primer pair.

View larger version (53K): [in this window] [in a new window]   FIG. 7. P1 12830 transgene copy numbers. Ten micrograms of DNA from each transgenic animal from nontransgenic littermates and from a nontransgenic animal to which 1, 2, 5, and 10 copies of P1 12830 DNA had been added were digested with PstI, Southern blotted, and hybridized to an rPLII cDNA probe. Comparisons of hybridization intensity between samples of the single PstI fragment were carried out using densitometry. P1 12830 copy numbers and estimated transgene copy numbers are shown below the lanes.

View larger version (61K): [in this window] [in a new window]   FIG. 8. Expression levels of rPLII and rPLP-B mRNA in the placentas of the P1 12830 transgenic mice. Total RNA was isolated from the placentas of transgenic animals 715–3, 715–4, and 715–6 and nontransgenic littermates. The d 16 rat placental RNA was a positive control for rPLII and rPLP-B. RT-PCR was carried out using either the PLII primer pair, IIF1-RNA/IIR2-RNA, or the PLP-B primer pair, BF1-RNA/BR9-RNA. Each primer pair amplified both rat and mouse cDNA to give a PCR product of the same size, which could be distinguished by restriction enzyme digestion. To compare endogenous mouse and rat transgene expression levels, one primer in each pair was end labeled with P32-ATP. One-microgram samples of RNA were used in each RT reaction and 1 µl of the RT reaction was used for RT-PCR. Then 20 µl of PCR product from 25 and 30 cycles of amplification were electrophoresed on a 2% agarose gel, dried, and exposed for autoradiography. A, PLII primer pair, IIF1-RNA/IIR2-RNA, amplified both rat and mouse PLII cDNAs to give a fragment of 404 bp. PCR products were digested with PvuII, which cleaved only the rat product to give two fragments of 225 and 179 bp. B, The forward primer, IIF1-RNA, was end labeled with P32-ATP as shown. After autoradiography, only the 404-bp mPLII PCR fragment and the 225-bp rPLII PvuII fragment were detected. C, PLP-B primer pair, BF1-RNA/BR9-RNA, amplified rat and mouse placental RNA to give a product of 526 bp. PCR products were digested with PstI, which cleaved only the mouse product to give two fragments of 373 and 153 bp. D, The reverse primer, BR9-RNA, was end labeled with P32-ATP. After autoradiography only the 526-bp rat PCR product and the 373-bp mPLP-B PstI fragment were detected. The signal intensity for each labeled rat fragment was quantified by densitometry, after suitable exposure times, and compared with the signal intensity of the labeled mouse fragment in the same lane, which was given a value of 1. Despite the fact that the PLII primer pair sequence is the rat sequence, the relative expression of rPLII to mPLII is very low in TG715–4 and 715–6. In these same animals the levels of rPLP-B and mPLP-B are similar. Neither rPLII nor rPLP-B was amplified in TG 715–3.

To test for ectopic expression of rPLII or rPLP-B, RNA was also isolated separately from the head, trunk, and abdominal regions of each of the transgenic fetuses. No PCR products, however, were seen for either the rPLII or rPLP-B gene in any of the fetuses after 30 cycles of amplification with labeled primers (data not shown). Although these results do not rule out expression in very localized regions of the fetus, as might be detected by in situ hybridization, they suggest that in these animals the transgenes were not expressed outside the placenta.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
The completion of the mouse genome sequence by the Mouse Genome Sequencing Consortium and the extensive annotation of the mouse PRL gene family on chromosome 13 (6) has provided definitive proof of the hypothesis that the rodent PRL family of genes forms a single large locus. Given the high degree of sequence homology and expression patterns between the identified mouse and rat PRL family members, it has long been speculated that their genomic organizations would also be very similar. The recent public release of the draft rat genome database by the Rat Genome Sequencing Consortium now provides the information for a direct comparison of the PRL loci in these closely related species. The rat genome sequence became available after we had completed our current study of the 80-kb P1 12830 clone, in which we have demonstrated a linkage of the rPLII, rPLP-I, and rPLP-B genes. The new genome information has allowed us to confirm our data, to determine the organization of the entire rat PRL locus and to compare its organization to the mouse PRL locus. The results of this comparison are shown in Fig. 9.

View larger version (29K): [in this window] [in a new window]   FIG. 9. A comparison of the rat and mouse PRL gene loci. The rat PRL locus (chromosome 17) is based on the sequence in the supercontig Rn17_650 as released by the Rat Genome Consortium in January 2003, with additional annotation, and on data presented in this article. The mouse PRL locus (chromosome 13) is based on the sequence of the supercontig Mm13.39618_30 in the mouse genome Build 30. The scale of both maps is in Mbp. The location of the rat genomic P1 clone discussed in this article, P1 12830, is indicated as a thick bar. Black dots indicate the locations of genes that are known to be expressed; white dots are potential genes or pseudogenes that are not known to be expressed but that encode PRL-related amino acid sequence. Dots to the right of the vertical axis are for genes transcribed downward and those on the left are for genes transcribed upward. In general, the genes in the two species are orthologs (with greater than 60% sequence identity) are found in the same order and are transcribed in the same relative directions. The rat genome sequence provides evidence for the rat versions of the PLI , ß, and and N genes recently described in the mouse (6 ). There is also sequence evidence for rat genes that are closely related to mPLF and mPLP-E (shown in italics), although it is unknown whether these genes are expressed in the rat. Some differences between the loci are the presence of the PLP-O gene in the mouse but not the rat and the PLP-D and PLP-H genes in rat but not in mouse. In addition, the rat PLIv gene, for which there is no equivalent in the mouse, is inserted into the middle of the PLP-C cluster but in the opposite orientation to neighboring genes.

Our analysis of the draft rat genome sequence provides evidence for rat orthologs of the recently described mouse (m)PLI , ß, and genes and for mPLP-N, although not for mPLP-O (6). All three rPLI genes are presumably expressed, but experiments, as were done in the mouse, would be needed to distinguish among transcripts from these highly similar genes. There is also evidence of genes for rat orthologs of proliferin (41) and mPLP-E (42, 43) (Fig. 9, italics). The mouse proliferin and mPLP-E proteins are of considerable interest because they are among the very few novel members of the PRL family for which functions have been described. Their putative roles in placental angiogenesis and megakaryocyte differentiation (5) have suggested that they would also be expressed in the rat, but there is no cDNA or EST evidence of this as yet.

The rat PRL locus contains genes that have not been described in the mouse. These include rPLP-D (33) and rPLP-H (35), although this latter gene is closely related in sequence and gene structure to the PLP-C subfamily and may represent an ortholog of one of the several mouse PLP-C genes. One striking difference between the rat and mouse loci is the rPL1-variant gene (11, 44), which is inserted into the middle of the oppositely transcribed PLP-C cluster in the rat (8, 45, 46). Given the general organization of the genes within the locus, rPL1-variant might have been expected to map near the PL1 gene itself. This gene has not been described in the mouse, and confirmation of its location within the locus must await further genome sequence information.

We also identified a number of regions in the rat locus, as illustrated by the open circles in Fig. 9, which show at least partial sequence homologies to other family members. These sequences may represent further authentic family members yet to be characterized, or they may be pseudogenes. Our identification of a portion of a pseudogene between the rPLII and rPLP-I genes is the first such example described in the PRL family. Members of the rodent PRL gene family have been identified largely by hybridization of cDNA libraries or EST database searches for homology with other known family members, neither of which approach would reveal a pseudogene. Why the P1 12830 subfragment containing the partial pseudogene sequence was detected by hybridization to labeled placental cDNA is unclear. Its sequence relatedness to rPLII may have been sufficient to allow hybridization. Several lines of evidence suggest that we have identified a pseudogene, rather than a new member of the family. Our sequencing data, which have now been confirmed by the rat genome sequence, indicate the presence of several frameshifts and stop codons within the putative coding regions. The RT-PCR results, which show no amplified product from early or late placental RNA, support the hypothesis that this sequence is not transcribed. BLAST comparisons to both nucleotide and protein databases indicate regions of homology to the PLII coding sequence of rat, mouse and hamster, but not other family members. No clones were identified when EST databases were searched. The location of the pseudogene adjacent to the authentic rPLII gene and its homology to rPLII follows the pattern of more closely related genes being linked and may be a reflection of the evolution of the locus.

The rPLP-I gene we identified on P1 12830 is the ortholog of the mouse gene also located between PLII and PLP-B. The mouse gene has been referred to as 1600016E11Rik (47) in the Ensembl mouse genome database but was recently confirmed to be mPLP-I (6). This PRL family member was first identified in the rat by Ishibashi and Imai (29) and, following the convention for naming members of the family, the designation "I" was assigned, another gene having previously been given the name rPLP-H (35). When the cDNA sequence was deposited in GenBank, however, it was incorrectly annotated as rPLP-H. The originally described rPLP-H gene is most closely related in sequence to the PLP-C subfamily and is located within the same region of the rat PRL locus as these genes. This confusion with nomenclature in the rat was further compounded when a cDNA, originally named rPLP-J by Ishibashi and Imai (29), was isolated by a second group and called rPLP-I (48). That gene has also been termed decidualin (36) and is located in both the mouse and rat loci between the PLI and PLII genes, to which it is more closely related. There are multiple accession numbers in GenBank for these various clones, and caution is needed when using this sequence information.

The original rPLP-I clone was isolated from a rat spleen cDNA library, and its expression in placenta was never investigated (29). Using RT-PCR, we demonstrated very low levels of expression of rPLP-I in placenta until between d 16 and d 18 of pregnancy, at which point RNA also becomes detectable by Northern blot. Expression of the mPLP-I is detectable by d 14 of pregnancy and highly expressed by d 16. These mPLP-I data are very similar to those recently published by Wiemers et al. (6), who also demonstrated, by in situ hybridization, that only the spongiotrophoblast cells of the mouse placenta express mPLP-I. The placental cell type(s) that expresses rPLP-I in the rat remains to be identified. rPLP-I is essentially undetectable in differentiated Rcho cells using RT-PCR, suggesting that the trophoblast giant cell is not the primary expressing cell type in the rat placenta.

More recently deposited sequence information in GenBank, which is correctly identified as rPLP-I, is very similar to that of Ishibashi and Imai, but the translated protein sequence differs from the original at its amino terminus (6). On comparison it appears that this difference is due to two extra nucleotides in the original sequence, which lead to the use of a different ATG as the initiator methionine codon and two shifts in reading frame. The sequence of our independent cDNA clone agrees with the most recent GenBank addition. Our data also indicate possible alternate splice variants of the rPLP-I gene. When rPLP-I RNA was amplified by RT-PCR for cloning, we found two products of different sizes. DNA sequencing confirmed that both contained the expected rPLP-I sequence but that the shorter fragment was missing 177 nucleotides in a region that we determined by comparison with the rat genome sequence to be in the second exon. This alternate splicing would lead to a deletion in the protein from Val 11, the first amino acid in the second exon, to Phe 69. Whether this truncated RNA is translated in placenta remains to be determined.

The P1 genomic clone studied here provided a means of testing in transgenic mice whether sequences flanking or within the native rPLII, rPLP-I, and rPLP-B genes were sufficient to reproduce the high-level, placental-specific expression of the endogenous genes. The expression of the endogenous mouse genes served as internal controls for these experiments. For these studies we chose to study F0 transgenics and selected d 14 of pregnancy for collection of the fetal/placental units to optimize for the expression of the PLII and PLP-B genes in both species. Developmentally this is equivalent to d 16 of pregnancy in the rat and the peak expression time for rPLII and rPLP-B in placenta (25, 28). Unlike the rat, the level of mPLP-B mRNA declines after d 14 of pregnancy in the mouse (14).

The rPLP-B gene on P1 12830 contains complete 5'-flanking DNA in addition to approximately 6 kb of 3'-flanking sequences. The levels of rPLP-B relative to mPLP-B mRNA in the placentas of the two expressing transgenic fetuses, 715–4 and 715–6, are similar to those of the mouse gene. Expression levels are approximately 40% (715–4) and 70% (715–6) of the endogenous mouse mRNA when corrected for copy number. By contrast, the level of rPLII relative to mPLII mRNA in both 715–4 and 715–6, is less than 1% when corrected for copy number. The rPLII gene on P1 12830 contains complete 3'-flanking and intronic sequences but only approximately 1 kb more 5'-flanking DNA than was previously tested in our luciferase reporter construct, which also showed very low expression (12). Unfortunately rPLP-I, the one gene for which complete 5'- and 3'-flanking sequences are present on P1 12830, does not express in either of these transgenics, although the mPLP-I gene itself is expressed. This is in fact consistent with the normal expression pattern in the rat which, as we show in this article, does not express until after d 16 of pregnancy. On the basis of this one time point, however, it is too soon to speculate whether the rat expression pattern is actually being maintained in the mouse placenta.

The third transgenic animal described in this study, 715–3, showed no expression of any of the three rat genes by PCR. Because 715–3 appeared to contain at most only one copy of the complete P1 12830 when DNA from extraembryonic membrane was tested, we considered whether this animal might be a mosaic for the transgene. We isolated DNA from the placenta and separately from the head, thorax, and abdominal regions of the fetus and analyzed for each of the genes on P1 12830, using PCR. DNA from all regions showed the presence of the three genes (data not shown), making it unlikely that transgene mosaicism could explain the lack of expression.

An alternative explanation would be that the transgene has been inserted into a region of chromatin that has silenced expression. Transgene silencing could potentially occur if, as has been shown for the hGH locus (23), a distant LCR not present on the P1 12830 clone is important for the regulation of gene expression in the PRL locus. It has previously been reported that as little as 3 kb of 5'-flanking DNA from the rat PRL gene itself was able to target reporter gene expression consistently to pituitary lactotrophs in transgenic mice (24). Because the PRL gene is at one end of the locus, it is possible that, at least for this gene, all regulatory elements are within close proximity, but this may not be true for the remaining PRL-related genes expressed during pregnancy. The organization of the PRL locus is considerably more complex than either of the well-studied human ß-globin and hGH gene loci (reviewed in Refs. 23 and 49). Gene order in the rodent PRL locus reflects the sequence relatedness of the genes, not the time or site of expression. If an LCR is a significant component of the regulation of the PRL gene family, it may be organized very differently, perhaps with elements dispersed among the genes.

This is the first study that tests expression of genes from the rat PRL family within their native genome context in transgenic mice. Even with the slight differences between expressing cell types and time of expression for some of the rat and mouse family members, the ability to compare expression of the endogenous mouse gene to that of a rat transgene provides a useful internal control for expression levels. Our transgenic studies confirm the importance of sequences flanking the PRL family of genes in regulating their developmental expression. In both the 715–4 and 715–6 transgenics, the identical placental expression patterns for the rat genes were seen: The rPLP-B gene was highly expressed, the rPLII gene weakly expressed, and the rPLP-I gene was not expressed. The finding that one transgenic animal did not express the rat genes still leaves open the possibility that distant LCR elements could have a role in the expression of the PRL gene family. More transgenics need to be examined at different times during pregnancy to explore this possibility further. The availability of the rat genome sequence, BAC clones that contain large segments of the rat PRL locus, and the ability to manipulate these clones in vitro (50) now provide a set of powerful tools for dissecting the factors that regulate the developmental expression of the rat PRL gene family.

	Acknowledgments

 
We thank Dr. Klaus Wrogemann (Department of Biochemistry and Medical Genetics, University of Manitoba) for the use of his pulse field gel electrophoresis apparatus and Ted Nylen for training us how to use it.

	Footnotes

 
This work was supported by a grant from the Canadian Institutes for Health Research (to M.L.D.). A.O. is a recipient of a Manitoba Institute of Child Health studentship.

Abbreviations: d/t PRP, Decidual/trophoblast PRL-related protein; EST, expressed sequence tag; h, human; m, mouse; PL, placental lactogen; PRL, prolactin; r, rat; rPLP, rat PRL-like protein; rPRP, rat proliferin-related protein; RT, reverse transcriptase.

Received May 14, 2003.

Accepted for publication July 25, 2003.

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