Linkage Arrangement of Human Placental Lactogen and Growth Hormone Genes*

Human placental lactogen (hPL) and growth hormone (hGH) are two hormones thought to have evolved from a common ancestral gene (along with prolactin), yet they have quite different functions and specificities. The nucleic acid sequences of the respective cDNAs of the two genes share considerable homology, as well as the existence of multiple forms of each gene within the genome. In this study we report on the linkage arrangement of several genes from this group. Two hPL-like genes as well as an hGH gene are shown to be linked within a 38-kilobase pair region of DNA. Linkage between a variant hGH gene and an hPL gene is also shown. The orientation and structural organization of these genes was previously established using 5'- and 3'-specific probes from a placental lactogen cDNA clone and detailed restriction endonuclease mapping. Restriction fragments from the overlapping clones were verified by comparison to digests of high molecular weight genomic DNA. In addition, the location of a specific class of repetitive DNA sequences, the Alu family, was mapped on these clones using the recombinant clone BLUR 8. All members of this multigene family have Alu repeat sequences either immediately flanking their 3' or 5' untranslated regions or within their intervening sequences.

Human placental lactogen (hPL) and growth hormone (hGH) are two hormones thought to have evolved from a common ancestral gene (along with prolactin), yet they have quite different functions and specificities. The nucleic acid sequences of the respective cDNAs of the two genes share considerable homology, as well as the existence of multiple forms of each gene within the genome. In this study we report on the linkage arrangement of several genes from this group. Two hPL-like genes as well as an hGH gene are shown to be linked within a 38-kilobase pair region of DNA. Linkage between a variant hGH gene and an hPL gene is also shown. The orientation and structural organization of these genes was previously established using 5'-and 3'specific probes from a placental lactogen cDNA clone and detailed restriction endonuclease mapping. Restriction fragments from the overlapping clones were verified by comparison to digests of high molecular weight genomic DNA. In addition, the location of a specific class of repetitive DNA sequences, the Alu family, was mapped on these clones using the recombinant clone BLUR 8. All members of this multigene family have Alu repeat sequences either immediately flanking their 3' or 5' untranslated regions or within their intervening sequences.
Human placental lactogen and human growth hormone are polypeptide hormones closely related in structure but different in both function and expression (1,2). Placental lactogen is expressed in increasing amounts during pregnancy, accounting for almost 5% of the total poly(A + ) RNA in the placenta during the third trimester of pregnancy (3,4). This hormone's function is not completely understood, but it is believed to be involved in supplying maternal nutrients to the fetus (5). Growth hormone is expressed in the anterior lobe of the pituitary and is involved in the regulation of growth and metabolism during development (6). Both of these hormones belong to a polypeptide hormone family that also includes the pituitary hormone prolactin and comprises the so-called "Prl" set of genes (7). This set of genes is believed to have evolved from a common ancestral gene by gene duplication (1,2,7) and recently, it has been demonstrated that the hPL and * This work was supported by Grants GM 23965, CA 16672, and CA 20124 from the United States Public Health Service and Grant G-267 from the Robert A. Welch Foundation. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
j National Institutes of Health predoctoral trainee (GM 07542). The abbreviations used are: hPL, human placental lactogen; hGH, human growth hormones; IVS, intervening sequence; kb, kilobase pairs; bp, base pairs. hGH genes are located on human chromosome 17 at band q22-24 (8,9). Recombinant DNA techniques facilitate the isolation of large genomic DNA fragments containing genes, their flanking regions, and regulatory elements that may be evolved in their differential and tissue-specific expression.
We have previously isolated and characterized seven recombinant clones containing four hPL-and three hGH-like genes. 2 In this article, we report on the close physical linkage of the hGH-and hPL-like genes within a single recombinant phage and between a series of overlapping phage clones. Detailed analysis of two closely linked hPL-like genes is presented, including the presence of an Alu family sequence within the first intron of an hPL-like gene. The nature of the Alu family sequences present in these recombinant clones has been examined by differential hybridization experiments. In addition, a conserved region of DNA found 2-3 kb from the 3' end of several members of this multigene family was identified.

EXPERIMENTAL PROCEDURES
A human chromosomal library (11) created by partial digestion with restriction endonucleases Hae III and Alu I and inserted into the Charon 4A vector using synthetic Eco RI linkers was screened using the hPL cDNA probe HCS-pBR322 (3) according to the method of Woo (12). Escherichia coli strain LE 392 was used in propagation of the plasmids. All procedures were carried out in compliance with National Institutes of Health guidelines.

Recombinant Probes and Nick Translation-The recombinant
clones HCS-pBR322, BLUR 2, and BLUR 8 (13) were used in these experiments. HCS-pBR322 contains a 550-bp insert complementary to lactogen mRNA and has previously been described (3). 5'-and 3'specific probes were prepared by digestion of HCS-pBR322 with HindIII and Xba 1.2 This allowed isolation of 150-bp 5' coding region and 400-bp 3' coding region fragments from a 6% polyacrylamide gel following elution by diffusion overnight at 37 °C (3). Labeling of DNA by nick translation was carried out according to the procedure of Lai et al. (14) using Amersham radionucleotides (specific activity of 2000-3000 Ci/mmol).
Phage Growth and Isolation-After plaque purification, recombinant phage were grown according to the procedure of Lai et al. (15).
Restriction Endonuclease Analysis of Clones-The DNA was digested with various restriction endonucleases (BRL, according to manufacturer's specifications), applied to 1 or 1.5% agarose horizontal gels run in regular strength TAE buffer (40 mM Tris-acetate, pH 8.0, and 2 mM EDTA), and subjected to electrophoresis for 12 h at 40 V. The gel was stained with ethidium bromide and transferred to nitrocellulose paper according to the Southern method (16) or by bidirectional transfer (17). Before hybridization the filter was treated with 2fold strength Denhardt's solution (6 x SSC, 0.04% Ficoll, 0.04% polyvinylpyrrolidone, 0.04% bovine serum albumin) for 24 h at 68 °C. Filters were then hybridized at 68 C overnight in 2-fold strength Denhardt's solution plus 0.5% sodium dodecyl sulfate and 1 mM EDTA. Genomic DNA digests were hybridized in the presence of the same solution plus 10% dextran sulfate. The filters were then washed two times each for 2 h at 68 °C in SSC (0.15 M NaCI and 0.015 M sodium citrate) plus 0.5% SDS. Labeled DNA bands were detected by exposure to x-ray film overnight in the presence of an intensifying screen (DuPont Instruments) at -80 C.
Plasmid Subclones and Transformation-Phage DNA was restricted with Eco RI, and separate bands were isolated by 1% agarose gel electrophoresis and electrophoretic elution of the particular fragment (18). The Eco RI fragments of interest were then cloned into the Eco RI site of pBR322, which had been treated with bacterial alkaline phosphatase (Worthington), using T4 DNA ligase (BRL). E. coli strain RR1 was transformed with the ligated DNA samples. Ampicillin-tetracycline-resistant colonies were selected and analyzed by the minilysate procedure (19). Specific plasmid subclones were then chosen and grown.
Human Genomic Blot-The Southern blot of genomic DNA was done using 20 Ig of high molecular weight human placental DNA in each restriction digest. Digestion conditions were the same as the phage digests, except that 2 units of enzume/gg of DNA and 2-h incubations were used. The digested DNAs were run on an 0.8% agarose (TAE) gel at 40 V overnight. The gel was transferred according to the method of Southern (16), and the resulting filter was treated as described above. The filter was hybridized with 2 x 107 cpm of HCS-pBR322 (108 cpm/pg of DNA) in the presence of 10% dextran sulfate (w/v). The filter was then washed sequentially in CHClI, 1 x SSC, and 0.5% sodium dodecyl sulfate at 68 "C.
End Labeling of DNA and DNA Sequence Analysis-Plasmid recombinant DNA, 40-80 Ig, was digested with an appropriate restriction endonuclease at 1 unit/tg of DNA for 2 h at 37 C in a 200-Yl reaction volume. The DNA was precipitated with 2.5 volumes of 95% EtOH in the presence of 0.3 M NaOAC, and the pellet was vacuum dried. The DNA fragments were treated with bacterial alkaline phosphatase and the 5' termini labeled with [y-32P]ATP (Amersham, 2000-3000 Ci/mmol), ATP, and T4 polynucleotide kinase (BRL) in a reaction volume of 30,ul (20). Briefly, 300-400 pCi of [y- 3 2 P]ATP were taken to dryness in a 1.5-ml Eppendorf tube. DNA was resuspended in 25 pl of 1 x kinase buffer (21) (stored as a 2 x kinase buffer stock at -20 C) and placed in a 0.5-ml Eppendorf tube containing the [y-32 P]ATP, vortexed, 5 l of T4 polynucleotide kinase was added, and the mixture was incubated at 37 C for 1 h. Terminally labeled DNA was then purified by polyacrylamide gel electrophoresis as described above.
Labeled DNA fragments were cleaved asymmetrically with a restriction endonuclease and isolated by preparative polyacrylamide gel electrophoresis. Fragments were eluted by placing the crushed gel slices in 0.1 x SSC at 37 C overnight and then subjecting it to chemical degradation according to the procedure of Maxam and 16 T Gilbert (21). Electrophoresis of degraded DNA fragments on thin polyacrylamide gels (25 and 10% or 20%) was carried out as described by Sanger and Coulson (22).

RESULTS AND DISCUSSION
Screening of Genomic Library and Identification of Positive Clones-A total of 900,000 recombinant phages were screened using 3 2 P-labeled hPL cDNA as a probe. Previous evidence indicated 93% sequence homology between the hPL and hGH cDNA (23), enabling detection of both genes with this probe. Seven clones were identified containing hPL, hGH, or hPL-and hGH-like gene sequences complementary to the lactogen probe. 2 Three of these clones were found to contain more than one member of the hGH-hPL multigene family. 2 In addition, three of these clones were found to have overlapping DNA sequences and span a 38-kb region. When the restriction endonuclease Eco RI was used, the four clones displayed different types of hybridization patterns with the lactogen probe (13). XH17 contained two hybridizing bands at 2.9 kb and 8.5 kb, while yH18 contained two hybridizing bands at 1.6 kb and 9.5 kb. Fiddes et al. (23) reported that large portions of the hPL and hGH gene sequences are contained in 2.8-kb and 2.6-kb Eco RI fragments, respectively. The two genes can be further identified by the presence of a Bgl II restriction site but no Xba I site in the 2.6-kb fragment (hGH), whereas the 2.9-kb (hPL) fragment contains an Xba I site (23). By these criteria, clone AH16 contains an hGH-like chromosomal gene and clone AH17 contains two hPL-like chromosomal gene sequences (Fig. 1). Additional restriction mapping and hybridization data for the 2.4-kb fragment contained in XH16 indicate it is similar to both hGH 1 , the expressed gene, and hGH 2 , the hGH variant gene (23, 24). but contains a different regional restriction map indicating the presence of a third hGH gene. 2 hGH 2 differs in nucleotide sequence from the hGHi gene by about 3%, including a Barn HI restriction site found in IVS D of the gene (Ref. 25, Fig. 2).
The third recombinant phage, XH18, contains a 17.5-kb human genomic DNA insert. Extensive overlapping between XH17 and AH18 is shown by several restriction endonuclease n , T T f.T I fragments. A 4.2-kb Xba I fragment that hybridizes strongly to the lactogen probe is contained in two of the clones, XH17 and XH18. This 4.2-kb Xba I fragment extends from approximately 200 bp to the right of the Eco RI restriction site that constitutes the 5' end of the 2.9-kb hPL 2 fragment to 4 kb to the left of the same Eco RI site and contains a major portion of the hPL 1 gene. Digests of XH17 and XH18 with restriction endonucleases Eco RI and Xba I result in a common 3.8-kb hybridizing band. 2 This fragment is part of the larger 4.2-kb Xba I fragment mapping from the left of the same Eco RI restriction site (Fig. 1). The majority of both the hPL 1 and hPL 2 chromosomal genes are contained in similar 4.2-kb Xba I fragments. The 9.5-kb Eco RI fragment extends from the 5' end of the 2.9-kb hPL 2 gene sequence and contains the entire hPLi chromosomal gene. It extends to the left toward the hGH 2 gene sequence. An Eco RI linker site 8.5 kb from the 5' end of the 2.9-kb hPL 2 sequence interrupts the 9.5-kb Eco RI fragment in AH17.
XH19 also contained two Eco RI fragments that hybridized to the lactogen cDNA 2 (Fig. 1). When this phage was digested with both Eco RI and Bam HI the 2.9-kb Eco RI fragment was reduced to 2.4 kb, and the 1.9-kb Eco RI fragment split into 0.9-kb and 1.0-kb fragments that hybridized to the probe. This indicated a Bam HI site near the 5' end of the 2.9 Eco RI fragment and a Ban HI site near the 3' end of the 1.9-kb Eco RI fragment (Fig. 2). It tentatively identifies the 1.9-kb Eco RI fragment as the hGH 2 gene previously described (23,24).
Linkage Arrangement of hPL and hGH Genes Within Recombinant Clones-Many multigene families with common evolutionary histories are found in linked groups. Initial experiments showed physical linkage of different family members within a single clone. The mapping and hybridization data presented previously 2 indicate linkage within several recombinant clones of hGH-and hPL-like genes. XH17 contains two gene sequences, the hPLI and hPL 2 genes. These two genes are separated by 2.0 kb of DNA and are in opposite orientations with respect to one another (Fig. 1).
The second recombinant clone, XH18, also contains two linked genes of this family. XH18 contains 1.5 kb of the 2.6-kb Eco RI fragment containing the hGH3 gene sequence. This gene does not contain the previously reported 3' Ban HI restriction site characteristic of the hGH 2 gene (23-25), but the regional map near its 3' end, as well as that shown for XHi6, differs from XH20 and XH21, believed to be the "true" hGH gene 2 ( Fig. 1; Ref. 26). This 1.5-kb Eco RI fragment containing the sequence termed hGH3 is linked to the 9.5-kb Eco RI fragment containing the hPL 1 gene. The hGH3 gene is in the same transcriptional orientation as the hPL 2 gene; therefore, the hPLI gene transcriptional orientation is opposite the hGH3 orientation. The two genes are separated by 12.5 kb of DNA (Fig. 1).
Clone XH19 also contains two linked genes. This clone contains a 1.9-kb Eco RI fragment with both a 3' Ban HI and a 3' Bgl II restriction site (Figs. 1 and 2), characteristics of the hGH 2 gene (23-25). The second gene is contained in a 2.9-kb Eco RI fragment and has a characteristic Xba I site in the coding region that corresponds to the cDNA sequence of lactogen mRNA. The two genes are in the same transcriptional orientation with respect to one another, as indicated by 5'-and 3'-specific probe hybridization experiments. The two genes are separated by 6.5 kb of DNA (Fig. 3).
Linkage Arrangement of hPL and hGH Genes from Overlapping Clones and Comparison with Genomic Blot Data-Linkage between several genes spanning greater than 20-30 kb is usually accomplished by isolating overlapping phage clones; an example is the human fi-globin gene family (27). The matching of numerous restriction sites and gene sequences is necessary to establish this type of linkage. Location of other DNA sequences, such as Alu family sequences, may also be used to establish linkage between distant genes. Examination of the various restriction maps of the hPL-hGH genomic clones revealed that three of the recombinant clones overlapped one another. This was subsequently confirmed by comparison with hybridizing fragments present in high molecular weight genomic DNA. The restriction maps of AH17 and XH18 have an 8.5-kb Eco RI fragment in common. This fragment contains six restriction endonuclease sites that are identical in both H17 and H18 (Fig. 3). The restriction fragments generated by cleavage at these sites hybridize to the same lactogen-specific probes (10). The positions of specific Alu family member repeats also map to the same location in both recombinant clones. 2 Therefore, it is apparent that XH17 and XH18 overlap in an 8.5-kb region (Figs. 1 and 3).
The 1.6-kb Eco RI fragment in XH18 containing the hGH3 sequence also appears to overlap a similar sequence found at the 3' end of AH16 (Fig. 3). The two genes have restriction sites common to the hGH3 gene. Establishing overlap between XH16 and AH18 is difficult, since the region they share in common is a gene sequence. A better method is to compare  in the duplication of the hGH and hPL family members. Therefore, a detailed examination of the flanking conserved sequences should provide insight into the evolutionary history of this gene family.