Differential placental hormone gene expression during pregnancy in a transgenic mouse containing the human growth hormone/chorionic somatomammotropin locus.

The human (h) growth hormone/chorionic somatomammotropin (GH/CS) gene locus presents a unique model to gain insight into the molecular mechanisms that have allowed a closely related family of genes to be expressed in two distinct cell lineages/tissues: pituitary somatotrophs and placental syncytiotrophoblasts. However, studies of external factors that regulate gene expression have been somewhat limited by (i) a lack of human cell lines expressing endogenous GH or CS appropriately; and (ii) the fact that the GH/CS locus is unique to primates and thus does not exist in rodents. In the current study, a transgenic (171 h GH/CS-TG) mouse was generated containing the intact hGH/CS gene cluster and hGH locus control region (LCR) in a 171-kilobase DNA fragment. Pituitary and placental-specific expression of hGH/CS RNA was detected at embryonic day (E) 18.5. Immunostaining of hGH was seen in somatotrophs of the anterior pituitary beginning in late gestation. The presence of hCS protein was detected in the placental labyrinth in trophoblasts functionally analogous to the syncytiotrophoblast of the chorionic villi. This pattern of gene expression is consistent with the presence of essential components of the hGH/CS LCR. Transcript levels for hCS-A, hCS-B and placental hGH-variant increased in 171 hGH/CS-TG placenta during gestation (E11.5-E18.5), as previously observed in human placental development. Throughout gestation, hCS-A RNA levels were proportionately higher, accounting for 91% of total CS RNA by E18.5, comparable to term human placenta. Finally, the previous correlation between the transcription factor AP-2alpha and hCS RNA expression observed in developing primary human cytotrophoblast cultures, was extended to pregnancy in the 171 hGH/CS-TG mouse. The 171 hGH/CS-TG mouse thus provides a model to investigate hGH/CS gene expression, including in pregnancy.

Identification of a placental-specific enhancer in the rat placental lactogen II gene that contains binding sites for members of the Ets and AP-1 (activator protein 1) families of transcription factors.

We previously identified a 3-kb proximal 5'-flanking region of the rat placental lactogen (rPLII) gene that is important for reporter gene transcription in the rat trophoblast cell line, Rcho, and targets expression to the placentas of transgenic mice. In our current studies we have used further deletion analysis and transfection studies in Rcho and GC cells to map more precisely the locations of regulatory elements involved in this placental expression. We show that sequences between - 1435 and -765 are necessary for minimal expression in Rcho cells and that there are negative regulatory elements between -3031 to -2838 and -1729 to -1435. Most importantly, we have identified a fragment between -1793 to -1729 that is essential for expression levels characteristic of the complete 3-kb 5'-region. When linked to the herpes simplex thymidine kinase minimal promoter, this fragment acts as an enhancing element in Rcho but not GC cells. Deoxyribonuclease I (DNAse I) protection and electrophoretic mobility shift assays with nuclear extracts and in vitro translated proteins identify binding sites for members of the activator protein-1 (AP-1) and Ets families of transcription factors. Site-directed mutagenesis of the individual AP-1- and Ets-binding sites leads to a partial loss of the enhancing activity; a double AP-1/Ets mutation leads to a complete loss of activity, demonstrating the functional importance of these sites. By these criteria, putative GATA-binding sites located within the enhancing fragment are not active. These new data suggest an important role for this enhancing fragment in rPLII placental giant cell expression and are the first to implicate a member of the Ets family in the regulation of this gene family.

Analysis of tissue- and hormone-specific regulation of the human prolactin-inducible protein/gross cystic disease fluid protein-15 gene in transgenic mice.

The human prolactin-inducible protein/gross cystic disease fluid protein-15 (PIP/GCDFP-15) gene is expressed in more than 90% of human breast cancer biopsies but not in the normal mammary gland. However, it is expressed in several normal human apocrine glands such as the lacrimal and salivary glands. In human breast cancer cell lines, the gene is regulated by a number of hormones including androgen and prolactin. It is not known whether gene expression in normal tissues is under similar hormonal control. To understand the mechanisms by which hormone- and tissue-specific expression of the human PIP/GCDFP-15 gene are regulated in vivo, we generated transgenic mice using a 13.7 kb genomic DNA fragment containing the entire 7 kb human gene, together with 2.9 kilobases of 5' and 3.8 kilobases of 3' flanking sequences. The human PIP/GCDFP-15 transgene was found to be expressed in both the lacrimal and salivary glands but was not expressed in the mammary glands of transgenic mice. This tissue-specific pattern of the transgene expression in the mouse was very similar to that of the endogenous human PIP/GCDFP-15 gene, and to the endogenous mouse,gene. In the mouse salivary glands, the transgene expression was highest in the parotid, considerably less in the submaxillary (submandibular) and absent in the sublingual glands. In the mouse lacrimal gland, as in the human breast cancer cell lines, the human PIP/GCDFP-15 transgene was also up-regulated by androgen. These studies demonstrate that the human gene with its 6.3 kb flanking sequences is able to confer gene expression in vivo in a tissue-specific and hormone-responsive manner.

The effects of epidermal growth factor/transforming growth factor alpha on the expression of placental lactogen I and II mRNAs in a rat choriocarcinoma cell line.

Pregnancy in the rat and mouse is characterized by a developmental switch in expression at midgestation between the related placental lactogens I and II (PLI, PLII) suggesting that these proteins have important but different roles. The factors that control this differential gene expression are poorly understood. In this paper we describe experiments which investigate the effects of EGF and TGFalpha on rPLI and rPLII mRNA levels in the rat choriocarcinoma cell line, Rcho. This cell line has been widely used as a model system for studying genes expressed in the rodent placental giant cell. We find that in these cultures both growth factors produce a two fold increase in endogenous rPLI mRNA levels, and a two fold decrease in rPLII mRNA levels. Using DNA transfection assays we have tested the effects of TGFalpha on the expression of hybrid rPLI and rPLII 5'flanking/luciferase reporter constructs in Rcho cells. The transfected rPLI/luciferase reporter constructs produce a similar two fold increase in reporter expression to that seen for the endogenous mRNA, suggesting that EGF/TGFalpha positively regulate rPLI mRNA levels by effects on gene transcription. Unlike the endogenous gene, however, the rPLII/ luciferase constructs show a five fold increase in rPLII-directed luciferase expression in the presence of TGFalpha. The effect of EGF/TGFalpha on placental rPLII mRNA levels appears to involve negative response element(s) located elsewhere in the gene to those regions tested.

Development, progression, and androgen-dependence of prostate tumors in probasin-large T antigen transgenic mice: a model for prostate cancer.

Probasin (PB) gene product is prostate-specific, epithelial cell in origin, and androgen-regulated. A large 12-kb promoter fragment of the PB gene (LPB) was linked to the simian virus 40 (SV40) large T antigen (Tag) deletion mutant (that removes the expression of the small t antigen) to deliver consistently high levels of transgene expression to the transgenic mouse prostate. Seven male founders, their male offspring, and all the male offspring from two female founders developed at least prostatic epithelial cell hyperplasia by 10 weeks of age, indicating that the incidence of transformation was 100%. Tumorigenesis in the LPB-Tag animals progressed in a manner similar to that observed in the human prostate. Initially, multifocal proliferating lesions were detected in the prostatic epithelium, which continued to progress into hyperplasia involving the entire epithelium and then low-grade dysplasia. Reactive stromal proliferation was induced and continued to develop throughout the progression to high-grade dysplasia, carcinoma in situ, and adenocarcinoma. Immunohistochemical studies indicated that most stromal cells stained positively for both androgen receptor and smooth muscle alpha-actin, suggesting that stromal overgrowth largely represented mesenchymal cells that had differentiated into smooth muscle cells. Epithelial cell transformation was accompanied by the down-regulation of differentiated function, as suggested by the loss of dorsolateral prostate-specific secretory proteins. Tumor growth was regarded as androgen-dependent because tumors regressed in animals castrated at 11 weeks of age, and androgen treatment restored both epithelial/stromal cell ratio and tumor growth. Furthermore, small populations of prostatic epithelial cells in castrated animals continued to proliferate, suggesting the potential for androgen-independent growth. Although prostatic metastasis to other organs was not observed, local invasion was detected. In summary, the LPB-Tag animal model is unique in that it is the only model generated with the Tag alone, thereby eliminating any influences of the small t antigen on prostate tumor formation. Moreover, this model undergoes molecular changes similar to those found in human prostate including: (a) the multi-focal nature of tumorigenesis, (b) the progressive histopathologic changes from low- to high-grade dysplasia similar to human prostatic intraepithelial neoplasia, (c) stimulation of reactive stromal proliferation, and (d) the androgen-dependent growth of the primary tumor. Thus, the LPB-Tag prostate tumor model will be useful for studying the sequential mechanisms underlying the development of multistep tumorigenesis.

Development, progression, and androgen-dependence of prostate tumors in probasin-large T antigen transgenic mice: a model for prostate cancer.

Probasin (PB) gene product is prostate-specific, epithelial cell in origin, and androgen-regulated. A large 12-kb promoter fragment of the PB gene (LPB) was linked to the simian virus 40 (SV40) large T antigen (Tag) deletion mutant (that removes the expression of the small t antigen) to deliver consistently high levels of transgene expression to the transgenic mouse prostate. Seven male founders, their male offspring, and all the male offspring from two female founders developed at least prostatic epithelial cell hyperplasia by 10 weeks of age, indicating that the incidence of transformation was 100%. Tumorigenesis in the LPB-Tag animals progressed in a manner similar to that observed in the human prostate. Initially, multifocal proliferating lesions were detected in the prostatic epithelium, which continued to progress into hyperplasia involving the entire epithelium and then low-grade dysplasia. Reactive stromal proliferation was induced and continued to develop throughout the progression to high-grade dysplasia, carcinoma in situ, and adenocarcinoma. Immunohistochemical studies indicated that most stromal cells stained positively for both androgen receptor and smooth muscle alpha-actin, suggesting that stromal overgrowth largely represented mesenchymal cells that had differentiated into smooth muscle cells. Epithelial cell transformation was accompanied by the down-regulation of differentiated function, as suggested by the loss of dorsolateral prostate-specific secretory proteins. Tumor growth was regarded as androgen-dependent because tumors regressed in animals castrated at 11 weeks of age, and androgen treatment restored both epithelial/stromal cell ratio and tumor growth. Furthermore, small populations of prostatic epithelial cells in castrated animals continued to proliferate, suggesting the potential for androgen-independent growth. Although prostatic metastasis to other organs was not observed, local invasion was detected. In summary, the LPB-Tag animal model is unique in that it is the only model generated with the Tag alone, thereby eliminating any influences of the small t antigen on prostate tumor formation. Moreover, this model undergoes molecular changes similar to those found in human prostate including: (a) the multi-focal nature of tumorigenesis, (b) the progressive histopathologic changes from low- to high-grade dysplasia similar to human prostatic intraepithelial neoplasia, (c) stimulation of reactive stromal proliferation, and (d) the androgen-dependent growth of the primary tumor. Thus, the LPB-Tag prostate tumor model will be useful for studying the sequential mechanisms underlying the development of multistep tumorigenesis.

Rat placental lactogen II gene: characterization of gene structure and placental-specific expression.

Rat placental lactogen II (rPLII) was the first described member of the rat PRL-like placental gene family in which nine novel proteins have now been identified. In this article, we present data on the isolation and characterization of the rPLII gene. Two genomic clones, GC I (18.5 kb) and GC II (9.4 kb), were isolated from an EMBL3 Sprague-Dawley rat liver genomic DNA library. GC I, which was used for further analysis, contains the entire coding region and extensive 5' and 3' flanking information. The rPLII gene, estimated to be 5.4 kb in size, has the same five-exon and four-intron structure and identical intron/exon splice sites and types as the rPRL gene. A major transcription start site 58 bp upstream of the initiator methionine codon and several minor sites 1-3 bp 5' and 3' of this site were identified by primer extension of day 18 placental messenger RNA. The rPLII gene has been localized to chromosome 17, using a series of hybrid cell lines derived from mouse hepatoma cells (MWTG3) and adult rat hepatocytes; this is the same chromosome designation as the PRL gene itself and other cloned placental members of this gene family. Luciferase reporter constructs containing 5' flanking DNA sequences were tested in transient transfection assays in the rat choriocarcinoma cell line, Rcho, and the rat pituitary GC cell line. Both a 4.5- and 3-kb 5' flanking sequence supported luciferase expression in the Rcho but not the GC cells. A 765-bp fragment showed no activity in either cell type. Transient transgenic mice, generated with the 3-kb 5' rPLII/luciferase construct, expressed varying amounts of luciferase expression in the placenta.

Large fragment of the probasin promoter targets high levels of transgene expression to the prostate of transgenic mice.

BACKGROUND: Androgen regulation and prostate-specific expression of targeted genes in transgenic mice can be controlled by a small DNA fragment of the probasin (PB) promoter (-426 to +28 base pairs, bp). Although the small PB fragment was sufficient to direct prostate-specific expression, the low levels of transgene expression suggested that important upstream regulatory sequences were missing. METHODS: To enhance transgene expression, a large fragment of the PB promoter (LPB, -11,500 to +28 bp) was isolated, linked to the bacterial chloramphenicol acetyl transferase (CAT) gene, and microinjected into CD1 mouse oocytes to generate transgenic mouse lines. RESULTS: As shown by the immunohistochemical studies, CAT gene expression was restricted to the prostatic epithelial cells in a tissue-specific manner. High levels of CAT gene expression were observed in two of the six LPB-CAT transgenic lines. In Line 1, developmental regulation of LPB-CAT was detected early, from 1 to 4 weeks of age, with the activity of CAT increasing from 3 to 40,936 dpm/min/mg protein. Upon sexual maturation and elevated serum androgen levels (7 weeks of age), a further 18-fold rise in CAT activity occurred. Hormone ablation by castration in mature mice dramatically reduced transgene expression, whereas treatment with androgens returned LPB-CAT expression to precastration levels. In contrast, treatment with glucocorticoids had no significant effect on CAT gene expression. Zinc treatment of the castrated animals also increased LPB-CAT expression three- to four-fold in two prostatic lobes. CONCLUSIONS: This study demonstrates that important regulatory DNA sequences located in the LPB fragment contribute to tissue-specific expression and greatly increase levels of transgene expression induced by androgens and zinc.

The rat probasin gene promoter directs hormonally and developmentally regulated expression of a heterologous gene specifically to the prostate in transgenic mice.

An expression cassette carrying 426 basepairs of the rat probasin (PB) gene promoter and 28 basepairs of 5'-untranslated region is sufficient to target the expression of the bacterial chloramphenicol acetyltransferase (CAT) gene specifically to the prostate in transgenic mice. The PB-CAT transgene was expressed in three of five (60%) independent lines of mice, and this expression, as reported previously for the endogenous rat gene, was male specific, restricted primarily to the lateral, dorsal, and ventral lobes of the prostate, with only very low levels of CAT activity detected in the anterior prostate and seminal vesicles. The developmental and hormonal regulation of the transgene also paralleled that reported for the rat gene, with a 70-fold increase in CAT activity in the mouse prostate observed between 2-7 weeks of age, a time corresponding to sexual maturation. PB-CAT activity in the prostate declined after castration to 3.5% of the precastration level, and the CAT activity in castrated males approached precastration levels when mice were supplemented with testosterone. Transgene expression in castrated males was not induced by dexamethasone. Coinjection of PB-CAT with a chicken lysozyme gene matrix attachment region resulted in their cointegration and further restricted the pattern of PB-CAT to the dorsolateral prostate, with suppressed expression observed in the ventral prostate. These studies demonstrate that a minimal rat probasin promoter can target heterologous gene expression specifically to the prostate in a developmentally and hormonally regulated fashion.

Rat prolactin-like protein A partial gene and promoter structure: promoter activity in placental and pituitary cells.

Rat prolactin-like protein A (rPLP-A) is a member of a rapidly expanding family of prolactin-related proteins that are expressed during pregnancy by the rat placenta according to specific developmental patterns. Although the factors involved in the pituitary-specific expression of the prolactin and growth hormone genes themselves have been extensively studied, essentially nothing is known of the factors responsible for the placental expression of these new family members. In this paper we describe the isolation of rPLP-A genomic clones, analyze a portion of the 5' flanking sequence of this gene and use the recently described rat choriocarcinoma cell line, Rcho, in transient transfection studies to show that a 975 base-pair (bp) fragment of 5' flanking sequence is sufficient to specify placental expression of the rPLP-A gene.

Human prolactin gene expression. The use of an alternative noncoding exon in decidua and the IM-9-P3 lymphoblast cell line.

The prolactin (PRL) gene is normally expressed in the anterior pituitary lactotrope and the decidualized stromal cell of the human endometrium. We have recently described the ectopic expression of the human PRL gene in a B-lymphoblastoid cell line, IM-9-P. The IM-9-P and decidual PRL mRNAs are approximately 150 nucleotides longer than the pituitary transcript. The protein coding sequence of the IM-9-P and decidual PRL cDNAs is identical to the pituitary PRL message, and the 3'-untranslated region (UTR) exhibited a 7-14-nucleotide elongation. The 5'-UTR of IM-9-P and decidual PRL cDNAs revealed an extension of 41 base pairs upstream of the normal transcription start site for the human pituitary PRL gene. This is preceded by an additional sequence that is not homologous to the DNA found 5' to the pituitary PRL cap site. Decidual and IM-9-P PRL mRNAs possess identical 5' ends having 5'-UTRs of 83-263 nucleotides longer than that of the pituitary mRNA. The decidual/IM-9-P-specific segment of the 5'-UTR is located as a new 5'-noncoding exon 5-7 kilobase pairs upstream of the human pituitary PRL gene, exon 1. The clonal IM-9-P3 cell line provides a unique and easily manageable resource with which to study decidual specific PRL gene transcription.

De novo synthesis and secretion of prolactin-like protein-B by rat placental explants.

Mid- to late-gestation rat placenta expresses three PRL-related mRNAs, rat placental lactogen-II (rPL-II), rat PRL-like protein-A (rPLP-A), and rat PRL-like protein-B (rPLP-B). The protein product of rPL-II mRNA has been characterized, and the protein products of the rPLP-A mRNA were recently identified. The mol wt of a nonsecreted nonglycosylated rPLP-B protein would be 27,145 based on the mRNA sequence. The present study is the first to report the identification of the rPLP-B protein. Antiserum was generated against a chemically synthesized oligopeptide inferred from a specific region of the rPLP-B cDNA. Three or four distinct proteins synthesized and secreted by rat basal zone explants (day 15 gestation) showed cross-reactivity with the rPLP-B antiserum. The relative mol wt of these immunoreactive proteins is approximately 30,000, with a pI varying from 6.1-6.6. De novo synthesized rPLP-B proteins were not secreted by the explant tissue in the presence of tunicamycin, suggesting that the proteins are glycosylated. These data are consistent with the presence of one potential N-glycosylation site derived from the rPLP-B mRNA sequence. The rPLP-B antiserum showed no cross-reactivity with proteins identified using antisera against rPLP-A, rPL-II, or human pregnancy-specific beta 1-glycoprotein. Immunocytochemical studies were carried out using paraffin sections from placentas of day 14 and 17 pregnant rats which were treated with anti-rPLP-B, followed by avidin-biotin-peroxidase complex. These experiments show perinuclear staining, which was localized in basophilic cytotrophoblast cells, confirming previous in situ mRNA hybridization studies. Although no physiological role has been established for rPLP-B, the synthesis and secretion of this protein by cells in contact only with maternal circulation suggest a hormonal role.

Cellular localization of rat placental lactogen II and rat prolactin-like proteins A and B by in situ hybridization.

In situ hybridizations using 35S-labelled antisense and sense RNA probes of rPLII, rPLP-A and rPLP-B were carried out on developing rat placenta to determine which cell types synthesized each specific mRNA. Cellular localization of the sites of synthesis of these placental RNAs would help to decide whether these proteins were functioning in the mother of the fetus. The cells of the basal zone are known to have access only to the maternal blood supply, while the labyrinth region is supplied by both maternal and fetal blood vessels. The data in this paper show that at day 12 of pregnancy the rPLII mRNA is synthesized in the primary and secondary giant cells. At later days, hybridization is seen in both the giant cells of the basal zone, and cells in the labyrinth, suggesting that rPLII has a function not only in the mother, but also in the fetus. The rPLP-A mRNA is synthesized in both the giant cells and the cytotrophoblasts of the basal zone. No hybridization is seen to any cells in the labyrinth, even at the later days when it appears that all cytotrophoblasts synthesize rPLP-A mRNA. The rPLP-B mRNA is synthesized exclusively by the cytophoblasts of the fetal placenta. Like rPLP-A, all these cells synthesize this mRNA in the late term placenta. The synthesis of the rPLP-A and rPLP-B mRNAs in cells which have access only to the maternal circulation suggest that they have a role in the mother.

A third prolactin-like protein expressed by the developing rat placenta: complementary deoxyribonucleic acid sequence and partial structure of the gene.

We have previously reported on the isolation and characterization of two PRL-related cDNA clones, rat placental lactogen II and rat PRL-like protein A which are among the more abundant mRNAs expressed int he late term rat placenta. In this paper we report the isolation and characterization of cDNA clones of a third abundant placental protein, the predicted amino acid sequence of which shows a 44% homology to rPRL. This protein which we have called rPRL-like protein B (rPLP-B) is different from, but related to, the other PRL related proteins which have been identified in the rat, mouse, and bovine placentas. Two rPLP-B mRNA transcripts of 0.9 and 1.2 kilobases are strongly expressed in essentially equal amounts from day 14 of pregnancy until term. Nucleotide sequence and hybrid select translation data predict one secreted, potentially glycosylated protein of approximately mol wt 27,000. Hybridization and primer extension studies show that the two transcripts differ in their 5'-untranslated regions. One of the cDNA clones isolated represents a portion of the unprocessed rPLP-B mRNA. All intron/exon boundaries in this clone are of the same splice class and occur in identical locations within the coding region as in the rPRL gene.

Cloning and characterization of a cDNA encoding a highly conserved, putative calcium binding protein, identified by an anti-prolactin receptor antiserum.

Using antisera raised against partially purified rabbit mammary gland prolactin receptor, we have isolated a cDNA from a T-47D human breast cancer cell expression library which encodes the putative calcium-binding protein, calcyclin. Since the protein encoded by this cDNA co-purified with the prolactin receptor, we have tentatively named it a prolactin receptor-associated protein (PRA). Hybrid selection and in vitro translation showed that the protein encoded by this cDNA was approximately 10 kDa, a result confirmed by the amino acid sequence derived after DNA sequencing analysis. The PRA gene product has significant sequence similarity to the S-100 proteins, the cystic fibrosis antigen, and p11 proteins. The PRA/calcyclin cDNA obtained from the T-47D human breast cancer cell library has 37 nucleotides more 5'-untranslated information than that of the calcyclin cDNA from human fibroblasts. Since the transcription start site for the calcyclin gene has been confirmed in fibroblasts, our data suggest that a different transcription start site may be used in the T-47D cells. Isolation and nucleotide sequencing of the rat PRA cDNA showed there was 96% predicted amino acid sequence similarity with the human PRA and confirmed the highly conserved nature of the PRA/calcyclin gene between species. The PRA is expressed in most but not all human breast cancer cell lines, e.g. MCF 7. Northern analyses of RNAs from rat tissue indicated that PRA mRNA levels vary widely. They are low in the liver, brain, testes, and ovary, moderate in skeletal muscle, and high in the lung, kidney, and uterus. The availability of prolactin receptor-positive human breast cancer cell lines, which do (T-47D) and do not (MCF 7) express the PRA/calcyclin gene, will allow the investigation of the role, if any, of PRA in prolactin action.

Isolation and characterization of a complementary DNA (galanin) clone from estrogen-induced pituitary tumor messenger RNA.

The administration of high levels of estrogen is a well established method for producing prolactin-secreting pituitary tumors in rodents but the mechanism of tumor induction is not clear. In this paper we describe a cDNA clone (pEIC) which has been isolated from an estrogen-induced pituitary tumor cDNA library. The mRNA transcript corresponding to the pEIC clone is 0.9 kilobase in length and is not detectable in normal pituitaries but is expressed as early as 3 h after estrogen stimulation. Nucleotide sequence analysis of two 700-base pair recombinant clones shows that they encode a 124-amino acid protein which is 70% identical to the porcine galanin precursor. The sequence of 29 amino acid residues coded for by the pEIC cDNA clone is 88% identical with porcine galanin with only three amino acid substitutions near the C terminus. This extensive homology suggests that the pEIC cDNA clone codes for rat galanin or a protein belonging to the galanin gene family. These results provide the first evidence of a physiological regulator (estrogen) of the expression of the galanin gene. They also imply that galanin is secreted by prolactin-secreting tumors. Because intracerebroventricular injection of galanin can stimulate prolactin secretion and galanin inhibits hypothalamic dopamine release, it is conceivable that galanin may play a role in the induction of prolactin-secreting tumors.

Identification, characterization, and regulation of a rat complementary deoxyribonucleic acid which encodes insulin-like growth factor-I.

A complementary DNA (cDNA) encoding rat insulin-like growth factor 1 (IGF-I) has been isolated from a kidney cDNA library. By analogy with the sequence of human and mouse preproIGF-IA this cDNA encodes rat preproIGF-I from amino acid minus 3 to amino acid 105. The predicted protein sequence shows 96% and 99% homology with the human and mouse preproIGF-I, respectively. Under stringent conditions the rat IGF-I cDNA hybridizes with at least three mRNA, messenger RNA species which have apparent sizes of 7, 1.8, and 0.7-1.1 kilobases. All three IGF-I transcripts are detectable in each of the normal rat tissues examined and the relative order of abundance in tissues from intact adult male rats is liver greater than lung greater than kidney greater than thymus greater than spleen greater than heart greater than skeletal muscle (quadriceps femoris) greater than testes greater than brain. The GH dependence of the IGF-I mRNAs was demonstrated by the administration of a single ip injection of human GH (hGH) to hypophysectomized (hypox) rats which resulted in an increase in all three transcripts in each of the tissues examined. The increase in IGF-1 mRNAs was most marked in skeletal and cardiac muscle (9.7- and 9.5-fold compared to hypox controls, respectively) and least marked in the brain. In the liver only a 4-fold increase in IGF-I expression was observed, possibly because of the relatively high level of IGF-I expression in the tissue in the hypox control rats. Each of the IGF-I messenger RNAs appeared to increase in parallel and the time course of IGF-I induction was similar in each tissue with maximal levels of IGF-I transcripts present 6 to 12 h after GH administration. A dose-dependent increase in IGF-I mRNAs was observed in most tissues of hypox rats treated for 10 days with hGH. Significant correlations between growth, as determined by body weight gain and IGF-I expression were observed in liver (r = 0.97), kidney (r = 0.90), quadriceps femoris (r = 0.95), diaphragm (r = 0.92), and thymus (r = 1.0). The IGF-I mRNA levels in tissues from rats that had been treated with the highest dose of hGH (90 microgram/rat X day) were similar to those observed in normal intact rats. This study confirms the highly conserved nature of the IGF-I precursor and provides clear evidence for the GH dependence of IGF-I gene expression in multiple tissues of the rat.

Isolation and identification of a cDNA clone of rat placental lactogen II.

The developing rat placenta expresses two placental lactogens at different stages of pregnancy: rat placental lactogen I from Days 11 to 13 of pregnancy and rat placental lactogen II (rPLII) from Day 12 to term. In this paper, we describe cDNA clones for rPLII, which have been isolated from a Day 18 rat placental cDNA library. The rPLII clones hybrid-select a mRNA which translates in vitro to a protein of 25,000 daltons. This protein is processed by dog pancreatic microsomes to a 22,000-dalton form, identical in size to rPLII isolated from pregnant rat serum. Both forms are precipitated by an anti-rPLII antiserum and an anti-ovine prolactin antiserum. The mRNA for rPLII is first expressed in Day 12 placenta and reaches a maximum at about Day 18 of pregnancy, in parallel with the appearance of the hormone in serum. Sequencing of the cDNA shows that, unlike human placental lactogen which is 85% homologous to human growth hormone at the amino acid level, rPLII is much more closely related to the prolactins. Thus, rPLII is 52% homologous to rat prolactin at the amino acid level, but only 34% related to rat growth hormone. This is the second placental lactogen to be fully characterized, and in the rat this hormone appears to have evolved by a route quite different from that which produced placental lactogen in humans.

Isolation of a novel prolactin-like cDNA clone from developing rat placenta.

The mid- to late-term rat placenta produces several moderately abundant proteins in a specific temporal manner, one of which we have identified as rat placental lactogen II (rPLII) (Duckworth, M. L., Kirk, K. L., and Friesen, H. G. (1986) J. Biol. Chem. 261, 10871-10878). In this paper, we describe the isolation and characterization of cDNA clones of another of these proteins, called rat prolactin-like protein A (rPLP-A) because of its homology with the prolactins. The single mRNA transcript corresponding to rPLP-A is 1 kilo-base in length and first appears at Day 14 of pregnancy, 2 days later than rPLII mRNA, and then increases and remains at high levels until term. The mRNA hybridizing to rPLP-A cDNA clones translates in vitro to a protein of 25,000 daltons which is processed by dog pancreatic microsomes to 27,000 daltons. The amino acid sequence deduced from the nucleotide sequence of the rPLP-A clones suggests that rPLP-A is a secreted protein of 196 amino acids with two potential glycosylation signals. The sequence is more than 40% homologous at the amino acid level to rat and human prolactins and rPLII. This study provides clear evidence for the existence of considerable amounts of undescribed prolactin-like proteins in late term rat placenta.

Rapid synthesis of oligodeoxyribonucleotides VI. Efficient, mechanised synthesis of heptadecadeoxyribonucleotides by an improved solid phase phosphotriester route.

Efficient mechanised synthesis of heptadecadeoxyribonucleotides has been achieved on an economically small scale by an improved solid phase phosphotriester method on a polydimethylacrylamide resin. Improvements were made in the preparation of dinucleotide building blocks, reaction conditions for oligonucleotide assembly and in purification of deprotected oligonucleotides by h.p.l.c. Several milligrams of pure heptadecamers were obtained. Two of the heptadecamers were designed for sequencing in opposite directions of DNA cloned in phage M13mp2.