Molecular cloning of mouse placental lactogen cDNA.

We have isolated a cDNA clone for the 23-kDa mouse placental lactogen II (mPL-II) from a phage lambda gt11 expression library containing cDNA synthesized from BALB/c placental RNA. Translation in vitro of placental mRNA selected by hybridization to the mPL-II cDNA clones yields a 26-kDa polypeptide that is the size of the expected precursor protein and that is immunoprecipitated with anti-mPL-II antiserum. The mPL-II cDNA clones hybridize to a 1.0-kilobase placental-specific mRNA. This mRNA, found in the fetal portion of the placenta, appears as early as day 10 of gestation and increases to a maximal level by day 12. The mPL-II cDNA nucleotide sequence has been determined. This sequence contains an open reading frame encoding a polypeptide of 222 amino acids with the amino-terminal 31 amino acids forming the signal sequence for secretion. The predicted secreted protein has 51% amino acid homology with mouse prolactin.     

Identification and characterization of a new member of the placental prolactin-like protein-C (PLP-C) subfamily, PLP-Cbeta

We have isolated a complementary DNA (cDNA) clone that encodes a new member of the PRL-like protein-C (PLP-C) subfamily of the PRL gene family. The clone was amplified from a 13.5-day-old mouse conceptus cDNA library by PCR using primers based on conserved regions of PLP-C sequences. The full-length cDNA encodes a predicted protein of 241 residues, which contains a putative signal sequence and 2 putative N-linked glycosylation sites. The predicted protein shares 55-66% amino acid identity with mouse PLP-Calpha and rat PLP-D, PLP-H, PLP-Cv, and PLP-C and also contains 6 homologously positioned cysteine residues. Thus, we named this protein PLP-Cbeta for consistency. We have also isolated rat PLP-Cbeta from rat placenta cDNA library. Surprisingly, two messenger RNA (mRNA) isoforms of rat PLP-Cbeta were isolated: one mRNA (rPLP-Cbeta) encodes a 241-amino acid product, but another mRNA (rPLP-Cbetadelta39) lacks 39 bases that encode for a region rich in aromatic amino acids. The 39-bp region corresponds to exon 3 of other PLP-C subfamily members, such as PLP-Calpha, PLP-Cv, and d/tPRP. It suggests that the two isoforms are probably generated by an alternative splicing from a single gene. RT-PCR analysis revealed that the rPLP-Cbeta form was dominantly expressed in placenta, although both isoforms are coexpressed during placentation. The mouse PLP-Cbeta mRNA expression, which was specific to the placenta, was first detected by Northern analysis on embryonic day 11.5 (E 11.5) and persisted until birth. However, in situ hybridization analysis revealed mPLP-Cbeta expression on E 10.5 in specific trophoblast subsets, such as giant cells and spongiotrophoblast cells. mPLP-Cbeta mRNA was detected in the labyrinthine zone on E 18.5, suggesting that spongiotrophoblast cells had penetrated the labyrinthotrophoblast zone. Consistent with the observed expression in trophoblast giant cells, PLP-Cbeta expression was also detected in in vitro differentiated Rcho-1 cells, which express the trophoblast giant cell phenotype. In summary, overall high amino acid identity (79%), the locations of cysteine residues, and consensus sites for N-linked glycosylation between mouse and rat PLP-Cbeta clearly indicate that PLP-Cbeta is a bona fide member of the PLP-C subfamily. The conservation between mouse and rat, the presence of alternative isoforms, and the pattern of expression during gestation suggest the biological significance of PLP-Cbeta during pregnancy.     

Characterization of the corticosteroid-binding globulin messenger ribonucleic acid response in the pregnant hamster

In this study we measured corticosteroid-binding globulin (CBG) mRNA levels in liver and various nonhepatic tissues of pregnant and nonpregnant hamsters. The N-terminal amino acid sequence (37 residues) of hamster CBG was determined and compared with published cDNA-deduced sequence information for rat and human CBG. Hamster CBG showed considerable sequence homology with both rat (70%) and human (59%) CBG. Because of the high level of homology, we were able to use a cRNA prepared from a rat CBG cDNA as a probe in Northern blot and solution hybridization analyses. Northern blots of hamster and rat liver RNA extracts revealed that the rat CBG cDNA probe hybridized to RNAs that were the same size in rats and hamsters. Further, the Northern blot showed that pregnant hamster liver contained substantially more CBG mRNA than nonpregnant hamster liver. The relative amounts of CBG mRNA in pregnant and nonpregnant hamster livers were compared using a solution hybridization assay. Slope-ratio analysis of the hybridization data revealed that pregnant hamster liver (day 14) contained 40-fold more CBG mRNA than nonpregnant hamster liver. When other tissues (kidney, spleen, small intestine, and decidual tissue) were assayed for CBG mRNA, a small amount of hybridization was detected by solution hybridization. However, Northern blot analysis of RNA extracts from nonhepatic tissues showed that the hybridizable sequences did not migrate at the same position as mature CBG mRNA. These results indicate that the observed increase in serum CBG during hamster pregnancy is largely attributable to an increase in hepatic CBG mRNA.     

Structure, expression, and functional analysis of a Na(+)-dependent glutamate/aspartate transporter from rat brain.

Transport systems specific for L-glutamate and L-aspartate play an important role in the termination of neurotransmitter signals at excitatory synapses. We describe here the structure and function of a 66-kDa glycoprotein that was purified from rat brain and identified as an L-glutamate/L-aspartate transporter (GLAST). A GLAST-specific cDNA clone was isolated from a rat brain cDNA library. The cDNA insert encodes a polypeptide with 543 amino acid residues (59,697 Da). The amino acid sequence of GLAST suggests a distinctive structure and membrane topology, with some conserved motifs also present in prokaryotic glutamate transporters. The transporter function has been verified by amino acid uptake studies in the Xenopus laevis oocyte system. GLAST is specific for L-glutamate and L-aspartate, shows strict dependence on Na+ ions, and is inhibited by DL-threo-3-hydroxy-aspartate. In situ hybridization reveals a strikingly high density of GLAST mRNA in the Purkinje cell layer of cerebellum, presumably in the Bergmann glia cells, and a less dense distribution throughout the cerebrum. These data suggest that GLAST may be involved in the regulation of neurotransmitter concentration in central nervous system.     

Isolation of a cDNA clone encoding pancreatic polypeptide.

We have isolated a cDNA clone encoding pancreatic polypeptide from a cDNA library constructed with RNA from an endocrine neoplasm of the human pancreas. The cDNA was inserted into plasmid pBR322 and the plasmid was cloned in Escherichia coli. Oligonucleotides (sequence in text) specific for the amino acid sequence (sequence in text) of pancreatic polypeptide were used as hybridization probes. The pancreatic polypeptide cDNA isolated was 465 base pairs long and encoded a peptide of 95 amino acids in the coding region. The 36-amino acid sequence of pancreatic polypeptide was flanked by a 29-amino acid putative leader sequence at the amino terminus and a connecting tripeptide (Gly-Lys-Arg) followed by a 27-amino acid peptide at the carboxyl terminus. The first 20 of the amino acids in the carboxyl-terminal heptacosapeptide were identical to the structure of human pancreatic icosapeptide with the single exception of an isoleucine substitution for valine in the 18th position. This alteration results from an A----G substitution in the nucleotide sequence of the cDNA and may represent a genetic variation or a point mutation in the pancreatic polypeptide cDNA.     

Distribution of vitamin D-dependent calcium-binding protein messenger ribonucleic acid in rat placenta and duodenum

The vitamin D-dependent calcium-binding protein (CaBP), cholecalcin or calbindin, is one of the best documented molecular expressions of 1,25-dihydroxyvitamin D, the hormonal metabolite of vitamin D. In this report, DNA/RNA hybridization assays have been used to examine cholecalcin (CaBP) mRNA production in the placenta and duodenum of 21-day pregnant rats. A cloned CaBP cDNA which codes for the rat intestinal 9000 mol wt cholecalcin (9KCaBP) was radiolabeled and used in hybridization assays to explore 1) the size and relative quantities of CaBP mRNA extractable from placenta and duodenum by molecular hybridization, and 2) the localization and quantification, by in situ hybridization histochemistry, of CaBP mRNA in specific cells in rat placenta and duodenum. Northern hybridization studies show that the [32P]cDNA sequence hybridizes to a single 500- to 600-nucleotide species in the placenta as in the duodenum and, therefore, demonstrate identical 9KCaBP mRNA processing in both tissues. Dot blot hybridization studies show that the concentration of 9KCaBP mRNA was greatest in the duodenum, while that of the inner (fetal) placenta was about 50% the duodenal level. Considerably less CaBP mRNA was found in the outer (maternal) placenta. The observed differences in 9KCaBP mRNA levels correlate well with the in vivo variations in 9KCaBP concentrations. In situ hybridization histochemistry using [3H]cDNA reveals that 9KCaBP mRNA visualized by silver grains was concentrated in the inner placenta over the cytoplasm of syncytial cells in the trophoblastic epithelium of the labyrinth and much less frequently in the cells of the outer placenta. In the duodenum, 9KCaBP mRNA was found only in the absorptive epithelial cells from the crypt region to the upper part of the villi. The silver grains were distributed throughout the cytoplasm of the columnar cells; they were densest in the perinuclear region and rarest in the nuclear region. The concentration was greater in the cells at the villous tips than in those of the crypts. This difference in 9KCaBP mRNA levels correlates well with the distribution of the protein itself along the villi. CaBP mRNA quantities detected by hybridization histochemistry showed greater labeling in the syncytial cells of the trophoblastic epithelium of the labyrinth than in the absorptive epithelial cells of the upper part of the villi (200% less), indicating accumulation of CaBP mRNA at a greater rate in the trophoblastic epithelium than in the absorptive epithelial cells. These results indicate that in the rat, 9KCaBP is synthesized in both the absorptive cells of the duodenum and the cells of the trophoblastic epithelium of the placenta.     

The luteotropic activity of rat placenta is not due to a chorionic gonadotropin

Placentae or uteri from pregnant rats (days 12-21) contained no detectable alpha-subunit of the glycoprotein hormones (CG, TSH, FSH, and LH) when assayed in either a rat or human alpha-RIA. The heads of rat fetuses contained increasing concentrations of alpha-subunit when assayed from days 12-20 of gestation (7.2-46 ng/g). Human term placenta contained large quantities of alpha-subunit (16,000 ng/g). alpha-Subunit was synthesized by the cell-free translation of poly(A)-enriched mRNA from mouse TSH-secreting pituitary tumor and human term placenta, but not from rat placentae or uterine implantation sites (days 11-21 of gestation). In addition, alpha mRNA was detected in mouse TSH-secreting pituitary tumor, rat pituitary, and human term placenta by hybridization to a 32P-labeled mouse alpha cDNA probe although no alpha mRNA could be detected in rat placentae (days 13-21 of gestation). The luteotropic activity found in pregnant rodents must be caused by a substance with a structure substantially distinct from any known gonadotropin.     

Construction and characterization of the alpha form of a cardiac myosin heavy chain cDNA clone and its developmental expression in the Syrian hamster.

A cDNA clone, pVHC1, was isolated from a Syrian hamster heart cDNA library and was compared to the rat alpha (pCMHC21) and beta (pCMHC5) ventricular myosin heavy chain cDNA clones. The DNA sequence and amino acid sequence deducted from the DNA show more homology with pCMHC21 than pCMHC5. This indicates that pVHC1 is an alpha ventricular myosin heavy chain cDNA clone. However, even though pVHC1 shows a high degree of nucleotide and amino acid conservation with the rat myosin heavy chain sequences, the carboxyl-terminal peptide and the 3'-untranslated region are highly divergent and specific for this cDNA clone. There appears to be an amino acid deletion in the 3' end of the hamster alpha myosin heavy chain as compared to the rat alpha myosin heavy chain. S1 nuclease mapping experiments have shown that the mRNA represented by this cDNA clone is scarcely expressed in neonatal development, but its expression increases with age and reaches maximal levels in adult life. This cDNA clone provides a useful tool to follow the myosin heavy chain mRNA changes during development and during the genesis of a cardiomyopathy, an autosomal recessive defect carried by the Syrian hamster.     

Isolation and sequence of a human cytochrome P-450 cDNA clone.

A previously reported cDNA clone [pP450(1)] coding for a phenobarbital-inducible cytochrome P-450 variant of rat liver microsomal membranes, designated P-450e(U.C.), was used as a specific hybridization probe to screen a human liver cDNA library. Restriction mapping showed that two of the colonies isolated contained plasmids coding for overlapping regions of the same cDNA sequence. The clone [pHP450(1)] having the longer cDNA insert (1.25 kilobase pairs) was sequenced. The homology between the rat and human cDNAs is 62% in their coding regions but is only random (24%) in the 3'-noncoding nucleotides. The amino acid sequence deduced from the human cDNA is 50% identical to that of P-450e(U.C.). The homology increases to 72% if conservative changes in amino acid residues are permitted. The hydropathy profile of the polypeptide encoded by pHP450(1) is almost identical to that of P-450e(U.C.). Regions known to be highly conserved in cytochrome P-450 isozymes isolated from rat, rabbit, and mouse were found to be conserved in the amino acid sequence derived from pHP450(1). Analysis by Southern blotting indicated that the human cytochrome P-450 encoded by pHP450(1) is part of a multigene family.     

Rat placental lactogen-I abolishes nocturnal prolactin surges in the pregnant rat

The twice-daily surges of prolactin (PRL) present during the first half of pregnancy abruptly terminate at midpregnancy concurrent with the appearance of high levels of placental lactogen-I (PL-I) in the blood. This study addressed the role PL-I and other pituitary or placental hormones have in terminating PRL surges in pregnant rats. Implantation of rat PL-I (rPL-I) or ovine PRL into the arcuate-median eminence area of the hypothalamus of day 7 pregnant rats totally eliminated nocturnal PRL surges on days 8 and 9. To assess the specificity of the inhibitory effects of hormones from the PRL-growth hormone (GH) family, rat growth hormone (rGH), human growth hormone (hGH), and rat prolactin-like protein-A (PLP-A) were tested. Only the lactogenic hormone, hGH, had any effect. Since lactogenic hormones may inhibit PRL by stimulation of dopamine synthesis and release into the hypophysial portal blood vessels leading to the anterior pituitary, the effect of these hormones on tyrosine hydroxylase (TH), the rate-limiting enzyme for the synthesis of dopamine activity, was determined. In pregnant rats, both ovine prolactin (oPRL) and hGH significantly increased (64%) TH activity, whereas rPL-I was less effective. In ovariectomized, bromocriptine-treated rats, both rPL-I and oPRL increased TH activity 207 and 151%, respectively. This supports the concept that termination of PRL surges at midpregnancy are owing to secretion of placental lactogens (PLs) from the placenta. However, the mechanism for the inhibition cannot be entirely attributed to an increase in tuberoinfundibular dopaminergic neuronal activity.     

Molecular cloning and developmental expression of the rat cardiac-specific isoform of troponin I

Troponin I is the subunit of the troponin complex in striated muscle which inhibits actomyosin ATPase activity. We have isolated a full-length cDNA clone for rat cardiac troponin I and determined its nucleic acid sequence. The amino acid sequence deduced from this clone shows 88%-92% similarity with previously reported amino acid sequences for rabbit (Wilkinson and Grand, 1978) and bovine (Leszyk et al.) cardiac troponin I. Examination of cardiac troponin I mRNA abundance during development revealed a 15-fold induction in its expression in the adult heart compared to that in embryonic (14 day) heart muscle. Furthermore, expression of cardiac troponin I mRNA was restricted to heart muscle and was not detected in skeletal muscle at any developmental stage.     

Absence of detectable chorionic gonadotropin subunit messenger ribonucleic acids in the rat placenta throughout gestation

We hypothesized that if a CG similar in structure to other glycoprotein hormones was present in the rat placenta, then mRNAs encoding its subunits would be detectable. To investigate the possible presence of a CG in the rat, we attempted to detect mRNAs encoding the alpha- and CG beta-like subunits in the placentae of timed pregnant rats by sensitive blot hybridization analyses using cloned cDNAs encoding alpha- and beta-subunits of LH derived from rat pituitary mRNA. No subunit-specific mRNAs in placentae of pregnant rats at 4-21 days gestation were detected at either high or low stringencies of hybridization. However, under similar conditions, subunit-specific mRNAs were readily observed in total pituitary RNA of normal as well as ovariectomized rats. Moreover, hybridization with a cDNA for alpha-tubulin, a major component of the cytoskeleton, yielded easily detectable bands in rat placental RNA. In addition, hybridization analysis, under low stringency conditions, of restriction enzyme digests of rat genomic DNA with a rat LH beta-cDNA, which would detect LH beta subunit-like genes, suggests the presence of a single gene that, in fact, encodes the rat LH beta-subunit. We conclude that mRNAs encoding for proteins structurally homologous to rat LH subunits are absent in the rat placenta and that only a single LH beta-like gene is present in the rat. The luteotropic activity associated with the rat placenta must be related to a gonadotropin-like hormone whose structure is dissimilar from that of CG.     

Modulation of sodium-channel mRNA levels in rat skeletal muscle.

Action potentials in many types of excitable cells result from changes in permeability to Na ions. Although these permeability changes in nerve and muscle are mediated by voltage-gated Na channels that are functionally similar, we found that the Na-channel gene expressed in skeletal muscle is different from the genes coding for two Na channels (type I and type II) in brain. Despite the structural differences between muscle and brain Na-channel genes, a cDNA clone derived from rat brain hybridizes to skeletal muscle Na-channel mRNA of approximately 9.5 kilobases. We used this cDNA probe to measure changes in Na-channel mRNA levels in skeletal muscle during development and following denervation. By blot hybridization analysis of electrophoretically fractionated RNA, we found that Na-channel mRNA can be detected as early as embryonic day 17 and that mRNA levels increase 2-fold between birth and postnatal day 35. Denervation of adult muscle causes a further 2- to 3-fold increase in muscle Na-channel mRNA levels, suggesting that expression of Na-channel genes in fast-twitch muscle may be regulated by the state of innervation.     

Dopamine transporter mRNA content in human substantia nigra decreases precipitously with age.

The dopamine transporter is the primary means of inactivating synaptic dopamine as well as a major site of action for psychostimulants (such as cocaine and amphetamine) and for neurotoxins that induce parkinsonism. In the present study, a human dopamine transporter partial cDNA clone obtained by polymerase chain reaction exhibited 87% and 89% identity at the nucleic acid and amino acid levels, respectively, with transmembrane domains 3-5 of the rat homolog. This clone was used to quantitate human dopamine transporter mRNA by nuclease protection assay. The postmortem content of dopamine transporter mRNA in the substantia nigrae of 18- to 57-yr-old subjects was relatively constant, while in subjects greater than 57 yr old, a precipitous (greater than 95%) decline in substantia nigra dopamine transporter mRNA was evident. In contrast, tyrosine hydroxylase mRNA in the same samples declined in a linear manner with increasing age. In situ hybridization experiments confirmed the profound loss of dopamine transporter gene expression in melanin-positive (presumptive dopamine) nigral neurons. These data may begin to shed light on compensatory changes occurring in human dopamine neurons during normal aging.     

Characterization of cDNA coding for human factor XIIIa.

A cDNA library prepared from human placenta has been screened for sequences coding for factor XIIIa, the enzymatically active subunit of the factor XIII complex that stabilizes blood clots through crosslinking of fibrin molecules. Two oligonucleotides, based on the amino acid sequences of tryptic peptides of factor XIIIa, were used as hybridization probes. Of 0.36 X 10(6) independent recombinants, 1 clone was identified that hybridized to both probes. The insert of 1704 base pairs coded for the amino-terminal 541 amino acid residues of the mature factor XIIIa molecule. Blot-hybridization analysis using this cDNA as a probe showed that the factor XIIIa mRNA from placenta has a size of approximately 4000 bases. The insert was used to rescreen cDNA libraries and to identify further factor XIIIa-specific sequences. The total length of the isolated factor XIIIa cDNA is 3905 bases, and it codes for a protein of 732 amino acids. In spite of the presence of factor XIII in blood plasma, we could not identify a leader sequence typical for secreted proteins.     

Cloning and expression of a cDNA encoding human sterol carrier protein 2.

We report the cloning and expression of a cDNA encoding human sterol carrier protein 2 (SCP2). The 1.3-kilobase (kb) cDNA contains an open reading frame which encompasses a 143-amino acid sequence which is 89% identical to the rat SCP2 amino acid sequence. The deduced amino acid sequence of the polypeptide reveals a 20-residue amino-terminal leader sequence in front of the mature polypeptide, which contains a carboxyl-terminal tripeptide (Ala-Lys-Leu) related to the peroxisome targeting sequence. The expressed cDNA in COS-7 cells yields a 15.3-kDa polypeptide and increased amounts of a 13.2-kDa polypeptide, both reacting with a specific rabbit antiserum to rat liver SCP2. The cDNA insert hybridizes with 3.2- and 1.8-kb mRNA species in human liver poly(A)+ RNA. In human fibroblasts and placenta the 1.8-kb mRNA was most abundant. Southern blot analysis suggests either that there are multiple copies of the SCP2 gene in the human genome or that the SCP2 gene is very large. Coexpression of the SCP2 cDNA with expression vectors for cholesterol side-chain cleavage enzyme and adrenodoxin resulted in a 2.5-fold enhancement of progestin synthesis over that obtained with expression of the steroidogenic enzyme system alone. These findings are concordant with the notion that SCP2 plays a role in regulating steroidogenesis, among other possible functions.