Proc. Natl. Acad. Sci. USA

Vol. 74, No. 3, pp. 1024-1027, March 1977 Biochemistry

Human placental lactogen mRNA and its structural genes during pregnancy: Quantitation with a complementary DNA (RNA.DNA hybridization/protein hormone/placental differentiation) DIANA MCWILLIAMS*, ROBERT C. CALLAHANt, AND IRVING BoIME* * Department of Obstetrics and Gynecology and Pharmacology, Washington University School of Medicine, St. Louis, Missouri 63110; and t Viral Leukemia and Lymphoma Branch, National Cancer Institute, Bethesda, Maryland 20014

Communicated by Oliver H. Lowry, January 10, 1977

ABSTRACT A complementary DNA (cDNA) strand was transcribed from human placental lactogen (hPL) mRNA. Based on alkaline sucrose gradient centrifugation, the size of the cDNA was about 8 S, which would represent at least 80% of the hPL mRNA. Previously we showed that four to five times more hFL was synthesized in cell-free extracts derived from term as compared to first trimester placentas. Hybridization of the cDNA with RNA derived from placental tissue revealed that there was about four times more hPL mRNA sequences in total RNA from term placenta than in a comparable quantity of total first trimester RNA. Only background hybridization was observed when the cDNA was incubated with RNA prepared from human kidney. To test if this differential accumulation of hPL mRNA was the result of an amplification of hPL genes, we hybridized the labeled cDNA with cellular DNA from first trimester and term placentas and with DNA isolated from human brain. In all cases, the amount of hPL sequences was approximately two copies per haploid genome. Thus, the enhanced synthesis of hPL mRNA appears to result from a transcriptional activation rather than an amplification of the hPL gene. The increase likely reflects placental differentiation in which the proportion of syncytial trophoblast increases at term.

population of hPL sequences in total RNA from term placenta than in total first trimester RNA. In addition, the data show that the number of hPL gene(s) per haploid genome is the same in term and first trimester tissue. METHODS Purification of hPL mRNA. Term placental RNA was purified on an oligo(dT)-cellulose column (2). Oligo(dT)-treated RNA (1 mg) was purified further on 5-20% sucrose gradients containing 10 mM Tris-HCI (pH 7.8) and 2 mM EDTA. The RNA was centrifuged in a SW-41 rotor at 32,000 rpm for 18 hr at 7° Fractions of 0.36 ml were collected and aliquots assayed in wheat germ extracts (2). The fraction enriched for directing prehPL synthesis was precipitated with ethanol and dissolved in sterile water. This RNA fraction was examined on acrylamide gel electrophoresis in formamide as described (2). Isolation of Placental DNA. Placental tissue was washed and minced through a tissue press (1). DNA was extracted from the tissue as described (6). Preparation of [3H]DNA. Reverse transcriptase (RNAdependent DNA polymerase), isolated and purified from avian myeloblastosis virus, was a gift from Dr. G. Todaro. It was used to synthesize [3H]cytidine-labeled DNA from hPL mRNA, that had been purified by sucrose gradient centrifugation, according to Benveniste et al. (6). The [3H]DNA was deproteinized, subjected to RNA hydrolysis, and dialyzed (6). The specific activity of the DNA product was about 107 cpm/,ug. Preparation of Nonrepetitive Cellular DNA. The cellular DNA of a rhabdosarcoma human cell line was labeled with [3H]thymidine (120 ,tCi/ml of culture medium) for 72 hr and extracted (6). DNA sequences not hybridizing to a Cot of 200 were isolated by fractionation on hydroxyapatite. (Cot is the initial concentration of DNA in mol of nucleotide/liter multiplied by time.) The specific activity of the DNA was 1.5 X 104

The biosynthesis of each of the human placental hormones, chorionic gonadotropin and placental lactogen (hPL), is differentially coupled to gestation and in each case the synthetic rate tends to parallel the blood level in vtivo. Whereas the synthesis of hPL is maximal at term, the peak of chorionic gonadotropin synthesis occurs at first trimester (1-5). Thus, the placenta represents a convenient and unique tissue for studying control of human hormonal genes under the influence of a variety of physiological stimuli. In order to quantitate hPL and chorionic gonadotropin structural genes and their corresponding mRNAs in placenta as well as in its malignant counterpart, choriocarcinoma, it would be very useful to synthesize complementary DNAs from these mRNAs. We have shown previously that there is four to five times more translatable hPL mRNA in term placenta than in a comparable quantity of first trimester tissue (1, 2). This difference may be the result of one or more of the following: (i) amplification of the number of hPL genes during gestation; (ii) activation of transcription without increase in the number of hPL genes; and (iil) a decrease in the synthesis of other proteins without change in synthesis of hPL. In other words, the fraction of hPL mRNA present might be greater at term because there is a selective decrease in the synthesis of specific mRNAs as compared to first trimester. Here we report on the synthesis of complementary DNA (cDNA) transcribed from relatively pure hPL mRNA. Studies with this cDNA reveal that there is about a 4-fold greater

cpm/Ag (7).

Alkaline Sucrose Gradients. A solution of [3H]cDNA was adjusted to a composition of 1.4 M NaCl, 0.6 M NaOH, and 0.01 M EDTA and was applied to 5-20% sucrose gradients adjusted with this solution. The gradient was centrifuged at 38,000 rpm in a SW-41 rotor for 20 hr at

200. Fractions of 0.5 ml were

collected, and 25-,ul aliquots from each fraction were assayed, in the presence of carrier calf thymus DNA, for acid-precipitable radioactivity. The appropriate fractions were pooled and neutralized with 10 M HC1. The sample was dialyzed against four changes of sterile water and then lyophilized. RNA-DNA Hybridization. Placental RNA and [3H]DNA were incubated at 630 for various times in reaction mixtures containing 0.01 M Tris-HCI (pH 7.5), 0.40 M NaCl, 0.1 mM EDTA, 0.02% sodium dodecyl sulfate, and approximately 16,000 cpm of [3H]DNA per ml. The ratio of RNA to [3H]DNA was 1 X 106. The hybridizations were started by heating reac-

Abbreviations: hPL, human placental lactogen; Cot (CAt), initial concentration of DNA (RNA) X time; 1 A260 unit, that amount of material that gives an absorbance of 1 when dissolved in 1 ml of solvent with a light path of 1 cm.

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Proc. Natl. Acad. Sci. USA 74 (1977)

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Table 1. Template specificity of reverse transcriptase of avian myeloblastosis virus

23S --

16S -12S---

Conditions of assay

Acid-precipitable 3H cpm/reaction

Complete Minus RNA Minus oligo(dT) Minus thymidine triphosphate

230,000 10,000 14,000 16,000

The reaction conditions were as described (6). Incubations were carried out in 1OO-Al reaction mixtures. The mRNA was purified as described in Materials and Methods and used in a concentration of 5 ,ug/ml.

q

FIG. 1. Analysis of hPL mRNA that had been purified by sucrose gradient centrifugation, in formamide polyacrylamide gel. Five micrograms of RNA was applied. The migration of 23S and 16S ribosomal RNA standards is shown.

tion mixtures at 1000 for 6 min, cooling, and incubating at 630 for up to 72 hr. After various time periods of incubation, 25-,ul aliquots were removed and the amount of hybridized DNA that was resistant to nuclease SI was measured. To control for possible inhibition of SI activity by the various RNA preparations used, 25-,ul aliquots were frozen immediately after heating the reaction mixture at 100°, adding SI, and digesting as described. The data are expressed in terms of Crt (8), which is calculated as A260 units/ml X hr/2. (One A260 unit is that amount of material dissolved in 1 ml of solvent that gives an absorbance of

prehPL

-RNA +RNA FIu. 2. Autoradiograph of a sodium dodecyl sulfate/polyacrylamide gel electropherogram of proteins synthesized in response to purified hPL mRNA. One microgram of RNA was added to a 150-il reaction mixture containing 30 pl of wheat germ S-30. Approximately 100,000 cpm (+RNA) and 20,000 cpm (-RNA) were applied to the gel. The migration of prehPL (molecular weight 25,000) is indicated.

1 when the light path is 1 cm.) Crt1/2 is the Crt at which hybridization of the cDNA is half completed. DNA-DNA hybridizations were carried out at 630 in reaction mixtures containing 0.01 M Tris-HCI (pH 7.4), 0.75 M NaCl, 0.2 mM EDTA, 0.02% sodium dodecyl sulfate, 10,000 cpm of [3H]DNA per ml, and 4 mg of cellular DNA per ml (6). The ratio of cellular DNA to [3H]DNA was 4 X 106. Hybridizations were started by heating the mixtures at 1000 for 10 min, cooling on ice to 40, and then incubating at 63'. At various times, 25-,41 aliquots were removed and frozen at -80° until digested by SI nuclease. As described above, Cot values are expressed in terms of A260 units/ml X hr/2, and were corrected to a monovalent cation concentration of 0.18 M (9).

RESULTS The hPL mRNA used as a template for the reverse transcriptase from avian myeloblastosis virus was from term placental tissue isolated by chromatography of total cellular RNA on oligo(dT)-cellulose and subsequent purification of the adsorbed RNA by sucrose density gradient centrifugation. The resulting RNA migrated essentially as one band at about 12-13 S on formamide polyacrylamide gels (Fig. 1). In the wheat germ cell-free system this RNA directed the synthesis of only one major protein, prehPL (Fig. 2). The material migrating ahead of prehPL represents endogenous proteins synthesized by the wheat germ. The synthesis of DNA with reverse transcriptase was markedly dependent upon the addition of mRNA, the reaction saturating at about 5 ug/ml of the purified mRNA (Table 1). The background level of DNA synthesis in the absence of RNA was about 5%. This mRNA-dependent synthesis of the DNA product also required the presence of oligo(dT) and each of the unlabeled deoxyribonucleoside triphosphates. The oligo(dT) dependence further indicates the presence of an adenine-rich sequence in hPL mRNA. The length of the cDNA product was assessed by subjecting it to an alkaline sucrose gradient centrifugation (Fig. 3). The single-stranded DNA product sedimented at about 8 S, which corresponds to a nucleotide length of approximately 850 bases (10). Because the size of hPL mRNA is about 1000 bases, it appears that most of the hPL mRNA has been transcribed. Subsequent experiments were performed using DNA pooled from fractions heavier than and including the 8 S peak from preparative alkaline gradients. The molecular complexity of the hPL cDNA was measured by following the kinetics of its hybridization with hPL mRNA that had been purified by sucrose gradient centrifugation (Fig. 4). The hybridization reaction of the cDNA with the mRNA occurs approximately over 2 decades of the Crt curve, with a Crtl/2 of 2.2 X i0-3. This indicates that the cDNA is comple-

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FIG. 3. Alkaline sucrose gradient analysis of [:HJDNA product. The solution of the DNA product was adjusted to 1.4 M NaCl, 0.6 M NaOH, and 0.01 M EDTA and then incubated at 370 for 30 min. The sample was then applied-to 12 ml of 5-20% sucrose gradient containing the same solution. Centrifugation was for 20 hr at 38,000 rpm at 200 in a SW-41 rotor. Fractions (0.5 ml) were collected, radioactivity was measured, and sedimentation values were calculated according to Studier (10). a major species of mRNA. As a standard, highly purified mouse globin mRNA and its cDNA (obtained from Dr. J. Ross) were hybridized under identical conditions; a CrtI/2 of 1 X 10-3 was obtained. Quantitation of hPL mRNA in Placental Tissue. Previous studies have shown that total term placental mRNA directs the synthesis of five times more prehPL in wheat germ cell-free extracts than does a comparable quantity of first trimester RNA (4). Further, it was demonstrated that this difference was not due to a differential recovery of RNA from the tissues nor was it the result of nuclease activity in first trimester tissue. However, it was not possible to eliminate the possibility that hPL mRNA is in a "nontranslatable" form in first trimester tissue and thus not detected in the cell-free system. To directly compare the relative concentration of hPL mRNA we have hybridized total cellular placental RNA with the hPL cDNA (Fig. 5). The Crti/2 value (Crt at which 50% of the cDNA is protected from SI nuclease digestion) is inversely proportional to the relative concentration of RNA complementary to the hPL cDNA. The Crtl/2 of the hybridization reaction with total term RNA is 0.6, as compared to 2.5 with total first trimester RNA. Thus, there is approximately four times more hPL mRNA in term than in first trimester tissue. Similarly, the total poly(A)-

mentary to

containing RNA from term tissue contained four times more hPL mRNA than that from first trimester tissue. This is consistent with the value obtained previously (1) and suggests that during the early development of the placenta, hPL mRNA is not sequestered in a nontranslatable form. Moreover, a comparison of the Crti/2 of the total poly(A)-containing term RNA (9 X 10-3) with that of hPL mRNA purified by sucrose gradient centrifugation (2.2 X 10-3) suggests that at term at least 20% of the poly(A)-containing RNA in the tissue is hPL mRNA. This is in agreement with the translation data that prehPL comprised 20% of the total mRNA-dependent protein synthesized in the wheat germ system. As expected, only background hybridization was observed with RNA from human kidney.

Measurement of Number of hPL Genes as Function of Gestation. The observed increase in the concentration of hPL mRNA in term placental tissue as compared to first trimester tissue suggested the possibility that this difference reflects an amplification of the hPL gene during gestation. We have investigated this point by measuring the number of genes encoding hPL in the cellular genome of first trimester and term placental tissue. Total cellular DNA isolated from first trimester and term tissues, as well as from human brain, was hybridized with hPL [3HJcDNA (Fig. 6). The Cotl/2 values for these hybridization reactions were 1100. Since the COt 12 for the selfannealing of human nonrepetitive cellular DNA under these conditions is 1800, the estimated frequency of the hPL gene is approximately 2 per haploid genome. An alternative method for the calculation of gene frequency is based on the final extent of hybridization of the cDNA with cellular DNA (11, 12). At completion of the hybridization reaction, the extent will be a function of the ratio of unlabeled cellular DNA sequences (those that are identical to the labeled probe) to the hPL cDNA. Using the final extent of hybridization observed in the experiment shown in Fig. 6, the calculated number of hPL genes per haploid genome was 2 to 3. Thus, the hPL genes are not amplified during gestation and are not highly repeated in the human cellular genome.

DISCUSSION The use of a [3H]DNA probe as an assay for mRNA levels depends upon the purity and specificity of the mRNA from which the DNA was transcribed. In the case of the mRNA encoding hPL, the RNA migrated as one band on formamide gels and was translated into only one protein. Furthermore, the population of hPL sequences in total cellular RNA from term placenta as compared to the same quantity of RNA from first trimester tissue parallels the differences in the amount of translatable hPL mRNA present in first trimester and term placentas. In addition, little, if any, hybridization was observed when [3H]DNA was incubated with RNA derived from a nonplacental human tissue. Based on these observations and the point that hPL mRNA comprises at least 20% of the translatable mRNA present in term placenta we believe that the majority of the DNA sequences are complementary to hPL mRNA. Data from alkaline sucrose gradients indicate a size of about 8 S (850 nucleotides) for the DNA product. This is somewhat smaller than what might be expected from the size of hPL

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Proc. Natl. Acad. Sci. USA 74 (1977)

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