In vitro biosynthesis of fish islet preprosomatostatin: evidence of processing and segregation of a high molecular weight precursor.

Evidence is presented for a precursor to somatostatin that is 10-12 times larger than the authentic secreted hormone. mRNA from angler fish (Lophius americanus) islets of Langerhans was translated in the wheat germ cell-free system and the products were identified by immunoprecipitation with specific antibodies to somatostatin followed by sodium dodecyl sulfate gel electrophoresis. One 18,000-dalton polypeptide was specifically immunoprecipitable. Competition experiments showed that authentic somatostatin competed with the 18,000-dalton molecule for antibody binding. When dog pancreas microsomal membranes were present during translation, an additional polypeptide of 16,000 daltons was also immunoprecipitable. Comparison of their tryptic peptides demonstrated that the 16,000-dalton polypeptide was derived from the 18,000-dalton one. Tryptic peptide analysis of somatostatin and the 18,000-dalton precursor demonstrated that the 18,000-dalton polypeptide contains the authentic somatostatin amino acid sequence and suggests that it is located at the carboxyl terminus of the precursor molecule and is preceded by a basic amino acid.     

Rabbit reticulocyte initiation factor 2 contains two polypeptide chains of molecular weights 48,000 and 38,000.

Eukaryotic initiation factor 2 (eIF-20) purified from rabbit reticulocyte lysates consists of equimolar amounts of two polypeptide chains of Mr 48,000 and 38,000. Determination of the molecular weight of the native factor gave a value which is consistent with a Mr of 86,000 indicating that the factor is composed of one Mr 48,000 and one Mr 38,000 polypeptide. The purified factor exhibited all the binding activities characteristic of eIF-2. The factor formed ternary complexes with Met-tRNAfMet and GTP; it bound GDP to form a binary complex; and it also possessed the property of binding a wide variety of RNA species, including reoviral mRNA, phage T3 mRNA, rRNAs, and tRNA. Furthermore, the ternary complex formed by purified eIF-2 interacted with the 40S ribosomal subunit in the presence of AUG codon to form a 40S initiation complex. These results indicate that all binding activities attributed to eIF-2 are contained in the 48,000- and 38,000-dalton polypeptides.     

腐食酪螨过敏原的分析鉴定与纯化

目的 分析、鉴定与纯化腐食酪螨过敏原组分。 方法 以十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）和蛋白质印迹（Western blotting）分析鉴定腐食酪螨蛋白质提取液中的蛋白质组分和过敏原组分，并通过阴离子交换层析和分子排阻色谱进一步纯化。 结果 腐食酪螨的蛋白质粗浸液电泳后可以辨认的蛋白质带共23条，相对分子质量（Mr）分别为177 000、118 000、107 000、70 000、67 000、60 000、52 000、45 000、41 000、40 000、38 000、37 000、35 000、27 000、23 000、22 000、18 000、17 000、16 000、15 000、14 000、13 000和12 000。Western blotting分析显示，腐食酪螨的5条致敏蛋白质带分别为Mr 128 000、67 000～70 000、36 000～37 000、18 000和16 000，其中前3条带特异性结合IgE阳性率均为100％，而Mr 18 000和Mr 16 000条带的阳性反应率分别为77.8%和44.44%。通过阴离子交换层析和分子排阻色谱从腐食酪螨粗浸液中分离到Mr 18 000的过敏原组分。 结论 腐食酪螨有4种主要过敏原及1种次要过敏原。     

Altered levels of laminin receptor mRNA in various human carcinoma cells that have different abilities to bind laminin.

The human laminin receptor was purified and molecularly cloned to investigate its biosynthetic regulation. Laminin receptor from normal and neoplastic tissue was preparatively affinity purified to homogeneity based on the high affinity of the receptor for laminin. The apparent molecular weight of the receptor from different carcinoma sources and from normal placental tissue is in the range of 68-72 kDa. Isoelectric focusing and two-dimensional gel electrophoresis indicated that the receptor protein consists of one major polypeptide chain with a pI value of 6.4 +/- 0.2. Laminin receptor cDNA clones were isolated after screening a human endothelial lambda gt11 cDNA library with a monoclonal antibody directed against a domain of the laminin receptor involved in ligand binding. Definitive identification of the cDNA clones was based on comparison of cDNA sequence with the amino acid sequence of a cyanogen bromide-generated octapeptide of purified placental laminin receptor. The laminin receptor mRNA is approximately 1700 bases long. The level of laminin receptor mRNA in a variety of human carcinoma-derived cell lines correlated with the number of laminin receptors on the cell surfaces of those cells. This suggests that the amount of laminin receptor mRNA may be a rate-limiting control step in the biosynthesis of the laminin receptor, and hence in the regulation of cellular attachment to basement membranes via laminin.     

Use of molecular hybridization to purify and analyze albumin messenger RNA from rat liver

A new procedure is described for purification of rat liver albumin mRNA. First a population of RNA molecules is enriched for albumin mRNA by immunoprecipitation of polysomes containing albumin nascent chains. Polyadenylylated RNA is prepared from immunoprecipitates, transcribed into complementary DNA, and shown to be enriched severalfold for a particular RNA frequency component. This enriched RNA component is then purified by molecular hybridization to a limited R(0)t value (product of RNA concentration and incubation time), under conditions in which only the most abundant sequence component is annealed. Potentially, this procedure can be employed for the purification of a wide variety of mRNAs present in lesser amounts in the cell.The isolated RNA appears to be a single frequency component by hybridization to complementary DNA transcribed from itself. This RNA is a 17S species and represents 5-8% of total cytoplasmic polyadenylylated RNA. In vitro translation of the purified RNA has shown that it codes for a single polypeptide that can be identified immunologically as albumin and migrates with rat serum albumin on sodium dodecyl sulfate/polyacrylamide gels. This albumin mRNA was determined to be essentially pure by comparing its kinetics of hybridization to those obtained with rabbit alpha + beta globin mRNA and its DNA complement. The sequence complexity of purified rat albumin mRNA corresponds to 5.9 x 10(5) daltons.     

Purification of low-abundance messenger RNAs from rat liver by polysome immunoadsorption.

We have purified three low-abundance hepatic mRNAs to near homogeneity by polysome immunoadsorption. The mRNAs coding for the precursor of ornithine transcarbamoylase [carbamoylphosphate:L-ornithine carbamoyltransferase, EC 2.1.3.3], the precursor of the beta-subunit of propionyl-CoA carboxylase [propionyl-CoA:carbon dioxide ligase (ADP-forming), EC 6.4.1.3], and cystathionine beta-synthase [L-serine hydro-lyase (adding homocysteine), EC 4.2.1.22], representing approximately 0.20, 0.02, and 0.015% of total hepatic mRNA, respectively, were purified 450- to 6,300-fold. We used the following steps: interaction of rat liver polysomes with an IgG fraction of monospecific antisera raised against each polypeptide; immobilization of polysome-antibody complexes on a protein A-Sepharose column; removal of the bulk of polysomes by extensive washing; dissociation of ribosomal subunits and elution of specific mRNA with EDTA; and isolation of the eluted mRNA by chromatography on an oligo(dT)-cellulose column. It seems likely that this procedure will permit isolation of other low-abundance mRNAs and subsequent cloning of their respective cDNAs.     

Purification of human platelet-derived growth factor.

Human platelets contain a polypeptide growth factor that stimulates the proliferation of connective tissue cells. Purification of this platelet-derived growth factor (PDGF) was accomplished by heat (100 degrees C) treatment of washed platelets and subsequent ion-exchange chromatography, gel filtration in 1 M acetic acid, isoelectric focusing, and preparative sodium dodecyl sulfate/polyacrylamide gel electrophoresis. PDGF has an isoelectric point of 9.8 and a molecular weight ranging from 13,000 to 16,000 as judged by gel filtration in 1 M acetic acid or analytical sodium dodecyl sulfate gel electrophoresis under reducing conditions. The specific activity of the purified PDGF is 20 million times greater than that found in unfractionated human serum. Purified PDGF stimulates replicative DNA synthesis and cell proliferation in quiescent density-arrested cultures of BALB/c 3T3 cells at concentrations of 1 ng/ml (0.1 nM).     

Physical association of pyrimidine dimer DNA glycosylase and apurinic/apyrimidinic DNA endonuclease essential for repair of ultraviolet-damaged DNA.

T4 endonuclease V [endodeoxyribonuclease (pyrimidine dimer), EC 3.1.25.1)], which is involved in repair of UV-damaged DNA, has been purified to apparent physical homogeneity. Incubation of UV-irradiated poly(dA).poly(dT) with the purified enzyme preparations resulted in production of alkali-labile apyrimidinic sites, followed by formation of nicks in the polymer. The activity to produce alkali-labile sites was optimal in a relatively broad pH range (pH 6.0-8.5), whereas the activity to form nicks had a narrow optimum near pH 6.5. By performing a limited reaction with T4 endonuclease V at pH 8.5, irradiated polymer was converted to an intermediate form that carried a large number of alkali-labile sites but only a few nicks. The intermediate was used as substrate for the assay of apurinic/apyrimidinic DNA endonuclease activity [endodeoxyribonuclease (apurinic or apyrimidinic, EC 2.1.25.2]. The two activities, a pyrimidine dimer DNA glycosylase and an apurinic/apyrimidinic DNA endonuclease, were copurified and found in enzyme preparations that contained only a 16,000-dalton polypeptide. An enzyme fraction from cells infected with bacteriophage T4v1, a mutant that is sensitive to UV radiation, was defective in both glycosylase and endonuclease activities. Moreover, occurrence of an amber mutation in the denV gene caused a simultaneous loss of the two activities, and suppression of the mutation rendered both activities partially active. These results strongly suggested that a DNA glycosylase specific for pyrimidine dimers and an apurinic/apyrimidinic DNA endonuclease reside in a single polypeptide chain coded by the denV gene of bacteriophage T4. Because the two activities exhibited different thermosensitivity, it was further suggested that conformation of the active sites for these activities may be different.     

Translation of rat liver fatty acid synthetase mRNA in a cell-free system derived from wheat germ

Total liver polysomes were isolated from rats that had fasted for 48 hr and that then had been re-fed a high-carbohydrate, fat-free diet for 20-24 hr. Indirect immunoprecipitation of the polysomes with purified antibody to rat liver fatty acid synthetase and deproteination on sodium dodecyl sulfate-containing sucrose gradients gave an RNA fraction which, when translated in a cell-free system derived from wheat germ, yielded a major polypeptide of apparent molecular weight 225,000 when the translation products were analyzed by polyacrylamide gel electrophoresis in sodium dodecyl sulfate. The polypeptide was specifically precipitated with antibody against rat liver fatty acid synthetase and competed with unlabeled fatty acid synthetase for binding to the antibody. It was somewhat smaller than native fatty acid synthetase subunits (molecular weight 240,000). The peptide accounted for approximately 65% of the radioactive, antibody-precipitable product, the remainder being peptides in the molecular weight range 100,000-150,000. Synthesis of the polypeptide was optimized with respect to K(+), Mg(2+), and spermine concentrations. The quantity of fatty acid synthetase mRNA obtained by the above procedure and measured by translation was a function of the nutritional state of the animal. The relative activity in fasting rats compared to rats that were re-fed for 12 hr was 1:12. The data suggest that rat liver fatty acid synthetase is synthesized as intact subunits from a large mRNA molecule or molecules.     

Sequence analysis of ripening-related cytochrome P-450 cDNAs from avocado fruit.

The ripening of avocado fruit is associated with the expression of a number of mRNAs concomitant with overt changes in texture and flavor. Two overlapping cDNAs for a mRNA that accumulates during ripening were identified. Sequence analysis of these two cDNAs revealed a polypeptide of 471 amino acids with characteristics of a typical P-450: an N-terminal hydrophobic membrane anchor, a conserved heme-binding domain in the C-terminal region, and patches of similarity to various P-450 family members. Further evidence that this polypeptide represents a cytochrome P-450 oxidase comes from the recent isolation and characterization of a cytochrome P-450 from ripe avocado mesocarp [O'Keefe, D. P. & Leto, K. J. (1989) Plant Physiol. 89, 1141-1149]. The N terminus of the predicted polypeptide in the cDNAs is identical to the N terminus of the purified avocado P-450. Gel blot analysis of RNA from fruit at various stages of ripening showed the accumulation of an 1800-nucleotide P-450 mRNA that hybridized to the P-450 cDNA. The P-450 protein predicted by the avocado cDNA sequence shares less than 40% positional identity with any known P-450 gene family. We propose therefore that it be placed in a separate family, P450LXXI, and that the corresponding gene from avocado be named cyp71A1.     

Synthesis of a major integral membrane polypeptide of rat liver peroxisomes on free polysomes.

The manner of synthesis and assembly of the peroxisomal membrane proteins is unknown. Understanding these processes is essential to an understanding of the formation of the organelle. We have investigated the biogenesis of the previously identified major 21.7-kDa integral peroxisomal membrane polypeptide [Fujiki, Y., Fowler, S., Shio, H., Hubbard, A. L. & Lazarow, P. B. (1982) J. Cell Biol. 93, 103-110]. This protein was purified to apparent homogeneity and used to elicit a rabbit antiserum. In immunoblotting analysis, antibody bound only to the 22-kDa membrane polypeptide present exclusively in peroxisomal membranes. Total rat liver RNA was translated in a nuclease-treated rabbit reticulocyte cell-free protein-synthesizing system. The in vitro translation product, isolated by means of the antibody and Staphylococcus aureus cells, comigrated with the mature 22-kDa polypeptide in NaDodSO4/PAGE. Analysis of the translation products of RNAs from free and membrane-bound polysomes indicated that the mRNA for the 22-kDa membrane polypeptide is found predominantly in free polysomes. The results imply post-translational insertion of the membrane polypeptide into the peroxisomal membrane without proteolytic processing and suggest that peroxisomes, like mitochondria and chloroplasts, form by fission from preexisting organelles.     

Molecular cloning of cDNA coding for the gamma subunit of Torpedo acetylcholine receptor.

From the electric organ of Torpedo californica, we purified mRNA that, when translated in vitro, produces polypeptides immunoprecipitable by antibodies against purified acetylcholine receptor. A novel cloning system [Okayama, H. & Berg, P. (1982) Mol. Cell. Biol. 2, 161-170] was used to produce a cDNA library from this mRNA. This library contained clones with receptor sequences identified by differential hybridization and hybridization-selection. We describe a clone of 2,030 base pairs with sequences appropriate for the amino-terminal amino acids of the gamma subunit of acetylcholine receptor. This clone contains 82 bases 5' of the codon for the amino-terminal amino acid of the mature protein. A portion of this sequence codes for a methionine followed by a 16-amino acid polypeptide that is contiguous to the amino-terminal amino acid of the mature protein and that has the characteristics of a leader peptide. The cDNA insert hybridizes to a 2,100-base RNA present in electric organ but not in the brain of T. californica.     

In vitro translation and characterization of a unique histidine-rich protein mRNA in the avian malaria parasite Plasmodium lophurae.

The histidine-rich protein (HRP) of the avian malaria parasite Plasmodium lophurae contains 70% histidine. It is found in dense cytoplasmic granules and during the erythrocytic cycle it accumulates to represent 10% of the dry weight of the parasite. In the present work the HRP mRNA was studied by in vitro translation and by the use of a polyhistidine oligonucleotide probe. The HRP mRNA contains 2,000-2,100 nucleotides encoding a protein with an apparent molecular weight of 50,000. In addition a HRP of molecular weight 35,000-40,000 is also produced in vitro, probably as a result of proteolytic cleavage of the molecular weight 50,000 polypeptide which corresponds to in vivo labeled and purified HRP. The HRP represents a much larger proportion of the in vitro products synthesized in the homologous cell-free system compared to the rabbit reticulocyte system, and it reflects more closely the pattern of protein synthesis seen in vivo. In addition, HRP mRNA is more abundant in polysomes isolated from young parasites than in polysomes from mature schizonts. These results indicate that the HRP accumulates as a result of amplified translation of its mRNA at certain stages of its erythrocytic cycle.     

RNase E activity is conferred by a single polypeptide: overexpression, purification, and properties of the ams/rne/hmp1 gene product.

Ribonuclease E, an enzyme that processes pre-5S rRNA from its precursor, is now believed to be the major endoribonuclease participating in mRNA turnover in Escherichia coli. The product of the ams/rne/hmp1 gene, which is required for RNase E activity, was overexpressed, purified to near homogeneity by electroelution from an SDS/polyacrylamide gel, and renatured. The purified polypeptide possesses nucleolytic activity in vitro with a specificity identical to that observed for crude RNase E preparations. In addition, both UV crosslinking and RNA-protein blotting unambiguously showed that the Ams/Rne/Hmp1 polypeptide has a high affinity for RNA. Our results demonstrate that RNase E activity is directly attributable to, and is an inherent property of, an RNA-binding protein, the ams/rne/hmp1 gene product.     

Octopine synthase mRNA isolated from sunflower crown gall callus is homologous to the Ti plasmid of Agrobacterium tumefaciens

We have shown that the structural gene for octopine synthase (a crown gall-specific enzyme) is located in a central portion of the T-DNA that came from the Ti plasmid of agrobacterium tumefaciens and is expressed after it has been transferred to the plant cells. Polyadenylylated RNA was prepared from polysomes isolated from an octopine-producing crown gall callus and purified by selective hybridization to one of five recombinant plasmids. Each such plasmid contained a different fragment of T-DNA of pTi-15955 (octopine-type Ti plasmid). Purified mRNA was translated in vitro in rabbit reticulocyte lysates, and the translation products were immunoprecipitated with antibody against octopine synthase. Total and immunoprecipitated products were characterized by their molecular weights. A polypeptide of M_r 40,000 (the same as authentic octopine synthase) was synthesized in vitro by crown gall mRNA selectively hybridized to three of the five fragments of T-DNA and precipitated with antibody against octopine synthase. This polypeptide was not immunoprecipitated with normal rabbit antibody nor was it synthesized when mRNA from the habituated callus was substituted. A mRNA 1500 bases long was detected when total mRNA was fractionated on an agarose gel, transferred to nitrocellulose, and used for hybridization to three of the five ³²P-labeled T-DNA fragments. This apparent mRNA for octopine synthase hybridized to the same three fragments of T-DNA as the mRNA for the M_r 40,000 polypeptide and was not detected in the habituated callus.     

Differential expression of rat pancreatic islet Beta-cell glucose transporter (GLUT 2), proinsulin and islet amyloid polypeptide genes after prolonged fasting,insulin-induced hypoglycaemia and dexamethasone treatment

The question posed by these studies was whether chronic adaptive changes in glucose-stimulated insulin secretion are accompanied by comparable changes in islet Beta-cell glucose transporter (GLUT 2) gene expression. Control, fasted (3-day), insulin-injected hypoglycaemic (5-day), and dexamethasone-treated (4-day) rats (n = 5 for each condition), were studied. After fasting significant decrements in proinsulin mRNA/microgram RNA (-32%, p < 0.05) and islet amyloid polypeptide mRNA/microgram RNA (-44%, p < 0.05) were observed, while there was no change in GLUT 2 mRNA/microgram RNA (-13%, p > 0.05). After insulin-induced hypoglycaemia, decrements in proinsulin mRNA/microgram RNA (-49%, p < 0.01) and islet amyloid polypeptide mRNA/microgram RNA (-44%, p < 0.01) were also observed, with no change in islet GLUT 2 mRNA/microgram RNA (-18%, p > 0.05). Dexamethasone treatment resulted in a marked stimulatory effect on proinsulin mRNA/microgram RNA (+236%, p < 0.001) and islet amyloid polypeptide mRNA/microgram RNA (+221%, p < 0.01), while again there was no change in islet GLUT 2 mRNA/microgram RNA (+0.3%, p > 0.05). Quantitative immunoblot analysis with a GLUT 2 specific antibody revealed no change in islet GLUT 2 protein with fasting, but a small decrease (-39 +/- 11%) in islet GLUT 2/microgram protein after insulin-induced hypoglycaemia. These results do not support the hypothesis that chronic changes in glucose-stimulated insulin secretion are accompanied by changes in GLUT 2 expression. In contrast to the lack of correlation with GLUT 2, there was a striking correlation between proinsulin and islet amyloid polypeptide mRNAs for all experimental conditions (r = 0.974, p < 0.001).(ABSTRACT TRUNCATED AT 250 WORDS)     

Coding nucleotide sequence of rat NADPH-cytochrome P-450 oxidoreductase cDNA and identification of flavin-binding domains.

The coding nucleotide sequence of the mRNA for NADPH-cytochrome P-450 oxidoreductase (NADPH:ferricytochrome oxidoreductase, EC 1.6.2.4) from rat liver was determined from two overlapping cDNA clones, pOR-7 and pOR-8, which together contain 2401 nucleotides complementary to rat liver oxidoreductase mRNA. The single open reading frame of 2034 nucleotides spanning these cDNAs codes for a 678 amino acid polypeptide with a molecular weight of 76,962. The deduced amino acid composition is in excellent agreement with that determined by direct amino acid analysis of purified rat liver P-450 oxidoreductase, and the amino-terminal region (residues 1-80) largely coincides with the amino-terminal sequence of the oxidoreductase isolated from rabbit liver. Comparison of the amino acid sequence to those of other flavoproteins revealed two separate domains that are likely to be involved in flavin binding: a long segment (residues 77-228) homologous with Desulfovibrio vulgaris flavodoxin, an FMN-containing protein, and a shorter segment (residues 452-477) homologous with the FAD-binding segment of fumarate reductase from Escherichia coli.     

Translation of thyroglobulin 33S messenger RNA as a means of determining thyroglobulin quaternary structure.

Thyroglobulin is a 19S protein of approximately 660,000 daltons and unknown quaternary structure. We have previously shown that a 33S mRNA purified from mammalian thyroids promoted synthesis in the Xenopus oocyte of a peptide immunologically related to thyroglobulin. Chemical identity to the native protein is now presented by means of a tryptic peptide analysis. Moreover, the 33S mRNA is shown to contain all the information required for the synthesis of a complete 19S thyroglobulin molecule. Gel filtration in Sepharose under denaturing conditions indicates that the reduced polypeptide encoded by the 33S mRNA is larger than 210,000 daltons. A model of a dimeric thyroglobulin with about 300,000 dalton subunits is presented.     

Maturation of catalase precursor proceeds to a different extent in glyoxysomes and leaf peroxisomes of pumpkin cotyledons

As an approach to study the mechanism of the microbody transition (glyoxysomes to leaf peroxisomes) in greening pumpkin cotyledons, catalase molecules were purified from the two different types of microbody and their structural properties were compared. The purified glyoxysomal catalase was found to consist of four identical subunits (55 kDa), whereas the leaf peroxisomal catalase contains two different forms of monomeric subunit (55 and 59 kDa). These different catalase species cross-reacted with the rabbit antibody raised against the glyoxysomal enzyme. During gel filtration on an Ultrogel AcA 34 column, the leaf peroxisomal 55-kDa polypeptide eluted slightly faster than the leaf peroxisomal 59-kDa polypeptide. The profile of catalase activities exactly paralleled the elution pattern of the 55-kDa molecules, which indicated that the 59-kDa polypeptide was enzymically inactive. Peptide mapping analysis using Staphylococcus aureus protease V8 showed that the glyoxysomal 55-kDa polypeptide was identical to the leaf peroxisomal 55-kDa species, whereas the leaf peroxisomal 59-kDa polypeptide had a different primary structure from the 55-kDa polypeptide. In an in vitro translation system directed by mRNA isolated from etiolated and green cotyledons, glyoxysomal and leaf peroxisomal catalases were synthesized as the identical 59-kDa polypeptide. From peptide mapping analysis, the in vitro-translated 59-kDa polypeptide was found to have a nearly identical primary structure to that of the leaf peroxisomal 59-kDa species. In vivo pulse-chase labeling experiments using etiolated cotyledons showed the conversion of the 59-kDa polypeptide to the 55-kDa molecular species. The overall results strongly indicate that the 59-kDa polypeptide is a precursor form of catalase in pumpkin cotyledons.     

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.