Xenopus fibrinogen: characterization of the mRNAs for the three subunits.

We purified and characterized the mRNAs coding for each of the three subunits of Xenopus fibrinogen. Purification was accomplished by electrophoretic separation of liver polyadenylated RNA in a fully denaturing gel, followed by recovery of the RNA from the gel via transfer to an ion-exchange membrane. This procedure yielded fractions which were highly enriched for the mRNAs for each of the fibrinogen chains. The fibrinogen mRNAs were identified by two methods: (i) in vitro translation followed by subunit-specific cleavage with the proteases thrombin and batroxobin; and (ii) cross-hybridization with cDNA clones for individual subunits of rat fibrinogen. The results demonstrate that the A alpha and gamma chains of frog fibrinogen are each coded by a single mRNA species. The A alpha mRNA is ca. 3,100 nucleotides in length, which is nearly twice the minimum size required to code for the A alpha precursor polypeptide. The gamma chain mRNA comprises about 1,600 bases and includes only a small untranslated region. In contrast, the B beta subunit is synthesized from two mRNAs, one of which is 2,500 and the other 1,800 nucleotides long. The 2,500-base mRNA includes a large noncoding region, whereas the smaller one is near the minimum required size. The larger B beta mRNA is ca, fivefold more abundant that the smaller species.     

Molecular cloning of cDNAs for the nerve-cell specific phosphoprotein, synapsin I.

To provide access to synapsin I-specific DNA sequences, we have constructed cDNA clones complementary to synapsin I mRNA isolated from rat brain. Synapsin I mRNA was specifically enriched by immunoadsorption of polysomes prepared from the brains of 10-14 day old rats. Employing this enriched mRNA, a cDNA library was constructed in pBR322 and screened by differential colony hybridization with single-stranded cDNA probes made from synapsin I mRNA and total polysomal poly(A)+ RNA. This screening procedure proved to be highly selective. Five independent recombinant plasmids which exhibited distinctly stronger hybridization with the synapsin I probe were characterized further by restriction mapping. All of the cDNA inserts gave restriction enzyme digestion patterns which could be aligned. In addition, some of the cDNA inserts were shown to contain poly(dA) sequences. Final identification of synapsin I cDNA clones relied on the ability of the cDNA inserts to hybridize specifically to synapsin I mRNA. Several plasmids were tested by positive hybridization selection. They specifically selected synapsin I mRNA which was identified by in vitro translation and immunoprecipitation of the translation products. The established cDNA clones were used for a blot-hybridization analysis of synapsin I mRNA. A fragment (1600 bases) from the longest cDNA clone hybridized with two discrete RNA species 5800 and 4500 bases long, in polyadenylated RNA from rat brain and PC12 cells. No hybridization was detected to RNA from rat liver, skeletal muscle or cardiac muscle.     

Effect of amines on fibrinogen synthesis

L-Epinephrine, serotonin, and isoproterenol stimulate the incorporation of [14C]leucine into thrombin-induced clottable protein; this stimulation was abolished by actinomycin D. The incorporation of 32P into total RNA of rat liver, the site of fibrinogen synthesis, was stimulated by epinephrine and was highest at 2 h after 32P administration. [14C]Orotic acid incorporation into polysomal RNA of liver was also increased significantly by epinephrine and serotonin. The immunoprecipitation of newly synthesized protein by monospecific antibody raised against pure rat fibrinogen clearly demonstrates that L-epinephrine increased fibrinogen formation in vivo under the experimental condition. Translation of poly (A)-containing RNA from total polysomal RNA clearly indicates that L-epinephrine increased mRNA specific for fibrinogen.     

Molecular cloning of cDNA for rat acyl-CoA oxidase

Poly(A+) RNA was prepared from hepatic free polysomes of rats which had been fed di(2-ethylhexyl) phthalate for the induction of peroxisomal beta-oxidation enzymes. This preparation was enriched for the mRNAs of these enzymes by sucrose density gradient centrifugation, and used for the synthesis of double-stranded cDNA. Recombinant plasmids were constructed from the cDNA and pBR322 by dG X dC-tailing method and used for the transformation of an Escherichia coli strain, chi 1776. By differential colony hybridization using [32P]cDNA of partially purified liver poly(A+) RNA from induced and noninduced rats as probes, and then by hybridization-selected translation, we obtained two clones with cDNA inserts which specifically selected acyl-CoA oxidase mRNA. On Northern blotting, both cDNA inserts hybridized to 3.8-kilobase RNA which was increased about 10-fold by di(2-ethylhexyl) phthalate treatment of the rats. The cleavage maps of the cDNA inserts showed they overlap with each other. We conclude that the above two recombinant plasmid clones contain cDNA sequences for rat acyl-CoA oxidase.     

Molecular cloning of cDNA for rat mitochondrial 3-oxoacyl-CoA thiolase

Messenger RNA of rat 3-oxoacyl-CoA thiolase (acetyl-CoA acyltransferase), a mitochondrial matrix enzyme involved in fatty acid beta-oxidation, was enriched by immunoprecipitation of rat liver free polysomes and recombinant plasmids were prepared from the enriched mRNA by a modification of the vector-primer method of Okayama and Berg. The transformants were initially screened for 3-oxoacyl-CoA thiolase cDNA sequences by differential colony hybridization with [32P]cDNAs, synthesized from the immunopurified and unpurified mRNAs. The cDNA clones for 3-oxoacyl-CoA thiolase were identified by hybrid-arrested translation and hybrid-selected translation. One of the clones, designated pT1-1, contained a 700-base insert and hybridized to a mRNA species of 1.6 X 10(3) bases in rat liver. The transformants were rescreened using the cDNA insert of pT1-1 as a hybridization probe and a clone (pT1-19) with a 1.5 X 10(3)-base insert was obtained. Activity and concentration of 3-oxoacyl-CoA thiolase mRNA were quantified by in vitro translation and dot-blot analysis using the cDNA insert as a hybridization probe. The level of translatable and hybridizable mRNA in rat liver was increased about 5.1-fold and 4.6-fold, respectively, after administration of di-(2-ethylhexyl)phthalate, a potent inducer of the enzyme. The 3-oxoacyl-CoA thiolase mRNA levels thus determined correlated closely with levels of the activity and amount of this enzyme.     

cDNA cloning of mRNAs induced in resistant barley during infection by Erysiphe graminis f.sp. Hordei

Summary Near-isogenic cultivars of Hordeum vulgare which differ for the Mlp gene for resistance to Erysiphe graminis f.sp. hordei were inoculated with race 3 of this pathogen and in vitro translation products of mRNA populations compared by 2-dimensional gel electrophoresis and fluorography. This revealed the presence of new mRNA species in infected leaves compared to non-inoculated controls. These new mRNA species were more abundant in resistant leaves than susceptible leaves. A cDNA library was prepared from poly(A)⁺RNA isolated from infected leaves carrying the Mlp gene for resistance (cvMlp). The library was screened by differential hybridization using [³²P]-labelled cDNA prepared from poly(A)⁺RNA of both control and infected leaves. Six cDNA clones showing greater hybridization to cDNA prepared from infected leaves were selected. These six cDNA clones hybridized to DNA isolated from barley leaves but not to DNA from conidia of the fungus. In Northern blot analysis of RNA from infected leaves the six cDNA clones each hybridized to mRNA species of different size. Translation products for three of the cDNA clones corresponded to infection-related translation products identified on 2-dimensional fluorograms. The cDNA clones were used to study the kinetics of host mRNA induction during infection of the near-isogenic cultivars of barley. The host mRNA species corresponding to the cDNA clones were induced prior to 24 h after inoculation during the primary penetration processes. In addition the mRNAs corresponding to four of the cDNA clones increased to greater amounts in cvMlp than in the near-isogenic susceptible cultivar (cvmlp) over a 2-d period following inoculation. These results suggest that the Mlp gene has a regulatory role in host gene expression resulting in enhanced expression of several host mRNA species following infection by the powdery mildew fungus.     

Isolation and characterization of a DNA sequence complementary to rat liver glutathione S-transferase B mRNA

Total rat liver poly(A+)-RNA has been isolated from phenobarbital-treated rats and fractionated on sucrose gradients to enrich for glutathione S-transferase B mRNA. Poly(A+)-RNA fractions were assayed for glutathione S-transferase B mRNA activity by in vitro translation and those fractions enriched in glutathione S-transferase B mRNA were used as a template for cDNA synthesis. The cDNA was cloned into the PstI site of pBR322 by G-C tailing. Bacterial clones harboring inserts complementary to glutathione S-transferase mRNA were identified by colony hybridization using a [32P]cDNA probe reverse transcribed from poly(A+)-RNA enriched significantly in glutathione S-transferase B mRNA and by hybrid-select translation. Two recombinant clones, pGTB6 and pGTB15 hybrid-selected the mRNAs specific for the Ya and Yc subunits, indicating these two mRNAs share significant sequence homology. Radiolabeled pGTB6 was utilized in RNA gel-blot experiments to determine that the size of glutathione S-transferase B mRNA is 980 nucleotides and the degree of induction of the mRNA in response to 3-methylcholanthrene administration is threefold.     

Molecular cloning and characterization of cDNA for androgen-repressible rat liver protein, SMP-2

SMP-2 is a rat liver protein whose synthesis is influenced by both androgens and aging. The steady-state level of its mRNA is repressed by the androgen. Compared to the adult male, SMP-2 mRNA is found in higher amounts in the prepubertal and senescent male rat livers which show relative androgen insensitivity. A cDNA library in the plasmid pBR322 was constructed from the female rat liver which contains a high level of SMP-2 mRNAs. Recombinant plasmids were screened by differential colony hybridization to 32P-labeled single-stranded cDNAs from adult female and adult male hepatic poly(A)+ RNAs. From a total of 3500 recombinant clones, 11 highly female specific clones were identified. From these female specific colonies the SMP-2 cDNA-containing plasmid (pSP11) was identified by its ability to select an mRNA species whose translation product is immunochemically and electrophoretically indistinguishable from SMP-2. This insert represents a 571-base pair portion of the SMP-2 cDNA. Rescreening of the library at a high colony density using the 32P-labeled cDNA insert of pSP11 identified several positive clones with larger inserts. Hybrid-selected mRNA translation again confirmed these clones to carry SMP-2 cDNA sequences. The plasmid pSP4a containing a 1040-base pair cDNA insert of SMP-2 was characterized by DNA sequence analysis. The size of the cDNA insert of pSP4a is close to the estimated size of the SMP-2 mRNA. The cDNA sequence provides an open reading frame of 282 amino acid residues. A comparison of the translated amino acid sequence with the protein sequences of NBRF-PIR, PSQNEW, and LOSALA data bases did not establish any sequence homology with known proteins. Northern blot analysis using the 32P-labeled cDNA insert of pSP4a confirms the androgenic repression of the SMP-2 mRNA.     

Cloning of DNA complementary to cytochrome P-450 induced by pregnenolone-16 alpha-carbonitrile. Characterization of its mRNA, gene, and induction response

The major form of cytochrome P-450 (P-450PCN) was isolated from rats administered pregnenolone-16 alpha-carbonitrile (PCN). Messenger RNA coding for P-450PCN was enriched by polysome immunoadsorption and utilized to construct a library of cDNA clones in pBR322. P-450PCN clones were isolated from this library by differential colony hybridization using [32P]cDNA probes transcribed from PCN-induced and PCN-induced P-450PCN immunoenriched poly(A) RNA. The P-450PCN clone with the largest cDNA insert (pP450PCN-10) was verified to contain sequences complementary to P-450PCN mRNA by hybrid selection-translation. pP450PCN-10 was composed of approximately 1900 base pairs and had a restriction map that overlapped at least 3 other cDNA clones selected by differential colony hybridization. Denaturing-agarose gel electrophoresis and nitrocellulose blot-hybridization using nick-translated 32P-labeled pP450PCN-10 indicated that pP450PCN mRNA is 2500 +/- 150 nucleotides in length; pP450PCN-10, therefore, represents approximately 76% of its corresponding mRNA sequence. Southern blot analysis of rat DNA using pP450PCN revealed that approximately 50 to 60 kilobases of DNA reacted with the PCN probe, suggesting the P-450PCN gene is either a very large gene or other genomic segments exist that react with the probe, such as pseudogenes or related P-450 genes that share homology. The mechanism of P-450PCN induction was examined by isolating poly(A) RNA at various times after steroid administration and quantitating for P-450PCN mRNA using pP450PCN-10 as a hybridization probe. PCN administration produced a rapid elevation of P-450PCN mRNA which reached maximal levels (7-fold above control) 12 h after administration. In contrast, cytochrome P-450b mRNA, which is readily induced by phenobarbital, was only slightly elevated (approximately 2-fold) after PCN administration.     

Tissue distribution of rat glutathione transferase subunit 7, a hepatoma marker.

Polyadenylated RNA isolated from NN-dimethyl-4-aminoazobenzene-induced rat hepatoma was used to prepare a cDNA library in lambda gt10. Full-length clones complementary to mRNA coding for glutathione transferase subunit 7 were isolated and one of these clones (pGSTr7) was fully characterized. In Northern blot analysis, mRNA hybridizing to 32P-labelled pGSTr7 was found in poly(A)-containing RNA isolated from seven normal rat tissues but not from testis and liver. A similar hybridizing mRNA species was also detected in human placental mRNA. The same probe, used in a Southern blot analysis of genomic DNA, suggests the presence of a multigene family in the rat.     

Analysis of fibrinogen genes in patients with congenital afibrinogenemia

Several cDNA clones coding for A alpha, B beta and gamma chains of fibrinogen have been isolated from a human liver cDNA library. They were selected by differential hybridization with probes raised against fractionated liver mRNA (positive probes) and muscle and albumin mRNA (negative probes), then firmly identified by positive hybridization selection. Three of these clones, encoding A alpha, B beta and gamma fibrinogen chain sequences, were further characterized by restriction mapping and used as probes to characterize fibrinogen mRNAs from adult and fetal liver and fibrinogen genes in normal individuals and two afibrinogenemic patients. The results indicate that there is a single copy of the fibrinogen genes which are present and grossly intact in afibrinogenemic DNA.     

Regulation of specific rat liver messenger ribonucleic acids by triiodothyronine

A plasmid cDNA library was constructed using poly(A+) RNA isolated from the livers of rats treated with 3,5,3'-triiodothyronine (T3) and fed a high carbohydrate diet. This library was screened by differential colony hybridization with [32P]cDNA probes made from hypothyroid and hyperthyroid rat liver poly(A+) RNA to obtain clones representing T3-inducible mRNAs. Using plasmid cDNAs to 4 different T3-inducible mRNAs, we have studied by hybridization assay the responses of these mRNAs to different thyroidal steady states and to a high carbohydrate diet. The fold of induction (hypothyroid to hyperthyroid) varied from about 4.0 (mRNA 5-8D) to 13.2 (mRNA 4-12B). The linearity of response with regard to nuclear receptor occupancy was estimated by assessing the relative mRNA levels in a euthyroid state. Three of the mRNAs demonstrated nonlinear responses with the largest portion of the induction occurring in the euthyroid to hyperthyroid transition. An induction by the high carbohydrate diet was clearly seen for only one mRNA (5-8D) suggesting that these two pathways of induction are independent. In a study of the response kinetics of each mRNA to a nuclear receptor saturating dose of T3 in hypothyroid animals, an increase was seen within 4 h (the earliest time point examined) for one of the mRNAs. The other 3 mRNAs did not increase significantly until 8 h after the T3 dose. Northern analysis showed a single mRNA corresponding to each of these 4 clones with sizes ranging from about 1375 to 7600 bases. Two mRNAs (5-9E and 4-12B) were shown by hybrid-selected translation to code for proteins of molecular mass of about 27 and 46 kDa, respectively. The availability of several different cDNA probes to T3 responsive liver mRNAs should facilitate future studies on the mechanism of action of this hormone.     

Molecular cloning of DNA sequences complementary to rat liver glucose-6-phosphate dehydrogenase mRNA. Nutritional regulation of mRNA levels

The nutritional regulation of rat liver glucose-6-phosphate dehydrogenase was studied using a cloned DNA complementary to glucose-6-phosphate dehydrogenase mRNA. The recombinant cDNA clones were isolated from a double-stranded cDNA library constructed from poly(A+) RNA immunoenriched for glucose-6-phosphate dehydrogenase mRNA. Immunoenrichment was accomplished by adsorption of polysomes with antibodies directed against glucose-6-phosphate dehydrogenase in conjunction with protein A-Sepharose and oligo(dT)-cellulose chromatography. Poly(A+) RNA encoding glucose-6-phosphate dehydrogenase was enriched approximately 20,000-fold using these procedures. Double-stranded cDNA was synthesized from the immunoenriched poly(A+) RNA and inserted into pBR322 using poly(dC)-poly(dG) tailing. Escherichia coli MC1061 was transformed, and colonies were screened for glucose-6-phosphate dehydrogenase cDNA sequences by differential colony hybridization. Plasmid DNA was purified from clones which gave positive signals, and the identity of the glucose-6-phosphate dehydrogenase clones was verified by hybrid-selected translation. A collection of glucose-6-phosphate dehydrogenase cDNA plasmids with overlapping restriction maps was obtained. Northern blot analysis of rat liver poly(A+) RNA using nick-translated, 32P-labeled cDNA inserts revealed that the glucose-6-phosphate dehydrogenase mRNA is 2.3 kilobases in length. RNA blot analysis showed that refeeding fasted rats a high carbohydrate diet results in a 13-fold increase in the amount of hybridizable hepatic glucose-6-phosphate dehydrogenase mRNA which parallels the increase in enzyme activity. These results suggest that the nutritional regulation of hepatic glucose-6-phosphate dehydrogenase occurs at a pretranslational level.     

Cloning of mRNA nucleotide sequences amplified during DNA replication in the regenerating liver

A library of double-stranded cDNA has been constructed using the mRNA of regenerating rat liver 20 hr after partial hepatectomy. The differential screening of the library with the regenerating liver specific and the resting-liver-specific single-stranded cDNA probes has identified 11 cDNA clones which sequences are preferentially expressed in regenerating rat liver. The RNA dot blot hybridization has shown that levels of RNA complementary to these clones are 3 to 8-fold higher in dividing cells as compared with resting cells.