Characterization and measurement of metallothionein messenger RNA of C57BL mouse liver

Liver poly(A)+RNA of Cd2+-treated C57BL mouse was characterized by cell-free translation, particularly intending to establish a procedure to measure the levels of messenger RNA coding for metallothioneins (MT-mRNA). Intact polysomes were obtained by Mg2+ precipitation from the liver cytoplasm of mice injected with 1 mg Cd2+/kg body wt. Poly(A)+RNA isolated from the polysomes was translated by a wheat germ cell-free system and the [35S]cysteine-labeled translation products were analyzed by sodium dodecyl sulfate (SDS)-15% polyacrylamide gel electrophoresis and fluorography. MTs were identified in the translation products directed by the RNA from the Cd2+-treated mice, but not in the translation products directed by the RNA from untreated mice. Relative incorporation of [35S]cysteine into MTs was determined by densitometrical quantification of the MT bands, and was found to be linear up to a RNA concentration of 150 micrograms/ml in the translation reaction mixture, showing that this system is suitable for the measurement of translatable MT-mRNA levels. Cd2+ stimulated the total levels of cell-free translation (1.4-fold at 20-60 micrograms/ml), not specifically to MT-mRNA. MT-mRNA sedimented at 9S in a sucrose gradient, and its size was comparable with rat and human MT-mRNAs.     

Thyroxine increases neonatal mouse submandibular gland mRNA-directed synthesis of epidermal growth factor

Thyroid hormones are known to modulate the concentrations of epidermal growth factor (EGF) in the mouse submandibular gland (SMG); this action is presumably mediated by the nuclear triiodothyronine receptor. To test the hypothesis that thyroid hormones act to increase SMG EGF concentrations by increasing the number of poly(A)+ -specific mRNA, poly(A)+ RNA was isolated from SMGs of neonatal mice which had been treated daily from birth through to 21 days of age with thyroxine (T4,0.4 microgram/g body weight). Poly(A)+ RNA also was extracted from SMGs of intact 21-day-old mice which had received vehicle alone. No significant differences in total nucleic acid, total RNA, or poly(A)+ RNA yields were noted between the two groups of animals. The isolated poly(A)+ RNAs from T4-treated and control mice were translated in an in vitro wheat germ system. Although no significant differences in efficiency of [35S]cysteine incorporation into trichloracetic acid precipitable material were noted between the two poly(A)+ RNA preparations, a significantly greater proportion of radioactivity was immunoprecipitable by anti-EGF antiserum in the translation medium derived from T4-treated mice (17.2 +/- 0.9%, mean +/- SEM) than in that of control mice (7.3 +/- 0.5%, P less than 0.001). Polyacrylamide gel electrophoresis of the immunoprecipitates (IMMP) revealed the presence of three radioactive bands with apparent relative masses (MrS) of 12,000, 9000, and 6000. The latter species comigrated with purified EGF, [125I]EGF, and an IMMP of a SMG extract. The translation product IMMPs following polyacrylamide gel electrophoresis were iodinated and digested with alpha-chymotrypsin.(ABSTRACT TRUNCATED AT 250 WORDS)     

Biochemical changes in progressive muscular dystrophy. XIV. Skeletal muscle myosin mRNA translatability in dystrophic mice

Variations in the content and translatability of the poly(A)+ RNA and mRNA molecules coding for myosin (M) were studied in the hind leg muscles of genetically dystrophic mice. The poly(A)+ RNA content of total skeletal muscle failed to increase normally during progression of the disease. M mRNA, isolated from dystrophic normally during progression of the disease. M mRNA, isolated from dystrophic murine muscle poly(A)+ RNA, was mostly found to be associated with the 26S RNA species. The translation of M mRNA in an in vitro heterologous wheat germ system was lower at 8 and 16 weeks in the dystrophic group as compared with the controls. Analysis of the translation products via sodium dodecyl sulfate-polyacrylamide gel electrophoresis, autoradiography, and densitometric autoradiographic tracing demonstrated the gradual disappearance of a protein band corresponding to M, the major component of skeletal muscle. cDNA was synthesized, using M mRNA that was isolated and purified from normal and dystrophic mouse muscle as a template. Total radioactivity was measured in some cDNA fractions produced from normal and dystrophic mouse muscle, while other fractions were utilized for separation and sizing of cDNA by disc gel electrophoresis. The cDNA from normal muscle was hybridized with M mRNA from normal and 16-week-old dystrophic mouse muscles. The cDNA probe, hybridization experiments, and studies involving the content and synthesis of M mRNA suggest that murine muscular dystrophy elicited a shorter species of mRNA or shorter sequences of the same species of mRNA coding for M. Not all poly(A)+ mRNA sequences coding for M, found in control mice, were present in their dystrophic counterparts.(ABSTRACT TRUNCATED AT 250 WORDS)     

Encephalomyocarditis virus RNA. II. Polyadenylic acid requirement for efficient translation.

Differentially polyadenylated subpopulatons of encephalomyocarditis (EMC) viral RNA were isolated by affinity chromatography on oligodeoxythymidylic acid-cellulose. Translation of these RNA fractions in several in vitro protein-synthesizing systems, isolated from Ehrlich ascites tumor cells, demonstrated that poly(A)+EMC viral RNA was translated two to three times more efficiently than poly(A)-EMC viral RNA. Sodium dodecyl sulfate-polyacrylamide gel electrophoretic analysis of the polypetides synthesized by the in vitro system in response to the different RNAs showed no detectable differences in the size or relative amount- of the translational products. mRNA saturation curves indicated that the in vitro systems were stimulated maximally by equivalent amounts of RNA, wheter it be poly(A)-or poly(A)+ EMC viral RNA. Time course experiments showed that the differences in translatability were more pronounced late in the reaction when reinitiation was required, and that by eliminating reinitiation with high salt the apparent effect of poly(A) on translation was diminished. Together, these results suggest that poly(A) may be required for efficient initiation and reinitiation of protein synthesis in the cell-free systems. This interpretation is discussed relative to earlier data.     

The preparation and characterization of locust vitellogenin messenger RNA and the synthesis of its complementary DNA.

Poly(A)+ (polyadenylated) RNA was isolated from vitellogenic female-locus fat-body by LiCl/urea extraction and poly(U)-Sepharose 4B affinity chromatography. Agarose-gel electrophoresis of this poly(A)+ RNA under denaturing conditions shows the presence of a high-molecular-weight species (greater than 31 S, 7100 nucleotides) as the major species, which is absent from the RNA prepared from male-locust fat-body. Inclusion of this poly(A)+ RNA in a mRNA-dependent reticulocyte-lysate system directs the synthesis of polypeptides that could be immunoprecipitated with monospecific antibodies against locust egg vitellin. DNA complementary (cDNA) to the poly(A)+ RNA was synthesized, and back-hybridization of the cDNA to its template reveals a major abundant species comprising about 45% of the total poly(A)+ RNA hybridizing with R0t 1/2 of 2 x 10(-2) mol . litre-1 . s. Abundant cDNA isolated from the total cDNA hybridizes to poly(A)+ RNA with a R0t 1/2 of 9 x 10(-3) mol . litre-1 . s. There are 9.1 x 10(3) copies of vitellogenin mRNA per cell of vitellogenic female-locust fat-body, comprising 55% of the poly(A)+ RNA and equivalent to 0.7% of total cellular RNA.     

Discrete poly(A)-RNA species from rat liver mitochondria are fragments of 16S mitochondrial rRNA carrying its 5′-termini

During electrophoresis in polyacrylamide gels containing 7M urea the major discrete components of preparations of rat liver mitochondrial poly(A)+ and poly(A)- RNA species have similar mobilities. Poly(A)- RNA components hybridize to the 16S rRNA gene of mtDNA. Analysis of 5'-terminal sequences of these components revealed their identity to the 5'-terminal sequence of 16S rRNA. These results show that poly(A)- RNA components are fragmentation products of 16S rRNA. Fragmentation occurs nonrandomly from the 3'-end of the original rRNA molecules and lead to formation of products with electrophoretic mobilities similar to those of poly(A)+ RNA components.     

Structure and function of rat liver polysome populations. II. Characterization of polyadenylate-containing mRNA associated with subpopulations of membrane-bound particles

Poly(A)+RNA fractions prepared from free and loosely and tightly membrane-bound polysome populations (poly(A)+RNAfree, poly(A)+RNAloose, and poly(A)+RNAtight) were used to drive cDNA in homologous and heterologous hybridization reactions. A large fraction by mass of sequences was shared among the three poly(A)+RNA populations, but shared sequences exhibited distinct frequency distributions within the different populations. 13-15 in vitro translation products of poly(A)+RNAfree and poly(A)+RNAloose detected by gel electrophoresis were shared. Most of these were produced in different relative quantities by the two RNA populations. Five or six higher mol wt polypeptides were produced by poly(A)+RNAloose that were not detected as products of either poly(A)+free or poly(A)+RNAtight. We suggest that loosely bound polysomes may not be artifactually derived as reflected in their quantitatively distinct poly(A)+RNA population. Two tightly membrane-bound RNP fractions were prepared from rat liver on the basis of their release from or retention on purified rough microsomes or a crude membrane fraction after in vitro disaggregation of polysomes with high-salt and puromycin. Homologous and heterologous hybridizations involving their poly(A)+RNA fractions revealed that a large portion by mass of sequences was shared but that these sequences exhibited distinct frequency distributions in the two fractions. The RNA fractions produced exhibited distinct frequency distributions in the two fractions. The RNA fractions produced an identical set of in vitro translation products but individual polypeptides were produced in different relative quantities. This indicates that the two RNP fractions do not arise by any random artifactual process and suggests that they may represent functionally distinct populations.     

In vitro synthesis of full-length influenza virus complementary RNA.

Influenza virus-specific RNA has been synthesized in vitro, using cytoplasmic or microsomal fractions of influenza virus-infected MDCK cells. The RNA polymerase activity was stimulated 5-30 times by priming with ApG. About 20-30% of the product was polyadenylated. Most of the in vitro product was of positive polarity, as shown by hybridization to strand specific probes and by T1 fingerprinting of the poly(A)+ and poly(A)- RNA segments encoding haemagglutinin and nucleoprotein. The size of poly(A)- RNA segments, determined on sequencing gels, was indistinguishable from that of virion RNA, whereas poly(A)+ RNA segments contain poly(A) tails approximately 50 nucleotides long. The size of in vitro synthesized RNA segments was also determined by gel electrophoresis of S1-treated double-stranded RNAs, obtained by hybridization of poly(A)+ or poly(A)- RNA fractions with excess of unlabelled virion RNA. The results of these experiments indicate that poly(A)- RNA contains full-length complementary RNA. This conclusion is further substantiated by the presence of additional oligonucleotides in the T1 fingerprints of in vitro synthesized poly(A)- haemagglutinin or nucleoprotein RNA, selected by hybridization to cloned DNA probes corresponding to the 3' termini of the genes.     

Changes in hepatic RNA, poly(A)+RNA, and poly(A)-RNA during the acute phase response to inflammation

The steady state changes in total rat hepatic cytoplasmic RNA, poly(A)+ RNA and poly(A)-RNA were assessed in response to turpentine induced inflammation. From 18 to 24 h after injury, cytoplasmic RNA doubled, while poly(A)+ RNA peaked at 24 h, 3.5 times over control animals. Cell-free translation showed significant increases in messenger RNA levels beginning at 18 h. Gel electrophoresis of translation products revealed significant increases in several polypeptides and a decrease in others. Poly(A)-RNA from control and injured rats translated to an insignificant level and the electrophoretic gel patterns of their proteins were similar. Furthermore, no change had occurred in the 3' poly(A)-sequences during the course of inflammation.     

Isolation and characterization of trout testis protamine mRNAs lacking poly (A)

Poly(A)+ protamine mRNA was isolated from trout testis cells in a very pure form, and artificial poly(A)- protamine mRNA molecules were derived from it by enzymatic deadenylation with RNAase H from calf thymus after hybridization with oligo(dT). The deadenylated protamine mRNA was found to be active in a wheat germ cell-free system and yielded a labeled product which co-migrated with authentic protamine. These deadenylated mRNA molecules were subsequently used as markers on denaturing polyacrylamide gels to identify and allow the purification of the poly(A)- protamine components known to exist in vivo in the total cellular poly(A)- RNA. RNA species of molecular weights similar to the enzymatically deadenylated subcomponents of protamine mRNA were observed in the natural poly(A)-RNA population of the testis cells. These naturally occurring poly(A)- protamine mRNAs were isolated by preparative gel electrophoresis and further characterized by 3H-poly(U) hybridization assay, by hybridization to complementary DNA made against highly purified poly(A)+ protamine mRNA, and by their ability to direct protamine synthesis in a cell-free system.     

Separation and characterization of low-molecular-weight nuclear RNA species that inhibit cell-free protein synthesis

Various RNA fractions were isolated from nuclei of 12-day lactating rat mammary glands and examined for their ability to inhibit cell-free protein synthesis. Although total nuclear RNA was generally inactive, material contained in the poly(A)+ nuclear RNA fraction and the low-molecular-weight RNA derived from total nuclear RNA by sucrose gradient centrifugation, inhibited the translation of several mRNAs but not poly(U) or poly(A). Separation of the small nuclear RNAs by preparative polyacrylamide-urea gel electrophoresis allowed the identification of at least three active inhibitor RNA species. These differed both with respect to their ability to inhibit protein synthesis, and in their mechanism of action. While two of the RNA species inhibited elongation the other inhibited initiation of protein synthesis.     

Gene activity during germination of spores of the fern, Onoclea sensibilis. Cell-free translation analysis of mRNA of spores and the effect of alpha-amanitin on spore germination

Poly(A)-RNA fractions of dormant, dark-imbibed (non-germinating) and photoinduced (germinating) spores of Onoclea sensibilis were poor templates in the rabbit reticulocyte lysate protein synthesizing system, but the translational efficiency of poly(A)+RNA was considerably higher than that of unfractionated RNA. Poly(A)+RNA isolated from photoinduced spores had a consistently higher translational efficiency than poly(A)+RNA from dark-imbibed spores. Analysis of the translation products by one-dimensional polyacrylamide gel electrophoresis showed no qualitative differences in the mRNA populations of dormant, dark-imbibed, and photoinduced spores. However, poly(A)+RNA from dark-imbibed spores appeared to encode in vitro fewer detectable polypeptides at a reduced intensity than photoinduced spores. A DNA clone encoding the large subunit of maize ribulose bisphosphate carboxylase hybridized at strong to moderate intensity to RNA isolated from dark-imbibed spores, indicating the absence of mRNA degradation. Although alpha-amanitin did not inhibit the germination of spores, the drug prevented the elongation of the rhizoid and protonemal initial with a concomitant effect on the synthesis of poly(A)+RNA. These results are consistent with the view that some form of translational control involving stored mRNA operates during dark-imbibition and photoinduced germination of spores.     

Translational properties of a non-polyadenylated oligo(U)-containing RNA fraction from wheat embryos

A non-polyadenylated oligo(U)-containing RNA (poly(A)- . oligo(U)+ RNA) fraction was isolated from wheat embryo cytoplasm and its properties were compared with those of polyadenylated RNA (poly(A)+ RNA) from the same source. Both RNA preparations were highly heterogeneous and effectively stimulated [14C]leucine incorporation in a wheat germ cell-free translation system. Electrophoretic patterns of the translation products appearing in the non-polyadenylated RNA- and polyadenylated RNA-supplemented translation assays, respectively, differed from each other. The non-polyadenylated RNA-specific translation products included, in particular, a series of high molecular weight polypeptides. It is concluded that a specific class of non-polyadenylated oligo(U)-containing mRNA species (other than histone mRNAs) occurs in the wheat embryo cells.