Poly(A+)mRNA sequences in free mRNP and polysomes of mouse ascites cells

The poly(A⁺)RNA of the free mRNP of mouse Taper ascites cell contains a very reduced number of different mRNA sequences compared to the polysome poly(A⁺)RNA. By the technique of mRNA:cDNA hybridization we have determined that the free mRNP contains approximately 400 different mRNA sequences while the polysomes contain about 9000 different mRNAs. The free mRNP poly(A⁺)RNA sequences are present in two abundance classes, the abundant free mRNP class containing 15 different mRNA sequences and the less abundant free mRNP class containing 400 different mRNAs. The polysome poly(A⁺)RNA consists of three abundance classes of 25, 500 and 8500 different mRNA sequences.Despite its intracellular location in RNP structures not directly involved in protein synthesis the poly(A⁺)RNA purified from the free RNP of these cells was a very effective template for protein synthesis in cell-free systems. Cell-free translation products of free mRNP and polysome poly(A⁺)RNAs were analyzed by two-dimensional gel electrophoresis. This analysis confirmed the hybridization result that the free mRNP poly(A⁺)RNA contained fewer sequences than polysomal poly(A⁺)RNA. The abundant free RNP-mRNA directed protein products were a subset of the polysome mRNA-directed protein products. The numbers of more abundant products of cell-free protein synthesis directed by the free RNP-mRNA and polysomal mRNA were in general agreement with the hybridization estimates of the number of sequences in the abundant classes of these two mRNA populations.     

Cell-free translation analysis of messenger RNA in echinoderm and amphibian early development

A wheat germ cell-free translation system has been used to analyze populations of abundant messenger RNA from sea urchin eggs and embryos and from amphibian oocytes and ovaries. We show directly that sea urchin eggs and embryos contain translatable mRNA of three general classes: poly(A)⁺ mRNA, poly(A)^? histone mRNA, and poly(A)^? nonhistone mRNA. Additionally, some histone synthesis appears to be promoted by poly(A)⁺ RNA. Sea urchin eggs seem to contain a higher proportion of prevalent poly(A)^? nonhistone mRNAS than do embryos. Some differences in the proteins encoded by poly(A)⁺ and poly(A)^? RNAs are detectable. Many coding sequences in the egg appear to be represented in both poly(A)⁺ and poly(A)^? RNAs, since the translation products of the two RNA classes exhibit many common bands when run on one-dimensional polyacrylamide gels. However, some of this overlap is probably due to fortuitous comigration of nonidentical proteins. Distinct stage-specific changes in the spectra of prevalent translatable mRNAs of all three classes occur, although many mRNAs are detectable throughout early development. Particularly striking is the presence of an egg poly(A)^? mRNA, encoding a 70,000–80,000 molecular weight protein, which is not detected in morula or later-stage embryos. In amphibian (Xenopus laevis and Triturus viridescens) ovary RNA, the translation assay detects the following three mRNA classes: poly(A)⁺ nonhistone mRNA, poly(A)^? histone mRNA, and poly(A)⁺ histone mRNA. Amphibian ovary RNA appearently lacks an abundant poly(A)^? nonhistone mRNA component of the magnitude detectable in sea urchin eggs. mRNA encoding histone-like proteins is found in the very earliest (small stage 1) oocytes of Xenopus as well as in later stage oocytes. During oogenesis there appear to be no striking qualitative changes in the spectra of prevalent translatable mRNAs which are detected by the cell-free translation assay.     

Poly(gamma-glutamylcysteinyl)glycine Synthesis in Datura innoxia and Binding with Cadmium : Role in Cadmium Tolerance

The effects of Cd on poly(γ-glutamylcysteinyl)glycine [(γEC)_nG] biosynthesis and formation of (γEC)_nG:Cd complexes were measured in two cell lines of Datura innoxia with differing Cd tolerance. In addition, RNA synthesis, protein synthesis, and GSH concentrations were measured during a 48 hour exposure to Cd. Exposure to 250 micromolar CdCl₂ was toxic to the sensitive line, whereas the tolerant line survived and grew in its presence. Cd-sensitive cells synthesized the same amount of (γEC)_nG as tolerant cells during an initial 24 hour exposure to 250 micromolar CdCl₂. However, rates of (γEC)_nG:Cd complex formation differed between the two cell lines with the sensitive cells forming complexes later than tolerant cells. In addition, the complexes formed by sensitive cells were of lower molecular weight than those of tolerant cells and did not bind all of the cellular Cd. Pulse-labeling of cells with l-[³⁵S]cysteine resulted in equivalent rates of incorporation into the (γEC)_nG of both cell lines during the initial 24 hours after Cd. Rates of protein and RNA synthesis were similar for both cell lines during the initial 8 hours after Cd but thereafter declined rapidly in sensitive cells. This was reflected by a decline in viability of sensitive cells. The GSH content of both cell lines declined rapidly upon exposure to Cd but was higher in sensitive cells throughout the experiment. These results show that the biosynthetic pathway for (γEC)_nG synthesis in sensitive cells is operational and that relative overproduction of (γEC)_nG is not the mechanism of Cd-tolerance in a Cd-tolerant cell line of D. innoxia. Rapid formation of (γEC)_nG:Cd complexes that bind all of the cellular Cd within 24 hours appears to correlate with tolerance in these cells.     

On the Role of Stored mRNA in Protein Synthesis in Embryonic Axes of Germinating Vigna unguiculata Seeds

Polyadenylated (poly A⁺) RNAs were prepared from both dry and incubated embryonic axes of Vigna unguiculata seeds and were translated by a wheat germ translation system. Analysis with gel electrophoresis and fluorography showed that translation products of poly A⁺ RNA from dry embryonic axes were nearly the same as those from 2-hour incubated axes but somewhat different from those of 4- to 24-hour incubated axes, and that translation products remained almost unchanged between the 4- and 24-hour stages of postimbibition. The results indicate the possibility that the stored mRNA (poly A⁺ RNA from dry embryonic axes) directs the protein synthesis required for early stages of germination. This is supported by comparison of the in vitro translation products of poly A⁺ RNAs with those of polysomal RNAs. Experiments with α-amanitin, a specific inhibitor of RNA polymerase II (J. Jendrisak 1980 J Biol Chem 255: 8529-8533), suggested that the synthesis of some of the stored mRNA species is resumed as early as 4 hours after the onset of imbibition.     

Regulation of Assimilatory Sulfate Reduction by Cadmium in Zea mays L

Plants cultivated with Cd can produce large amounts of phytochelatins. Since these compounds contain much cysteine, these plants should have an increased rate of assimilatory sulfate reduction, the biosynthetic pathway leading to cysteine. To test this prediction, the effect of Cd on growth, sulfate assimilation in vivo and extractable activity of two enzymes of sulfate reduction, ATP-sulfurylase (EC 2.7.7.4) and adenosine 5′-phosphosulfate sulfotransferase were measured in maize (Zea mays L.) seedlings. For comparison, nitrate reductase activity was determined. In 9-day-old cultures, the increase in fresh and dry weight was significantly inhibited by 50 micromolar and more Cd in the roots and by 100 and 200 micromolar in the shoots. Seedlings cultivated with 50 micromolar Cd for 5 days incorporated more label from ³⁵SO₄²⁻ into higher molecular weight compounds than did controls, indicating that the predicted increase in the rate of assimilatory sulfate reduction took place. Consistent with this finding, an increased level of the extractable activity of both ATP-sulfurylase and adenosine 5′-phosphosulfate sulfotransferase was measured in the roots of these plants at 50 micromolar Cd and at higher concentrations. This effect was reversible after removal of Cd from the nutrient solution. In the leaves, a significant positive effect of Cd was detected at 5 micromolar for ATP-sulfurylase and at 5 and 20 micromolar for adenosine 5′-phosphosulfate sulfotransferase. At higher Cd concentrations, both enzyme activities were at levels below the control. Nitrate reductase (EC 1.6.6.1) activity decreased at 50 micromolar or more Cd in the roots and was similarly affected as ATP-sulfurylase activity in the primary leaves.     

Fate of glyoxysomal malate dehydrogenase from watermelon after heterologous in-vivo translation in xenopus oocytes

Total poly A⁺-mRNA from watermelon cotyledons was translated in Xenopus laevis oocytes. Watermelon glyoxysomal malate dehydrogenase was found as its higher molecular weight precursor (pre-gMDH) and accumulates over at least 48 hours of translation. Organelle separation and immunocytochemistry located the watermelon pre-gMDH in the cytosol of the oocyte. The heterologous translation product from oocytes can be imported into isolated glyoxysomes from endosperm of castor bean. This import was correct in terms of protection against proteolysis and cleavage of the presequence within the limits of accuracy. We conclude that watermelon pre-gMDH accumulates in mRNA-injected oocytes as an import competent cytosolic precursor.     

Interactive effects of cadmium and carbon nanotubes on the growth and metal accumulation in a halophyte Spartina alterniflora (Poaceae)

A study quantifying the interactive effects of cadmium (Cd) and carbon nanotubes (CNTs) on plant growth and Cd accumulation of pot-cultured Spartina alterniflora was conducted. The experiment consisted of two Cd levels (50, 200 mg kg^?1) as well as two CNTs levels (800, 2,400 mg kg^?1). As expected, CNTs alleviated higher Cd stress (200 mg kg^?1) due to restored shoot growth reduction, retrieved water content and resumed plant height. Furthermore, CNTs mitigated the deleterious effects of Cd stress through improving K⁺ and Ca²⁺ contents, while reducing Na⁺/K⁺ and Na⁺/Ca²⁺ ratios, regardless of the level of Cd stress. The proline contents in combined Cd and CNTs treatments were lower than Cd alone, suggesting that CNTs could reduce production of organic solutes under Cd stress. The results also showed higher Cd accumulation in roots than shoots, and both were improved by CNTs, except inhibition in roots under higher Cd stress (200 mg kg^?1). It appears that CNTs may not significantly affect negative Cd effects on growth of S. alterniflora, but improve total Cd accumulation under lower Cd stress (50 mg kg^?1). However, under higher Cd stress (200 mg kg^?1), CNTs restored the reduced plant growth, improved and reduced Cd accumulation in shoots and roots, respectively. Therefore, the effects of CNTs on plant growth and Cd accumulation are different, and levels of Cd stress should be considered when evaluating the combined application of CNTs and S. alterniflora on phytoremediation of Cd pollution.     

Rat liver glutathione S-transferase B: the functional mRNAs specific for the Ya Yc subunits are induced differentially by phenobarbital

Liver poly(A⁺)-RNA isolated from untreated and phenobarbital-treated rats has been translated in the rabbit reticulocyte cell-free system in order to examine the kinetics of induction of the translatable mRNAs encoding each subunit of glutathione S-transferase B. Translatable glutathione S-transferase B mRNA levels were maximally elevated at 16 to 24 h after a single injection of phenobarbital. Interestingly, the functional mRNA specific for the low-molecular-weight subunit was elevated markedly by phenobarbital administration whereas the mRNA specific for the high-molecular-weight subunit was only increased slightly. Our data suggest that different mRNAs direct the synthesis of the two subunits of glutathione S-transferase B and that these two mRNAs are under independent regulation.     

Isoenzymes of glucose 6-phosphate dehydrogenase from the plant fraction of soybean nodules

Two isoenzymes of glucose 6-phosphate dehydrogenase (EC 1.1.1.49) have been separated from the plant fraction of soybean (Glycine max L. Merr. cv Williams) nodules by a procedure involving (NH₄)₂SO₄ gradient fractionation, gel chromatography, chromatofocusing, and affinity chromatography. The isoenzymes, which have been termed glucose 6-phosphate dehydrogenases I and II, were specific for NADP⁺ and glucose 6-phosphate and had optimum activity at pH 8.5 and pH 8.1, respectively. Both isoenzymes were labile in the absence of NADP⁺. The apparent molecular weight of glucose 6-phosphate dehydrogenases I and II at pH 8.3 was estimated by gel chromatography to be approximately 110,000 in the absence of NADP⁺ and double this size in the presence of NADP⁺. The apparent molecular weight did not increase when glucose 6-phosphate was added with NADP⁺ at pH 8.3. Both isoenzymes had very similar kinetic properties, displaying positive cooperativity in their interaction with NADP⁺ and negative cooperativity with glucose 6-phosphate. The isoenzymes had half-maximal activity at approximately 10 micromolar NADP⁺ and 70 to 100 micromolar glucose 6-phosphate. NADPH was a potent inhibitor of both of the soybean nodule glucose 6-phosphate dehydrogenases.     

Cell-free synthesis of epoxide hydrase by liver poly (A+)-RNA isolated from phenobarbital treated rats

Total liver RNA has been isolated from normal and 8 day phenobarbital treated rats by guanidine thiocyanate β-mercaptoethanol extraction and fractionated by oligo (dT)-cellulose chromatography to yield poly (A⁺)-RNA. Poly (A⁺)-RNA from normal and phenobarbital treated rats have similar translational activity in the rabbit reticulocyte cell-free system. However major alterations occurred in the polypeptide products directed by these two classes of RNA. The translation products directed by 8 day phenobarbital poly(A⁺)-RNA were immunoprecipitated with rabbit IgG prepared against purified rat liver epoxide hydrase. The immunoprecipitate was subjected to SDS-polyacrylamide gel electrophoresis and the radioactive products detected by fluorography. Analysis of the fluorogram revealed that the major immunoprecipitable product co-electrophoresed with purified epoxide hydrase. These data suggest that the primary translation product of epoxide hydrase messenger RNA has the same molecular weight as the mature form of the enzyme.     

Interconversions of aglycone and host-selective toxin from Helminthosporium sacchari

Helminthosporium sacchari, a fungus that causes disease in sugarcane, produces oligosaccharide-sesquiterpene toxins (HS toxins A, B, and C) that are required for infection and disease development. Two free sesquiterpenes were isolated from mycelium but not from culture fluids of the fungus. One sesquiterpene was identified by HPLC and mass spectrometry as an aglycone of HS toxin C and could be obtained by enzymatic hydrolysis of this toxin. The other sesquiterpene appeared to be the 2-keto form of the first compound. The aglycone from toxin C hydrolysis was labelled with tritium by successive treatments with active manganese dioxide, sodium boro[³H]hydride, and lithium aluminium hydride. The labelled compound was fed to cultures of H. sacchari, radioactivity was incorporated into HS toxin C and into lower molecular weight homologues. The results suggest a metabolic route (aglycone → metabolite Y, → HS toxin → metabolite X) for the biosynthesis of HS toxin; metabolites X and Y are lower molecular weight homologues of the toxin.     

Changes in Cotyledon mRNA during Ethylene Inhibition of Floral Induction in Pharbitis nil Strain Violet

Floral induction in seedlings of Pharbitis nil strain Violet, with one cotyledon removed, was manipulated by applying various ethylene treatments to the remaining cotyledon during a 16 hour inductive dark period. Exposure of cotyledons to ethylene (100 microliters per liter) for 4 hours at different times during the dark period inhibited flowering to some extent, with inhibition being greater towards the end of the dark period. RNA from cotyledons given a 16 hour dark period (induced) or exposed to 100 microliters per liter ethylene throughout the dark period, which completely inhibited flowering, was examined. The poly(A)⁺RNA was translated in vitro using a wheat germ system, and the resulting translation products were analyzed by two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis. There were substantial qualitative and quantitative differences between the poly(A)⁺RNA extracted from induced cotyledons and that from those exposed to ethylene throughout the dark period. Some of these changes are similar to those observed when flowering was inhibited by photoperiodic treatments (M Lay-Yee, RM Sachs, MS Reid 1987 Planta. In press). The significance of these findings to our understanding of the molecular control of flower induction is discussed.     

Casein and alpha-lactalbumin messenger RNAs during the development of mouse mammary gland. Isolation, partial purification, and translation in a cell-free system

Messenger RNAs for the milk proteins, casein and α-lactalbumin, were isolated and partially purified from lactating mouse mammary glands by oligo(dT)cellulose chromatography followed by sucrose density gradient centrifugation. The translation of poly(A)⁺ mRNA in a wheat germ cell-free system yielded three casein polypeptides and a putative precursor form of α-lactalbumin which were precipitated by specific antibodies and characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The casein polypeptides synthesized in vitro had a molecular weight that was no greater than that of the caseins in mouse milk. The presence of individual casein mRNAs coding for these polypeptides was demonstrated by the translation of various fractions of mRNA obtained by sucrose density gradient centrifugation of poly(A)⁺ mRNA. Casein mRNA activity increased about 250-fold between midpregnancy and the 10th–12th days of lactation, amounting to 50–60% of the total mRNA activity in that tissue. A similar study of α-lactalbumin mRNA showed an increase during lactation amounting to 0.2–0.4% of the total mRNA activity, which corresponds to the percentage of α-lactalbumin in total mouse milk protein.     

Kinetic study of protein unfolding and refolding using urea gradient electrophoresis

When total cytoplasmic RNA from mouse Friend cells is fractionated using oligo(dT)-cellulose or poly(U)-Sepharose chromatography, approximately 20% of the messenger RNA activity (as measured in the reticulocyte lysate cell-free system) remains in the unbound fraction, even though this contains < 0.5% of the poly(A) (as measured by titration with poly(U)). This RNA, operationally defined as poly(A)^?, is found almost entirely in polysome structures in vivo. Its major translation products, as shown by one-dimensional sodium dodecyl sulphate-containing gels, are the histones and actin. Two-dimensional gels (isoelectric focusing: sodium dodecyl sulphate/gel electrophoresis) show that, with the exception of the mRNAs coding for histones, poly(A)^? mRNA encodes similar proteins to poly(A)⁺ mRNA, though in very different abundances. This is directly confirmed by the arrest of the translation of the abundant poly(A)^? mRNAs after hybridization with a complementary DNA transcribed from poly(A)⁺ RNA.RNA sequences which are rare in the poly(A)⁺ RNA are also found in poly(A)^? RNA, as shown by hybridizing a cDNA transcribed from poly(A)⁺ RNA to total and poly(A)^? polysomal RNA. That this does not simply represent a flow-through of poly(A)⁺ RNA is indicated by (i) the lack of poly(A) by hybridizing to poly(U) in this fraction, (ii) the fact that further passage through poly(U)-Sepharose does not remove the hybridizing sequences, (iii) the very different quantitative distribution of proteins encoded by poly(A)⁺ and poly(A)^? RNAs. We also think that it does not result from removal of poly(A) from polyadenylated RNAs during extraction because RNAs prepared using the minimum of manipulations give similar results. The distribution of both total mRNA and α and β globin mRNAs between poly(A)⁺ and poly(A)^? RNA does not change significantly during the dimethyl sulphoxide-induced differentiation of Friend cells.     

Identification of barley and wheat cDNA clones related to the high-Mr polypeptides of wheat gluten

We have identified 3 cDNA clones related to the high-M_r group of storage proteins in barley endosperm, the D-hordeins. A cDNA library has been constructed from wheat endosperm poly(A⁺)-RNA and screened using one of the D-hordein cDNA clones. Two wheat clones which cross-hybridised to the barley clone have been identified, by hybrid-release translation and nucleotide sequence analysis, as partial copies of mRNAs encoding the high-M_r gluten polypeptides of wheat.     

Changes in Gene Expression during Tomato Fruit Ripening

Total proteins from pericarp tissue of different chronological ages from normally ripening tomato (Lycopersicon esculentum Mill. cv Rutgers) fruits and from fruits of the isogenic ripening-impaired mutants rin, nor, and Nr were extracted and separated by sodium dodecylsulfate-polyacrylamide gel electrophoresis. Analysis of the stained bands revealed increases in 5 polypeptides (94, 44, 34, 20, and 12 kilodaltons), decreases in 12 polypeptides (106, 98, 88, 76, 64, 52, 48, 45, 36, 28, 25, and 15 kilodaltons), and fluctuations in 5 polypeptides (85, 60, 26, 21, and 16 kilodaltons) as normal ripening proceeded. Several polypeptides present in ripening normal pericarp exhibited very low or undetectable levels in developing mutant pericarp. Total RNAs extracted from various stages of Rutgers pericarp and from 60 to 65 days old rin, nor, and Nr pericarp were fractionated into poly(A)⁺ and poly(A)⁻ RNAs. Peak levels of total RNA, poly(A)⁺ RNA, and poly(A)⁺ RNA as percent of total RNA occurred between the mature green to breaker stages of normal pericarp. In vitro translation of poly(A)⁺ RNAs from normal pericarp in rabbit reticulocyte lysates revealed increases in mRNAs for 9 polypeptides (116, 89, 70, 42, 38, 33, 31, 29, and 26 kilodaltons), decreases in mRNAs for 2 polypeptides (41 and 35 kilodaltons), and fluctuations in mRNAs for 5 polypeptides (156, 53, 39, 30, and 14 kilodaltons) during normal ripening. Analysis of two-dimensional separation of in vitro translated polypeptides from poly(A)⁺ RNAs isolated from different developmental stages revealed even more extensive changes in mRNA populations during ripening. In addition, a polygalacturonase precursor (54 kilodaltons) was immunoprecipitated from breaker, turning, red ripe, and 65 days old Nr in vitro translation products.     

Reduction of turgor induces rapid changes in leaf translatable RNA

The turgor of pea (Pisum sativum) leaves was reduced by exposing excised pea shoots to a stream of 23°C air for 20 min. Poly(A)⁺ RNA was isolated from control and wilted shoots, translated in vitro and radiolabeled translation products separated by electrophoresis on two-dimensional (isoelectric focusing-sodium dodecyl sulfate) polyacrylamide gels. This analysis showed that the levels of several poly(A)⁺ RNAs increased in wilted plants. Most of the poly(A)⁺ RNAs induced in wilted plants did not accumulate in response to heat shock or exogenously applied ABA even though endogenous ABA levels were found to increase in shoots 30 min after wilting and by 4 h had increased 50-fold (1 versus 0.02 microgram per gram fresh weight). A λgt10 cDNA library was constructed using poly(A)⁺ RNA from wilted shoots which had been incubated for 4 hours. Differential screening of the library identified four clones corresponding to poly(A)⁺ RNAs which are induced in wilted shoots.     

Characterization of poly(A+)RNA in free messenger ribonucleoprotein and polysomes of mouse Taper ascites cells

Cytoplasmic extracts of mouse Taper ascites cells were centrifuged on sucrose gradients to give 0–80 S, monosome, and polysome fractions. CsCl equilibrium density centrifugation of formaldehyde-fixed material from the 0–80 S fraction demonstrated that the messenger RNA in the 0–80 S fraction was in the form of free ribonucleoprotein. The size of the poly(A⁺)RNA and the size of the poly(A) segments of these molecules were shown to be very similar in both the free mRNP² and polysome fractions. The labeling kinetics of the free mRNP poly(A⁺)RNA was similar to that of the polysomal poly(A⁺)RNA.The free mRNP poly(A⁺)RNA efficiently stimulated protein synthesis in the wheat germ cell-free system, supporting the view that it was mRNA. Two-dimensional gel electrophoresis was used to analyze the proteins whose synthesis was directed by free mRNP and polysomal poly(A⁺)RNA. The free mRNP poly(A⁺)RNA directed the synthesis of a simpler set of abundant protein products than did the polysomal poly(A⁺)RNA. Most of the free mRNP abundant protein products were also present in the polysomal products, though obvious quantitative differences were evident, indicating that each individual mRNA had its own characteristic distribution between polysomes and the translationally inactive RNP form.