Phytohormonal regulation of S-adenosylmethionine synthetase by gibberellic acid in wheat aleurones.

Gibberellic acid (GA3) brought about a 3-fold stimulation of AdoMet synthetase activity in wheat aleurones. At the qualitative level, three isozymes of AdoMet synthetase were observed by DE-52 chromatography in GA3-treated wheat aleurones. In contrast, the control wheat aleurones showed a single isozyme. Thus the phytohormone (GA3, 1 microM) induced two additional isozymes of AdoMet synthetase in wheat aleurones. The activity of all the three isozymes in GA3-treated aleurones was considerably decreased by the simultaneous presence of abscisic acid (ABA, 10 microM). Cycloheximide (20 micrograms/ml) also significantly lowered the levels of the three isozymes of AdoMet synthetase in Ga3-treated aleurones, thereby suggesting the requirement of de-novo protein synthesis for the complete induction of isozymes. However, wheat aleurones excised from embryonated wheat seeds, did not require the application of GA3 for the induction of two additional isozymes of AdoMet synthetase. Apparently, the transport of GA3 from the embryo to aleurones induced two new isozymes of AdoMet synthetase. Three isozymes of AdoMet synthetase were also observed in wheat embryos excised from germinated wheat grains, without exogenous application of GA3. The molecular weight of all the three isozymes of AdoMet synthetase in wheat system is 181,000. The molecular weight of the subunit of the enzyme is 84,000. The dimeric nature of AdoMet synthetase was established by SDS-PAGE analysis of the purified enzyme. In-vitro hybridization of two flanking isozymic peaks I and III by NaCl-freeze-thaw method resulted in the appearance of an additional middle activity peak (isozyme II). However, no additional isozymic peaks were generated when isozymic peaks I and III were individually given a freeze-thaw treatment. Thus the flanking isozymic peaks I and III represent homodimers that differed in their net charge. In contrast, the middle isozymic activity peak II, when subjected to NaCl-freeze-thaw treatments yielded two additional isozymic peaks, I and III, thereby suggesting its heterodimeric nature. We envisage that the three isozymes in GA3-treated wheat aleurone layers are formed by the random dimerization of two classes of enzyme subunits. The two enzyme subunits which differ in their net charge could be the product of two genes of AdoMet synthetase (SAM1 and SAM2). Based on this assumption, we propose that a single isozyme I in water imbibed control wheat aleurones is the product of SAM1 gene of AdoMet synthetase. The occurrence of three isozymes in GA3-treated aleurones could be ascribed to the expression of an alternate gene of AdoMet synthetase (SAM2 gene).(ABSTRACT TRUNCATED AT 400 WORDS)     

Isolation of DNA polymerase alpha from germinated wheat embryos

DNA polymerase alpha from germinated wheat embryos was purified by ammonium sulphate fractionation, chromatography on DEAE-Toyopearl, followed by phosphocellulose and heparin Sepharose columns. The specific activity of the purified enzyme was more than 60 000 units/mg. It belongs to the alpha-type according to the large molecular mass, high sensitivity to NEM, aphidicoline, 200 mM KCl, low sensitivity to ethidium bromide and the absence of inhibition by ddTTP. DNA polymerase alpha consists of four subunits as shown by SDS-PAGE and seems to be homogeneous under non-denaturating conditions.Abbreviations ddTTP dideoxythymidine triphosphate - NEM N-ethylmaleimide - PMSF phenylmethylsulfonyl fluoride     

Phytohormonal regulation of S-adenosylmethionine synthetase and S-adenosylmethionine levels in dwarf pea epicotyls.

A significant stimulation (2- to 2.5-fold) of AdoMet synthetase was witnessed in glibberellicd acid (GA3, 1 microM)-treated epicotyls of the dwarf pea (Pisum sativum). This was accompanied by a 2.4-fold increase in the endogenous pool of S-adenosylmethionine. Both abscisic acid (10 microM) and cycloheximide (20 micrograms/ml) inhibited the GA3-mediated enhancement of AdoMet synthetase activity. Three isozymes of AdoMet synthetase were detected in GA3-treated epicotyls, whereas a single activity peak was observed in controls. Thus, GA3 seems to control the induction of two new isozymes of AdoMet synthetase in the dwarf pea. By contrast, the tall pea exhibited three isozymes of AdoMet synthetase even in the absence of GA3 treatment. High concentration of L-methionine (2 mM) mimicked the GA3-elicited induction of two new isozymes of AdoMet synthetase in dwarf pea epicotyls.     

Developmental regulation of the synthesis of proteins encoded by stored mRNA in radish embryos

Major radish (Raphanus sativus L. cv National) proteins synthesized at the beginning of germination have been characterized by their migration in two-dimensional electrophoresis.     

Wheat embryo ribonucleates. XIV. Mass isolation of mRNA from wheat germ and comparison of its translational capacity with that of mRNA from imbibing wheat embryos

Commercially milled wheat germ is shown to be a convenient source material for facile recovery of mass (milligram) quantities of highly purified poly(A)-rich RNA. This poly(A)-rich RNA is efficiently translated in a nuclease-treated extract of rabbit reticulocytes. By sucrose density gradient fractionation of bulk poly(A)-rich RNA from wheat germ, it has been possible to show that there is a direct relationship between the molecular weights of the polypeptide products of cell-free synthesis and the molecular weights of the wheat mRNA molecules which program their synthesis. As assessed by SDS -- polyacrylamide gel electrophoresis, the same array of polypeptides is synthesized when nuclease-treated reticulocyte extract is programmed by poly(A)-rich RNA from either commercially supplied or laboratory-prepared wheat embryos. Significantly, there are gross quantitative if not qualitative differences between the translational capacities of poly(A)-rich RNA from dry and imbibing wheat embryos, and the possible importance of these differences for interpreting a changing pattern of polypeptide synthesis in imbibing wheat embryos is the subject of a brief discussion.     

Assay and regulation of S-adenosylmethionine synthetase in Saccharomyces cerevisiae and Candida utilis.

A simple and sensitive assay for S-adenosylmethionine (SAM) synthetase is described which depends on the quantitative separation of the product, [14CH3]S-adenosylmethionine, from the substrate, L-[14CH3]methionine, on a Bio-Rex 70 column. L-Methionine protects the enzyme during preparation of cell extracts by sonic treatment but causes repression of enzyme activity during growth of Candida utilis. The presence of 5 mM methionine in the growth medium repressed SAM synthetase specific activity threefold compared to the specific acitivity of the enzyme isolated from cells grown in unsupplemented medium. Conversely, the presence of methionine in the growth medium resulted in an 80-fold increase in the intracellular concentration of SAM as compared to the Sam accumulated intracellularly in unsupplemented cultures.     

Translation of hepatic mRNA in extracts from wheat germ embryos

Extracts from wheat embryos have been used to study the incorporation of amino acids into TCA insoluble products using hepatic mRNA fractions. The properties of this system are described and compared to the incorporation obtained with poly U and rabbit globin mRNA. SDS-acrylamide gel analysis showed that the major polypeptide synthesized with globin mRNA co-migrates with rabbit globin (15 500 daltons). Rat liver products were numerous, with molecular weights from less than 10000 to greater than 65000 daltons. The KCl concentration for maximum incorporation into TCA precipitable polypeptides with hepatic mRNA was not the optimum KCl concentration for synthesis of complete products.     

Translation in vitro and regulation of mouse liver S-adenosylmethionine synthetase messenger RNA

Total RNA was isolated from adult mouse liver tissues. The alpha- and beta-form isozymes of S-adenosylmethionine synthetase existing in liver were synthesized in a reticulocyte lysate cell-free system under the direction of total RNA and were immunoprecipitated with antibody to the beta-form. The newly synthesized and the in vivo labeled S-adenosylmethionine synthetase subunits were compared by SDS-polyacrylamide gel electrophoresis. Both the alpha- and beta-forms consist of the same size Mr 48 000 subunit. The level of the beta-form mRNA activity in mouse liver was shown to increase following intraperitoneal transplantation of Ehrlich ascites tumor cells and the changes in the mRNA activity parallel those in the cellular level of S-adenosylmethionine synthetase beta.     

Post-transcriptional regulation of protein synthesis in Ilyanassa embryos and isolated polar lobes

The experimental removal of the polar lobe, an anucleate cytoplasmic protrusion formed in preparation for the first cleavage, from the egg of Ilyanassa obsoleta results in grossly abnormal embryonic development. In experiments reported here normal and delobed embryos, as well as isolated polar lobes, were incubated with [³⁵S]methionine for 4 hr beginning at the completion of the first cleavage or 21 hr later during epiboly. Proteins were extracted and examined by fluorography after resolution by two-dimensional polyacrylamide gel electrophoresis. In normal embryos the synthesis of several proteins begins or ends between the two stages investigated. In isolated polar lobes a subset of these developmental changes in protein synthesis occurs, indicating that the regulation of these events is independent of concomitant nuclear activity and probably involves selective regulation of the translation of mRNA stored in the eggs. The patterns of protein synthesis in normal embryos and delobed embryos are qualitatively extremely similar, though quantitative differences are also observed. No proteins can be detected which are synthesized exclusively in polar lobes.     

Post-transcriptional regulation of RNase-L expression is mediated by the 3'-untranslated region of its mRNA

RNase-L mediates critical cellular functions including antiviral, pro-apoptotic, and tumor suppressive activities; accordingly, its expression must be tightly regulated. Little is known about the control of RNASEL expression; therefore, we examined the potential regulatory role of a conserved 3'-untranslated region (3'-UTR) in its mRNA. The 3'-UTR mediated a potent decrease in the stability of RNase-L mRNA, and of a chimeric beta-globin-3'-UTR reporter mRNA. AU-rich elements (AREs) are cis-acting regulatory regions that modulate mRNA stability. Eight AREs were identified in the RNase-L 3'-UTR, and deletion analysis identified positive and negative regulatory regions associated with distinct AREs. In particular, AREs 7 and 8 served a strong positive regulatory function. HuR is an ARE-binding protein that stabilizes ARE-containing mRNAs, and a predicted HuR binding site was identified in the region comprising AREs 7 and 8. Co-transfection of HuR and RNase-L enhanced RNase-L expression and mRNA stability in a manner that was dependent on this 3'-UTR region. Immunoprecipitation demonstrated that RNase-L mRNA associates with a HuR containing complex in intact cells. Activation of endogenous HuR by cell stress, or during myoblast differentiation, increased RNase-L expression, suggesting that RNase-L mRNA is a physiologic target for HuR. HuR-dependent regulation of RNase-L enhanced its antiviral activity demonstrating the functional significance of this regulation. These findings identify a novel mechanism of RNase-L regulation mediated by its 3'-UTR.     

Primary structure of alanyl-tRNA synthetase and the regulation of its mRNA levels in Bombyx mori.

cDNA clones encoding Bombyx mori alanyl-tRNA synthetase were isolated from a library in lambda gt11 using antibody, synthetic oligonucleotides, and a characterized cDNA as probes. Analysis of the sequence revealed significant homology between the B. mori and Escherichia coli alanyl-tRNA synthetases, particularly in their amino-terminal domains. Northern blot analysis indicated that the mRNA for alanyl-tRNA synthetase is 3.8 kilobase pairs in mRNA isolated from posterior silk gland, middle silk gland, and ovarian tissue. Steady-state levels of alanyl-tRNA synthetase mRNA in the posterior silk gland increased in the first 48 h of the fifth larval instar, decreasing gradually thereafter. In the middle silk gland, alanyl-tRNA synthetase mRNA peaked at 72 h of the fifth larval instar, declining to undetectable levels by 120 h. Genomic Southern blot analysis using a nick-translated cDNA probe revealed hybridization to single fragments when B. mori genomic DNA was digested with various restriction endonucleases.     

Temporal, spatial and hormonal regulation of the S-adenosylmethionine synthetase gene in petunia

Gibberellic acid (GA₃) promotes corolla elongation and pigmentation in petunia flowers. We have previously shown that G.A₃ induces pigmentation by activating specific genes of the anthocyanin biosynthetic pathway. The aim of the present work was to examine whether GA₃ induces also the expression of genes from other metabolic pathways in petunia corollas that may be associated with growth. Recently we reported the cloning of the petunia sam gene coding for S -adenosylmethionine synthetase (SAM-S). In the present work we show that sam expression is induced by GA₃ in both corollas and stems. The expression of the gene was correlated with corolla elongation. GA₃ and the cylokinin, N -6-benzyladenine (BA) promoted corolla growth and sam expression, whereas abscisic acid (ABA) inhibited corolla elongation and repressed sam mRNA accumulation. An analysis of sam expression in stems indicated a high level in young, elongating internodes and a very low level in the mature, non-elongating stem zone. The results of the present study show that the effect of GA₃ on gene expression in the corolla of petunia, is not restricted to the anthocyanin biosynthetic pathway, they also suggest a possible role for sam in GA₃-induced corolla and stem elongation.