Development of RNase activity in embryonic axes of germinating pea seeds

RNase activity in embryonic pea axes increased in parallel withthe rise of RNA synthesis as germination proceeded. The developmentof this enzymatic activity was modified antagonistically byapplication of GA₃ and ABA and inhibited severely by treatmentwith CH. Sedimentation analysis of ³H-adenosine-labeled RNAindicated that the synthesis of all types of RNA species isuniformly stimulated by GA₃ and inhibited by ABA. However, 5-FUtreatments, which severely inhibited the synthesis of rRNA,with a slight effect on that of mRNA, had no appreciable effecton the development of RNase activity in the axes. These resultsindicate that active RNA synthesis during germination is independentof the development of RNase activity and that the de novo synthesisof RNases may be controlled by the synthesis of their specificmRNAs. Among the three types of RNase (RNase I, II and III) detectedin the embryonic axes, RNase III showed a sharp increase inactivity with embryo growth and the activity of this enzymewas mainly associated with the endoplasmic reticulum. (Received June 5, 1978; )     

Effect of abscisic acid and auxin on ribonuclease during ageing of bean endocarp tissue sections

The activity of RNase increases rapidly upon cutting sectionsof bean (Phascolus vulgaris L. var. Kentucky Wonder) endocarp,peaks within 4 to 8 hr and then declines. This rapid developmentof RNase activity is inhibited by cycloheximide. Auxin (naphthaleneaceticacid, NAA) accelerates the rate of decline of RNase. Abscisicacid (ABA) enhances the level of RNase between 4 and 24 hr,associated with a decline in RNA, and this effect of ABA isobscured in the presence of auxin. ¹ This work was supported by National Science Foundation Grant(GB-8316) to J. A. Sacher. ² On leave from Laboratorio di Radiobiochimica ed EcofisiologiaVegetale, C. N. R., Roma, (Italy), with a Fellowship supportedby North Atlantic Threaty Organization. ³ Present address: Instituto di Botanica, Universita di Ban,Bari, 70126, Italy. (Received April 1, 1971; )     

Demonstratioin of de novo synthesis of RNase in Rhoeo leaf sections by deuterium oxide labeling

The increase in RNase activity that occurs in fresh cut Rhoeodiscolor leaf sections was followed in sections aged in wateror deuterium oxide for 24 hr. The increase in RNase activityis probably due to de novo synthesis, since the buoyant densityof the RNase from the deuterium labelled tissue was significantlyhigher (1.10 to 1.88%) than that of the controls. (Received June 29, 1973; )     

Thyroxine in Early Strobilation in Aurelia aurita

Radiochromatographic studies of ¹³¹I-treated Aurelia polypsrevealed synthesis of three compounds tentatively identifiedas monoiodotyrosine (MIT), diiodotyrosine (DIT), and thyroxine(T₄). One compound, MIT, is found within 8 hr after ¹³¹I administration,before the detection of DIT or T₄ which appear within 24 hr.T₄ is not usually detected after 48 hr although MIT and DITwere found up to the segmentation period. None of the compoundswere detected in ephyrae treated with ¹³¹I for 24 hr. Administration of low dosages of the goitrogens, thiourea,,propylthiouracil, and potassium thiocyanate, in conjunctionwith iodide, prevented strobilation induction. Radiochromatographyof jellyfish given the goitrogens and ¹³¹I revealed a reduceduptake of iodide and an impairment of the synthesis of the iodinatedcompounds. Jellyfish use thyroxine directly for strobilatioa inductionas demonstrated by ¹³¹I - labeled T₄ administration. The T₄was detected in the polyps up to the 48-hr period of strobilationduring which time some of the T₄ was excreted into the medium,as was some ¹³¹I. The fact that T₄, synthesis has thus far been found only instrobilating forms of Aurelia suggests that T₄ is involved primarilywith the differentiation of new structures which occurs duringstrobilation.     

RNA synthesis required for DNA replication in Vicia seed embryos

The synthesis of DNA and RNA during germination of Vicia seedswas examined. Incorporation of ³H-thymidine into DNA reacheda maximum at about 32 hr after the beginning of imbibition,and RNA synthesis was shown to precede DNA replication. Sedimentationanalyses of ³H-uridine-labeled RNAs indicated that the embryossynthesize all types of rRNA, heterodisperse RNA and 4–5SRNA before and also during the phase of DNA replication. Actinomycin-treatments at lower concentrations (50 or 100 µg/ml)resulted in the specific inhibition of rRNA synthesis. Suchinhibition did not lead to a large reduction in ³H-thymidineincorporation during the replication phase. However, DNA synthesiswas drastically inhibited by a higher level (200 µg/ml)of actinomycin D. The results strongly suggest the involvementof synthesis of heterodisperse RNA in DNA replication. (Received May 28, 1976; )     

Microheterogeneity of N-glycosylation on a stylar self-incompatibility glycoprotein of Nicotiana alata

Gametophytic self-incompatibility, a mechanism that preventsinbreeding in some families of flowering plants, is mediatedby the products of a single genetic locus, the S-locus. Theproducts of the S-gene in the female sexual tissues of Nicotianaalata are an allelic series of glycoproteins with RNase activity.In this study, we report on the microheterogeneity of N-linkedglycosylation at the four potential N-glycosylation sites ofthe S²-glycoprotein. The S-glycoproteins from N.alata containfrom one to five potential N-glycosylation sites based on theconsensus sequence Asn-Xaa-Ser/Thr. The S²-glycoprotein containsfour potential N-glycosylation sites at Asn²⁷, Asn³⁷, Asn¹³⁸and Asn¹⁵⁰, designated sites I, n, IV and V, respectively. SiteIII is absent from the S₂-glycoprotein. Analysis of glycopeptidesgenerated from the S₂-glycoprotein by trypsin and chymotrypsindigestions revealed the types of glycans and the degree of microheterogeneitypresent at each site. Sites I (Asn²⁷) and IV (Asn¹³⁸) displaymicroheterogeneity, site II (Asn³⁷) contains only a single typeof N-glycan, and site V (Asn¹⁵⁰) is not glycosylated. The microheterogeneityobserved at site I on the S₂-glycoprotein is the same as thatobserved at the only site, site I, on the Srglycoprotein (Woodwardet al., Glycobiology, 2, 241-250, 1992). Since the N-glycosylationconsensus sequence at site I is conserved in all S-glycoproteinsfrom other species of self-incompatible solanaceous plants,glycosylation at this site may be important to their function.No other post-translational modifications (e.g. O-glycosylation,phosphorylation) were detected on the S₂-glycoprotein. fertilization microheterogeneity N-glycans plants RNase     

A multiple mutant of Escherichia coli lacking the exoribonucleases RNase II, RNase D, and RNase BN

A multiple mutant strain of Escherichia coli containing mutations affecting the exoribonucleases, RNase II, RNase D, and RNase BN, and also the endonuclease, RNase I, was constructed by P1-mediated transduction. Extracts of the mutant strain were lacking the aforementioned RNase activities. The multiple mutant displayed normal growth in both rich and minimal media at a variety of temperatures, recovered from starvation essentially as the wild-type parent, and could support the growth of a variety of bacteriophages. In addition, RNA synthesis was normal and no precursor RNA accumulation was observed. The properties of the mutant strain indicate that the three exoribonucleases are not essential for the viability of E. coli. The implications of these findings to our understanding of RNA processing and degradation are discussed.     

Correlation between the Starch Level and the Rate of Starch Synthesis during the Developmental Cycle of Chlorella ellipsoidea

The starch content as well as the rate of photosynthetic starchformation in Chlorella ellipsoidea was studied throughout thecell cycle. The starch level in Chlorella cells rose markedlyduring the growing phase in the light, but it started to decreaseafter about 14 to 16 hr regardless of illumination. The rateof starch synthesis, measured by the level of ¹⁴C-incorporationinto starch, increased rapidly in the growing phase until 10hr, and decreased promptly thereafter, even in the light. From these results, it was concluded that both the cellularlevel of starch and the rate of starch synthesis were a functionnot only of the light regime, but also of the stage of celldevelopment. ³ Present address: Yamada High School, Yamada-machi, Iwate Pref.028-13, Japan. (Received October 12, 1981; Accepted May 12, 1982)     

Increased enzymatic activity of the neutral ribonuclease II-inhibitor system of liver and muscle after the administration of polyinosinylyl-polycytidylyl complex to mice

Administration of the synthetic, double-stranded polynucleotide, (I)_n·(C)_n, to mice in the range from 10–100 μg per g body weight results 24 hr later in increases approaching 2-fold in total RNase II (EC 3.1.4.22) activity levels in liver and skeletal muscle. Free RNase inhibitor levels fall so that free RNase II activities approach 5-fold increases. This response of RNase II may be part of the general defensive response against infectious agents exhibited by mammalian and avian cells and could also account for accompanying effects on inhibition of cellular protein synthesis which are caused by agents such as viruses and (I)_n·(C)_n.     

Inhibition of the import of mitochondrial proteins by RNase

RNase treatment of a cell-free translation system prevents transport of mitochondrial precursor proteins from that system into isolated yeast mitochondria. This inhibition depends on the presence of ribosomes in the reticulocyte lysate; if they are cleared by centrifugation, RNase treatment does not specifically inhibit protein uptake by mitochondria. Since protein import can occur in the absence of polyribosomes, RNase treatment does not degrade a structure essential for this process. Rather, the inhibition may be an effect of degraded ribosomes.     

Effect of cycloheximide and cordycepin on auxin-induced elongation and {beta}-glucan degradation of noncellulosic polysaccharides of Avena coleoptile cell wall

The effect of cycloheximide (10^–5 M) and cordycepin (10^–4M) used as protein and RNA synthesis inhibitors, respectively,on auxin action in noncellulosic ß-glucan degradationof Avena coleoptile cell wall was investigated. Both depressedauxin-induced ßglucan degradation of the cell wallas well as auxin-induced elongation and cell wall loosening,suggesting that the process of ß-glucan degradationof the cell wall is closely associated with cell wall looseningand that auxin enhances the activity of an enzyme for ß-glucandegradation through de novo synthesis of RNA and protein butnot through activation of the enzyme in situ. Kinetic studywith the inhibitors showed that RNA metabolism involved in ß-glucandegradation was stimulated by auxin treatment of only 15 minwhile a longer lag phase (about 1 hr) existed for the synthesisof the enzyme. (Received December 16, 1978; )     

Anatomical and physiological aspects of developmental processes of adventitious root formation in Azukia cuttings

In Azukia stem cuttings, root primordia always appeared in theinterfascicular regions between the endodermis and the interfascicularcambium. Transverse cell divisions were observed as the first eventsin the process of root formation. They began to occur 10 hrafter cuttings had been made and were restricted to the interfascicularregions about 1 mm above the basal cut end of the cutting. Ineach of interfascicular region, 10 to 20 cells divided. Transversedivisions were followed by longitudinal divisions, which beganto occur 18 hr after cuttings had been made. The early process of root primordium formation is distinguishedby the following three phases: the first phase during whichno cell division occurs (0–8 hr), the second phase duringwhich transverse cell divisions occur (8–16 hr) and thethird phase during which longitudinal divisions occur (16–24hr). Cuttings in each phase responded differently to test substances. ¹Supported in part by Grant No. 139011 from the Ministry ofEducation, Japan. ² Present address: Junior College of Toyo University, Hakusan,Bunkyo-ku, Tokyo 112, Japan. (Received October 24, 1977; )     

Swapping the domains of exoribonucleases RNase II and RNase R: conferring upon RNase II the ability to degrade ds RNA

RNase II and RNase R are the two E. coli exoribonucleases that belong to the RNase II super family of enzymes. They degrade RNA hydrolytically in the 3' to 5' direction in a processive and sequence independent manner. However, while RNase R is capable of degrading structured RNAs, the RNase II activity is impaired by dsRNAs. The final end-product of these two enzymes is also different, being 4 nt for RNase II and 2 nt for RNase R. RNase II and RNase R share structural properties, including 60% of amino acid sequence similarity and have a similar modular domain organization: two N-terminal cold shock domains (CSD1 and CSD2), one central RNB catalytic domain, and one C-terminal S1 domain. We have constructed hybrid proteins by swapping the domains between RNase II and RNase R to determine which are the responsible for the differences observed between RNase R and RNase II. The results obtained show that the S1 and RNB domains from RNase R in an RNase II context allow the degradation of double-stranded substrates and the appearance of the 2 nt long end-product. Moreover, the degradation of structured RNAs becomes tail-independent when the RNB domain from RNase R is no longer associated with the RNA binding domains (CSD and S1) of the genuine protein. Finally, we show that the RNase R C-terminal Lysine-rich region is involved in the degradation of double-stranded substrates in an RNase II context, probably by unwinding the substrate before it enters into the catalytic cavity.     

Intraspecific variation in the light/dark modulation of ribulose 1,5-bisphosphate carboxylase-oxygenase activity in soybean

A comparative study was made of the inhibition of ribulose-1,5-bisphosphatecarboxylase-oxygenase (Rubisco) amongst six cultivars of Glycinemax L. Merr., associated with synthesis of 2-carboxyarabinitol1-phosphate (CA1P) during darkness. Significantly lower meanvalues of dark inhibition of Rubisco were observed in soybeancv. Davis than in cvs Bragg, Cobb, Hardee, Gordon, and Kirby.The CA1P synthesis/degradation cycle during dark/light transitionsremained operational in cv. Bragg plants grown at low irradiance(40 µmol photons m–2 s–1). However, CA1P synthesisand degradation rates were slower in the dark (t_0.5 = 240 versus25 min), and light (t_0.5 = 20 versus 3.8 min) respectively,as compared to plants grown at higher irradiance (550 µmolphotons m^–2 s^–1). In addition, the activation stateof Rubisco in low-light-grown plants showed only a small declineafter a transition to darkness. We conclude that (a) cultivar-dependentvariation occurs amongst soybeans with respect to CAlP regulationof Rubisco, and (b) soybeans acclimated to low irradiance maydepend more on CA1P synthesis/degradation to regulate Rubisco,and less on changes in the enzyme activation state. Key words: Activation state, Glycine max, photosynthesis, Rubisco, 2-carboxyarabinitol 1-phosphate     

Yeast mitochondrial RNase P, RNase Z and the RNA degradosome are part of a stable supercomplex

Initial steps in the synthesis of functional tRNAs require 5'- and 3'-processing of precursor tRNAs (pre-tRNAs), which in yeast mitochondria are achieved by two endonucleases, RNase P and RNase Z. In this study, using a combination of detergent-free Blue Native Gel Electrophoresis, proteomics and in vitro testing of pre-tRNA maturation, we reveal the physical association of these plus other mitochondrial activities in a large, stable complex of 136 proteins. It contains a total of seven proteins involved in RNA processing including RNase P and RNase Z, five out of six subunits of the mitochondrial RNA degradosome, components of the fatty acid synthesis pathway, translation, metabolism and protein folding. At the RNA level, there are the small and large rRNA subunits and RNase P RNA. Surprisingly, this complex is absent in an oar1Δ deletion mutant of the type II fatty acid synthesis pathway, supporting a recently published functional link between pre-tRNA processing and the FAS II pathway--apparently by integration into a large complex as we demonstrate here. Finally, the question of mt-RNase P localization within mitochondria was investigated, by GFP-tracing of a known protein subunit (Rpm2p). We find that about equal fractions of RNase P are soluble versus membrane-attached.