The mode of incorporation of 6-benzylaminopurine into tobacco callus transfer ribonucleic Acid: a double labeling determination

Cytokinin-dependent tobacco (Nicotiana tabacum) tissue was grown in the presence of N⁶-benzyladenine (6-benzylaminopurine) labeled in the phenyl ring with ³H and in the 8-position of the purine ring with ¹⁴C. The ³H/¹⁴C ratio of N⁶-benzyladenosine recovered from labeled tobacco callus transfer RNA preparations was compared with the corresponding ratio for N⁶-benzyladenine supplied in the medium. By this means, with suitable controls, the incorporation of the synthetic cytokinin, N⁶-benzyladenine, into the transfer RNA of tobacco callus tissue was shown to involve the intact moiety. The observed level of incorporation was up to one N⁶-benzyladenosine molecule per 10⁴ transfer RNA molecules.     

Kinetin incorporated into tobacco callus ribosomal RNA and transfer RNA preparations

Kinetin, N⁶-furfuryladenine, was incorporated into tobacco (nicotiana tabacum L., var. Wis. No. 38) callus RNA isolated from rapidly growing tissue cultured in the presence of N⁶-furfuryladenine-8-¹⁴C or unlabeled kinetin. Approximately 0.7% of the radioactivity in the labeled kinetin added to the medium was recovered as N⁶-furfuryladenosine (fr⁶A) in the rRNA and tRNA preparations from the tobacco callus. The rRNA contained over 90% of these fr⁶ A moieties. The extent of kinetin incorporation was four times greater than that observed for N⁶-benzyladenine. The radiochemical purity of the recovered fr⁶ A was confirmed by three successive chromatographic purifications on Sephadex columns (LH-20 eluted with 35% ethanol, G-10 eluted with 20% ethanol, and LH-20 eluted with water). A cytokinin-active ribonucleoside with elution volumes corresponding to fr⁶ A was isolated from the tobacco callus rRNA preparation. This compound was analyzed by gas-liquid chromatography and rigorously characterized as N⁶-furfuryladenosine by gas-liquid chromatography-mass spectrometry of the trimethylsilyl derivative.     

Specific degradation of a plant leucyl transfer ribonucleic Acid by a factor in the homologous synthetase preparation

Partially purified aminoacyl synthetase preparations from pea roots (Pisum sativum L. var. Alaska) contain a heat-labile factor which can degrade leucyl-tRNA₆^leu to a new species. The singular electrophoretic and chromatographic mobilities, the isoprenoid nucleoside content, and the charging characteristics of the new species (designated leucyl-tRNA_L^leu), suggest that it is a fragment of tRNA₆^leu containing at least that portion of the original molecule extending from the 3′ terminus to the anticodon. Conversion appears to be highly specific since neither bulk tRNA, the other leucine tRNA subspecies, nor tyrosine, phenylalanine, or tryptophan tRNAs are susceptible to degradation during incubation with the synthetase preparation.     

Fingerprinting nonradioactive ribonucleic acid with the aid of polynucleotide phosphorylase

We describe a method for obtaining radioactive fingerprints from nonradioactive ribonucleic acid. Fragments derived by T1 ribonuclease digestion of RNA are dephosphorylated with bacterial alkaline phosphatase. When these fragments are used as primers for the reaction of primer dependent polynucleotide phosphorylase with [α-³²P]GDP in the presence of T1 ribonuclease the 3′-hydroxyl group of each fragment becomes phosphorylated. The degree of phosphorylation is reasonably uniform. The method has been applied to T1 ribonuclease digests of Escherichia coli tRNA^Met_f; the oligonucleotides were further analyzed by spleen phosphodiesterase digestion. In a similar manner fingerprints of pancreatic ribonuclease digests of RNA can be obtained, when [α-³²P]UDP, polynucleotide phosphorylase and pancreatic ribonuclease are used.     

Incorporation of mevalonic Acid into ribosylzeatin in tobacco callus ribonucleic Acid preparations

The incorporation of ¹⁴C-2-mevalonic acid into transfer RNA and ribosomal RNA (high molecular weight RNA) in rapidly growing, cytokinin-dependent tobacco (Nicotiana tabacum var. Wisconsin No. 38) callus cultures has been investigated. Approximately 40% of the label incorporated into transfer RNA was present in a ribonucleoside with chromatographic properties identical to those of cis-ribosylzeatin. The remainder of the label in the transfer RNA appears to be nonspecific incorporation resulting from degradation and metabolism of ¹⁴C-2-mevalonic acid by the tobacco callus tissue. Although the total radioactivity incorporated into ribosomal RNA was roughly the same as in transfer RNA, the specific radioactivity of the transfer RNA was about four times higher than that of the ribosomal RNA, and the ribosomal RNA labeling could be distinguished from the cytokinin labeling observed in transfer RNA. The distributions of the ¹⁴C-2-mevalonic acid label and cytokinin activity in tobacco callus transfer RNA fractionated by benzoylated diethylaminoethylcellulose chromatography indicate that at least two cytokinin-containing transfer RNA species are present in this tissue.     

Cytokinin inhibition of Arabidopsis root growth: An examination of genotype, cytokinin activity, and N6-benzyladenine metabolism

The effects of cytokinins on the in vitro growth of the roots of Arabidopsis thaliana seedlings were examined. Root growth was inhibited in a manner dependent upon the type of cytokinin compound, the cytokinin concentration, the Arabidopsis genotype, and the duration of exposure to cytokinin. For the cytokinins N ⁶-benzyladenine (BA), isopentenyl adenine (iP), or dihydrozeatin (DHZ), the concentration required for 50% root growth inhibition differed for each cytokinin and in each of three Arabidopsis genotypes tested. iP was the most active cytokinin in inhibiting the root growth of the Ler-0 genotype, whereas iP and BA had equal activity when tested with the Col-2 and Columbia genotypes. DHZ had the lowest activity of the three cytokinins tested in all three genotypes. A brief 1-day exposure of seeds to a root-inhibiting concentration of BA increased root growth compared with seedlings grown without BA; exposure to BA for 3–6 days inhibited root growth. BA metabolism was evaluated after 6 h and 1, 3, and 6 days in Columbia seedlings. BA, N ⁶-benzyladenosine (BAR), and N ⁶-benzyladenosine-5-monophosphate (BAMP) decreased with time, whereas N ⁶-benzyladenine-7--d-glucopyranoside (BA-7-G) and N ⁶-benzyladenine-9--d-glucopyranoside (BA-9-G) accumulated in the growing seedlings. Seven aromatic cytokinins were compared at 5 m for their effect on Col-3 root growth. BA, BAR, N ⁶-(m-hydroxybenzylamino)adenine, and N ⁶-(o-hydroxybenzylamino)adenine were highly effective in inhibiting root growth, whereas N ⁶-(p-hydroxybenzylamino)adenine produced only a slight decrease in root growth. BA-7-G and BA-9-G did not affect root growth.Abbreviations BA N ⁶-benzyladenine - iP isopentenyl-adenine - DHZ dihydrozeatin - BAR N ⁶-benzyladenosine - BAMP N ⁶-benzyladenosine 5-monophosphate - BA-7-G N ⁶-benzyladenine-7--d-glucopyranoside - BA-9-G N ⁶-benzyladenine-9--d-glucopyranoside - m-OH BA N ⁶-(m-hydroxybenzylamino)adenine - o-OH BA N ⁶-(o-hydroxybenzylamino)adenine - p-OH BA N ⁶-(p-hyrdoxybenzylamino)adenine - HPLC high performance liquid chromatography - gFW grams fresh weight     

The application of PEI-cellulose thin-layer chromatography for the resolution of large oligonucleotide fragments of transfer ribonucleic acids.

Large oligonucleotide fragments from tRNA were separated on PEI-cellulose tle using stepwise gradients of increased concentrations of LiCl (containing 0.3 m Tris-HCl and 7.5 m urea at pH 7.9) or Li-formate (containing 7.5 m urea at pH 3.5). These large oligonucleotides, obtained by cleavage of tRNA with nuclease S₁, aniline-NaOH, or partial ribonuclease T₁ digestion and separated on PEI-cellulose, were analyzed by three different methods. The first method entailed elution and total base analysis by the tritium-postlabeling technique; the second involved complete ribonuclease T₁ digestion in situ, contact transfer to another PEI-cellulose tle plate, and two-dimensional tle fingerprinting; the third employed complete digestion in situ with ribonuclease T₁ and bacterial alkaline phosphatase, followed by the elution, periodate oxidation, introduction of a tritium into 3′-terminus, and subsequent two-dimensional PEI-cellulose fingerprinting. These techniques can aid in the determination of the complete nucleotide sequence of tRNA when only small quantities of pure tRNAs (less than 10 A₂₆₀ units) are available or when the tRNAs are not amenable to in vivo radioactive labeling.     

The occurrence and distribution of poly(a) ribonucleic Acid in soybean

The occurrence and distribution of poly(A) sequences in the RNA of soybean (Glycine max var. Wayne) have been studied. Only one of the two species of AMP-rich RNA contains poly(A). D-RNA does not contain detectable poly(A) sequences. The TB-RNA is the poly(A) RNA in this system. At least a part (up to 50% or more) of the mRNA in polyribosomes contains a poly(A) sequence. The poly(A) RNA is heterodisperse in size but has a mean size of approximately 18S (2,000 nucleotides) in urea and formamide gels. The poly(A) fragment resulting from ribonuclease A and T₁ digestion migrates as a broad band overlapping the 4 to 5.8S regions of the gels with a mean size of somewhat greater than 5S. No evidence was found for the occurrence of a discrete oligo(A) fragment in the poly(A) RNA; however, oligonucleotides which migrate faster than the poly(A) fraction were observed in preparations which were not bound to oligo(dT) cellulose prior to electrophoresis. This oligonucleotide region was enriched in AMP (up to about 65%) as would be expected after ribonuclease A and T₁ digestion.     

Studies on the fractionation of total transfer ribonucleic acid and purification of an alanine transfer ribonucleic acid from chick embryo

Chick embryo tRNA, prepared by a simple large-scale method, was fractionated on three different ion-exchange columns. In all cases simple chromatographic patterns for various tRNA species were observed, indicating the presence of only a few major species of tRNA for each amino acid. By repeated chromatography one species of alanine tRNA was purified to approx. 80% purity. T₁ ribonuclease digest of this purified tRNA gave a simple chromatographic pattern. Because of the simplicity of the method of preparation of tRNA from this readily available source and the presence of only a few species of tRNA for each amino acid, chick embryo is suited for the study of tRNA and its various functions in higher systems.     

Methylation of TMV RNA

Several plant and animal viral RNAs contain a tRNA like structure at their 3′ ends. In this communication we show that tobacco mosaic virus (TMV) RNA is an acceptable substrate for a specific tRNA methyltransferase. Using a crude preparation of $\text{E. coli}$ ribothymidine (rT) forming uracil methylase and (methyl ³H) S-adenosyl-L-methionine (SAM) as a methyl donor, 0.7 moles of methyl group is incorporated per mole of TMV RNA in 10 hours at 30°C. Upon T₂ RNAse digestion of the labeled RNA, all of the radioactivity was found to be in TMP. T₁ RNAse digestion of ³H methylated TMV RNA showed that all of the label was located in a tetranucleotide which co-migrated with authentic TpψpCpGp, an oligonucleotide characteristically found in normal cellular tRNA.The use of this specific methyl transferase reaction may provide a simple assay for the detection of tRNA like structures in large RNAs.     

Molecular integrity of plant ribosomal ribonucleic Acid

Thermal denaturation of plant ribosomal RNA followed by gel fractionation shows that although a large percentage of molecules contain breaks in the polynucleotide chain, 25S and 18S RNAs do exist as unique molecular species. Values for the rate constant of hydrolysis under routine denaturing conditions are of the order of 10⁻⁷ to 10⁻⁸ sec^{− 1} and these are shown not to be a result of ribonuclease activity. This high rate of hydrolysis and the use of insensitive fractionation procedures may account for the reported absence of a 25S rRNA molecule and its apparent conversion to a molecule similar in size to 18S RNA.     

Endogenous cytokinins in the ribosomal RNA of higher plants

Endogenous cytokinin-active ribonucleosides were isolated from the rRNA and tRNA of pea epicotyls (Pisum sativum L., var Alaska) and of wheat germ (Triticum aestivum). The RNA preparations were analyzed for cytokinins by enzymic hydrolysis, ethyl acetate extraction, and Sephadex LH-20 fractionation in several solvents. Tentative identification of the cytokinins was based on cochromatography with synthetic cytokinin standards in several systems and on activity in the tobacco bioassay. Both the rRNA and tRNA from 10 day old pea epicotyls contained ribosylzeatin, isopentenyladenosine, and 2-methylthioribosylzeatin. The latter compound was the most active fraction in the pea rRNA, but was the least active fraction in the tRNA, where isopentenyladenosine activity was predominant. The 2-methylthioribosylzeatin from pea rRNA was identified by gas chromatography-mass spectrometry. Wheat germ rRNA contained cis and trans ribosylzeatin and 2-methylthioribosylzeatin. The tRNA contained isopentenyladenosine in addition. The specific cytokinin activity (activity per A₂₆₀ unit) of the tRNA was over forty times that of the rRNA. Significant contamination of the rRNA preparations by cytokinin-containing tRNA is considered unlikely on the basis of quantitative differences in the cytokinin content of the rRNA and tRNA preparations, electrophoretic analysis of rRNA purity and cytokinin analysis of fractionated oligonucleotide digests.     

Terminal-sequence studies of high-molecular-weight ribonucleic acid. The reaction of periodate-oxidized ribonucleosides, 5′-ribonucleotides and ribonucleic acid with isoniazid

1. The reaction products of isoniazid with periodate-oxidized ribonucleosides and 5′-ribonucleotides have been characterized as the monohydrazones. 2. The stability, chromatographic and electrophoretic properties of the hydrazones are described. 3. ³H-labelled isoniazid was shown to react with the 5′-linked terminal adenosine and cytidine groups of periodate-oxidized Escherichia coli transfer RNA. One mole of isoniazid reacts with 27×10³g. of the transfer RNA. 4. One mole of ³H-labelled isoniazid reacts with approx. 10⁶g. of rabbit-reticulocyte ribosomal RNA. After fractionation of the RNA into its two components and treating the fractionated material with pancreatic ribonuclease and ribonuclease T₁ evidence is presented for the existence of two 5′-linked terminal sequences in the 30s fraction and only one sequence in the 17s fraction. 5. The application of this method to determining terminal sequences of high-molecular-weight RNA is discussed.     

Enzyme immunoassays of N 6-benzyladenine and N 6-(meta-hydroxybenzyl)adenine cytokinins

Enzyme-linked immunosorbent assays (ELISAs) were developed for determination of N ⁶-benzyladenosine, N ⁶-(meta-hydroxybenzyl)adenosine, and structurally related cytokinins. The use of the ELISAs allowed detection over the range of 0.05–70 pmol for N ⁶-benzyladenine and 0.01–20 pmol for the N ⁶-(meta-hydroxybenzyl)adenine cytokinins. Polyclonal antibodies used in the assays were specific for N ⁶-benzyladenine and N ⁶-(meta-hydroxybenzyl)adenine and their corresponding N ⁹-substituted derivatives. By the use of internal standardization, dilution assays, authentic [2-³H]cytokinin recovery markers, and immunohistograms, the ELISAs have been shown to be applicable for the estimation of N ⁶-benzyladenine and N ⁶-(meta-hydroxybenzyl)adenine-type cytokinins in plant tissues. For the analysis of cytokinins in the tissues of young poplar leaves and Solarium teratoma shoot culture, the extracts were fractionated by high performance liquid chromatography (HPLC) and the fractions analyzed by ELISAs. Immunohistogram ELISA analysis of fractions from different HPLC systems indicated major peaks of immunoreactivity co-chromatographing with the labeled and unlabeled standards of N ⁶-benzyladenine, N ⁶-meta-hydroxybenzyl)adenine, and their N ⁹-glycosides in these tissues.Abbreviations ELISA enzyme-linked immunosorbent assay - FW fresh weight - (mOH)[9R]BAP N ⁶-(meta-hydroxybenzyl)adenosine - HPLC high performance liquid chromatography - TBS Tris-buffered saline - TEAA triethylammonium acetate - [9R]BAP N ⁶-benzyladenosine     

Photoreduction of Sulfite by Spinach Leaf Preparations in the Presence of Grana System

The β-anomer of N⁶-benzyladenosine has been long known as an artificial cytokinin.¹⁾ Recently, however, this compound was isolated from a cytokinin-autotrophic cell culture of anise, Pimpinella anisum L.²⁾ Thus, this compound now constitutes the third member of the naturally-occurring cytokinins possessing an N⁶-benzyladenine structure, together with N⁶-(O-hydroxybenzyl)adenosine isolated from leaves of Poplus robusta³⁾ and N⁶-(o-hy-droxybenzyl)-2-methylthio-9-β-d-glucofuranosyl-adenine isolated from fruits of Zantedeschia aethio-pica.⁴⁾ No information has been available concerning whether or not the a-anomer of N⁶-benzyladenosine possesses cytokinin activity.

We report here the synthesis and cytokinin activity of N⁶-benzyl-α-adenosine (N⁶-benzyl-9-α-d-ribofuranosyl adenine).     

The synthesis and biological properties of 8-azido-N 6-benzyladenine,a potential photoaffinity reagent for cytokinin

A cytokinin photoaffinity reagent, 8-azido-N ⁶-benzyladenine (8N₃BA), was synthesized from 8-bromoadenosine via azide replacement, benzylation at N–1, rearrangement to the N-6-benzyl derivative and acid hydrolysis. The compound thus obtained was found to have full cytokinin activity in the moss and tobacco cell-suspension bioassays. Photolysis of 8N₃BA was accomplished with long and short-wavelength ultraviolet light and produced compounds which had very little or no biological activity in the two bioassays. In-vivo photolysis of 8N₃BA caused loss of the cytokinin activity of this compound in moss protonemata. This result was similar to earlier ones where the biological response of moss protonemata to benzyladenine was reversed following removal of the hormone by a short rinse with water.Abbreviations BA N ⁶-benzyladenine - 8N₃BA 8-azido-N ⁶-benzyladenine - PMR proton magnetic resonance - TLC thin-layer chromatography - UV ultraviolet In partial fulfillment of requirements for the Ph.D. degree at Michigan State University     

Identification of recognition residues for ligation-based detection and quantitation of pseudouridine and N6-methyladenosine

Over 100 chemical types of RNA modifications have been identified in thousands of sites in all three domains of life. Recent data suggest that modifications function synergistically to mediate biological function, and that cells may coordinately modulate modification levels for regulatory purposes. However, this area of RNA biology remains largely unexplored due to the lack of robust, high-throughput methods to quantify the extent of modification at specific sites. Recently, we developed a facile enzymatic ligation-based method for detection and quantitation of methylated 2′-hydroxyl groups within RNA. Here we exploit the principles of molecular recognition and nucleic acid chemistry to establish the experimental parameters for ligation-based detection and quantitation of pseudouridine (Ψ) and N⁶-methyladenosine (m⁶A), two abundant modifications in eukaryotic rRNA/tRNA and mRNA, respectively. Detection of pseudouridylation at several sites in the large subunit rRNA derived from yeast demonstrates the feasibility of the approach for analysis of pseudouridylation in biological RNA samples.     

Isoprenylation of transfer ribonucleic acid

A two-fold difference in the total N⁶-(Δ²-isopentenyl) adenosine content was found between the serine accepting tRNA fractions from adult and embryonic bovine liver. Elution profiles of benzoylated DEAE cellulose showed three peaks of adult tRNA were capable of accepting serine. Using gas-liquid chromatography, each peak had measurable amounts of N⁶-(Δ²-isopentenyl) adenosine. When the same techniques were applied to embryonic bovine tRNA, three peaks accepted serine; however, only one peak contained N⁶-(Δ²-isopentenyl) adenosine. These results can be interpreted to indicate that adult and embryonic tRNA differ in the N⁶-(Δ²-isopentenyl) adenosine content of tRNA^ser.     

Rex1p deficiency leads to accumulation of precursor initiator tRNAMet and polyadenylation of substrate RNAs in Saccharomyces cerevisiae

A synthetic genetic array was used to identify lethal and slow-growth phenotypes produced when a mutation in TRM6, which encodes a tRNA modification enzyme subunit, was combined with the deletion of any non-essential gene in Saccharomyces cerevisiae. We found that deletion of the REX1 gene resulted in a slow-growth phenotype in the trm6-504 strain. Previously, REX1 was shown to be involved in processing the 3′ ends of 5S rRNA and the dimeric tRNA^Arg-tRNA^Asp. In this study, we have discovered a requirement for Rex1p in processing the 3′ end of tRNA_i^Met precursors and show that precursor tRNA_i^Met accumulates in a trm6-504 rex1Δ strain. Loss of Rex1p results in polyadenylation of its substrates, including tRNA_i^Met, suggesting that defects in 3′ end processing can activate the nuclear surveillance pathway. Finally, purified Rex1p displays Mg²⁺-dependent ribonuclease activity in vitro, and the enzyme is inactivated by mutation of two highly conserved amino acids.