Dihydroxyboryl-substituted methacrylic polymer for the column chromatographic separation of mononucleotides, oligonucleotides, and transfer ribonucleic acid

Schott, Herbert; Rudloff, Eberhard; Schmidt, Paul G.; Roychoudhury, Ranajit; H, Koessel

doi:10.1021/bi00729a022

Cited by 119 publications

(48 citation statements)

References 5 publications

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“…The boronate gel column was used to distinguish the molecules with cis-diol structures from others (19,20). Under standard conditions, 150 [lI of sample containing 50 ,ug of oligonucleotide marker in 1 M TEAB (pH 8.2) was charged to 2 ml of boronate gel equilibrated with 1 M TEAB (pH 8.2) at 4°C.…”

Section: Methodsmentioning

confidence: 99%

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Discontinuous DNA replication of Drosophila melanogaster is primed by octaribonucleotide primer.

Kitani¹,

Yoda²,

Okazaki³

1984

Mol. Cell. Biol.

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To investigate the precise structure of eucaryotic primer RNA made in vivo, short DNA chains isolated from nuclei of Drosophila melanogaster embryos were analyzed. Post-labeling of 5' ends of short DNA chains with polynucleotide kinase and [_Y-32P]ATP revealed that 7% of the DNA fragments were covalently linked with mono-to octaribonucleotide primers at their 5' ends. Octaribonucleotides, the major component (ca. 30%), formed the cap structure in the reaction with vaccinia guanylyltransferase and [o-32P]GTP, indicating that they were the intact primer RNA with tri-(or di-) phosphate termini, and the shorter ribooligomers were degradation intermediates. The intact primers started with purine (A/G ratio, 4:1), and the starting few ribonucleotide residues were rich in A.Primer RNA molecules for discontinuous DNA replication of eucaryotic genomes were extensively studied by using isolated nuclei of papovavirus-infected and noninfected mammalian cells (4,8,18,28,29). Chain length of the primer RNA was approximately decanucleotide, synthesis of the primer was started with pppA or pppG, and nucleotide sequence at the RNA-DNA junction was random (18). Recently, DNA primase activities associated with purified DNA polymerase a of various eucaryotic systems were found, and the size of primer RNA was approximately decanucleotide in most cases (1, 2,9,22,26). Primer RNA molecules in vivo were analyzed by using a human lymphoblastoid cell line (27) and a simian virus 40-infected monkey cell line (6). Their chain length varied from mono-up to approximately nonanucleotide, and it was suggested that the longest primer molecules probably corresponded to the primary products, whereas the shorter molecules were in various stages of excision process.The purposes of this work were to detect intact primer RNA molecules in eucaryotic cells, to determine their exact sizes, and to clarify whether any characteristic nucleotide sequences were present. Rigorous investigations on these points have not been done previously with eucaryotic primer RNA made in vivo. Early embryos of Drosophila inelanogaster are used for the analyses because their rapid rate of nuclear division suggests an abundance of nascent chains (10,17 A residues, though lesser so than the in vitro primer (32), and the 3'-terminal region is rich in A and U residues. These results raise the possibility that primer synthesis starts preferentially on a T-rich region on the template strands. MATERIALS AND METHODSReagents and enzymes. 32P, (carrier-free), G(5')pppA and G(5')pppG, nitrocellulose, nuclease P1, nuclease SW, and vaccinia guanylyltransferase (capping enzyme) have been described previously (32). Pancreatic DNase I, T4 polynucleotide kinase, Escherichia coli alkaline phosphatase, and T4 DNA polymerase have been described previously (15).[_-32PJATP and [a-3 PJGTP were prepared as described previously (32). Dihydroxyboryl Bio-Gel P-60 (boronate gel) was prepared by the method of Okayama et al. (16).Purification and terminal labeling of short DNA fragments. D. melanogaster...

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Section: Methodsmentioning

confidence: 99%

“…The boronate gel with TEAB system has a high affinity to relatively short oligoribonucleotides, but not enough affinity to tRNA (20). Therefore, a morpholine system showing high affinity to tRNA (20) The sample was adjusted to match buffer A at a nucleic acid concentration lower than 0.25 mg/ml.…”

Section: Methodsmentioning

confidence: 99%

Discontinuous DNA replication of Drosophila melanogaster is primed by octaribonucleotide primer.

Kitani¹,

Yoda²,

Okazaki³

1984

Mol. Cell. Biol.

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“…Recently we developed covalent chromatography for ADP-ribosylated proteins using a dihydroxyboryl polyacrylamide bead column which was originally used for separation of nucleic acids (22,23). A highly specific interaction of the borate residue with the cis-diol portion of the ADP-ribose bound to proteins in the presence of 6 M guanidine-HCI enabled us to selectively isolate ADP-ribosylated proteins from unmodified proteins irrespective of chain length.…”

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confidence: 99%

Purification of ADP-ribosylated nuclear proteins by covalent chromatography on dihydroxyboryl polyacrylamide beads and their characterization.

Okayama¹,

Ueda²,

Hayaishi³

1978

Proc. Natl. Acad. Sci. U.S.A.

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Nuclear proteins modified by mono or poly ADP-ribosylation were selectively isolated and purified by covalent chromatography on a dihydroxyboryl polyacrylamide bead column that specifically interacts with cis-diol-containing compounds. From rat liver nuclei that had been incubated with NAD+, histones and some nonhistone proteins were extracted with 0.25 M HCL. Approximately 60% of the ADP-ribose incorporated into 20% trichloroacetic acid-precipitable material was recovered in this extract. The ADP-ribosylated material was then isolated from the extract by covalent chromatography on a borate gel column and further purified by carboxymethylcellulose column chromatography. As judged by electrophoretic mobilities in various gel systems and by amino acid compositions, approximately 50% of the ADP-ribose recovered in the carboxymethylcellulose fractions was associated with several nonhistone proteins with molecular weights of 2-6 X 104, while 35% and 15% were associated with histones H2B and HI, respectively. Since the average chain length of the polymer bound to any of these proteins was less than two ADP-ribosyl units, the percentage distribution reflects the number of ADP-ribosylated sites rather than the chain length.Poly ADP-ribosylation is one of the post-translational modifications of nuclear proteins in eukaryotes (1)(2)(3)(4)(5) and has been suggested to be involved in the regulation of various nuclear functions, such as DNA synthesis, histone function, and cell differentiation (6-9).Since the first report from our laboratory in 1968 (10) that a large part of the ADP-ribose incorporated into CC13COOH-insoluble material from NAD+ was covalently attached to histones, this unique group of basic proteins has been demonstrated to be the major acceptors of ADP-ribose in vitro (11-15) as well as in vivo (16)(17)(18). Recently some nonhistone chromosomal proteins were also suggested to be ADP-ribosylated (19)(20)(21). Definitive characterization of the acceptor proteins, however, has not been successful because purification of these proteins, especially of the nonhistone acceptors, proved to be extremely difficult. The major obstacles in purifying these acceptor proteins were the heterogeneity of acceptors induced by variable chain lengths of the bound polymer and their tendency to aggregate with one another.Recently we developed covalent chromatography for ADP-ribosylated proteins using a dihydroxyboryl polyacrylamide bead column which was originally used for separation of nucleic acids (22, 23). A highly specific interaction of the borate residue with the cis-diol portion of the ADP-ribose bound to proteins in the presence of 6 M guanidine-HCI enabled us to selectively isolate ADP-ribosylated proteins from unmodified proteins irrespective of chain length. Preparation of Dihydroxyboryl Bio-Gel P-60. Bio-Gel P-60 (crosslinked polyacrylamide beads) was initially converted to a hydrazide derivative. Aminophenyl boronic acid was then coupled to the derivative via the acyl azide by the method of Inman and Dintzis...

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“…Levallorphan tartrate was a gift from W. Scott, Hoffman-LaRoche, Nutley, NJ. Boric acid gel (particle size 0.1-0.4 mm) was obtained from Aldrich Chemical Co. and was pretreated with acetone as described (16). Nucleotides other than ppGpp were obtained from P-L Biochemicals.…”

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confidence: 99%

“…Nucleotides containing-free 2'-and 3'-OH groups were separated from 2'-or -3'-substituted nucleotides by a modified boric acid gel chromatographic method (16). The.boric acid gel column (0.6 X 18' cm) was preequilibrated with 0.95 M triethylamine-HCO3, pH 9/50 mM Mg(OAc)2.…”

mentioning

confidence: 99%

In vitro degradation of guanosine 5'-diphosphate, 3'-diphosphate.

Sy¹

1977

Proc. Natl. Acad. Sci. U.S.A.

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The degradation of guanosine 5'-diphosphate,3'-diphosphate (ppGpp) by the "crude" ribosomal fraction of Escherichia coli CP 78 (rel+, spoT+) was demonstrated and characterized. When the 3'-pyrophosphoryl group of ppGpp was hydrolyzed, the primary degradation product was 5'-GDP. PhospboryIation of ppGpp to guanosine 5'-triphosphate,3'-diphosphate (pppGpp) prior to degradation was not necessary. The degradation process required Mn2+ and was inhibited by EDTA. Levallorphan, an inhibitor of in vivo ppGpp degradation, also inhibited ppGpp degradation by the crude ribosome. Thiostrepton and tetracycline did not have any inhibitory effect, indicating that the reaction is not a reversal of pyrophosphorylation catalyzed by the stringent factor/ribosome complex.Crude ribosome fractions from E. coli NF161 and NF162, both spoT, contained little degrading activity, but similar fractions of E. coli CP79, a reL4-and spoT+ strain, contained ppGpp degrading activity.Guanosine 5'-diphosphate,3'-diphosphate (ppGpp) is a pleotropic effector that regulates various metabolic pathways (1) as well as the transcription of certain operons during nutritional stress (2) in bacterial cells. Bacteria have therefore developed a sensitive balance of biosynthesis and degradation in controlling the cellular concentration of ppGpp (3). The biosynthesis of this important nucleotide during amino acid deprivation was found to be a ribosome-dependent process (4), the basic mechanism of which is that the stringent factor, ATP: GTP(GDP) 3'-pyrophosphotransferase, is activated by a ribosome-mRNA complex when the aminoacyl-tRNA site of the ribosome is occupied by a codon-specified, uncharged tRNA. A nonribosome-dependent synthesis of ppGpp has also been found (5, 6).The degradation process of ppGpp has not been well defined due to the lack of an in vitro cell-free system. In vivo studies have shown that it is very rapid (7,8) and is controlled by the availability of an energy source (9), but studies with spoTmutant, which has a 10-fold slower degradation rate, have led to conflicting theories as to its degradation pathway (10-13). I report here on the characterization of an in vitro cell-free ppGpp degradation system. METHODS Materials. Escherichia coli CP78 (arg-, his-, leu -, thr, thi-, relA+, spoT+), CP79 (arg-, his-, leu-, thr-, thi-, relA-, spoT+), NF161 (met-, arg-, relA+, spoT-), and NF162 (met , arg-, relA , spoT-), kindly supplied by N. Fiil of the University of Copenhagen, were grown in a yeast extract/phosphate medium (14).[3H]ppGpp (8.1 Ci/mmol) was prepared as described (15).[3H]GDP and [3H]GTP were purchased from New England Nuclear. Levallorphan tartrate was a gift from W. Scott, Hoffman-LaRoche, Nutley, NJ. Boric acid gel (particle size 0.1-0.4 mm) was obtained from Aldrich Chemical Co. and was pretreated with acetone as described (16). Nucleotides other than ppGpp were obtained from P-L Biochemicals.Preparation of Crude Ribosomdl Fraction. E. coli CP78 cells from the late logarithmic phase were harvested and washed once with buf...

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Dihydroxyboryl-substituted methacrylic polymer for the column chromatographic separation of mononucleotides, oligonucleotides, and transfer ribonucleic acid

Cited by 119 publications

References 5 publications

Discontinuous DNA replication of Drosophila melanogaster is primed by octaribonucleotide primer.

Discontinuous DNA replication of Drosophila melanogaster is primed by octaribonucleotide primer.

Purification of ADP-ribosylated nuclear proteins by covalent chromatography on dihydroxyboryl polyacrylamide beads and their characterization.

In vitro degradation of guanosine 5'-diphosphate, 3'-diphosphate.

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