A fractionating column for analysis of nucleic acids

Mandell, Joseph D.; Hershey, Alfred Day

doi:10.1016/0003-2697(60)90020-8

Cited by 1,371 publications

(242 citation statements)

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“…(2), and the amount was determined by using the ninhydrin method (17). Nucleic 15 to 5 C for the first 2 weeks followed by three successive 2-week periods during which the temperature was lowered for 2 hr during the dark period to -2.0 C, -4.5 C and -6.5 C, respectively. The hardiness, moisture content of leaves, protein content, and RNase activity were determined at 2-week intervals throughout the study.…”

Section: General Methodsmentioning

confidence: 99%

Nucleic Acid and Protein Changes in Relation to Cold Acclimation and Freezing Injury of Korean Boxwood Leaves

Gusta

Weiser

1972

Plant Physiol.

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Quantitative and qualitative differences in nucleic acids of Korean boxwood (Buxus microphylla var. Koreana) leaves were determined by methylated albumin kieselguhr chromatography at different levels of cold hardiness. During cold acclimation there was an increase in RNA, mainly ribosomal RNA, with little or no change in DNA. The increase in ribosomal RNA was closely paralleled by an increase in water soluble and membrane bound proteins. As cold hardiness increased, ribonuclease activity declined.Exposure of hardy boxwood plants to warm temperatures resulted in a rapid loss in cold resistance and a rapid synthesis of nucleic acids as judged by ' (13,14,16, 25).While this is an attractive idea, it is possible that observed autumnal increases in proteins and nucleic acids are due to reduced degradation by nucleases and proteases during sample preparation rather than increased synthesis rates. RNase activity has been shown to decrease in woody plant tissues during the autumn (8), and substantial nucleic acid degradation has been observed in boxwood leaves during lyophilization (5). Another possibility is that the nucleic acid and protein increases are real, but that these changes bear a coincidental rather than a causal relationship to cold acclimation. For example, proteins and nucleic acids may increase in the autumn as a result of a build up of precursor pools in the living bark of deciduous plants when leaves senesce and abscise.This study was designed to investigate these possibilities by examining the changes in proteins and different classes of RNA during cold acclimation in Korean boxwood leaves. The fate of nucleic acids following lethal freezing was also examined. Korean boxwood is a broad-leaved evergreen shrub which retains its leaves throughout the autumn and winter when major changes in hardiness occur. MATERIALS AND METHODSThe four general types of studies which were conducted involved: (a) characterization of cold acclimation of Korean boxwood leaves (Buxus microphylla var. Koreana) in the field with concurrent measurements of changes in nucleic acids, proteins, and RNase activity; (b) similar studies during acclimation at different photoperiods in controlled environment chambers; (c) examination of changes in the metabolism of nucleic acids during short term dehardening and rehardening when hardy plants were exposed to alternating periods of high and low temperatures; (d) a study of the fate of nucleic acids in leaves following exposure to a killing frost. GENERAL METHODSLeaf Hardiness Evaluations. The leaves of boxwood were the basic experimental unit for all analytical and hardiness evaluations in this study. The overwintering leaves are mature, differentiated organs which do not undergo cell division or other visible signs of growth and development during the autumn, winter, and spring when their cold resistance changes significantly. Since Korean boxwood is an evergreen species, this choice of material reduces the problem of a major build up in precursor pools such as that which occurs...

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Section: General Methodsmentioning

confidence: 99%

Nucleic Acid and Protein Changes in Relation to Cold Acclimation and Freezing Injury of Korean Boxwood Leaves

Gusta

Weiser

1972

Plant Physiol.

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“…The crude nucleic acid pellet was washed three times with 66% ethanol containing 1 % potassium acetate, dissolved in 0.3 M NaCl in 0.05 M KH2PO4 (pH 6.7), and fractionated on MAK columns (28). After addition of the sample, the column was washed with 200 ml of 0.3 M NaCl in 0.05 M KH2PO4 (pH 6.7), and elution was done at 37 C with a 300-or 400-ml linear gradient of NaCl from 0.3 M to 1.6 M in 0.05 M KH2PO4, pH 6.7.…”

Section: Methodsmentioning

confidence: 99%

Characterization of Rapidly Labeled Ribonucleic Acid from Dwarf Peas

Johri

Varner

1970

Plant Physiol.

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The ribonucleic acid synthesized by excised shoots of dwarf pea (Pisum sativum L. cv. Progress No. 9) during short labeling periods has been characterized. Thirty per cent of the total 32Pi incorporated in 1 hour is found in the ribosomal fraction. This labeled RNA was polydisperse (6-18 Svedberg units) and after chromatography on a methylated albumin-kieselguhr column about 80% of the radioactivity appeared in two peaks. One of these appeared on the shoulder of heavy ribosomal RNA ("mRNA") while the other was tenaciously bound to the column (TB-RNA).In the presence of high NaCl concentration, about half of the polydisperse RNA interacted with ribosomal RNA and eluted as "mRNA" while the remainder eluted as TB-RNA.This interaction in the presence of salt seems to result in the alteration of secondary structure because the "mRNA" fraction had a high sedimentation coefficient (45-50 Svedberg units). The polydisperse RNA approaches DNA in low cytidylate and guanylate content. After short periods of labeling TB-RNA showed higher adenylate content than "mRNA." The radioactivity from the "mRNA" peak can be chased, and these counts may represent a class of shortlived messenger RNA molecules with an average half-life of 10 to 15 minutes. The other component, TB-RNA, could not be chased and accumulated radioactivity during the chase period.In bacteria and viruses the information for the sequence of amino acids in a polypeptide chain is encoded into messenger RNA. Presence of a similar fraction has been suggested in some higher plants (5,6,21,26,39). All of our knowledge about messenger RNA from higher plants is presumptive and based on analogy to microorganisms; in no case has this fraction been purified or characterized, nor has its rate of synthesis or its stability been studied.We chose a dwarf pea as the experimental material because we have been investigating the RNA synthesized by nuclei isolated from shoots of this plant (19), and the knowledge obtained from the system in vivo may be applicable to the results obtained with isolated nuclei. MATERIALS AND METHODS Uptake and Incorporation of Labeled Precursors. Dwarf pea seeds (Pisum sativum L., cv. Progress No. 9) were surfacesterilized for 7 to 10 min in 1% sodium hypochlorite, implanted in 500-ml Erlenmeyer flasks containing moist, sterile vermiculite, and allowed to germinate at 23 C in continuous light. At the end of 5 or 6 days, shoots 0.8 to 1 cm long were excised and 10 or 15 shoots were incubated in 25-ml Erlenmeyer flasks under aseptic conditions with labeled uridine or 32Pj (carrier-free H3u2PO4, neutralized) in 2 ml of water containing 100 ,ug of chloramphenicol. Flasks were transferred to a shaker, and, after incubation for various times, the shoots were washed thoroughly in ice-cold 0.05 M KH2PO4 and deionized water and were then extracted for nucleic acids.In experiments designed to study the relationship between uptake and incorporation, the labeled shoots were homogenized in ethanol, 80% (v/v,) and the nucleic acids were determined (33). The ...

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“…After a 30-min incubation at 37 "C, the mixture was heated for 15 min at 45 " C ; the DNA was purified by phenol extraction [14] [14], had a specific radioactivity of 240000 dis. min-' pg-'.…”

Section: Doubly Labelled S'-oh/s'-phosphate Nicked D N Amentioning

confidence: 99%

Properties of the Main Endonuclease Specific for Apurinic Sites of Escherichia coli (Endonuclease VI)

Gossard

Verly

1978

European Journal of Biochemistry

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The main endonuclease for apurinic sites of Eschrrichia coli (endonuclease VI) has no action on normal strands, either in double-stranded or single-stranded DNA, or on alkylated sites. The enzyme has an optimum pH at 8.5, is inhibited by EDTA and needs Mgz+ for its activity; it has a half-life of 7 min at 40 "C. A purified preparation of endonuclease VI, free of endonuclease I1 activity, contained exonuclease 111 ; the two activities (endonuclease VI and exonuclease 111) copurified and were inactivated with the same half-lives at 40 "C. Endonuclease VI cuts the DNA strands on the 5' side of the apurinic sites giving a 3'-OH and a 5'-phosphate, and exonuclease 111, working afterwards, leaves the apurinic site in the DNA molecule; this apurinic site can subsequently be removed by DNA polymerase I. The details of the excision of apurinic sites in vitro from DNA by endonuclease VI/exonuclease 111, DNA polymerase I and ligase, are described; it is suggested that exonuclease 111 works as an antiligase to facilitate the DNA repair.An enzyme which hydrolyzes a phosphoester bond near apurinic sites was found in Escherichia c d i by Verly and Paquette [l]. This enzyme, which has no action on normal DNA strands or on alkylated sites [ 2 ] , has been completely purified by Verly and Rassart[3]; it is a monomeric protein of M , = 32000. Depurinated DNA has been repaired in vitro by Verly et al. [4] with three enzymes: the endonuclease specific for apurinic sites from E. coli, DNA polymerase I and the four deoxynucleoside triphosphates, ligase and its coenzyme. Mutants of E. coli lacking DNA polymerase I are very sensitive to methylmethanesulfonate and it is known that the lethal action of this alkylating agent is due mostly to the depurination of DNA [5]. On the other hand, however, mutations in gene sthA, which codes for the endonuclease specific for apurinic sites, only slightly increase the sensitivity of E. coli to methylmethanesulfonate [6]; this is most likely due to the fact that E. coli possesses two enzymes active on apurinic sites: the main enzyme which we have isolated

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A fractionating column for analysis of nucleic acids

Cited by 1,371 publications

References 10 publications

Nucleic Acid and Protein Changes in Relation to Cold Acclimation and Freezing Injury of Korean Boxwood Leaves

Nucleic Acid and Protein Changes in Relation to Cold Acclimation and Freezing Injury of Korean Boxwood Leaves

Characterization of Rapidly Labeled Ribonucleic Acid from Dwarf Peas

Properties of the Main Endonuclease Specific for Apurinic Sites of Escherichia coli (Endonuclease VI)

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