Rapid group separations of nucleotides and related compounds on silica columns

Lothrop, Clinton D.; Uziel, Mayo

doi:10.1016/0003-2697(80)90025-1

Cited by 17 publications

(3 citation statements)

References 15 publications

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“…Silica chromatography. The nucleotides in the cell extract were separated from the nucleosides and purine and pyrimidine bases on 1.0-ml silica columns (Waters Associates, Inc.) as described by Lothrop and Uziel (20,21). The columns were washed with 5.0 ml of distilled water and then with 5.0 ml of 90% acetonitrile (Mallinckrodt) in water.…”

Section: Methodsmentioning

confidence: 99%

Alterations in nucleotide content of human lung fibroblasts infected with Mycoplasma pneumoniae

Upchurch¹,

Gabridge²

1982

Infect Immun

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The nucleotide content of normal MRC-5 human lung fibroblasts and fibroblasts infected with Mycoplasma pneumoniae PI 1428 was determined. Nucleotides from control and infected fibroblasts were extracted with 5% trichloracetic acid. After neutralization of the extracts, the nucleotides in the extracts were separated by anion-exchange chromatography. Significant differences were found between the nucleotide content of the control and infected cells. Nucleotide triphosphate levels were twofold higher in the control fibroblasts than in the infected fibroblasts 4 h after the initiation of infection. At the same time, nucleotide diphosphate and monophosphate levels were higher in the infected fibroblasts than in the control fibroblasts. Determination of the energy charge ratio for each set of nucleotides (adenosine, guanosine, cytidine, and uridine) demonstrated a shift of nucleotide content in the infected fibroblasts. Immediately after infection, the energy charge for each set of nucleotides was higher for the control fibroblasts than it was for the infected fibroblasts. This pattern continued throughout the infection period with only minor exceptions. The work presented here indicates a loss of energy charge in fibroblasts infected with M. pneumoniae and may help to explain some of the metabolic changes and cell damage which accompany infection.

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Section: Methodsmentioning

confidence: 99%

Alterations in nucleotide content of human lung fibroblasts infected with Mycoplasma pneumoniae

Upchurch¹,

Gabridge²

1982

Infect Immun

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“…2'-Omethyl ribonucleoside fractions were separated from enzymatically digested tRNAs for independent examination, using a modification of a previously published method (31). A stainless steel column (250 by 4.6 mm) was dry packed with Porasil A and washed with 10 mM ammonium borate (pH 9) until the effluent was alkaline.…”

Section: -Thiouridine (S2u) and N22'-o-dimethylguanosine (M2gm)mentioning

confidence: 99%

Posttranscriptional modification of tRNA in thermophilic archaea (Archaebacteria)

et al. 1991

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Nucleoside modification has been studied in unfractionated tRNA from 11 thermophilic archaea (archaebacteria), including phylogenetically diverse representatives of thermophilic methanogens and sulfur-metabolizing hyperthermophiles which grow optimafly in the temperature range of 56 (Thermoplasma acidophilum) to 105°C (Pyrodictium occultum), and for comparison from the most thermophilic bacterium (eubacterium) known, Thermnotoga narilima (80WC). Nine nucleosides are found to be unique to the archaea, six of which are structurally novel in being modified both in the base and by methylation in ribose and occur primarily in tRNA from the extreme thermophiles in the Crenarchaeota of the archaeal phylogenetic tree. 2-Thiothymine occurs in tRNA from Thermococcus sp., and constitutes the only known occurrence of the thymine moiety in archaeal RNA, in contrast to its near-ubiquitous presence in tRNA from bacteria and eukarya. A total of 33 modified nucleosides are rigorously characterized in archaeal tRNA in the present study, demonstrating that the structural range of posttranscriptional modifications in archaeal tRNA is more extensive than previously known. From a phylogenetic standpoint, certain tRNA modifications occur in the archaea which are otherwise unique to either the bacterial or eukaryal domain, although the overall patterns of modification are more typical of eukaryotes than bacteria.Posttranscriptional processing of tRNA produces a variety of structurally modified nucleosides, some of which have been shown to be associated with a range of biological functions, including maintenance of translational fidelity and efficiency, codon usage, tRNA-protein interactions, and adaptation to cellular stress (9). More than 75 different nucleotides are presently known in tRNA from all sources, with modifications occurring mostly in the base and less commonly by methylation at 0-2' in ribose. Both the chemical nature and sequence locations of individual modifications are highly selective (for reviews, see references 9, 19, and 32), with numerous distinct differences exhibited among the three primary phylogenetic domains, Archaea, Bacteria, and Eucarya (formerly termed archaebacteria, eubacteria, and eukaryotes, respectively [53]). Knowledge of tRNA modification in thermophiles is important as an initial step in understanding structure-stability relationships in the nucleic acids of these remarkable organisms, which grow optimally around the boiling point of water (43), and in identifying domain-or kingdom-specific nucleoside modifications which may serve as phylogenetic markers. Additionally, knowledge of the distributions of modified nucleosides will be useful in later sequencing studies, particularly in avoiding misidentifications when structurally new nucleosides are encountered.Among archaeal microorganisms, tRNA from Halobacterium volcanji has been the most extensively studied (18,20),

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“…Even for the highly sensitive determination of ATP and c-AMP by biolunimescence it is necessary to purify the samples either on charcoal (Azam & Hodson 1977) or ion exchangers (Francko & Wetzel 1980). Group separation of nucleotides and nucleosides on a s h c a column has been reported by Lohtrop & Uziel (1980), so that obtaining purified and 10 to 100-fold concentrated samples of nucleotides from seawater seems feasible. Once these sample pre-treatments are realized, the full range of nucleotides in seawater can be measured by HPLC.…”

Section: Importance Of Organic Phosphorus To Phaeocystis Pouchetiimentioning

confidence: 99%

Determination of nucleosides and nucleotides in seawater by HPLC; application to phosphatase activity in cultures of the alga Phaeocystis pouchetii

Admiraal¹,

Veldhuis²

1987

Mar. Ecol. Prog. Ser.

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Nucleosides and nucleotides, dissolved in artificial and natural seawater, were separated by HPLC (High Pressure Liquid Chromatography) on a C-18 reversed phase column. Test compounds in concentrations as low as 5 to 25 nM were directly detectable by UV-absorption. Determinations of nucleosides and nucleosidemonophosphates were used to analyse the alkaline phosphatase activity (APA) in cultures of Phaeocystis pouchetii. Phosphate-depleted cultures of this colonial phytoplankter hydrolysed uridine mono-phosphate at a rate 7 times higher than 3-0-methylfluorescein phosphate, a commonly used artificial test substance for APA. The uridine, cytidine and guanosine moieties of nucleoside monophosphates were partially assimilated by the cultures, probably in connection with enzymatic hydrolysis. The application of HPLC analysis of cellular and dissolved nucleotides in both cultures and natural communities of planktonic organisms is discussed.

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Rapid group separations of nucleotides and related compounds on silica columns

Cited by 17 publications

References 15 publications

Alterations in nucleotide content of human lung fibroblasts infected with Mycoplasma pneumoniae

Alterations in nucleotide content of human lung fibroblasts infected with Mycoplasma pneumoniae

Posttranscriptional modification of tRNA in thermophilic archaea (Archaebacteria)

Determination of nucleosides and nucleotides in seawater by HPLC; application to phosphatase activity in cultures of the alga Phaeocystis pouchetii

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