Kenneth C. Kuo

Major and modified nucleosides in tRNA hydrolysates by high-performance liquid chromatography.

We describe a high-performance liquid chromatographic analytical method that can be readily placed in operation, and which is particularly well suited to scientists investigating tRNA structure, biosynthesis, and function, and for the determination of major and modified nucleosides of tRNA. The method is characterized by the following features: (1) Sensitivity at the nanogram level; (2) High chromatographic resolution and selectivity; (3) Direct measurement of nucleosides with accuracy and precision; (4) Analysis is non-destructive and the high capacity of this chromatographic system allows easy isolation of pure nucleosides for further characterization; (5) Rapid separation and measurement in ca. 1 h after hydrolysis to nucleosides; and (6) Quantitation without use of radiolabeled compounds; however, labeled compounds are readily isolated and measured.

Large-scale methylation patterns in the nuclear genomes of plants

Methylation was investigated in compositional fractions of nuclear DNA preparations (50-100 kb in size) from five plants (onion, maize, rye, pea and tobacco), and was found to increase from GC-poor to GC-rich fractions. This methylation gradient showed different patterns in different plants and appears, therefore, to represent a novel, characteristic genome feature which concerns the noncoding, intergenic sequences that make up the bulk of the plant genomes investigated and mainly consist of repetitive sequences. The structural and functional implications of these results are discussed.

Eukaryotic tRNAsPro: Primary structure of the anticodon loop; presence of 5-carbamoylmethyluridine or inosine as the first nucleoside of the anticodon

The modified nucleoside U*, located in the first position of the anticodon of yeast, chicken liver and bovine liver tRNA(Pro) (anticodon U*GG), has been determined by means of TLC, HPLC, ultraviolet spectrum and gas chromatography-mass spectrometry. The structure was established as 5-carbamoylmethyluridine (ncm5U). In addition, we report on the primary structures of the above-mentioned tRNAs as well as those which have the IGG anticodon. In yeast, the two tRNA(Pro) (anticodons U*GG and IGG) differ by eight nucleotides, whereas in chicken and in bovine liver, both anticodons are carried by the same body tRNA with one posttranscriptional exception at position 32, where pseudouridine is associated with ncm5U (position 34) in tRNA(Pro) (U*GG) and 2-O-methylpseudouridine is associated with inosine (position 34) in tRNA(Pro) (IGG).

High Performance Liquid Chromatography of Nucleosides in RNA and DNA

Abstract Reversed-phase high performance liquid chromatography has been developed and used effectively as a research tool for the quantitative analysis of major and modified nucleosides present in RNAs, DNAs, and physiological fluids. Gehrke et al. (5, 6, 24, 28) have shown that RP-HPLC is especially well suited for the analysis of the array of modified nucleosides found in tRNA, as more than forty nucleosides can be resolved and quantitatively determined in a single analysis. Coupled with our rapid, quantitative and straightforward enzymatic hydrolysis protocol, RP-HPLC compositional analyses can be directly performed on microgram quantities of either unfractionated or isoaccepting tRNAs. This method is applicable to the comparison of nucleoside compositions of tRNAs from parental and mutant organisms. In addition, Gehrke and Kuo (31, 33) have developed a highly precise RP-HPLC method for the analysis of the methylated nucleoside present in DNA, 5-methyldeoxycytidine, which has been used in collaborative...

Separation of Fullerenes C60, C70, and C76-84 on Polystyrene Divinylbenzene Columns

Abstract New HPLC methods have been developed to effectively separate and recover essentially pure amounts of C60 and C70 fullerenes from the lower MW contaminants and higher MW fullerenes co-extracted from carbon soot. Separation with toluene/methylene chloride mobile phase results from different degrees of interaction of conjugated π-electrons of the fullerenes and the polystyrene divinylbenzene (PSDVB) resin stationary phase. The presence of either toluene or CS2 in the mobile phase attenuates π-electron interactions. The separation mechanism becomes predominantly size exclusion chromatography for a CS2 concentration in the mobile phase ≥ 60%. Excellent separation of C60, C70 and C76–84 is obtained with the analytical Envirosep-ABC(tm) columns in less than 15 minutes. Substituted fullerene reaction products are also well separated from their parent compounds. The effects of column size and PSDVB pore size on analytical separation, along with the effects of pore size, mobile phase, and sample size on se...

RESTRICTION ENZYME ANALYSIS AND CLONING OF HIGH MOLECULAR WEIGHT GENOMIC DNA ISOLATED FROM CHLORELLA SOROKINIANA (CHLOROPHYTA)1

High molecular weight (50–70 kb) genomic DNA was isolated from the eukaryotic green alga, Chlorella sorokiniana spec. nov, (formerly Chlorella pyrenoidosa Chick, strain 7‐11‐05), for restriction endonuclease digestion studies and for preparation of a genomic DNA library. Twenty restriction endonucleases were examined for their abilities to digest this DNA. Nine of the endonucleases gave nearly complete digestion of the DNA, whereas 11 gave only partial digestion. Additional purification steps to remove possible contamination by proteins, RNAs, or polysaccharides did not improve digestion. Digestion studies with pairs of endonuclease isoschizomers, of which one member was sensitive to base methylation, suggested that 5‐methylcytosine might be responsible Jor inhibition of certain endonucleases. Analysis of the DNA showed it to contain 63% GC and to have a high content (5.1 mol %) of 5‐methylcytostne but no other methylated or unusual bases. Evidence indicates that this high 5‐methylcytosine content, which is a characteristic of higher plant genomic DNA rather than of eukaryotic microorganisms, interfered with the cloning of restriction fragments (or fragments produced by mechanical shearing) of C. sorokiniana genomic DNA in standard bacterial host‐strains. Escherichia coli strain K803, which is a permissive host for cloning highly methylated DNA from higher plants, also permitted the cloning of a complete genomic library of 15–20 kb Mbol restriction fragments inserted into the BamHI site of the γ vector, EMBL 3. This C. sorokiniana genomic library appears to be the first genomic‐library constructed for any species of Chlorella.

Chapter 1 Progress and Future Prospects of Modified Nucleosides as Biological Markers of Cancer

Publisher Summary This chapter discusses the topic of nucleic acid components as potential biological markers; specifically, modified nucleosides. There have been indications for more than 30 years that cancer patients excrete elevated levels of methylated purines and pyrimidines as well as other modified bases and nucleosides. The origin of these compounds was obscure until the discovery of the modification of transfer RNA. Investigators have also attempted to elucidate cancer markers that could be utilized to predict which patients are more likely to respond to treatment and which patients have a worse prognosis. High performance liquid chromatography with diode array detection (HPLC-UV) has emerged as one of the most popular and powerful techniques for studying the constituents of nucleic acids, especially in complex samples such as physiological fluids and cell extracts. Research on tRNA catabolites in urine and serum/plasma has concentrated on HPLC analysis of the modified nucleosides following isolation of the nucleosides by boronate gel affinity chromatography as the nucleosides are generally the major tRNA catabolic excretion products and are easily isolated by the boronate gel.

Chapter 12 Serum Nucleoside Chromatography for Classification of Lung Cancer Patients and Controls

Publisher Summary This chapter describes serum nucleoside chromatography for classification of lung cancer patients and controls. Modified ribonucleosides derived predominantly from transfer RNA and ribosomal RNA, are known to be excreted in abnormal amounts in the urine of cancer patients. The molecular mechanisms of elevated excretion are unclear. Extract of neoplastic tissues have aberrant tRNA methyltransferases, and it has been suggested that the high turnover of tRNA is because of the rapid degradation of aberrantly modified tRNAs. Experimental evidence indicates that methylation of tRNA occurs only after-synthesis of the intact macro-molecule. No kinases have been found that will re-incorporate these compounds into tRNA, and consequently, they are excreted following metabolic degradation of tRNA. A characteristic of small cell carcinoma (SCC) is its tendency toward early and distant metastases estimated to occur in 70% of patients at the time of diagnosis. This very typical behavior of SCC creates problems in accurately defining tumor burden, and in estimating extent of disease despite technical advances in modern radiological techniques for demonstrating the presence of tumor in organs or tissues. As a consequence, difficulties are frequently encountered not only at initial staging but in correctly quantifying response and assessing sites of recurrent or metastatic disease.

A Dedication and Thanks

Publisher Summary The publication, “Journal of Chromatography Library Volume–45B,” is dedicated to the efforts of those researchers across the world who are searching for a better understanding of roles and functions of modified nucleosides found in all nucleic acids, the complexities and fidelity of protein synthesis, conformational effects, codon–anticodon interactions, and the development of new research tools to probe the biological significance of these important macromolecules.