Paul F. Agris

Quantitative enzymatic hydrolysis of tRNAs : Reversed-phase high-performance liquid chromatography of tRNA nucleosides☆

A rapid quantitative method for enzymatic hydrolysis of microgram amounts of tRNA has been developed, specifically to take full advantage of our precise, accurate, and selective reversed-phase high-performance liquid chromatographic (HPLC) system for separation and measurement of the major and modified nucleosides in tRNA. After study of several enzyme systems, nuclease P1 and bacterial alkaline phosphatase were selected and the hydrolysis parameters were systematically studied. Optimized hydrolysis conditions give quantitative hydrolysis in 2 h and this short incubation time prevents loss of unstable nucleosides. The chromatographic system can tolerate relatively high levels of protein in the sample allowing high enzyme--substrate ratios and direct injection of hydrolysates. This enzymatic hydrolysis--HPLC method is the best described to date for quantitative determination of the nucleoside composition of tRNAs and has already provided important information for investigation of the role of modification in the function of RNAs.

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.

Deteriorating collagen synthesis and cell ultrastructure accompanying senescence of human normal and Werner's syndrome fibroblast cell strains

Abstract Ultrastructural changes that accompanied senescence, in vitro, of six normal fibroblast cell strains were found to correspond to the morphological characteristics of a Werners syndrome progeroid cell strain: greatly increased size of midnuclear diameters; heavy vacuolation; maturation of predominantly primary to secondary lysosomes; increase in multilobed nuclei; dilation of mitochondrial cristae; dilation of rough endoplasmic reticulum and the filling of intermembrane space with electron-dense particles presumably protein; and vacuolar accumulation of fibrous material which was later discharged into the medium. This fibrous material was produced only by late-passage fibroblasts and Werners syndrome fibroblasts and had an ultrastructure dissimilar from that of normal collagen fibers produced by exponentially-growing cultures. The fibrous debris had a high subunit molecular weight (approx. 200000 D), an amino acid composition that included hydroxyproline, hydroxylysine, and relatively large amounts of proline and glycine, and was degraded by collagenase. This protein is believed to be a form of procollagen produced by either incorrect maturation of mRNA or incomplete processing of procollagen.

High-performance liquid chromatography and mass spectrometry of transfer RNA bases for isotopic abundance

Abstract We present a quantitative analysis of the incorporation of stable isotopes into nucleic acids for verification of the site and determination of the abundance of the label. High-performance liquid chromatography and mass spectrometry of nucleic acid bases were used for quantitation of isotopic enrichment, with only μg amounts of available RNA. Conditions for acid hydrolysis of tRNA were optimized for quantitative yield of bases without destruction, and optimum conditions for the reversed-phase high-performance liquid chromatographic separation of the bases were determined. Three tRNA preparations, 13 C-enriched in vivo by incorporation of [ 13 C 2 ]adenine, [ 13 C 2 ]uracil and [ 13 C]methyl groups from methionine, were subjected to these procedures, followed by mass spectrometry of the bases. In natural abundance, 13 C is 1.08 atom%; in these tRNA preparations we found: 27% of all adenine was labeled at position 2 with 13 C atoms; 43 atom% 13 C at position 2 of uracil and 45 atom% 13 C at position 2 of cytosine; and 56.9 atom% 13 C at the methyl of thymine, respectively. The techniques described are important to the study of nucleic acid biosynthesis, modification and structure by nuclear magnetic resonance spectroscopy.

Purification of transfer RNA species by single-step ion-exchange high-performance liquid chromatography

Anion-exchange high-performance liquid chromatography (HPLC) methods have been developed for the purification and concentration of milligram quantities of tRNA. A Waters Protein Pak DEAE 5PW 150 x 21.5 mm I.D. column was utilized for the separation of tRNA species. The chromatographic conditions chosen created non-denaturing conditions for separating the different species: 0.1 M Tris buffer (pH 7.6) at 25 degrees C, with a 0.25 M to 0.4 M sodium chloride gradient, using a 170-min gradient. The gradient form could be adjusted for optimizing purification (to over 85%) of the tRNA species of interest. The same DEAE packing in a smaller column was found to be effective for concentrating solutions of the purified tRNA. Fifty-fold concentration and recoveries above 90% have been obtained by this method. These methods were successfully applied to the purification of individual tRNA species from both Escherichia coli and yeast.

Long-term in vitro cell culture of sinclair swine melanoma

SummaryThe melanoma of Sinclair swine exhibits several characteristics similar to human melanoma but demonstrates an unusually high incidence of spontaneous regression. A total of 66 finite cell lines derived from 21 swine melanotic lesions, both cutaneous and visceral, were studied in vitro over their life spans of up to 14 months. The growth characteristics of the cultures varied with the age of the swine from which the tumors were obtained. Cell cultures of tumors obtained from swine aged less than 2 months grew steadily in culture with a population-doubling time of 120 to 180 hr until growth and division ceased after a maximum of 25 to 35 population doublings (6 to 8 passages). Cell culture of tumors obtained from swine aged 3 months or older showed a biphasic growth pattern with an early slow growth rate (population-doubling time 120 to 160 hr), which shifted after 3 to 6 passages to a faster rate (80 to 110 hr population-doubling time) until termination of growth and division after a maximum of 75 to 85 population doublings (18 to 20 passages). The cultures were morphologically heterogeneous including cuboidal, spindle and dendritic cell types. Electron microscopy showed classic melanosomes only in the primary and passage 1 cultures although vesicular inclusions were numerous in later-passage cells. However, continued melanin synthesis was indicated by the spectroscopic characteristics of material obtained from medium of passage 8 cultures and by DOPA staining of cultures as advanced as passage 18.

Chapter 8 High Performance Liquid Chromatography of Cap Structures and Nucleoside Composition of MRNAs

Publisher Summary The chapter describes high performance liquid chromatography (HPLC) of cap structures and nucleoside composition of MRNAs The chapter discusses the development of a rapid, efficient, sensitive, and quantitative procedure for analyzing messenger RNA (mRNA) cap 0 and 1 structures without the need for radio labels. The method is adaptable to cap 2 structures and to the m 2 2 m 7 G cap structures of small nuclear RNAs. This method uses reversed-phase HPLC for separating caps from nucleosides with the use of either a stepwise buffer system or a gradient curve buffer system. Identification of cap structures is done by retention time of known reference caps and total UV spectra of the HPLC peaks. Quantitation is accomplished by using an internal standard method with established relative molar response factors (RMRs) of all nine caps. An anion-exchange technique has been developed to separate cap structures from other nucleosides that result from nuclease P1 and bacterial alkaline phosphatase (BAP) hydrolysis of total crude RNA. Potential uses of an accurate quantitative chromatographic method for separation and measurement of cap structures include screening of mutants for cap production; study of cap production in the cell cycle; investigation of the presence of different caps before and after viral infection, transformation during differentiation, and after hormonal changes; identification and quantitation of cap in single species mRNA; and studies on the half-lives of mRNA and caps.

Small nuclear ribonucleoprotein antigens are absent from 10S translation inhibitory ribonucleoprotein but present in cytoplasmic messenger ribonucleoprotein and polysomes

A cytoplasmic 10S ribonucleoprotein particle (iRNP), which is isolated from chick embryonic muscle, is a potent inhibitor of mRNA translation in vitro and contains a 4S translation inhibitory RNA species (iRNA). The iRNP particle shows similarity in size to the small nuclear ribonucleoprotein (snRNP) particles. Certain autoimmune disease patients contain antibodies directed against snRNP antigenic determinants. The possibility that iRNP may be related to the small nuclear particles was tested by immunoreactivity with monospecific autoimmune antibodies to six antigenic determinants (Sm, RNP, PM-1, SS-A (Ro), SS-B (La), and Scl-70). By Ouchterlony immunodiffusion assays, the cytoplasmic 10S iRNP did not show any immunoreactivity. Also, a more sensitive hemagglutination inhibition assay for detecting Sm and RNP antigens failed to show reactivity with the 10S iRNP. Thus, the 10S iRNP particles are distinct from the similarly sized snRNP. However, free and polysomal messenger ribonucleoprotein (mRNP) particles and polysomes also isolated from chick embryonic muscle and analyzed by Ouchterlony immunodiffusion and hemagglutination inhibition for the presence of the antigenic determinants showed reactivity to Sm and RNP autoantibodies, but were not antigenic for the other four antibodies. Some of the Sm antigenic peptides of mRNP particles and polysomes were identical to those purified from calf thymus nuclear extract, as judged by Western blot analysis. The association of Sm with free and polysomal mRNP and polysomes suggests that Sm may be involved in some cytoplasmic aspects of mRNA metabolism, in addition to a nuclear function in mRNA processing.

Metabolism of macromolecular methyl groups in Escherichia coli: Whole cell NMR spectroscopy

Carbon-l 3 NMR spectra of ‘3C-methyI-enriched, intact and viable K. coZi cells have been obtained and shown to be useful in studies of the in vivo metabolism of this molecular group. Methyl resonances from whole cell spectra were shown to originate from nondialyzable, macromolecular material present in subcellular fractions and not due to free cellular [met@Z‘3CJmethionine or other dialyzable compounds. Comparisons with the methyl resonances of several cellular proteins, membrane constituents, and other compounds have been made. 13C-Enriched methyl groups were ‘chased’ from their original sites in late log phase cultures when cells were placed in minimal medium with [methyl-“2C]methionine. Methyl, originally assigned to locations in protein, as bound to the sulfur of methionine, was ‘chased’ to an exclusively membrane site bound to nitrogen in the quaternary amine of phosphatidyl choline. Specific enrichment of carbon-13 is essential to carbon NMR studies of transient metabolites which may exist in the metabolic processes for only short periods of time and small molecules or macromolecules that exist at very low concentrations in the cell [ 1,2]. Thus, cell growth in media containing a macromolecular precursor or a metabolite that is 90t atom % enriched in carbon-13 at a particular carbon is highly advantageous for studies in vivo. The label is restricted

Chapter 6 Three Dimensional Dynamic Structure of Transfer Rna by Nuclear Magnetic Resonance Spectroscopy

Publisher Summary This chapter presents a study on the three-dimensional dynamic structure of transfer RNA (tRNA) by nuclear magnetic resonance (NMR) spectroscopy. 13 C-enriched, native modified nucleosides are used as probes, whose aromatic and proton resonance chemical shifts and Nuclear Overhauser Effect (NOE) values give access to internucleotide distances of bases not involved in Watson–Crick pairs. In Escherichia coli ( E. coli ) , tRNA multiple peaks for the methyl groups of T in tRNA Phe and tRNA Tyr , and Gm in the latter, indicates multiple structural forms for the regions of the molecule probed. These multiple forms coalesced to a single structure upon the addition of MgCl 2 . The studies on carbon NMR signals of yeast tRNA Phe are correlated with their corresponding proton resonances via a two-dimensional carbon-proton correlation experiment. Although analysis of the protein perturbation of tRNA conformation is difficult, studies of other effectors of tRNA site-specific structure are quite successful.