Biosynthesis of the modified nucleoside Q in transfer RNA.

During biosynthesis of the modified nucleoside Q, 7-(4,5-DIHYDROXYL-1-1-CYCLOPENTEN-3-YL-AMINOMETHYL)-7-DEAZAGUANOSINE, IN TRNA, the carbon atom at position 8 in precursor molecule guanine was expelled together with the nitrogen atom N-7 in a fashion similar to that in the biosynthesis of the nucleoside antibiotic toyocamycin.     

Isolation of Escherichia coli precursor tRNAs containing modified nucleoside Q.

Affinity chromatography based on the complex formation of the modified nucleoside Q with boronic acid has been applied to the isolation of specific tRNA precursors containing this modified nucleoside. When [32P]RNA isolated from an Escherichia coli strain containing a thermolabile ribonuclease P was chromatographed on dihydroxyboryl-substituted cellulose, the precursors for asparagine, aspartate, histidine, and tyrosine tRNA were specifically retained. All precursors were monomeric. The nucleotide sequences of four asparagine tRNA precursors were determined.     

Inhibition of nucleoside Q formation in transfer ribonucleic acid during methionine starvation of relaxed-control Escherichia coli.

The elution profiles of Asp-tRNA from unstarved and starved cultures of a relaxed-control (Rel-) strain of Escherichia coli were compared by reversed-phase chromatography. Methionine starvation results in the appearance of several additional species of Asp-tRNA which are not observed with starvation for leucine or histidine. By the criterion of cyanogen bromide-effected shifts in chromatographic elution position, a large portion of the tRNAAsp synthesized in methionine-starved cells lacks the normal Q nucleoside. By the same criterion, virtually all of the tRNAAsp from unstarved, leucine-starved, and histidine-starved cells contain Q. We conclude that methionine starvation prevents the formation of the norma Q nucleoside in Rel- E. coli.     

Detection of nucleoside Q precursor in methyl-deficient E.coli tRNA.

32P-Labeled tRNAAsn was isolated from methyl-deficient E. coli tRNA. Nucleotide sequence analysis showed that tRNAAsn contains three derivatives of the Q nucleoside, possibly Q precursors, in addition to guanosine in the first position of the anticodon. One of the Q precursors was isolated on a large scale. Its UV spectra were identical with those of normal Q, indicating that 7-deazaguanosine structure having a side chain at position C-7 is complete in the Q precursor. No radioactivity was incorporated into Q or Q precursors from either [methyl-14C]methionine, [1-14C]methionine or [U-14C]methionine, showing that methionine was not directly involved in the formation of Q.     

Demonstration of a tertiary interaction in solution between the extra arm and the D-stem in two different transfer RNA''s by NMR.

According to the X-ray structure of yeast tRNAPhe at 2.5 A resolution, a hydrogen bond is formed between m7G46 and G22. By removal of this m7G46-residue we demonstrate that this interaction is present in solution as well. Comparison of the 1H 360 MHz NMR spectra of intact yeast tRNAPhe and its m7G-excised derivative locates the position of this tertiary H-bond at 12.5 ppm downfield from DSS. Additional evidence for the presence of this interaction in solution comes from a comparison of 1H NMR spectra of E. coli tRNAf1Met and E. coli tRNAf3Met, which differ only in a single position in the extra arm. In tRNAf3Met residue 47 is a m7G-residue, whereas in tRNAf3Met it is A, resulting in the absence of the m7G47 - G23 - C13 triple interaction, characteristic of tRNAf1Met. The resonance position of this tertiary interaction in tRNAf1Met is located around -13.6 ppm, a chemical shift difference of 1.1 ppm with respect to the position observed for tRNAPhe. The origin of this chemical shift difference is discussed in relation to the structure of their respective augmented D-helices.     

Maturation of a hypermodified nucleoside in transfer RNA.

E. coli C6 rel- met- cys- was cultured in a fully supplemented medium and in media lacking cysteine or methionine. tRNA isolated from the three cultures containted, respectively, a normal complement of modified nucleosides; a deficiency in thiolated nucleosides and a deficiency in methylated nucleosides. Both sulfur-deficient tRNA and methyl-deficient tRNA contained large amounts of N-6- (delta-2-isopentenyl) adenosine and small amounts of the 2-methylthio derivative. Methyl-deficient tRNA contained, in addition a large amount of a cytokinin active, differently modified nucleoside that is believed to be a sulfur derivative of N6-(delta-2-isopentenyl) adenosine. The structure of this compound is unknown. When methly-deficient tRNA and the precusor the tRNA-Tyr su3-+ A25 were enzymatically methylated in vitro, methyl groups were incorporated into derivatives of isopentenyladenosine. These results indicate that the biosynthesis of the 2-methylthio derivative of isopentenyladenosine may occur in a sequential manner, i.e., thiolation of isopentenyladenosine followed by methylation.     

The nucleoside derivatives possessing sedative and hypnotic effects.

N3-Substituted oxopyrimidines and their related compounds were synthesized. The central nervous system (CNS) depressant activities of the compounds such as hypnotic activity and barbiturate-induced narcosis have been evaluated. N3-Benzyl, o,m,p-xylyl, alpha-phenylethyl, phenacyl substituted and related oxopyrimidine nucleosides exhibited potent hypnotic activity by intracerebroventricular (i.c.v.) administration to mice. The results indicate that hypnotic action of oxopyrimidine nucleoside derivatives might relate to functional group at the N3 position on oxopyrimidine ring and stereospecificity of sugar moiety.     

Pyrimidine nucleoside, pseudouridine, and modified nucleoside excretion by growing and resting fibroblasts.

We are examining the relationship of RNA metabolism and de novo pyrimidine synthesis as parameters of malignant transformation. These initial experiments on normal hamster embryo fibroblasts have shown that excreted nucleosides are markers for intracellular RNA metabolism. We employed affinity chromatography to concentrate the nucleosides in the medium and sensitive column chromatographic procedures to quantitatively measure them. The excretion of pyrimidine nucleoside from hamster embryo fibroblasts in sulture was found to be dependent on the growth state of the cells, with the greatest accumulation occurring cell quiescence. The major nucleoside excretion products, uridine and cytidine, were both normal end products of RNA metabolism and the major nucleoside excretion products from cultured cells. The modified nucleosides N-1-methylguanosine, N-2-methylguanosine, N-2-dimethylguanosine, N-4-acetylcytidine, N-1-methylinosine, pseudouridine, N-1-methyladenosine, N-3-methylcytidine, and 5-methyleycytidine were found, as were several unidentified nucleosides.     

Synthesis and anti-HIV activity of unusual nucleoside oxanosine derivatives.

A series of the oxanosine and carbocyclic oxanosine derivatives were synthesized to evaluate for their anti-HIV activity. Compound 1, 7 and 9 showed weak anti-HIV activities.     

Dependence of the nucleoside effect on linear energy transfer.

L929 cells were irradiated by cyclotron-produced neutrons and by 14.8 MeV monoenergetic neutrons. For comparison cells were also irradiated by 60Co gamma rays. Following irradiation cells were treated by an equimolar solution of deoxyribonucleosides, and the effect on cell survival measured. Results show that nucleoside treatment was efficient after low-LET irradiation: gamma ray survival curves were altered by deoxyribonucleosides in terms of significantly increased extrapolation numbers only, but without Do change. Cells irradiated by neutrons from either of the two sources did not respond to nucleoside treatment, and consequently their survival curves remained unaltered. These results show that the nucleoside effect does occur after low-LET irradiation, but apparently not following high-LET irradiation. Since deoxyribonucleosides as well as other cell breakdown products are released in irradiated and necrotic tumours due to massive cell destruction, such a nucleoside effect could possibly enhance the cell survival and thus effect the result of radiotherapy. Absence of the nucleoside effect in case of high-LET irradiation may therefore be an additional potential gain from neutrons in radiotherapy.     

Alkaline stability of guanosine and some of its derivatives modified by the carcinogen N-acetoxyacetylaminofluorene.

The alkaline treatment of Guo, dGuo, dGMP and denatured DNA modified by N-acetoxyacetylaminofluorene (N-AcO-AAF) was performed in 0.1 M NaOH at 40 degrees C. The kinetics of the reaction were followed by ultraviolet absorption and by chromatographic methods and were found different for the four products under study. Circular dichroism spectra show differences in the environment of acetylaminofluorene residue in these products. The alkaline treatment of Guo-AAF (and dGuo-AAF) leads to the formation of three products. These products were separated by thin layer chromatography and by HPLC and were characterized by spectroscopic methods. One is the already known unstable Guo-AF (and respectively dGuo-AF) (1). The other two products are relatively stable products of the transformation of Guo-AF (or dGuo-AF). These last ones present almost identical ultraviolet absorption spectra, but very different circular dichroism spectra.     

RNA recombination in animal and plant viruses.

An increasing number of animal and plant viruses have been shown to undergo RNA-RNA recombination, which is defined as the exchange of genetic information between nonsegmented RNAs. Only some of these viruses have been shown to undergo recombination in experimental infection of tissue culture, animals, and plants. However, a survey of viral RNA structure and sequences suggests that many RNA viruses were derived form homologous or nonhomologous recombination between viruses or between viruses and cellular genes during natural viral evolution. The high frequency and widespread nature of RNA recombination indicate that this phenomenon plays a more significant role in the biology of RNA viruses than was previously recognized. Three types of RNA recombination are defined: homologous recombination; aberrant homologous recombination, which results in sequence duplication, insertion, or deletion during recombination; and nonhomologous (illegitimate) recombination, which does not involve sequence homology. RNA recombination has been shown to occur by a copy choice mechanism in some viruses. A model for this recombination mechanism is presented.