Under-modified Y base in a tRHAPhe isoacceptor observed in tumor cells.

A fluorescent wye (Ye) was isolated from tRHAPhe specific to Ehrlich ascites cells. The structure was determined to be alpha-amino-beta-hydroxy-4,9,-dihydro-4,6-dimethyl-9-oxo-1-H-imidazo(1,2-alpha)purine-7-butyric acid: namely the compound lacking methyl carboxyl and methyl groups and thus is an under-modified precursor of hydroxy-Y base present in normal liver tRNAPhe.     

Incorporation of lysine into Y base of phenylalanine tRNA in Vero cells.

Vero cells, a line derived from African green monkey kidney, contains a hypermodified base, called Y, adjacent to the 3' end of the anticodon of tRNAPhe. Two types of evidence are presented suggesting that lysine is involved in biosynthesis of Y base in these cells. First, when Vero cells are starved for lysine, a new, early-eluting species of tRNAPhe which lacks the fully modified Y base can be detected by reversed phase chromatography (RPC-5). After addition of lysine to the medium, this new species disappears. Second, when these cells are grown in low-lysine medium and then exposed to [3H]lysine, radioactivity from the lysine comigrates with tRNAPhe. The Y base can be selectively excised from tRNAPhe by incubation at pH 2.9, and extracted into ethyl acetate. Thin-layer chromatography of acid-excised material from these cells reveals that lysine-derived radioactivity comigrates with genuine Y base from calf liver tRNAPhe and the acid-excised tRNA no longer contains radioactivity. These results are consistent with the model that lysine is a structural precursor of Y base in tRNAPhe of Vero cells.     

Primary structure of yeast mitochondrial DNA-coded phenylalanine-tRNA.

Mitochondrial tRNAPhe from Saccharomyces cerevisiae isolated by two-dimensional gel electrophoresis was sequenced by fingerprinting uniformly labeled 32 P-tRNA as well as by 5'-end postlabeling techniques. Its sequence was found to be: pG-C-U-U-U-U-A-U-A-G-C-U-U-A-G-D-G-G-D-A-A-A-G-C-m22G-A-U-A-A-A-phi-U-G-A-A-m1G-A-phi-U-U-A-U-U-U-A-C-A-U-G-U-A-G-U-phi-C-G-A-U-U-C-U-C-A-U-U-A-A-G-G-G-C-A-C-C-A. The secondary structure we propose, in order to maximize base pairing in the phiC stem and to allow tertiary interaction between G15 and C46, excludes U50 from base pairing giving a bulge in the phiC stem. No conclusion can be drawn concerning the endosymbiotic theory of mitochondria evolution by comparing the primary structure of mt. tRNAPhe with other sequenced tRNAsPhe. This mt.tRNAPhe lacks some of the structural elements reported to be involved in the yeast cytoplasmic phenylalanyl-tRNA ligase recognition site and cannot be aminoacylated by purified yeast cytoplasmic phenylalanyl-tRNA ligase.     

Molecular cloning and sequencing of pheU, a gene for Escherichia coli tRNAPhe.

A recombinant plasmid (designated pID2) carrying the E. coli gene for tRNAPhe has been isolated from a plasmid bank constructed by the ligation of a total EcoRI digest of E. coli K12 DNA into the EcoRI site of pACYC184 DNA. The plasmid was selected by virtue of its ability to complement a temperature-sensitive lesion in the gene (PheS) for the alpha-subunit of phenylalanyl-tRNA synthetase. Crude tRNA isolated from such transformants exhibited elevated levels of phenylalanine acceptor activity. The tRNAPhe gene has been localized within the first 300 base pairs of a 3.6 kb SalI fragment of pID2. The sequence of the gene and its flanking regions is presented.     

Identification of tertiary base pair resonances in the nuclear magnetic resonance spectra of transfer ribonucleic acid.

The low-field hydrogen-bond ring NH proton nuclear magnetic resonance (NMR) spectra of several transfer ribonucleic acids (tRNAs) related to yeast tRNAPhe have been examined in detail. Several resonances are sensitive to magnesium ion and temperature, suggesting that they are derived from tertiary base pairs. These same resonances cannot be attributed to cloverleaf base pairs as shown by experimental assignment and ring current shift calculation of the secondary base pair resonances. The crystal structure of yeast tRNAPhe reveals at least six tertiary base pairs involving ring NH hydrogen bonds, which we conclude are responsible for the extra resonances observed in the low-field NMR spectrum. In several tRNAs with the same tertiary folding potential and dihydrouridine helix sequence as yeast tRNAPhe, the extra resonances from tertiary base pairs are observed at the same position in the spectrum.     

Nucleotide sequence of tRNAPhe from the seeds of lupin (Lupinus luteus). Comparison of the major species with wheat germ tRNAPhe.

The nucleotide sequence of tRNAPhe of yellow lupin seeds (Lupinus luteus) is deduced from the composition of pancreatic and T1 ribonuclease digestion products and compared with tRNAPhe of wheat germ. Major lupin tRNAPhe, unlike pea tRNAPhe, differs from wheat germ tRNAPhe in the first base pair of stem TpsiC ("e").     

The effect of chemical modification of 3-(3-amino-3-carboxypropyl)uridine on tRNA function.

The minor base 3-(3-amino-3-carboxypropyl)uridine (acp3U) in Escherichia coli tRNAPhe was acylated with the N-hydroxysuccinimide esters of acetic, phenoxy-acetic, and naphthoxyacetic acid, as well as the ester of 5-dimethylaminonaphthalene-1-sulfonyl (dansyl)-glycine. The derivatives of tRNAPhe formed were all capable of accepting phenylalanine. There were only minor effects on the kinetic parameters of these derivatives for E. coli phenylalanyl-tRNA synthetase. There was no effect on the ability of tRNAPhe to participate in poly(U)- or poly(ACU)-directed polypeptide synthesis or in the poly(U)-stimulated binding to E. coli ribosomes. The rate of photodynamic cross-linking of 4-Srd 8 to Cyd 13 was decreased in tRNAs containing the acetyl and dansyl-glycyl derivatives of acp3U, indicating that acylation of this base may perturb the tertiary structure of the tRNA. This base in tRNAPhe does not appear to play any role in the known biological functions of tRNAPhe.     

Procedure for C2 deuteration of nucleic acids and determination of A psi 31 pseudouridine conformation by nuclear Overhauser effect in yeast tRNAPhe

Nuclear Overhauser effect (NOE) combined with semispecific deuteration provides a general strategy for identification of exchangeable protons in nucleic base pairs, and has been extended to NOEs involving purine C2 protons in tRNA. Deuterated tri-ethyl orthoformate was condensed with 5(4)-amino imidazole 4(5)-carboxamide to yield C2 deuterated hypoxanthine. C2 deuterated hypoxanthine was fed to a purine requiring mutant of yeast and C2 deuterated yeast tRNAPhe was isolated. This C2 deuterated tRNAPhe was used to identify A psi 31 and U8-A14. A psi 31 was found to be bonded through N1H. The utility of C2 deuteration in nucleic acid NMR is thus demonstrated.     

S-(1,2-dicarboxyethyl)glutathione and activity for its synthesis in rat tissues

The contents of S-(1,2-dicarboxyethyl)glutathione (DCE-GS) in several tissues of rat were determined by HPLC. The peptide was present at concentrations (nmol/g tissue) of 119 in lens, 71.6 in liver, and 27.4 in heart. It was, however, not detected in spleen, kidney, cerebrum, or cerebellum. In rat liver, DCE-GS was located primarily in the cytosolic fraction. The substrates for the enzymic synthesis of DCE-GS were GSH and L-malate. In rats, the DCE-GS-synthesizing activity was found to be highest in the liver and in the cytosol of rat liver subcellular fractions. The DCE-GS-synthesizing enzyme was partially purified from rat liver cytosolic fraction by ammonium sulfate fractionation, Phenyl Superose chromatography, hydroxyapatite chromatography, and gel filtration. The molecular mass of the enzyme was estimated to be 53 kDa by gel filtration and SDS-PAGE, showing it to be a monomeric protein. The Km values for GSH and L-malate were 2.3 and 4.0 mM at 37 degrees C, respectively. The enzyme did not utilize 1-chloro-2,4-dinitrobenzene, 1,2-dichloro-4-nitrobenzene, p-nitrophenyl bromide, trans-4-phenyl-3-buten-2-one, or p-nitrobenzyl chloride, which were substrates for previously characterized glutathione S-transferases. The isolated enzyme preparation showed no fumarase activity, which supported the conclusion that the formation of DCE-GS was not the result of a nonenzymic reaction following the synthesis of fumarate from L-malate by the isolated enzyme. The N-terminal amino acid of this polypeptide was presumably blocked since no sequence was obtained by automatic sequencing after electro-blotting onto a siliconized-glass fiber (SGF) sheet.     

Proton nuclear magnetic resonance of minor nucleosides in yeast phenylalanine transfer ribonucleic acid. Conformational changes as a consequence of aminoacylation, removal of the Y base, and codon--anticodon interaction.

The assignments of the resonances of the methyl and methylene groups belonging to the residues dihydro-uridine-16 and -17 (C5 and C6), dimethylguanosine-26, N-2-methylguanosine-10, and 7-methylguanosine-46 of yeast tRNAPhe at low temperature are reported. Observing the high-field proton NMR spectral region at different temperatures, the effects of aminoacylation, removal of the Y base, and codon-anticodon interaction on the tertiary structure of yeast tRNAPhe were investigated. The following are the results of this study. (1) The two dihydrouridine residues of tRNAPhe have different environments in aqueous solution: dihydro-uridine-16 is more shielded than dihydrouridine-17. (2) The ribothymidine residue from the fragment (47--76) of yeast tRNAPhe and from a tRNA with a partially disrupted structure exhibits multiple conformations arising from different stacking modes between the ribothymidine-54 and the guanosine-53 residue. (3) Upon aminoacylation the type of guanosine-53 interaction with ribothymidine-54 in the tRNAPhe changes. (4) Removal of the Y base from the anticodon loop of yeast tRNAPhe weakens the thermal stability of the tertiary interactions. (5) The interaction of two complementary anticodons in the absence of proteins and of ribosomes results in stabilization of the tertiary structure. Codon-anticodon interaction dependent rearrangement of the tertiary structure of yeast tRNAPhe was not observed. The spin-lattice relaxation times of the methyl and methylene groups of the minor nucleosides in yeast tRNAPhe demonstrate that the minor nucleosides undergo rotational reorientation (tau c) in the nano-second range. The observed differences in these tau c values indicate a similarity of structure of tRNAPhe in solution and in crystalline form.     

Photolabile and paramagnetic derivatives of the nucleoside X and of Escherichia coli tRNAPhe.

The synthesis of N3-[3-L-(5-azido-2-nitrobenzamido)-3-carboxypropyl]uridine (4b) and N3-[3-carboxy-3-L-(2,2,5,5-tetramethyl-3-pyrroline-3-carbonylamino)propyl]uridine Npyr-oxyl (4c) starting from the nucleoside X (4a) and the appropriate N-hydroxysuccinimide ester 1 or 2 is described. After acylation of tRNAPhe from E. coli (5a) with 1 or 2, the photolabile tRNAPhe derivative 5b and the paramagnetic tRNAPhe derivative 5c could be isolated. The position of modification in the polynucleotide chain was elucidated by comparison of the ribonuclease II/alkaline phosphatase digestion products of the substituted and unsubstituted tRNAPhe samples, and was identified as being exclusively the amino group of the nucleoside X in position 47 of E. coli tRNAPhe.     

Stereoselective metabolism of anthracene and phenanthrene by the fungus Cunninghamella elegans.

The fungus Cunninghamella elegans oxidized anthracene and phenanthrene to form predominately trans-dihydrodiols. The metabolites were isolated by reversed-phase high-pressure liquid chromatography for structural and conformational analyses. Comparison of the circular dichroism spectrum of the fungal trans-1,2-dihydroxy-1,2-dihydroanthracene to that formed by rat liver microsomes indicated that the major enantiomer of the trans-1,2-dihydroxy-1,2-dihydroanthracene formed by C. elegans had an S,S absolute stereochemistry, which is opposite to the predominately 1R,2R dihydrodiol formed by rat liver microsomes. C. elegans oxidized phenanthrene primarily in the 1,2-positions to form trans-1,2-dihydroxy-1,2-dihydrophenanthrene. In addition, a minor amount of trans-3,4-dihydroxy-3,4-dihydrophenanthrene was detected. Metabolism at the K-region (9,10-positions) of phenanthrene was not detected. Comparison of the circular dichroism spectra of the phenanthrene trans-1,2- and trans-3,4-dihydrodiols formed by C. elegans to those formed by mammalian enzymes indicated that each of the dihydrodiols formed by C. elegans had an S,S absolute configuration. The results indicate that there are differences in both the regio- and stereoselective metabolism of anthracene and phenanthrene between the fungus C. elegans and rat liver microsomes.     

Endogenous oxidative DNA base modifications analysed with repair enzymes and GC/MS technique

GC/MS technique was used to identify endogenous levels of oxidatively modified DNA bases. To avoid possible artefact formation we used Fpg and Endo III endonucleases instead of acid hydrolysis to liberate the base products from unmodified DNA samples. Several different DNA preparations were used: (i) commercial calf thymus DNA, (ii) DNA isolated from rat liver, (iii) DNA isolated from human lymphocytes and (iv) nuclei isolated from rat liver. In all DNA samples used in our assays the most efficiently removed bases by Fpg protein are FapyG and FapyA although 8-oxoG was also detected in all preparations. The amount of 8-oxoG in human lymphocytes and in rat liver DNA was 3 and 2 per 10⁷ bases, respectively. It is reasonable to postulate that the presented method is one of the techniques which should be used to reveal the enigma of endogenous, oxidative DNA damage.     

Endogenous oxidative DNA base modifications analysed with repair enzymes and GC/MS technique

GC/MS technique was used to identify endogenous levels of oxidatively modified DNA bases. To avoid possible artefact formation we used Fpg and Endo III endonucleases instead of acid hydrolysis to liberate the base products from unmodified DNA samples. Several different DNA preparations were used: (i) commercial calf thymus DNA, (ii) DNA isolated from rat liver, (iii) DNA isolated from human lymphocytes and (iv) nuclei isolated from rat liver. In all DNA samples used in our assays the most efficiently removed bases by Fpg protein are FapyG and FapyA although 8-oxoG was also detected in all preparations. The amount of 8-oxoG in human lymphocytes and in rat liver DNA was 3 and 2 per 10(7)bases, respectively. It is reasonable to postulate that the presented method is one of the techniques which should be used to reveal the enigma of endogenous, oxidative DNA damage.     

Isolation and anaylsis of (Gp)nXp sequences of rat liver 5S RNA by means of restricted ribonucleas T2 hydrolysis

Essentual difficulties arise when base number in oligoguanylic blocks and location of these blocks along the polynucleotide chain need to be determined in the course of determination of the nucleotide sequences in ribonucleic acids. To overcome this difficulty it is suggested to take advantage of a recently discovered resistance of phosphodiester bond between kethoxalated G and its 3'-neighbour against T(2) RNase hydrolysis 1,2. The approach is illustrated by analysis of 5S RNA from rat liver. Sequences of general formula (Gp)(n)Xp were isolated from T(2) RNase hydrolysate of 5 S RNA rapidly and quantitatively. The information obtained greatly facilitates the whole procedure of sequencing. It is expected that the method proposed would be effective for analysis of 5 S and 4 S RNA and for highmolecular weight fragments of ribosomal and viral RNAs.     

Tumor-specific, hypomodified phenylalanyl-tRNA is utilized in translation in preference to the fully modified isoacceptor of normal cells

Phenylalanine transfer RNA (tRNAPhe) of mammalian tissues contains the hypermodified guanine derivative Y (Wye) adjacent to the 3'-end of the anticodon and two O-methylated bases in the 5' portion of the anticodon loop. These positions are hypomodified in a variety of tumor cells including a mouse neuroblastoma. The normal and tumor-specific Phe-tRNAPhe iso-acceptors were prepared from mouse liver and mouse neuroblastoma cells and compared for their activity in incorporating phenylalanine into each phenylalanine site of rabbit globin in a reticulocyte cell-free protein synthesizing system. The hypomodified Phe-tRNAPhe of neuroblastoma cells is generally preferred to the fully modified tRNAPhe of liver in globin synthesis by about 15%. This preference is the same in the translation of both phenylalanine codons, UUC and UUU, but the ratios of incorporation by the Phe-tRNAPhe species vary from site to site within a 2-fold range. Only 2 of 16 phenylalanine residues are donated preferentially by the fully modified Phe-tRNAPhe. One such residue occurs in beta-42, the second of two tandem phenylalanine residues (both encoded by UUC), while the hypomodified isoacceptor is preferred in translation of the first residue. This result indicates that the translation of tandem residues is particularly affected by the tRNAs available. Since the tumor-specific hypomodified Phe-tRNAPhe is generally utilized preferntially, it appears that the bulky Y base and/or other modifications of normal tRNAPhe may modulate protein synthesis and that tumor cells may achieve a growth advantage if their tRNAPhe is hypomodified.     

40 S subunits from rat liver ribosomes contain two codon-dependent sites for transfer RNA

40 S subunits from rat liver ribosomes are able to bind, after heat activation, two molecules of either Phe-tRNAPhe, Ac-Phe-tRNAPhe or deacylated tRNAPhe. Addition of 60 S subunits to the quaternary complex 40 S.poly(U).(Phe-tRNAPhe)2 results in quantitative formation of (Phe)2-tRNAPhe. This indicates that the two binding sites for tRNA on 40 S subunits should be considered as the constituent of P and A sites of 80 S ribosomes.     

A phenylalanine tRNA gene from Neurospora crassa: conservation of secondary structure involving an intervening sequence.

We have isolated and sequenced a tRNAPhe gene from Neurospora crassa. Hybridization analyses suggest that trnaPhe is the only tRNA encoded on the cloned 5 kb DNA fragment. The tRNAPhe gene contains an intervening sequence 16 nucleotides in length located one nucleotide 3' to the anticodon position. The tRNAPhe coding region of Neurospora and yeast are 91% conserved, whereas their intervening sequences are only 50% identical. The pattern of sequence conservation is consistent with a proposed secondary structure for the tRNA precursor in which the anticodon is base paired with the middle of the intervening sequence and the splice points are located in adjacent single-stranded loops. The DNA sequence following the tRNAPhe coding region is similar to sequences following other genes transcribed by RNA polymerase III in that it is AT-rich and includes a tract of A residues in the coding strand. In contrast, the sequence preceding the Neurospora tRNAPhe coding region does not resemble sequences preceding other sequenced tRNA genes.