Changes in de novo DNA (cytosine-5-)-methyltransferase activity in oncogenically susceptible rat target tissues induced by N-methyl-N-nitrosourea.

The activity of de novo DNA (cytosine-5-)-methyltransferase (DNA methylase) in various rat tissues after administration of a single dose of N-methyl-N-nitrosourea (MNU) has been analyzed. The total and specific activities of the DNA methylase of the brain, where tumor induction is important, are increased. In kidney, the DNA methylase activity first increases up to 16 h and decreases afterwards. Liver DNA methylase activity does not change. This organ is not susceptible to MNU induced cancers. Because organs in which the DNA methylase activity is high or increased after MNU are more prone to carcinogenesis by this compound, we argue that there is a relationship between the effects of MNU and DNA methylase activity.

Deficiency of queuine, a highly modified purine base, in transfer RNAs from primary and metastatic ovarian malignant tumors in women.

The tRNAs from rapidly growing tissues, particularly from neoplasia, often exhibit queuine deficiency. In order to check whether different kinds of ovarian tumors display queuine deficiencies we have analyzed tRNA samples from 16 ovarian malignancies. The tRNAs from histologically normal myometrium (4 samples) and myoma (6 samples) were taken as healthy tissue and benign tumor references. Queuine deficiency was determined by an exchange assay using [8-3H]guanine and tRNA:guanine transglycosylase from Escherichia coli. The mean values of queuine deficiencies in tRNAs were: 10.95 +/- 2.21 (SD) pmol/A260 in gonadal and germ cell tumors (5 cases); 23.75 +/- 7.89 pmol/A260 in primary epithelial tumors (9 cases); and 34.58 +/- 7.18 pmol/A260 in metastatic tumors (2 cases). These values displayed statistically significant differences (P = 0.0003, Kruskal-Wallis test). The queuine deficiencies in tRNAs significantly increased when moving from well-differentiated through moderately differentiated to poorly differentiated tumors, with the highest values found in poorly differentiated metastatic tumors (P = 0.0002, Kruskal-Wallis test). Queuine deficiency determination in tRNAs is proposed as a factor for clinical outcome prognosis of ovarian malignancies.

The primary structure of rabbit, calf and bovine liver tRNAPhe.

Highly purified tRNAPhe from rabbit liver, calf liver and bovine liver were completely digested with pancreatic ribonuclease and ribonuclease T1. The oligonucleotides were separated and identified. The tRNAPhe from rabbit liver and calf liver were partially cleaved with ribonuclease T1 or by action of lead acetate. We describe the analyses of the large fragments and the derivation of the primary structure of these mammalian tRNAsPhe.

Pairing properties of the methylester of 5-carboxymethyl uridine in the wobble position of yeast tRNA3Arg.

At optimum magnesium concentration (10 mM) both yeast tRNA1Arg and tRNA3Arg are able to bind to poly (A,G) and A-G-A in presence of Escherichia coli robisomes. With A-G-G only tRNA1Arg ginds, wherea tRNA3Arg (anticodon mcm5 U-C-U) is not bound. This result means that the methylcarboxymethyl substituant in position 5 of U prevents its wobble with G.

Specific cleavages of pure tRNAs by plumbous ions.

After renaturation some pure tRNAs were submitted to the action of lead acetate 1 . 10(-3) M at pH 7.3 and at 37 degrees C in the presence of either 1 M or 0.5 M NaCl. These tRNAs were specifically cleaved by Pb2+. The exact cleavage points were determined by analysing the oligonucleotides obtained from three yeast tRNAs. In 1 M NaCl, tRNA(Phe) is cleaved after the hUp17 and partially cleaved after Cp73. In 0.5 M NaCl, there are cleaveages after hUp16, hUp17 as well as a partial one after pGP1. In 1 M NaCl tRNA(Asp) is not cleaved, whereas in 0.5 M NaCl 50% of the molecules are cleaved in the anticodon region after Up35, 14% after hUp19 and 6% after hUp16. In 1 M NaCl tRNA(Val) is cleaved in the hU loop: 40% after hUp16 and 60% after CP17. The action of lead on five other pure tRNAs was studied on the analytical scale only, by polyacrylamide gel electrophoresis. They could be classified into two familites, one cleaved mainly in the hU loop, the other in the anticodon loop. The minimal concentrations of Pb2+ required for cleavage were determined for several tRNAs, the most sensitive of which, yeast tRNA(Val), being still cleaved with a concentration of 5 . 10(-6) M in 0.15 M NaCl. Although the cleavage often occurs after hUp, poly (hU) is less sensitive than poly(U). This and other results indicate that cleavages depend more on the conformation than the sequence of the polynucleotide chain, bends in the tertiary structure being lead-sensitive sites. Finally, the amino acid acceptance activities of cleaved tRNA(Phe) and tRNA(Asp) were determined.

Use of a dot blot hybridization method for identification of pure tRNA species on different membranes.

The characterization of a tRNA in purification procedures usually involves aminoacylation assays but recently, the hybridization by dot blot with specific oligonucleotides as probes has been used for the tRNA identification. We present here an optimization of a dot blot hybridization method for the tRNA detection by comparing the efficiency of eight different nylon membranes. Neutral 0.22 microns porosity membranes (Nytran, Biodine A) give the best detection efficiency when small quantities of material (less than 40 ng of tRNA) are dotted on filter; by contrast, neutral 0.45 microns porosity membranes (such as Hybond N) are the most efficient when larger quantities of tRNA are dotted on the filter. The described technique allows to detect less than 20 pg of a pure tRNA species. Its use in the identification of Saccharomyces cerevisiae initiator tRNA(Met) in counter-current distribution fractions is shown.

Primary structure of bovine liver tRNATrp.

Purified tRNATrp from bovine liver, accepting 1700 pmol tryptophan per A260nm unit, was completely digested with pancreatic ribonuclease and T1 ribonuclease. The sequences of the resulting oligonucleotides were determined and the primary structure of the tRNA was deduced. These analyses showed numerous incomplete post-transcriptional modifications, and several positions heterogenously occupied by two different nucleotides, which lead us to think that in bovine liver there exist a mixture of several tRNATrp.

A calorimetric investigation of melting of tRNAAsp from brewer's yeast.

The thermodynamics of tRNAAsp unfolding was studied using a precision scanning microcalorimeter. The overall heat of melting was found to be about 55 J/g irrespective of the ionic strength and magnesium activity. The analysis of complex melting curves obtained in the absence of Mg2+ reveals four successive two-state transitions. The first was identified as the cooperative melting of the tertiary structure and the D region and the others as the melting of individual helical arms.

The primary structure of six leucine isoacceptor tRNAs of yellow lupin seeds. The structural requirements for amber tRNA suppressor activity.

Six tRNA(Leu) isoacceptors from yellow lupin seeds were purified, sequenced, and their readthrough properties over the UAG stop codon were tested using TMV RNA as a messenger. The tested tRNAs(Leu) did not show amber suppressor activity. The partial structure of tRNA(Gln), a minor species in yellow lupin, was also determined. Comparison of the nucleotide sequence of all known isoacceptors of tRNA(Tyr), tRNA(Gln) and tRNA(Leu) from plants, mammals and ciliates enabled us to find general structural requirements for tRNA to be a UAG suppressor. From the partial sequence of lupin tRNA(Gln) we suggest that it will have readthrough properties.

Neurospora crassa mitochondrial transfer RNAs.

Total mitochondrial tRNA from Neurospora crassa was characterized by base composition analysis, one- and two-dimensional gel electrophoreses and reversed-phase chromatography on RPC5. The guanosine + cytidine content was about 43%, as compared to 60% for cytoplasmic tRNA. The modified nucleoside content was low and about the same as that of total yeast mitochondrial tRNA, though the G + C content is very different. We found psi, T, hU, t6A, m1G, M2G, m22G. Neither the eukaryotic "Y" base, nor the prokaryotic s4U were present. On two-dimensional polyacrylamide gel electropherograms about 25 species were separated. One species for phenylalanine, two for leucine and two for methionine could be located. Neurospora crassa mitochondrial tRNA does not hybridize with yeast mitochondrial DNA.

Overproduction and purification of native and queuine-lacking Escherichia coli tRNA(Asp). Role of the wobble base in tRNA(Asp) acylation.

Escherichia coli tRNA(Asp) was overproduced in E. coli up to 15-fold from a synthetic tRNA(Asp) gene placed in a plasmid under the dependence of an isopropyl-beta,D-thiogalactopyranoside-inducible promoter. Purification to nearly homogeneity (95%) was achieved after two HPLC DEAE-cellulose columns. E. coli tRNA(Asp)[G34] (having guanine instead of queuine at position 34) was obtained by the same procedure except that it was overproduced in a strain lacking the enzyme responsible for queuine modification. Nucleoside analysis showed that, except for the replacement of Q34 by G34 in mutant-derived tRNA(Asp), the base modification levels of both tRNAs are the same as those in wild-type E. coli tRNA(Asp). Kinetic properties of tRNA(Asp)[Q34] and [G34] with yeast AspRS compared to those in the homologous reactions in yeast and E. coli clearly indicate that the major identity elements are the same in both organisms: the conserved discriminant base and the anticodon triplet. In connection with this, we explored by site-directed mutagenesis the functional role of the interactions which, as revealed by the crystallographic structure, occur between the wobble base of yeast tRNA(Asp) and two residues of yeast AspRS. Their absence strongly affected aspartylation and the kd of tRNA(Asp). Each contact individually restores almost completely the wild-type acylation properties of the enzyme; thus, wobble base recognition in yeast appears to be more protected against mutational events than in E. coli, where only one contact is thought to occur at position 34.

Postlabeling: a sensitive method for studying DNA adducts and their role in carcinogenesis.

The covalent binding of xenobiotics to DNA is an important trigger of the multistage process that leads to carcinogenesis. 32P-postlabeling represents a highly sensitive method for biomonitoring exposure to genotoxic agents and for cancer risk assessment; it is capable of detecting less than one DNA adduct per human genome. Recent improvements to the technique have shown that the resistance of adducted DNA to enzyme digestion may lead to an overestimation of the number of different adducts present in a sample.

Isolation and characterization of the yeast aspartyl-tRNA synthetase gene.

A yeast genomic library in Escherichia coli, constructed by insertion of Sau3A restriction fragments into the hybrid Saccharomyces cerevisiae-E. coli plasmid pFL1, was screened by a radioimmunoassay (RIA) for colonies expressing yeast aspartyl-tRNA synthetase (AspRS). Four clones were isolated by this technique. Data obtained by Southern and restriction analysis of the inserts showed a common 3.8-kb BamHI restriction fragment which, when inserted into the plasmid pFL1, gave a positive RIA. Several controls showed that this 3.8-kb insert codes for the entire AspRS: (i) S. cerevisiae transformed by the PFL1 plasmid carrying the 3.8-kb fragment overproduces AspRS activity by a factor of ten compared to the wild-type yeast strain; and (ii) a new protein with electrophoretic behaviour similar to AspRS and immuno-reactive toward anti-AspRS appears in crude extracts of transformed yeast and E. coli.

Native bovine selenocysteine tRNA(Sec) secondary structure as probed by two plant single-strand-specific nucleases.

Two single-strand-specific nucleases, discovered in plants, have been used to investigate the secondary and tertiary structures of the native bovine liver selenocysteine tRNA(Sec). To check the possible influence of nucleotide modifications on these structures, we compared the results obtained with the fully modified tRNA to the unmodified transcript prepared by in vitro T7 transcription of the Xenopus laevis tRNA(Sec) gene. We found that the structures in solution of the native tRNA(Sec) and the transcript are very similar despite some differences in accessibility to the enzymatic probes. Indeed, the modified anticodon-loop of native bovine tRNA(Sec), containing 5-methylcarboxymethyluridine (mcm5U34) and N6-isopentenyladenosine (i6A37), is less accessible to Rn nuclease than that of the transcript: the intensity of bands representing cuts at A36 and A38 is much lower as compared to those of the transcript, whereas no cuts were found at the level of i6A37 in the anticodon loop of the native molecule. Surprisingly, the variable arm of the native molecule has been found to be more susceptible to single-strand-specific nuclease action, suggesting a looser structure of the variable arm in native bovine tRNA(Sec) than in the transcript.

In vitro enzymatic methylation of DNA substituted by N-2-aminofluorene.

Both the initial velocity and the overall methylation of DNA substituted by aminofluorene, by a rat liver DNA(cytosine-5-)-methyltransferase, are increased as compared to native DNA. The Km and Vmax of the modified DNA for the enzyme increase as a function of the extent of modification. The carcinogen may induce a secondary structure favouring the 'walking' of the enzyme along the DNA. The hypermethylation caused by this carcinogen could have a significance in gene activity, cellular differentiation and cancer induction.

Codon reading patterns in Saccharomyces cerevisiae mitochondria based on sequences of mitochondrial tRNAs.

The sequences of Saccharomyces cerevisiae mitochondrial tRNA Arg1, tRNA Arg2, tRNA Gly, tRNA Lys2, tRNA Leu amd tRNA Pro are reported. Special structural features were found in tRNA Pro, which has A8, C21, A48 instead of the constant residues U8, A21 and pyrimidine 48, and in tRNA Lys2, which has a U excluded from base-paring and bulging out from the TpsiC stem. The tRNA Arg1, tRBA Lys2 and tRNA Leu, which belong to two-codon families ending in a purine, have a modified uridine in the wobble position, which prevents misreading of C and U. It is likely to be 5-carboxymethylaminomethyluridine. tRNA Gly and tRNA Pro have an unmodified uridine in the wobble position allowing the reading of all four codons of a four-codon family. However, tRNA Arg2, which is a minor species and belongs to the CGN four-codon family, has an unmodified A in the wobble position. This unusual feature raises the problem of the mechanism by which the codons CGA, CGG and CGC are recognized.

The primary structure of yeast mitochondrial tyrosine tRNA.

The mitochondrial tyrosine tRNA from Saccharomyces cerevisiae has been sequenced. It has two interesting structural features: (i) it lacks two semi-invariant purine residues in the D-loop which are involved in tertiary interactions in the yeast cytoplasmic tRNAPhe; (ii) it has a large variable loop and therefore resembles procaryotic tRNAsTyr rather than eucaryotic cytoplasmic ones.

Kinetic properties of pure overproduced Bacillus subtilis phenylalanyl-tRNA synthetase do not favour its in vivo inhibition by ochratoxin A.

Ochratoxine A (OTA) inhibits growth of Bacillus subtilis at pHs below 7. Since OTA is a phenylalanine analogue, this effect could be due to inhibition of phenylalanine-tRNA synthetase (PheRS) by competition of this mycotoxin with the amino acid. Homogeneous PheRS was purified from Bacillus subtilis and from E. coli transformed with the PheRS gene. The latter produced about 40 times more PheRS than B. subtilis. The Km and Ki values of PheRS, respectively, for phenylalanine and OTA were measured and their concentrations within the cell determined. It appears that the concentration of OTA in the cell, in spite of a 25-fold accumulation, remained too low to significantly compete with phenylalanine. This does not suggest PheRS to be the target of OTA in cell growth and protein synthesis inhibition in Bacillus subtilis. It was also shown that the 2-3-fold increase of PheRS in OTA-treated cells is not due to phenylalanine-controlled attenuation regulation.

Yeast serine isoacceptor tRNAs: variations of their content as a function of growth conditions and primary structure of the minor tRNA(Ser)GCU.

The primary structure of Saccharomyces cerevisiae tRNA(Ser)GCU is presented (EMBL database accession No. X74268 S. cerevisiae tRNA-Ser). In addition, quantitation of the relative amounts of serine isoaccepting tRNAs in yeast grown on different media showed that the minor tRNA(Ser)GCU decreased while the major tRNA(Ser)AGA increased as the growth rate and the cellular protein content increased. The minor species, tRNA(Ser)CGA and tRNA(Ser)UGA, were not separated by our gel system, however, taken together they appeared to vary in the same way as tRNA(Ser)GCU. These data suggest a growth rate dependence of yeast tRNAs similar to that previously described for E. coli tRNAs.

Enzymatic methylation of DNA and poly(dG-dC) X poly(dG-dC) modified by 4-acetoxyaminoquinoline-1-oxide, the ultimate carcinogen of 4-nitroquinoline-1-oxide.

Both the initial velocity and the overall methylation of Ac-4HAQO modified DNA by a calf brain DNA (cytosine-5-)-methyltransferase are increased as compared to native DNA. The affinity of the modified DNA for the enzyme decreases as a function of the extent of the modification. Heat-denatured, single-stranded DNA shows exactly the opposite results: the more it is modified, the less it is methylated. The poly(dG-dC) X poly(dG-dC) modified by 4NQO is as well methylated as the non-modified one. The carcinogen may induce a tertiary structure favouring the 'walking' of the enzyme along the DNA. The hypermethylation caused by this carcinogen could have a significance in gene activity and cellular differentiation.