Méthylations biologiques chez le crabe Carcinus maenas; inhibition in vitro par une fraction purifiée extraite des glandes androgènes

Extracts from muscles, testis, seminal vesicles and ovaries of the Crab, Carcinus maenas, have been studied in vitro, in presence of [¹⁴C]-methyl S-adenosylmethionine, with an E. coli tRNA as methyl acceptor. The highest level of methylases is found in the testis. It has been reported previously that a purified fraction extracted from the androgenic glands of Carcinus maenas inhibits the vitellogenesis in ovaries. We now show that the same fraction inhibits tRNA methylation in an extract of testis as methylase; a 50 per cent inhibition is obtained with about 10 μg of a purified fraction corresponding to 15 glands. With an enzymatic preparation from the ovaries, a 50 per cent inhibition of the tRNA methylase is observed with the purified extract from 4 glands.     

A smiple specific assay for uracil tRNA methylases of enteric bacteria: use of ribothymidine-deficient tRNA

A simple quantitative assay that is about 95% specific for uracil tRNA methylases of E. coli and A. aerogenes has been developed. tRNA was isolated from a strain of E. coli carrying the trm^? mutation. These organisms have a low level of uracil methylase and consequently produce tRNA with a selective deficiency of ribothymidine. This RNA acted as a specific substrate for uracil tRNA methylases, when exposed to cell extracts from E. coli or A. aerogenes containing tRNA-methylating enzymes of multiple specificities. This assay can be used to screen organisms for trm^? mutations and for studies with inhibitors.     

Selective inhibition of uracil tRNA methylases of E. coli by ethionine.

L-ethionine has been found to inhibit uracil tRNA methylating enzymes in vitro under conditions where methylation of other tRNA bases is unaffected. No selective inhibitor for uracil tRNA methylases has been identified previously. 15 mM L-ethionine or 30 mM D,L-ethionine caused about 40% inhibition of tRNA methylation catalyzed by enzyme extracts from E. coli B or E. coli M3S (mixtures of methylases for uracil, guanine, cytosine, and adenine) but did not inhibit the activity of preparations from an E. coli mutant that lacks uracil tRNA methylase. Analysis of the 14CH3 bases in methyl-deficient E. coli tRNA after its in vitro methylation with E. coli B3 enzymes in the presence or absence of ethionine showed that ethionine inhibited 14CH3 transfer to uracil in tRNA, but did not diminish significantly the 14CH3 transfer to other tRNA bases. Under similar conditions 0.6 mM S-adenosylethionine and 0.2 mM ethylthioadenosine inhibited the overall tRNA base methylating activity of E. coli B preparations about 50% but neither of these ethionine metabolites preferentially inhibited uracil methylation. Ethionine was not competitive with S-adenosyl methionine. Uracil methylation was not inhibited by alanine, valine, or ethionine sulfoxide. It is suggested that the thymine deficiency that we found earlier in tRNA from ethionine-treated E. coli B cells, resulted from base specific inhibition by the amino acid, ethionine, of uracil tRNA methylation in vivo.     

Transfer ribonucleic acid methylase activity in adult and fœtal rat liver

Foetal rat liver extracts were found to have higher tRNA methylase activities than corresponding extracts of adult liver. When the specific activities were expressed per mg of liver or per mg of protein, the foetal tRNA methylating enzymes were respectively 2.5 and 6 times higher than those of adult livers.The presence of an inhibitor in adult liver can be excluded, since the same recoveries of total tRNA methylase activity were obtained after partial purification of both adult and foetal liver extracts: yields were close to 100 per cent.The apparent Km's for the substrates in the methylating reactions were the same when tRNA methylases from either adult or foetal liver were used: values were 0.2 μM for Escherichia coli tRNA and 2.1 μM for S-adenosyl-l-methionine.After T₁-T₂ ribonuclease digestion of an in vitro methylated tRNA, similar methyl nucleotide patterns were observed in foetal and adult enzymatic extracts.It is concluded that the same tRNA methylase pool is present in adult and foetal liver. In addition, it is hypothesized that the different reaction rates exhibited by these enzymes might be due to the tRNA functional requirements rather than to the presence of a tRNA methylase inhibitor.     

Fractionation and specificity of DNA methylases from the Escherichia coli SK cells

Summary Specificity of DNA methylation enzymes from the E. coli SK cells and conditions for their separation have been investigated. Column chromatography on carboxymethylcellulose permits fractionation of methylase activity into six discrete peaks whose specificity to the methylated base has been determined in vitro with H³-SAM as precursor. All methylases specific for adenine produced 6-methylaminopurine, all methylases specific for cytosine yielded 5-methylcytosine.The first enzymatic activity peak containing cytosine methylase free of traces of adenine-methyiating activity (E₁), and the second peak containing both the enzymes (E₂) were not adsorbed on the ion exchanger and went off the column with the effluent (column buffer). Adenine specific methylase E₂ is retarded to a small extent during the passage through the column. The second adenine methylases (W) was characterized by weak bonds with the ion exchanger and was removed when washing the column with column buffer. The elution with NaCl gradient produced successively three enzymatic activity peaks: adenine methylase (G_I), cytosine methylase (G_II), and one more adenine methylase (G_III) removed from the column by 0.16 m, 0.24 m and 0.43 m NaCl respectively.Using a new modification of the complementary methylation test, the specificity with regard to recognition site was examined for all enzymes, except for W and G_III, which were extremely unstable. The adenine methylases E₂ and G_I and the cytosine methylases E₁ and G_II were shown to recognize different sites and to be different enzymes. In view of the drastic differences in their chromatographic behaviour and physical stability, the adenine methylases W and G_III are probably also different enzymes.     

Transfer Ribonucleic Acid Methylases During the Germination of Neurospora crassa

Transfer ribonucleic acid (tRNA) methylases were studied during the germination of spores in Neurospora crassa. The total methylase capacity and base specific tRNA methylase activities were determined in extracts from cells harvested at various stages of germination. Germinated conidia have a 65% higher methylase capacity than ungerminated conidia. Three predominant methylase activities were found in the extracts, and the relative amount of each activity was different at the various stages. Enzymes from vegetative cells catalyzed significant hypermethylation of tRNA from conidia, whereas conidial enzymes were much less active on tRNA from vegetative cells. The results indicate differences in the tRNA methylase content and tRNA species of conidia and vegetative cells.     

Origin and formation of 1,7-dimethylguanosine in tRNA chemical and enzymatic methylation

tRNA chemical methylation: 1. 1,7-Dimethylguanosine was found in in vivo methylated tRNA from liver and kidney of rat after exposure to a low dose of dimethylnitrosamine (4 mg/kg body weight). 2. At 4 h after dimethylnitrosamine administration, the 1,7-dimethylguanosine:7-methylguanine ratio (product ratio) for liver and kidney tRNA was 0.017 and 0.091, respectively. At 24 h after dimethylnitrosamine administration, the product ratio was lower in both hepatic and renal tRNA. 3. When dimethylnitrosamine was given in four separate daily injections, the product ratio in hepatic tRNA 4 h after the last dose was the same as for the same total dose given by a single injection, but in renal tRNA it was lower. No dialkyl compound was found in liver and kidney tRNA 24 h after the last multiple injection. tRNA enzymatic methylation: 1. Base analyses of Escherichia coli B tRNA methylated in vitro, by using S-adenosylmethionine as physiological methyl donor and enzyme preparations from liver and kidney of normal rat, indicated that 1,7-dimethylguanosine was also a product of enzymatic methylation. 2. The amount of 1,7-dimethylguanosine formed by kidney enzyme preparation was 3-times that produced by the liver extract. 3. A second type of enzymatic methylation assay where chemically methylated tRNA was used as substrate indicated that the 7-methylguanosine residues in the nucleic acid are not the substrate of the methylase activity forming the 1,7-dimethylguanosine moieties. Analogous data were obtained for the origin of 1,7-dimethylguanosine residues in tRNA chemical methylation by dimethyl sulphate.     

tRNA methylases from HeLa cells: purification and properties of an adenine-1-methylase and a guanine-N2-methylase

Two enzymes (methylases) that catalyze the transfer of methyl groups from S-adenosyl-l-methionine to tRNA (prepared from Escherichia coli) have been partially purified from extracts of HeLa cells. One catalyzes the methylation of adenine residues of the tRNA to give 1-methyladenine units and the other is responsible for the conversion of guanine residues to N²-methylguanine and N²,N²-dimethylguanine (and may be a mixture of two enzymes). Activities of these relatively unstable enzymes could be maintained by storage at ?20 °C in the presence of 50% glycerol. Substrate specificity studies have revealed that bacterial tRNA (E. coli, Bacillus subtilis) can be used as substrate, whereas tRNA of animal origin (HeLa cells, rat liver) cannot be used. Of the specific tRNA's tested, E. coli tRNA^fMet was used as substrate by both enzymes. E. coli tRNA^Tyr was used by the adenine-1-methylase but not by the guanine-N²-methylase. The adenine-1-methylase catalyzed the transfer of approximately one methyl group per mole of either tRNA^fMet or tRNA^Tyr offered as substrate; in the presence of the guanine-N²-methylase 1 mole of E. coli tRNA^fMet accepted 1 mole of methyl. Studies with the use of both enzymes established that enzymic methylation of the guanine site of E. coli tRNA^fMet did not interfere with subsequent methylation of an adenine residue and neither did prior methylation of adenine interfere with the subsequent methylation of a guanine residue. In the presence of both enzymes, approximately 2 moles of methyl groups were accepted by 1 mole of the E. coli tRNA^fMet.     

The absence of ribothymidine in specific eukaryotic transfer RNAs. I. Glycine and threonine tRNAs of wheat embryo

Ribothymidine, generally considered a universal nucleotide in tRNA, is completely absent in five specific wheat embryo tRNAs. These consist of two species of glycine tRNA and three species of threonine tRNA. These tRNAs, all extensively purified, are acceptable substrates for $\text{E. coli}$ - ribothymidine forming-uracil methylase, which produces one mole of ribothymidine per mole of tRNA. These five tRNAs account for about 90% of the wheat embryo tRNAs which are substrates for this methylase. Nucleotide sequence analysis of one of these tRNAs, tRNA^Gly_I, confirmed both the complete absence of ribothymidine at position 23 from the 3′end, and the presence of uridine at that site instead. In addition, it is shown that methylation with $\text{E. coli}$ uracil methylase quantitatively converts uridine at position 23 to ribothymidine, while no other uridine in the molecule is affected.Using $\text{E. coli}$ uracil methylase as an assay we have detected this class of ribothymidine lacking tRNA, in each case consisting of a few specific species, in other higher organisms, such as wheat seedling, fetal calf liver and beef liver, in addition to wheat embryo. We could not detect this class of tRNA in $\text{E. coli}$ or yeast tRNA.     

Methyl-deficient mammalian transfer RNA: II. Homologous methylation in vitro of liver tRNA from normal and ethionine-fed rats: ethionine effect on 5-methyl-cytidine synthesis in vivo

Following hydroxyapatite chromatography, rat liver tRNA methylase activity was assayed with liver tRNA from normal rats and with methyl-deficient liver tRNA from ethionine-fed rats. The difference in homologous methylation between normal and methyl-deficient tRNA was maximal in certain fractions in presence of cadaverine, and much less in presence of Mg⁺⁺ or Mg⁺⁺ plus cadaverine. These methylase fractions, which contained endogenous tRNA, were used for preparative homologous methylation of added normal and methyl-deficient tRNA in presence of 30 mM cadaverine. The ¹⁴C-methylated tRNA was digested with RNase T₂ and the resulting methylated mononucleotides were characterized and quantitated after twodimensional thinlayer chromatography and autoradiography. The major products of homologous tRNA methylation were m⁵C and m¹A. However, the methylase fraction used here did not catalyze the formation of m⁶₂A with m⁶₂A-deficient tRNA as substrate.- In addition to the previously described, analytically detectable m⁶₂A-deficiency, a partial m⁵C-deficiency was demonstrated in liver tRNA from ethionine-fed rats by measuring the methylacceptance in vitro. In presence of cadaverine, with the methylase fraction used here, methyl-deficient tRNA from ethionine-fed rats was a twofold more efficient methyl-acceptor in vitro than normal liver tRNA, while endogenous tRNA isolated from the methylase fraction was a threefold more efficient methyl-acceptor than normal liver tRNA. Homologous methylation of normal tRNA, as observed here, has not been described before.     

Further investigation of the increased transfer ribonucleic acid methylase activity in tumours of the mouse colon

1. Extracts prepared from tumours of the mouse colon induced by 1,2-dimethylhydrazine were considerably more active in catalysing the methylation of tRNA than were extracts from normal colon. The enhanced activity was observed when both unfractionated ;methyl-deficient' tRNA and purified tRNA preparations from yeast and bacteria were used as substrates for methylation. 2. The methylated bases produced in these reactions were identified. There were no differences between the products of the reaction catalysed by extracts of tumour and normal colon. 3. The increased activity of tRNA methylases was not due to the presence in the extracts of stimulatory or inhibitory molecules of low molecular weight such as polyamines or S-adenosylhomocysteine. 4. Other enzymes concerned with tRNA metabolism (RNA polymerase, ATP-tRNA adenylyltransferase, aminoacyl-tRNA ligases) were also increased in activity in the tumour tissue. 5. The extent of methylation of a limiting amount of tRNA was greater when tumour extracts were compared with controls, but in no case was it possible to achieve a stoicheiometric methylation of the purified tRNA preparations used as substrates, and the tumour extracts were not able to methylate tRNA obtained from normal mouse colon. We conclude that the tumours contained greater activities of tRNA methylases but that there was no evidence for changes in the specificity of these enzymes during neoplastic growth.     

DNA methylation and Dc8-GUS transgene expression in carrot (Daucus carota L.)

Summary DNA methylation has been associated with gene activity in differentiating and developing plant tissues. The objective of this study was to determine the involvement of methylation in the expression of a gene transferred into carrot (Daucus carota L.) tissues by particle bombardment. Expression of the Dc8-GUS gene construct in response to treatments with 5-azacytidine (S-azaC) and to in vitro methylation by methylases was investigated by histochemical assay of GUS activity. The 5-azaC treatment increased the frequency of Dc8-driven GUS expression in both calli and somatic embryos. The increase occurred with treatment either to E. coli containing the plasmid insert or to the carrot tissues before bombardment. GUS expression, increased by the 5-azaC treatment, was enhanced by ABA treatment of both calli and somatic embryos and was more prominent in the latter. Increased digestion of the 5-azaC-treated plasmid DNA with EcoRII suggested that demethylation had occurred. In vitro methylation of Dc8-GUS by methylases generally resulted in a lower frequency of GUS expression. SssI methylase completely inhibited GUS expression. The level of GUS expression was correlated with the extent of methylation of the plasmid.Abbreviations ABA Abscisic Acid - 5-azaC 5-azacytidine - GUS -glucuronidase - Dc8 carrot promoter     

Protein methylation in normal and cystic fibrosis fibroblasts

Human-cultured fibroblasts contain protein methylase activities. These activities were determined and the enzymatic products were identified after acid hydrolysis of the protein substrate for protein methylases I (arginine) and III (lysine) and by organic solvent extraction of the methanol produced by alkaline treatment of the protein substrate (for the protein methylase II). A methylation of histidine residues of proteins occurs in cultured fibroblasts. Protein methylase activities were unmodified in the cystic fibrosis fibroblasts as compared to the control cells.     

The methylation of transfer ribonucleic acid during regeneration of the liver

Transfer ribonucleic acid¹ is methylated after the molecule is synthesized; at least eight enzymes are involved in the transfer of methyl groups (derived from methionine). The time courses of methylation and synthesis of tRNA during rat liver regeneration have been compared in an in vivo radioisotopic study, using 6-orotic acid-¹⁴C and ³H-methyl-L-methionine as precursors in double label pulses. Liver regeneration is a synchronized system in which biochemical events of the cell cycle are separable. Transfer RNA methylation increase precedes by several hours tRNA synthesis during regeneration, although the curves overlap. A ratio of the relative rate of methylation to the relative rate of synthesis has been made; that curve positively correlates with the rise and fall of protein synthesis during regeneration. It is clear that methylation and synthesis of tRNA are only weakly coupled; changing methyl content of the tRNA "pool" resulting from differential tRNA methylase and polymerase activities may regulate the rate of protein synthesis in the cell cycle at the translational level. The "pool sizes" of uridine monophosphate (UMP) and S-adenosylmethionine (SAM) were measured indirectly; UMP and SAM were isolated from perchloric acid supernatants and their specific activities were computed. Differential changes in radioactivity available to tRNA methylases and polymerases are not a source of artifact. That is, the control of both the synthesis and methylation of tRNA is at the enzyme level in vivo, rather than at some enzymatic step prior to those enzymatic reactions.     

A genetic and functional analysis of the unusually large variable region in the M·Alu I DNA-(cytosine C5 )-methyltransferase

The M·AluI DNA-(cytosine C5)-methyltransferase (5mC methylase) acts on the sequence 5′-AGCT-3′. The amino acid sequences of known 5mC methylases contain ten conserved motifs, with a variable region between Motifs VIII and IX that contains one or more “target-recognizing domains” (TRDs) responsible for DNA sequence specificity. Monospecific 5mC methylases are believed to have only one TRD, while multispecific 5mC methylases have as many as five. M·AluI has the second-largest variable region of all known 5mC methylases, and sequence analysis reveals five candidate TRDs. In testing whether M·AluI is in fact monospecific it was found that AGCT methylation represents only 80–90% of the methylating activity of this enzyme, while control experiments with the enzyme M·HhaI gave no unexplained activity. Because individual TRDs can be deleted from multispecific methylases without general loss of activity, a series of insertion and deletion mutants of the M·AluI variable region were prepared. All deletions that removed more than single amino acids from the variable region caused significant loss of activity; a sensitive in vivo assay for methylase activity based on McrBC restriction suggested that the central portion of the variable region is particularly important. In some cases, multispecific methylases can accommodate a TRD from another multispecific methylase, thereby acquiring an additional specificity. When TRDs were moved from a multispecific methylase into two different locations in the variable region of M·AluI, all hybrid enzymes had greatly reduced activity and no new specificities. M·AluI thus behaves in most respects as a monospecific methylase despite the remarkable size of its variable region.     

The host specificity system inEscherichia coli SK

E. coli SK has its own enzyme system providing DNA host specificity which differs from the known types of specificity inE. coli K12 andE. coli B. Modification and restriction are observed when the PBVI or PBV3 phages are transferred fromE. coli SK toE. coli B or K12 (and back).A methylase has been isolated fromE. coli SK cells and partly purified. This methylase catalyzesin vitro transfer of the labelled methyl groups from S-adenosylmethionine (SAM) to DNA of both phage and tissue origin which gives rise to 5-methylcytosine (5MC) and 6-methylaminopurine (6MAP). The methylase preparations isolated from the cells at the stationary growth have proved to be 1.5–1.7 times as active as the enzyme from the cells at the logarithmic growth stage. The extract ofE. coli SK cells infected with the phage S_D cannot methylate DNAin vitro. This fact is due tode novo synthesis of the enzyme which disintegrates SAM down to 5-methylthioadenosine (5MTA) and homoserine (HS). This enzyme is not found in the cells infected with the S_D phage in the presence of chloroamphenicole. The activity of the enzyme which disintegrates SAM is the highest between the 4th and the 5th minutes of infection. Thus it may be assumed that this enzyme, most probably, is an early virus specific protein and preventsin vivo methylation of the phage DNA.     

S-adenosylhomocysteine hydrolases as the primary target enzymes in androgen regulation of methylation complexes

tRNA methylation complexes consisting of S-adenosylmethionine (AdoMet) synthetase, tRNA methylases, and S-adenosylhomocysteine (AdoHcy) hydrolase have been prepared from rat Novikoff hepatoma cells. The existence of the ternary enzyme complex is supported by dissociation and reconstitution of the ternany tRNA methylation complexes. In rat prostate and testis, two isozymes each for AdoMet synthetase and AdoHcy hydrolase are detected. The K_m (methionine) values for the two AdoMet synthetases are 3.1 and 23.7 μm and the K_m (adenosine) values for the two AdoHcy hydrolases are 0.33 and 1.8 μm. Correspondingly, two groups of methylation complexes are detectable, sedimenting in a sucrose gradient as 7 S and 8 S. The 7 S complexes are composed of AdoMet synthetase and AdoHcy hydrolase with the higher K_m values, and the 8 S complexes are composed of the respective isozymes with the lower K_m values. tRNA methylation complexes belong to the 8 S group. In hormone-depleted rat prostates and testes following hypophysectomy, the specific activities of AdoMet synthetases, tRNA methylases, and AdoHcy hydrolases are decreased severely, but are restored promptly after administration of testosterone. Thus, methylation enzymes are responsive to the regulation by steroid hormone. AdoHcy hydrolases from hormone-depleted tissues are unstable, and ternary tRNA methylation complexes are easily dissociable into individual activities. The stability of AdoHcy hydrolases is markedly improved by testosterone, and the integrity of ternary tRNA methylation complexes is maintained in the presence of testosterone. These results suggest that AdoHcy hydrolases are the primary target enzymes in adrogen regulation of methylation complexes.     

Increased kasugamycin sensitivity in Escherichia coli caused by the presence of an inducible erythromycin resistance (erm) gene of Streptococcus pyogenes

Summary An inducible erythromycin resistance gene (erm) of Streptococcus pyogenes was introduced into Escherichia coli by transformation with a plasmid. The recipient E. coli cells were either kasugamycin sensitive (wildtype) or kasugamycin resistant (ksgA). The MIC values of erythromycin increased from 150 g/ml to>3000 g/ml for E. coli. An extract of transformed cells, particularly a high-salt ribosomal wash, contained an enzyme that was able to methylate 23S rRNA from untransformed cells in vitro; however, 23S rRNA from transformed cells was not a substrate for methylation by such an extract. 165 rRNA and 30S ribosomal subunits of either the wild type or a kasugamycin resistant (ksgA) mutant were not methylated in vitro. Transformation of E. coli by the erm-containing plasmid led to a reduction of the MIC values for kasugamycin. This happened in wild-type as well as in ksgA cells. However, in vitro experiments with purified ksgA encoded methylase demonstrated that also in erm transformed E. coli, the ksgA encoded enzyme was active in wild-type, but not in ksgA cells. It was also shown by in vitro experiments that ribosomes from erm ksgA cells have become sensitive to kasugamycin. Our experiments show that in vivo methylation of 23S rRNA, presumably of the adenosine at position 2058, leads to enhanced resistance to erythromycin and to reduced resistance to kasugamycin. This, together with previous data, argues for a close proximity of the two sites on the ribosome that are substrates for adenosine dimenthylation.Abbreviations MLS macrolide, lincosamide, streptogramin B     

Methylation of intact chromosomes by bacterial methylases in agarose plugs suitable for pulsed-field electrophoresis. Methylation of intact chromosomes in agarose by methylases

Conditions were determined for the methylation of intact yeast chromosomes by EcoRI, HhaI, and MspI bacterial methylases using an endonuclease protection assay while the chromosomes were embedded in agarose plugs suitable for transverse-field electrophoresis. Parameters were also established for the methylation of human chromosomes by EcoRI methylase. Methylation of embedded chromosomes by EcoRI methylase required prewashes with EDTA. EcoRI, HhaI, and MspI methylases showed optimal activity when nonacetylated bovine serum albumin, high levels of S-adenosylmethionine, and high levels of methylase were used. The use of bacterial methylases for methylation of embedded chromosomes will allow investigators to normalize variations in cellular DNA methylation prior to restriction and create new and rare endonuclease recognition sites which will facilitate the detection of chromosomal alterations and deletions.