Translation ability of mitochondrial tRNAsSer with unusual secondary structures in an in vitro translation system of bovine mitochondria.

BACKGROUND: Metazoan mitochondrial (mt) tRNAs are structurally quite different from the canonical cloverleaf secondary structure. The mammalian mt tRNASerGCU for AGY codons (Y = C or U) lacks the entire D arm, whereas tRNASerUGA for UCN codons (N = A, G, C or U) has an extended anti-codon stem. It has been a long-standing problem to prove experimentally how these tRNAsSer work in the mt translation system. RESULTS: To solve the above-mentioned problem, we examined their translational abilities in an in vitro bovine mitochondrial translation system using transcripts of altered tRNASer analogues derived from bovine mitochondria. Both tRNASer analogues had almost the same ability to form ternary complexes with mt EF-Tu and GTP. The D-arm-lacking tRNASer GCU analogue had considerably lower translational activity than the tRNASerUGA analogue and produced mostly short oligopeptides, up to a tetramer. In addition, tRNASerGCU analogue was disfavoured by the ribosome when other tRNAs capable of decoding the cognate codon were available. CONCLUSION: Both mt tRNASerGCU and tRNASerUGA analogues with unusual secondary structure were found to be capable of translation on the ribosome. However, the tRNASerGCU analogue has some molecular disadvantage on the ribosome, which probably derives from the lack of a D arm.

Changing the amino acid specificity of yeast tyrosyl-tRNA synthetase by genetic engineering.

In an attempt to generate mutant aminoacyl-tRNA synthetases capable of charging non-canonical amino acids, a series of yeast tyrosyl-tRNA synthetase (TyrRS) mutants was constructed by site-specific mutagenesis of putative active site residues, which were deduced by analogy with those of Bacillus stearothermophilus TyrRS. Among these mutants, one with the replacement of tyrosine at position 43 by glycine, "Y43G," was found to be able to utilize several 3-substituted tyrosine analogues as substrates for aminoacylation. The catalytic efficiency (k(cat)/K(m)) of mutant Y43G for aminoacylation with L-tyrosine was about 400-fold decreased as compared to that of the wild-type TyrRS. On the other hand, the ability to utilize 3-iodo-L-tyrosine was newly generated in this mutant TyrRS, since the wild-type TyrRS could not accept 3-iodo-L-tyrosine at all under physiological conditions. This mutant TyrRS should serve as a new tool for site-specific incorporation of non-canonical amino acids, such as those in 3-substituted tyrosine analogues, into proteins in an appropriate translation system in vivo or in vitro.

Analysis of GABA(A)- and GABA(B)-receptor mediated effects on intracellular Ca(2+) in DRG hybrid neurones.

1. Using pharmacological analysis and fura-2 spectrofluorimetry, we examined the effects of gamma-aminobutyric acid (GABA) and related substances on intracellular Ca(2+) concentration ([Ca(2+)]i) of hybrid neurones, called MD3 cells. The cell line was produced by fusion between a mouse neuroblastoma cell and a mouse dorsal root ganglion (DRG) neurone. 2. MD3 cells exhibited DRG neurone-like properties, such as immunoreactivity to microtubule-associated protein-2 and neurofilament proteins. Bath applications of capsaicin and alpha, beta-methylene adenosine triphosphate reversibly increased [Ca(2+)]i. However, repeated applications of capsaicin were much less effective. 3. Pressure applications of GABA (100 microM), (Z)-3-[(aminoiminomethyl) thio] prop-2-enoic acid sulphate (ZAPA; 100 microM), an agonist at low affinity GABA(A)-receptors, or KCl (25 mM), transiently increased [Ca(2+)]i. 4. Bath application of bicuculline (100 nM - 100 microM), but not picrotoxinin (10 - 25 microM), antagonized GABA-induced increases in [Ca(2+)]i in a concentration-dependent manner (IC(50)=9.3 microM). 5. Ca(2+)-free perfusion reversibly abolished GABA-evoked increases in [Ca(2+)]i. Nifedipine and nimodipine eliminated GABA-evoked increases in [Ca(2+)]i. These results imply GABA response dependence on extracellular Ca(2+). 6. Baclofen (500 nM - 100 microM) activation of GABA(B)-receptors reversibly attenuated KCl-induced increases in [Ca(2+)]i in a concentration-dependent manner (EC(50)=1.8 microM). 2-hydroxy-saclofen (1 - 20 microM) antagonized the baclofen-depression of the KCl-induced increase in [Ca(2+)]i. 7. In conclusion, GABA(A)-receptor activation had effects similar to depolarization by high external K(+), initiating Ca(2+) influx through high voltage-activated channels, thereby transiently elevating [Ca(2+)]i. GABA(B)-receptor activation reduced Ca(2+) influx evoked by depolarization, possibly at Ca(2+)-channel sites in MD3 cells.

Cell-free translation reconstituted with purified components.

We have developed a protein-synthesizing system reconstituted from recombinant tagged protein factors purified to homogeneity. The system was able to produce protein at a rate of about 160 microg/ml/h in a batch mode without the need for any supplementary apparatus. The protein products were easily purified within 1 h using affinity chromatography to remove the tagged protein factors. Moreover, omission of a release factor allowed efficient incorporation of an unnatural amino acid using suppressor transfer RNA (tRNA).

Anticancer molecular mechanism of 3'-ethynylcytidine (ECyd).

The antitumor ribonucleoside analogue 1-(3-C-ethynyl-beta-D-ribo-pentofuranosyl)cytosine (ECyd), synthesized in 1995, has strong antitumor activity. In mouse mammary tumor FM3A cells, ECyd was rapidly phosphorylated to ECyd-triphosphate (ECTP) as the final product, strongly inhibiting RNA synthesis. The ultimate metabolite of ECyd, ECTP, is stable in cultured FM3A cells with a half-life of 21 hr; ECyd is on a "closed" metabolic pathway to ECTP. Deaminated ECyd derivatives were minor metabolites in the cells treated with Ecyd; therefore cytidine forms probably were not converted to uridine forms at the nucleoside or nucleotide stage. The characteristics of ECyd may be important for the antitumor activity. RNA polymerase in the nucleus was inhibited competitively by ECTP; the ki value was 21 nM. ECyd induced DNA and 28S ribosomal RNA fragnetations. The cleavage pattern of rRNA resembled in that mediated by RNase L. The results suggested that RNase L related mechanisms might be involved in the antitumor activity of ECyd.

Evidence that 3'-phosphorylated polyphosphoinositides are generated at the nuclear surface: use of immunostaining technique with monoclonal antibodies specific for PI 3,4-P(2).

Phosphatidylinositol (PI) 3,4-P(2) is a phosphoinositide that has been shown to be important for signal transduction in growth factor stimulation. We have produced monoclonal antibodies specific for PI 3,4-P(2), which were able to detect PI 3,4-P(2) generated in 293T cells treated with H(2)O(2), or in MKN45/BD110 cells expressing activated PI 3-kinase in immunostaining. Prolonged treatment with 0.05% Tween 20 resulted in detection of staining not only at the plasma membrane, but also at the nuclear surface, indicating that 3'-phosphorylated phosphoinositides can be generated and function in the nucleus.

Characterization and tRNA recognition of mammalian mitochondrial seryl-tRNA synthetase.

Animal mitochondrial protein synthesis systems contain two serine tRNAs (tRNAs(Ser)) corresponding to the codons AGY and UCN, each possessing an unusual secondary structure; the former lacks the entire D arm, and the latter has a slightly different cloverleaf structure. To elucidate whether these two tRNAs(Ser) can be recognized by the single animal mitochondrial seryl-tRNA synthetase (mt SerRS), we purified mt SerRS from bovine liver 2400-fold and showed that it can aminoacylate both of them. Specific interaction between mt SerRS and either of the tRNAs(Ser) was also observed in a gel retardation assay. cDNA cloning of bovine mt SerRS revealed that the deduced amino acid sequence of the enzyme contains 518 amino acid residues. The cDNAs of human and mouse mt SerRS were obtained by reverse transcription-polymerase chain reaction and expressed sequence tag data base searches. Elaborate inspection of primary sequences of mammalian mt SerRSs revealed diversity in the N-terminal domain responsible for tRNA recognition, indicating that the recognition mechanism of mammalian mt SerRS differs considerably from that of its prokaryotic counterpart. In addition, the human mt SerRS gene was found to be located on chromosome 19q13.1, to which the autosomal deafness locus DFNA4 is mapped.

Escherichia coli proline tRNA: structure and recognition sites for prolyl-tRNA synthetase.

A major proline tRNA was purified from bulk Escherichia coli A19 tRNA by affinity chromatography with a biotinylated DNA probe. Its nucleotide sequence including modified nucleotides was determined by the post-labelling technique. In order to study the recognition sites of this proline tRNA for prolyl-tRNA synthetase, various mutant transcripts were prepared using an in vitro transcription system with T7 RNA polymerase. Based on the results of in vitro kinetic analyses of mutant transcripts, it was concluded that the second and third letters, G35 and G36, of the anticodon, G37 of the anticodon loop, the discriminator base A73, G72 of the acceptor stem, G49 and U17A that existed in the corner of an L-shaped structure are the recognition sites of proline tRNA for prolyl-tRNA synthetase.

Cytotoxic mechanisms of inhibitor of RNA synthesis, 1-(3-C-ethynyl-beta-D-ribo-pentofuranosyl)cytosine (ECyd, TAS-106).

We investigated the molecular mechanisms of cell death induced by 1-(3-C-ethynyl-beta-D-ribo-pentofuranosyl)cytosine (ECyd, TAS-106: Figure 1), a potent inhibitor of RNA synthesis, using mouse mammary tumor FM3A cells and human fibrosarcoma HT1080 cells. ECyd induced the characteristics of apoptosis on these cells, such as morphological changes, DNA fragmentations and caspase-3-like protease activation. General caspases inhibitor (Z-Asp-CH2-DCB) inhibited cell death. Interestingly, we also found that ECyd induced rRNA fragmentation with the size of 3.2, 2.8 and 1.5 kb, and which might be caused by inhibition of RNA synthesis. rRNA fragmentation was mainly occurred in D8 domain of 28 S rRNA, and the end of 5'-terminal sequence of 1.5 kb fragment was C3220pC3221p or C3221pG3222p, that was identical to the recognition sequence of RNase L. Furthermore, the fragmentation patterns of rRNA digested with RNase L resembled that of ECyd treated cells in shape. These results indicate that antitumor mechanisms of ECyd are involved in activation of RNase L. rRNA fragmentation may be one of the death events as a result of inhibition of RNA synthesis and play an important role in the antitumor activity of ECyd.

Cytotoxic mechanism of 1-(3-C-ethynyl-beta-D-ribo-pentofuranosyl)cytosine (ECyd).

The molecular mechanism of cell death induced by 1-(3-C-ethynyl-beta-D-ribo-pentofuranosyl)cytosine (ECyd: Figure 1), a potent inhibitor of RNA synthesis, was performed using mouse mammary tumor FM3A cells and human fibrosarcoma HT1080 cells. ECyd induced the characteristics of apoptosis on these cells, such as morphological changes, DNA fragmentations (Figure 2), and caspase-3-like protease activation. General caspases inhibitor (Z-Asp-CH2-DCB) inhibited these changes and cell death. We also found that ECyd induced DNA and 28S ribosomal RNA (rRNA) fragmentations. Though the mechanisms of rRNA fragmentations haven't revealed, it suggests that translational function of the treated cells should be disturbed. These results indicate that antitumor mechanism of ECyd are characteristics of apoptosis on the cells and rRNA fragmentations is one of the death events resulted inhibition of RNA synthesis.

Co-expression of yeast amber suppressor tRNATyr and tyrosyl-tRNA synthetase in Escherichia coli: possibility to expand the genetic code.

An efficient system was developed for the co-expression of a yeast tRNATyr/tyrosyl-tRNA synthetase (TyrRS) pair in Escherichia coli. Analysis of suppression patterns using several sets of E. coli and lambda phage mutants indicated that the expressed yeast suppressor tRNATyr was aminoacylated only with tyrosine by its cognate yeast TyrRS and not by E. coli TyrRS or other aminoacyl-tRNA synthetases. This extra tRNA/TyrRS pair is expected to be a key bridgehead for developing an in vivo system for the site-directed incorporation of unnatural amino acids into proteins.

Usefulness of scintigraphy with Tc-99m phytate for the diagnosis of alcoholic foamy degeneration.

PURPOSE: Alcoholic foamy degeneration (AFD) is a liver disease causing temporary hepatocyte dysfunction. The prognosis is usually good, but liver biopsy is needed for diagnosis. We report the usefulness of liver-spleen scintigraphy with the radiocolloid Tc-99m phytate for the diagnosis of AFD. PATIENTS AND METHODS: We used liver scintigraphy with Tc-99m phytate to study three patients with AFD diagnosed on the basis of findings from a liver biopsy. RESULTS: Liver-spleen scintigraphy showed hepatomegaly and splenomegaly, and bone marrow was visible, but radioisotope uptake by the liver was uniform. CONCLUSIONS: This pattern of scintigraphic findings is different from that reported for patients with alcoholic fatty livers or severe alcoholic hepatitis, and seems to be specific for AFD.

Assignment of imino proton signals of G-C base pairs and magnesium ion binding: an NMR study of bovine mitochondrial tRNA(SerGCU) lacking the entire D arm.

The mammalian mitochondrial tRNA(SerGCU) (mt tRNA[SerGCU]) has a unique structure in that it lacks the whole D arm. To elucidate its higher-order structure, we synthesized unmodified bovine mt tRNA(SerGCU) using T7 RNA polymerase and measured its 1H-NMR spectrum in the imino proton region. Although the imino proton signals heavily overlapped, we succeeded in assigning all the seven imino proton signals of the G-C base pairs by a combination of base replacement and 15N-labeling of the G residues of a whole tRNA molecule or of the 3'-half fragment. The results indicate that the tRNA possesses the secondary structure that has been supposed on the basis of biochemical studies. Analysis of the effect of the magnesium concentration on the G-C pairs suggests that the acceptor and T stems do not form a co-axial helix, and that the core region of the tRNA does not interact with magnesium ions. These features are significantly different from those of canonical tRNAs. Despite this, it is very likely that the tRNA as a whole takes a nearly L-shape tertiary structure.

Mechanistic studies of the translational elongation cycle in mammalian mitochondria.

Polyclonal antibodies have been prepared against both components of the bovine liver mitochondrial translational elongation factor Tu and Ts complex (EF-Tu x Ts(mt)). The antibodies against EF-Tu(mt) cross-react somewhat with Escherichia coli EF-Tu and wheat germ EF-1alpha. The antibodies against EF-Ts(mt) cross-react little, if at all, with E. coli EF-Ts or with EF-Ts from Euglena gracilis chloroplasts. These polyclonal antibodies have been used to investigate the relative amounts of EF-Tu(mt) and EF-Ts(mt) in bovine liver mitochondria and in cultured cells. The results of this analysis suggest that there is a 1:1 ratio of EF-Tu(mt) to EF-Ts(mt) in mammalian mitochondria. Intermediate complexes formed during the elongation cycle of protein synthesis in bovine liver mitochondria have also been investigated. The EF-Tu x Ts(mt) complex is quite resistant to dissociation by guanine nucleotides. This complex will, however, dissociate in the presence of GTP and Phe-tRNA resulting in the formation of a ternary complex comparable to that observed in prokaryotes. Kinetic data suggest that the use of the ternary complex in chain elongation increases the rate of Phe-tRNA binding to ribosomes, suggesting that it is a true intermediate in the elongation cycle. Sucrose gradient analysis indicates that the binding of EF-Tu(mt) to ribosomes can be detected in the presence of Phe-tRNA and a non-hydrolyzable analog of GTP. These results suggest that, in contrast to previous thinking, the basic features of the elongation cycle in mammalian mitochondria are quite similar to those in prokaryotes.

Changes in results of Gallium-67-citrate scanning after interferon therapy for chronic hepatitis C.

UNLABELLED: Gallium-67 scanning is useful for early diagnosis and grading of interstitial lung disease. In a study of the side effects of interferon (IFN) on the lungs of patients with chronic hepatitis C, we performed 67Ga scanning before and after IFN therapy. METHODS: The 66 subjects who underwent at least one scanning, before IFN therapy, were 8 patients with chronic persistent hepatitis (CPH), 21 with chronic aggressive hepatitis 2A (CAH-2A), 25 with chronic aggressive hepatitis 2B (CAH-2B) and 12 with cirrhosis. All had underlying hepatitis C viral infection. Of those patients, 20 were examined again within 1 mo after IFN therapy. Patients received an intravenous injection of 340 MBq 67Ga-citrate and were imaged 72 hr later. ROIs were established for anterior views of the lungs (Lu), liver (Li) and soft tissue of the upper arm as background (B). The counts per unit size of each region of interest were used in calculation of the ratios Lu/B and Li/B. RESULTS: The medians of Lu/B were 2.46 in CPH, 2.56 in CAH-2A, 2.50 in CAH-2B and 2.47 in cirrhosis. These differences were not statistically significant. The medians of Li/B were 6.42 in CPH, 6.14 in CAH-2A, 5.11 in CAH-2B and 4.03 in cirrhosis. The differences between the median Li/B of cirrhotic patients and the medians for patients with CPH, CAH-2A and CAH-2B were significant. After therapy, Lu/B was higher than before in 16 of the 20 patients and lower in the four other patients; the overall rise was significant (Wilcoxon rank-sum test). Li/B was higher than before in 11 of the 20 patients and lower in the nine other patients. CONCLUSION: IFN caused uptake of the radionuclide to increase in most patients. This method showed changes in the accumulation of 67Ga-citrate that could have been missed if the results had been inspected by eye. IFN can cause interstitial lung disease, but unlike other drugs with this side effect, the onset seems to be gradual enough to be detected quantitatively by 67Ga scanning.

Use of biotinylated-cysteinyl-tRNA as a non-RI probe in protein synthesis.

A convenient method for the preparation of biotinylated aminoacyl-tRNA to use in the non-radioisotopic (non-RI) detection of cell-free translation products was developed. After aminoacylation of E. coli tRNA(Cys) with L-cystein, its sulfhydryl group was modified with N-(6-[Biotinamide]hexyl)-3'-(2'-pyridyl dithio) propionamide or 1-Biotin amido-4-(4'-[maleimidomethyl] cyclohexane-carboxamido) butane. These biotin-labelled cysteinyl-tRNA are expected to function as the non-RI probe for protein synthesis equally to or even better than the biotinylated lysyl-tRNA which is now commercially available.

Highly specific and efficient cleavage of squid tRNA(Lys) catalyzed by magnesium ions.

Two lysine isoacceptor tRNAs corresponding to the codons AAA and AAG, respectively, were isolated from squid (Loligo bleekeri), and their nucleotide sequences were determined. During this analysis, we discovered that the tRNA with the anticodon CUU was efficiently cleaved at a specific site in the presence of magnesium ions, whereas the tRNA with the anticodon UUU was not. Cleavage occurred almost exclusively at the phosphodiester linkage between G15 and D16 (p16). The most remarkable feature of this cleavage reaction is that the end product was not a 2',3'-cyclic phosphate but was mainly a 3'-phosphate. Thus, this reaction was distinct from the well characterized cleavage of yeast tRNA(Phe) by lead and from reactions catalyzed by various other metalloribozymes. The presence of a cytidine residue at position 60 was required for efficient cleavage but was not crucial for the reaction, and the entire tRNA molecule had to be intact for this specific and efficient cleavage reaction. The present study provides evidence that there exists a new catalytic mechanism for cleavage of tRNA that exploits biologically ubiquitous ions rather than toxic, nonessential ions such as lead.

The ability of bovine mitochondrial transfer RNAMet to decode AUG and AUA codons.

The ability of bovine mitochondrial tRNA(Met) with the anticodon f5CAU (where f5C is 5-formylcytidine) to decode AUG and AUA codons was examined in a codon-dependent ribosomal binding assay. The AUG codon stimulated the binding of Met-tRNA(Met) to mitochondrial ribosomes in the presence of EF-Tu/TSmt. In contrast, the AUA codon did not promote the binding to mitochondrial Met-tRNA to the ribosome. To investigate the translation of the AUG and AUA codons more fully, an in vitro translation system from bovine liver mitochondria was developed. The activity of this system was greatly enhanced by the addition of 1 mM spermine and reached about half the activity observed with a comparable translational system from E coli. Two types of mRNA containing either AUG or AUA codons were synthesized using T7 RNA polymerase to transcribe their chemically synthesized genes. In the E coli system, the AUG-containing mRNA was translated as Met and the AUA-containing mRNA was translated as Ile. The AUG-containing mRNA but not the AUA-containing mRNA was translated as Met by the mitochondrial translational system. The process by which the AUA codon is translated as Met in the mitochondrial system remains to be clarified.

Higher-order structure of bovine mitochondrial tRNA(SerUGA): chemical modification and computer modeling.

On the basis of enzymatic probing and phylogenetic comparison, we have previously proposed that mammalian mitochondrial tRNA(sSer) (anticodon UGA) possess a slightly altered cloverleaf structure in which only one nucleotide exists between the acceptor stem and D stem (usually two nucleotides) and the anticodon stem consists of six base pairs (usually five base pairs) [Yokogawa et al. (1991) Nucleic Acids Res. 19, 6101-6105]. To ascertain whether such tRNA(sSer) can be folded into a normal L-shaped tertiary structure, the higher-order structure of bovine mitochondrial tRNA(SerUGA) was examined by chemical probing using dimethylsulfate and diethylpyrocarbonate, and on the basis of the results a tertiary structure model was obtained by computer modeling. It was found that a one-base-pair elongation in the anticodon stem was compensated for by multiple-base deletions in the D and extra loop regions of the tRNA(SerUGA), which resulted in preservation of an L-shaped tertiary structure similar to that of conventional tRNAs. By summarizing the findings, the general structural requirements of mitochondrial tRNAs necessary for their functioning in the mitochondrial translation system are considered.