ISOACCEPTOR tRNAs FOR GLUTAMATE, GLUTAMINE, ASPARTATE AND ASPARAGINE IN CALF BRAIN

Abstract— Glutamyl, glutaminyl. aspartyl and asparaginyl tRNAs of calf brain were analysed by reverse phase chromatography for isoacceptor tRNAs. The radioactivity profiles revealed two peaks for gluta-mate. three for glutamine, two for aspartate and one for asparagine. Comparison of brain tRNAs with tRNAs from other sources showed that glutamate and aspartate tRNAs of brain closely resembled a majority of other tRNAs in the number and relative abundance of isoacceptors. Glutamine and asparagine tRNAs from different sources exhibited more marked differences.     

Aminoacylation of rat liver transfer RNA with homologous and heterologous enzyme systems during aging

Total tRNA extracted from livers of young (7 +/- 1 weeks), adult (40 +/- 1 weeks) and old (80 +/- 1 weeks) rats showed quantitative variation with age, being maximal in adults. Young and old animals yielded almost the same level of tRNAs. Quantitative changes in tRNAs were also observed from the study of amino acid acceptor activity using homologous enzyme i.e., aminoacyl-tRNA synthetase preparations from rat liver of the same age group. Quantitative variation followed the trend of qualitative variation. When tRNA was amino-acylated with a heterologous enzyme system, i.e., synthetase preparation from rat liver of another age group, age-related variation in aminoacyl-tRNA did not follow a pattern similar to that in the case of the homologous enzyme system. Young and adult synthetase enzymes showed maximum affinity for their homologous tRNAs but synthetases from old rat liver did not show any specific affinity for "old" tRNAs. This shows that apart from tRNAs, enzyme activity also changes with age.     

Prepubertal bovine oocyte: A negative model for studying oocyte developmental competence

To identify potential markers of maturation quality, differences in developmental capacity between cow and calf oocytes were compared in parallel with their constitutive and neosynthetic protein profiles before and after in vitro maturation (IVM). A comparison was also made between the protein profiles of follicular fluid (FF) from calf and cow ovaries. The effect of epidermal growth factor (EGF) during IVM on the subsequent development of prepubertal calf oocytes was examined. The effect of the presence of fetal calf serum (FCS) during development of embryos originating from calf oocytes was also examined. No differences were noted between the constitutive proteins of cow and calf oocytes and only a minor modification was observed before IVM in the pattern of neosynthesized proteins (presence of a band of 37 kD and a slight increase in the intensity of band of 78 kD in cow as compared to calf oocytes). However, the comparison of constitutive protein profiles from calf and cow FF demonstrated quantitative (the bands of 34 and 45 kD were more intense for cow than for calf) differences. EGF receptors (EGF-R) were demonstrated on cumulus—oocytes complexes (COCs) by immunofluorescence. There was no difference in intensity between cow and calf COCs. Furthermore, the addition of EGF during IVM of calf oocytes dramatically stimulated cumulus expansion and significantly increased the cleavage rate at 72 h post-insemination (82% vs 67%), as well as the proportion of embryos at the 5- to 8-cell stage at this time (54% vs 43%). Also, blastocyst yields at day 6 (11% vs 5%) and at day 8 (17% vs 10%) were significantly higher in the presence of EGF P < 0.05). The addition of FCS to synthetic oviduct fluid droplets at day 2 of culture (48 hpi) had no effect on cleavage, blastocyst yield, or blastocyst cell number. In conclusion, differences in developmental ability between calf and cow oocytes would appear to be not solely linked to differences in oocyte protein patterns. It is likely that the FF, which constitutes the microenvironment in which the oocyte develops, plays a major modulating role in determining the fate of the oocyte/follicle. © 1996 Wiley-Liss, Inc.     

Chromatographic Analyses of Isoaccepting tRNAs from Avian Myeloblastosis Virus

Avian myeloblastosis virus (AMV) 4S RNA was tested for amino acid acceptor activity for 18 of the 20 amino acids. A nonrandom distribution of viral tRNAs was found compared with tRNA from normal liver or from AMV-infected leukemic myeloblasts, confirming previous reports. Methionine and proline tRNAs were considerably enriched, whereas glutamic acid, glutamine, serine, tyrosine, and valine tRNAs were markedly depleted in AMV relative to homologous cellular tRNAs. The seven AMV tRNAs with the greatest amino acid acceptance capacities, which were in order methionine, proline, lysine, arginine, histidine, isoleucine, and threonine tRNAs, were compared with homologous tRNAs from leukemic myeloblasts and liver by reversed-phase 5 chromatography. Of the 25 isoaccepting chromatographic fractions identified, no tRNA species unique to AMV was detected. Only methionyl-tRNA showed a substantial quantitative variation in isoaccepting species compared with the host cell. Thus, viral selectivity for amino acid-specific tRNAs is not, generally, paralleled by selectivity for individual isoaccepting tRNA species. Qualitative differences in arginyl- and histidyl-tRNA isoaccepting species were discovered in virus and leukemic myeloblasts compared with liver. This indicates the existence of structural differences in these tRNA species which could be related to virus replication or expression.     

Kinetics of nuclear maturation and protein profiles of oocytes from prepubertal and adult cattle during in vitro maturation

The aim of this present study was to compare the kinetics of nuclear maturation between calf and cow oocytes in order to determine if there are differences between the 2 groups which could explain their disparate developmental capacity. The constitutive and neosynthetic protein patterns of cow and calf oocytes and of their corresponding cumulus cells were also compared during in vitro maturation. A total of 397 calf oocytes and 406 cow oocytes was matured in M199 + 10 ng/mL EGF. The first group of oocytes (n = 30) was immediately fixed and stained after removal from the follicle, and represent 0 h. The remaining oocytes were removed from the maturation medium at 4, 8, 12, 16, 20 and 24 h respectively. Half were denuded, fixed and stained for nuclear status; while the remainder were radiolabeled with methionine-(35S). Immediately after isolation, all the oocytes were at the GV stage. By 8 h, GVBD had occurred in most oocytes (calf: 97%; cow: 100%) and some had reached pro-metaphase I (calf: 49%; cow: 51%). By 12 h, most of the oocytes were at metaphase I (calf: 84%; cow: 94%). By 16 h, 54% of calf oocytes had reached telophase I or beyond compared with 71% of cow oocytes. This difference between the 2 groups became significant by 20 h, with 89% of cow oocytes (P < 0.05) at metaphase II and 71% of calf oocytes. By 24 h of culture, GVBD had occurred in all cases. Most oocytes completed meiosis I and were arrested at metaphase II with the first polar body extruded (calf: 72%; cow: 86%). No differences were noted in the constitutive and the neosynthetic protein profiles of cumulus cells in relation to the age of animal. Changes in neosynthetic protein patterns were observed both in cow and calf cumulus during IVM, and several proteins showed stage-specific synthesis. For the constitutive protein patterns of cow and calf oocytes, there were quantitative (38 and 40 kD) and qualitative (4, 10, 16, 17, 24, 25 and 26 kD) differences between the 2 groups. Only a few differences were observed in neosynthetic proteins between cow and calf oocytes, but there were changes in relation to nuclear status both in cow and calf oocytes. In conclusion, the difference in developmental capacity between cow and calf oocytes may be explained by a difference in the kinetics of nuclear maturation, which was significant at 20 h of culture (with 89% of cow oocytes at metaphase II and 71% of calf oocytes). At the biochemical level, our results indicate that nuclear progression during in vitro maturation of bovine oocytes is linked to changes in protein synthesis by the oocyte itself, while cumulus protein synthesis may either stimulate or modulate the process of oocyte maturation.     

Synthesis of elastin and procallagen by cells from embryonic aorta. Differences in the role of hydroxyproline and the effects of proline analogs on the secretion of the two proteins.

Transfer ribonucleic acid (tRNA) profiles in human lymphocytes stimulated by various mitogens have been compared with profiles from nonstimulated cells and from leukemic cells using reversed-phase chromatography. Comparisons of [³H]- or [¹⁴C]uridine- or [³²P]phosphate-labeled tRNAs showed that the greatest changes in tRNA composition upon phytohemagglutinin (PHA) stimulation occurred in the first 8 h after mitogen addition. Stimulation of lymphocytes by pokeweed mitogen, anti-human immunoglobulin, or bacterial lipopolysaccharide resulted in tRNA species which showed distinct differences from each other and also from the tRNAs produced by phytohemagglutinin stimulation. Leukemic lymphocyte tRNAs showed the most extensive differences in profile when compared with chromatograms from non-neoplastic cells stimulated by a variety of mitogens. Specific isoaccepting species of tyrosyl-, aspartyl-, and phenylalanyl-tRNAs were also compared in PHA-stimulated and resting lymphocytes and no differences were found. When these same species were studied in leukemic cells, tyrosyl-tRNA profiles were shifted to elute at a lower salt concentration, while the aspartyl-tRNA profile showed a new peak not present in noncancerous cells.     

Comparison of rat liver and Walker 256 carcinosarcoma tRNAs.

The complete nucleotide sequences of both rat liver and Walker 256 mammary carcinosarcoma tRNAAsn reveal that they are identical except for the nucleotide present in the wobble position of the anticodon loop. The rat liver tRNAAsn contains the Q nucleoside, whereas the tumour tRNAAsn contains an unmodified guanosine. The tRNAs from both tissues also show significant quantitative differences in the chromatographic mobilities for isoaccepting species of tRNAAsp, tRNAAsn, tRNAHis and tRNATyr. In addition, chromatographic shifts upon cyanogen bromide treatment and analyses of the alkaline hydrolysates of these tRNAs demonstrate that those of tumour origin contain significantly less Q and Q nucleoside than their normal rat liver counterparts.     

Nucleotide sequences of animal mitochondrial tRNAs(Met) possibly recognizing both AUG and AUA codons.

To elucidate the role of modified nucleosides of tRNA in mitochondrial translation systems, especially with regard to their codon recognition, we purified mitochondrial tRNAs(Met) isolated from liver of frog, chicken and rat, and determined their nucleotide sequences. All of these tRNAs(Met) were found to possess 5-formylcytidine in the first letter of the anticodon, which is known to be prerequisite for bovine mt tRNA(Met) to decode AUA codon as well as AUG codon. These tRNA possesses two pseudeuridines in similar positions, and only chicken tRNA(Met) had ribothymidine at the first position of the T-loop, which is always found in the usual tRNAs. Considering that AUA codon is used as five times frequently as AUG codon in these animal mitochondrial genomes, it is deduced that 5-formylcytidine at the wobble position is essential for the recognition of both AUA and AUG codons.     

Antibodies to N6-(delta2-isopentenyl) adenosine and its nucleotide: interaction with purified tRNAs and with bases, nucleosides and nucleotides of the isopentenyladenosine family.

The interaction of antibodies directed toward N6-(delta2-isopentenyl)adenosine, i6Ado, or its nucleotide with related bases, nucleosides, nucleotides and purified tRNAs is described. The selectivity of the antibody preparation was tested in inhibition experiments utilizing a sensitive radioimmunoassay to quantitate the binding of [3H]i6Ado to the antibody. Purified tRNAs containing various modified nucleosides adjacent to the 3'-end of the anticodon were tested to provide information about the selectivity of the antibody preparation toward nucleotides in this position of the tRNA chain. Antibodies directed against the nucleotide hapten were used to purify tRNAs which contain i6Ado and to quantitate the amount of that nucleotide. The same order of selectivity was expressed whether the nucleotides were free or in a tRNA molecule. Interaction of the antibody with compounds from the i6Ado family demonstrated dominance of the hydrophobic isopentenyl group and the importance of positional differences of modifications.     

Reversed-phase boronate chromatography for the separation of O-methylribose nucleosides and aminoacyl-tRNAs

Boronate forms an anionic complex with the cis-2′,3′ hydroxyls of unsubstituted ribonucleosides and the 3′-terminal adenosine of unacylated tRNAs, but not with ribosesubstituted nucleosides such as 2′-O-methylnucleosides and aminoacyl-tRNAs. We have synthesized phenyl boronates with hydrophobic side chains of about 1-nm-long and coated inert 10-μm solid beads of polychlorotrifluoroethylene with this material. This matrix complexes easily with compounds containing free cis-hydroxyls, but not with their O-alkyl or O-acyl derivatives. This permits the separation of mammalian and bacterial amino-acyl-tRNAs from uncharged tRNAs and O-methyl nucleosides from ribose-unsubstituted nucleosides in one chromatographic step, as the substituted members of each group do not undergo boronate complex formation and are thus not as much retarded in passing through the column. Complex formation between ribofuranoses and the boronate matrix appears to be enhanced by the hydrophobic “tail” of the boronate compound, by the high ionic environment of the solvent, and by the hydrophobic nature of the inert support. This method of one-step purification of tRNAs on reversed-phase boronate columns has been tested for several tRNAs specific for amino acids of different hydrophobicity and ionic character. The results indicate that each tRNA tested can be purified with appreciable purity (70–95%) and high yield (80%). However, recovery of the queuine base containing aminoacyl-tRNAs is only about 6% of the applied material. Several other boronate matrices have also been synthesized using cellulose, agarose. Sepharose, or porous glass beads as the inert support with different lengths of the spacer arm. Cellulose with a 1-nm-long spacer arm is satisfactory not only for the separation of aminoacyl-tRNAs and O-methylribose nucleosides, but also for the separation as a group of tRNAs containing the base of Q, queuine. However, other inert supports are unsatisfactory because of a non-specific binding of the tRNAs.     

Size and charge heterogeneity of rat tissue ferritins.

Ferritins purified from horse spleen and from rat liver, kidney, heart and hepatoma were analyzed by quantitative polyacrylamide gel electrophoresis. From the migration characteristics of these ferritins at several gel concentrations, Ferguson plots were constructed and the molecular sizes and charges (apparent valences) together with their statistical variability were obtained by applying Rodbard computer programs to the data. Finally, ellipses were drawn describing the 95% confidence limits of these data for size and charge and were used to identify those ferritins that differed in size and/or charge. By these criteria, many of the tissue ferritins were differentiated from one another in terms of their molecular size and/or charge. Among the various tissue ferritin monomers, the molecular sizes were essentially similar (420 000-490 000) except for the two heart ferritins which were larger (530 000 and 626 000, respectively). However, the estimated charges on rat liver, kidney and hepatoma monomers (30-38 net protons per molecule) differed from that of spleen monomer (51 net protons per molecule) while the larger rat heart ferritin also had a greater charge (83 net protons) than the smaller (40 net protons). Apoferritins prepared chemically by removal of iron from the holoferritins had migration properties indistinguishable from the parent holoferritins. The migration properties of minor (dimeric) ferritin bands on the gels were compared with those of the monomer bands. The molecular sizes of the minor bands were larger than those of the major bands, and were not inconsistent with a doubling in size. However, charge differences varied, being either similar for major and minor forms (spleen ferritin), approximately twice for the minor form (rat hepatoma ferritin) or five times greater for the minor form (rat liver ferritin). These differences in behavior were confirmed by using minimally sieving gels, on which the major bands of horse spleen ferritin failed to separate whereas those of rat liver ferritin were readily separable. It is concluded that dimers of ferritins from different tissues may associate in different ways.     

Synthesis of transfer ribonucleic acids with uridine or 2'-O-methylribothymidine at position 54 in developing Dictyostelium discoideum

In amoebae of Dictyostelium discoideum the ribothymidine (rT) content of tRNA is 0.9 mol%, but decreases progressively during development into spores. To elucidate which nucleosides replace rT at position 54 in developmental tRNA we have characterized 'vegetative' and 'developmental' tRNAs from the slime mould. Specific tRNAs were separated by two-dimensional gel electrophoresis. During early developmental stages, all tRNA species that could be separated by this method were newly synthesized. A new tRNA with uridine in place of rT and having an electrophoretic mobility similar to 'vegetative' tRNAAsn was detected during the early preaggregation stage. This 'development' tRNA was also extracted from purified polysomes. When development proceeds from preaggregation to postaggregation, tRNAs accumulate with 2'-O-methylribothymidine in place of rT. We suggest that these developmental tRNAs are important for the synthesis of specific developmental proteins.     

Identification of modified nucleosides in intact transfer ribonucleic acid by pyrolysis-electron impact-collisional activation mass spectrometry.

A novel mass spectrometric method has been developed for the detection and identification of dihydrouridine, ribothymidine, 4-thiouridine, and 7-methylguanosine in Escherichia coli tRNAs. The method utilizes (a) Pyrolysis-Electron Impact-Mass Spectrometry (PYEIMS), a procedure which releases the purine and pyrimidine bases from the intact, underivatized tRNA molecule. The mass spectrum exhibits intense peaks for the bases deriving from the common nucleosides in tRNA as well as peaks of much lower intensity at mass values expected for the bases from modified components known to be present in the tRNA; and, (b) Collisional Activation Mass Spectrometry (CAMS), a technique which permits the isolation of a single ion species from a complex mass spectrum. Subsequent fragmentation of that species yields a characteristic collisional activation spectrum. Such analyses of the ion species that were presumed to originate from H2Urd, rThd, 4SUrd, and 7MeGuo in the tRNA were used to define the structure and, thus, the identity of each component. Attributes of the PYEICAMS technique are that (a) precise structural elucidation of minor nucleosides present in tRNAs at the 1 - 4% level is obtained; (b) the high order of sensitivity allows the analysis to be done on microgram amounts of tRNA; and (c) there is no requirement for enzymatic or chemical hydrolysis of the tRNA or for subsequent chromatographic separation methods.     

Ubiquity of selenium-containing tRNA in plants

Sodium[⁷⁵Se]selenite supplemented culture of Chlamydomonas, wild carrot, tobacco, bamboo, and rice cells as well as mung bean and soybean seedlings incorporated, without exception, ⁷⁵Se into tRNAs. The content of ⁷⁵Se-labeled tRNAs ranged from 0.04 to 1.89% of the total tRNAs in these seven plant species. [⁷⁵Se]tRNA samples of wild carrot and mung bean were fractionated into six or seven seleno-tRNA species by chromatography on RPC-5 column. Samples of tobacco, bamboo and Chlamydomonas each exhibited only a single seleno-tRNA species with a close interspecific resemblance in the elution position among the three samples. All these [⁷⁵Se]tRNAs contained a new, not yet identified ⁷⁵Se-labeled nucleoside, whose retention time on HPLC was distinctly different from that of the previously reported bacterial selenonucleosides. [⁷⁵Se]tRNA samples of rice, tobacco, bamboo, mung bean and Chlamydomonas also contained one or two minor ⁷⁵Se-labeled nucleosides. These results suggest that (1) selenium-containing tRNAs appear to be widespread in the plant kingdom and (2) a new, not yet characterized selenonucleoside might be universal in plants.     

Alterations of tRNA modification in mammalian systems: the effect of ethionine.

The relationship between the modification of tRNA and its ability to act as a substrate for homologous tRNA modification enzymes in vitro was studied. The tRNA extracted from the livers of rats was active as a substrate for in vitro methylation with extracts from normal rat liver 19 h after treatment with L-ethionine (35 mg/100 g/24 h). After 4 weeks of feeding a diet containing o.25% DL-ethionine, the tRNA was a poor substrate for methylation in vitro, even though it was deficient in methylated nucleosides. Only 18% and 7% of the available sites could be methylated after 67 h and 4 weeks, respectively, of ethionine treatment. 3-(3-amino-3-carboxypropyl)uridine, a nucleoside that is also synthesized from S-adenosylmethionine, was assayed in individual tRNAs by their reactivity with the N-hydroxysuccinimide ester of phenoxyacetic acid. The reactivity of tRNAIle, tRNAAsn, and tRNAThr was decreased by treatment with ethionine at 67 h as well as at 2 and 4 weeks, although no difference could be detected at 19 h.     

Alterations of purine salvage pathways during differentiation of rat heart myoblasts towards myocytes

Enzyme activities of purine catabolism and salvage, the concentrations of high-energy phosphates and the reutilisation of purine bases and purine nucleosides were studied in rat heart myoblasts and myocytes. Rat heart myoblasts H9c2(2-1) were grown in Dulbecco's modified Eagle's minimum essential medium supplemented with 10% fetal calf serum. Reduction of fetal calf serum to 2% for 1 week resulted in a differentiation into myocytes with respect to their morphological features and their enzyme pattern. In differentiated myocytes, activity of 5'-nucleotidase was increased more than 2-fold, and AMP deaminase and creatine kinase activities were more than 10-fold elevated. The concentration of creatine phosphate in differentiated myocytes was doubled compared to that in myoblasts. The uptake into myoblasts and myocytes and the incorporation into adenine nucleotides was highest using adenosine, inosine and adenine uptake rates were intermediate, and hypoxanthine was utilised least. Differentiation of myoblasts into myocytes resulted in a slightly lower overall uptake of adenosine and adenine, whereas about 40% more inosine and hypoxanthine were utilised by myocytes. Increasing the phosphate concentration in the incubation medium up to 50 mmol/l resulted in a stimulation of uptake of all purine compounds tested. This stimulation was more pronounced in myoblasts.     

Nucleotide sequences of serine tRNAs from Bacillus subtilis.

Three B. subitilis serine tRNAs were sequenced including modified nucleosides. All the serine tRNAs contained 1-methyl-adenosine in the D-loop. As other characteristic modified nucleosides, 5-methoxyuridine was found in the first letter of the anticodon in the tRNA(UGA).