Amount Of tRNA Research Articles

The tRNA(Gly) T10003C mutation in mitochondrial haplogroup M11b in a Chinese family with diabetes decreases the steady-state level of tRNA(Gly), increases aberrant reactive oxygen species production, and reduces mitochondrial membrane potential.

Mitochondrial diabetes originates mainly from mutations located in maternally transmitted, mitochondrial tRNA-coding genes. In a genetic screening program of type 2 diabetes conducted with a Chinese Han population, we found one family with suggestive maternally transmitted diabetes. The proband's mitochondrial genome was analyzed using DNA sequencing. Total 42 known nucleoside changes and 1 novel variant were identified, and the entire mitochondrial DNA sequence was assigned to haplogroup M11b. Phylogenetic analysis showed that a homoplasmic mutation, 10003T>C transition, occurred at the highly conserved site in the gene encoding tRNA(Gly). Using a transmitochondrial cybrid cell line harboring this mutation, we observed that the steady-state level of tRNA(Gly) significantly affected and the amount of tRNA(Gly) decreased by 97%, production of reactive oxygen species was enhanced, and mitochondrial membrane potential, mtDNA copy number and cellular oxygen consumption rate were remarkably decreased compared with wild-type cybrid cells. The homoplasmic 10003T>C mutation in the mitochondrial tRNA(Gly) gene suggested to be as a pathogenesis-related mutation which might contribute to the maternal inherited diabetes in the Han Chinese family.

Expanding a tyrosyl-tRNA synthetase assay to other aminoacyl-tRNA synthetases.

Aminoacyl-tRNA synthetases catalyze the attachment of amino acids to their cognate tRNAs. In general, aminoacyl-tRNA synthetase assays require stoichiometric amounts of tRNA, which limits their sensitivity while increasing their cost. This requirement for stoichiometric amounts of tRNA can be alleviated if the aminoacyl-tRNA product is cleaved following the tRNA aminoacylation reaction, regenerating the free tRNA substrate. This data article is related to the research article entitled “A continuous tyrosyl-tRNA synthetase assay that regenerates the tRNA substrate” in which this approach is used to develop a continuous spectrophotometric assay for tyrosyl-tRNA synthetase [1]. Here we present enzymes that can be used to cleave the aminoacyl-tRNA product for at least 16 of the 20 naturally occurring amino acids. These enzymes can be used to extend the tyrosyl-tRNA synthetase assay to other aminoacyl-tRNA synthetases.

Immunity factors for two related tRNAGln targeting killer toxins distinguish cognate and non-cognate toxic subunits

The cytoplasmic virus-like element pWR1A from Debaryomyces robertsiae encodes a toxin (DrT) with similarities to the Pichia acaciae killer toxin PaT, which acts by importing a toxin subunit (PaOrf2) with tRNA anticodon nuclease activity into target cells. As for PaT, loss of the tRNA methyltransferase Trm9 or overexpression of tRNA(Gln) increases DrT resistance and the amount of tRNA(Gln) is reduced upon toxin exposure or upon induced intracellular expression of the toxic DrT subunit gene DrORF3, indicating DrT and PaT to share the same in vivo target. Consistent with a specific tRNase activity of DrOrf3, the protein cleaves tRNA(Gln) but not tRNA(Glu) in vitro. Heterologous cytoplasmic expression identified DrOrf5 as the DrT specific immunity factor; it confers resistance to exogenous DrT as well as to intracellular expression of DrOrf3 and prevents tRNA depletion by the latter. The PaT immunity factor PaOrf4, a homologue of DrOrf5 disables intracellular action of both toxins. However, the DrT protection level mediated by PaOrf4 is reduced compared to DrOrf5, implying a recognition mechanism for the cognate toxic subunit, leading to incomplete toxicity suppression of similar, but non-cognate toxic subunits.

Tissue-Specific Differences in Human Transfer RNA Expression

Over 450 transfer RNA (tRNA) genes have been annotated in the human genome. Reliable quantitation of tRNA levels in human samples using microarray methods presents a technical challenge. We have developed a microarray method to quantify tRNAs based on a fluorescent dye-labeling technique. The first-generation tRNA microarray consists of 42 probes for nuclear encoded tRNAs and 21 probes for mitochondrial encoded tRNAs. These probes cover tRNAs for all 20 amino acids and 11 isoacceptor families. Using this array, we report that the amounts of tRNA within the total cellular RNA vary widely among eight different human tissues. The brain expresses higher overall levels of nuclear encoded tRNAs than every tissue examined but one and higher levels of mitochondrial encoded tRNAs than every tissue examined. We found tissue-specific differences in the expression of individual tRNA species, and tRNAs decoding amino acids with similar chemical properties exhibited coordinated expression in distinct tissue types. Relative tRNA abundance exhibits a statistically significant correlation to the codon usage of a collection of highly expressed, tissue-specific genes in a subset of tissues or tRNA isoacceptors. Our findings demonstrate the existence of tissue-specific expression of tRNA species that strongly implicates a role for tRNA heterogeneity in regulating translation and possibly additional processes in vertebrate organisms.

The reliability of in Vivo structure-function analysis of tRNA aminoacylation

The G·U wobble base-pair in the acceptor helix of Escherichia coli tRNA Ala is critical for aminoacylation by the alanine synthetase. Previous work by several groups probed the mechanism of enzyme recognition of G·U by a structure-function analysis of mutant tRNAs using either a cell assay (amber suppressor tRNA) or a test tube assay (phage T7 tRNA substrate and purified enzyme). However, the aminoacylation capacity of particular mutant tRNAs was about 10 4-fold higher in the cell assay. This led us to scrutinize the cell assay to determine if any parameter exaggerates the extent of aminoacylation in mutants forming substantial amounts of alanyl-tRNA Ala. In doing so, we have refined and developed experimental designs to analyze tRNA function. We examined the level of aminoacylation of amber suppressor tRNA Ala with respect to the method of isolating aminoacyl-tRNA, the rate of cell growth, the cellular levels of alanine synthetase and elongation factor TU (EF-Tu), the amount of tRNA and the characteristics of EF-Tu binding. Within the precision of our measurements, none of these parameters varied in a way that could significantly amplify cellular alanyl-tRNA Ala. A key observation is that the extent of aminoacylation of tRNA Ala was independent of tRNA Ala concentration over a 75-fold range. Therefore, the cellular assay of tRNA Ala reflects the substrate quality of the molecule for formation of alanyl-tRNA Ala. These experiments support the authenticity of the cellular assay and imply that a condition or factor present in the cell assay may be absent in the test tube assay.

The roles of the human immunodeficiency virus type 1 Pol protein and the primer binding site in the placement of primer tRNA(3Lys) onto viral genomic RNA.

Factors that modulate the placement of primer tRNA(3Lys) onto the viral RNA genome in human immunodeficiency virus type 1 (HIV-1) were investigated through analysis of reverse-transcribed products that are extended from the tRNA(3Lys) primer. Mutations were introduced into the HIV-1 pol gene to result in the appearance of a stop codon in the open reading frame of the reverse transcriptase (RT) gene. These constructs, BH10-RT1 and BH10-RT2, yielded viruses with truncated Pol proteins. Alternatively, we altered the sequences involved in frameshifting by generating the construct BH10-FS. With each of these mutated viruses, we found that the primer tRNA(3Lys) that was placed onto viral genomic RNA was present in an unextended state. In contrast, as expected, tRNA(3Lys) in the case of wild-type BH10 virus had been extended by 2 bases. Furthermore, the amount of tRNA(3Lys) that was placed onto viral RNA in mutated viruses was significantly less than that placed in the wild-type virus. We also generated a mutant within the polymerase-active site of RT (D185H) (Asp-->His) that eliminated RT polymerase activity. We found that the placement of primer tRNA(3Lys) onto viral genomic RNA was independent of enzyme function; however, the tRNA(3Lys) that was placed was present in an unextended state due to the loss of RT activity. In contrast, the elimination of protease activity through a D25A (Asp-->Ala) point mutation in the protease-active site (construct BH10-PR) did cause a drop in the efficiency of tRNA(3Lys) placement. In this situation, a proportion of the placed tRNA(3Lys) was found to be extended by 2 bases, although not to the extent found with wild-type virus (BH10), due to a decrease in RT activity associated with unprocessed Gag-Pol protein that could not be cleaved because of the loss of protease activity. We also investigated the role of the primer binding site (PBS) in the placement of tRNA(3Lys) through a series of 2-, 4-, and 8-nucleotide (nt) deletions at the 3' end of the PBS, i.e., BH10-PBS2, BH10-PBS4, and BH10-PBS8, respectively. In mutated viruses BH10-PBS2 and BH10-PBS4, the 2-base-extended form of tRNA(3Lys) was still detected. However, less primer tRNA(3Lys) was placed onto viral genomic RNA as more nucleotides were deleted until the percentage of placement seen with wild-type BH10 virus dropped to only 4% in the virus with 8 nt deleted (BH10-PBS8). Consistently, these mutated viruses possessed decreased initial replication capacity compared with that of the wild-type virus, with the extent of incapacity corresponding to the size of the deletion. However, after several days, an increase in replication potential was accompanied by a reversion to a wild-type PBS.

Suppression of the serT42 mutation with modified tRNA(1Ser) and tRNA(5Ser) genes.

Serine tRNA gene derivatives with altered anticodons were introduced to the temperature-sensitive serT42 mutant, whose tRNA(1Ser) shows a base substitution of A10 for wild type G10. When a low copy number vector-system was used, the growth and beta-galactosidase synthetic activity of the serT42 mutant were restored by complementation with the tRNA(5Ser) (T34) gene or the tRNA(1Ser) (G34) gene as well as the tRNA(1Ser) (wt) gene, but not with tRNA(5Ser) (wt), tRNA(1Ser) (A34) or tRNA(1Ser) (C34) genes at 42 degrees C. When multicopy vectors were used, the transformation even with tRNA(1Ser) (A10) gene restored the growth and beta-galactosidase synthetic activity at 42 degrees C. The tRNA(1Ser) (A10) showed no thermosensitivity in serine acceptor activity by in vitro assay. At 42 degrees C, the amount of tRNA(1Ser) (A10) in the serT42 mutant was almost the same as those in the wild type. The nucleotides in the tRNA(1Ser) (A10) were found to be fully modified like those in the wild type tRNA(1Ser). Both of the tRNAs transcribed from tRNA(5Ser) (T34) and tRNA(1Ser) (G34) genes showed serine acceptor activity. Modified nucleosides of these tRNAs were also analyzed.

Transcriptional patterns of the mutL-miaA superoperon of Escherichia coli K-12 suggest a model for posttranscriptional regulation

The complex amiB-mutL-miaA-hfq-hflX-hflK-hflC superoperon of E coli contains important genes for several fundamental cellular processes, including cell-wall hydrolysis ( amiB), DNA repair ( mutL), tRNA modification ( miaA) and proteolysis ( hflX-hflK-hflC). We report here the transcriptional pattern and possible posttranscriptional regulation of mutL, miaA and hfq genes of this superoperon. RNase protection analysis of mRNA transcribed from the bacterial chromosome demonstrated that there is co-transcription of mutl and miaA. In addition, two internal promoters, P miaA and P1 hfq were identified and mapped to 201 and 837 nucleotides upstream from the respective translation start sites. P miaA contains poor matches to the −10 and −35 regions of the sigma-70 RNA polymerase concensus sequences, but it contains multiple potential Fis-binding sites and an upstream AT-rich region with poly(A) sequences. The basic arrangement of Fis-binding sites followed by an AT rich region is shared with promoters for rRNA operons and some of the tRNA and tRNA modification genes. As part of an initial study of mutL and miaA regulation, we measured transcript amounts in isogenic rne, rnc and rne rnc double mutants which are deficient in RNase E, RNase III or both. The amounts of steady state level mutL-miaA cotranscript, P miaA transcript and Pl hfq transcript increased eight-, nine- and three-fold respectively in an rne3071 mutant when compared to the rne + parent. In contrast, amounts of the three transcripts were the same in an rnc105 mutant and its rnc + parent. These results indicate that mutL, miaA, and hfq expression could be regulated by multiple mechanisms, including degree of cotranscription from upstream genes, modulation of internal promoter strength, and by RNase E activity. A model is presented for RNase E-mediated posttranscriptional regulation that may coordinated mutL expression with replication and miaA with tRNA amounts under different growth conditions, especially during nutrient upshifts.

Relationship of the queuine content of transfer ribonucleic acids to histopathological grading and survival in human lung cancer : Huang B-S, Wu R-T, Chien K-Y. Graduate Clinical Medicine Institute, National Yang-Ming Medical College, Taipei. Cancer Res 1992;52:4696–4700

To elucidate the significance of tRNA hypomodified with queuine to the grade of malignancies in human solid tumors, the amount of tRNA having guanosine in place of queuosine was determined in human lung cancer and normal lung tissue, by exchanging the unmodified guanosine residue for [3H]guanine. The reaction is catalyzed by guanine:queuine tRNA transglycosylase. Total tRNA was extracted from 23 different lung cancer specimens and the precursor of isoacceptor tRNA that contains guanine instead of queuine in the first or wobble position of the anticodon [(Q-)tRNA] content was determined. In 12 cases the (Q-)-tRNA was determined in normal lung tissues as well. In each individual, the (Q-)tRNA content in lung cancer tissue was higher than that of the normal lung tissue. The (Q-)tRNA content was not correlated to the surgicopathological staging of the patients but was highly correlated to the histopathological classification of the tumors. The amounts of (Q-)-tRNA were 1.75 +/- 0.67 (SD), 2.36 +/- 0.89, 3.77 +/- 1.39, 5.18 +/- 2.32, and 7.65 +/- 1.34 pmol/A260 in normal, well, moderately, moderately to poorly, and poorly differentiated tumors. The difference from normal to moderately differentiated or less differentiated groups was significant (P less than 0.05). In 10 patients with (Q-)tRNA higher than 3.5 pmol/A260, their cancers relapsed and only 2 were alive after 4 years. In 11 patients with (Q-)tRNA less than 3.5 pmol/A260 in their lung cancer tissues, 7 patients were still alive without any evidence of disease, 3 were dead, and 1 had recurrence of disease. These results, taken together with other previous studies, suggest that a decreased queuosine content of tRNA may be a general feature of neoplasms and may be useful for grading malignancy and perhaps also for the prediction of survival in human lung cancer.

Ribonucleoside analysis by reversed-phase high-performance liquid chromatography

Over the past fifteen years we have developed and refined the analytical chromatographic methodologies using reversed-phase high-performance liquid chromatography and UV—photodiode array detection (RPLC—UV) for the detection and measurement of the major and modified nucleosides in nucleic acids and biological fluids. RPLC—UV nucleoside analysis as it has now evolved is a powerful new research tool to aid investigators in the fields of biochemical and biomedical research. This RPLC—UV nucleoside method can resolve more than 65 nucleosides in a single analysis with “run-to-run” peak retention variations of less than 1%. A complete nucleoside composition can be obtained from as little as 0.5 μg RNA. Identification and confirmation of nucleosides can be made from the highly reproducible retention times and from the characteristic UV spectrum from a few picomoles ( ca. 1 ng) of nucleoside. In this paper we introduce standard RPLC—UV methodologies for the analysis of nucleosides and nucleoside composition of RNAs. The chromatographic protocols and standard nucleoside columns are presented and the essential requirements necessary in the HPLC instrumentation are described. Three optimized RPLC systems were developed, each with particular emphasis placed on resolution, speed, or sensitivity. In addition, three unfractionated tRNAs were selected as sources of reference nucleosides are for assessment of the performance of the chromatography. From these tRNAs, a large array of nucleosides were characterized which are used in standardization and calibration of the method. Also discussed is the use of a diode-array detector for enhancement of the reliability of nucleoside identification and accuracy of measurement. An extended enzymatic hydrolysis protocol for the liberation of exotically modified nucleosides in tRNAs is also described. Chromatographic retention times and UV spectra for a large number of ribonucleosides are tabulated. The RPLC—UV ribonucleoside analytical protocols are capable of quantifying 31 nucleosides. Approximately 1 μg of an isoaccepting tRNA, or 20 μg of unfractioned tRNA are needed for quantitative analysis. With this amount of tRNA, the percent relative error of measurement of the four major nucleosides is less than 2%, and for the modified nucleosides about 5%. As little as 0.2 μg of pure isoaccepting tRNA can be analyzed, but at the expense of precision as at this low sample size a 20–30% relative error for modified nucleosides is to be expected. For quantitation of the modified nucleosides in rRNA, which contains much less modification than tRNAs, 10–100 μg of sample are needed per injection. This amount is within the loading capacity of a regular analytical column (25 cm × 4.6 mm silica based C 18 column). However, with this quantity injected, caution is required to ensure that the response for the four major nucleosides is within the linear range of the detector and data reduction system. Quantitative data from the analysis of 16S and 23S rRNA are given. Examples are presented of some unique and interesting applications of this nucleoside methodology to biochemical and biomedical investigations.

The detection of natural opal suppressor seryl-tRNA in Escherichia coli by the dot blot hybridization and its phosphorylation by a tRNA kinase

It was believed that there was no natural suppressor tRNA in Escherichia coli, however, it has been suggested that selC, relating to the synthesis of formate dehydrogenase of a selenoprotein [(1988) Nature 331, 723–725], codes for tRNA, even though the presence of tRNA has not yet been demonstrated. We detected the product of selC in the tRNA preparation of the E. coli MC 4100 strain by the dot blot hybridization method with a DNA probe (ACCGCTGGCGGC) corresponding to the extra arm of selC tRNA. Two hybridization peaks were found in the chromatographic pattern from Sephadex A50. The amount of tRNA was estimated to be about 0.03% of the total tRNA. The suppressor [ 3H]seryl-tRNA was phosphorylated by a tRNA kinase in E. coli B. These results suggest that the opal suppressor seryl-tRNA in E. coli should be converted to selenocysteyl-tRNA through phosphoseryl-tRNA, and occurs in vertebrates as a general phenomenon.

Comparative study of 5′ flanking sequences of eukaryotic genes: possible functional implications

We investigated the influence of the transcription product, tRNA, on the transcriptional efficiency of its structural gene in a Xenopus laevis in vitro system. Addition of tRNA to the assays leads to a decreased transcriptional efficiency. While genes with high transcriptional efficiencies react strongly to the addition of bulk tRNA, those with low efficiencies react less markedly. One of the genes tested contains an unusual 5′ flanking sequence—a tRNA-pseudogene—leading to an oversized product in this system. The amount of this product is positively influenced when a low dose of tRNA is added, but the addition of a larger amount of tRNA decreases its transcriptional efficiency. We also present a comparative computer study of published eukaryotic tRNA gene data. In most tRNA genes, there is a box consisting of four purines between two pyrimidines in the 5′ flanking sequence. This box is surrounded by or is part of repeating four-nucletide sequences. In addition there is a slight homology of the 5′ flank with its following structural gene.

Queuine in plants and plant tRNAs: Differences between embryonic tissue and mature leaves

In eubacterial and eukaryotic tRNAs specific for Asn, Asp, His and Tyr the modified deazaguanosinederivative queuosine occurs in position 34, the first position of the anticodon. Analysis of unfractionated tRNAs from wheat and from tobacco leaves shows that these tRNAs contain high amounts of guanosine (G) in place of queuosine (Q). This was measured by the exchange of G34 for [(3)H]guanine catalysed by the specific tRNA guanine transglycosylase from E. coli. Upon gel electrophoretic separation of the labeled tRNAs, seven Q-deficient tRNA species including isoacceptors are detectable. Two are identified as cytoplasmic tRNAs(Tyr) and tRNA(Asp) and two represent chloroplast tRNA(Tyr) isoacceptors. In contrast to leaf cytoplasm and chloroplasts, wheat germ has low amounts of tRNAs with G34 in place of Q.A new enzymatic assay is described for quantitation of free queuine in cells and tissues. Analysis of queuine in plant tissues shows that wheat germ contains about 200 ng queuine per g wet weight. In wheat and tobacco leaves queuine is present, if at all, in amounts lower than 10 ng/g wet weight. The absence of Q in tRNAs from plant leaves is therefore caused by a deficiency of queuine. Tobacco cells cultivated in a synthetic medium without added queuine do not contain Q in tRNA, indicating that these rapidly growing cells do not synthesize queuine de novo.

Developmental studies of the first step of the initiation of brain protein synthesis, role for initiation factor 2

Developmental changes at the level of initiation step of translation in the rat brain were studied. The level of deacylated tRNA i met in rat brain was measured at two stages of postnatal development. Although the amount of tRNA was slightly lower in adult than in young (4 day old) rats, the charging capacity of initiator tRNA i met in vitro was similar at both ages. No differences during development were found in methionyl-tRNA synthetase activity, which throws doubt on its possible participation in regulation of the initiation step. When assayed in the ribosomal salt wash protein fractions, initiation factor 2 activity decreased during brain development, and increased activities were detected in the supernatant of the microsomal fractions. The decrease in elF-2 activity paralleled the observed decrease in the rat of overall protein synthesis or initiation activity in vitro, suggesting that the regulation of the initiation step of translation during brain development may be tightly linked to changes in initiation factor 2 activity in brain tissue.

Quantitative enzymatic hydrolysis of tRNAs : Reversed-phase high-performance liquid chromatography of tRNA nucleosides

A rapid quantitative method for enzymatic hydrolysis of microgram amounts of tRNA has been developed, specifically to take full advantage of our precise, accurate, and selective reversed-phase high-performance liquid chromatographic (HPLC) system for separation and measurement of the major and modified nucleosides in tRNA. After study of several enzyme systems, nuclease P1 and bacterial alkaline phosphatase were selected and the hydrolysis parameters were systematically studied. Optimized hydrolysis conditions give quantitative hydrolysis in 2 h and this short incubation time prevents loss of unstable nucleosides. The chromatographic system can tolerate relatively high levels of protein in the sample allowing high enzyme—substrate ratios and direct injection of hydrolysates. This enzymatic hydrolysis—HPLC method is the best described to date for quantitative determination of the nucleoside composition of tRNAs and has already provided important information for investigation of the role of modification in the function of RNAs.

Cell growth and tRNA 4lys synthesis in mouse 3T3 cells

The RPC-5 chromatographic profiles of lys-tRNA were analyzed during the growth of 3T3 cells in culture. An inverse relationship was seen between tRNA 2 lys and tRNA 4 lys which was markedly influenced by medium changes. This interchange of tRNA 2 lys and tRNA 4 lys could be controlled by altering the levels of serum in the medium, or more precisely by altering the serum to cell ratio. A different change in lys-tRNA distribution was seen when the cells reached confluency. The amounts of tRNA 2 lys, tRNA 3 lys and tRNA 4 lys all decreased with a corresponding increase in either tRNA 5 lys or tRNA 6 lys. An identical change in lys-tRNA could be produced by shifting sparse cells into a medium containing 10% calf plasma instead of 10% serum. Both tRNA lys profiles and cell growth were returned to normal when the cells were returned to medium with 10% fetal calf serum (FCS) or 10% calf plasma and fibroblast growth factor (FGF). A third alteration in tRNA lys profiles was seen by the addition of cAMP to the cultures. A decrease in tRNA 5 lys and a corresponding increase in tRNA 6 lys was seen upon the addition of 10 −3 M db-cAMP and was accentuated by the simultaneous addition of 10 −3 M methyl isobutylxanthine. These data are consistent with an ordered sequence of tRNA lys modification involving tRNA 2 lys, tRNA 3 lys, tRNA 4 lys, tRNA 5B lys and tRNA 6 lys. Several of the factors which control proliferation appear to control the activity of different tRNA-modifying enzymes in this tRNA lys pathway thereby controlling the levels of tRNA 4 lys, a tRNA previously shown to correlate directly with the proliferative rate of cells.

TRNA methylation A rapid and simple method for determination of total radioactivity and methylated base distribution in the same sample

A simple and rapid method is described for the analysis of in vitro methylated tRNA. The total radioactivity is first determined after trichloroacetic acid-precipitation and filtration. Then the tRNA is hydrolyzed while still on the filter, and the bases are separated by thin-layer chromatography. This method eliminates the need for duplicate sample preparation, and has proved especially useful when the amount of tRNA and/or tRNA methylase is limited.

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.

Nicotiana glauca × Nicotiana langsdorffii tumor hybrid: growth, morphology, polyamines and nucleic acids in vitro

Callus of the tumor hybrid of Nicotiana glauca × N. langsdorffii grew better on agar in flasks than on liquid medium in flasks or on agar medium in Petri dishes. Asymmetric callus without roots produced small leaves and parenchyma cells were the most common type of cell. Few meristematic clusters were present, but these were very active during exponential and deceleration growth phases. The volume of their nucleoli, which were large and stained intensely, was used as a marker of the cell cycle. Shortly after transplantation the tissue divided synchronously, but thereafter it became asynchronous. An investigation of nucleic acids and polyamines showed that subcultures initiated a rapid synthesis and accumulation of DNA; thereafter the levels of tRNA and rRNA increased, especially in the deceleration phase, the amount of tRNA always being higher than rRNA. The polyamines putrescine and spermidine are always in larger amounts than in the normal tissue, and spermine could be detected in trace amounts. Their metabolism is correlated with arginine levels, the most important precursor of putrescine. Polyamine levels increased several fold during the deceleration phase, their increase being positively correlated with increased levels of nucleic acids, mainly during the very beginning of the subculture and, then during the deceleration phase.

Regulation of biosynthesis of aminoacyl-tRNA synthetases and of tRNA in Escherichia coli: III. Biochemical characterization of regulatory mutants affecting leucyl-tRNA synthetase levels

We have investigated the consequences of two mutations, leu X and leu Y, which affect the level of leucyl-tRNA synthetase on the amount of tRNA and other aminoacyl-tRNA synthetases and on the expression of the ilv and leu operons in Escherichia coli . Neither mutation appears to alter the cellular concentrations of other aminoacyl-tRNA synthetases or tRNA isoacceptor families. It was shown that leucyl-tRNA rather than leucyl-tRNA synthetase is the defective control element responsible for derepression of the leu and ilv operon in leu S31 strains. Steady-state levels of leucine, isoleucine, valine, ppGpp, pppGpp and leucyl-tRNA, and the rate of protein synthesis have been measured in leu S + , leu S − , leu S − leu X − , and leu S − leu Y − strains. The levels of magic spot compounds appear to correlate with the extent that protein synthesis is arrested rather than the concentration of limiting leucyl-tRNA.