Cell growth and tRNA-lys4 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 tRNA₂^lys and tRNA₄^lys which was markedly influenced by medium changes. This interchange of tRNA₂^lys and tRNA₄^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 tRNA₂^lys, tRNA₃^lys and tRNA₄^lys all decreased with a corresponding increase in either tRNA₅^lys or tRNA₆^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 tRNA₅^lys and a corresponding increase in tRNA₆^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 tRNA₂^lys, tRNA₃^lys, tRNA₄^lys, tRNA_5B^lys and tRNA₆^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 tRNA₄^lys, a tRNA previously shown to correlate directly with the proliferative rate of cells.     

Lys-tRNA4 levels and cell division in mouse 3T3 cells

tRNA₄^lys is an isoaccepting tRNA^lys which has been proposed as a necessary requirement for cell division in mammalian cells. We have measured the levels of this tRNA^lys during the growth cycle of mouse 3T3 fibroblasts. High levels of tRNA₄^lys were seen throughout exponential growth. However, a marked decrease in tRNA₄^lys occurred 24 h before the cells became confluent. This decrease was observed in three different 3T3 cell lines, but was not seen in a transformed 3T3 cell line. Trypsinization and replating of contact-inhibited cells returned tRNA₄^lys to the levels characteristic of exponential cells. Data from these and other cell lines show a direct relationship between the levels of tRNA₄^lys and the growth rate of cells in culture.     

Lys-tRNA4 and cell division : Changes in lys-tRNA during the growth of mouse L cells

The presence of one isoaccepting tRNA species, lys-tRNA₄, has been correlated with the ability of mammalian cells to carry out cell division. The levels of this tRNA were measured during the growth of mouse L cells in culture. RPC-5 profiles of the lys-tRNA population of mouse L cells were determined at 9 points during the growth of mouse L cells in culture. The level of lys-tRNA₄ was constant at 13–14% of the total lys-tRNA population during log growth, increased to 18% when the cells became confluent and then returned to 13–14% when the cells were held at confluency. These results are in contrast to other cell lines where lys-tRNA₄ decreased at confluency. Confluent cultures of mouse L cells retained full proliferative potential since the addition of medium containing 30% serum led to renewed exponential growth. Another lys-tRNA species, lys-tRNA_5A, was present in variable amounts depending upon the nutritive state of the cells. The appearance of lys-tRNA_5A correlated with a decrease in lys-tRNA₅. These data suggest that lys-tRNA_5A is a modified lys-tRNA₅ which appears when the medium becomes depleted.     

Primary colonic epithelial cell culture of the rabbit producing prostaglandins

We have established primary colonic epithelial cell culture from adult rabbits and examined effects of anti-inflammatory drugs on prostaglandin (PG) E₂ production. Colonic epithelium of adult rabbits was scraped and minced into small pieces. They were incubated for isolation in Hanks' balanced salt solution with 0.35 % collagenase and Earle's solution with 1 mM EDTA. Isolated cells were cultured in Coon's modified Ham's F-12 medium with 10 % fetal bovine serum and antibiotics on collagen coated cell wells. The medium was refed twice a week. The production of PGs was assessed by high pressure liquid chromatography (HPLC). PGE₂ and PGF_2α were measured by radioimmunoassay. Within 24 hours after inoculation, the cell clumps attached to the surface of the wells and cells began to spread out and grow. Monolayer cultures became confluent in 4 days. Phase contrast microscopy showed that these cells consisted of a homogeneous population of epithelial cells with large oval nuclei, polyhedral shape, and organized sheet-like growth pattern. HPLC profile showed synthesis of 6-keto-PGF_1α, thromboxane B₂, PGF_2α, PGE₂, and PGD₂ by cultured cells. Quantitatively, 117±7 ng/mg-protein/hour PGE₂ by 7.4±0.7 ng/mg-protein/hour PGF_2α were produced. While hydrocortisone (10⁻⁴-10⁻² M) did not show a significant effect on PGE₂ production, indomethacin (10⁻⁸-10⁻⁶ M), and 5-aminosalicylic acid (2×10⁻⁴-5×10^{−3 M}) inhibited PGE₂ production. We have established relatively convenient procedure for primary culture of colonic epithelial cells from adult rabbits. Different actions of anti-inflammatory drugs on PGE₂ synthesis suggest that these cultured cells might be a good tool for the various cellular functional studies of normal colonic epithelial cells.     

Role of mitogen-induced calcium influx in the control of the cell cycle in Balb-c 3T3 fibroblasts

The role of mitogen-activated calcium influx from the extracellular medium in the control of cell proliferation was studied in Balb-c 3T3 fibroblasts. Stimulation of serum-deprived, quiescent cells with 10% foetal calf serum (FCS) induced a long-lasting (up to 70 min elevation of intracellular free calcium concentration ([Ca²⁺]_i). Both the sustained [Ca ²⁺]_i increase and the related inward current, described in a previous paper [Lovisolo D. Munaron L. Baccino FM. Bonelli G. (1992) Potassium and calcium currents activated by foetal calf serum in Balb-c 3T3 fibroblasts. Biochim. Biophys. Acta, 1104, 73–82], could be abolished either by chelation of extracellular calcium with EGTA or by SKF 96365, an imidazole derivative that can block receptor-activated calcium channels. The effect of the abolition of these ionic signals on FCS-induced proliferation was investigated by adding either EGTA or SK&F 96365 to the culture medium during the first hours of stimulation of quiescent cells with 10% FCS. As measured after 24 h, a 22% inhibition of growth was observed when SK&F 96365 was added for the first hour, and stronger inhibitions, up to 56%, were obtained by adding the blocker for the first 2 or 4 h. Similar effects were observed with addition of 3 mM EGTA, though the inhibition was less marked for the 4 h treatment. By contrast, incubation with either substance in the next 4 h of serum stimulation did not influence cell growth, except for a slight inhibition observed when SKF 96365 was applied from the 4^th to the 8^th hour. The reduction in growth resulting from the abolition of the early calcium influx was paralleled by an accumulation of cells in the G₂/M phase. Both growth inhibition and G₂/M accumulation were reversible, since after further 24 h in 10% FCS cells had fully recovered the exponential growth. These data indicate that the early calcium influx seen in response to mitogen stimulation develops on a timescale long enough to play a significant role in cell cycle progression, and that its block in the early G1 phase can lead to a reduction of proliferation by arresting cells in later stages of the cycle.     

The influence of serum components on the growth and mutation of Chinese hamster cells in medium containing aminopterin

When seeded in small numbers in medium containing 10^?6M aminopterin and fetal calf serum, V₇₉ Chinese hamster cells required dialyzable components from the serum for growth. However, the cells grew in medium containing 10^?6M aminopterin and dialyzed serum, provided that the medium was supplemented with 10^?5M hypoxanthine and sufficient 5·10^?6M) thymidine. A growth-inhibitory property of some batches of dialyzed serum was abolished on heating the serum for 30 min at 56°. Three lines of V₇₉ cells which lacked detectable hypoxanthine-guanine phosphoribosyl transferase (HGPRT) activity were seleccted in medium containing 8-azaguanine (8-AzG). In two of these, no spontaneous reversion to the HGPRT⁺ phenotype was detectable, and these cells did not cooperate metabolically with HGPRT⁺ cells to prevent the growth of the latter in HAT medium. One of the HGPRT^? lines showed a high rate of spontaneous reversion (118/10⁵ cells) in medium containing undialyzed serum. However, in medium containing dialyzed serum the spontaneous reversion rate fell to $\text{4}\text{10}{}^5\text{cells}$, suggesting that the revertants arising in medium containing undialyzed serum were biochemically heterogeneous.     

UDP-galactose inhibition of BALB/3T12-3 cell growth : Requirement for medium galactosyltransferase activity

Incubation of BALB/3T12-3 cells with uridine diphosphate galactose (UDP-gal) resulted in a concentration-dependent inhibition of cell growth when cells were cultured in calf serum-supplemented Dulbecco's modified Eagle medium (CS-DMEM). Cell growth was completely inhibited by 5 mM UDP-gal with an ID₅₀ of 0.75 mM. This inhibitory effect was reversible. Other nucleotide-sugars, as well as galactose, glucose, and galactose-1-phosphate had no effect on cell growth. UDP-gal had no effect on cell growth when cells were cultured in heat-inactivated calf serum containing DMEM (HICS-DMEM) suggesting that a serum enzyme activity was responsible for the inhibition observed in CS-DMEM. No significant difference could be detected by descending chromatography in the degradation of UDP-gal during 96 h of incubation in CS-DMEM and in HICS-DMEM. Furthermore, the potential breakdown products of UDP-gal had no effect on cell growth when added directly to 3T12 cultures. When cells were incubated with 5 mM UDP-gal+5 mM CDP-choline (a potent inhibitor of pyrophosphatase activity), complete inhibition of cell growth was still observed. However, if cells were incubated with 5 mM UDP-gal+UDP (which inhibited calf serum galactosyltransferase activity), no inhibition of cell growth was observed over that found for UDP alone, suggesting that galactosyltransferase and not pyrophosphatase activity mediated the effect of UDP-gal on cell growth. A direct effect of UDP-gal on cells was suggested by (a) normal growth of cells in UDP-gal-conditioned medium (preincubated with UDP-gal for 24 h followed by dialysis to remove UDP-gal); (b) 3-fold greater incorporation of [³H]galactose from UDP-[³H]gal into cells grown in CS-DMEM than in HICS-DMEM. These studies suggest that the inhibition of 3T12 cell growth by exogenous UDP-gal may be due to alteration of cell surface glycoconjugates by extracellular galactosyltransferase activity.     

Relationship among the genes, enzymes, and intermediates of the biosynthetic pathway of lysine in Saccharomyces

Summary Genetic lesions of 10 of the lysine loci in Saccharomyces have been provisionally correlated with the various enzymatic steps of the homocitric acid pathway for the biosynthesis of lysine. The characterization of mutational blocks in different mutants was made on the basis of the accumulation of appropriate intermediates, lack of enzymatic activity, and feeding experiments. Genetic lesions in mutants lys ₄, lys₇, lys₈, and lys ₁₂ correspond to the biochemical steps between homocitric acid and -amino-adipic acid. Similarly the genetic lesions in lys ₁, lys₂, lys₅, lys₉, lys₁₃, and lys ₁₄ correspond to the intermediate steps between -aminoadipic acid and lysine. Lysine-genes are not clustered, instead are mapped on different chromosomes of Saccharomyces. Some of the intermediate steps in the biosynthesis of lysine correspond to two or more unlinked genes.Supported by NSF Grant GB 28558X, Lilly Research Foundation, and Faculty Research Committee, Miami University.     

Estrogen induction of progestophilins in rat estrogen-sensitive cells grown in media supplemented with sera from castrated rats and from rats bearing an α-fetoprotein-secreting hepatoma

The purpose of this work was to study the effect of α-fetoprotein (AFP) over cell multiplication and the induction of an estradiol-17β (E₂)-dependent marker, i.e., progestophilins in E-sensitive cells C₂9RAP derived from a W/Fu rat pituitary tumor. These cells proliferate in isogeneic hosts under the influence of E₂, while they proliferate in culture regardless of the presence of E₂. C₂9RAP cells were grown in medium supplemented with 10% horse serum. Progestophilin levels were measured 48 h after adding serum (20% horse, or castrated rat, or AFP-secreting tumor-bearing rat) and estrogen to the 10% horse serum-supplemented medium in which the cells were growing. Maximal induction of progestophilins was obtained at 3 × 10⁻¹⁰ M E₂ in cells grown in medium containing horse or castrated rat serum. In contrast, maximal induction of progestophilins required 3 × 10⁻⁸ M E₂ in cells grown in medium supplemented with the serum of Morris hepatoma 7777-bearing rats. This serum contained AFP levels comparable to those present at birth in the rat. 11-Methoxy-17β ethynylestradiol (R₂₈₅₈), a synthetic estrogen with little affinity for AFP, was also tested for its ability to induce progestophilins. The degree of maximal induction of progestophilins expressed as percentage of the respective control, was similar for all experimental groups, both with E₂ and with R₂₈₅₈.In addition, we compared the free E₂ levels in the culture medium with the progestophilin levels and the cell proliferation rate. We found that the progestophilin levels were maximal at free E₂ concentrations above 11 pg E₂/ml, whereas there was no correlation between the free E₂ levels and the proliferation rate. Moreover, the proliferation rate of cells in medium supplemented with horse or castrated rat serum was maximal at concentrations of free E₂ below 0.4 pg/ml, whereas cell proliferation was inhibited with hepatoma serum even at concentrations of free E₂ of 44 pg/ml. We conclude that the effect of hepatoma serum on the E₂ induction of progestophilins seems to be mediated by the effect of AFP on the availability of free estrogen, since it is abolished by the addition of both natural and synthetic estrogens. The inhibitory effect of hepatoma serum upon cell proliferation is not reversed by estrogens and thus seems to be mediated by mechanisms other than E₂ trapping by AFP.     

Ascorbate potentiates serum-induced S phase entry of quiescent 3T3 cells

Summary Arrested BALB/c 3T3 cells were induced to the G₀-G₁ transition by fetal calf serum (FCS) and S phase entry was measured by [³H]thymidine incorporation as an index of DNA synthesis. [³H]Thymidine uptake was proportional to FCS concentration. Ascorbate (ASC) itself was unable to increase DNA synthesis in these cells but potentiated it in the presence of both 1% and 10% FCS. [³H]Thymidine uptake profile was similar with and without ASC, and showing at 24 h an ASC stimulation of 69% in the presence of 1% FCS and 58% with 10% FCS. These data are discussed in reference to the participation of ASC on plasma membrane energization for membrane translocations and transport.Abbreviations ASC ascorbate - FCS fetal calf serum     

Mutagenic effect of ultraviolet radiation on the non-acid-fast strain ofMycobacterium phlei

The mutability of the PN strain ofMycobacterium phlei was examined after induction of auxotrophic mutants and of STM and VM-resistant mutants, by UV irradiation. A total of 30 auxotrophic mutants were isolated, most of them amino acid-dependent five purine-dependent, and one uracil-dependent. To induce the mutants higher UV doses had to be used so that the survival of cells in the original suspension would not exceed a few per cent. For further genetic work use can be made of 8 auxotrophic mutants (PN try^?ura^?, PN arg^?ura^?, PN ileu^?val^?, PN ileu^?, PN leu^?, PN lys^?, PN lys^?-VM^r, PN val^?), these showing a low frequency of spontaneous reversions. No spontaneous auxotrophic mutants have been found. The frequency of STM and VM-resistant mutants is increased upon UV irradiation, a post-irradiation incubation in a liquid medium without the drug being essential for their phenotypic expression. The highest increase of the number of these mutants is attained after 48 h of post-irradiation incubation and it has been found that, within a certain experimental scatter, the same frequency increase is found on using a complete or a minimal liquid medium. The frequency of spontaneous STM-resistant mutants lies within 5.8×10^?6–8.8×10^?6, of those VM-resistant between 3.1×10^?5 and 4.1×10^?5. The highest frequency of induced STM-resistant mutants lies between 3.0×10^?5 and 9.3×10^?5 and of VM-resistant mutants between 1.1×10^?4 and 2.2×10^?4     

Oxysterol activation of arachidonic acid release and protaglindin E2 biosynthesis in NRK 49F cells is partially dependent on protein kinase activity

We previously demonstrated that the oxysterol potentiation of arachidonic acid release and prostaglandin biosynthesis induced by foetal calf serum activation of normal rat kidney (NRK) cells (fibroblastic clone 49F) was not related to a direct effect of oxysterols on cell free Ca²⁺ level. Since both Ca²⁺ variations and protein C are involved in arachidonic acid release in some models, we looked for a possible modulation by protein C in the oxysterol effect on arachidonic acid release. We show that when the phorbol ester 12-O-tetradecanoyl-phorbol-13acetate (TPA), a protein kinase C activator, was added to the culture medium, the oxyterol effect on arachidonic acid release and prostaglandin synthesis clearly increased. Moreover, the effect of TPA was dose-dependent and TPA EC₅₀ (4 × 10⁻⁹ M) was unchanged in the presence of the oxysterol. Preincubation of cells with TPA for 24 h prevented the arachidonic acid release induced by TPA alone, whereas the oxysterol effect was decreased but not abolished. In the absence of serum, TPA and ionomycin added together induced the same noticeable (arachidonic acid) release and PGE₂ synthesis as serum alone. Nevertheless, the potentiating effect of cholest-5-ene-3β,25-diol was much higher when serum itself was used to activate NRK cells than it was in the present serum-mimicking experimental conditions. Thus, the presence of growth factors is probably required to obtain a full oxysterol effect. We conclude that the oxysterol effect was synergistic with, but not fully dependent on, protein kinase C and Ca²⁺ ion fluxes, therefore oxysterols could affed earlier events triggered by serum growth factor binding to their cell membrane receptors.     

Novel Temperature-Sensitive Mutants of Escherichia coli That Are Unable To Grow in the Absence of Wild-Type tRNA6Leu

Escherichia coli has only a single copy of a gene for tRNA₆^Leu (Y. Komine et al., J. Mol. Biol. 212:579–598, 1990). The anticodon of this tRNA is CAA (the wobble position C is modified to O²-methylcytidine), and it recognizes the codon UUG. Since UUG is also recognized by tRNA₄^Leu, which has UAA (the wobble position U is modified to 5-carboxymethylaminomethyl-O²-methyluridine) as its anticodon, tRNA₆^Leu is not essential for protein synthesis. The BT63 strain has a mutation in the anticodon of tRNA₆^Leu with a change from CAA to CUA, which results in the amber suppressor activity of this strain (supP, Su⁺6). We isolated 18 temperature-sensitive (ts) mutants of the BT63 strain whose temperature sensitivity was complemented by introduction of the wild-type gene for tRNA₆^Leu. These tRNA₆^Leu-requiring mutants were classified into two groups. The 10 group I mutants had a mutation in the miaA gene, whose product is involved in a modification of tRNAs that stabilizes codon-anticodon interactions. Overexpression of the gene for tRNA₄^Leu restored the growth of group I mutants at 42°C. Replacement of the CUG codon with UUG reduced the efficiency of translation in group I mutants. These results suggest that unmodified tRNA₄^Leu poorly recognizes the UUG codon at 42°C and that the wild-type tRNA₆^Leu is required for translation in order to maintain cell viability. The mutations in the six group II mutants were complemented by introduction of the gidA gene, which may be involved in cell division. The reduced efficiency of translation caused by replacement of the CUG codon with UUG was also observed in group II mutants. The mechanism of requirement for tRNA₆^Leu remains to be investigated.In the universal genetic code, 61 sense codons correspond to 20 amino acids, and the various tRNA species mediate the flow of information from the genetic code to amino acid sequences. Since codon-anticodon interactions permit wobble pairing at the third position, 32 tRNAs, including tRNA^fMet, should theoretically be sufficient for a complete translation system. Although some organisms have fewer tRNAs (1), most have abundant tRNA species and multiple copies of major tRNAs. For example, Escherichia coli has 86 genes for tRNA (79 genes identified in reference 14, 6 new ones reported in reference 3, and one fMet tRNA at positions 2945406 to 2945482) that encode 46 different amino acid acceptor species. Although abundant genes for tRNAs are probably required for efficient translation, the significance of the apparently nonessential tRNAs has not been examined.E. coli has five isoaccepting species of tRNA^Leu. According to the wobble rule, tRNA₁^Leu recognizes only the CUG codon. The CUG codon is also recognized by tRNA₃^Leu (tRNA₂^Leu) and thus tRNA₁^Leu may not be essential for protein synthesis. Similarly, tRNA₆^Leu is supposed to recognize only the UUG codon, but tRNA₄^Leu can recognize both UUA and UUG codons. Thus, tRNA₆^Leu appears to be dispensable. The existence of an amber suppressor mutation of tRNA₆^Leu (supP, Su⁺6) supports this possibility. tRNA₆^Leu is encoded by a single-copy gene, leuX (supP), and Su⁺6 has a mutation in the anticodon, which suggests loss of the ability to recognize UUG (26). Why are so many species of tRNA^Leu required? Holmes et al. (12) examined the utilization of the isoaccepting species of tRNA^Leu in protein synthesis and showed that utilization differs depending on the growth medium; in minimal medium, isoacceptors tRNA₂^Leu (cited as tRNA₃^Leu; see Materials and Methods) and tRNA₄^Leu are the predominant species that are found bound to ribosomes, but an increased relative level of tRNA₁^Leu is found bound to ribosomes in rich medium. The existence of tRNA₆^Leu is puzzling. This isoaccepting tRNA accounts for approximately 10% of the tRNA^Leu in total-cell extracts. However, little if any tRNA₆^Leu is found on ribosomes in vivo, and it is also only weakly active in protein synthesis in vitro with mRNA from E. coli (12). It thus appears that tRNA₆^Leu is only minimally involved in protein synthesis in E. coli.To investigate the role of tRNA₆^Leu in E. coli, we attempted to isolate tRNA₆^Leu-requiring mutants from an Su⁺6 strain. These mutants required wild-type tRNA₆^Leu for survival at a nonpermissive temperature. We report here the isolation and the characterization of these mutants.     

The serum growth and survival requirements of SV40-transformed 3T3 cells

Summary Simian virus 40-transformed 3T3 cells are dependent on serum for survival and growth. This growth activity can be separated on a pH 2 Sephadex G100 column into two fractions: a high molecular weight activity and a low molecular weight substance that has recently been characterized as containing as its major agent, biotin. To replace the remainder of the serum requirement, hormones and other growth factors were tested. Both insulin at high, nonphysiological concentrations (200 to 500 ng/ml) and transferrin (5×10⁻⁸ M) stimulate the growth rate in low serum medium (0.3% v/v bovine calf serum DME) individually and, when added together, are nearly as growth enhancing as 10% serum. The need for the residual serum in this medium can be eliminated by the use of crystalline trypsin during trypsinization. Under these serum-free conditions, biotin and transferrin supplementation provide for moderately good growth (20 to 30 hr population doubling time, 1×10⁶ cells/3.2-cm dish final cell density). Insulin addition further stimulates the growth rate (16 to 20 hr) and the final density (1.5×10⁶ cells). Although the protein growth factors, EGF (0.5 to 1.0 ng/ml) and FGF (4 to 10 ng/ml), also appear to enhance growth individually and additively, their effects are slight and very variable. Nevertheless, the complete serum-free medium (DME supplemented with biotin, transferrin, insulin, EGF and FGF) yields growth comparable but still inferior to 10% serum supplementation (14-versus 12-hr population doubling time, 1 to 2×10⁶ versus 2 to 3×10⁶ cells final cell density). This work was supported by NIH Grant CA 20040.     

tRNAHis 5-methylcytidine levels increase in response to several growth arrest conditions in Saccharomyces cerevisiae

tRNAs are highly modified, each with a unique set of modifications. Several reports suggest that tRNAs are hypomodified or, in some cases, hypermodified under different growth conditions and in certain cancers. We previously demonstrated that yeast strains depleted of tRNA^His guanylyltransferase accumulate uncharged tRNA^His lacking the G₋₁ residue and subsequently accumulate additional 5-methylcytidine (m⁵C) at residues C₄₈ and C₅₀ of tRNA^His, due to the activity of the m⁵C-methyltransferase Trm4. We show here that the increase in tRNA^His m⁵C levels does not require loss of Thg1, loss of G₋₁ of tRNA^His, or cell death but is associated with growth arrest following different stress conditions. We find substantially increased tRNA^His m⁵C levels after temperature-sensitive strains are grown at nonpermissive temperature, and after wild-type strains are grown to stationary phase, starved for required amino acids, or treated with rapamycin. We observe more modest accumulations of m⁵C in tRNA^His after starvation for glucose and after starvation for uracil. In virtually all cases examined, the additional m⁵C on tRNA^His occurs while cells are fully viable, and the increase is neither due to the GCN4 pathway, nor to increased Trm4 levels. Moreover, the increased m⁵C appears specific to tRNA^His, as tRNA^Val(AAC) and tRNA^Gly(GCC) have much reduced additional m⁵C during these growth arrest conditions, although they also have C₄₈ and C₅₀ and are capable of having increased m⁵C levels. Thus, tRNA^His m⁵C levels are unusually responsive to yeast growth conditions, although the significance of this additional m⁵C remains unclear.     

Conformational Change of the L-Shaped tRNAPhe Molecule

Abstract

Fluorophore of proflavine was introduced onto the 3′-terminal ribose moiety of yeast tRNA^Phe. The distance between the fluorophore and the fluorescent Y base in the anticodon of yeast tRNA^Phe was measured by a singlet-singlet energy transfer. Conformational changes of tRNA^Phe with binding of tRNA^Glu ₂, which has the anticodon UUC complementary to the anticodon GAA of tRNA^Phe, were investigated. The distance obtained at the ionic strength of 100 mM K⁺ and 10 mM Mg²⁺ is very close to the distance from x-ray diffraction, while the distance obtained in the presence of tRNA^Glu ₂ is significantly smaller. Further, using a fluorescent probe of 4-bromomethl-7-methoxycoumarin introduced onto pseudouridine residue Ψ₅₅ in the TΨC loop of tRNA^Phe, Stern-Volmer quenching experiments for the probe with or without added tRNA^Glu ₂were carried out. The results showed greater access of the probe to the quencher with added tRNA^Glu ₂. These results suggest that both arms of the L-shaped tRNA structure tend to bend inside with binding of tRNA^Glu ₂ and some structural collapse occurs at the corner of the L-shaped structure.     

Structural relationship between tRNA2 Lys and tRNA4 Lys from mouse lymphoma cells

Summary Mouse lymphoma cells have three major isoaccepting lysine tRNAs. Two of these isoacceptors, tRNA₂ ^Lys and tRNA₄ ^Lys, were sequenced by rapid gel or chromatogram readout methods. They have the same primary sequence but differ in two modified nucleotides. tRNA₄ ^Lys has an unmodified uridine replacing one dihydrouridine and an unidentified nucleotide, t⁶A^*, replacing t⁶A. This unidentified nucleotide is not a hypomodified form of t⁶A. Thus, tRNA4ys is not a simple precursor of tRNA₂ ^Lys. Both tRNAs have an unidentified nucleotide, U^**, in the third position of the anticodon. Also, partial sequences of minor homologs of tRNA₂ ^Lys and tRNA₄ ^Lys were obtained. The distinctions between tRNA₂ ^Lys and tRNA₄ ^Lys may be part of significant cellular roles as illustrated by the differential effects of these isoacceptors on the synthesis by lysyl-tRNA synthetase of diadenosine-5,5-P₁,P₄-tetraphosphate, a putative signal in DNA replication.     

Chromate reduction by <Emphasis Type="Italic">Bacillus megaterium</Emphasis> TKW3 isolated from marine sediments

Summary Bacillus megaterium strain TKW3 was isolated from multiple-metal-contaminated marine sediments of Tokwawan, Hong Kong SAR. This facultative aerobe utilized arabinose, mannitol, N-acetylglucosamine, maltose, caprate, citrate, butyrate or lactate as the sole source of carbon and energy for growth.B. megaterium TKW3 reduced highly toxic and soluble Cr⁶⁺ (as CrO₄²⁻) into almost non-toxic and insoluble Cr³⁺ under aerobic conditions. Complete reduction of 0.20 mM Cr⁶⁺ by B. megaterium TKW3 was achieved within 360 h. Initial Cr⁶⁺ concentration below 0.90 mM or inoculum less than 10⁷ cells ml⁻¹ did not have significant effect on ⁶⁺ reduction, while the residue Cr⁶⁺ concentration was the lowest at 10⁷ cells ml⁻¹. Cr⁶⁺ reduction by this strain was inhibited by high levels of NaCl (55%). B. megaterium TKW3 was also resistant to other oxyanions including 0.34 mM Cr₂O₇²⁻ 0.32 mM AsO₄³⁻, 0.58 mM SeO₃²⁻ and 0.53 mM SeO₄²⁻, and reduced soluble Se⁴⁺ (as SeO₃²⁻) to insoluble red amorphous Se⁰. B. megaterium TKW3 might have potential application in bioremediation of Cr-laden sediments associated with other oxyanions.