An Unusual Pattern of Tritiated Thymidine Incorporation in Euglena

SYNOPSIS. Thymidine-methyl-H² is incorporated into the cytoplasm of Euglena . The label is non-nuclear and not in DNA; evidence for its presence in RNA and protein is presented. Only Euglena which maintain the potentiality to develop chloroplasts show this incorporation; it was not observed in streptomycin. uv, benadryl, O-methyl threonine or heat "bleached" Euglena , nor in Astasia longa.
Preliminary incorporation experiments show that exogenous pyrimidines are not utilized as nucleic acid precursors in Euglena in general. However, the tritiated purines are incorporated into DNA and RNA. The use of thymidine to localize DNA autoradiographically in Euglena is completely excluded.     

DNA Synthesis and Tritiated Thymidine Incorporation by Heterotrophic Freshwater Bacteria in Continuous Culture

Continuous cultivation of heterotrophic freshwater bacteria was used to assess the relationship between DNA synthesis and tritiated thymidine incorporation. The bacteria were grown on a yeast extract medium with generation times of 0.25 to 3.7 days. In six different continuous cultures, each inoculated with a grazer-free mixed bacterial sample from Lake Vechten (The Netherlands), tritiated thymidine incorporation into a cold trichloroacetic acid precipitate and bacterial cell production were measured simultaneously. Empirical conversion factors were determined by division of both parameters. They ranged from 0.25 × 10¹⁸ to 1.31 × 10¹⁸ cells mol of tritiated thymidine^-1 (mean, 0.60 × 10¹⁸ cells mol of tritiated thymidine^-1). In addition, DNA concentrations were measured by fluorometry with Hoechst 33258. The validity of this technique was confirmed. Down to a generation time of 0.67 day, bacterial DNA content showed little variation, with values of 3.8 to 4.9 fg of DNA cell^-1. Theoretical conversion factors, which can be derived from DNA content under several assumptions, were between 0.26 × 10¹⁸ and 0.34 × 10¹⁸ cells mol of thymidine^-1 (mean, 0.30 × 10¹⁸ cells mol of thymidine^-1). Isotope dilution was considered the main factor in the observed discrepancy between the conversion factors. In all experiments, a tritiated thymidine concentration of 20 nM was used. Control experiments indicated maximum incorporation at this concentration. It was therefore concluded that the observed difference resulted from intracellular isotope dilution which cannot be detected by current techniques for isotope dilution analysis.     

Enhanced Incorporation of Tritium into Glycolate during Photosynthesis by Tobacco Leaf Tissue in the Presence of Tritiated Water

Tobacco (Nicotiana tabacum var. Havana Seed) leaf discs were allowed to photosynthesize for 3 to 20 minutes in the presence of ¹⁴CO₂ and ³H₂O. Several metabolites of the Calvin cycle and photorespiratory pathway were isolated and purified and the ³H:¹⁴C values measured. Glycolate had a 5- to 10-fold higher ³H:¹⁴C than the Calvin cycle intermediate 3-phosphoglyceric acid, or its end product sucrose. The glycolate oxidase inhibitor α-hydroxy-2-pyridinemethanesulfonic acid caused glycolate to accumulate in the tissue and lowered the ³H:¹⁴C in glycolate to a value similar to that in 3-phosphoglyceric acid. Phosphoglycolate, a possible precursor of glycolate arising from the Calvin cycle, exhibited a ³H:¹⁴C value similar to 3-phosphoglyceric acid under all conditions. The finding of a ³H enrichment in glycolate suggests that another source of glycolate, possibly the reduction of glyoxylate, exists in leaf tissue. Analyses of incorporation of ³H into the pro-2R and pro-2S hydrogens of glycolate, in the presence and absence of α-hydroxy-2-pyridinemethanesulfonic acid, suggest an alternative source of glycolate. Biochemical mechanisms to account for ³H enrichment into glycolate are evaluated.     

氚标记胸腺嘧啶掺入法在土壤细菌生长速率研究中的应用

氚标记胸腺嘧啶掺入法因其原理明确、方法简单易行、重复性好等特点在测定细菌生长速率中发挥着重要的作用。然而,相比较医学和水生生态学领域,该方法在土壤生态学研究中的应用还较少,尤其是我国此方面的研究尚未见报道。就氚标记胸腺嘧啶掺入法的原理、操作流程、影响因素、计算方法及其在土壤生态学中的应用等方面进行了综述,为该方法在我国土壤生态学领域的推广应用提供参考。     

Thymidine Incorporation by the Microbial Community of Standing Dead Spartina alterniflora

Thymidine incorporation by the microbial community on standing dead leaves of Spartina alterniflora did not obey many of the assumptions inherent in the use of the technique in planktonic systems. Incorporation rates of [methly-³H]thymidine were nonsaturable over a wide concentration range (10¹ to 10⁵ nM). Owing to metabolism by both fungi and bacteria, a major fraction of the radiolabel (mean, 48%) appeared in protein. Extraction of the radiolabeled macromolecules was inefficient, averaging 8.8%. Based on an empirically derived conversion factor, 4 × 10¹⁸ cells · mol of thymidine⁻¹, doubling times ranged from 4 to 69 h for the epiphytic bacterial assemblage.     

Differential Uptake of Tritiated Thymidine into Hetero- and Euchromatin in Melanoplus and Secale

Grasshoppers of the species Melanoplus differentialis were injected with tritium-labelled thymidine. At intervals thereafter autoradiographic stripping film was applied over Feulgen squashes and sections. In this species during early prophase of meiosis the sex chromosome forms a heterochromatic block large enough to be resolved in tritium autoradiographs. A study of the squash preparations reveals that the sex chromosome is synthesizing DNA at a different period of time from the euchromatic autosomes. Since there is a developmental sequence of spermatocyte cysts along the testicular tubes it is possible from the sections to show that the heterochromatin synthesizes DNA later than does the euchromatin. To find out whether the results obtained in Melanoplus were characteristic of heterochromatin in general, young seedlings of rye were grown in a tritiated thymidine solution and Feulgen squashes were made as for Melanoplus. In rye leaf nuclei there is a large block of heterochromatin constituted by the proximal regions of the chromosomes and a euchromatic one formed by the median and distal regions of the same chromosomes. Here also the heterochromatin synthesizes DNA at a different period of time from the euchromatin. It is concluded that in rye the asynchrony of synthesis occurs within each chromosome. Counts of silver grains over the two types of chromatin in nuclei of Melanoplus and Secale disclosed that the number of grains per unit area was two to three times higher over the heterochromatin. To check the DNA content, Feulgen photometric measurements were made of Melanoplus nuclei at the same stage. The Feulgen and grain counts agree in showing that the heterochromatin contains two to three times more DNA per unit area than the euchromatin.     

Multiple Heavy Metal Tolerance of Soil Bacterial Communities and Its Measurement by a Thymidine Incorporation Technique

A thymidine incorporation technique was used to determine the tolerance of a soil bacterial community to Cu, Cd, Zn, Ni, and Pb. An agricultural soil was artificially contaminated in our laboratory with individual metals at three different concentrations, and the results were compared with the results obtained by using the plate count technique. Thymidine incorporation was found to be a simple and rapid method for measuring tolerance. Data obtained by this technique were very reproducible. A linear relationship was found between changes in community tolerance levels obtained by the thymidine incorporation and plate count techniques (r = 0.732, P < 0.001). An increase in tolerance to the metal added to soil was observed for the bacterial community obtained from each polluted soil compared with the community obtained from unpolluted soil. The only exception was when Pb was added; no indication of Pb tolerance was found. An increase in the tolerance to metals other than the metal originally added to soil was also observed, indicating that there was multiple heavy metal tolerance at the community level. Thus, Cu pollution, in addition to increasing tolerance to Cu, also induced tolerance to Zn, Cd, and Ni. Zn and Cd pollution increased community tolerance to all five metals. Ni amendment increased tolerance to Ni the most but also increased community tolerance to Zn and, to lesser degrees, increased community tolerance to Pb and Cd. In soils polluted with Pb increased tolerance to other metals was found in the following order: Ni > Cd > Zn > Cu. We found significant positive relationships between changes in Cd, Zn, and Pb tolerance and, to a lesser degree, between changes in Pb and Ni tolerance when all metals and amendment levels were compared. The magnitude of the increase in heavy metal tolerance was found to be linearly related to the logarithm of the metal concentration added to the soil. Threshold tolerance concentrations were estimated from these linear relationships, and changes in tolerance could be detected at levels of soil contamination similar to those reported previously to result in changes in the phospholipid fatty acid pattern (Å. Frostegård, A. Tunlid, and E. Bååth, Appl. Environ. Microbiol. 59: 3605-3617, 1993).     

Thymidine and Thymine Incorporation into Deoxyribonucleic Acid: Inhibition and Repression by Uridine of Thymidine Phosphorylase of Escherichia coli

Thymidine is poorly incorporated into deoxyribonucleic acid (DNA) of Escherichia coli. Its incorporation is greatly increased by uridine, which acts in two ways. Primarily, uridine competitively inhibits thymidine phosphorylase (E.C.2.4.4), and thereby prevents the degradation of thymidine to thymine which is not incorporated into normally growing E. coli. Uridine also inhibits induction of the enzyme by thymidine. It prevents the actual inducer, probably a deoxyribose phosphate, from being formed rather than competing for a site on the repressor. The inhibition of thymidine phosphorylase by uridine also accounts for inhibition by uracil compounds of thymine incorporation into thymine-requiring mutants. Deoxyadenosine also increases the incorporation of thymidine, by competitively inhibiting thymidine phosphorylase. Deoxyadenosine induces the enzyme, in contrast to uridine. But this is offset by a transfer of deoxyribose from deoxyadenosine to thymine. Thus, deoxyadenosine permits incorporation of thymine into DNA, even in cells induced for thymidine phosphorylase. This incorporation of thymine in the presence of deoxyadenosine did not occur in a thymidine phosphorylase-negative mutant; thus, the utilization of thymine seems to proceed by way of thymidine phosphorylase, followed by thymidine kinase. These results are consistent with the data of others in suggesting that wild-type E. coli cells fail to utilize thymine because they lack a pool of deoxyribose phosphates, the latter being necessary for conversion of thymine to thymidine by thymidine phosphorylase.     

Adaptation of Aquatic Microbial Communities to Quaternary Ammonium Compounds

The effects of long-chain (C₁₂ to C₁₈) quaternary ammonium compounds (QACs) on the density, heterotrophic activity, and biodegradation capabilities of heterotrophic bacteria were examined in situ in a lake ecosystem. Monoalkyl and dialkyl substituted QACs were tested over a range of concentrations (0.001 to 10 mg/liter) in both acute (3 h) and chronic (21 day) exposures. In general, none of the QACs tested had significant adverse effects on bacterial densities in either acute or chronic studies. However, significant decreases in bacterial heterotrophic activity were noted in acute studies at QAC concentrations from 0.1 to 10 mg/liter. Chronic exposure of lake microbial communities to a specific monoalkyl QAC resulted in an adaptive response and recovery of heterotrophic activity. No-observable-effect level in the adapted populations was >10 mg/liter. Chronic exposure also resulted in significant increases in the number and activity of bacteria capable of biodegrading the material. The increase in biodegradation capability was observed at low (microgram per liter) concentrations which are approximately the same as realistic environmental levels. In general, our studies indicated that exposure of lake microbial communities to QACs results in the development of adapted communities which are less sensitive to potential toxic effects and more active in the biodegradation of these materials.     

Incorporation of Tritium from Thymidine-methyl-H3 into DNA,RNA, Lipids and Protein in Logarithmic and Stationary Phase Tetrahymena pyriformis,Strain W*

SYNOPSIS. Numerous reports may be found in the literature on cytoplasmic and non-DNA utilization of tritium from H³-thymidine. Such reports underscore the need to clarify the metabolic fate of H³-thymidine. This investigation outlines the fate of thymidinemethyl-H³ (TMH³) in logarithmic phase and stationary phase Tetrahymena pyriformis, strain W. Isotope identification by liquid scintillation spectrometry in chemically derived fractions of log phase cultures grown thruout the initial 48 hours of population growth with TMH³ revealed the majority of the radioactivity (90% of intracellular recovery) to be in the DNA fraction. The remainder of the intracellular label was recovered in the acid soluble fraction, lipid fraction, and a small amount in the RNA and cell residue. On chromatographs, tritium appeared only in the thymine moiety of the nucleic acid derivatives. Hence in dividing cells, thymidine-methyl-H³ is “essentially” specific for DNA at the dosage used although some incorporation into other compounds was detected. Fractionation of the lipid extract from the above experiment on a florisil column localized most of the label to the triglyceride and phospholipid fractions with some recovery in the cholesterol-esters. Similar scintillation counting of the various fractions of early stationary phase cells incubated for the last 48 hours of culture with TMH³ revealed limited tritium distribution in all fractions.     

Adaptation of Aquatic Microbial Communities to Hg2+ Stress

The mechanism of adaptation to Hg²⁺ in four aquatic habitats was studied by correlating microbially mediated Hg²⁺ volatilization with the adaptive state of the exposed communities. Community diversity, heterotrophic activity, and Hg²⁺ resistance measurements indicated that adaptation of all four communities was stimulated by preexposure to Hg²⁺. In saline water communities, adaptation was associated with rapid volatilization after an initial lag period. This mechanism, however, did not promote adaptation in a freshwater sample, in which Hg²⁺ was volatilized slowly, regardless of the resistance level of the microbial community. Distribution of the mer operon among representative colonies of the communities was not related to adaptation to Hg²⁺. Thus, although volatilization enabled some microbial communities to sustain their functions in Hg²⁺-stressed environments, it was not mediated by the genes that serve as a model system in molecular studies of bacterial resistance to mercurials.     

Specificity and Efficiency of Thymidine Incorporation in Escherichia coli Lacking Thymidine Phosphorylase

A mutant of Escherichia coli lacking the catabolic enzyme thymidine phosphorylase readily incorporates exogenous thymidine into deoxyribonucleic acid (DNA) even when provided at concentrations as low as 0.2 mug/ml. Incorporation by this prototrophic strain occurs specifically into DNA, since, with radioactively labeled thymidine, (i) more than 98% is incorporated into alkali-stable material, (ii) at least 90% is recovered as thymine after brief formic acid hydrolysis, and (iii) at least 90% is incorporated into material with the buoyant density of DNA. During growth in medium containing thymidine, the bacteria obtain approximately half of their DNA thymines from the exogenous thymidine and half from endogenous synthesis. The thymines and cytosines of DNA can be simultaneously and specifically labeled by thymidine-2-(14)C and uridine-5-(3)H, respectively. The mutant, which does not degrade thymidine, retains the ability to degrade the thymidine analogue 5-bromodeoxyuridine.     

Effects of Alkaline Phosphatase Activity on Nucleotide Measurements in Aquatic Microbial Communities

Alkaline phosphatase (APase) activity was detected in aquatic microbial assemblages from the subtropics to Antarctica. The occurrence of APase in environmental nucleotide extracts was shown to significantly affect the measured concentrations of cellular nucleotides (adenosine triphosphate, adenosine diphosphate, adenosine monophosphate, guanosine triphosphate, uridine triphosphate, and cytidine triphosphate), adenylate energy charge, and guanosine triphosphate/adenosine triphosphate ratios, when conventional methods of nucleotide extraction were employed. Under the reaction conditions specified in this report, the initial rate of hydrolysis of adenosine triphosphate was directly proportional to the activity of APase in the sample extracts and consequently can be used as a sensitive measure of APase activity. A method was devised for obtaining reliable nucleotide measurements in naturally occurring microbial populations containing elevated levels of APase activity. The metabolic significance of APase activity in microbial cells is discussed, and it is concluded that the occurrence and regulation of APase in nature is dependent upon microscale inorganic phosphate limitation of the autochthonous microbial communities.     

Inhibition of Growth and of Thymidine Incorporation into DNA in Tetrahymena by Chlorpromazine,Pimozide and Penfluridol1

The antipsychotic drugs chlorpromazine, pimozide, and penfluridol caused a 50% inhibition of growth of Tetrahymena at concentrations of 4.5, 5.5, and 1.5 μM, respectively. The degree of growth inhibition was dependent on the concentration of cells; higher drug concentrations were needed to produce inhibition of denser cell cultures. Binding studies with penfluridol showed that 50% growth inhibition resulted when approximately 50 μmoles of drug were bound per 10⁶ cells. A 20-min preincubation of cells with chlorpromazine (14.7 μM) inhibited DNA synthesis by 46%, and with penfluridol (4 μM) DNA synthesis was inhibited by 27%. The incorporation of labeled thymidine into the thymidine triphosphate pool was inhibited by chlorpromazine but not by penfluridol, indicating that the drugs produce their growth inhibitory effects by different mechanisms. TDP kinase activity was demonstrated in a particle-free fraction of the cells. Its enzymatic activity was not affected by added chlorpromazine, penfluridol, or calmodulin, suggesting that inhibition of DNA synthesis by these drugs may be a consequence of growth inhibition.     

Ligand Incorporation into Protein Microcrystals for MicroED by On-Grid Soaking

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In Vitro Incorporation of Selenomethionine into Protein by Vigna radiata Polysomes

Vigna radiata polysomes efficiently incorporated [⁷⁵Se]selenomethionine, [¹⁴C]methionine, and [¹⁴C]leucine in vitro. The optimal conditions for translation were determined to be 4.8 millimolar Mg²⁺, 182 millimolar K⁺, and pH 7.4. The rates of incorporation of [⁷⁵Se]selenomethionine and [¹⁴C]methionine were similar when measured separately, but [⁷⁵Se]selenomethionine incorporation was 35% less than [¹⁴C]methionine incorporation when both amino acids were present in equal molar concentrations. Polyacrylamide gel electrophoresis of the hot trichloroacetic acid precipitable translation products demonstrated synthesis of high molecular weight labeled proteins in the presence of [⁷⁵Se]selenomethionine or [³⁵S]methionine. No major differences in molecular weights could be detected in the electrophoretic profiles. Utilization of selenomethionine during translation by Vigna radiata polysomes establishes a route for the assimilation of selenomethionine by plants susceptible to selenium toxicity.     

Incorporation of tritiated adenosine into mouse ovum RNA

The total RNA of ovulated mouse ova has been examined by polyacrylamide gel electrophoresis. The amount of RNA present in the two main peaks observed, 28 S and 18 S ribosomal RNA, has been estimated as 0.20 ng.The RNA of ovulated mouse ova was labeled by exposure of growing mouse oocytes to adenosine-8-³H in vivo. For this purpose a small volume of a concentrated solution of the precursor was injected into the ovarian bursa, and ova were collected by superovulation at various subsequent times. The major growth phase of the oocyte is known to lie between 20 and 6 days before ovulation. Significant incorporation into egg RNA was observed when bursal injection was performed between 19 and 7 days, but not between 5 days and 1 day before ovulation.The types of labeled RNA in ova ovulated at five intervals between 19 and 7 days after bursal injection of adenosine-8-³H or uridine-5,6-³H were analyzed by polyacrylamide gel electrophoresis. The distribution of label on the gels demonstrated that the bulk of the label appeared in ribosomal RNA and transfer RNA. In addition labeled heterogeneous RNA was estimated to represent 10–15% of the total incorporation.