Differential Mutation Production by the Decay of Incorporated Tritium Compounds in Escherichia coli

We have studied the differential mutation production by the decay of incorporated tritium compounds in E. coli (WWU) using DNA-seeking precursors (H³-thymidine), RNA-seeking precursors (H³-uracil, H³-uridine), and protein-seeking precursors (H³-histidine, H³-proline). In particular we have determined the reversion frequency of an arginine locus. The reversion frequency is measured in units of revertants/surviving bacteria/H³ decay, and has an average value of 1.84 × 10^-8 for H³-uridine and H³-uracil, 0.67 × 10^-8 for H³-thymidine, and 0.28 × 10^-8 for H³-proline and H³-histidine. Thus, the revertants are produced most effectively by H³ decays when the label is introduced in the form of an RNA precursor. The macromolecular distribution of the label shows that 5 to 8 per cent of the H³-uridine or H³-uracil is incorporated into DNA.     

The Exchange of RNA and Protein During Conjugation in Tetrahymena*

SYNOPSIS. Exchange of cytoplasm in Tetrahymena pyriformis, syngen 1, has been demonstrated by growing cells of 1 mating type in medium supplemented with H³-uridine or H³-histidine, washing, mixing with cells of an unlabeled, starved mating type, sampling conjugants at different times, and preparing autoradiographs. It was found that cytoplasmic interchange begins soon after the mates unite, and has become extensive before the end of the 1st prezygotic prophase (micronuclear crescent stage). When the RNA in one mating type had been labeled with H³-uridine, the activity was distributed almost evenly between the mates by late stages of conjugation. These results are consistent with electron micrographs of this syngen showing small pores in the attachment region of the mates, and many free ribosomes in the cytoplasm (8,11). By contrast, when protein in one mating type had been labeled with H³-histidine, these cells at late conjugation remained about twice as active as their originally unlabeled mates, presumably because of the physical characteristics of some structures which incorporated the amino acid (for example, cilia and membranes of the cell surface; cytoplasmic bodies, such as mitochondria, larger than the pores). That the radioactivity in the originally unlabeled cells came from their mates and not from the environment is indicated by the continued presence of inactive non-conjugants after 1 and 2 days in the mating type mixtures. Other cells which did acquire small amounts of active cytoplasm probably had engaged in abortive conjugation, separating from labeled mates before forming and exchanging pronuclei.     

Effects of Decay of Incorporated H3-Thymidine on Bacteria

The killing efficiency due to the decay of incorporated H³-thymidine in three mutants of E. coli strain 15: 15_T-, 15_T-L-, and 15_T-U- has been determined. This efficiency is comparable to that previously determined by others for P³² decay. The killing efficiency has been determined as a function of H³-thymidine specific activity, storage media and storage temperature. We have observed a latent killing effect that causes lethality under certain conditions. The kinetics of latent killing have been examined at several temperatures. Finally, mutation production induced by H³-thymidine decays was shown to occur. The results are consistent with the idea that inactivation and mutations may be caused by a process in the nuclear transmutation that is not associated with β-particle ionization damage.     

Tritium labelling and cytochemistry of extra DNA in Acheta

Females of Acheta domesticus were injected with H³-thymidine and H³-uridine at various stages of development in order to study DNA and RNA synthesis in the DNA body present in the oocytes. Staining with alkaline fast green, azure B and the Feulgen reaction were employed as cytochemical tests. The following main results were obtained.

1. The DNA body appears in the oogonia at interphase as a Feulgen positive spherical structure 2 microns in diameter and is seen in subsequent mitotic divisions as a slightly smaller structure of variable shape. H³-thymidine autoradiography discloses that the DNA present in this body is synthesised at a different time from the chromosomal DNA.
2. At interphase and during the early prophase of meiosis the DNA body increases in size becoming a large Feulgen positive sphere 6 microns in diameter. Small nucleoli are present within this body. The DNA of the body is complexed with histone as revealed by alkaline fast green staining. H³-thymidine labelling discloses that it is at these stages that the bulk of the DNA synthesis takes place in the body.
3. Every oocyte contains a DNA body, and no body of comparable size or shape seems to be present in the male meiotic prophase.
4. At pachytene and diplotene the DNA body acquires the appearance of a “puff”. Two zones can be distinguished inside the DNA body: (1) an inner core of DNA and an outer shell of RNA. The inner core is Feulgen positive and stains light green with azure B, the outer shell is Feulgen negative and stains purple-violet with azure B, as does the cytoplasm. From the inner DNA core many Feulgen positive fibrils radiate into the outer RNA shell. These fibrils appear unstained or slightly greenish with Azure B, forming a transparent network in a purple-violet background. This gives the body the typical appearance of a “puff”. H³-uridine incorporation reveals that the RNA synthesis occurs in the outer RNA shell of the body and in the chromosomes. RNase treatment removes the H³-uridine incorporated into these regions.
5. At the end of diplotene the DNA body starts to disintegrate. The DNA core breaks up into minor components and the outer RNA zone also begins to disintegrate. By late diplotene the whole body has vanished, releasing DNA, histone and RNA into the nucleus. Subsequently the nuclear envelope disintegrates as it regularly does at the end of prophase of meiosis.
6. The simplest interpretation of the above results is that the DNA body represents hundreds of copies of the genes of the nucleolar organizing region.

     

DNA synthesis in the giant salivary chromosomes of Drosophila virilis prior to pupation

Both two-wavelength microspectrophotometry of Feulgen-stained whole nuclei and autoradiography of H³-thymidine incorporation by giant salivary chromosomes in Drosophila virilis demonstrate a net decrease in the relative rate of salivary DNA synthesis during the late third instar and prepupal stages of development. Amounts of DNA-Feulgen per nucleus were distributed into several classes, the means of which closely approximated values projected as geometric multiples of the basic somatic DNA level estimated from hemocyte nuclei of the same larvae. Comparison of DNA polytene class frequencies showed no statistical difference between male larvae of different development stages, although female prepupae showed a greater frequency of nuclei in higher polytene classes when compared to male prepupae of the same age. Comparison of chromosomal H³-thymidine incorporation with previously described H³-histidine incorporation suggests that the amino acid labeling, which reaches a maximum during the prepupal period, has a physiological significance distinct from chromosomal endoreplication.     

H3-THYMIDINE DERIVATIVE POOLS IN RELATION TO MACRONUCLEAR DNA SYNTHESIS IN TETRAHYMENA PYRIFORMIS

The formation of a soluble H³-thymidine derivative pool has been examined in Tetrahymena pyriformis as a function of macronuclear DNA synthesis during the cell life cycle. An autoradiographic technique which allows the detection of water-soluble materials within a cell has shown that these cells do not take up and retain exogenous H³-thymidine during G1 or G2. Uptake of H³-thymidine is restricted to the S period of the cell cycle. Additional autoradiographic experiments show, however, that a soluble pool of H³-thymidine derivatives persists from the end of one DNA synthesis period to the beginning of the next synthesis period in the subsequent cell cycle. Since this persisting pool cannot be labeled with H³-thymidine, the pool does not turn over during non-S periods.     

Macromolecule synthesis leading to cell division in Tetrahymena pyriformis after replacement of required amino acids

Summary Tetrahymena pyriformis W were brought to a nonmultiplying state by removal of required amino acids from their growth medium. After amino-acid replacement, the incorporation rates of H³-uridine, H³-thymidine and H³-leucine were measured by the autoradiographic method. Following amino-acid replacement, the first response was detected in RNA synthesis, then protein synthesis, then DNA synthesis and, lastly, in cell division. Amino-acid deprived cells showed a 23% net increase in DNA content, a result supporting the view of others that protein synthesis is not necessary for the initiation of DNA synthesis but is necessary for the maintainance of DNA synthesis.     

Raman spectra of polypeptides containing L-histidine residues and tautomerism of imidazole side chain

Raman spectra were measured for poly(L -histidine) in H₂O, poly(L -histidine-d₂ and -d₃) in D₂O, L -histidine in H₂O, L -histidine-d₃ (and d₄) in D₂O, and 4-methylimidazole in H₂O with various pH (or pD) values. The Raman scattering peaks observed for these samples were ascribed to the neutral and positively charged imidazole groups on the basis of the spectral changes due to the pH variation and to the deuterium substitution of the imino protons. The vibrational modes of these peaks were deduced from the normal coordinate analysis made on the positively charged and neutral 4-ethylimidazoles. The Raman scattering peaks from the imidazole groups in the neutral form clearly indicate that these imidazole groups exist in the equilibrium between the two tautomeric forms, the 1-N protonated from (tautomer I) and the 3-N protonated one (tautomer II). For example, the breathing vibration of the 1-N protonated form is observed at 1282 cm^?1 for L -histidine and at 1304 cm^?1 for 4-methylimidazole, while the breathing vibration of the 3-N protonated form is observed at 1260 cm^?1 for L -histidine and 4-methylimidazole. From the temperature dependence of the relative intensities of the tautomer I peak to that of the tautomer II, it was concluded that the tautomer I is energetically more stable than the tautomer II, and the ΔH value is 1.0 ± 0.3 kcal/mol for L -histidine and 0.4 ± 0.1 kcal/mol for 4-methylimidazole. Poly(L -histidine) with the neutral imidazole side chains shows the amide I peak at 1672 cm^?1, indicating that the sample assumes the antiparallel pleated-sheet structure. Poly(L -Ala⁷⁵L -His²⁵) and poly(L -Ala⁵⁰L -His⁵⁰) were found to take the α-helical and β-form conformations, respectively.     

THYMIDINE DEGRADATION PRODUCTS IN PLANT TISSUES LABELED WITH TRITIATED THYMIDINE

A study of the metabolic pathways of H³-thymidine utilization in buds of Lilium longiflorum and root tips of Vicia faba was undertaken in order to obtain information that might explain the binding of H³ from H³-thymidine in the cytoplasm of these plants. H³-thymidine was administered for various periods of time, the tissues were fixed and processed in the manner routinely used in preparation for sectioning and autoradiography, and the radioactivity removed in this way from the tissues was determined. It was found that the ethanol/acetic acid fixative contained the major portion of the radioactivity. Analysis of this extract by paper chromatography showed that the radioactivity was distributed among various degradation products of thymidine, principally β-ureidoisobutyric acid and β-aminoisobutyric acid. Time course experiments with Vicia showed that these degradation products rapidly appeared in the tissue during incubation with H³-thymidine, while H³-thymine appeared in the incubation medium. Preliminary studies indicated that Vicia root tips incubated with H³-dihydrothymine for 24 hours would bind a small amount of H³ non-specifically in the cells. It seems unlikely that utilization of degradation products of H³-thymidine is sufficient to explain labeling which is concentrated in the cytoplasm.     

SYNTHETIC CAPACITIES OF CHROMOSOME FRAGMENTS CORRELATED WITH THEIR ABILITY TO MAINTAIN NUCLEOLAR MATERIAL

Onion (Allium cepa) and bean (Vicia faba) root tip cells containing many micronuclei, derived from x-ray-induced chromosome fragments, were exposed to H³-thymidine and H³-cytidine to determine the ability of such fragments to undergo DNA and RNA synthesis. Only a few micronuclei in onion and many in bean roots synthesize nucleic acid simultaneously with their main nuclei. A few micronuclei labeled with H³-thymidine undergo mitotic chromosome condensation along with the main nuclei, while the unlabeled ones never do so. The onset of nucleic acid synthesis as well as mitosis in micronuclei appears to be under generalized cellular control. Although all chromosomes and chromosome fragments at telophase give a positive reaction for a silver stainable nucleolar fraction, in the subsequent interphase only some micronuclei, derived from such chromosome fragments, are found to maintain nucleoli; others lose them with time. Those micronuclei which maintain nucleoli, perhaps due to the presence of specific chromosomal regions, are also active in DNA and RNA synthesis. These results are compatible with the concept that nucleoli and associated chromosome regions play an important role in the primary biosynthetic processes of the cell.     

EVIDENCE FOR CYTOPLASMIC DNA IN ROOT CELLS OF NICOTIANA

Sterile root cultures from Nicotiana tabacum were grown with H³-thymidine added to the medium for various intervals. Incorporation of the labeled nucleoside into nuclear DNA occurred in a fraction of the nuclei which increased with time. In addition, the cytoplasm of all cells incorporated enough tritium to be readily detected by autoradiography. The tritium was not removed by hydrolysis in 1 N HCl at 60°C for 10 minutes, but was removed by digestion in a DNase solution which also removed nuclear DNA. The amount of tritium in the cytoplasm increased during the first 2 hours, but did not appear to increase significantly during the following 5 hours. If the roots were transferred to unlabeled medium after 2 hours, the label was diluted faster than expected by growth without turnover of the labeled component. If FUdR was added to the unlabeled medium, the depletion occurred faster during the first 6 hours, but later appeared to level off so that at 10 hours these cultures did not differ from those incubated without FUdR. However, the addition of an excess of unlabeled carrier had no effect on the rate of depletion of the cytoplasmic label. Actinomycin D, which inhibited the incorporation of H³-cytidine into RNA in the root tips, had no effect on the incorporation of H³-thymidine into the cytoplasmic component. However, Mitomycin C or a high concentration of deoxyadenosine inhibited the incorporation of H³-thymidine into the cytoplasmic component as well as into the nuclear DNA. It is concluded that H³-thymidine is incorporated into a cytoplasmic fraction which has the characteristics of DNA, with a measurable rate of turnover. This fraction is synthesized regardless of whether or not the nucleus is synthesizing DNA. Although the function of cytoplasmic fraction is not yet known, it does not appear to be that of supplying precursors for the synthesis of the nuclear DNA.     

Thioglycollate stimulus modifies lymphocyte metabolism and proliferation. A comparison with lymphocyte activation by walker 256 tumour implantation

Key enzyme activities of glycolysis, the pentose-phosphate pathway, the Krebs' cycle and glutaminolysis were measured in lymphocytes obtained from the control (CC), thioglycollate-injected (TG) and Walker 256 tumour-implanted (WT) groups, non-immune and immune inflammatory stimuli, respectively. The rates of incorporation of [2-¹⁴C]-thymidine and [5-³H]-uridine into cultured lymphocytes were also determined. The results indicated that the rates of both [2-¹⁴C]-thymidine and [5-³H]-uridine incorporation were enhanced in lymphocytes obtained from thioglycollate-injected (by an average of 80 per cent) and tumour-implanted animals (by 2·4-fold) as compared to control rats. Lymphocyte hexokinase activity diminished both in the TG (23 per cent) and WT (61 per cent) groups, whereas glucose 6-phosphate dehydrogenase activity was not altered due to the non-immune inflammatory stimulus, being reduced (23 per cent) in WT rats as compared to CC. The activity of lymphocyte citrate synthase was lowered by thioglycollate (39 per cent) and tumour-implantation (46 per cent). In contrast, glutaminase activity was augmented in lymphocytes from the TG (41 per cent) and was not modified in the WT groups. Taken as a whole, the presence of the Walker 256 tumour did not affect the capacity for glutamine utilization but depressed glucose metabolism in these cells. On the other hand, the non-immune inflammatory stimulus suppressed the activities of glycolysis and the Krebs' cycle and enhanced that of glutaminolysis in lymphocytes.     

ELECTRON MICROSCOPIC EXAMINATION OF THE SITES OF NUCLEAR RNA SYNTHESIS DURING AMPHIBIAN EMBRYOGENESIS

The site of H³-uridine incorporation and the fate of labeled RNA during early embryo-genesis of the newt Triturus pyrrhogaster were studied with electron microscopic autoradiography. Isolated ectodermal and mesodermal tissues from the embryos were treated in H³-uridine for 3 hours and cultured in cold solution for various periods before fixation with OsO₄ and embedding in Epon. At the blastula stage, the only structural component of the nucleus seen in electron micrographs is a mass of chromatin fibrils. At the early gastrula stage, the primary nucleoli originate as small dense fibrous bodies within the chromatin material. These dense fibrous nucleoli enlarge during successive developmental stages by the acquisition of granular components 150 A in diameter, which form a layer around them. Simultaneously larger granules (300 to 500 A) appear in the chromatin, and they fill the interchromatin spaces by the tail bud stage. Autoradiographic examination has demonstrated that nuclear RNA synthesis takes place in both the nucleolus and the chromatin, with the former consistently showing more label per unit area than the latter. When changes in the distribution pattern of radioactivity were studied 3 to 24 hours after immersion in isotope at each developmental stage, the following results were obtained. Labeled RNA is first localized in the fibrous region of the nucleolus and in the peripheral region of chromatin material. After longer culture in non-radioactive medium, labeled materials also appear in the granular region of the nucleolus and in the interchromatin areas. Further incubation gives labeling in cytoplasm.     

Autoradiographic Studies of H3-Thymidine Uptake in Entamoeba histolytica in CLG Medium*

SYNOPSIS. Entamoeba histolytica grown with H³-thymidine in CLG medium took up tritium into DNase-sensitive material in the nucleus and cytoplasm. The distribution of nuclear activity indicated that the entire nucleus, including the peripheral chromatin, may possess DNA; previous investigators reported DNA only in the endosome. The penicillin-inhibited bacterial associate (Bacteroides sp.) used in the CLG medium incorporated tritium from H³-thymidine into autoradiographically detectable DNase-sensitive material. Autoradiographs of amebae fed bacteria prelabeled with H³-thymidine also revealed some nuclear and cytoplasmic label. Thus, the amount of cytoplasmic label due to ingested, prelabeled bacterial DNA and/or actual biosynthesis of cytoplasmic DNA by the amebae themselves, is not known. Also, at least some of the nuclear DNA of amebae is synthesized from ingested bacteria, or, more likely, from bacterial degradation products.     

Enhancing Effect of Zinc on <Emphasis Type="SmallCaps">l</Emphasis>-Histidine Transport in Rat Lung Microvascular Endothelial Cells

The aim of this study was to examine enhancing effect of l-histidine into cultured rat lung microvascular endothelial cells (LMECs), which constitute the gas–blood barrier. Uptake of l-histidine into LMECs markedly increased with the addition of ZnSO₄ (0.1 mmol/L), and this enhanced uptake of l-histidine was drastically reduced in the presence of the Na⁺-independent system L substrate, 2-amino-2-norbornanecarboxylic acid (BCH). However, the uptake of l-histidine together with ZnSO₄ was not reduced by the addition of metabolic inhibitor, 2,4-dinitrophenol, or sodium ion replacement. Moreover, the addition of the system N-substrate, l-glutamic acid γ-monohydroxamate did not significantly decrease the uptake of l-histidine with 143 mmol/L Na ⁺ + 1 mmol/L BCH. These results indicated that system-N transporter does not play a role in the uptake of l-histidine in the presence of ZnSO₄, suggesting that only system-L transporter is involved in the uptake of l-histidine, although l-histidine in the absence of ZnSO₄ was taken up by at least two pathways of Na⁺-dependent system-N and Na⁺-independent system-L processes into rat LMECs. The uptake of l-histidine into rat LMECs in the presence of ZnSO₄ was also found to be unaffected by pH (5.0–7.4), indicating that uptake of l-histidine into LMECs by the addition of zinc may not be involved in the H⁺-coupled transporters.     

Tritiated-histidine incorporation into Drosophila salivary chromosomes

An autoradiographic study of H³-histidine incorporation into nonhistone protein of explanted larval salivary gland chromosomes of D. virilis showed patterns of incorporation that were dependent upon the stage of larval development. The sequence of changes in the development of several puffs in a specific chromosomal region was followed using the appearance of pigment in the anterior spiracles as a means of larval staging. H³-histidine incorporation into these puffs in prepupae occurred as the puffs were regressing in size and protein staining. Acid extraction of histone and nucleic acid failed to alter the character of the autographs; presumably a non-histone protein is involved in the H³-histidine incorporation. Other puff sites in the same prepupal chromosomes showed various patterns of isotopic amino acid incorporation indicating that the pattern reported for a specific region may not be true for all puff sites.     

Breakdown of DNA in X-Irradiated Escherichia coli

A comparison of differences in incorporation and loss of radio-activity between two strains of Escherichia coli shows that: (a) three times as much irradiation is necessary to produce the same reduction in incorporation of H³-thymidine in B/r, the resistant strain, as in B_{s - 1}, the sensitive one; (b) radioactivity is lost from the DNA of previously labeled bacteria during the first few cell generations after X-ray exposure, and even though the initial rate of loss is similar for all strains, the sensitive one loses much more label; (c) loss of DNA is a complicated function of dose. Losses increase with dose up to 25 or 50 kr in both strains; with higher doses, losses decrease in B_{s - 1} but are unchanged in B/r. Since in both strains labeled RNA is retained in irradiated cells, lysis has not occurred but the DNA is broken down into small pieces which leak from each cell. Losses from either strain do not occur at ice-bath temperature, indicating that breakdown is a function of metabolic processes. A proposed mechanism for X-ray damage and repair is advanced.     

Isolation and characterization of the haemocytes of Calliphora vicina on density gradients of Ficoll

Discontinuous gradients of Ficoll have been used in an equilibrium density analysis of the haemocytes of Calliphora vicina. Using histochemical criteria, it was shown that the acid phosphatase-containing haemocytes decreased in mean density during larval life. Enzymatic analysis, and an analysis of the density distribution of labelled haemocytes at various times after an injection of [H³]-thymidine, provided evidence that a dense, replicating population of cells had been separated from a non-replicating acid phosphatase-containing population. The latter gained increasing amounts of the lysosomal enzymes acid phosphatase and protease as they aged.     

DNA replication patterns in somatic chromosomes of Leptodactylus ocellatus (Amphibia,Anura)

The morphology and pattern of replication in the somatic chromosomes of Leptodactylus ocellatus (Amphibia, Anura) was studied by means of H³-thymidine autoradiography. A total of 300 metaphases from leukocyte cultures and 200 metaphases from spleen cell cultures were analysed.The diploid chromosome number in Leptodactylus ocellatus is 22. The pairs 1, 2, 3, 4, 7 and 8 could be easily identified on the basis of their size, centromere position, and location of secondary constrictions. In 30% of metaphases the pair 10 could be recognized on account of an end-to-end homologous association, which originated from a satellite fusion.The continuous H³-thymidine labelings carried out in the last 10, 5 and 3 hours of a culture indicated that the G₂ period was 3.5 hours. The labeled metaphases were divided in two groups. In the first one all those cells showing radioactivity along the entire length of every chromosome were included. The second group was formed by metaphases with extensive unlabeled chromosome regions. The former and the latter group were identified as representatives of the intermediate and final stages of the S period, respectively.The pattern of chromosome labeling indicates that secondary constrictions are associated with late replicating regions. However, the presence of chromosome areas, which in spite of being late in finishing duplication did not bear any kind of constriction, suggests that regions other than those associated with constrictions also may replicate late. No interchromosomal asynchrony of replication at the end of the S period was noticed. However, very often in pair 10 one chromosome had about two times as much labeling as its homologue. No sex-linked differences in chromosome morphology or in patterns of chromosome replication could be noticed.