Effect of DNA inhibitors upon DNA synthesis and development of Drosophila embryos

Staged wildtype embryos of Drosophila melanogaster were permeabilized and then subjected to a short pulse of either methyl-3H-thymidine, one of four different inhibitors of DNA synthesis (mitomycin C, 5-fluorouracil, nalidixic acid, or 1-beta-D-arabino-furanosylcytosine-5'-monophosphate), or a combination of both. The incorporation of methyl-3H-thymidine into acid insoluble material was at a maximum during the first half-hour of embryogenesis, after which the incorporation dropped to half the initial value and remained constant throughout the remainder of development. There was no correlation between the rate of incorporation of methyl-3H-thymidine into DNA and the known periods of high mitotic activity. The time course of the estimated specific activity of the DNA newly synthesized in vivo closely paralleled the known changes in the DNA polymerase activity determined in vitro. The known periods of high mitotic activity in the embryo (0-3 hours, 5-12 hours) agree with the periods of maximal sensitivity of the embryo to the inhibitors of DNA synthesis. All four DNA inhibitors affected the incorporation of methyl-3H-thymidine into DNA, although they did not affect it in any simplistic manner. Inhibitor treatment during early cleavage stages resulted in arrested development, treatment during late cleavage and blastoderm stages resulted in abnormal development, and treatment during late blastoderm and early gastrula resulted in normal development. The major phenotypic abnormality caused by the inhibitors is an abnormal distribution of blastoderm cells. As judged by the ID50 values, the embryos remained very sensitive to the effects of the inhibitors until the stages of head and body segmentation, when they then very rapidly became insensitive.     

Localization and synthesis of the tumor protein oncomodulin in extraembryonic tissues of the fetal rat

The calcium-binding protein oncomodulin, previously found only in tumors, has been detected during rat development. Specific antisera to purified rat hepatoma oncomodulin (MW 11,500) were used to detect oncomodulin by radioimmunoassay (RIA) and by avidin-biotin-peroxidase complex (ABC) immunohistochemistry. Using RIA, oncomodulin was found to increase in placenta from below the limits of detection (2 ng/mg protein) on Day 13 to approximately 25 ng/mg on Day 16 of pregnancy, and to remain high through to the end of gestation. Determinations on separated inner and outer placenta showed the increase to be greater in the outer placenta (basal zone and decidua) than in the inner placenta (labyrinth). The ABC technique on paraffin sections produced positive staining for oncomodulin throughout the placenta, with the most intense staining occurring in the outer placenta (cytotrophoblast and giant cells of the basal zone). Parietal and visceral yolk sac, and amnion also stained positively, while fetal organs did not. Oncomodulin synthesis measured by [35S]methionine incorporation into immunoprecipitates occurred in isolated inner and outer placenta, whole placenta, the separated trophectoderm and endoderm of the parietal yolk sac, and amnion. No oncomodulin synthesis could be measured in visceral yolk sac, fetal liver, or 16-day embryo. This occurrence in developing and transformed tissues demonstrates that oncomodulin is an oncodevelopmental protein.     

Effect of polyamine limitation on DNA synthesis and development of mouse preimplantation embryos in vitro

In-vitro treatment of preimplantation mouse embryos with spermine and spermidine biosynthesis inhibitor, methylglyoxal-bis-(guanylhydrazone) (MGBG), arrested embryo development at the 8-cell or morula stage. In addition, the embryo DNA synthetic rate, as measured by [3H]thymidine incorporation, was strongly inhibited. The inhibition of blastocyst formation and DNA synthesis by MGBG was readily reversible by an exogenous supply of spermine and/or spermidine to the culture medium. DL-alpha-Methylornithine or DL-alpha-difluoromethylornithine (alpha-DFMO), inhibitors of putrescine biosynthesis, had no effect on embryos cultured for 1 or 2 days, but on the 3rd day embryo DNA synthesis was significantly depressed in the presence of alpha-DFMO. These observations suggest that, during early development of the preimplantation mouse embryo, spermine and spermidine are involved in regulation of embryo growth and DNA synthesis. They may also indicate a role of putrescine at a later stage of mouse embryo development.     

Relationship between fatty acid binding proteins,acetyl-CoA formation and fatty acid synthesis in developing human placenta

The relationship between fatty acid binding proteins, ATP citrate lyase activity and fatty acid synthesis in developing human placenta has been studied. Fatty acid binding proteins reverse the inhibitory efect of palmitoyl-CoA and oleate on ATP citrate lyase and fatty acid synthesis. In the absence of these inhibitors fatty acid binding proteins activate ATP citrate lyase and stimulate [ 1-¹⁴ C] acetate incorporation into placental fatty acids indicating binding of endogenous inhibitors by these proteins. Thus these proteins regulate the supply of acetyl-CoA as well as the synthesis of fatty acids from that substrates. As gestation proceeds and more lipids are required by the developing placenta fatty acid binding protein content, activity of ATP citrate lyase and rate of fatty acid synthesis increase indicating a cause and efect relationship between the demand of lipids and supply of precursor fatty acids during human placental development.     

Anomalous neural differentiation induced by 5-bromo-2'-deoxyuridine during organogenesis in the rat

The influence of 5-bromo-2'-deoxyuridine (BrdU) on rat embryo development and neurogenesis was investigated using a rat conceptus culture system during organogenesis (pregnancy days 10-13). The embryos and visceral yolk sacs of conceptuses cultured with BrdU were examined for overall growth, morphological anomalies, incorporation of radiolabeled BrdU into DNA, and neurotransmitter enzyme activities in embryos. In addition, neural tubes from cultured whole embryos were isolated and mechanically dissociated into fragments and cultured again to assess neural cell differentiation into neuron-like cells. BrdU was found to incorporate differentially into embryonic and visceral yolk sac DNA with simultaneous stage-specific retardation and anomalous organogenesis in proportion to the increasing concentrations used. Neural tube differentiation of cultured embryos was markedly altered, and there were morphologically distinct neural anomalies. The neurite outgrowth from neuroblast cells (type 1) of explanted spinal neural tube fragments from BrdU-treated embryos was markedly reduced in length and number compared to those from similar areas of embryos grown without BrdU. In contrast, BrdU at the same doses did not affect differentiation of a number of neural tissue-related enzymes. These results indicate that BrdU incorporation into DNA of primordial embryonic cells significantly affects neurogenesis and differentiation of neurites from neuroblasts, which is a specific neural cytodifferentiation characteristic of neuronal cells.     

Synthesis of ribonucleic acid by isolated rat liver mitochondria

Rat liver mitochondria isolated in sucrose-N-tris(hydroxymethyl)methyl-2-aminoethane-sulphonic acid (TES) incorporated [(3)H]UTP into RNA for 1h. Incorporation was inhibited 50% by 1mug of actinomycin D/ml, 1mug of acriflavine/ml and 0.5mug of ethidium bromide/ml but was insensitive to rifampicin, rifamycin SV, streptovarcin and deoxyribonuclease. After the first 10min of incubation, the synthesis was insensitive to ribonuclease. RNA synthesis by mitochondria isolated in sucrose-EDTA was insensitive to actinomycin D and sensitive to ribonuclease during the first 10min of the incubation but thereafter the sensitivities were the same as for mitochondria isolated in sucrose-TES. In a hypo-osmotic medium the relative extent of incorporation of the four ribonucleoside triphosphates into RNA was CTP>UTP=ATP>GTP. In an iso-osmotic medium the incorporation of CTP and GTP decreased. All four nucleotides were incorporated into RNA in a DNA-dependent process, as indicated by the inhibition by actinomycin D. In addition, CTP and ATP were incorporated into the CCA end of mitochondrial tRNA. ATP was also incorporated into an unidentified acid-insoluble compound, which hydrolysed in alkali to a product that was not ATP, ADP or 5'- or 2(3')-AMP. Atractyloside inhibited the incorporation of ATP into RNA with 50% inhibition at 2-3nmol/mg of protein. The [(3)H]UTP-labelled RNA had peaks of 16S and 13S characteristic of mitochondrial rRNA. In addition a peak at 20-21S was observed as well as heterogeneous RNA sedimenting throughout the gradient. The synthesis of all these species was inhibited by actinomycin D, indicating that rat liver mitochondrial DNA codes for mitochondrial rRNA as well as other as yet unidentified species.     

Exogenous DNA transcription in cells with their native DNA inhibited. 1. DNA incorporation into homologous and heterologous cells.

The incorporation of mouse S-EAC DNA into homologous normal cells (mouse embryo secondary cultures), and into heterologous cancer cells (TC-SV40 line), with both systems having their native DNA blocked by BrUdR incorporation, was studied. 3H-TdR-DNA was inoculated with DEAE-D to protect it and to potentiate its incorporation, the process being autoradiograohically controlled. The amount of incorporated DNA was radioisotopically determined, and the incorporation process was studied by analysing the fractions obtained after density gradient centrifugation separation of the inoculated cells DNA. Receptivity was greater in those cells inoculated with DEAE-D-protected DNA. The incorporation was slightly greater for cells whose DNA had been blocked by BrUdR incorporation, and for homologous with respect to heterologous cells. In those cells inoculated while the DNA blockade was incomplete, part of the inoculated DNA became incorporated into the cell genome (L-H chains). However, in the completely blocked cells it could not be determined if the incorporation occurred in a lysogenic-like or in an episomic-like form.     

Synthesis of 8-(2-4 dinitrophenyl 2-6 aminohexyl) amino-adenosine 5' triphosphate: biological properties and potential uses.

We have synthetised 8-(2-4 dinitrophenyl 2-6 aminohexyl) amino-adenosine 5' triphosphate (in short : rATP-DNP), a derivative of ATP which carries a dinitrophenyl group. We show that rATP-DNP is a substrate for calf thymus deoxynucleotidyl terminal transferase (EC 2.7.7.31) and E. coli DNA polymerase I (Kornberg polymerase EC 2.7.7.7.). It can therefore be incorporated into DNA molecules by elongation from 3' ends or by nick translation. The incorporated dinitrophenyl group can be recognized by specific antibodies which can then be detected by anti-antibodies coupled to an enzyme. DNP groups could also be introduced into DNA after enzymatic incorporation of 8-aminohexyl adenosine 5' triphosphate and reaction with 1-fluoro-2-4-dinitrobenzene. Thus, DNA molecules carrying DNP groups can ultimately be revealed by enzymatic coloured reactions. Potential uses of this enzymatic labelling as a substitute to the radioactive detection of nucleic acids, are discussed.     

In vitro synthesis of simian virus 40 DNA. I. Synthesis by a soluble extract from infected CV1 cells.

Simian Virus 40 (SV40) DNA replication was studied in vitro using cell free extracts prepared from SV40 infected CV1 cells. The cells were fractionated into a soluble cytoplasmic fraction and nuclei. The nuclei were lysed with high salt and used to prepare a soluble nuclear fraction. Both fractions displayed DNA polymerase activity as measured with activated calf thymus DNA. However, only the cytoplasmic fraction was active when SV40 DNA comonent I molecules were used as template. Under these conditions, the cytoplasmic extract was shown to catalyse the SV40 DNA dependent, in vitro incorporation of the four deoxyribonucleotides into DNA molecules which had, at both neutral and alkaline pH, the same sedimentation behavior as authentic SV40 DNA component I and component II molecules. Optimal Mg++ concentration was 5-8 mM. Incorporation of label into DNA component I molecules showed an initial lag of about 15 min., after which it was linear with time for up to 5 hrs at 32 degrees. Incorporation into DNA component II molecules proceeded without obvious lag and reached a plateau after approximately 2 hrs of incubation. It is concluded that the cytoplasmic extract supports the in vitro synthesis of SV40 DNA and that DNA component II molecules appear to be a precursor to DNA component I molecules in the reaction. Labeling of viral DNA molecules was highly dependent on ATP and on an ATP generating system. In the absence of ATP and of the energy generating system, incorporation occurred but both template and newly synthesized DNA molecules were extensively degraded.     

Comparison of the metabolic properties of plastids isolated from developing leaves or embryos of Brassica napus L

Plastids isolated from developing leaves and embryos of oilseed rape (Brassica napus L.) were incubated with substrates in the light or the dark, with or without exogenous ATP. Incorporation of HCO^-3, and carbon from a range of substrates into fatty acids and/or starch by leaf chloroplasts was absolutely light-dependent and was unaffected by provision of ATP. Incorporation of HCO^-3 into fatty acids and/or starch by embryo plastids was also light-dependent. However, the light-dependent rates attained, when expressed on a comparable basis, were less than 32% of those from Glc6P (plus ATP), which was the most effective substrate for starch and fatty acid synthesis. In the light alone the rates of carbon incorporation from Glc6P, pyruvate and acetate into fatty acids, and from Glc6P into starch by embryo plastids were less than 27% of the respective ATP-dependent (dark) rates. Light had no effect on these ATP-dependent rates of synthesis by embryo plastids. While transporter activities for both glucose and Glc6P were present in embryo plastids, leaf chloroplasts did not have the latter activity. It is concluded that light at in vivo levels can contribute energy to carbon metabolism in embryo plastids. However, this contribution is likely to be small and these plastids are therefore largely dependent upon interaction with the cytosol for the ATP, reducing power and carbon precursors that are required for maximal rates of starch and fatty acid synthesis.     

Biochemistry of terminal deoxynucleotidyltransferase: mechanism of inhibition by adenosine 5'-triphosphate

The polymerization of deoxyribunucleoside triphosphate catalyzed by terminal deoxyribonucleotidyltransferase (TdT, EC 2.7.7.31) is severely inhibited by the addition of ribonucleoside triphosphates, ATP being the most potent inhibitor. Examination of the inhibitory effect of ATP using oligo(dA)12-18 as well as activated DNA as primers revealed that (a) ATP inhibition is not due to its addition onto a 3'-OH primer terminus ad judged by the lack of incorporation of labeled ATP, although under similar conditions incorporation of GTP can be demonstrated, (b) a consistent degree of inhibition was noted independent of primer or enzyme concentration; (c) addition of ATP to an ongoing reaction promptly reduces the rate of polymerization; (d) kinetic studies indicate a competitive (with respect to substrate deoxy triphosphate) pattern of inhibition; (e) addition of excess deoxyribotriphosphate promptly relieves the inhibition. Unlike ATP, other ribotriphosphates yield a mixed pattern of inhibition partly mediated by competitive mechanisms. GTP and CTP and to a minor extent UTP are incorporated into DNA in the presence or absence of deoxy triphosphate. Furthermore, addition of ATP also inhibits incorporation of GTP and CTP.     

On the role of ATP hydrolysis in RecA protein-mediated DNA strand exchange. I. Bypassing a short heterologous insert in one DNA substrate.

RecA protein promotes a substantial DNA strand exchange reaction in the presence of adenosine 5'-O-3-(thio)triphosphate (ATP gamma S) (Menetski, J.P., Bear, D.G., and Kowalczykowski, S.C. (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 21-25), calling into question the role of ATP hydrolysis in the strand exchange reaction. Here, we demonstrate that the ATP gamma S-mediated reaction can go to completion when the duplex DNA substrate is only 1.3 kilobase pairs in length. The ATP gamma S-mediated reaction, however, is completely blocked by a 52-base pair heterologous insertion in either DNA substrate. This same barrier is readily bypassed when ATP replaces ATP gamma S. This indicates that at least one function of recA-mediated ATP hydrolysis is to bypass structural barriers in one or both DNA substrates during strand exchange. This suggests that ATP hydrolysis is directly coupled to the branch migration phase of strand exchange, not to promote strand exchange between homologous DNA substrates during recombination, but instead to facilitate the bypass of structural barriers likely to be encountered during recombinational DNA repair.     

Effects of culture conditions on the synthesis of human chorionic gonadotropin by placental organ cultures.

Culture conditions for maintaining first trimester human placenta in organ culture, which enhance the secretion of human chorionic gonadotropin (hCG), are described. Nutrient medium, oxygen tension and Gelfoam support matrix influence the synthesis of hCG by these cultures. Placental tissue remained viable for the duration of experiments (12 days) as judged by the incorporation of tritiated thymidine into DNA and the lack of release of incorporated [125I]iododeoxyuridine. Optimal conditions for hCG synthesis in placental organ culture included an atmosphere of 95% air and 5% Co2 (approximately 20% O2), CMRL 1066 medium containing fetal human or bovine serum, insulin, hydrocortisone and retinal acetate. Multiple pieces of placenta could be cultured in the same dish with an additive effect on hCG secretion. The functional responsiveness of these placental cultures was demonstrated by modulation of hCG synthesis with theophylline and 3'5' dibutyryl cyclic AMP.     

Genome multiplication in the trophoblasts and glandular epithelium of endometrium during embryo implantation and placentation of silver fox

Dynamics of genome multiplication during establishment of interrelations between the trophoblast and the glandular epithelium of endometrium was studied in the course of placenta formation in the silver fox. Endometrium response on the embryo implantation exhibits some features of inflammation. In the course of placenta formation the trophoblast gains access to the endometrial glandular epithelium zone, while the endometrial blood vessels grow the other way into the expanding trophoblast zone. The trophoblast gradually replaces the whole epithelium and part of the stroma of the endometrium, closely adjoining the endometrial vessels but not disrupting them. Cytophometric DNA measurements in the trophoblast nuclei have shown that most of the nuclei are polyploid: predominantly 4c-64c, occasionally 128c and 256c. Polyploidy of the trophoblast may result from various types of polyploidizing mitoses. Cytophotometric DNA measurements in mitotic figures have revealed mitoses with DNA amounts equal to 4c (2n), 8c (4n), and 16c (8n), which indicates that trophoblast cells in the silver fox placenta are able to enter mitosis prior to the octaploid level. Higher degrees of polyploidy in the trophoblast cells may be achieved presumably by endoreduplication. In the silver fox polyploidization of uterine grandular epithelial cells during placentation occurs until the level of 8c. Thus, the tissue-specific response of the uterus to the implanting embryo is an active proliferation and polyploidization of the glandular epithelium, rather than formation of a population of polyploid decidual cells (i.e. connective tissue cells). Using the silver fox endotheliochorial placenta as an example, a regularity has been confirmed that cells of both maternal and fetal origin are polyploid in sites of their contact in placenta, which might be of protective significance in the contact of allogenic organisms.     

Nascent DNA chains synthesized in reversibly permeable cells of mouse thymocytes

Freshly prepared thymocytes continue to synthesize DNA under hypotonic conditions in the presence of 4.5% dextran T-150, the four deoxyribonucleoside triphosphates and ATP. Permeable cells could seal the membrane in a serum-enriched medium within a few hours. 2'-Deoxycytidine 5'-triphosphate is effectively substituted by 5-mercuri-2'-deoxycytidine 5'-triphosphate as a substrate. The newly synthesized mercurated DNA can be separated from cellular DNA and RNA on a thiol-agarose affinity matrix. The rate of incorporation of [3H]thymidine triphosphate into permeable cells is the same as that of the incorporation of [3H]thymidine into intact cells, corresponding to approximately 30% of the rate in vivo. Synthesis in permeable cells reflects DNA replication shown by inhibitors such as 1-beta-D-arabinofuranosylcytosine 5'-triphosphate (aCTP), nalidixic acid and novobiocin and by density shift experiments. More than 80% of the newly synthesized low-molecular-mass DNA, 8-60 nucleotides in length, consists of RNA-linked DNA. This conclusion is based on phosphorylation with [gamma-32]ATP and polynucleotide kinase and rephosphorylation after alkaline hydrolysis. The 5' end of RNA consists of adenylate, guanylate, cytidylate and uridylate residues in a ratio of 4:3:1.5:1.5.     

Positional cues for the starch/lipid balance in maize kernels and resource partitioning to the embryo

This study tests the hypotheses that in vivo oxygen levels inside developing maize grains locally affect assimilate partitioning and ATP distribution within the kernel. These questions were addressed through combined topographical analysis (O2- and ATP-mapping), metabolite profiling, and isotope flux analysis. Internal and external oxygen levels were also experimentally altered. Under ambient conditions, mean O2 concentration immediately inside starchy endosperm dropped to only 1.4% of atmospheric saturation (approximately 3.8 microm), but was 10-fold higher in the oil-storing embryo. Increasing the O2 supply to intact kernels stimulated their O2 demand, shifted ATP localization within the kernel, and elevated their ATP/ADP ratio. Enhanced O2 availability also increased steady-state levels of glycolytic intermediates and those of the citric acid cycle, as well as some related pools of free amino acids. Subsequent analyses indicated that starch formation within endosperm, but not lipid biosynthesis within embryo, was adapted to the endogenous low oxygen. Increasing the O2 supply did not change ADP-glucose levels, activity of ADP-glucose pyrophosphorylase, 13C-labeling of ADP-glucose, or flux of 14C-sucrose into starch. In contrast, enhanced O2 availability increased 14C-label uptake into the embryo, 13C-labeling of acetyl-coenzyme A, and finally 14C-incorporation into lipids. Lipid accumulation in embryo appeared highest in regions with higher ATP. Consistent with labeling data, a decrease in O2 supply most strongly affected the embryo, whereas rising O2 levels expanded ATP-rich zones toward the starch-storing endosperm and the scutellar part of embryo. The latter might be responsible for higher 14C-label uptake into the embryo and flux toward lipid. Collectively, data indicate that the in vivo oxygen distribution in maize kernels markedly affects ATP gradients, metabolite levels, and favors assimilate partitioning toward starch within the O2-depleted endosperm. Clear advantages are thus evident for peripheral localization of the protein and lipid storing structures in maize kernels.     

Effects of colchicine on nucleic acid metabolism during metamorphosis of Tenebrio molitor L. and in some mammalian tissues

1. Administration of 10mug. of colchicine/pupa of the beetle Tenebrio molitor L. arrests its differentiation, the pupa remaining alive for 2-3 weeks. 2. The same concentration of colchicine inhibits DNA synthesis and stimulates RNA synthesis (as shown by incorporation into the nucleic acids of labelled adenine, labelled uridine and labelled thymidine). The effects of colchicine on nucleic acid metabolism are first detected 3 days after its administration to first-day pupae. 3. No effects of colchicine are seen on [1-(14)C]glycine incorporation into protein in vivo. 4. Relatively high concentrations of colchicine (e.g. 10mm) suppress incorporation of [8-(14)C]adenine into RNA in dorsal abdominal wall in vitro. Such concentrations have no effect on its incorporation into acid-soluble nucleotides. 5. Colchicine (1mm) suppresses incorporation of [8-(14)C]adenine into DNA to a greater extent than into RNA in various mammalian tissues in vitro (e.g. rat spleen, regenerating rat liver, rat embryo, guinea-pig intestinal mucosa, Ehrlich ascites cells). Colchicine (1mm) has no effect on the rate of respiration of, or on incorporation of radioactivity into acid-soluble nucleotides in, the mammalian tissues tested. 6. Further evidence indicates complex-formation between colchicine and DNA, and it is suggested that the effect of colchicine in suppressing DNA synthesis is due to its combination with the DNA primer (template).     

Inhibition by 1-beta-D-arabinofuranosylcytosine of the incorporation of N-acetylneuraminic acid into glycolipids and glycoproteins of hamster embryo cells

1-β-D-Arabinofuranosylcytosine which interferes with DNA synthesis in bacteria and mammalian cells and brings about transformation of hamster embryo fibroblasts, has been found to inhibit the incorporation of N-Acetylneuraminic acid into glycolipids and glycoproteins of both normal and transformed hamster embryo cells in tissue culture. Three hours after commencement of treatment (10^?3M ara-C), incorporation of [¹⁴C] thymidine into DNA was inhibited by 95 per cent, while incorporation of [³H] D-glycosamine (precursor of sialic acid) into glycolipids and glycoproteins was inhibited by 85 per cent. At 24 hours, the inhibition of incorporation of the two labelled components was 83 and 80 per cent respectively. In homogenates of both cell types, incorporation of [¹⁴C] N-acetylneuraminic acid was competitively inhibited by ara-CMP. Ara-C was found to have no effect on the incorporation of [¹⁴C] choline into phospholipids of cells grown in tissue culture. These results suggest that interference with DNA synthesis by ara-C may not be the only factor involved in cell transformation by this substance.     

黄顶菊的大孢子发生、雌配子体与胚胎发育

用常规石蜡制片对黄顶菊(Flaveria bidentis(L.) Kuntze)大孢子发生、雌配子体和胚胎的发育过程进行了观察.黄顶菊雌蕊柱头二裂,2心皮,1室,单胚珠,基生胎座,单珠被,薄珠心,倒生胚珠,具发达的珠被绒毡层.珠心表皮下分化出孢原细胞,孢原细胞直接发育为大孢子母细胞,大孢子母细胞减数分裂形成直列四分体...