N uptake, assimilation and isotopic fractioning control δ 15N dynamics in plant DNA: A heavy labelling experiment on Brassica napus L.

In last decades, a large body of evidence clarified nitrogen isotope composition (δ15N) patterns in plant leaves, roots and metabolites, showing isotopic fractionation along N uptake and assimilation pathways, in relation to N source and use efficiency, also suggesting 15N depletion in plant DNA. Here we present a manipulative experiment on Brassica napus var. oleracea, where we monitored δ 15N of purified, lyophilized DNA and source leaf and root materials, over a 60-days growth period starting at d 60 after germination, in plants initially supplied with a heavy labelled (δ 15NAir-N2 = 2100 mUr) ammonium nitrate solution covering nutrient requirements for the whole observation period (470 mg N per plant) and controlling for the labelled N species (ṄH4, ṄO3 and both). Dynamics of Isotopic Ratio Mass Spectrometry (IRMS) data for the three treatments showed that: (1) leaf and root δ 15N dynamics strictly depend on the labelled chemical species, with ṄH4, ṄO3 and ṄH4ṄO3 plants initially showing higher, lower and intermediate values, respectively, then converging due to the progressive NH4+ depletion from the nutrient solution; (2) in ṄH4ṄO3, where δ15N was not affected by the labelled chemical species, we did not observe isotopic fractionation associated to inorganic N uptake; (3) δ15N values in roots compared to leaves did not fully support patterns predicted by differences in assimilation rates of NH4+ and NO3-; (4) DNA is depleted in 15N compared to the total N pools of roots and leaves, likely due to enzymatic discrimination during purine biosynthesis. In conclusion, while our experimental setup did not allow to assess the fractionation coefficient (ε) associated to DNA bases biosynthesis, this is the first study specifically reporting on dynamics of specific plant molecular pools such as nucleic acids over a long observation period with a heavy labelling technique.

Immuno-electron microscopic identification of Methanosarcina spp. in anaerobic digester fluid

Whole cell antibodies against Methanosarcina mazei strain S6 were used with protein A – colloidal gold to identify bacteria in thin sections of samples from anaerobic methane producing digesters. It was possible to identify bacteria at the genus level and to show relatedness at the species and strain levels. Heavy labelling was observed on thin sections of the immunogenic strain S6. Lighter labelling was observed with pure cultures of M. mazei strain LYC. Pure cultures of Methanococcus voltae or Methanobacterium thermoautotrophicum did not label. In samples from mesophilic sewage digesters, sarcinal colonies of bacteria similar to Methanosarcina showed heavy labelling per cell while colonies from 55 °C digesters show lighter labelling. A few free cocci, which were released from the sarcinal colonies, also labelled. Labelling was not observed on other bacterial forms in either set of digesters. These studies indicate that a collection of bacterial antibodies can be used to identify and map bacteria in situ in mixed samples.

Altered labelling of the cell surface and intracellular organelles with [3H]mannose in enucleated amoebae

The production, transport, and disposition of material labelled with [3H]mannose were studied in microsurgically enucleated and control amoebae. Cells were injected with the precursor and samples were prepared for electron-microscope radioautography at intervals, up to 24 h later. Control cells showed heavy labelling of the rough endoplasmic reticulum and the Golgi apparatus at early intervals after injection. Later, labelling of groups of small vesicles increased, and the percentage of grains over the cell surface peaked 12 h after administration of the precursor. Two major changes were detected in enucleate amoebae. First, the kinetics of labelling of cell organelles with [3H]mannose were altered in the absence of the nucleus. The Golgi apparatus and cell surface both displayed maximal labelling at later intervals in enucleates, and the percentage of grains over the rough endoplasmic reticulum varied less with time in enucleated than in control cells. Second, the distribution of radioactivity was altered. A greater percentage of grains was associated with lysosomes in enucleates than in control cells. The change in the kinetics of labelling of the endoplasmic reticulum, Golgi apparatus and cell surface indicates that intracellular transport of surface material was slower in the absence of the nucleus. It is suggested that this is related to the decreased motility of enucleate cells.

Calculation of the rate of gluconeogenesis from the incorporation of 14C atoms from labelled bicarbonate or acetate

The rate of gluconeogenesis in vivo may be estimated by the incorporation of 14C atoms from a labelled precursor into plasma glucose or by introducing 14C atoms into the pathway of gluconeogenesis at known stages by metabolites which in themselves do not contribute to the net synthesis of glucose (e.g., bicarbonate or acetate). The purpose of the investigation was to examine some of the assumptions involved in the calculation of gluconeogenic flux by the second approach. [2- 14C]acetate or NaH 14CO3 was infused to dogs, and the specific activity (SA) of glucose, bicarbonate CO2, urea, and lactate in the plasma was followed. The incorporation of 14C atoms from [2- 14C]acetate into glucose allows the calculation of the degree of underestimation of glucose formation due to "metabolic exchange" in the hepatic oxaloacetate pool. The possible error introduced into this calculation by the incorporation of 14C atoms from 14CO2 (a product of acetate oxidation) was found to be negligible, but the heavy labelling of plasma lactate may possibly affect the estimate of metabolic exchange. It is proposed that in the calculation of the rate of gluconeogenesis from infused NaHCO3 the SA of hepatocellular and not of plasma bicarbonate CO2 should be related to that of plasma glucose. This latter is expected to equal the SA of plasma urea, since the sole precursor of its C atom is hepatocellular CO2. The rate of gluconeogenesis estimated from the SA(glucose)/SA(urea) ratio and a previously estimated correction factor for metabolic exchange was 51% of the glucose production in the postabsorptive state. The nearly identical SA(urea)/SA(CO2) ratios, irrespective of the tracer infused, indicated that plasma CO2 is a major precursor of urea C and that a large fraction of injected acetate is oxidized by extrahepatic tissues.

Some trends in the evolution of very large chromosomes

The general features of the arrangement and cytological distribution of repeated sequences in animal chromosomes are reviewed. These features include internal repetitiveness, conservation of clearly functional sequences, rapid divergence of certain classes of repeated sequence with subsequent fixation of families of diverged sequences, and well defined cytological localization of large blocks of sequences in specific parts of the chromosome set. Moderately or ‘middle’ repetitive (m.r.) sequences constitute a large part of the genomes of higher organisms, they seem to accumulate in a balanced manner within chromosome sets, they are mainly responsible for genome growth, and they are interspersed with sequences of other kinds. Little is known about their cytological distribution. Four experiments are described, each of which aimed to locate middle repetitive sequences in the chromosomes of a salamander and a newt. Tritiated m.r. DNA from Plethodon cinereus binds in a non-random fashion to the meiotic diplotene and mitotic chromosomes of the same species, suggesting a non-random distribution of m.r. sequences on these chromosomes. The same DNA, hybridized in situ to the RNA transcripts on the loops of lampbrush chromosomes, produces light and widespread labelling of many loops, but intense labelling of six pairs of loops, each of which lies near to a centromere. Similar experiments in which newt m.r. DNA was hybridized in situ to newt lampbrush chromosomes showed heavy labelling of about 30 loop pairs on each of the long heteromorphic arms of chromosome I, but very little labelling elsewhere. Autoradiographs of newt mitotic chromosomes hybridized with newt m.r. DNA showed rather even labelling of all chromosomes including chromosome I. The significance of the heavy labelling of lampbrush loops near centromeres and on the heteromorphic arms of the newt chromosome I is discussed in relation to possible cytological and molecular mechanisms for generating and preserving families of tandemly linked and cytologically localized m.r. sequences.

A Massive System of Microtubules Associated with Cytoplasmic Movement in Telotrophic Ovarioles

The telotrophic ovary of Notonecta glauca glauca consists of 7 ovarioles. Each ovariole comprises, from front to rear, a terminal filament, a trophic region, a prefollicular region, and a series of 10-15 follicles of progressively increasing size The trophic region is largely syncytial and is made up of polyploid trophic nuclei packed around a central trophic core The cytoplasm of the trophic core is continuous with the cytoplasm of each oocyte through a system of trophic tubes. There is one trophic tube per oocyte. The trophic nuclei have large nucleoli. There are a few small nucleoli in the oocyte nuclei The cytoplasm of the trophic core, the trophic tubes, and the oocytes is rich in RNA. Autoradiographs of sections of ovarioles fixed 2 h after injection of [3H]uridine into animals show label over the trophic nuclei only. Eight-hour autoradiographs show heavy labelling of the trophic region and label over the front ends of the trophic tubes, but little label over the posterior regions of the tubes or the oocyte cytoplasm. Later autoradiographs mdicate that label gradually spreads backwards from the trophic core, along the trophic tubes, and progressively builds up in the oocyte cytoplasm These observations are thought to indicate synthesis of RNA in the trophic region and movement of RNA from the trophic core along the trophic tubes to the oocytes The trophic core and tubes show brilliant positive form birefringence with respect to their lengths. This birefringence can be reduced by keeping animals at 2 °C for 12 h, and eliminated by placing ovarioles in 1 % colchicine for 6 h. Electron micrographs of thin sections of ovarioles show that trophic core and tubes are densely and uniformly packed with ribosomes and microtubules The latter are lined up along the trophic tubes. There are about 30000 microtubules evident in a TS through a trophic tube 15µm wide. Lengths of microtubules up to 2µm have been observed. Ribosomes are packed between the microtubules but are excluded from regions where the spacing between adjacent microtubules is less than 25 nm The contribution of the trophic region to the oocytes and the role of the microtubules in maintaining or facilitating the movement of ribosomes along the trophic tubes is discussed

Organization and Development of the Macronuclear Anlage in Stylonychia Notophora Stokes

The development of the macronuclear Anlage of Stylonychia notophora was studied by means of cytochemical and autoradiographic techniques. In early stages of its differentiation, the Anlage has its chromatin distributed homogeneously. Nine to ten hours later chromosomes are noticed; the nucleus is possibly diploid. Following this, the number of chromosomes increases progressively resulting in polyploidy. Later, chromosomes uncoil and polytenization follows. After consolidation and shrinkage of these chromosomes, the Anlage splits into two bodies. Five series of replication bands quickly pass through each and the vegetative macronucleus results. Synthesis of RNA by the new macronucleus starts soon after the shrinkage stage. The old macronuclei break up into small spherical bodies which are gradually resorbed in the cytoplasm. When [3H]thymidine is provided continuously to the exconjugants, there is little uptake into the Anlage until the old macronucleus is completely resorbed. Only after this, heavy labelling is noticed. Fate of the previously labelled macronuclei during conjugation shows that their breakdown products are re-utilized for the development of the new macronucleus.