Ribonucleic acid labelling and nucleotide pools during compensatory renal hypertrophy

During the first 48h of compensatory renal hypertrophy induced by unilateral nephrectomy, RNA content per cell increased by 20-40%. During this period, rates of RNA synthesis derived from the rates of labelling of UTP and RNA after a single injection of [5-(3)H]uridine showed no change in the rate of RNA synthesis (3.1nmol of UTP incorporated into RNA/min per mg of RNA). ATP and ADP pools were not changed. The rate of RNA synthesis was considerably in excess of the increment of total RNA appearing in the kidneys. With [5-(3)H]uridine as label, only continuous infusion for 24h could produce an increase (60%) in the specific radioactivity of renal rRNA in mice with contralateral nephrectomies. With a single injection of [methyl-(3)H]methionine used to identify methyl groups inserted into newly synthesized rRNA, the specific radioactivity of this rRNA was unchanged 5h after contralateral nephrectomy, increased by 60% at 9-48h, and returned to normal values at 120h. Most RNA synthesized in both nephrectomized and sham-nephrectomized mice has a short half-life. Since total cellular RNA content increases in compensatory hypertrophy despite unchanged rates of rRNA synthesis, the accretion of RNA might involve conservation of ribosomal precursor RNA or a change in rate of degradation of mature rRNA.     

Cytotoxic effects of dexamethasone restricted to noncycling,early G1-phase cells of L1210 leukemia

The cytostatic and cytolytic effects of dexamethasone were studied as functions of cell cycle position in mouse L1210 leukemia cells. To this end, the cells were separated according to size by sedimentation at unit gravity in a specially designed sedimentation chamber. The fractions were analyzed by radioautography and flow cytophotometry. The size-distributions obtained by 1g sedimentation coincided with cell-cycle age distribution. With increasing fraction number, samples highly enriched in G1, S, and G2/M cells, respectively were obtained: the smallest cells being in early G1 and the largest in mitosis. In the presence of dexamethasone (10^?6-10^?5 M), growth slowed down after a few cell cycles and the cells accumulated in early G1 phase. Lytic cell kill by continued exposure to the drug was confined to the fractions containing the small, early G1-phase cells. These fractions were also enriched in noncycling cells that were not labeled by prolonged exposure to ³H-thymidine. After removal of dexamethasone, the cells in S and G2/M phase completed cell cycle traverse but were retarded again in the G1 and early S phase of the next division cycle. The data suggest a memory effect for previous drug exposure. It is concluded that the cytostatic and cytolytic effects of dexamethasone are separate, though not unrelated events. Cytolysis is confined to the noncycling cells that in untreated populations can exit from the dividing compartment during a transitional phase of about 60 minutes subsequent to mitotic division. The cytostatic effects potentiate cytolysis by accumulating the cells in the early G1 phase and thus increasing the probability of their transit to the G0 compartment, sensitive for drug-mediated cytolysis.     

Expression of Frankia nif genes in nodules of Alnus glutinosa

Expression of Frankia genes involved in nitrogen fixation was studied in Alnus glutinosa nodules using the in situ hybridization technique. The results show that high level expression of nif genes does not occur immediately upon infection of cortical cells by Frankia. Also, only in the infected cells near the tips of the nodule lobes, nif genes are expressed at high levels. In the majority of infected cells, nif gene expression is rather low.     

High-resolution mapping on pachytene chromosomes and extended DNA fibres by fluorescencein-situ hybridisation

This article describes two protocols for high-resolution physical mapping of DNA sequences in tomato using fluorescencein situ hybridisation (FISH). The first technique involves FISH to spread chromosomes from pollen mother cells at pachytene and proves to be an excellent method for assigning DNA sequences to chromosome regions at a resolution of up to a few hundred kilobase. An even higher resolution was obtained for extended DNA fibre, prepared from interphase nuclei and used as hybridising component. This technique permits strong enhancement of physical map resolution to values of a few kilobase. The power of both methods simultaneously applied for the same material was demonstrated with the combination of the telomeric repeat and the tomato specific telomere-associated repeat TGR1 as example.     

Promotive and inhibitory effects of diverse arabinogalactan proteins on Daucus carota L. somatic embryogenesis

In the 3-d-old 2-mm root tip of Pisum sativum L. cv. Lincoln the percentage of actively proliferating cells is estimated to be 70%. The remaining cells are non-cycling and arrested with 2C and 4C DNA content in G₀ and in G₂Q, respectively. In this work we studied the kinetic significance of these quiescent cells, using the sorting capabilities of flow cytometry and immunofluorescence techniques to detect the proliferation marker PCNA (proliferating cell nuclear antigen) inside cells within the different cell-cycle compartments. While in animal cells, PCNA is present at a high level only in actively proliferating cells, in 3-d-old pea root tips 95% of the cells are PCNA-positive. After flow cytometry and sorting of pea non-cycling nuclear populations, all G₂Q nuclei appeared strongly PCNA-positive, indicating that these cells had recently left the cell cycle. By contrast, most G₀ nuclei showed a low level of PCNA immunofluorescence intensity, as measured by image analysis, with about 25% of the nuclei being PCNA-negative. This small percentage was found to correspond to root cap cells, as could be observed in the root tip section. These are the only cells in the root apical region which are fully differentiated and which, therefore, lack the competence to enter the cell cycle. In contrast, the more or less PCNA-positive G₀ nuclei could represent a kinetically heterogeneous population of cells competent to proliferate, but which have either recently left the cell cycle or are progressing to the G₀-G₁ transition. Received: 6 November 1996 / Accepted: 14 January 1997     

Expression of the JIM8 cell wall epitope in carrot somatic embryogenesis

Certain single cells in carrot (Daucus carota L.) suspension cultures react with the monoclonal antibody JIM8, and it has been proposed that these cells represent a transitional stage in somatic embryo formation. Shortly after isolation of the single cells by sieving, up to 80% of the cells react with JIM8. Within 4 d, JIM8 labelling becomes restricted to 1% of the single cells. To obtain evidence for the proposed correlation between expression of the JIM8 cell wall epitope and somatic embryo formation the developmental fate of carrot single cells labelled with JIM8 was determined by cell tracking. The results, obtained by recording 43 000 cells, show that only few JIM8-labelled cells give rise to embryos, and most somatic embryos develop from cells devoid of the JIM8 cell wall epitope. We therefore conclude that the presence of the JIM8 cell wall epitope does not coincide with the ability of single suspension cells to form embryos.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - AGP arabino galactan protein - B5-0 Gamborg's B5 medium - B5-0.2 Gamborg's B5 medium supplemented with 0.2 M 2,4-D - FITC fluoresceïn isothiocyanate - PBS phosphate-buffered saline     

Liposome-mediated transfer of YAC DNA to tobacco cells

This article describes a set of protocols—for retrofitting, transformation and purification—that together enable the delivery of full-sized YAC-DNA to plant cells. To be able to equip YACs of interest with plant selectable markers, we have constructed a retrofitting vector that carriesnptII anduidA. Furthermore, we established a transformation protocol for plant protoplasts that is sufficiently efficient to support transfer of high-molecular-weight DNA. In this protocol lipofection is combined with PEG-mediated direct gene transfer. Large amounts of purified DNA are necessary for lipofection. To obtain sufficient quantities of concentrated, purified YAC-DNA, we used an optimized two-step, gel-purification method. Transient expression of a YAC-bornuidA demonstrates that both retrofitting vector and transformation protocol are effective.