Three-dimensional graphical reconstruction from HVEM stereoviews of biological specimens by means of a microcomputer

A microcomputer reconstruction technique has been developed in order to permit a larger exploitation of stereomicroscopy. The microcomputer facility consists of a digitizing tablet, a microcomputer, a graphics terminal, a graphics plotter and a printer. The technique has been applied to the study of HVEM stereopairs, performed by recording two images of the same area of a specimen (thick section of araldite-embedded leech ganglion neurons), tilted relative to the beam axis through an angle 0/20 degrees. Coordinates of N conjugate points of interest, expressed in a common reference system were obtained with the help of a digitizing tablet and the misorientation between the two images was determined by a method based on a least square technique. New projections of the object on different planes are provided by the microcomputer facility. Also the microcomputer method permits to obtain new stereopairs drawings, in various orientations and slices from a three-dimensional reconstruction of the object oriented in any direction in space. The method permits to obtain computed anaglyph drawings, printed here, which are stereoviews of the same object.     

Isolation and characterization of the yeast aspartyl-tRNA synthetase gene

A yeast genomic library in Escherichia coli, constructed by insertion of Sau3A restriction fragments into the hybrid Saccharomyces cerevisiae-E. coli plasmid pFL1, was screened by a radioimmunoassay (RIA) for colonies expressing yeast aspartyl-tRNA synthetase (AspRS). Four clones were isolated by this technique. Data obtained by Southern and restriction analysis of the inserts showed a common 3.8-kb BamHI restriction fragment which, when inserted into the plasmid pFLl, gave a positive RIA. Several controls showed that this 3.8-kb insert codes for the entire AspRS : (i) S. cerevisiae transformed by the PFL1 plasmid carrying the 3.8-kb fragment overproduces AspRS activity by a factor of ten compared to the wild-type yeast strain; and (ii) a new protein with electrophoretic behaviour similar to AspRS and immuno-reactive toward anti-AspRS appears in crude extracts of transformed yeast and E. coli.     

The growth of Saccharomyces cerevisiae CBS 426 on mixtures of glucose and succinic acid: A model

Saccharomyces cerevisiae CBS 426 was grown in continuous culture in a defined medium with a mixture of glucose and succinic acid as the carbon source. Growth on succinic acid was possible after long adaptation periods. The flows of glucose, succinic acid, oxygen, carbon dioxide, and biomass to and from the system were measured. It proved necessary to expand our previous model to accommodate the active transport of succinic acid by the cell. The values found for the efficiency of the oxidative phosphorylation (P/O) and the amount of ATP needed for production of biomass from monomers gave the same values as found for substrate mixtures taken up passively.     

Enrichment and characterization of the mRNAs of four aminoacyl-tRNA synthetases from yeast.

We have partially purified the messenger RNAs for yeast arginyl-, aspartyl-, valyl-, alpha and beta subunits of phenylalanyl-tRNA synthetases in order to study their biosynthesis and ultimately, to isolate their genes. Sucrose gradient fractionation of poly U-Sepharose selected mRNAs resulted in a ten fold enrichment of the in vitro translation activity of these mRNAs. The translation products of messenger RNAs for arginyl- and valyl-tRNA synthetases have the same molecular weight as the purified enzymes; translation of aspartyl-tRNA synthetase messenger RNA yielded a 68 kD molecular weight polypeptide (while the purified cristallisable enzyme appears as a 64-66 kD doublet, which, as we showed is a proteolysis product). The translation of the mRNAs for alpha and beta phenylalanyl-tRNA synthetase gave polypeptides having the same molecular weight as those obtained from the purified enzyme, but the major translation products are slightly heavier, indicating that they may be translated as precursors. As estimated from centrifugation experiments mRNAs of arginyl-, aspartyl-, alpha and beta subunits of phenylalanyl-tRNA synthetase were 1700-2000 nucleotides long, indicating that alpha and beta are translated from two different mRNAs.     

Effect of chromatic errors in microscopy on the visualization of multi-color fluorescence in situ hybridization

The relevant microscopical conditions for the optimal visualization of ratio-color FISH stained cells were investigated. Special attention was given to the influence of the type of illumination, (semi)-critical vs. K?hler type illumination, in combination with the use of multi-band excitation and emission filters, on the registration of the colors of ratio labelled probes. Due to chromatic errors, many collecting lenses were found to cause a wavelength dependent excitation pattern with critical illumination. This resulted in a change of the observed color of microscopic objects when stained with a mixture of two dyes and excited with a dual band pass filter. A quantitative study of this effect for semi-critical illumination of FISH ratio-labelled chromosomes revealed a difference of 20% between highest and lowest ratio values depending on the position of the object in the microscopic field vs. only 2.5% for K?hler type of illumination. The impact of these errors on the identification of ratio-labelled probes and on the sensitivity of comparative genomic hybridization (CGH) to detect gene amplifications or losses is discussed. Standard preparations consisting of solutions of defined mixtures of fluorescent dyes or objects stained with defined ratios of fluorophores, are proposed to correct for the errors observed.     

Widespread formation of intracellular calcium carbonates by the bloom-forming cyanobacterium Microcystis

The formation of intracellular amorphous calcium carbonates (iACC) has been recently observed in a few cultured strains of Microcystis, a potentially toxic bloom-forming cyanobacterium found worldwide in freshwater ecosystems. If iACC-forming Microcystis are abundant within blooms, they may represent a significant amount of particulate Ca. Here, we investigate the significance of iACC biomineralization by Microcystis. First, the presence of iACC-forming Microcystis cells has been detected in several eutrophic lakes, indicating that this phenomenon occurs under environmental conditions. Second, some genotypic (presence/absence of ccyA, a marker gene of iACC biomineralization) and phenotypic (presence/absence of iACC) diversity have been detected within a collection of strains isolated from one single lake. This illustrates that this trait is frequent but also variable within Microcystis even at a single locality. Finally, one-third of publicly available genomes of Microcystis were shown to contain the ccyA gene, revealing a wide geographic and phylogenetic distribution within the genus. Overall, the present work shows that the formation of iACC by Microcystis is common under environmental conditions. While its biological function remains undetermined, this process should be further considered regarding the biology of Microcystis and implications on the Ca geochemical cycle in freshwater environments.     

Reflection contrast microscopy of ultrathin sections in immunocytochemical localization studies: a versatile technique bridging electron microscopy with light microscopy

Reflection contrast microscopy (RCM) of ultrathin sections was recently introduced as a sensitive technique for visualization with enhanced definition in immunogold histochemistry. Experience of using RCM as a major tool in immunocytochemical research in different fields is summarized, e.g. oncology, nephrology and embryology. The sensitive visualization of immunocytochemical labels, gold particles or peroxidase-diaminobenzidine deposits in or on ultrathin sections, by RCM instead of electron microscopy is demonstrated. RCM of ultrathin sections is an adequate light microscopical alternative for immunoelectron microscopy, since an overview of both label and tissue is obtained with a high image definition and high contrast of label. In the studies presented, RCM is shown to provide a better gradation in staining intensity and staining pattern than other light microscopical methods. Moreover, a precise localization of multiple labels is obtained with this method. Besides the applications shown, ultrathin section visualization by RCM is very useful for correlative light- and electron microscopical studies of fine structures. Commercially available fluorescence microscopes can be adapted for proper RCM functioning; an adaptation scheme and list of microscopes tested is provided.