Studies on ultrastructure and host range of aChlorella attacking virus

Summary A tail-less polygonal virus with a prominent capsid of about 140–150 nm in diameter and about 14–15 nm in thickness has been isolated from a freshwater pond. It shows a marked host specificity in attacking only an endosymbioticChlorella sp. isolated fromParamecium bursaria (Ciliata). Viral replication starts in the algal cytoplasm and both autospores and old cells are lysed. The ecology of the virus in the freshwater habitat is discussed. Screening tests for further phycoviruses were not successful.     

Structure-activity relationships of the yeast alpha-factor

The yeast Saccharomyces cerevisiae produces a peptide pheromone, termed the alpha-factor, as a prelude to sexual conjugation. Haploid MAT alpha-cells, but not haploid MAT a-cells or MAT a/alpha-diploids, produce this tridecapeptide of the structure: Trp-His-Trp-Leu-Gln-Leu-Lys-Pro-Gly-Gln-Pro-Met-Tyr. Structural analogues of the alpha-factor have been prepared with alterations in many of the residues, derivatized peptides have been synthesized, and truncated and elongated peptides have been studied. These peptides have been analyzed for their biological activities by various assays. Mutants of S. cerevisiae have been isolated that do not respond to alpha-factor or are supersensitive to the pheromone and its analogues. The mating system of S. cerevisiae provides a powerful model in which genetics, biochemistry, and molecular biology can be used to unravel the mysteries of peptide hormone structure and function.     

Attachment of DNA to nucleolar and nuclear skeletal structures as visualized by Kleinschmidt molecular spreading

Co-isolated residual nuclear shells and residual nucleoli from membrane-depleted rat liver nuclei were spread according to Kleinschmidt's method. Comparison of the spread residual structures isolated from nuclear shells and spread pore complex-lamina isolated from nuclear envelopes showed that these residual structures are morphologically identical. Furthermore, our nuclear shell isolation procedure allowed visualization of DNA strands bound to a granular component of the lamina. The fragmentation of nuclear shells allowed us to obtain well-spread nucleolar remnants, in which we observed DNA strands anchored on a residual nucleolar network attached to the lamina. The different molecular features revealed by the spreading of residual nucleolar structures suggest that both non-transcribing nucleolar DNA and active ribosomal genes are linked to the nucleolar network. Although the exact nature of this network remains to be defined, the results of the present study strongly suggest that the DNA molecules of the chromosomes bearing ribosomal genes have many sites of attachment to a non-chromatin nucleolar network which can be referred to as a nucleolar skeletal complex.     

Changes in ascorbic acid content and ascorbate peroxidase activity during the development of Acetabularia mediterranea

We cloned ras-related sequences from goldfish genomic libraries constructed as recombinants using the lambda phage. Restriction enzyme mapping of the clones obtained revealed three kinds of ras-related sequences among approximately 350,000 genomic clones. One of these clones was partially sequenced. Comparison with the nucleotide sequences of mammalian ras genes showed that the determined sequences covered the predicted amino acid coding regions and parts of the intervening regions. The predicted amino acid sequences of the cloned ras-related goldfish gene suggested that the coding region is localized separately in DNA, and that its exon-intron boundaries are exactly the same as those of corresponding mammalian genes. The nucleotide and amino acid sequences of the goldfish ras-related gene may have extensive homologies to mammalian p 21 protein. Among the three mammalian ras proteins, the predicted amino acid sequence of the sequenced ras-related goldfish clone is most closely homologous (96%) to the Kirsten ras protein. Differences in the predicted amino acid sequence were greatest in the sequence predicted from the fourth exon; fewer differences were found in the sequence from the third exon, and only slight or no differences were found in the sequence predicted for the first and second exons. The 12th and 61st amino acids from the N-terminal of the protein, which are thought to be critical positions for GTP binding and catalysis, are both conserved in the goldfish protein.(ABSTRACT TRUNCATED AT 250 WORDS)     

Influence of very low doses of ionizing radiation on Synechococcus lividus metabolism during the initial growth phase

Previous results from this laboratory have shown that very low chronic doses of gamma radiation can stimulate proliferation of the Cyanobacterium Synechococcus lividus. This modification of cell proliferation occurred during the first doubling. In this paper, we have compared the metabolism of cells cultivated in a normal environment or under chronic irradiation. Incubation of the cells in a new medium induced a high superoxide dismutase (EC 1.15.1.1, SOD) activity at the 18th hour and a degradation of phycocyanin, thus demonstrating that cells were submitted to a photooxidative stress. This increase in superoxide dismutase activity was followed by concomittant peaks of glutathione reductase (EC 1.6.4.2, GR) and glucose-6-phosphate dehydrogenase (EC 1.1.1.49, G6P-DH) at the 24th hour. Irradiated cultures at a dose of 53.5 mGray/year show an earlier and higher peak of SOD, GR, and G6P-DH. In a second stage, cultures showed an earlier onset of photosynthesis under irradiation, as evidenced by an increase in pigment content and an enhancement of glyceraldehyde-3-phosphate dehydrogenase (EC 1.2.1.13, GAP-DH). These results show that the radiostimulation is related to the activation of enzymes protecting against peroxides that were induced under oxidative circumstances and to the activation of a glucose catabolism via the oxidative pentose phosphate pathway.Abbreviations mGy milli-Gray - SOD superoxide dismutase - G6P-DH glucose-6-phosphate dehydrogenase - GAP-DH glycer-aldehyde-3-phosphate dehydrogenase - GSSG oxidized glutathione     

The proα2 (V) collagen gene (COL5A2) maps to 2q14→2q32, syntenic to the proα1 (III) collagen locus (COL3A1)

Summary A recombinant probe specific for the pro2 chain of human Type V collagen has been used for the localization of the corresponding gene (COL5A2) to chromosome 2. Regional mapping by in situ hybridization and analysis of DNA from humanxrodent cell lines indicated that COL5A2 is confined within the segment 2q142q32, thus syntenic to the pro1 (III) collagen gene (COL3A1).     

Receptor-mediated particle uptake by liver macrophages. The galactose-particle receptor mediates uptake via coated and also non-coated structures

The endocytosis pathways of particles with terminal beta-D-galactosyl groups were studied in isolated rat Kupffer cells by electron microscopy. Colloidal gold particles of sizes 5, 17 and 50 nm were coated with asialofetuin (ASF) and isolated liver macrophages were allowed to bind (at 4 degrees C) or take up (at 37 degrees C) these ligands. Particles of all three sizes were bound via the galactose-particle receptor as shown by carbohydrate inhibition experiments and were ingested effectively. But, whereas ASF-gold particles of sizes 5 and 17 nm are taken up via the coated pit/coated vesicle pathway, the 50 nm particles are not. These enter the cell via non-coated endocytic vacuoles. All three particle sizes are transported to the same lysosomal compartment. These observations demonstrate that at least in macrophages one receptor is capable to mediate endocytosis via two different pathways depending on ligand size and/or valency.     

Production and purification of monoclonal antibodies to Pisum and Avena phytochrome

At least nine monoclonal antibodies against phytochrome from Pisum sativum L. and 20 against phytochrome from Avena sativa L. have been obtained from mouse hybridomas that were produced by fusion of spleen cells with SP 2/O-Ag14 myeloma cells. Hybridomas were selected and cloned in a single step by plating on a semisolid methylcellulose medium. Eight antibodies to Pisum and one to Avena phytochrome were immunopurified from hybridoma medium or ascitic fluid. When necessary, secreted antibodies were verified to be against phytochrome by demonstrating to be against phytochrome by demonstrating immunoadsorption of phytochrome, detected as loss of photoactivity and-or by appearance of the approx. 120,000-dalton phytochrome band upon sodium dodecyl sulfate polyacrylamide gel electrophoresis.     

Purification and characterization of hydroxypyruvate reductase from cucumber cotyledons

Hydroxypyruvate reductase (HPR), a marker enzyme of peroxisomes, has been purified to homogeneity from cotyledons of light-grown cucumber seedlings (Cucumis sativus var. Improved Long Green). In addition, the peroxisomal location of both HPR and serine-glyoxylate aminotransferase has been confirmed in cucumber cotyledons. The isolation procedure involved Polymin-P precipitation, a two-step precipitation with ammonium sulfate (35 and 50% saturation), affinity chromatography on Cibacron Blueagarose, and ion-exchange chromatography on DEAE-cellulose. HPR was purified 541-fold to a final specific activity of 525 ± 19 micromoles per minute per milligram of protein. Enzyme homogeneity was established by native and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The native molecular weight was 91 to 95 kilodaltons, approximately double the apparent subunit molecular weight of 40,500 ± 1,400. With hydroxypyruvate as substrate, the pH optimum was 7.1 and K_m values were 62 ± 6 and 5.8 ± 0.7 micromolar for hydroxypyruvate and NADH, respectively. With glyoxylate as substrate, the pH optimum was 6.0, and the K_m values for glyoxylate and NADH were 5700 ± 600 and 2.9 ± 0.5 micromolar, respectively. Antibodies to HPR were raised in mice (by the ascites tumor method) and in rabbits, and their monospecificity was demonstrated by a modified Western blot immunodetection technique.