B. Parisi

Specific and reversible inhibition by aphidicolin of the α‐like DNA polymerase of plant cells

Aphidicolin, a tetracyclic diterpenoid obtained from Cephalosporium aphidicola [ 1 ] is an inhibitor of the replication of nuclear and viral DNA in animal cells [2,3]. The drug interferes specifically with the replicative [4-l l] DNA polymerase a! of animal cells or with the virus-induced replicative DNA polymerase [3], while it has no effect on the &polymerase [3,12] which is most likely involved in DNA repair [7,8,13], nor on the y-polimerase [3,12], involved in animal mitochondrial DNA synthesis [7,8]. Plant cells contain at least two distinct DNA polymerases; we have named them a-like [ 151 and y-like [ 161 because their properties closely resemble those of the (Yand y-polymerases present in animal cells [4]. The a-like DNA polymerase is the most abundant in cultured plant cells and responds to changes in the rate of cell multiplication [ 151, whereas the y-like DNA polymerase is present in chloroplasts [ 161. This paper describes the effect of aphidicolin both in vivo, on cultured plant cells, and, in vitro, on the activity of the a-like and r-like plant DNA polymerases. The results show that aphidicolin depresses the incorporation of thymidine into the DNA of cultured plant cells whereas it has no effect on RNA and protein synthesis. The effect is reversible since DNA synthesis and cell growth resume upon removal of the drug. In vitro the drug inhibits the activity of the a-like but not of the chloroplasts, r-like DNA polymerase. The inhibition is competitive with dCTP in analogy with the mode of inhibition of the animal 01 polymerase [14,17]. The results, besides adding further evidence on the similarity between the a-like plant DNA polymerase

Species specificity in protein synthesis

The phenomenon of species specificity in poly U-directed cell-free protein synthesis (whereby combinations of ribosomes of one organism with supernatant enzymes from a different one are sometimes inactive) was studied in the case of the amino acid-incorporating systems prepared from Escherichia coli, Bacillus subtilis, Saccharomyces cerevisiae , castor bean seedlings and rat liver. These organisms could be classified into at least two groups, one including B. subtilis and E. coli , and the other S. cerevisiae , castor bean seedlings and rat liver. It appears that within each group, the combination of ribosomes and supernatant enzymes extracted from different organisms gave active synthesis, whereas the permutations of ribosomes and enzymes prepared from organisms belonging to the two different groups gave very little if any synthesis. The mechanism of incompatibility was analyzed in detail in the E. coli -castor bean seedlings combination. It was found that the inability to synthesize polypeptides was not due to any of the following factors: the synthesis of phenylalanyl-sRNA; the source of the phenylalanyl-sRNA used; the selective degradation of heterologous phenylalanyl-sRNA; the binding of phenylalanyl-sRNA to ribosomes; the presence of specific inhibitory activities. The tentative conclusion is drawn that failure to observe protein synthesis in mixed systems is due to the lack of interaction between ribosomes of a given size (70 or 80 s) and polymerizing enzymes extracted from organisms containing ribosomes of different size.

Selection and nuclear DNA analysis of cell hybrids between Daucus carota and Oryza sativa

In order to study the fate of the parental genomes in somatic cell hybrids between distantly related species, protoplasts from cultured cells of Daucus carota and Oryza sativa were fused. Selective conditions resulted, exclusively, in the growth of hybrid colonies which combined the capacity to multiply of carrot cells with the natural resistance to A2CA of rice cells. A methodology for measuring the relative contribution of the parental cells to the hybrid nuclear genome has been worked out. This is based both on hybridization of nuclear DNA bound to nitrocellulose filters (dot hybridization) with radioactively labelled nuclear DNA from one of the parents and on agarose gel fractionation of nuclear DNA digested with restriction endonucleases. The dot hybridization analysis, performed on one of the D. carota x O. sativa cell hybrids, showed that the major portion of the nuclear genome is homologous with the carrot partner, with rice contributing only a minor fraction, along with the selected resistance gene(s). The homology was confirmed after agarose gel fractionation of restriction endonuclease BamHI-digested nuclear DNA. Furthermore, strong homology at the level of gene expression between hybrid and carrot cells was shown by polyacrilamide gel electrophoresis of total soluble proteins.

Genetic markers in cultured plant cells: Differential sensitivities to amethopterin, azetidine-2-carboxylic acid and hydroxyurea

Abstract The effects of three growth inhibitors, amethopterin, L-azetidine-2-carboxylic acid and hydroxyurea, have been tested on cell cultures from 6 plant species. The results show that naturally-occurring differences in drug sensitivity are present and can be easily demonstrated by assaying the effects of the drugs on the growth of cells in suspension culture under strictly-controlled experimental conditions. Similar differences are also evident in cells of the plants from which the cultures were derived. In order to determine the usefulness of these different sensitivities as selective tools, a culture flask was devised in which the effect of the drugs on a mixed culture of two cell species can be determined.

Characterization of a carrot cell line resistant to azetidine-2-carboxylic acid

Abstract The mechanism of resistance of a carrot cell line to azetidine-2-carboxylic acid (AZCA) was investigated. Two main alterations were observed in the resistant cells: an over-production of proline and of some other amino acids and a reduced rate of uptake either of AZCA or of other amino acids tested. This latter phenomenon appears to be a secondary effect due to the increased internal amino acid concentration, which inhibits the activity of the amino acid carrier(s).

Functional roles of the plant α‐like and γ‐like DNA polymerases

Plant cells are endowed with two distinct DNA polymerases [ 1,2] whose properties closely resemble those of the DNA polymerases cr and y present in animal cells [3,4]. The plant DNA polymerases have consequently been named o-like [ 1 ] and y-like [2]. The B-like DNA polymerase activity is the most abundant in cultured plant cells [l ] and responds to changes in the rate of cell multiplication, whereas experiments with spinach leaves have shown that the y-like DNA polymerase is present in the chloroplast 121. A DNA polymerase has also been isolated from the mitochondria of wheat embryos [S]. Spinach mitochondria may also contain a DNA polymerase whose properties are partially different from those of the y-like DNA polymerase isolated from the chloroplasts of the same cells and are similar to those of the wheat embryo enzyme (unpublished). However, no evidence is available as yet on the existence in plant cells of a DNA repair enzyme similar to the DNA polymerase p of mammalian cells ]4,6]. By analogy with animal cells, the assignment of functions to the DNA polymerases in plant cells is hampered by the lack of conditional mutants defective in DNA synthesis. Thus, we approach this problem by exploiting the properties of aphidicolin and of ethidium bromide. Aphidicolin [7] specifically inhibits the cr-like DNA polymerase purified from plant cells 181, while the

Liposome-mediated transfer of DNA to carrot protoplasts: A biochemical and autoradiographic analysis

Abstract The interaction between positively charged multimellar liposomes loaded with [ 3 H]DNA from Bacillus subtilis , and carrot protoplasts has been studied. Molecular sieving analysis of DNA recovered from liposomes after incubation with protoplasts demonstrates that these liposomes can efficiently protect DNA from nucleases present in protoplast suspensions. Autoradiographic analysis of sectioned protoplasts reveals that liposomes can mediate, although at low efficiency, transfer of DNA inside the protoplasts. Transfer efficiency appears to be highly improved by treating with polyethylene glycol protoplasts preincubated with liposomes.

Presence of two sets of ribosome-specific transfer factors in the cell-free extracts from the non-photosynthetic alga Prototheca zopfii

Abstract Preparations of polymerizing enzymes from the non-photosynthetic alga Prototheca zopfii possess two transfer factors G, one active on the ribosomes of the 70 s type ( Escherichia coli ) and one active on those of the 80 s type ( Saccharomyces cerevisiae ). Evidence is presented in favour of the presence also of two ribosome-specific transfer factors T. The G-like activity for E. coli ribosomes present in the polymerizing enzymes from the yeast S. cerevisiae (Ciferri, Parisi, Perani & Grandi, 1968) has been demonstrated to be due to the presence of a transfer factor G, specific for ribosomes of the 70 s type that may be separated from that active on ribosomes of the 80 s type.

Dihydrofolate reductase from Daucus carota cell suspension cultures: purification, molecular and kinetic characterization

SummaryThe purification of dihydrofolate reductase (5, 6, 7, 8 tetrahydrofolate: NADP+ oxidoreductase, E.C.: 1.5.1.3) from Daucus carota to apparent homogeneity, is described. The enzyme is a soluble protein with a molecular weight of 183 000±2 500, composed of identical subunits of 58 400±1 000. The enzyme is only weakly recognized by antibodies against human DHFR. The carrot DHFR is characterized by a pH optimum of 5.9, Km values for dihydrofolate and NADPH of 3.7 μM and 2.2 μM, respectively and a turnover number of 4 750 or 1 500 when referring to the 183 K form or the 58 K monomer, respectively. Molecular and kinetic properties are remarkably different from those reported for the soybean enzyme. Sensitivity to methotrexate is similar to that of bacterial and mammalian enzymes while sensitivity to trimethoprim and dihydrotriazine is intermediate between the two groups of organisms.

Origin of the ribosome specific factors responsible for peptide chain elongation in yeast

The factors responsible for polypeptide chain elongation are specific for ribosomes of either the 70 S or the 80 S type [l--4]. Two different sets of elongation factors, one specific for 70 S ribosomes and one for 80 S ribosomes, are present in eukaryotic organisms: in Ne~rosporu