Molecular cloning of cDNA for rat L-type pyruvate kinase and aldolase B

Two double-stranded cDNA recombinant pBR322 plasmid libraries were constructed starting from high carbohydrate diet rat liver poly(A)+ mRNA, either fractionated by denaturing sucrose gradient centrifugation for the cloning of L-type pyruvate kinase cDNA, or nonfractionated for aldolase B. Both libraries were screened with single-stranded cDNA probes reverse transcribed from fasted or high carbohydrate diet rat liver mRNAs. mRNAs from fasted animals were also fractionated by sucrose gradient centrifugation and mRNAs from the fed animals were, in addition, further purified by high performance liquid gel filtration chromatography. Those clones hybridizing with the "positive" probe (from animals fed the high carbohydrate diet) and not with the "negative" one (from fasted animals) were preselected and their plasmid DNA was purified and analyzed by positive hybridization-selection. Thirty of 4500 bacteria colonies transformed by recombinant plasmids were preselected by differential screening for pyruvate kinase, and 8 of 864 colonies for aldolase B. Twenty-two recombinant plasmids for pyruvate kinase and two for aldolase B were shown to contain specific cDNA inserts by positive hybridization-selection. Plasmids DNAs of some pyruvate kinase and aldolase B clones (whose inserts ranged from 700 to 1050 bases in length) were labeled by nick translation and used as probes for Northern blot hybridization. The pyruvate kinase cDNA probes recognized mainly a 3400-base RNA species which was detected in high carbohydrate diet rat liver, but not in fasted rat liver and in tissues which do not synthesize L-type pyruvate kinase. In addition, some pyruvate kinase probes hybridized with minor RNA species of about 2000 bases in length, only observed after carbohydrate diet. For aldolase B, the recombinant plasmid DNA hybridized with a single RNA species of 1750 bases. This RNA, detected in kidney, small intestine and liver, was induced by a high carbohydrate diet and increased with liver development. The rat probe cross-hybridized with human aldolase B messenger RNA.     

Irreversible Activation of the Opiate Receptor of Neuroblastoma × Glioma Hybrid Cells by an Alkylating Benzomorphan Derivative

The benozomorphan derivative (-)-2-[2-(p-bromoacetamidophenyl)ethyl]-5,9 alpha-dimethyl-2'-hydroxy-6,7-benzomorphan (BAB), capable of reacting with nucleophilic groups, acts on neuroblastoma X glioma hybrid cells as a potent, irreversible opiate agonist. Its potency in inhibiting the increase in cellular cyclic AMP, evoked by prostaglandin E1, is comparable to that of Leu-enkephalin. This also applies to its capacity to compete with [3H]D-Ala2-Met-enkephalinamide ([3H]DAEA) in binding on cell membrane preparations. The comparatively lower potency of (-)-2-[2-(p-acetamidophenyl)-ethyl]-5,9 alpha-dimethly-2'-hydroxy-5,7-benzomorphan (AB), which differs from BAB in the substitution of the bromoacetamido group by an acetamido group, is of the same order of magnitude as that of morphine. The covalent interaction of BAB with the opiate receptors is deduced from the observations that (1) it is not possible to wash away this compound from the receptors, (2) the potency of BAB in inhibiting the specific binding of [3H]DAEA increases with prolonged preincubation time, and (3) AB behaves as a reversible agonist.     

The 60S ribosomal subunit is altered in the skeletal muscle of dystrophic hamsters

Polysomes from the skeletal muscle of normal and dystrophic hamsters were dissociated into ribosomal subunits by treatment with puromycin and the subunits from both strains were reassociated in all possible combinations. When their protein synthesis activity was assayed in a poly(U)-directed cell-free system at a low magnesium concentration, the reassociated ribosomes from dystrophic hamsters were less active than the ribosomes from control animals. The ribosomal defect is a property of the 60S subunit and is due to a ribosomal component rather than to abnormal binding of a non-ribosomal protein.     

Comparison of the localization of nucleic acid synthesis during bud formation on leaf fragments and on intact undetached leaves ofBegonia rex Putz.

The budding capacity ofBegonia rex leaf fragments is well known; that of undetached leaves has been shown by us only recently after treating the leaves with 6γγ DMAAP. Benzyladenine is as effective as 6γγ DMAAP in stimulating budding. Lower temperatures (17°, 22–12°, 12°) are also capable of inducing bud formation but only after a small cut has been made in a main vein of the undetached leaf. Root formation can also be provoked on undetached leaves which have a cut in the main leaf vein by higher temperatures (24–22°) or by an IAA treatment. Differences in the first stages of bud formation on leaf fragments and on undetached leaves are observed using histochemical and histoautoradiographic techniques.     

Mycoplasma-like bodies in plants infected with potato witches' broom disease and the response of plants to tetracycline treatment

The virus origin of a Czechoslovak isolate of potato witches' broom disease is discounted: electron micrographs of ultrathin section of phloem tissues from plants infected with potato witches' broom disease demonstratedMycoplasma-like bodies, spherical or elongated showing binary fission and fragmentation. The minute corpuscles have a diameter of about 50–60 nm, the largest bodies of 1000 nm. The width of elongated filamentous structures is about 200 nm, most oval bodies have a diameter of 250 nm. A weak tetracycline treatment of diseased plants causes a delay of symptom development; a strong dose of tetracycline (applied by means of the wick method into the stem) inhibits symptom appearance completely. Tetracycline produces a phytotoxic effect inhibiting the growth of tomato plants and causing (at higher concentrations) necrosis and death of these plants. There is a note in the paper dealing with the term “mycoplasma”. The word mycoplasma in the sense ofEriksson (1897) or ofMereschkowsky (1910) does not correspond to the genus nameMycoplasma Nowak (1929).     

Ribonucleic Acid Synthesis During the Differentiation of Sporangia in the Water Mold Achlya

The role of ribonucleic acid (RNA) synthesis in the development of sporangia in the saprolegniaceous mold Achlya was studied. Methods were developed for growing and treating large populations of mycelia so that the hyphal tips would differentiate into sporangia with considerable synchrony. Under the starvation conditions imposed for the differentiation of sporangia, net RNA, deoxyribonucleic acid (DNA), and protein synthesis ceased. However, incorporation of radioactive precursors into RNA continued at a high rate throughout the period of differentiation, showing that the enzymatic mechanism for RNA synthesis was still in an active state. Actinomycin D inhibited the differentiation of sporangia and the incorporation of labeled precursors into RNA. The level of actinomycin used did not inhibit the normal outgrowth and branching of the mycelia that occurred during differentiation. Thus, DNA-dependent RNA synthesis was required for the differentiation of sporangia. Sucrose gradient analysis of newly synthesized RNA showed that only the ribosomal and soluble fractions of RNA were labeled during vegetative growth. During the differentiation of sporangia, ribosomal and soluble RNA fractions were also labeled, and, in addition, a heterodisperse fraction of labeled RNA which was heavier than ribosomal RNA appeared; this fraction was not evident in the newly synthesized RNA from vegetative mycelia.     

Etude ultrastructurale de l'endoplasme et des vacuoles chez deux types de Dinoflagellés appartenant aux genres Noctiluca (Suriray) et Blastodinium (Chatton)

Résumé L'ultrastructure des éléments cytoplasmiques de deux Dinoflagellés a révélé des différences fondamentales directement liées à leur mode de vie.Chez Noctiluca (Dinoflagellé libre, incolore), les corps de Golgi jouent un rôle important dans la formation des sphères archoplasmiques au moment des divisions et lors de l'élaboration de l'endoglée. Les mitochondries de grande taille, parfois géantes, ont des crêtes nombreuses et une matrice réduite. Elles offrent souvent des relations de contiguïté avec les granulations lipidiques et l'enveloppe nucléaire; elles sont très abondantes dans le tentacule du trophozoïte. Il n'existe pas de plastes. Les inclusions lipidiques abondent ainsi que les trichocystes fibreux et les poches mucifères dont le contenu est polysaccharidique.Chez Blastodinium (Dinoflagellé parasite, coloré sauf une espèce), les corps de Golgi sont très réduits dans le trophocyte, assez rares mais de structure classique dans les sporocytes. Les mitochondries sont de petite taille, pauvres en crêtes et ont une matrice importante chez le trophocyte; elles sont plus grandes dans les sporocytes. Les plastes bien développés, périphériques, possèdent des lamelles stromatiques formées de trois thylakoïdes chez les sporocytes; par contre, chez l'individu végétatif, le plus souvent incolore et relativement protégé de la lumière par plusieurs rangées de sporocytes, les plastes, de grande taille ont un aspect dégénéré: lamelles stromatiques rares, thylakoïdes tassés et stroma important; ce sont des étioplastes. Le cas de Blastodinium contortum hyalinum toujours incolore, est particulier: les plastes existent encore mais sont totalement dégénérés. Il y a très peu de lipides dans le trophocyte, mais ils sont par contre abondants dans les sporocytes. Il existe des trichocystes fibreux et chez l'individu végétatif, des figures myéliniques.Ces importantes variations structurales illustrent parfaitement, dans l'échelle évolutive des Dinoflagellés, l'exemple de deux modes de vie très différents.

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Fine structure of endoplasm and vacuoles in two types of dinoflagellates of the genus Noctiluca and Blastodinium

Summary The fine structure of cytoplasmic elements of two Dinoflagellates reveals fundamental differences directly linked to their way of life.In Noctiluca, free-living, colourless dinoflagellate, the Golgi bodies play an important role in the formation of archoplasmic spheres at the moment of division and when the endoglea is elaborated. The mitochondria of great size, sometimes giant, have very abundant cristae and a reduced matrix. They often show relations of contiguity with the lipid granules and the nuclear envelope; they are very abundant in the tentacle of the trophozoïd. There are no plastids. The lipid inclusions are very abundant and so are fibrous trichocysts and muciferous bags whose content is polysaccharidic.In Blastodinium, parasitic dinoflagellate, coloured except for one species, the Golgi bodies are much reduced in the trophocyte, rather rare but of classical structure in the sporocytes. The mitochondria are of small size, with rare cristae and have an enlarged matrix in the trophocyte; they are bigger in the sporocytes. The plastids are well developed, peripheral, and have stromatic lamellae formed of three thylakoïds in the sporocytes. On the other hand, in the vegetative individual, usually colourless since it is protected from the light by many rows of sporocytes, the large plastids have a degenerated aspect, rare stromatic lamellae, packed thylakoïds and enlarged stroma; they are etioplastids. The case of Blastodinium contortum hyalinum, always colourless, is special: the plastids still exist but are completely degenerated. There are very few lipids in the trophocyte but abundant ones in the sporocytes. Some fibrous trichocysts occur, and in the vegetative individual, some myelinic vacuoles.These important structural variations illustrate very well the example of two very different ways of life, in the evolutionary tree of dinoflagellates.

Cet article fait partie d'un travail d'ensemble constituant une Thèse de Doctorat d'Etat intitulée: La cytologie et la division de deux types de Dinoflagellés: Noctiluca Suriray (genre libre) et Blastodinium Chatton (genre parasite).     

Phosphorus disturbances associated with potato leafroll virus infection

Potato leafroll virus induces considerable disturbances of phosphorus metabolism in leafroll-infected potato plants. Each variety reacts differently in this respect on the infection, to some extent being dependent on the susceptibility of the respective variety to leafroll and to the season as well. In varieties particularly susceptible to leafroll as in Apta and Sieglinde there is a decrease of total P after infection. On the contrary, varieties showing medium susceptibility such as varieties Tatranka, Rita and Ambra react to leafroll infection with a considerable increase of total P. In the comparatively less susceptible variety Krasava, there is no change in the level of total P after infection. In autumn, however, a slight decrease of total P even in the medium susceptible variety Tatranka, and the less susceptible variety, Krasava, can be found. Our results shed a new light on existing controversies in literature regarding phosphorus content in leafroll-infected potato plants. When the changes in the contents of various phosphorus fractions in the infected plants are compared we can observe a tendency of lowmolecular fractions to rise. Among the different phosphorus fractions one common feature could be seen, except for the hypersensitive resistant variety Apta, namely, that the acid-soluble organic phosphates in the stems of all varieties examined, were considerably higher than in healthy stems.     

Biosynthesis of methionine in an ethionine-resistant strain ofCandida utilis

The strainCandida utilis T 20 adapted to a high concentration of ethionine, excretes considerable amounts of methionine in a synthetic medium, about 40 times as much as the original non-adapted strain. At the same time, the amount of methionine in yeast cells incrncreased, predominantly in the pool (9 times as much as in the control). This ability to produce greater amounts of methionine in the pool or to excrete it into the medium is not permanent, since after 5 passages on agar with ut ethionine the amount of methionine was practically not increased as compared with the original non-adapted strain. An increase in free methionine and of methionine excreted into the medium was found on cultivating the strain in a molasses-containing medium, too.