Histone-poly(A) hybrid molecules as tools to block nuclear pores

Histone-poly(A) hybrid molecules were used for transport experiments with resealed nuclear envelopes and after attachment of a cleavable cross-linker (SASD) to identify nuclear proteins. In contrast to histones, the hybrid molecules cannot be accumulated in resealed nuclear envelopes, and in contrast to poly(A), the export of hybrids from preloaded nuclear envelopes is completely impaired. The experiments strongly confirm the existence of poly(A) as an export signal in mRNA which counteracts the nuclear location signals (NLS) in histones. The contradicting transport signals in the hybrid molecules impair translocation through the nuclear pore complex. The failure to accumulate hybrid molecules into resealed nuclear envelopes results from the covalent attachment of polyadenylic acid to histones in a strict 11 molar ratio. This was demonstrated in control transport experiments where radiolabeled histones were simply mixed with nonlabeled poly(A) or radiolabeled poly(A) mixed with nonlabeled histones. In comparison, control uptake experiments with histones covalently linked to a single UMP-mononucleotide are strongly enhanced. Such controls exclude the conceivable possibility of a simple masking of the nuclear location signal in the histones by the covalent attached poly(A) moiety. Photoreactive histone-poly(A) hybrid analogs serve to identify nuclear envelope proteins-presumably in the nuclear pore-with molecular weights of 110, 80, and 71.4 kDa.     

Gene-enzyme relationships of aromatic acid biosynthesis in Bacillus subtilis

Mutants have been isolated which correspond to every step concerned with the biosynthesis of the aromatic amino acids in Bacillus subtilis. Each mutant has been characterized, and the lesion it bore was analyzed by deoxyribonucleic acid transformation and PBS-1 mediated transduction. The biochemical analysis revealed that each of the mutations appears to have affected a single enzyme, except for two groups of pleiotropic mutations. All aroF mutants (chorismic acid synthetase) lack dehydroquinic acid synthetase (aroB) activity. The gene that specifies aroB is closely linked to the gene coding for the aroF enzyme. Both genes are a part of the aro cluster. Mutants lacking chorismate mutase activity also lack d-arabino-heptulosonic acid-7-phosphate synthetase and shikimate kinase activity, presumably as a result of these three activities forming a multi-enzyme complex. Another mutant, previously undescribed, had been isolated. The affected gene codes for the tyrosine and phenylalanine aminotransferase activity. All of the mutations have been located on the B. subtilis genome except those in the genes specifying shikimate kinase activity and tyrosine-phenylalanine aminotransferase activity.     

Mitochondrial inner membrane in hypothyroidism.

Inner membrane vesicles obtained from the liver mitochondria of hypothyroid and normal rats were compared. In the vesicles from hypothyroid rats the rate of ATP synthesis at 30 °C is 35–50% less than the normal rate. The Arrhenius profile for phosphorylation by such vesicles lacks the discontinuity at 21 °C that is seen with vesicles from normal rats; at temperatures below about 9 °C the rate of phosphorylation by vesicles from hypothyroid rats is not lower than normal. The phospholipids of hypothyroid vesicles contain higher mole fractions of 18:2 (linoleic), 18:3 (linolenic or γ-linolenic), and 20:3 (eicosatrienoic) and lower fractions of 20:4 (arachidonic), 22:3 (docosatrienoic), and 22:4 (docosatetraenoic); the unsaturation index, mainly due to 20:4, is 10% less than in normal vesicles. Injecting hypothyroid rats with l-thyroxine 3 days before preparation of vesicles corrects the relative contents of these unsaturated fatty acids as well as the Arrhenius profile and also increases the phosphorylation rate at 30 °C. Respiration and cytochrome a content do not differ for membrane vesicles prepared from livers of rats in the various thyroid states. A metabolic defect in unsaturated fatty acid metabolism in hypothyroidism may be involved in the function of the inner mitochondrial membrane.     

Control of tryptophan biosynthesis by the methyltryptophan resistance gene in Bacillus subtilis

5-Methyltryptophan-resistant mutants derived from Bacillus subtilis strain 168 synthesize all of the tryptophan biosynthetic enzymes constitutively and excrete tryptophan. These mutants can be divided into three classes: class 1, low enzyme level and low rate of tryptophan excretion; class 2, high enzyme level and intermediate rate of tryptophan excretion; and class 3, high enzyme level and high rate of tryptophan excretion. A bradytrophic requirement for phenylalanine is correlated with the rate of tryptophan excretion. The phenylalanine requirement is relieved when the rate of tryptophan excretion is reduced by either (i) lowering the level of the tryptophan enzymes, (ii) reducing the supply of a tryptophan precursor (chorismate), or (iii) stopping tryptophan synthesis by a mutational block in the pathway. All of the mutants map in a region of the chromosome previously reported as the mtr locus. Our data show that synthesis of the tryptophan enzymes is controlled through the mtr locus but not influenced by precursors of tryptophan.     

Citric acid cycle: gene-enzyme relationships in Bacillus subtilis

The genetic location of mutations affecting the citric acid cycle and the properties of mutants of Bacillus subtilis possessing these mutations have been examined. Genes coding for the component enzymes of the cycle were found to be unlinked to each other and thus do not form an operon. The mutational defect in a mutant lacking fumarase mapped between thr-5 and cysB3. Mutations causing inability to produce isocitrate dehydrogenase and succinate dehydrogenase were found to map between argA11 and leu-1. The alpha-ketoglutarate dehydrogenase mutations were mapped at the terminal end of the B. subtilis chromosome through a weak linkage in phage PBS-1 transduction of one class of these mutations of ilvA2 and metB4. A second class of alpha-ketoglutarate dehydrogenase mutations mapped closer to ilvA2 and metB4 but still terminal with respect to these markers. Aconitaseless mutants possessed mutations that could not be linked to any of the known transducing segments of the chromosome. An effect of mutation conferring loss of one enzyme of the cycle on the specific activity of the other enzymes in the cycle was observed.     

Chromosomal Location and Properties of Radiation Sensitivity Mutations in Bacillus subtilis

Transformation and transduction crosses involving recA1, recB2, and urv-1 mutations have shown that these mutations belong to three distinct unlinked genetic loci. The precise position of these loci on the Bacillus subtilis chromosome map has been determined. The behavior of recB2 strains in transformation studies suggested a dominance of recB2(+) function over recB2 and an early expression of this phenotype during transformation. Strains bearing two ultraviolet sensitivity markers possess a phenotype characteristic of the marker with the most adverse effect on recombination. The possibility that the effects of the two mutations are additive was also considered. Results are also presented which show that a phage-induced enzyme is not responsible for the high transducibility of recA1 strains.     

Adenine nucleotide translocation in liver mitochondria of hypothyroid rats.

In measurements using a disc filtration method, liver mitochondria obtained from hypothyroid rats translocate external ADP at 0 °C via the atractyloside-sensitive carrier much more slowly than do mitochondria from normal rats, confirming the findings of Portnay et al. (Biochem. Biophys. Res. Commun. 55, 17, 1973). The hypothyroid mitochondria contain 60% more ATP + ADP than do mitochondria from normals, but the excess nucleotides are not exchangeable and so do not contribute to translocation. A decrease in the first-order rate constant accounts for the decreased velocity. Neither a decrease in the number of translocator sites nor changes in ADP phosphorylation or ATPase activity seem to account for the abnormal kinetics of translocation. Although the filtration method limits the maximal translocation rate observed in normal mitochondria at temperatures above 17 °C that induce a fluid membrane state, no such transition is seen in mitochondria from hypothyroid rats up to 35 °C, indicating that the translocator is in an altered environment in hypothyroidism. Injecting a hypothyroid rat once with l-thyroxine corrects the abnormal compartmentation and produces a temperature-rate relationship like that in normal mitochondria in 3 days, a period which would accommodate the hormone actions reported on translation, membrane phospholipid synthesis, or fatty acid desaturation.     

SUBEPIDERMAL AIR SPACES: BASIS FOR THE PHENOTYPIC EXPRESSION OF THE ARGENTEUM MUTANT OF PISUM

The Argenteum (Arg) mutant of pea is characterized by a gray-green, silvery cast of the leaves and stipules. Light and electron microscopy showed that the nature of the silvery cast was due to extensive intercellular air space between the epidermis and palisade parenchyma. Adaxial epidermal strips from mutant leaves were free of palisade cell wall fragments, whereas similar strips from normal leaves had remnants of the torn palisade cells attached to the epidermal cell walls. The periphery of the epidermal-palisade cell wall interfaces showed a more electron-opaque middle lamella in the normal leaves than in the Arg mutant leaves.     

Adhesion of culture cells to their substratum

The attachment of cells to culture dishes has been investigated by replica techniques and scanning electron microscopy. Cells were removed from their substratum either in a stream of medium or by micromanipulation. In the most usual case of interphase cells one finds many small points of attachment uniformly distributed over the whole cell underside. Attachment sites are also found under the lamellipodia and at the trailing edge of the cells. A large portion of the cell underside is sometimes left behind. This is probably because the sole plate is reinforced by cytoskeletal elements, and does not necessarily indicate the presence of large adhesion points. The distal ends of the retraction fibers formed as the cells round-up in trypsin, or in the cold, represent the attachment points of the cell to the substratum. Agents which tend to stabilize microtubules greatly slow cell detachment by proteolytic agents. The primary effect of trypsin is not on the glue which holds the cells to the substratum but rather on the cell shape itself, affecting the rigidity of cytoskeletal elements.