Expression of thymidylate synthetase activity in Bacillus subtilis upon integration of a cloned gene from Escherichia coli

The gene from Escherichia coli encoding thymidylate synthetase was cloned in the plasmid pBR322. The resulting chimeric plasmid, pER2, was effective in transforming both E. coli and Bacillus subtilis to thymine prototrophy. Uncloned linear E. coli chromosomal DNA was unable to transform thymine-requiring strains of B. subtilis to thymine independence. Linearization of the chimeric plasmid, pER2, with restriction enzymes markedly diminished its ability to transform B. subtilis auxotrophs. The Thy+ transformants derived from the transformation of B. subtilis with pER2 DNA did not contain detectable extrachromosomal DNA as demonstrated by Southern hybridization patterns and centrifugation in CsCl gradients of DNA isolated from B. subtilis colonies transformed with the chimeric plasmid. We conclude that the DNA from the chimeric plasmid was integrated into the chromosome of B. subtilis, demonstrating that extensive homology is not required for the integration of foreign DNA. This is the first reported case of a gene from a Gram-negative bacterium functioning in a Gram-positive organism.     

Electric field promotion of the bacteriorhodopsin BR570 to BR412 photoconversion in films of Halobacterium halobium purple membranes

The combined action of electric field (10⁵–10⁷ V · m^?1) and light (380–580 nm, 80 W · m^?2) activating the photoenergetic reaction of bacteriorhodopsin (BR) in dry films of purple membranes from Halobacterium halobium was studied. A new stimulating effect of the field on the BR₄₁₂ intermediate accumulation in the normal photochromic cycle of BR₅₇₀ has been observed. The formation of the product BR₄₁₂ is supposed to be accompanied by specific rearrangements of certain charged, polar and polarizable groups in the BR pigment-protein matrix. Such an intrinsic polarization could be promoted by an external electric field, the displacement vector of those groups being oriented in the direction of the field. The dielectric polarization properties of the purple membranes have been demonstrated by electret-thermal analysis.     

Generation of H-2-reactive T cell lines that bear the 5936 idiotype(s)

The present experiments showed 1) that it was possible to produce mouse T cell lines against MHC determinants with a relatively high success rate by stimulation of purified T cells with allogeneic cells in the presence of irradiated syngeneic spleen cells; 2) that these lines could be led to react against selected H-2 specificities; 3) that only T cell lines established from Ig-1b allotype mice contained 5936-Id+ T cells (5936-Idiotypes are defined by an antiserum against B6 anti-CBA IgG produced in rabbit no 5936, which was tolerant to mouse gamma-globulin); and 4) that antigenic determinants coded by IAk genes induce the 5936-Idiotype(s). The latter data are in accordance with the 5936-idiotype characteristics of primary MLC T blasts. All T cell lines contained both specific MLC-responding cells and cytolytic cells. However, studies on the functional capacity of 5936-Id+ T cells from both primary MLC and the T cell lines showed that neither MLC-responding cells nor cytolytic cells directed against H-2Kk, IAk, or H-2Dk were 5936-Id+. Thus, 5936-Id+ T cells may be regulator cells induced by IAk antigens.     

Rapid assay for cyclic AMP and cyclic GMP phosphodiesterases.

A rapid highly sensitive assay for cyclic AMP phosphodiesterase has been devised. After a 5-min incubation, cyclic AMP is readily resolved from 5′-AMP, adenosine, and inosine by ion-exchange thin-layer chromatography on 1.3 × 6.5-cm strips of PEI-cellulose for 7 to 8 min. This procedure combines the accuracy of the standard paper chromatography assay (1) with the speed of ion-exchange resin techniques (2), while surmounting some of the major drawbacks of the other two methods (3). Since chromatography on PEI-cellulose efficiently resolves cyclic GMP, 5′-GMP, and guanosine, this methodology has also been adapted to the measurement of cyclic GMP hydrolysis.     

Plasma catecholamines in man are not influenced by the inhibition of prostaglandin synthesis.

Indomethacin has been reported to potentiate the release of noradrenaline from sympathetic nerve endings in vitro and to increase urinary noradrenaline excretion in rats. We have studied the influence of indomethacin on plasma catecholamine levels in 10 normal men, using measurement of plasma renin activity (PRA) as an index of the pharmacodynamic effect of indomethacin. Both in the supine and standing positions indomethacin failed to alter the plasma concentrations of noradrenaline, adrenaline or dopamine, while PRA was markedly suppressed. It is concluded that in the intact human indomethacin does not influence catecholamine concentrations.     

Influence of trace elements and nitrogen sources on versicolorin production by a mutant strain of Aspergillus parasiticus

A mutant strain of Aspergillus parasiticus blocked in aflatoxin biosynthesis accumulates versicolorin A and versicolorin C. The effect of trace elements on the growth and versicolorin production by this strain was studied in a defined medium. The omission of manganese was slightly stimulatory to versicolorin production; when zinc was omitted from the medium, no detectable versicolorins were produced. Experiments on nitrogen sources in a highsucrose medium indicated that fourfold to fivefold increases in versicolorin yields could be obtained by substituting 3 ml/l corn steep liquor or 0.1 M NH₄NO₃ for the 0.023 M (NH₄)₂SO₃ used previously as the nitrogen source in studies on versicolorin production by this strain. These improved yields will facilitate attempts to accumulate enough versicolorin A and versicolorin C for toxicity and carcinogenicity testing. Chromatographic profiles of mycelial extracts of cultures grown in a defined medium with 0.1 M NH₄NO₃ as the nitrogen source revealed 2 previously unrecognized compounds. The accumulation of these new metabolites in a mutant blocked in aflatoxin production may indicate that they are biosynthetically related to aflatoxin.     

Plant pyruvate dehydrogenase complex: analysis of the kinetic properties and metabolite regulation

The pyruvate dehydrogenase complex was isolated from the mitochondria of broccoli florets and shown to be similar in its reaction mechanism to the complexes from other sources. Three families of parallel lines were obtained for the initial velocity patterns, indicating a multisite ping-pong mechanism. The apparent K_m values obtained were 321 ± 18, 148 ± 13, and 7.2 ± 0.51 μm for pyruvate, NAD⁺, and CoA, respectively. Product inhibition studies using acetyl-CoA and NADH yielded results which were in agreement with those predicted by the multisite ping-pong mechanism. Acetyl-CoA and NADH were found to be competitive inhibitors versus CoA and NAD⁺, respectively. All other substrate-product combinations showed uncompetitive inhibition patterns, except for acetyl-CoA versus NAD⁺. Among various metabolites tested, only hydroxypyruvate (K_i = 0.11 mM) and glyoxylate (K_i = 3.27 mM) were found to be capable of inhibiting the broccoli enzyme to a significant degree. Initial velocity patterns using Mg²⁺? or Ca²⁺-thiamine pyrophosphate and pyruvate as the variable substrate were found to be consistent with an equilibrium ordered mechanism where Mg? or Ca-thiamine pyrophosphate bind first, with dissociation constants of 33.8 and 3 μm, respectively. The Mg- or Ca-thiamine pyrophosphate complexes also dissociated rapidly from the enzyme complex.     

Characterization of the interaction of reticuloendotheliosis virus with the avian lymphoid system.

Splenic lymphocytes from chickens infected with reticuloendotheliosis virus (REV) are cytostatically impaired in their ability to undergo mitogen-induced blastogenesis ([³H]TdR uptake and proliferation), but are fully capable of eliciting cytotoxic reactions against allogeneic, ⁵¹Chromium-labeled chicken erythrocytes. Spleen cells from birds with reticuloendotheliosis (RE_s) are able to suppress DNA synthesis of normal splenic lymphocytes (N_s), but are unable to inhibit ¹[³H]TdR uptake by chick embryo fibroblasts. The suppression of the N_s mitogenic response is not restricted by major histocompatibility (B-locus) differences between populations of RE_s suppressor and N_s target cells. Moreover, infection of birds with an attenuated form of REV, which replicates in the host but does not cause tumorigenesis, also leads to suppression of phytohemagglutinin-induced, [³H]TdR uptake by host lymphocytes. These results are discussed in terms of the interaction between viral-infected/transformed cells and host defense mechanisms.