Construction of a gene bank and identification of the ribulose bisphosphate carboxylase/oxygenase genes from the nutritionally versatile cyanobacterium Chlorogloeopsis fritschii

A gene bank of the nutritionally versatile, nitrogen-fixing cyanobacterium Chlorogloeopsis fritschii was constructed in 082/xxlarge955.gif" alt="lambda" align="BASELINE" BORDER="0"> Charon 4A. 2,800 recombinants containing 10013;20 kbp C. fritschii DNA fragments were screened by Southern hybridization using probes containing the genes for the large (LSU) and small (SSU) subunits of ribulose bisphosphate carboxylase/oxygenase (RuBisCO) from Anacystis nidulans. A single recombinant plaque (082/xxlarge955.gif" alt="lambda" align="BASELINE" BORDER="0">CDG1) containing a 10.9 kbp EcoR1 fragment from C. fritschii hybridized to both the LSU and SSU probes, indicating a possible linkage of these RuBisCO genes in C. fritschii. RuBisCO activity and protein were detected in 082/xxlarge955.gif" alt="lambda" align="BASELINE" BORDER="0">CDG1 lysates of Escherichia coli. Hybridization was also obtained between C. fritschii DNA and the LSU probe from Chlamydomonas reinhardtii, although no homology was detected using the LSU probe from maize or the SSU probe from pea.Abbreviations RuBisCO d-ribulose 1,5-bisphosphate carboxylase/oxygenase - RuBP d-ribulose 1,5-bisphosphate - LSU large subunit of RuBisCO - SSU small subunit of RuBisCO - SDS sodium dodecyl sulphate - DOC deoxycholate     

Synthesis of phage-specific transfer RNA molecules by vibriophage phi 149

32P-Labelled tRNA was isolated from uninfected and phage phi 149-infected Vibrio cholerae cells. These tRNA preparations were then hybridised with DNA isolated from phage phi 149. Significant hybridisation was observed only with tRNA from phage phi 149-infected cells. This strongly suggests that infection of classical vibrio with phage phi 149 results in the synthesis of phage-specific tRNA molecules.     

Secretion of the sweet-tasting plant protein thaumatin byStreptomyces lividans

Summary To produce and direct the export inStreptomyces lividans of the sweet plant protein thaumatin, thaumatin II cDNA was fused in the correct reading frame to the 03762106n5831/xxlarge946.gif" alt="beta" align="MIDDLE" BORDER="0">-galactosidase leader peptide, under the control of the 03762106n5831/xxlarge946.gif" alt="beta" align="MIDDLE" BORDER="0">-galactosidase promoter and ribosome binding site. The export of the recombinant thaumatin may allow the correct formation of the thaumatin disulfide bonds. The recombinant thaumatin was purified from the medium on an S-Sepharose column and detected with western blots by sheep 03762106n5831/xxlarge945.gif" alt="agr" align="BASELINE" BORDER="0">-thaumatin antibodies. The recombinant thaumatin was the same size as authentic thaumatin and changed position on an acrylamide gel in response to reduction by 2-mercaptoethanol in the same manner.     

Inhibition of hexose transport in the human erythrocyte by 5, 5′-dithiobis(2-nitrobenzoic acid): Role of an exofacial carrier sulfhydryl group

Summary The sulfhydryl reagent 5, 50675m753u564w14/xxlarge8242.gif" alt="prime" align="BASELINE" BORDER="0">-dithiobis (2-nitrobenzoic acid) (DTNB) was used to study the functional role of an exofacial sulfhydryl group on the human erythrocyte hexose carrier. Above 1mm DTNB rapidly inhibited erythrocyte 3-O-methylglucose influx, but only to about half of control rates. Efflux was also inhibited, but to a lesser extent. Uptake inhibition was completely reversed by incubation and washing with 10mm cysteine, whereas it was only partially reduced by washing in buffer alone, suggesting both covalent and noncovalent interactions. The covalent thiol-reversible reaction of DTNB occurred on the exofacial carrier, since (i) penetration of DTNB into cells was minimal, (ii) blockade of potential uptake via the anion transporter did not affect DTNB-induced hexose transport inhibition, and (iii) DTNB protected from transport inhibition by the impermeant sulfhydryl reagent glutathione-maleimide-I. Maltose at 120mm accelerated the covalent transport inhibition induced by DTNB, whereas 6.5 0675m753u564w14/xxlarge956.gif" alt="mgr" align="MIDDLE" BORDER="0">m cytochalasin B had the opposite effect, indicating under the one-site carrier model that the reactive sulfhydryl is on the outward-facing carrier but not in the substrate-binding site. In contrast to glutathione-maleimide-I, however, DTNB did not restrict the ability of the carrier to reorient inwardly, since it did not affect equilibrium cytochalasin B binding. Thus, carrier conformation determines exposure of the exofacial carrier sulfydryl, but reaction of this group may not always 0675m753u564w14/xxlarge8220.gif" alt="ldquo" align="MIDDLE" BORDER="0">lock0675m753u564w14/xxlarge8221.gif" alt="rdquo" align="MIDDLE" BORDER="0"> the carrier in an outward-facing conformation.     

Entropy as a factor in the binding of γ-aminobutyric acid and nipecotic acid to the γ-aminobutyric acid transport system

Nipecotic acid is one of the most potent competitive inhibitors and alternative substrates for the high-affinity 0">-aminobutyric acid transport system in neurons, but the structural basis of this potency is unclear. Because 0">-aminobutyrate is a highly flexible molecule in solution, it would be expected to lose rotational entropy upon binding to the transport system, a change which does not favor binding. Nipecotic acid, in contrast, is a much less flexible molecule, and one would expect the loss of conformational entropy upon binding to be smaller thus favoring the binding of nipecotic acid over 0">-aminobutyric acid. To investigate this possibility, the thermodynamic parameters, 0">G°, 0">H°, and 0">S°, were determined for the binding of 0">-aminobutyrate and nipecotic acid to the high affinity GABA transport system in synaptosomes. In keeping with expectations, the apparent entropy change for nipecotic acid binding (112±13 J·K^013;1) was more favorable than the apparent entropy change for 0">-aminobutyric acid binding (61.3±6.6 J·K^013;1). The results suggest that restricted conformation per se is an important contributory factor to the affinity of nipecotic acid for the high-affinity transport system for 0">-aminobutyric acid.This work was conducted when both authors were at the Department of Chemistry, University of Maryland, College Park.Special issue dedicated to Dr. Elling Kvamme.     

Study of amino acid formation during palmitate oxidation in rat brain mitochondria

The interrelation of palmitate oxidation with amino acid formation in rat brain mitochondria has been investigated in purified mitochondria of nonsynaptic origin by measuring the formation of aspartate, 00j1l58378/xxlarge945.gif" alt="agr" align="BASELINE" BORDER="0">-ketoglutarate, and glutamate during palmitate oxidation, and also by assaying¹⁴C-products of [1-¹⁴C]palmitate oxidation. Oxidation of palmitate (or [1-¹⁴C]palmitate) resulted in the formation of aspartate (or¹⁴C-aspartate), and the oxidation was inhibited by aminooxyacetate (an inhibitor of transaminase), Palmitate oxidation also resulted in 00j1l58378/xxlarge945.gif" alt="agr" align="BASELINE" BORDER="0">-ketoglutarate formation, which was sensitive to the effect of aminooxyacetate. Addition of NH₄Cl was found to increase¹⁴C-products and formation of 00j1l58378/xxlarge945.gif" alt="agr" align="BASELINE" BORDER="0">-ketoglutarate, whereas glutamate formation was not increased unless the rate of palmitate oxidation was reduced by 50% by aminooxyacetate or 00j1l58378/xxlarge945.gif" alt="agr" align="BASELINE" BORDER="0">-ketoglutarate was added exogenously. Exogenous 00j1l58378/xxlarge945.gif" alt="agr" align="BASELINE" BORDER="0">-ketoglutarate was found to decrease¹⁴C-products, but not aspartate formation. These results indicated that palmitate oxidation was closely related to aspartate formation via aspartate aminotransferase. During palmitate oxidation without aminooxyacetate or added 00j1l58378/xxlarge945.gif" alt="agr" align="BASELINE" BORDER="0">-ketoglutarate, however, 00j1l58378/xxlarge945.gif" alt="agr" align="BASELINE" BORDER="0">-ketoglutarate was not available for glutamate formation via glutamate dehydrogenase. We discuss the possibility that this was because (a) oxidative decarboxylation of 00j1l58378/xxlarge945.gif" alt="agr" align="BASELINE" BORDER="0">-ketoglutarate to form succinyl-CoA was favored over glutamate formation for the competition for 00j1l58378/xxlarge945.gif" alt="agr" align="BASELINE" BORDER="0">-ketoglutarate in the same pool, and (b) the pool of 00j1l58378/xxlarge945.gif" alt="agr" align="BASELINE" BORDER="0">-ketoglutarate produced in the aspartate aminotransferase reaction did not serve as substrate for glutamate formation.     

Genetic approaches for studying rhizosphere colonization

Most bacterial traits involved in colonization of plant roots are yet to be defined. Studies were initiated to identify genes in Pseudomonas which play significant roles in this process. The general approach is to use transposons to construct collections of insertion mutants, each of which is then screened for alterations in its interactions with the host plant. In one study a Tn5 derivative containing a constitutively expressed 0k24405/xxlarge946.gif" alt="beta" align="MIDDLE" BORDER="0">-galactosidase (lacZ) gene was used to generate a collection of insertion mutants which could be distinguished from the wild-type parent on X-gal plates. Each mutant was examined for its ability to colonize wheat seedlings in the presence of the wild-type parent. Mutants which gave wild-type:mutant ratio of 20:1 or greater were obtained. In a second study a Tn5 derivative which carries a promoterless lacZ gene located near one end of the transposon was constructed. Expression of the lacZ gene depends on the presence of an active promoter outside of the transposon in the correct orientation. Insertion mutants generated with this transposon were examined for changes in 0k24405/xxlarge946.gif" alt="beta" align="MIDDLE" BORDER="0">-galactosidase expression in the presence and absence of plant root exudate. A number of mutants which showed differential lacZ expression have been identified.     

β-Oxidation enzymes in the mitochondria of Arum and oilseed rape

072v050583/xxlarge946.gif" alt="beta" align="MIDDLE" BORDER="0">-Oxidation enzymes were detected both in the mitochondria and microbodies of Arum maculatum L. spadices and Brassica napus L. seeds. It is apparent that the mitochondrial membrane barrier, which remains intact after sucrose-density-gradient centrifugation, prevents rapid access of acyl-GoA substrates to matrix 072v050583/xxlarge946.gif" alt="beta" align="MIDDLE" BORDER="0">oxidation tes. Thus intact mitochondria showed little 072v050583/xxlarge946.gif" alt="beta" align="MIDDLE" BORDER="0">-oxidation enzyme activity. Rupturing of the mitochondrial membrane allowed rapid access of acyl CoAs to matrix sites. Consequently, in ruptured mitochondria, high 072v050583/xxlarge946.gif" alt="beta" align="MIDDLE" BORDER="0">-oxidation enzyme activities were measured.C. Masterson thanks the Science and Engineering Research Council for the award of a postgraduate student maintenance grant. D.R. Thomas and C. Wood thank their relatives for continuing financial support. The authors also thank West Cumberland Farmers Ltd., Hexham, UK for their gift of oilseed rape seeds.