Importance of the galE gene on the virulence of Pasteurella multocida

The galE gene of Pasteurella multocida has been isolated by complementing galE-defective mutants of Salmonella typhimurium with a plasmid library of this organism. The complete nucleotide sequence of the P. multocida galE gene consists of 1017 nucleotides, encoding a predicted polypeptide of 339 amino acids. The deduced amino acid sequence displayed the highest identity (85%) to the GalE protein of Haemophilus influenzae. However, the gene organization surrounding the galE locus was different from that of H. influenzae. A galE-defective mutant of P. multocida was obtained by replacement of the active galE gene by a copy inactivated in vitro. The resulting galE mutant was highly attenuated as seen in a biological test carried out in a mouse model.     

The Effects of Fluctuations in the Nutrient Supply on the Expression of Five Members of the AGL17 Clade of MADS-Box Genes in Rice

The ANR1 MADS-box gene in Arabidopsis is a key gene involved in regulating lateral root development in response to the external nitrate supply. There are five ANR1-like genes in Oryza sativa, OsMADS23, OsMADS25, OsMADS27, OsMADS57 and OsMADS61, all of which belong to the AGL17 clade. Here we have investigated the responsiveness of these genes to fluctuations in nitrogen (N), phosphorus (P) and sulfur (S) mineral nutrient supply. The MADS-box genes have been shown to have a range of responses to the nutrient supply. The expression of OsMADS61 was transiently induced by N deprivation but was not affected by re-supply with various N sources. The expression of OsMADS25 and OsMADS27 was induced by re-supplying with NO₃ ⁻ and NH₄NO₃, but downregulated by NH₄ ⁺. The expression of OsMADS57 was significantly downregulated by N starvation and upregulated by 3 h NO₃ ⁻ re-supply. OsMADS23 was the only gene that showed no response to either N starvation nor NO₃ ⁻ re-supply. OsMADS57 was the only gene not regulated by P fluctuation whereas the expression of OsMADS23, OsMADS25 and OsMADS27 was downregulated by P starvation and P re-supply. In contrast, all five ANR1-related genes were significantly upregulated by S starvation. Our results also indicated that there were interactions among nitrate, sulphate and phosphate transporters in rice.     

A New Interpretation of the Dynamic Changes of the Potassium Conductance in the Squid Giant Axon

The solutions, n(t), of the differential equation dn/dt = α (1 - n) n (4 - 6n + 4n² - n³) - βn² (4 - 6n + 4n² - n³) in which α and β are instantaneous functions of membrane potential, are shown to fit with good accuracy the time courses of the rise of potassium conductance during depolarizing steps in clamp potential, found experimentally by Hodgkin and Huxley and by Cole and Moore. The equation is derived by analysing the dynamic behaviour of a system consisting of a square array of interacting pores. The possible role of Ca⁺⁺ ions in this system is discussed.     

Analysis of the Bacillus subtilis recO gene: RecO forms part of the RecFLOR function

The deduced protein product of the Bacillus subtilis gene yqfI, which is 255 residues long, shares homology (25% identity) with the Escherichia coli RecO protein. A null allele of yqfI, when present in an otherwise Rec⁺ B. subtilis strain, causes cells to become highly sensitive to DNA-damaging agents, and plasmid transformation (intramolecular recombination) is reduced by 25-fold while chromosomal transformation (intermolecular recombination) is only moderately affected (2.5-fold reduction). Therefore, the yqfI gene was renamed recO and its null allele is referred to as recO1. The recO1 mutation was introduced into recombination-deficient strains representative of the epistatic groups α (recF, recR and recL strains), β (addA5 addB72), γ (recH342) and ? (recU40). The recO mutation did not affect the sensitivity of recF, recR or recL cells to DNA-damaging agents, increased the sensitivity of recU and addAB cells and abolished the DNA repair capacity of recH cells. The recO mutation did not affect intermolecular recombination in recF, recL, recH or recU cells, but reduced (by about 9-fold) the incidence of intermolecular recombination in addAB cells. The recO mutation did not affect intramolecular recombination in the addAB, recU, recF or recL cells, but reduced it by about 75-fold in recH cells. The defects caused by the recO1 mutation can be partially suppressed by a common suppressor of the recF, recL and recR phenotypes. We therefore assigned recO to epistatic group α and predict that the RecO protein acts at the same stage of recombination as the RecF, RecL and RecR proteins, in a RecFLOR complex.     

An electrophysiological study of the membrane properties of the immature and mature oocyte of the Batstar,Patiria miniata

Immature oocyte membrane properties of a starfish, Patiria miniata, were investigated by microelectrode techniques. The resting membrane potential in artificial seawater (ASW) was ?78.5 ± 6.7 mV (n = 61, inside negative). This was mainly accounted for by a selective permeability to potassium ions. Potassium ion-selective microelectrodes were used to measure intracellular K⁺ ion activity, which was 350 mM. The sodium to potassium permeability ratio was 0.02 ± 0.01 (n = 4). The current-voltage relation was nonlinear. The I–V curve included both areas of inward and outward rectification. The dependence of inward rectification upon the K⁺ ion electrochemical gradient was demonstrated. The membrane was capable of a regenerative action potential due to permeability changes for Ca²⁺ and Na⁺ ions. The Ca and Na components of the action potential were identified. The Ca component was reversibly suppressed by cobalt and irreversibly blocked by D-600. The Na component was tetrodotoxin (TTX) insensitive. The excitable response of P. miniata oocytes is similar to that described by Miyazaki et al. (1975a) for those of the starfish Asterina pectinifera.Immature oocytes were stimulated to mature with 10^?5M 1-methyladenine (1-MA) during continuous monitoring of the membrane potential. The resting potential in ASW became more inside negative during maturation. This change of the passive membrane property of the oocyte may be accounted for by the increased selectivity to K⁺ ions. The specific membrane resistance near the resting potential increased from 4.2 ± 1.4 to 21 ± 8.7 kΩ·cm² (n = 15) during maturation, while the specific membrane capacitance decreased slightly from 2 ± 0.5 to 1.7 ± 0.6 μF/cm² (n = 5). Maturation had little effect upon the active membrane properties.     

Regulation of the regulatory gene for the arabinose pathway,araC

The promoter of the araC gene was fused to the structural genes of the lac operon using the techniques described in the preceding paper. The resulting fusion strains were used to study the regulation of the araC gene by assaying the fused lac gene products. It was found that the expression of the fused lac genes was repressed by the product of the araC gene and was regulated by the cyclic AMP catabolite control system. This implies that the araC gene itself is repressed by its own product and is catabolite regulated. These findings introduce a new level of complexity in the regulation of the arabinose pathway of Escherichia coli.     

Three highly divergent subfamilies of the impala transposable element coexist in the genome of the fungus Fusarium oxysporum

The transposable element impala is a member of the widespread superfamily of Tc1-mariner transposons, identified in the genome of the plant pathogenic fungus Fusarium oxysporum. This element is present in a low copy number and is actively transposed in the F.?oxysporum strain F24 that is pathogenic for melons. The structure of the impala family was investigated by cloning and sequencing all the genomic copies. The analysis revealed that this family is composed of full-length and truncated copies. Four copies contained a long open reading frame that could potentially encode a transposase of 340 amino acids. The presence of conserved functional domains (a nuclear localisation signal, a catalytic DDE domain and a DNA-binding domain) suggests that these four copies may be autonomous elements. Sequence comparisons and phylogenetic analysis of the impala copies defined three subfamilies, which differ by a high level of nucleotide polymorphism (around 20%). The coexistence of these divergent subfamilies in the same genome may indicate that the impala family is of ancient origin and/or that it arose by successive horizontal transmission events.     

Organization of the nar genes at the chlZ locus

The two membrane-bound respiratory nitrate reductases of Escherichia coli are encoded by distinct operons at two different loci, chlC and chlZ, on the chromosome. The chlZ locus includes a narK homologue, narU, encoding a nitrite extrusion protein, and narZYWV encoding nitrate reductase Z. No apparent homologue to the narXL operon has been found. Homology between narU and narK on the one hand and narZYWV and narGHJI on the other hand is limited to the coding regions.     

On the amino acids involved in the ATPase site of mitochondrial F1 and the implication of the subunit C.

The data on the pH dependence of the K_m for Mg-ATP and the V_m of the ATPase of pig heart mitochondrial F₁ indicate the presence of two groups of different pK's which modify the enzyme activity. The first pK at pH 9.6 ± 0.2 may be related to the possible presence of arginine and/or tyrosine residues in the ATPase site; the second pK at pH 7.2 ± 0.2 could be due to the presence of a histidine residue in the ATPase site or to the involvement of amino groups in the ATPase site. The inhibition induced by photooxidation in the presence of Rose Bengal is not pH dependent in the pH range corresponding to the pK of histidine. The inhibition induced by diethylpyrocarbonate cannot be reversed by hydroxylamine and the characteristics of this inhibition rather correspond to the reaction of the inhibitor with amino groups. Pyridoxal phosphate also inhibits the ATPase activity of F₁ by reaction with amino groups. The presence of ATP or phosphate partially protects against the inhibition induced by diethylpyrocarbonate or pyridoxal phosphate, which indicates that amino groups may be directly or indirectly involved in the binding of nucleotide and phosphate to F₁. Glutaraldehyde also inhibits the enzyme by reacting with amino groups and inducing a crosslinking of the subunits. The disappearance of subunit C is well correlated with the decrease of ATPase activity, indicating that subunit C is essential in the ATPase activity.     

A modifier of the Bg element of the Bg-rbg transposable element system of maize

A modifier of the Bg autonomous element of the Bg-rbg system of transposable elements has been found in the genotype of the inbred maize line 346. In the presence of this modifier (designated Mbg), the frequency of reversion of mutable allele o2-lf in combination with the Bg-lf element increases by 7–24 times. An increase in the Mbg dosage by three times increases the o2-lf reversion frequency by a factor of about two. The joint presence of Mbg and Bg-lf in the same genotype before meiosis is necessary for the expression of the Mbg modifying effect. The possible nature and mechanism of action of the novel modifier are discussed.     

A CIS-Dominant Mutation in ASPERGILLUS NIDULANS Affecting the Expression of the amdS Gene in the Presence of Mutations in the Unlinked Gene, amdA

A mutant producing very high levels of the acetamidase enzyme encoded by the amdS gene has been isolated in a strain containing the amdA7 mutation, which itself causes high levels of this enzyme. Genetic analysis has shown that this mutation, designated amdI66, is adjacent to the amdS gene and is cis-dominant in its effect. The amdI66 mutation has little effect on amdS expression when present in strains not containing the amdA7 mutation. Two other amdA mutations investigated also interact with the amdI66 mutation to result in high acetamidase levels. No interaction between amdI66 and any of the other putative regulatory genes affecting amdS expression has been observed. The amdI66 mutation has been located by fine structure mapping at the extreme end of the controlling region, which has previously been defined by genetic mapping (Hynes 1979). Analysis of this region has been extended by mapping new mutations resulting in loss of amdS expression. One of these defines the most extreme site capable of mutation to loss of gene function found so far.     

Investigation of the cyt gene in Bacillus thuringiensis and the biological activities of Bt isolates from the soil of China

The presence of cyt genes was investigated in 80 type strains of Bacillus thuringiensis and 143 isolates obtained from soil samples of China by PCR amplification using two pairs of primers for the cyt1 and cyt2 genes. Three type strains of serotypes H11ac, H14 and H36, eight isolates belonging to H3, H14, H18 and H21, and one isolate of unknown serotype harbored cyt genes. We also tested the cytolytic activity for mammal cells, the hemolytic activity for sheep erythrocytes and insecticidal activity against mosquitoes of five isolates that contained cyt genes but did not belong to B. thuringiensis serovar israelensis. The protein profiles of the five isolates were different from those of the type strains of B. thuringiensis serovar israelensis, and among the five isolates, only Y-5 showed mosquitocidal activity against larvae of Culex quinquefasciatus. All five of the isolates exhibited hemolytic activity, but only three could cause the cell death of A549 cells. The cytopathological changes induced by NX-4 in some A549 cells were characterized with cell-ballooning.     

Characterizing the effects of the protein environment on the reduction potentials of metalloproteins

The reduction potentials of electron transfer proteins are critically determined by the degree of burial of the redox site within the protein and the degree of permanent polarization of the polypeptide around the redox site. Although continuum electrostatics calculations of protein structures can predict the net effect of these factors, quantifying each individual contribution is a difficult task. Here, the burial of the redox site is characterized by a dielectric radius R _p (a Born-type radius for the protein), the polarization of the polypeptide is characterized by an electret potential ? _p (the average electrostatic potential at the metal atoms), and an electret-dielectric spheres (EDS) model of the entire protein is then defined in terms of R _p and ? _p. The EDS model shows that for a protein with a redox site of charge Q, the dielectric response free energy is a function of Q ², while the electret energy is a function of Q. In addition, R _p and ? _p are shown to be characteristics of the fold of a protein and are predictive of the most likely redox couple for redox sites that undergo different redox couples.     

Enantiomeric Composition of the trans-Dihydrodiols Produced from Phenanthrene by Fungi

The trans-dihydrodiols produced during the metabolism of phenanthrene by Cunninghamella elegans, Syncephalastrum racemosum, and Phanerochaete chrysosporium were purified by high-performance liquid chromatography (HPLC). The enantiomeric compositions and optical purities of the trans-dihydrodiols were determined to compare interspecific differences in the regio- and stereoselectivity of the fungal enzymes. Circular dichroism spectra of the trans-dihydrodiols were obtained, and the enantiomeric composition of each preparation was analyzed by HPLC with a chiral stationary-phase column. The phenanthrene trans-1,2-dihydrodiol produced by C. elegans was a mixture of the 1R,2R and 1S,2S enantiomers in variable proportions. The phenanthrene trans-3,4-dihydrodiol produced by P. chrysosporium was the optically pure 3R,4R enantiomer, but that produced by S. racemosum was a 68:32 mixture of the 3R,4R and 3S,4S enantiomers. The phenanthrene trans-9,10-dihydrodiol produced by P. chrysosporium was predominantly the 9S,10S enantiomer, but those produced by C. elegans and S. racemosum were predominantly the 9R,10R enantiomer. The results indicate that although different fungi may exhibit similar regioselectivity, there still may be differences in stereoselectivity that depend on the species and the cultural conditions.     

Studies on the reactivity of the essential cysteine of thymidylate synthetase

The reaction of one of the four cysteinyl residues of thymidylate synthetase from methotrexate-resistant Lactobacillus casei with a variety of sulfhydryl reagents results in complete inhibition of the enzyme. Kinetic studies indicate that the rates of reactivity of the reagents tested are N-ethylmaleimide > iodoacetamide > N-(iodoacetylaminoethyl)-S-naphthylamine-1-sulfonic acid > iodoacetic acid. The enzyme is also inactivated by 5-Hg-deoxyuridylate, a compound which reacts stoichiometrically with a single cysteine. Unlike the other reagents, the inhibition produced by this compound can be completely reversed by added thiols. The same cysteine appears to react with all of the sulfhydryl reagents, as shown by competition experiments and by protection against inactivation by deoxyuridylate. Even at a 100-fold excess of the alkylating agents, only one of the four cysteines in the native enzyme was reactive, attesting to the uniqueness of this residue. Carboxypeptidase A inactivation of the enzyme does not affect either the binding of deoxyuridylate to the enzyme or the reactivity of N-ethylmaleimide with the “catalytic” cysteine. Under denaturing conditions, all four cysteinyl residues react with N-ethylmaleimide or iodoacetate, as shown by identifying the reaction products by amino acid analysis. The covalent ternary complex [(+)5,10-methylenetetrahydrofolate-5-fluorodeoxyuridylate-thymidylate synthetase] (molar ratio = 2:2:1) revealed only two cysteinyl residues capable of reacting with N-ethylmaleimide or iodoacetate upon denaturation. From these data, it appears that one cysteine is involved in the binding of deoxyuridylate and that two of the enzyme's four cysteines are responsible for binding 5-fluorodeoxyuridylate in the ternary complex.     

Suppression of the Biocontrol Agent Trichoderma harzianum by Mycelium of the Arbuscular Mycorrhizal Fungus Glomus intraradices in Root-Free Soil

Trichoderma harzianum is an effective biocontrol agent against several fungal soilborne plant pathogens. However, possible adverse effects of this fungus on arbuscular mycorrhizal fungi might be a drawback in its use in plant protection. The objective of the present work was to examine the interaction between Glomus intraradices and T. harzianum in soil. The use of a compartmented growth system with root-free soil compartments enabled us to study fungal interactions without the interfering effects of roots. Growth of the fungi was monitored by measuring hyphal length and population densities, while specific fatty acid signatures were used as indicators of living fungal biomass. Hyphal ³³P transport and β-glucuronidase (GUS) activity were used to monitor activity of G. intraradices and a GUS-transformed strain of T. harzianum, respectively. As growth and metabolism of T. harzianum are requirements for antagonism, the impact of wheat bran, added as an organic nutrient source for T. harzianum, was investigated. The presence of T. harzianum in root-free soil reduced root colonization by G. intraradices. The external hyphal length density of G. intraradices was reduced by the presence of T. harzianum in combination with wheat bran, but the living hyphal biomass, measured as the content of a membrane fatty acid, was not reduced. Hyphal ³³P transport by G. intraradices also was not affected by T. harzianum. This suggests that T. harzianum exploited the dead mycelium but not the living biomass of G. intraradices. The presence of external mycelium of G. intraradices suppressed T. harzianum population development and GUS activity. Stimulation of the hyphal biomass of G. intraradices by organic amendment suggests that nutrient competition is a likely means of interaction. In conclusion, it seemed that growth of and phosphorus uptake by the external mycelium of G. intraradices were not affected by the antagonistic fungus T. harzianum; in contrast, T. harzianum was adversely affected by G. intraradices.     

Stress and salicylic acid induce the expression of PnFT2 in the regulation of the stress-induced flowering of Pharbitis nil

Poor nutrition and low temperature stress treatments induced flowering in the Japanese morning glory Pharbitis nil (synonym Ipomoea nil) cv. Violet. The expression of PnFT2, one of two homologs of the floral pathway integrator gene FLOWERING LOCUS T (FT), was induced by stress, whereas the expression of both PnFT1 and PnFT2 was induced by a short-day treatment. There was no positive correlation between the flowering response and the homolog expression of another floral pathway integrator gene SUPPRESSOR OF OVEREXPRESSION OF CO1 and genes upstream of PnFT, such as CONSTANS. In another cultivar, Tendan, flowering and PnFT2 expression were not induced by poor nutrition stress. Aminooxyacetic acid (AOA), a phenylalanine ammonia-lyase inhibitor, inhibited the flowering and PnFT2 expression induced by poor nutrition stress in Violet. Salicylic acid (SA) eliminated the inhibitory effects of AOA. SA enhanced PnFT2 expression under the poor nutrition stress but not under non-stress conditions. These results suggest that SA induces PnFT2 expression, which in turn induces flowering; SA on its own, however, may not be sufficient for induction.     

A new method for degradation of the protein part of glycoproteins: isolation of the carbohydrate chains of asialofetuin

A new method has been developed for degrading the protein part of several glycoproteins, whilst leaving the carbohydrate portion virtually intact apart from partial degradation at the reducing end. The method is based upon stabilization of the glycosidic linkages of the sugar residues by trifluoroacetyl groups and subsequent cleavage of the peptide bonds by transamidation. The two reactions are carried out in a mixture of trifluoroacetic anhydride and trifluoroacetic acid. After O- and N-detrifluoroacetylation, the carbohydrate portion can be isolated and re-N-acetylated. The applicability of the method is demonstrated by the isolation from asialofetuin of the carbohydrate chains that are attached by N- and O-glycosyl links.