Cloning of aroG, the gene coding for phospho-2-keto-3-deoxy-heptonate aldolase(phe), in Escherichia coli K-12, and subcloning of the aroG promoter and operator in a promoter-detecting plasmid

Defective transducing phages carrying aroG, the structural gene for phenylalanine (phe)-inhibitable phospho-2-keto-heptonate aldolase (EC 4.1.2.15; previously known as 3-deoxy-D-arabinoheptulosonate-7-phosphate synthetase[phe]), have been isolated, and DNA from two of these phages has been used to construct a restriction map of the region from att lambda to aroG. A 7.6-kb PstI-HindIII fragment from one of these phages was cloned into pBR322 and shown to contain aroG. The location of aroG within the 7.6 kb was established by subcloning and Tn3 transpositional mutagenesis. A fragment carrying the aroG promoter and operator has been cloned into a high copy number promoter-cloning vector (pMC489), and the resulting aroGpo-LacZ' (alpha) fusion subcloned in a low copy number vector. Strains with this fusion on the low copy number vector exhibit negative regulation of beta-galactosidase expression by both phenylalanine and tryptophan and positive regulation by tyrosine in a tyrR+ background.     

Sequence heterogeneity and anomalous electrophoretic mobility of kinetoplast minicircle DNA from Leishmania tarentolae

Several unit-length minicircles from the kinetoplast DNA of Leishmania tarentolae were cloned into pBR322 and into M13 phage vectors. The complete nucleotide sequences of three different partially homologous minicircles were obtained. The molecules contained a region of approx. 80% sequence homology extending for 160–270 bp and a region unique to each minicircle. A 14-mer was found to be conserved in all kinetoplast minicircle sequences reported to date. The frequency distributions of various minicircle sequence classes in L. tarentolae were obtained by quantitative gel electrophoresis and by examination of the “T ladder” patterns of minicircles randomly cloned into M13 at several sites. By these methods we could assign approx. 50% of the total minicircle DNA into a minimum of five sequence classes. A sequence-dependent polyacrylamide gel migration abnormality was observed with several minicircle fragments both cloned and uncloned. The abnormality was dependent on the presence of a portion of the conserved region of the minicircle.     

A new tubulin-binding protein

A new brain protein is described which forms an insoluble complex with tubulin, with concomitant stoichiometric hydrolysis of GTP. The complex contains a maximum of one tubulin-binding protein (MW 52,500) per two tubulin dimers. The tubulin-binding protein (TBP) does not compete with colchicine, but in the presence of microtubule-associated proteins tubulin appeared less accessible to it. Proteins such as TBP might sequester tubulin and thereby function either to inhibit indiscriminate polymerization, or to promote ordered nucleation by maintaining high local concentrations.     

Monoclonal antibodies against beta-galactoside-binding lectin of chick embryo recognizes conformational change of the antigen

Monoclonal antibodies against chick embryonic beta-galactoside-binding lectin were obtained. One of the monoclonal antibodies was ineffective in Western blotting and seemed to be unable to bind the SDS-denatured lectin. When the native lectin was dotted on a nitrocellulose filter and subjected to denaturation by treatment with SDS, urea or heat, binding of this antibody no longer occurred, though other monoclonal antibodies bound normally. This antibody seems to have been raised against an epitope which is destroyed upon denaturation.     

Volume regulation in the early proximal tubule of theNecturus kidney

Summary The ability of early proximal tubule cells of theNecturus kidney to regulate volume was evaluated using light microscopy, video analysis and conventional microelectrodes.Necturus proximal tubule cells regulate volume in both hyperand hyposmotic solutions. Volume regulation in hyperosmotic fluids is HCO ₃ ^– dependent and is associated with a decrease in the relative K⁺ conductance of the basolateral cell membrane and a decrease in the resistance ratio,R _a /R _bl . Volume regulation in hyposmotic solutions is also dependent upon the presence of HCO ₃ ^– but is also inhibited by 2mm Ba²⁺ in the basolateral solution. Hyposmotic regulation is accompanied by an increase in the relative K⁺ conductance of the basolateral cell membrane and an increase inR _a /R _bl . Neither hypo- nor hyposmotic regulation have any affect on the depolarization of the basolateral cell membrane potential induced by HCO ₃ ^– removal. We conclude that volume regulation in the early proximal tubule of the kidney involves both HCO ₃ ^– -dependent transport systems and the basolateral K⁺ conductance.     

Development and Significance of Cysteamine and Propionitrile Models of Duodenal Ulcer

Cysteamine is widely used in rodents to induce duodenal ulcer. Herein, the pathogenesis of duodenal ulceration in its earliest stages was reviewed using findings from cysteamine-and propionitrile-induced duodenal ulcer in rodent models, especially taking into account changes in the secretion of gastric acid, duodenal and pancreatic bicarbonate as well asgastroduodenal motility. The effect of cysteamine-HCl in inducing ulcers in rats is circadian rhythm-dependent. The effect is greatest from just before the end of diurnal rest to just after the start of nocturnal activity. The chronobiologic effect may be in part due to the circadian rhythm-dependent increased gastric acid production from cysteamine. Titratable acidity was found to be twice as great in the gastric juice of rodents when cysteamine was given by injection at 2000 (just after the start of nocturnal activity) in comparison to when given at 0800 or 1200 (at the beginning or middle span of daily rest). Further studies have shown that adrenalectomy of rats 7 days before cysteamine administration obliterated the observed circadian susceptibility to ulcer formation. Duodenal ulceration, at least in the cysteamine model, appears to be under chronobiologic neuroendocrine control or influence, seemingly mediated by the adrenal glands.     

The molecular mechanism of the bicarbonate effect at the plastoquinone reductase site of photosynthesis

It has been known for some time that bicarbonate reverses the inhibition, by formate under HCO₃ ^--depletion conditions, of electron transport in thylakoid membranes. It has been shown that the major effect is on the electron acceptor side of photosystem II, at the site of plastoquinone reduction. After presenting a historical introduction, and a minireview of the bicarbonate effect, we present a hypothesis on how HCO₃ ^- functions in vivo as (a) a proton donor to the plastoquinone reductase site in the D1-D2 protein; and (b) a ligand to Fe²⁺ in the Q_A-Fe-Q_B complex that keeps the D1-D2 proteins in their proper functional conformation. They key points of the hypothesis are: (1) HCO₃ ^- forms a salt bridge between Fe²⁺ and the D2 protein. The carboxyl group of HCO₃ ^- is a bidentate ligand to Fe²⁺, while the hydroxyl group H-bonds to a protein residue. (2) A second HCO₃ ^- is involved in protonating a histidine near the Q_B site to stabilize the negative charge on Q_B. HCO₃ ^- provides a rapidly available source of H⁺ for this purpose. (3) After donation of a H⁺, CO₃ ^2- is replaced by another HCO₃ ^-. The high pKa of CO₃ ^2- ensures rapid reprotonation from the bulk phase. (4) An intramembrane pool of HCO₃ ^- is in equilibrium with a large number of low affinity sites. This pool is a H⁺ buffering domain functionally connecting the external bulk phase with the quinones. The low affinity sites buffer the intrathylakoid [HCO₃ ^-] against fluctuations in the intracellular CO₂. (5) Low pH and high ionic strength are suggested to disrupt the HCO₃ ^- salt bridge between Fe²⁺ and D₂. The resulting conformational change exposes the intramembrane HCO₃ ^- pool and low affinity sites to the bulk phase.Two contrasting hypotheses for the action of formate are: (a) it functions to remove bicarbonate, and the low electron transport left in such samples is due to the left-over (or endogenous) bicarbonate in the system; or (b) bicarbonate is less of an inhibitor and so appears to relieve the inhibition by formate. Hypothesis (a) implies that HCO₃ ^- is an essential requirement for electron transport through the plastoquinones (bound plastoquinones Q_A and Q_B and the plastoquinone pool) of photosystem II. Hypothesis (b) implies that HCO₃ ^- does not play any significant role in vivo. Our conclusion is that hypothesis (a) is correct and HCO₃ ^- is an essential requirement for electron transport on the electron acceptor side of PS II. This is based on several observations: (i) since HCO₃ ^-, not CO₂, is the active species involved (Blubaugh and Govindjee 1986), the calculated concentration of this species (220 M at pH 8, pH of the stroma) is much higher than the calculated dissociation constant (Kd) of 35–60 M; thus, the likelihood of bound HCO₃ ^- in ambient air is high; (ii) studies on HCO₃ ^- effect in thylakoid samples with different chlorophyll concentrations suggest that the left-over (or endogenous) electron flow in bicarbonate-depleted chloroplasts is due to left-over (or endogenous) HCO₃ ^- remaining bound to the system (Blubaugh 1987).Abbreviations DCMU 3-(3,4-dichlorophenyl)-1, 1-dimethylurea (common name: diuron) - PSII photosystem II - Q_A first plastoquinone electron acceptor of PSII - Q_B second plastoquinone acceptor of PS II     

Photosynthesis and apparent affinity for dissolved inorganic carbon by cells and chloroplasts of Chlamydomonas reinhardtii grown at high and low CO2 concentrations

Chloroplasts with high rates of photosynthetic O₂ evolution (up to 120 mol O₂· (mg Chl)^-1·h^-1 compared with 130 mol O₂· (mg Chl)^-1·h^-1 of whole cells) were isolated from Chlamydomonas reinhardtii cells grown in high and low CO₂ concentrations using autolysine-digitonin treatment. At 25° C and pH=7.8, no O₂ uptake could be observed in the dark by high- and low-CO₂ adapted chloroplasts. Light saturation of photosynthetic net oxygen evolution was reached at 800 mol photons·m^-2·s^-1 for high- and low-CO₂ adapted chloroplasts, a value which was almost identical to that observed for whole cells. Dissolved inorganic carbon (DIC) saturation of photosynthesis was reached between 200–300 M for low-CO₂ adapted chloroplasts, whereas high-CO₂ adapted chloroplasts were not saturated even at 700 M DIC. The concentrations of DIC required to reach half-saturated rates of net O₂ evolution (K_m(DIC)) was 31.1 and 156 M DIC for low- and high-CO₂ adapted chloroplasts, respectively. These results demonstrate that the CO₂ concentration provided during growth influenced the photosynthetic characteristics at the whole cell as well as at the chloroplast level.Abbreviations Chl chlorophyll - DIC dissolved inorganic carbon - K_m(DIC) coneentration of dissolved inorganic carbon required for the rate of half maximal net O₂ evolution - PFR photon fluence rate - SPGM silicasol-PVP-gradient medium     

Inorganic-carbon uptake by a small-celled strain of Stichococcus bacillaris

Air-grown cells of a marine, small-celled (2 m diameter) strain of Stichococcus bacillaris contained appreciable carbonic-anhydrase activity but this was repressed when cells were grown on air enriched with 5% (v/v) CO₂. Assay of carbonic-anhydrase activity using intact cells and cell extracts showed all activity was intracellular in this Stichococcus strain. Measurement of inorganic-carbon-dependent photosynthetic O₂ evolution at pH 5.0, where CO₂ is the predominant form of inorganic carbon, showed that the concentration of inorganic carbon required for half-maximal rate of photosynthetic O₂ evolution [K_0.5(CO₂)] was 4.0 M for both air- and CO₂-grown cells. At pH 8.3 the K_0.5(CO₂) was 0.3 mM for air-grown and 0.6 mM for CO₂-grown cells. Sodium ions did not enhance bicarbonate utilization. Measurement of the internal inorganic-carbon pool (HCO ₃ ^– +CO₂) by the silicone-oil-layer centrifugal filtering technique showed that air- and CO₂-grown cells were able to concentrate inorganic carbon up to 20-fold in relation to the external medium at pH 5.0 but not at pH 8.3. In this alga the high affinity for CO₂ and inorganic-carbon accumulation in CO₂- and air-grown cells results from active CO₂ transport that is not dependent on carbonic-anhydrase activity.Abbreviation Hepes 4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid     

Stress studies in lentil (Lens esculenta Moench)

The response of soil exchangeable sodium percentage levels to nitrate reductase activity, nitrite reductase activity, free proline, DNA, RNA, chlorophyll a and b contents and yield components in lentil (Lens esculenta Moench)cv. PL 406 was studied in a replicated pot experiment. All the biochemical observations were recorded at four growth stages i.e. 30, 60, 90 and 120 days after sowing (DAS). Germination occurred up to exhangeable sodium percentage of 30, but plants survived only up to 25. With increasing exchangeable sodium percentage, there was a continuous decrease in chlorophyll a and b content, nitrate and nitrite reductase enzyme activities and DNA and RNA content. Increasing level of sodicity enhanced the free proline content up to 60 DAS, after which values fell.Number of pods per plant, 1000 grain weight and grain yield were significantly reduced with increasing level of sodicity, but the number of grains per pod was not affected.