Co-immunolocalization of topoisomerase II and chloroplast DNA in developing,dividing and mature wheat chloroplasts

This paper describes the first localization of immunofluorescence of topoisomerase II in developing chloroplasts. In order to investigate the relationship between topoisomerase II and chloroplast DNA (ctDNA) replication during chloroplast development the 7-day-old wheat leaf was used. Topoisomerase II was immunolabelled and fluorescein tagged and the ctDNA simultaneously stained with 4,6-diamidino-2-phenylindole (DAPI) in the same sections. Topoisomerase II was detected at every stage of chloroplast development and maximal levels of topoisomerase II were found in chloroplasts at the time of ctDNA replication. Topoisomerase II was localized around the plastid periphery, exactly mirroring the position of the ctDNA. After chloroplast division both topoisomerase II and ctDNA are seen to be restricted to small discrete areas within the plastid, but at different sites. These findings strongly suggest a role for topoisomerase II in ctDNA decatenation prior to chloroplast division.     

The structure of agarose gels,and the accommodation within them of irregularly shaped particles

This paper shows how the number of cross-linkage points (nodes) in a random reticulum, such as an ideal polysaccharide gel, may be calculated in accordance with mathematical principles. The influence of nodal configurations upon the statistical geometry of the reticulum is discussed, and it is shown from experimental evidence that the nodal configurations in agarose gel are nonrandom. A method is given for calculating the accommodation probability of an irregularly shaped particle in a reticulum, which is relevant to the theory of gel chromatography and to the distribution of cells in tissues permeating a network of capillaries or veins.     

Characterisation in vivo of the reactive thiol groups of the lactose permease from Escherichia coli and a mutant; exposure, reactivity and the effects of substrate binding

The reactivity and accessibility of the reactive thiol groups of the native lactose permease and a mutant have been studied in a number of circumstances and with a number of reagents, in particular using the specific thiol-disulphide exchange reaction. Seven different reactive states of the thiol in the native protein have been characterised by their different second-order rate constants. Interconversion between these states is dependent on the magnitude of the protonmotive force, pH and substrate binding. In the absence of galactoside, reactivity is controlled by an ionisation with apparent pKa 9.3. This pKa is not affected by the protonmotive force, but it is lowered in the presence of external galactoside. The conformation adopted by the permease when in equilibrium with saturating galactoside appears to be different from that of the intermediate that accumulates during net turnover. In the former state, the reactivity of the thiol group is depressed, whereas in the latter state it is enhanced. The thiol group of the native protein is buried in a hydrophobic environment that has a dielectric constant considerably lower than that of water. The environment is not greatly perturbed by changes in the magnitude of the protonmotive force, but it is affected by the binding of galactoside. In a strain which carries the YUN mutation (Wilson, T.H. and Kusch, M. (1972) Biochim. Biophys. Acta 255, 786-797), two reactive thiols were characterised. The more reactive of the two is more exposed than the thiol group of the native molecule and is in an environment that has a dielectric constant close to that of water. The less reactive thiol appears to be more deeply buried than that of the native protein. Thus the mutation appears to produce a conformation change in the central portion of the polypeptide chain that results in greater exposure of the reactive thiol to the aqueous environment.     

Isolation of cDNA clones for fourteen nuclear-encoded thylakoid membrane proteins

Summary Spinach cDNA libraries, made from polyadenylated seedling RNA, have been constructed in pBR322 and the expression vector gt11. Recombinant plasmids or phage for 14 intrinsic and peripheral thylakoid membrane proteins and one stromal protein have been identified. They encode components containing antigenic determinants against the lysine-rich 34 kd, the 23 kd and 16 kd proteins all associated with the water-splitting apparatus of the photosystem II reaction center, the ATP synthase subunits gamma, delta and CF_o-II, the Rieske Fe/S protein of the cytochrome b/f complex, subunits 2, 3, 5 and 6 of the photosystem I reaction center, plastocyanin, ferredoxin oxidoreductase, chlorophyll a/b-binding apoproteins of the lightharvesting complex associated with photosystem II, and the small subunit of the stromal enzyme ribulose bisphosphate corboxylase/oxygenase. The cDNA inserts lack complementarity to plastid DNA but hybridize to restricted nuclear DNA as well as to discrete poly A⁺-mRNA species. The precursor products obtained after translation of hybrid selected RNA fractions in a wheat germ assay are imported and processed by isolated unbroken spinach chloroplasts. The imported components comigrate with the respective authentic proteins.     

Biosynthesis of the Macrocyclic Diterpene Casbene in Castor Bean (Ricinus communis L.) Seedlings : The Purification and Properties of Farnesyl Transferase from Elicited Seedlings

Farnesyl transferase (farnesyl pyrophosphate: isopentenyl pyrophosphate farnesyl transferase; geranylgeranyl pyrophosphate synthetase) was purified at least 400-fold from extracts of castor bean (Ricinus communis L.) seedlings that were elicited by exposure for 10 h to Rhizopus stolonifer spores. The purified enzyme was free of isopentenyl pyrophosphate isomerase and phosphatase activities which interfere with prenyl transferase assays. The purified enzyme showed a broad optimum for farnesyl transfer between pH 8 and 9. The molecular weight of the enzyme was estimated to be 72,000 ± 3,000 from its behavior on a calibrated G-100 Sephadex molecular sieving column. Mg²⁺ ion at 4 millimolar gave the greatest stimulation of activity; Mn²⁺ ion gave a small stimulation at 0.5 millimolar, but was inhibitory at higher concentrations. Farnesyl pyrophosphate (K_m = 0.5 micromolar) in combination with isopentenyl pyrophosphate (K_m = 3.5 micromolar) was the most effective substrate for the production of geranylgeranyl pyrophosphate. Geranyl pyrophosphate (K_m = 24 micromolar) could replace farnesyl pyrophosphate as the allylic pyrophosphate substrate, but dimethylallyl pyrophosphate was not utilized by the enzyme. One peak of farnesyl transferase activity (geranylgeranyl pyrophosphate synthetase) and two peaks of geranyl transferase activity (farnesyl pyrophosphate synthetases) from extracts of whole elicited seedlings were resolved by DEAE A-25 Sephadex sievorptive ion exchange chromatography. These results suggest that the pathway for geranylgeranyl pyrophosphate synthesis in elicited castor bean seedlings involves the successive actions of two enzymes—a geranyl transferase which utilizes dimethylallypyrophosphate and isopentenyl pyrophosphate as substrates and a farnesyl transferase which utilizes the farnesyl pyrophosphate produced in the first step and isopentenyl pyrophosphate as substrates.     

A probability matrix for the identification of species of Vibrio and related genera

A matrix for the probabilistic identification of species of Vibrio and related genera has been constructed using the data from 1091 strains collected throughout the world and classified. Thirty-eight phenons are included in the matrix, 31 of these represent previously identified species or biovars and seven represent phenons which could not be identified and may represent new species. The identification matrix incorporates 81 characters although a subset of 30 tests can be used to distinguish the 38 phenons from each other. The additional 51 tests were included to assist the identification of some strains for which the initial 30 tests were inadequate. No significant cluster overlap was found at the 5% level and the identification score for the Hypothetical Median Organism of each cluster exceeded 0.9999 in all cases.     

A tentative national reference procedure for isolation and enumeration of Escherichia coli from bivalve molluscan shellfish by most probable number method

In the UK several quantitative methods exist for the examination of bivalve molluscan shellfish for sewage contamination. These methods include roll tubes, pour plates and most probable number (MPN) techniques, but there is no national standard method. A comparative study was made of the most commonly used methods for detection of Escherichia coli in bivalve shellfish. Schemes employing solid media, such as the roll tube and pour plate methods, underestimated faecal contamination in shellfish tissue compared with a liquid MPN multiple test-tube method using minerals-modified-glutamate broth (MMGB) as primary enrichment medium. The composition of MMGB apparently permits repair of sublethally injured cells of E. coli. Incorporation of resuscitation stages into the pour plate technique did not yield higher counts. A standardized MPN technique for examination of bivalve molluscan shellfish for E. coli content is proposed as a possible national reference procedure pending further collaborative assessment.     

On a mechanism of cardiac electrical stability. The fractal hypothesis.

Electrical activation of the ventricles via the His-Purkinje system is represented on the body surface by a waveform with a broad range of frequency components. We speculate that this process is mediated by current flow through a fractal-like conduction network and therefore that the broadband spectrum of the depolarization waveform should be scaled as a power-law distribution. The prediction is confirmed by Fourier analysis of electrocardiographic data from healthy men. This observation suggests a new dynamical link between nonlinear (fractal) structure and nonlinear function in a stable physiologic system.     

A Salmonella typhimurium strain defective in uracil catabolism and beta-alanine synthesis

A selection procedure for uracil catabolism mutant strains involving indicator dye plates was developed. Using this method, a strain defective in uracil catabolism has been isolated in Salmonella typhimurium that was temperature-sensitive at 42 degrees C where it required low concentrations of N-carbamoyl-beta-alanine, beta-alanine or pantothenic acid for growth. An extract of the mutant strain degraded uracil at 37 degrees C at a significantly diminished rate compared to that observed for the wild-type strain under the same growth conditions. The conversion of dihydrouracil to N-carbamoyl-beta-alanine was blocked at all temperatures examined in the mutant strain. By means of genetic analysis, the mutant strain was determined to be defective at two genetic loci. Transduction studies with bacteriophage P22 indicated that the panD gene is mutated in this strain, accounting for its beta-alanine requirement. Episomal transfers between Escherichia coli and the mutant strain provided evidence that the defect in uracil catabolism was located in another region of the S. typhimurium chromosome.     

Discrimination between the effects of X-ray irradiation of the mouse oocyte and uterus on the induction of dominant lethals and congenital anomalies. II. Localised irradiation experiments

In order to evaluate whether irradiation of the postimplantation maternal environment contributed to the induction of postimplantation mortality or congenital anomalies, mouse ovaries were surgically exteriorised and selectively irradiated or shielded in a specially constructed apparatus. The results show that exposure of the mouse abdomen and uterus to 3.70 Gy X-rays, 15-21 days prior to conception, has no significant effect on the incidence of either postimplantation mortality or congenital anomalies. Exposure of the ovaries to 3.27 Gy X-rays during the same period, however, increased the frequency of both postimplantation mortality and congenital anomalies.