Metal ion coordination to 2′ functionality of guanosine mediates substrate-guanosine coupling in group I ribozymes: implications for conserved role of metal ions and for variability in RNA folding in ribozyme catalysis

Divalent metal ions are necessary in the self splicing reaction of group I introns, and we report that metal interaction to the 2′ position of guanosine for the Azoarcus ribozyme is required for catalysis. Moreover, this metal coordination promotes the guanosine-substrate coupled binding to the ribozyme, which is another conserved feature seen across phylogenetic boundaries. Typically there is a 4-9-fold difference in binding of G to E_free versus E · S. In the Tetrahymena ribozyme’s case this substrate-guanosine communication was attributed to conformational change(s) that lead to cooperative binding of the two cofactors which is almost nonexistent at low temperatures (4 °C). In the prokaryotic Azoarcus ribozyme we also see a 4-5-fold difference in binding of the guanosine/substrate to E_free versus E · G or E · S at 10 °C that is attributed to guanosine-substrate coupling. This coupling is diminished when the metal (Mg²⁺) coordination to the 2′ is disrupted with use of 2′-amino-2′-deoxyguanosine. The coupling is restored when softer Mn²⁺ ions are added to the buffer. This evidence generalizes a model for group I ribozyme catalysis that involves metal coordination to the 2′ position of guanosine. However, we see one striking difference in that the guanosine-substrate coupling is reversed. In the Azoarcus system (10 °C) the guanosine/substrate binds 5-fold more tightly to E_free than to E · S or E · G, which is the opposite for Tetrahymena even when the later is run at 4 °C. One implication for this difference in coupling is that the Azoarcus is in a folded state well accommodated for guanosine or substrate binding. This initial binding actually causes a conformational change that retards the subsequent binding of the second cofactor, which contrasts what was found for the Tetrahymena ribozyme. These results indicate that while the role for the metal ions in the chemical catalysis is conserved across phylogenetic boundaries, there is variability in the folding pattern of the ribozyme that leads to phosphoryl transfer.     

STUDIES ON THE METABOLISM OF THE AUTOTROPHIC BACTERIA : III. THE NATURE OF THE ENERGY STORAGE MATERIAL ACTIVE IN THE CHEMOSYNTHETIC PROCESS

In the autotrophic bacterium, Thiobacillus thiooxidans, the oxidation of sulfur is coupled to transfers of phosphate from the medium to the cells. CO₂ fixation is coupled to transfers of inorganic phosphate from the cells to the medium and is dependent, in the absence of concomitant sulfur oxidation, upon the amount of phosphate previously taken up during sulfur oxidation. The energy reservoir, which is formed by sulfur oxidation in the absence of CO₂ and which can be released for the fixation of CO₂ under conditions which do not permit sulfur oxidation, is a phosphorylated compound and the data suggest that the energy is stored in the cell as phosphate bond energy. It is possible to oxidize sulfur at a constant rate for hours in the absence of CO₂. The phosphate energy formed during this process is probably released by cell phosphotases. It is possible to inhibit these phosphotases by means of inorganic phosphate and thus to inhibit sulfur oxidation in the absence of CO₂. In the presence of CO₂, where alternative uses for the phosphate energy are available, the inhibition is relieved. Sulfur oxidation (energy input) is coupled, not to CO₂ fixation, but to phosphate esterification. CO₂ fixation (energy utilization) is coupled with phosphate release.     

Lac repressor-operator interaction. 8. Lactose is an anti-inducer of the lac operon

Lactose is shown to be an effective anti-inducer of the lac operon both in vivo and in vitro. When lactose is used as a carbon source, the synthesis of β-galaetosidase in Escheriahia coli is not fully induced. Moreover, lactose is able to partially inhibit induction by isopropyl-(β-d-thiogalactoside in strains synthesizing inactive as well as active β-galactosidase. These effects in vivo are not due to catabolite repression by the glucose derived from lactose. These in vivo results suggest that lactose is acting as an anti-inducer. This is confirmed in vitro by showing that lactose binds to the lac represser and stabilizes the represser-operator complex.     

The chromosomes of the Filariae

An understanding of the nature of the chromosomes of the filariae is expected to greatly assist the future interpretation of genome data. Filarial development is not eutelic, and there does not seem to be a fixed number of cell divisions in the way that there is in Caenorhabditis. It is not clear whether the chromosomes of the filariae have localized centromeres or whether they are holocentric. Sex determination is by a chromosomal "balance" X0 system in most filariae, but in some Onchocercidae there has been a chromosomal fusion to create a neo-XY system. It is presumed that the molecular basis of sex determination in filariae is similar to Caenorhabditis. The ancestral karyotype of the filariae is probably 5A+X0, but in some Onchocercidae this has been reduced to 4A+XY, and in O. volvulus and O. gibsoni it has been further reduced to 3A+XY. Onchocerca volvulus and O. gibsoni both have supernumary (B-) chromosomes and in O. volvulus there is a single active nucleolus organising region near the middle of the long autosome.     

The Q-cycle reviewed: How well does a monomeric mechanism of the bc1 complex account for the function of a dimeric complex?

Recent progress in understanding the Q-cycle mechanism of the bc₁ complex is reviewed. The data strongly support a mechanism in which the Q_o-site operates through a reaction in which the first electron transfer from ubiquinol to the oxidized iron–sulfur protein is the rate-determining step for the overall process. The reaction involves a proton-coupled electron transfer down a hydrogen bond between the ubiquinol and a histidine ligand of the [2Fe–2S] cluster, in which the unfavorable protonic configuration contributes a substantial part of the activation barrier. The reaction is endergonic, and the products are an unstable ubisemiquinone at the Q_o-site, and the reduced iron–sulfur protein, the extrinsic mobile domain of which is now free to dissociate and move away from the site to deliver an electron to cyt c₁ and liberate the H⁺. When oxidation of the semiquinone is prevented, it participates in bypass reactions, including superoxide generation if O₂ is available. When the b-heme chain is available as an acceptor, the semiquinone is oxidized in a process in which the proton is passed to the glutamate of the conserved -PEWY- sequence, and the semiquinone anion passes its electron to heme b_L to form the product ubiquinone. The rate is rapid compared to the limiting reaction, and would require movement of the semiquinone closer to heme b_L to enhance the rate constant. The acceptor reactions at the Q_i-site are still controversial, but likely involve a “two-electron gate” in which a stable semiquinone stores an electron. Possible mechanisms to explain the cyt b₁₅₀ phenomenon are discussed, and the information from pulsed-EPR studies about the structure of the intermediate state is reviewed.The mechanism discussed is applicable to a monomeric bc₁ complex. We discuss evidence in the literature that has been interpreted as shown that the dimeric structure participates in a more complicated mechanism involving electron transfer across the dimer interface. We show from myxothiazol titrations and mutational analysis of Tyr-199, which is at the interface between monomers, that no such inter-monomer electron transfer is detected at the level of the b_L hemes. We show from analysis of strains with mutations at Asn-221 that there are coulombic interactions between the b-hemes in a monomer. The data can also be interpreted as showing similar coulombic interaction across the dimer interface, and we discuss mechanistic implications.     

New skull of Lesothosaurus (Dinosauria: Ornithischia) from the Upper Elliot Formation (Lower Jurassic) of southern Africa

A new ornithischian skull from the Elliot Formation of southern Africa is described. The specimen is compared in detail with the fabrosaurid Lesothosaurus diagnosticus. It actually shares many characters with specimens of the syntypes of this species or specimens referred to it. It is nevertheless not identical to any of these specimens and it is, moreover, remarkably larger than them. The possibility of attributing this specimen to a so far undescribed ‘large fabrosaur’ from the same formation is discussed. It is concluded that the specimen in question in this paper, while being ascribable to the genus Lesothosaurus, cannot be determined to a specific level until the existence of two fabrosaurid species in the ‘Stormberg Group’ is demonstrated and their range of morphological and size variation is properly appraised.     

A small sequence in domain v of the mitochondrial large ribosomal RNA restores <Emphasis Type="Italic">Drosophila melanogaster</Emphasis> pole cell determination in uv-irradiated embryos

The mechanism by which the mitochondrial large rRNA is involved in the restoration of the pole cell-forming ability in Drosophila embryos is still unknown. We identified a 15-ribonucleotide sequence which is conserved from the protobacterium Wolbachia to the higher eukaryotes in domain V of the mitochondrial large rRNA. This short sequence is sufficient to restore pole cell determination in UV-irradiated Drosophila embryos. Here, we provide evidence that the conserved 15-base sequence is sufficient to restore luciferase activity in vitro. Moreover, we show that the internal GAGA sequence is involved in protein binding and that mutations in this tetranucleotide affect the sequence’s ability to restore luciferase activity. The obtained results lead us to propose that mtlrRNA may be involved either in damaged protein reactivation or in protein biosynthesis during pole cell determination.     

Studies in the floral morphology and anatomy of nymphaeales

Floral morphology ofBrasenia schreberi Gmel. andCabomba caroliniana A. Gray was observed chiefly from an anatomical point of view. The receptacle ofB. schreberi is rather flat and a vascular plexus is observable in the mature flower. The vasculature in this plexus is so complex taht it is not easy to trace its structure in detail. by observation on small buds, it can be seen that the receptacular vasculature consists of a girdling bundle in the basal area and usually nine receptacular strands from which traces to the petals and stamens branch off. The vasculature in the receptacle is reconstructed and diagramatically shown as though split longitudinally and spread out in one plane. Floral vasculature inCabomba caroliniana is simpler, and is probably related to the smaller number of stamens and carpels. It also has a girdling bundle at the bottom of receptacle and this vasculature is suggested to be derived by simplification from aBrasenia-type vasculature. Evidence from floral anatomy suggests that these two genera are closely related. InNymphaea, a vascular plexus in the receptacle is also observed (Moseley, 1961; Ito 1983). The plexus ofBrasenia andNymphaea are not the same in their construction. Nevertheless, their fundamental floral vasculature is comparable and it is preferable to place them in the same family or same order.     

Analysis of morphogenetic mutants of hydra

Summary A mutant ofHydra attenuata is analysed, theaberrant, which is distinct from the wild type in having a smaller head with fewer tentacles and only half the number of head-specific cells. The rate of head and foot regeneration and the doubling time are slower inaberrants than in normal hydra.The lower head-forming potential is paralleled by a reduced concentration of head-specific morphogens: compared to the wild type, in theaberrant the concentration of head activator is reduced to 70% in the head and to 50% in the body, the concentration of head inhibitor is reduced to 50% in the head and to 80% in the body. Theaberrant is more sensitive (3 times) to added head activator and less sensitive (>5 times) to added head inhibitor than the wild type.The slower rate of foot regeneration is paralleled by a lower content of foot-specific morphogens: compared to the wild type, in theaberrant the foot activator is reduced to 40% and the foot inhibitor to 70%.     

The Role of Organic-matter-bound Chlorine in the Chlorine Cycle: A Case Study of the Stubbetorp Catchment,Sweden

The objective of this study is to construct a balanced chlorine budget for a small forested catchment, focusing on the interaction between chloride (Cl_inorg) and organic-matter-bound chlorine (Cl_org). Data from the actual catchment are combined with secondary data from other sites to elucidate more clearly which parts of the cycle are fairly well known and which are more or less unknown. The budget calculations show that the principal input and output fluxes of Cl in the catchment are inorganic but that the main pool is Cl_org in the soil. In addition, the budget calculations suggest that a considerable portion of Cl_inorg in soil is transformed to Cl_org and subsequently leached to deeper soil layers, that net mineralization of Cl_org takes place in soil, preferably in deeper soil layers, and that degrading organic matter is a major source of Cl_inorg in runoff. The loss of Cl_org through runoff is small to negligible in relation to other fluxes. It appears as if dry deposition of Cl_inorg is at risk of being underestimated if Cl_inorg is assumed to be conservative in soil. The pool of organic-matter-bound chlorine in soil is considerably larger than the annual flux of chloride through the system. The estimates suggest that the amount of Cl_org in the upper 40 cm of the soil at the investigated site is approximately twice as large as the Cl_inorg. Furthermore, the amount of Cl_org biomass is small in relation to the occurrence of Cl_org in soil. Finally, the estimates indicate that the transport of volatile Cl_org from the soil to the atmosphere may influence the chlorine cycle.     

The POU-domain protein Pdm3 regulates axonal targeting of R neurons in the Drosophila ellipsoid body

The ability of axons to project correctly to the target is essential for the formation of complex neural networks. The intrinsic regulation of this process is still unclear. Here, we show that POU domain motif 3 (Pdm3) is required in ring (R) neurons to control precise axon targeting to the Drosophila ellipsoid body (EB). Pdm3 is expressed in neurons of the central nervous system in larvae and adults and required for the normal development of the EB of the central complex in the adult brain. The normal EB structure is abolished in pdm3 mutants, and this phenotype is rescued by pdm3 expression in R neurons, suggesting that the defect in axonal targeting of R neurons is the major cause in EB malformation in pdm3 mutants. We show that cell fate determination, dendritic arborization, and initial axon projection of R neurons are normal while the axonal targeting to the EB is defective in pdm3 mutants. © 2011 Wiley Periodicals, Inc. Develop Neurobiol, 2012     

Catabolism of biphenyl by Pseudomonas sp. NCIB 10643 and Nocardia sp. NCIB 10503

Summary The metabolism of biphenyl by Pseudomonas sp. NCIB 10643 is reported in detail; that of Nocardia sp. NCIB 10503 is briefly investigated. Both organisms dissimilate biphenyl by the same route via oxidation to 2,3-dihydroxybiphenyl, meta cleavage to a product identified as 2-hydroxy-6-oxo-phenylhexa-2,4-dienoate which is then cleaved to give benzoate. Benzoate is a deadend metabolite in the pseudomonad but in the nocardia is further catabolised to catechol and thence to cis, cis-muconate. The enzymes involved in the individual steps of the proposed pathway have been assayed. The proposed pathway differs from that previously suggested for Pseudomonas sp. NCIB 10643 but is the same as found in other pseudomonads. This is the first report of catabolism of biphenyl in an actinomycete.     

Metabolism of C-Maltose in Avena fatua Seeds During Germination

Non-dormant and dormant seeds of Avena fatua metabolize ¹⁴C-maltose in different ways: in non-dormant seeds, ¹⁴C-maltose administered to the endosperm is readily converted to sucrose in the scutellum and translocated to the embryo; in dormant seeds, little sucrose is synthesized from ¹⁴C-maltose, and maltose and glucose tend to accumulate in the endosperm. It is suggested that biosynthesis of sucrose is essential for effective transport of the endosperm reserve to the embryonic axis in germinating seeds.     

Use of field observations to characterise genotypic flowering responses to photoperiod and temperature: a soyabean exemplar

Thirty-nine accessions of soyabean [Glycine max (L.) Merrill] and 1 of wild annual soyabean (Glycine soja L.) were sown at two sites in Taiwan in 1989 and 1990 and on six occasions during 1990 at one site in Queensland, Australia. On two of the occasions in Australia additional treatments extended natural daylengths by 0.5 h and 2 h. The number of days from sowing for the first flower to appear on 50% of the plants in each treatment was recorded (f), and from these values the rate of progress towards flowering (1/f) was related to temperature and photoperiod. In photoperiod-insensitive accessions it was confirmed that the rate is linearly related to temperature at least up to about 29°C. In photoperiod-sensitive genotypes this is also the case in shorter daylengths but when the critical photoperiod (P _c) is exceeded flowering is delayed. This delay increases with photoperiod until a ceiling photoperiod (P _ce) is reached. Between P _c and P _ce, 1/f is linearly related to both temperature (positive) and photoperiod (negative), but in photoperiods longer than P _ce there is no further response to either factor. The resulting triple-intersecting-plane response surface can be defined by six genetically-determined coefficients, the values of which are environment-independent but predict time to flower in any environment, and thus quantify the genotype x environment interaction. By this means the field data were used to characterise the photothermal responses of all 40 accessions. The outcome of this characterisation in conjunction with an analysis of the world-wide range of photothermal environments in which soyabean crops are grown lead to the following conclusions: (1) photoperiod-insensitivity is essential in soyabean crops in temperate latitudes, but such genotypes flower too rapidly for satisfactory yields in the tropics; (2) photoperiod-sensitivity appears to be essential to delay flowering sufficiently to allow adequate biomass accumulation in the warm climates of the tropics; (3) contrary to a widely held view, some degree of photoperiod-sensitivity is also needed in the tropics if crop-duration homeostasis is required where there is variation in sowing dates (this is achieved through a photoperiod-controlled delay in flowering which counteracts the seasonal increase in temperature that is correlated with increase in day-length); and (4) a greater degree of photoperiod-sensitivity is necessary to provide maturity-date homeostasis for variable sowing dates — a valuable attribute in regions of uncertain rainfall. Since the triple-intersecting-plane response model used here also applies to other species, the use of field data to characterise the photothermal responses of other crops is discussed briefly.     

EVOLUTIONARY TRENDS IN DION (ZAMIACEAE)

Distribution, ecology, and morphology of the species of Dion are presented. The genus Dion is distributed in Mexico and in Honduras, where it is generally scattered and restricted to rocky, steep habitats. On the basis mainly of the morphology of the seeds, of the seedlings, and of the female cones, three natural groups were recognized. The first group, which is the most distinct morphologically, is made up of D. mejiae endemic to Honduras. The second group is made up of D. spinulosum and D. rzedowskii, whose distribution is restricted to a very small area in Mexico. They appear to be the only Dion spp. to be adapted to moist and shaded habitats. The third group is made up of the remaining spp. of Dion, all Mexican in distribution. They appear to be well adapted to open and dry habitats generally. Stems, female cones, and seeds show the trends to decreasing size from south to north in this group. A key of the genus is given.     

On a formal definition of organization

A mathematic definition is proposed to account for the intuitive features of what is usually meant by organization. To account for both functional and structural aspects of organization the rate at which information content of a system changes in time is examined. It can be shown that Shannon's expression for ambiguity in a channel has two different meanings according to whether one is interested in the information transmitted in the channel or in the information transmitted to the observer from a whole system in which the channel is a part of a redundant communication network. This was applied in a previous work to show that the effects of noise on the information content H of a system result in two kinds of ambiguities, “autonomy-producing” and “destructive” leading to increase and decrease in H, respectively. By making use of this observation and Shannon's definition of redundancy R, a single equation for $\text{d}\text{H}\text{dt}$ is proposed to define organization on the basis of a kinetics of change of information content of a system under the effects of environmental noise-producing factors accumulated in time. It is shown how these factors, obviously responsible for a decrease in H, i.e. a “disorganizing” effect, can be responsible also—under certain conditions and up to a certain time or “dose” of noise—for an initial increase in H interpreted as a process of “self”-organization. The autonomy-producing ambiguity is expressed by a term of decrease in redundancy, while a second term in the equation, of decrease in maximum-non-redundant-information content expresses the destructive ambiguity. A given organization is defined at least by three parameters, which determine the main features of its characteristic function H(t). One of them is the initial information content H₀ and has a structural meaning. A second parameter, with a dimension of time, has the meaning of a functional reliability, related to the overall resistance of the system to noise-producing factors. The third parameter, namely the initial redundancy R₀, is both structural and functional in character, since structural redundancy is known to help insure reliability. Various conditions on these parameters lead to various kinds of organizations, with and without self-organizing properties.     

Zellwandregeneration und Peroxidase-Isoenzym-Synthese isolierter Protoplasten von Nicotiana tabacum L.

Summary Mesophyll-protoplasts of tobacco show increasing peroxidase-activity immediately after isolation. This is due to an enhancement of activity of the constitutive isoenzymes of G_III (=slow migrating cathodic group) and to a new formation of G_II-isoenzymes (=slow migrating anodic group). (G_II is not present in intact leaves.) As both processes are inhibited by actinomycin and actidion it is assumed that there is a new synthesis of peroxidase enzymes —The peroxidase reaction is independent of the further development of the protoplasts, as was evidenced by culturing protoplasts in different media which regulate the development of the protoplasts. Peroxidase reaction is always the same whether or not there is cell-wall synthesis and cell division. This leads to the conclusion that peroxidases in this case have no relation to synthesis of primary cell-walls. On the other hand they could be related to the dedifferentiation processes that always take place in isolated protoplasts.In the protoplasts G_II is localized in the cytoplasma as G_III is, because G_II appears before cell walls are synthesised and there is no lack of G_II isoenzymes when protoplasts are remazerated after having formed new cell walls.G_I (fast migrating anodic group), which is not detectable in isolated protoplasts, appears again after small calluses have developed out of protoplasts. Therefore as far as function is concerned G_I is quite different from G_II and G_III. The results confirm the hypothesis that G_I is localized in intercellular spaces only. It is discussed whether all of the isoenzymes of peroxidase detectable in crude extracts are cytoplasmic ones.