Influence of sequence and distance between two operators on interaction with the lac repressor

The influence of additional operator or pseudooperator sequences on the lactose repressor-operator interaction has been investigated. Results of kinetic and equilibrium binding measurements suggest an important in vivo role for the Z-gene pseudooperator in repressor-operator binding; the formation of a ternary, looped complex is indicated by the influence of secondary operator sites on binding parameters. Although the binding affinity of the Z-gene pseudooperator [Oz] is only approximately 1/30 that observed for the primary operator [O], the binding affinity to DNA containing both Oz and O is significantly higher than either sequence alone or the sum of the two. This synergistic effect is enhanced further by replacing the pseudooperator sequence [Oz] with the primary operator sequence and results in an even stronger ternary complex in plasmids with duplicate primary sites. The distance between the center of the two primary operators affects the formation of a ternary complex in the linear DNA molecules. Decreased dissociation rate constants were observed with spacing of operator-like sequences between 300 and 500 base pairs (bp). Minimal influence of the second operator on repressor binding is observed when the operators are separated by approximately 4000 or approximately 100 bp. The significant influence of distance on kinetic and equilibrium parameters was demonstrated by measurements on plasmid pRW1511 [Oi-O-PL-Oz] cleaved with restriction enzymes either in the polylinker region to place Oi-O and Oz on opposite ends of the linear plasmid or outside this region to maintain the sites within 500 bp. These results are consistent with the formation of operator-repressor-pseudooperator ternary complex to generate a looped DNA structure.     

Influence of supercoiling and sequence context on operator DNA binding with lac repressor

The dissociation of the repressor-operator complex from a series of negatively supercoiled plasmid DNAs was examined as a function of the sequence context, orientation, and spacing. The plasmids were grouped into four classes, each with common sequence context. The highest dissociation rate constants were observed for the plasmids containing only a single operator (or pseudooperator) sequence, while approximately 10-fold lower rate constants were measured for plasmids with the I gene pseudooperator in conjunction with either the Z gene pseudooperator or the primary operator. Comparison of the behavior of these two classes of plasmids demonstrated the importance of two operator sequences and supported a model of DNA loop formation to stabilize the repressor-operator complex (Whitson, P. A., and Matthews, K. S. (1986) Biochemistry 25, 3845-3852; Whitson, P. A., Olson, J. S., and Matthews, K. S. (1986) Biochemistry 25, 3852-3858; Whitson, P. A., Hsieh, W. T., Wells, R. D., and Matthews, K. S. (1987) J. Biol. Chem. 262, 4943-4946; Kr?mer, H., Niem?ller, M., Amouyal, M., Revet, B., von Wilcken-Bergmann, B., and Müller-Hill, B. (1987) EMBO J. 6, 1481-1491). The third class, with intermediate dissociation rate constants, was comprised of plasmids which contained the primary operator and the higher affinity pseudooperator normally located in the Z gene. Neither the additional presence of the I gene pseudooperator nor the orientation of the primary operator relative to the Z gene pseudooperator significantly affected the dissociation rate constants. The binding characteristics of this group of plasmids demonstrated the essential role of the Z gene pseudooperator in the formation of intramolecular ternary complex and suggested an in vivo function for this pseudooperator. Plasmids containing two primary operator sequences were the class with lowest dissociation rate constants from lac repressor, and minimal effects of salt or spacing on dissociation of this class were observed. These data are consistent with formation of an intramolecular complex with a looped DNA segment stabilized by the combination of increased local concentration of binding sites and torsional stresses on the DNA which favor binding in supercoiled DNA.     

Resolution of the fluorescence equilibrium unfolding profile of trp aporepressor using single tryptophan mutants.

Single tryptophan mutants of the trp aporepressor, tryptophan 19-->phenylalanine (W19F) and tryptophan 99-->phenylalanine (W99F), were used in this study to resolve the individual steady-state and time-resolved fluorescence urea unfolding profiles of the two tryptophan residues in this highly intertwined, dimeric protein. The wild-type protein exhibits a large increase in fluorescence intensity and lifetime, as well as a large red shift in the steady-state fluorescence emission spectrum, upon unfolding by urea (Lane, A.N. & Jardetsky, O., 1987, Eur. J. Biochem. 164, 389-396; Gittelman, M.S. & Matthews, C.R., 1990, Biochemistry 29, 7011-7020; Fernando, T. & Royer, C.A., 1992, Biochemistry 31, 6683-6691). Unfolding of the W19F mutant demonstrated that Trp 99 undergoes a large increase in intensity and a red shift upon exposure to solvent. Lifetime studies revealed that the contribution of the dominant 0.5-ns component of this tryptophan tends toward zero with increasing urea, whereas the longer lifetime components increase in importance. This lifting of the quenching of Trp 99 may be due to disruption of the interaction between the two subunits upon denaturation, which abolishes the interaction of Trp 99 on one subunit with the amide quenching group of Asn 32 on the other subunit (Royer, C.A., 1992, Biophys. J. 63, 741-750). On the other hand, Trp 19 is quenched in response to unfolding in the W99F mutant. Exposure to solvent of Trp 19, which is buried at the hydrophobic dimer interface in the native protein, results in a large red shift of the average steady-state emission.(ABSTRACT TRUNCATED AT 250 WORDS)     

Fitting the HIV Epidemic in Zambia: A Two-Sex Micro-Simulation Model

Background

In describing and understanding how the HIV epidemic spreads in African countries, previous studies have not taken into account the detailed periods at risk. This study is based on a micro-simulation model (individual-based) of the spread of the HIV epidemic in the population of Zambia, where women tend to marry early and where divorces are not frequent. The main target of the model was to fit the HIV seroprevalence profiles by age and sex observed at the Demographic and Health Survey conducted in 2001.

Methods and Findings

A two-sex micro-simulation model of HIV transmission was developed. Particular attention was paid to precise age-specific estimates of exposure to risk through the modelling of the formation and dissolution of relationships: marriage (stable union), casual partnership, and commercial sex. HIV transmission was exclusively heterosexual for adults or vertical (mother-to-child) for children. Three stages of HIV infection were taken into account. All parameters were derived from empirical population-based data. Results show that basic parameters could not explain the dynamics of the HIV epidemic in Zambia. In order to fit the age and sex patterns, several assumptions were made: differential susceptibility of young women to HIV infection, differential susceptibility or larger number of encounters for male clients of commercial sex workers, and higher transmission rate. The model allowed to quantify the role of each type of relationship in HIV transmission, the proportion of infections occurring at each stage of disease progression, and the net reproduction rate of the epidemic (R ₀ = 1.95).

Conclusions

The simulation model reproduced the dynamics of the HIV epidemic in Zambia, and fitted the age and sex pattern of HIV seroprevalence in 2001. The same model could be used to measure the effect of changing behaviour in the future.     

The kinetics of reactions around the cytochrome bf complex studied in an isolated system

The kinetics of oxidation and reduction of P700, plastocyanin, cytochrome f and cytochrome b-563 were studied in a reconstituted system consisting of Photosystem I particles, cytochrome bf complex and plastocyanin, all derived from pea leaf chloroplasts. Decyl plastoquinol was the reductant of the bf complex. Turnovers of the system were initiated by laser flashes. The reaction between oxidised P700 and plastocyanin was non-homogeneous in that a second-order rate coefficient of c. 5×10^–7 M^–1 s^–1 applied to 80% of the P700⁺ and c. 0.7×10⁷ M^–1 s^–1 to the remainder. In the presence of bf complex, but without quinol, the electron transfer between cytochrome f and oxidised plastocyanin could be described by a second-order rate coefficient of c. 4×10⁷ M^–1 s^–1 (forward), and c. 1.6×10⁷ M^–1 s^–1 (reverse). The equilibrium coefficient was thus 2.5. Unexpectedly, there was little reduction of cytochrome f ⁺ or plastocyanin⁺ by electrons from the Rieske centre. With added quinol, reduction of cytochrome b-563 occurred. Concomitantly, electrons appeared in the oxidised species. It was inferred that either the Rieske centre was not involved in the high-potential chain of electron transfer events, or that, only in the presence of quinol, electrons were quickly passed from the Rieske centre to cytochrome f ⁺. Additionally, the presence of quinol altered the equilibrium coefficient for the cyt f/PC interaction from 2.5 to c. 5. The reaction between quinol and the bf complex was describable by a second-order rate coefficient of about 3×10⁶ M^–1 s^–1. The pattern of the redox reactions around the bf complex could be simulated in detail with a Q-cycle model as previously found for chloroplasts.Abbreviations AQS anthraquinone sulphonate - cyt cytochrome - cyt b-563(H) high-potential cyt b-563 - cyt b-563(L) low potential cyt b-563 - FeS(R) the Rieske protein of the cyt bf complex, containing an Fe₂S₂ centre - PC plastocyanin - PS photosystem - P700 reaction centre in PS I     

Development of an expressed sequence tag (EST) resource for wheat (Triticum aestivum L.): EST generation, unigene analysis, probe selection and bioinformatics for a 16,000-locus bin-delineated map

This report describes the rationale, approaches, organization, and resource development leading to a large-scale deletion bin map of the hexaploid (2n = 6x = 42) wheat genome (Triticum aestivum L.). Accompanying reports in this issue detail results from chromosome bin-mapping of expressed sequence tags (ESTs) representing genes onto the seven homoeologous chromosome groups and a global analysis of the entire mapped wheat EST data set. Among the resources developed were the first extensive public wheat EST collection (113,220 ESTs). Described are protocols for sequencing, sequence processing, EST nomenclature, and the assembly of ESTs into contigs. These contigs plus singletons (unassembled ESTs) were used for selection of distinct sequence motif unigenes. Selected ESTs were rearrayed, validated by 5′ and 3′ sequencing, and amplified for probing a series of wheat aneuploid and deletion stocks. Images and data for all Southern hybridizations were deposited in databases and were used by the coordinators for each of the seven homoeologous chromosome groups to validate the mapping results. Results from this project have established the foundation for future developments in wheat genomics.     

Rate of succession in restored wetlands and the role of site context

Question: Are changes in plant species composition, functional group composition and rates of species turnover consistent among early successional wetlands, and what is the role of landscape context in determining the rate of succession? Location: Twenty‐four restored wetlands in Illinois, USA. Methods: We use 4 years of vegetation sampling data from each site to describe successional trends and rates of species turnover in wetlands. We quantify: (1) the rate at which composition changes from early‐successional to late‐successional species and functional groups, as indicated by site movement in ordination space over time, and (2) the rate of change in the colonization and local extinction of individual species. We correlate the pace of succession to site area, isolation and surrounding land cover. Results: Some commonalities in successional trends were evident among sites. Annual species were replaced by clonal perennials, and colonization rates declined over time. However, differences among sites outweighed site age in determining species composition, and the pace of succession was influenced by a site's landscape setting. Rates of species turnover were higher in smaller wetlands. In addition, wetlands in agricultural landscapes underwent succession more rapidly, as indicated by a rapid increase in dominance by late‐successional plants. Conclusions: Although the outcome of plant community succession in restored wetlands was somewhat predictable, species composition and the pace of succession varied among sites. The ability of restoration practitioners to accelerate succession through active manipulation may be contingent upon landscape context.     

Naturally Occurring Triggers that Induce Apoptosis-Like Programmed Cell Death in Plasmodium berghei Ookinetes

Several protozoan parasites have been shown to undergo a form of programmed cell death that exhibits morphological features associated with metazoan apoptosis. These include the rodent malaria parasite, Plasmodium berghei. Malaria zygotes develop in the mosquito midgut lumen, forming motile ookinetes. Up to 50% of these exhibit phenotypic markers of apoptosis; as do those grown in culture. We hypothesised that naturally occurring signals induce many ookinetes to undergo apoptosis before midgut traversal. To determine whether nitric oxide and reactive oxygen species act as such triggers, ookinetes were cultured with donors of these molecules. Exposure to the nitric oxide donor SNP induced a significant increase in ookinetes with condensed nuclear chromatin, activated caspase-like molecules and translocation of phosphatidylserine that was dose and time related. Results from an assay that detects the potential-dependent accumulation of aggregates of JC-1 in mitochondria suggested that nitric oxide does not operate via loss of mitochondrial membrane potential. L-DOPA (reactive oxygen species donor) also caused apoptosis in a dose and time dependent manner. Removal of white blood cells significantly decreased ookinetes exhibiting a marker of apoptosis in vitro. Inhibition of the activity of nitric oxide synthase in the mosquito midgut epithelium using L-NAME significantly decreased the proportion of apoptotic ookinetes and increased the number of oocysts that developed. Introduction of a nitric oxide donor into the blood meal had no effect on mosquito longevity but did reduce prevalence and intensity of infection. Thus, nitric oxide and reactive oxygen species are triggers of apoptosis in Plasmodium ookinetes. They occur naturally in the mosquito midgut lumen, sourced from infected blood and mosquito tissue. Up regulation of mosquito nitric oxide synthase activity has potential as a transmission blocking strategy.     

Spectral analysis of the DNA targeting bisalkylaminoanthraquinone DRAQ5 in intact living cells.

BACKGROUND: We report on the potential DNA binding modes and spectral characteristics of the cell-permeant far red fluorescent DNA dye, DRAQ5, in solution and bound within intact cells. Our aim was to determine the constraints for its use in flow cytometry and bioimaging. METHODS: Solution characteristics and quantum yields were determined by spectroscopy. DRAQ5 binding to nuclear DNA was analyzed using fluorescence quenching of Hoechst 33342 dye, emission profiling by flow cytometry, and spectral confocal laser scanning microscopy of the complex DRAQ5 emission spectrum. Cell cycle profiling utilized an EGFP-cyclin B1 reporter as an independent marker of cell age. Molecular modeling was used to explore the modes of DNA binding. RESULTS: DRAQ5 showed a low quantum yield in solution and a spectral shift upon DNA binding, but no significant fluorescence enhancement. DRAQ5 caused a reduction in the fluorescence intensity of Hoechst 33342 in live cells prelabeled with the UV excitable dye, consistent with molecular modeling that suggests AT preference and an engagement of the minor groove. In vivo spectral analysis of DRAQ5 demonstrated shifts to longer wavelengths upon binding with DNA. Analysis of spectral windows of the dual emission peaks at 681 and 707 nm in cells showed that cell cycle compartment recognition was independent of the far red-near IR emission wavelengths monitored. CONCLUSIONS: The study provides new clues to modes of DNA binding of the modified anthraquinone molecule in vivo, and its AT base-pair selectivity. The combination of low quantum yield but high DNA affinity explains the favorable signal-to-noise profile of DRAQ5-nuclear fluorescence. The robust nature of cell cycle reporting using DRAQ5, even when restricted spectral windows are selected, facilitates the analysis of encroaching spectral emissions from other fluorescent reporters, including GFP-tagged proteins.     

Intracellular heteroplasmy for disease-associated point mutations in mtDNA: implications for disease expression and evidence for mitotic segregation of heteroplasmic units of mtDNA

Studies in vitro have shown that a respiratorydeficient phenotype is expressed by cells when the proportion of mtDNA with a disease-associated mutation exceeds a threshold level, but analysis of tissues from patients with mitochondrial encephalomyopathy, lactic acidosis, and strokelike episodes (MELAS) have failed to show a consistent relationship between the degree of heteroplasmy and biochemical expression of the defect. One possible explanation for this phenomenon is that there is variation of heteroplasmy between individual cells that is not adequately reflected by the mean heteroplasmy for a tissue. We have confirmed this by study of fibroblast clones from subjects heteroplasmic for the MELAS 3243 (A G) mtDNA mutation. Similar observations were made with fibroblast clones derived from two subjects heteroplasmic for the 11778 (GA) mtDNA mutation of Leber's hereditary optic neuropathy. For the MELAS 3243 mutation, the distribution of mutant mtDNA between different cells was not randomly distributed about the mean, suggesting that selection against cells with high proportions of mutant mtDNA had occurred. To explore the way in which heteroplasmic mtDNA segregates in mitosis we followed the distribution of heteroplasmy between clones over approximately 15 generations. There was either no change or a decrease in the variance of intercellular heteroplasmy for the MELAS 3243 mutation, which is most consistent with segregation of heteroplasmic units of multiple mtDNA molecules in mitosis. After mitochondria from one of the MELAS 3243 fibroblast cultures were transferred to a mitochondrial DNA-free (⁰) cell line derived from osteosarcoma cells by cytoplast fusion, the mean level and intercellular distribution of heteroplasmy was unchanged. We interpret this as evidence that somatic segregation (rather than nuclear background or cell differentiation state) is the primary determinant of the level of heteroplasmy.