Organization of repetitive DNAs and the genomic regions carrying ribosomal RNA,<Emphasis Type="Italic"> cob</Emphasis>, and<Emphasis Type="Italic"> atp9</Emphasis> genes in the cucurbit mitochondrial genomes

Plants in the genus Cucumis (cucumber and melon) have the largest mitochondrial genomes known among all plants, due in part to the accumulation of repetitive DNAs of varying complexities. Recombination among these repetitive DNAs should produce highly rearranged mitochondrial genomes relative to the smaller mitochondrial genomes of related plants. We cloned and sequenced mitochondrial genomic regions near the rRNA, atp9 and cob genes from cucumber, melon, squash and watermelon (all members of the Cucurbitaceae family), and compared to the previously sequenced mitochondrial genomes of Arabidopsis thaliana and sugar beet to study the distribution and arrangement of coding and repetitive DNAs. Cucumber and melon had regions of concentrated repetitive DNAs spread throughout the sequenced regions; few repetitive DNAs were revealed in the mitochondrial genomes of A. thaliana, sugar beet, squash and watermelon. Recombination among these repetitive DNAs most likely produced unique arrangements of the rrn18 and rrn5 genes in the genus Cucumis. Cucumber mitochondrial DNA had more pockets of dispersed direct and inverted repeats than melon and the other plants, and we did not reveal repetitive sequences significantly contributing to mitochondrial genome expansion in both cucumber and melon.Disclaimer. Names are necessary to report factually on available data; however, the U.S. Department of Agriculture (USDA) neither guarantees nor warrants the standard of the product, and the use of the name by USDA implies no approval of the product to the exclusion of others that may also be suitable.Communicated by R. Hagemann     

Identification of single tiny seeds ofOrobanche using RAPD analysis

The tiny seeds of parasitic weeds of the genusOrobanche can be identified by using RAPD markers. A simple procedure for DNA extraction from single seeds, 10 μg each, followed by RAPD-PCR and using specific DNA markers, leads to species identification. Seeds of five different species could be identified using this method.     

HT-29 human colon cancer cell proliferation is regulated by cytosolic phospholipase A2α dependent PGE2via both PKA and PKB pathways

Cytosolic phospholipase A₂α (cPLA₂α) up-regulation has been reported in human colorectal cancer cells, thus we aimed to elucidate its role in the proliferation of the human colorectal cancer cell line, HT-29. EGF caused a rapid activation of cPLA₂α which coincided with a significant increase in cell proliferation. The inhibition of cPLA₂α activity by pyrrophenone or by antisense oligonucleotide against cPLA₂α (AS) or inhibition of prostaglandin E₂ (PGE₂) production by indomethacin resulted with inhibition of cell proliferation, that was restored by addition of PGE₂. The secreted PGE₂ activated both protein kinase A (PKA) and PKB/Akt pathways via the EP2 and EP4 receptors. Either, the PKA inhibitor (H-89) or the PKB/Akt inhibitor (Ly294002) caused a partial inhibition of cell proliferation which was restored by PGE₂. But, inhibited proliferation in the presence of both inhibitors could not be restored by addition of PGE₂. AS or H-89, but not Ly294002, inhibited CREB activation, suggesting that CREB activation is mediated by PKA. AS or Ly294002, but not H-89, decreased PKB/Akt activation as well as the nuclear localization of β-catenin and cyclin D1 and increased the plasma membrane localization of β-catenin with E-cadherin, suggesting that these processes are regulated by the PKB pathway. Similarly, Caco-2 cells exhibited cPLA₂α dependent proliferation via activation of both PKA and PKB/Akt pathways. In conclusion, our findings suggest that the regulation of HT-29 proliferation is mediated by cPLA₂α-dependent PGE₂ production. PGE₂via EP induces CREB phosphorylation by the PKA pathway and regulates β-catenin and cyclin D1 cellular localization by PKB/Akt pathway.     

Base-CP proteasome can serve as a platform for stepwise lid formation

26S proteasome, a major regulatory protease in eukaryotes, consists of a 20S proteolytic core particle (CP) capped by a 19S regulatory particle (RP). The 19S RP is divisible into base and lid sub-complexes. Even within the lid, subunits have been demarcated into two modules: module 1 (Rpn5, Rpn6, Rpn8, Rpn9 and Rpn11), which interacts with both CP and base sub-complexes and module 2 (Rpn3, Rpn7, Rpn12 and Rpn15) that is attached mainly to module 1. We now show that suppression of RPN11 expression halted lid assembly yet enabled the base and 20S CP to pre-assemble and form a base-CP. A key role for Regulatory particle non-ATPase 11 (Rpn11) in bridging lid module 1 and module 2 subunits together is inferred from observing defective proteasomes in rpn11–m1, a mutant expressing a truncated form of Rpn11 and displaying mitochondrial phenotypes. An incomplete lid made up of five module 1 subunits attached to base-CP was identified in proteasomes isolated from this mutant. Re-introducing the C-terminal portion of Rpn11 enabled recruitment of missing module 2 subunits. In vitro, module 1 was reconstituted stepwise, initiated by Rpn11–Rpn8 heterodimerization. Upon recruitment of Rpn6, the module 1 intermediate was competent to lock into base-CP and reconstitute an incomplete 26S proteasome. Thus, base-CP can serve as a platform for gradual incorporation of lid, along a proteasome assembly pathway. Identification of proteasome intermediates and reconstitution of minimal functional units should clarify aspects of the inner workings of this machine and how multiple catalytic processes are synchronized within the 26S proteasome holoenzymes.     

Comparison of coupling of humans to electric and magnetic fields with frequencies between 100 Hz and 100 kHz

Recent laboratory and epidemiological results have stimulated interest in the hypothesis that human beings may exhibit biological responses to magnetic and/or electric field transients with frequencies in the range between 100 Hz and 100 kHz. Much can be learned about the response of a system to a transient stimulation by understanding its response to sinusoidal disturbances over the entire frequency range of interest. Thus, the main effort of this paper was to compare the strengths of the electric fields induced in homogeneous ellipsoidal models by uniform 100 Hz through 100 kHz electric and magnetic fields. Over this frequency range, external electric fields of about 25–2000 V/m (depending primarily on the orientation of the body relative to the field) are required to induce electric fields inside models of adults and children that are similar in strength to those induced by an external 1 μT magnetic field. Additional analysis indicates that electric fields induced by uniform external electric and magnetic fields and by the nonuniform electric and magnetic fields produced by idealized point sources will not differ by more than a factor of two until the sources are brought close to the body. Published data on electric and magnetic field transients in residential environments indicate that, for most field orientations, the magnetic component will induce stronger electric fields inside adults and children than the electric component. This conclusion is also true for the currents induced in humans by typical levels of 60 Hz electric and magnetic fields in U.S. residences. Bioelectromagnetics 18:67–76, 1997. © 1997 Wiley-Liss, Inc.     

Identification and Localization of a Rickettsia sp. in Bemisia tabaci (Homoptera: Aleyrodidae)

Whiteflies (Homoptera: Aleyrodidae) are sap-sucking insects that harbor “Candidatus Portiera aleyrodidarum,” an obligatory symbiotic bacterium which is housed in a special organ called the bacteriome. These insects are also home for a diverse facultative microbial community which may include Hamiltonella, Arsenophonus, Fritchea, Wolbachia, and Cardinium spp. In this study, the bacteria associated with a B biotype of the sweet potato whitefly Bemisia tabaci were characterized using molecular fingerprinting techniques, and a Rickettsia sp. was detected for the first time in this insect family. Rickettsia sp. distribution, transmission and localization were studied using PCR and fluorescence in situ hybridizations (FISH). Rickettsia was found in all 20 Israeli B. tabaci populations screened but not in all individuals within each population. A FISH analysis of B. tabaci eggs, nymphs, and adults revealed a unique concentration of Rickettsia around the gut and follicle cells, as well as a random distribution in the hemolymph. We postulate that the Rickettsia enters the oocyte together with the bacteriocytes, leaves these symbiont-housing cells when the egg is laid, multiplies and spreads throughout the egg during embryogenesis and, subsequently, disperses throughout the body of the hatching nymph, excluding the bacteriomes. Although the role Rickettsia plays in the biology of the whitefly is currently unknown, the vertical transmission on the one hand and the partial within-population infection on the other suggest a phenotype that is advantageous under certain conditions but may be deleterious enough to prevent fixation under others.     

Effect of norleucine on growth,protein and catechol oxidase synthesis in tobacco suspension cultures

Tobacco cells were grown in artificial media with defined amino acid composition. In such media, the addition of methionine or norleucine caused increases in the specific activity of the catechol oxidase, while in the normal medium norleucine depressed it. The differences of the effect of norleucine on synthesis of catechol oxidase and on cell growth is demonstrated, as is the reversibility of the norleucine effect by methionine. The incorporation of norleucine into a purified enzyme fraction is shown. The change in the electrophoretic patterns of the enzyme during growth in the absence and presence of norleucine was followed. [¹⁴C]-Leucine incorporation by control and norleucine treated cells was examined and it was shown that protein synthesis in the norleucine treated cells was markedly changed and total incorporation reduced. Incorporation into soluble protein was reduced, but increased in the 20 000 g precipitate fraction. Nevertheless use of autoradiography indicates that some catechol oxidase is apparently synthesised in the presence of norleucine.     

Combining bulk segregation analysis and microarrays for mapping of the pH trait in melon

The availability of sequence information for many plants has opened the way to advanced genetic analysis in many non-model plants. Nevertheless, exploration of genetic variation on a large scale and its use as a tool for the identification of traits of interest are still rare. In this study, we combined a bulk segregation approach with our own-designed microarrays to map the pH locus that influences fruit pH in melon. Using these technologies, we identified a set of markers that are genetically linked to the pH trait. Further analysis using a set of melon cultivars demonstrated that some of these markers are tightly linked to the pH trait throughout our germplasm collection. These results validate the utility of combining microarray technology with a bulk segregation approach in mapping traits of interest in non-model plants.     

A conservation and biophysics guided stochastic approach to refining docked multimeric proteins

Background

We introduce a protein docking refinement method that accepts complexes consisting of any number of monomeric units. The method uses a scoring function based on a tight coupling between evolutionary conservation, geometry and physico-chemical interactions. Understanding the role of protein complexes in the basic biology of organisms heavily relies on the detection of protein complexes and their structures. Different computational docking methods are developed for this purpose, however, these methods are often not accurate and their results need to be further refined to improve the geometry and the energy of the resulting complexes. Also, despite the fact that complexes in nature often have more than two monomers, most docking methods focus on dimers since the computational complexity increases exponentially due to the addition of monomeric units.

Results

Our results show that the refinement scheme can efficiently handle complexes with more than two monomers by biasing the results towards complexes with native interactions, filtering out false positive results. Our refined complexes have better IRMSDs with respect to the known complexes and lower energies than those initial docked structures.

Conclusions

Evolutionary conservation information allows us to bias our results towards possible functional interfaces, and the probabilistic selection scheme helps us to escape local energy minima. We aim to incorporate our refinement method in a larger framework which also enables docking of multimeric complexes given only monomeric structures.

     

Host-pathogen interplay and the evolution of bacterial effectors

Many bacterial pathogens require a type III secretion system (T3SS) and suite of type III secreted effectors (T3SEs) to successfully colonize their hosts, extract nutrients and consequently cause disease. T3SEs, in particular, are key components of the bacterial arsenal, as they function directly inside the host to disrupt or suppress critical components of the defence network. The development of host defence and surveillance systems imposes intense selective pressures on these bacterial virulence factors, resulting in a host–pathogen co-evolutionary arms race. This arms race leaves its genetic signature in the pattern and structure of natural genetic variation found in T3SEs, thereby permitting us to infer the specific evolutionary processes and pressures driving these interactions. In this review, we summarize our current knowledge of T3SS-mediated host–pathogen co-evolution. We examine the evolution of the T3SS and the T3SEs that traverse it, in both plant and animal pathosystems, and discuss the processes that maintain these important pathogenicity determinants within pathogen populations. We go on to examine the possible origins of T3SEs, the mechanisms that give rise to new T3SEs and the processes that underlie their evolution.