Molecular drift of the bride of sevenless (boss) gene in Drosophila

DNA sequences were determined for three to five alleles of the bride-of- sevenless (boss) gene in each of four species of Drosophila. The product of boss is a transmembrane receptor for a ligand coded by the sevenless gene that triggers differentiation of the R7 photoreceptor cell in the compound eye. Population parameters affecting the rate and pattern of molecular evolution of boss were estimated from the multinomial configurations of nucleotide polymorphisms of synonymous codons. The time of divergence between D. melanogaster and D. simulans was estimated as approximately 1 Myr, that between D. teissieri and D. yakuba as approximately 0.75 Myr, and that between the two pairs of sibling species as approximately 2 Myr. (The boss genes themselves have estimated divergence times approximately 50% greater than the species divergence times.) The effective size of the species was estimated as approximately 5 x 10(6), and the average mutation rate was estimated as 1-2 x 10(-9)/nucleotide/generation. The ratio of amino acid polymorphisms within species to fixed differences between species suggests that approximately 25% of all possible single-step amino acid replacements in the boss gene product may be selectively neutral or nearly neutral. The data also imply that random genetic drift has been responsible for virtually all of the observed differences in the portion of the boss gene analyzed among the four species.      

The contractile basis of amoeboid movement: V. The control of gelation, solation, and contraction in extracts from dictyostelium discoideum

Motile extracts have been prepared from Dictyostelium discoideum by homogenization and differential centrifugation at 4 degrees C in a stabilization solution (60). These extracts gelled on warming to 25 degrees Celsius and contracted in response to micromolar Ca++ or a pH in excess of 7.0. Optimal gelation occurred in a solution containing 2.5 mM ethylene glycol-bis (β-aminoethyl ether)N,N,N',N'-tetraacetate (EGTA), 2.5 mM piperazine-N-N'-bis [2-ethane sulfonic acid] (PIPES), 1 mM MgC1(2), 1 mM ATP, and 20 mM KCI at ph 7.0 (relaxation solution), while micromolar levels of Ca++ inhibited gelation. Conditions that solated the gel elicited contraction of extracts containing myosin. This was true regardless of whether chemical (micromolar Ca++, pH >7.0, cytochalasin B, elevated concentrations of KCI, MgC1(2), and sucrose) or physical (pressure, mechanical stress, and cold) means were used to induce solation. Myosin was definitely required for contraction. During Ca++-or pH-elicited contraction: (a) actin, myosin, and a 95,000-dalton polypeptide were concentrated in the contracted extract; (b) the gelation activity was recovered in the material sqeezed out the contracting extract;(c) electron microscopy demonstrated that the number of free, recognizable F-actin filaments increased; (d) the actomyosin MgATPase activity was stimulated by 4- to 10-fold. In the absense of myosin the Dictyostelium extract did not contract, while gelation proceeded normally. During solation of the gel in the absense of myosin: (a) electron microscopy demonstrated that the number of free, recognizable F- actin filaments increased; (b) solation-dependent contraction of the extract and the Ca++-stimulated MgATPase activity were reconstituted by adding puried Dictyostelium myosin. Actin purified from the Dictyostelium extract did not gel (at 2 mg/ml), while low concentrations of actin (0.7-2 mg/ml) that contained several contaminating components underwent rapid Ca++ regulated gelation. These results indicated : (a) gelation in Dictyostelium extracts involves a specific Ca++-sensitive interaction between actin and several other components; (b) myosin is an absolute requirement for contraction of the extract; (c) actin-myosin interactions capable of producing force for movement are prevented in the gel, while solation of the gel by either physical or chemical means results in the release of F-actin capable of interaction with myosin and subsequent contraction. The effectiveness of physical agents in producting contraction suggests that the regulation of contraction by the gel is structural in nature.     

Saikosaponin-d,a novel SERCA inhibitor,induces autophagic cell death in apoptosis-defective cells

Autophagy is an important cellular process that controls cells in a normal homeostatic state by recycling nutrients to maintain cellular energy levels for cell survival via the turnover of proteins and damaged organelles. However, persistent activation of autophagy can lead to excessive depletion of cellular organelles and essential proteins, leading to caspase-independent autophagic cell death. As such, inducing cell death through this autophagic mechanism could be an alternative approach to the treatment of cancers. Recently, we have identified a novel autophagic inducer, saikosaponin-d (Ssd), from a medicinal plant that induces autophagy in various types of cancer cells through the formation of autophagosomes as measured by GFP-LC3 puncta formation. By computational virtual docking analysis, biochemical assays and advanced live-cell imaging techniques, Ssd was shown to increase cytosolic calcium level via direct inhibition of sarcoplasmic/endoplasmic reticulum Ca²⁺ ATPase pump, leading to autophagy induction through the activation of the Ca²⁺/calmodulin-dependent kinase kinase–AMP-activated protein kinase–mammalian target of rapamycin pathway. In addition, Ssd treatment causes the disruption of calcium homeostasis, which induces endoplasmic reticulum stress as well as the unfolded protein responses pathway. Ssd also proved to be a potent cytotoxic agent in apoptosis-defective or apoptosis-resistant mouse embryonic fibroblast cells, which either lack caspases 3, 7 or 8 or had the Bax-Bak double knockout. These results provide a detailed understanding of the mechanism of action of Ssd, as a novel autophagic inducer, which has the potential of being developed into an anti-cancer agent for targeting apoptosis-resistant cancer cells.     

A new topological method to measure protein structure similarity

A method for the quantitative evaluation of structural similarity between protein pairs is developed that makes use of a Delaunay-based topological mapping. The result of the mapping is a three-dimensional array which is representative of the global structural topology and whose elements can be used to construe an integral scoring scheme. This scoring scheme was tested for its dependence on the protein length difference in a pairwise comparison, its ability to provide a reasonable means for structural similarity comparison within a family of structural neighbors of similar length, and its sensitivity to the differences in protein conformation. It is shown that such a topological evaluation of similarity is capable of providing insight into these points of interest. Protein structure comparison using the method is computationally efficient and the topological scores, although providing different information about protein similarity, correlate well with the distance root-mean-square deviation values calculated by rigid-body structural alignment.     

Evidence for microbial Fe(III) reduction in anoxic, mining-impacted lake sediments (Lake Coeur d'Alene, Idaho)

Mining-impacted sediments of Lake Coeur d'Alene, Idaho, contain more than 10% metals on a dry weight basis, approximately 80% of which is iron. Since iron (hydr)oxides adsorb toxic, ore-associated elements, such as arsenic, iron (hydr)oxide reduction may in part control the mobility and bioavailability of these elements. Geochemical and microbiological data were collected to examine the ecological role of dissimilatory Fe(III)-reducing bacteria in this habitat. The concentration of mild-acid-extractable Fe(II) increased with sediment depth up to 50 g kg(-1), suggesting that iron reduction has occurred recently. The maximum concentrations of dissolved Fe(II) in interstitial water (41 mg liter(-1)) occurred 10 to 15 cm beneath the sediment-water interface, suggesting that sulfidogenesis may not be the predominant terminal electron-accepting process in this environment and that dissolved Fe(II) arises from biological reductive dissolution of iron (hydr)oxides. The concentration of sedimentary magnetite (Fe(3)O(4)), a common product of bacterial Fe(III) hydroxide reduction, was as much as 15.5 g kg(-1). Most-probable-number enrichment cultures revealed that the mean density of Fe(III)-reducing bacteria was 8.3 x 10(5) cells g (dry weight) of sediment(-1). Two new strains of dissimilatory Fe(III)-reducing bacteria were isolated from surface sediments. Collectively, the results of this study support the hypothesis that dissimilatory reduction of iron has been and continues to be an important biogeochemical process in the environment examined.     

High rate of DNA loss in the Drosophila melanogaster and Drosophila virilis species groups

We recently proposed that patterns of evolution of non-LTR retrotransposable elements can be used to study patterns of spontaneous mutation. Transposition of non-LTR retrotransposable elements commonly results in creation of 5' truncated, "dead-on-arrival" copies. These inactive copies are effectively pseudogenes and, according to the neutral theory, their molecular evolution ought to reflect rates and patterns of spontaneous mutation. Maximum parsimony can be used to separate the evolution of active lineages of a non-LTR element from the fate of the "dead-on-arrival" insertions and to directly assess the relative frequencies of different types of spontaneous mutations. We applied this approach using a non-LTR element, Helena, in the Drosophila virilis group and have demonstrated a surprisingly high incidence of large deletions and the virtual absence of insertions. Based on these results, we suggested that Drosophila in general may exhibit a high rate of spontaneous large deletions and have hypothesized that such a high rate of DNA loss may help to explain the puzzling dearth of bona fide pseudogenes in Drosophila. We also speculated that variation in the rate of spontaneous deletion may contribute to the divergence of genome size in different taxa by affecting the amount of superfluous "junk" DNA such as, for example, pseudogenes or long introns. In this paper, we extend our analysis to the D. melanogaster subgroup, which last shared a common ancestor with the D. virilis group approximately 40 MYA. In a different region of the same transposable element, Helena, we demonstrate that inactive copies accumulate deletions in species of the D. melanogaster subgroup at a rate very similar to that of the D. virilis group. These results strongly suggest that the high rate of DNA loss is a general feature of Drosophila and not a peculiar property of a particular stretch of DNA in a particular species group.      

Arabidopsis has two redundant Cullin3 proteins that are essential for embryo development and that interact with RBX1 and BTB proteins to form multisubunit E3 ubiquitin ligase complexes in vivo

Cullin-based E3 ubiquitin ligases play important roles in the regulation of diverse developmental processes and environmental responses in eukaryotic organisms. Recently, it was shown in Schizosaccharomyces pombe, Caenorhabditis elegans, and mammals that Cullin3 (CUL3) directly associates with RBX1 and BTB domain proteins in vivo to form a new family of E3 ligases, with the BTB protein subunit functioning in substrate recognition. Here, we demonstrate that Arabidopsis thaliana has two redundant CUL3 (AtCUL3) genes that are essential for embryo development. Besides supporting anticipated specific AtCUL3 interactions with the RING protein AtRBX1 and representative Arabidopsis proteins containing a BTB domain in vitro, we show that AtCUL3 cofractionates and specifically associates with AtRBX1 and a representative BTB protein in vivo. Similar to the AtCUL1 subunit of the SKP1-CUL1-F-box protein-type E3 ligases, the AtCUL3 subunit of the BTB-containing E3 ligase complexes is subjected to modification and possible regulation by the ubiquitin-like protein Related to Ubiquitin in vivo. Together with the presence of large numbers of BTB proteins with diverse structural features and expression patterns, our data suggest that Arabidopsis has conserved AtCUL3-RBX1-BTB protein E3 ubiquitin ligases to target diverse protein substrates for degradation by the ubiquitin/proteasome pathway.