Purification of a plant cell wall fibronectin-like adhesion protein involved in plant response to salt stress

The structural role of extracellular-matrix (ECM) has been recognized in both plants and animals as a support and anchorage-inducing cell behavior. Unlike the animal ECM proteins, the proteins that have been identified in plant ECM have not yet been purified from whole plants and cell wall. As several immunological data indicate the presence of animal ECM-like proteins in plants cell wall, especially under salt stress or water deficit, we propose a protocol to purify a fibronectin-like protein from the cell wall of epicotyls of young germinating peas. The process consists of a combination of gelatin and heparin affinity chromatography, close to the classical one used for human blood plasma fibronectin purification. Proteins with affinity for gelatin and heparin, immunologically related to human fibronectin, are found in the cell wall of epicotyls grown under salt stress or not. Total amount of purified proteins is 3-4 times more enriched in salt stressed epicotyls. SDS-PAGE and Western blot with antibodies directed against human blood plasma fibronectin give evidence that the cell wall proteins purified by gelatin/heparin affinity chromatography are closely related to human fibronectin. The present protocol leads us to purify 17 (control) or 65 (salt stress) micrograms of protein per g of fresh starting material. Our results suggest that plant cell wall proteins can provide better anchorage of the cell to its cell-wall during salt stress or water deficit and could be considered not only as cell adhesion but also as signaling molecules.     

Why do good hunters have higher reproductive success?

Anecdotal evidence from many hunter-gatherer societies suggests that successful hunters experience higher prestige and greater reproductive success. Detailed quantitative data on these patterns are now available for five widely dispersed cases (Ache, Hadza, !Kung, Lamalera, and Meriam) and indicate that better hunters exhibit higher age-corrected reproductive success than other men in their social group. Leading explanations to account for this pattern are: (1) direct provisioning of hunters’ wives and offspring, (2) dyadic reciprocity, (3) indirect reciprocity, (4) costly signaling, and (5) phenotypic correlation. I examine the qualitative and quantitative evidence bearing on these explanations and conclude that although none can be definitively rejected, extensive and apparently unconditional sharing of large game somewhat weakens the first three explanations. The costly signaling explanation has support in some cases, although the exact nature of the benefits gained from mating or allying with or deferring to better hunters needs further study. For comments on earlier drafts, I thank Monique Borgerhoff Mulder, Mike Gurven, Ray Hames, Kristen Hawkes, Kim Hill, Robert Kelly, Frank Marlowe, John Patton, and Polly Wiessner. Rebecca Bliege Bird and Douglas W. Bird invited me to collaborate in the Meriam research and (along with Del Passi of Mer) collected the data on Meriam demography. Geoff Kushnick and Matt Wimmer ably assisted with coding and statistical analysis of these data. Eric Alden Smith (PhD 1980, Cornell University) is a professor of anthropology at the University of Washington, Seattle. His research interests include the links between production and reproduction, the ecology and evolution of collective action, and politics in small-scale societies. He has conducted fieldwork among Inuit on Hudson Bay, Meriam in Torres Strait, and Mardu Aborigines in the Australian Western Desert.     

Mouse embryonic stem cells efficiently lipofected with nuclear localization peptide result in a high yield of chimeric mice and retain germline transmission potency

Embryonic stem (ES) cells are an important tool in developmental biology, genomics, and transgenic methods, as well as in potential clinical applications such as gene therapy or tissue engineering. Electroporation is the standard transfection method for mouse ES (mES) cells because lipofection is quite inefficient. It is also unclear if mES cells treated with cationic lipids maintain pluripotency. We have developed a simple lipofection method for high efficiency transfection and stable transgene expression by employing the nonclassical nuclear localization signal M9 derived from the heterogeneous nuclear ribonucleoprotein A1. In contrast to using 20 microg DNA for 10 x 10(6) cells via electroporation which resulted in 10-20 positive cells/mm2, M9-assisted lipofection of 2 x 10(5) cells with 2 microg DNA resulted in > 150 positive cells/mm2. Electroporation produced only 0.16% EGFP positive cells with fluorescence intensity (FI) > 1000 by FACS assay, while M9-lipofection produced 36-fold more highly EGFP positive cells (5.75%) with FI > 1000. Using 2.5 x 10(6) ES cells and 6 microg linearized DNA followed by selection with G418, electroporation yielded 17 EGFP expressing colonies, while M9-assisted lipofection yielded 72 EGFP expressing colonies. The mES cells that stably expressed EGFP following M9-assisted lipofection yielded > 66% chimeric mice (8 of 12) and contributed efficiently to the germline. In an example of gene targeting, a knock-in mouse was produced from an ES clone screened from 200 G418-resistant colonies generated via M9-assisted lipofection. To our knowledge, this is the first report of generation of transgenic or knock-in mice obtained from lipofected mES cells and this method may facilitate large scale genomic studies of ES developmental biology or large scale generation of mouse models of human disease.     

The Drosophila tctex-1 light chain is dispensable for essential cytoplasmic dynein functions but is required during spermatid differentiation

Variations in subunit composition and modification have been proposed to regulate the multiple functions of cytoplasmic dynein. Here, we examine the role of the Drosophila ortholog of tctex-1, the 14-kDa dynein light chain. We show that the 14-kDa light chain is a bona fide component of Drosophila cytoplasmic dynein and use P element excision to generate flies that completely lack this dynein subunit. Remarkably, the null mutant is viable and the only observed defect is complete male sterility. During spermatid differentiation, the 14-kDa light chain is required for the localization of a nuclear "cap" of cytoplasmic dynein and for proper attachment between the sperm nucleus and flagellar basal body. Our results provide evidence that the function of the 14-kDa light chain in Drosophila is distinct from other dynein subunits and is not required for any essential functions in early development or in the adult organism.     

Technologies to support the creation of complex systems models--using StarLogo software with students

Research on complex, adaptive systems has made significant advances in recent years in the study of natural and social phenomena that exhibit random variation and selection, resulting in learning or evolution. Unfortunately, students (including K-12, undergraduate and graduate) in most biology programs have little opportunity to explore complex systems during the course of their studies. StarLogo and the Adventures in Modeling Curriculum [Adventures in Modeling: Exploring Complex, Dynamic Systems with StarLogo. Teachers College Press, New York] provide an easily accessible entry point into complex systems modeling for students and other novice modelers. These specialized tools can provide powerful insights into the dynamics of systems and create opportunities to explore challenging and meaningful domains in the biological sciences. Specific applications to epidemiological and ecological systems are explored, including the often debated topic of the evolution of reduced attack rates in predator-prey systems.     

Repair of hydrolytic DNA deamination damage in thermophilic bacteria: cloning and characterization of a Vsr endonuclease homolog from Bacillus stearothermophilus

Hydrolytic deamination of 5-methyl cytosine in double stranded DNA results in formation of a T/G mismatch that—if left unrepaired—leads to a C→T transition mutation in half of the progeny. In addition to several mismatch-specific glycosylases that have been found in both pro- and eukaryotes to channel this lesion into base excision repair by removing the T from the mismatch, Vsr endonuclease from Escherichia coli has been described which initiates repair by an endonucleolytic strand incision 5′ to the mismatched T. We have isolated a gene coding for a homolog of E.coli Vsr endonuclease from the thermophilic bacterium Bacillus stearothermophilus H3 (Vsr.Bst) using a method that allows PCR amplification with degenerated primers of gene segments which code for only one highly conserved amino acid region. Vsr.Bst was produced heterologously in E.coli and purified to apparent homogeneity. Vsr.Bst specifically incises heteroduplex DNA with a preference for T/G mismatches. The selectivity of Vsr.Bst for the sequence context of the T/G mismatch appears less pronounced than for Vsr.Eco.     

The DNA sequence of chromosome I of an African trypanosome: gene content,chromosome organisation,recombination and polymorphism

The African trypanosome, Trypanosoma brucei, causes sleeping sickness in humans in sub-Saharan Africa. Here we report the sequence and analysis of the 1.1 Mb chromosome I, which encodes approximately 400 predicted genes organised into directional clusters, of which more than 100 are located in the largest cluster of 250 kb. A 160-kb region consists primarily of three gene families of unknown function, one of which contains a hotspot for retroelement insertion. We also identify five novel gene families. Indeed, almost 20% of predicted genes are members of families. In some cases, tandemly arrayed genes are 99–100% identical, suggesting an active process of amplification and gene conversion. One end of the chromosome consists of a putative bloodstream-form variant surface glycoprotein (VSG) gene expression site that appears truncated and degenerate. The other chromosome end carries VSG and expression site-associated genes and pseudogenes over 50 kb of subtelomeric sequence where, unusually, the telomere-proximal VSG gene is oriented away from the telomere. Our analysis includes the cataloguing of minor genetic variations between the chromosome I homologues and an estimate of crossing-over frequency during genetic exchange. Genetic polymorphisms are exceptionally rare in sequences located within and around the strand-switches between several gene clusters.     

Crystal structure of a wild-type Cre recombinase-loxP synapse reveals a novel spacer conformation suggesting an alternative mechanism for DNA cleavage activation

Escherichia coli phage P1 Cre recombinase catalyzes the site-specific recombination of DNA containing loxP sites. We report here two crystal structures of a wild-type Cre recombinase–loxP synaptic complex corresponding to two distinct reaction states: an initial pre-cleavage complex, trapped using a phosphorothioate modification at the cleavable scissile bond that prevents the recombination reaction, and a 3′-phosphotyrosine protein–DNA intermediate resulting from the first strand cleavage. In contrast to previously determined Cre complexes, both structures contain a full tetrameric complex in the asymmetric unit, unequivocally showing that the anti-parallel arrangement of the loxP sites is an intrinsic property of the Cre–loxP recombination synapse. The conformation of the spacer is different to the one observed for the symmetrized loxS site: a kink next to the scissile phosphate in the top strand of the pre-cleavage complex leads to unstacking of the TpG step and a widening of the minor groove. This side of the spacer is interacting with a ‘cleavage-competent’ Cre subunit, suggesting that the first cleavage occurs at the ApT step in the top strand. This is further confirmed by the structure of the 3′-phosphotyrosine intermediate, where the DNA is cleaved in the top strands and covalently linked to the ‘cleavage-competent’ subunits. The cleavage is followed by a movement of the C-terminal part containing the attacking Y324 and the helix N interacting with the ‘non-cleaving’ subunit. This rearrangement could be responsible for the interconversion of Cre subunits. Our results also suggest that the Cre-induced kink next to the scissile phosphodiester activates the DNA for cleavage at this position and facilitates strand transfer.