The herbicide glyphosate became widely used in the United States and other parts of the world after the commercialization of glyphosate-resistant crops. These crops have constitutive overexpression of a glyphosate-insensitive form of the herbicide target site gene, 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS). Increased use of glyphosate over multiple years imposes selective genetic pressure on weed populations. We investigated recently discovered glyphosate-resistant Amaranthus palmeri populations from Georgia, in comparison with normally sensitive populations. EPSPS enzyme activity from resistant and susceptible plants was equally inhibited by glyphosate, which led us to use quantitative PCR to measure relative copy numbers of the EPSPS gene. Genomes of resistant plants contained from 5-fold to more than 160-fold more copies of the EPSPS gene than did genomes of susceptible plants. Quantitative RT-PCR on cDNA revealed that EPSPS expression was positively correlated with genomic EPSPS relative copy number. Immunoblot analyses showed that increased EPSPS protein level also correlated with EPSPS genomic copy number. EPSPS gene amplification was heritable, correlated with resistance in pseudo-F 2 populations, and is proposed to be the molecular basis of glyphosate resistance. FISH revealed that EPSPS genes were present on every chromosome and, therefore, gene amplification was likely not caused by unequal chromosome crossing over. This occurrence of gene amplification as an herbicide resistance mechanism in a naturally occurring weed population is particularly significant because it could threaten the sustainable use of glyphosate-resistant crop technology.5-enolpyruvylshikimate-3-phosphate synthase | herbicide resistance | mobile genetic element | evolution | Palmer amaranth

No abstract

Sex determination in papaya is controlled by a recently evolved XY chromosome pair, with two slightly different Y chromosomes controlling the development of males (Y) and hermaphrodites (Y h ).To study the events of early sex chromosome evolution, we sequenced the hermaphrodite-specific region of the Y h chromosome (HSY) and its X counterpart, yielding an 8.1-megabase (Mb) HSY pseudomolecule, and a 3.5-Mb sequence for the corresponding X region. The HSY is larger than the X region, mostly due to retrotransposon insertions. The papaya HSY differs from the X region by two large-scale inversions, the first of which likely caused the recombination suppression between the X and Y h chromosomes, followed by numerous additional chromosomal rearrangements. Altogether, including the X and/or HSY regions, 124 transcription units were annotated, including 50 functional pairs present in both the X and HSY. Ten HSY genes had functional homologs elsewhere in the papaya autosomal regions, suggesting movement of genes onto the HSY, whereas the X region had none. Sequence divergence between 70 transcripts shared by the X and HSY revealed two evolutionary strata in the X chromosome, corresponding to the two inversions on the HSY, the older of which evolved about 7.0 million years ago. Gene content differences between the HSY and X are greatest in the older stratum, whereas the gene content and order of the collinear regions are identical. Our findings support theoretical models of early sex chromosome evolution.Carica papaya | DNA sequencing | molecular evolution | sex chromosomes S ex chromosomes have evolved independently in diverse lineages of animals and plants, and new dioecious species are still evolving (1, 2). Evidence of homology between nascent sex chromosome pairs in flowering plants and fish (3-6) supports the notion that sex chromosomes evolved from autosomes that gained sex determination genes. The key event in sex chromosome evolution is the suppression of recombination between the sex-determining regions of ancestrally homologous chromosome pairs, which limits one chromosome of the pair to one sex, producing XY (male heterogametic) or ZW (female heterogametic) systems. Evolutionary models predict that a lack of recombination allows for Y-or W-specific characteristics to accumulate, through the reduced efficacy of selection on these chromosomes (7,8), leading to the Y and W chromosomes accumulating deleterious mutations and transposable elements, and ultimately undergoing genetic degeneration, through the loss of genes or gene functions, as observed in mammals, Drosophila, birds, fishes, and snakes (9, 10). In some animals and plants, the greater number of mitotic cell divisions in spermatogenesis than oogenesis also leads to Y chromosomes having a higher mutation rate than autosomes or X chromosomes (11)(12)(13)(14) and is predicted to further contribute to greater changes of the evolving Y (or W) chromosome than the X (or Z) chromosome.To test these predictions of repetitive sequence accumulation, chromosoma...

Integrin receptors play an important role during cell migration by mediating linkages and transmitting forces between the extracellular matrix and the actin cytoskeleton. The mechanisms by which these linkages are regulated and released during migration are not well understood. We show here that cell-permeable inhibitors of the calcium-dependent protease calpain inhibit both ␤1 and ␤3 integrin-mediated cell migration. Calpain inhibition specifically stabilizes peripheral focal adhesions, increases adhesiveness, and decreases the rate of cell detachment. Furthermore, these inhibitors alter the fate of integrin receptors at the rear of the cell during migration. A Chinese hamster ovary cell line expressing low levels of calpain I also shows reduced migration rates with similar morphological changes, further implicating calpain in this process. Taken together, the data suggest that calpain inhibition modulates cell migration by stabilizing cytoskeletal linkages and decreasing the rate of retraction of the cell's rear. Inhibiting calpain-mediated proteolysis may therefore be a potential therapeutic approach to control pathological cell migration such as tumor metastasis.Cell migration requires a dynamic interaction between a cell, its substratum, and the actin cytoskeleton. Integrin receptors, which are ␣␤ heterodimers present on the cell surface, play an important role during cell migration by mediating these interactions and transmitting forces between the extracellular matrix and the actin cytoskeleton (1, 2). The mechanisms by which these linkages are regulated and released at the cell's rear during migration are not well understood. Previous studies implicate calcium transients in adhesive release in neutrophils migrating on both fibronectin and vitronectin. However, calcineurin mediates the calcium-dependent release of adhesions at the cell's rear in neutrophils migrating on vitronectin but not on fibronectin (3, 4). The specificity of the calcineurin effect for vitronectin and the ␣v␤3 integrin suggests that other calciumdependent mechanisms are also likely to contribute to detachment during migration.The calcium-dependent protease calpain is an attractive candidate to be a calcium-responsive regulator of cell migration. It localizes to focal adhesions and cleaves many focal adhesionrelated proteins including integrin receptors, focal adhesion kinase, and talin (5-8). Calpain is a cysteine protease with two characterized isoforms, calpain I (-calpain) and II (m-calpain). Both contain an 80-kDa catalytic subunit and a 30-kDa regulatory domain. Activation requires calcium concentrations in the micromolar range and millimolar range for calpain I and II, respectively (9, 10). The increases in calcium seen in migrating cells appear to be within the range to support activation of calpain (3). In this study we use genetic and inhibitor studies to show that inhibition of the calcium-dependent protease calpain reduces both ␤1 and ␤3 integrin-mediated migration. EXPERIMENTAL PROCEDURES Cells and Reagents-CHO1 KI cells ...

Centromeres in most higher eukaryotes are composed of long arrays of satellite repeats. By contrast, most newly formed centromeres (neocentromeres) do not contain satellite repeats and instead include DNA sequences representative of the genome. An unknown question in centromere evolution is how satellite repeat-based centromeres evolve from neocentromeres. We conducted a genome-wide characterization of sequences associated with CENH3 nucleosomes in potato (Solanum tuberosum). Five potato centromeres (Cen4, Cen6, Cen10, Cen11, and Cen12) consisted primarily of single-or low-copy DNA sequences. No satellite repeats were identified in these five centromeres. At least one transcribed gene was associated with CENH3 nucleosomes. Thus, these five centromeres structurally resemble neocentromeres. By contrast, six potato centromeres (Cen1, Cen2, Cen3, Cen5, Cen7, and Cen8) contained megabase-sized satellite repeat arrays that are unique to individual centromeres. The satellite repeat arrays likely span the entire functional cores of these six centromeres. At least four of the centromeric repeats were amplified from retrotransposon-related sequences and were not detected in Solanum species closely related to potato. The presence of two distinct types of centromeres, coupled with the boom-and-bust cycles of centromeric satellite repeats in Solanum species, suggests that repeat-based centromeres can rapidly evolve from neocentromeres by de novo amplification and insertion of satellite repeats in the CENH3 domains.