IntDOT interactions with core- and arm-type sites of the conjugative transposon CTnDOT

CTnDOT is a Bacteroides conjugative transposon (CTn) that has facilitated the spread of antibiotic resistances among bacteria in the human gut in recent years. Although the integrase encoded by CTnDOT (IntDOT) carries the C-terminal set of conserved amino acids that is characteristic of the tyrosine family of recombinases, the reaction it catalyzes involves a novel step that creates a short region of heterology at the joined ends of the element during recombination. Also, in contrast to tyrosine recombinases, IntDOT catalyzes a reaction that is not site specific. To determine what types of contacts IntDOT makes with the DNA during excision and integration, we first developed an agarose gel-based assay for CTnDOT recombination, which facilitated the purification of the native IntDOT protein. The partially purified IntDOT was then used for DNase I footprinting analysis of the integration site attDOT and the excision sites attL and attR. Our results indicate that CTnDOT has five or six arm sites that are likely to be involved in forming higher-order nucleoprotein complexes necessary for synapsis. In addition, there are four core sites that flank the sites of strand exchange during recombination. Thus, despite the fact that the reaction catalyzed by IntDOT appears to be different from that typically catalyzed by tyrosine recombinases, the protein-DNA interactions required for higher-order structures and recombination appear to be similar.     

Thiol-based regulation of redox-active glutamate-cysteine ligase from Arabidopsis thaliana

Glutathione biosynthesis is a key component in the network of plant stress responses that counteract oxidative damage and maintain intracellular redox environment. Using a combination of mass spectrometry and site-directed mutagenesis, we examined the response of Arabidopsis thaliana glutamate-cysteine ligase (GCL) to changes in redox environment. Mass spectrometry identified two disulfide bonds (Cys186-Cys406 and Cys349-Cys364) in GCL. Mutation of either Cys-349 or Cys-364 to a Ser reduced reaction rate by twofold, but substitution of a Ser for either Cys-186 or Cys-406 decreased activity by 20-fold and abrogated the response to changes in redox environment. Redox titrations show that the regulatory disulfide bond has a midpoint potential comparable with other known redox-responsive plant proteins. Mutation of Cys-102, Cys-251, Cys-349, or Cys-364 did not alter the response to redox environment, indicating that modulation of activity depends on the Cys186-Cys406 disulfide bond. In vivo analysis of GCL in Arabidopsis root extracts revealed that multiple oxidative stresses altered the distribution of oxidized (active) and reduced (inactive) enzyme and that this change correlated with increased GCL activity. The thiol-based regulation of GCL provides a posttranslational mechanism for modulating enzyme activity in response to in vivo redox environment and suggests a role for oxidative signaling in the maintenance of glutathione homeostasis in plants.     

Seed and seedling ecology of piñon and juniper species in the pygmy woodlands of western North America

Knowledge of the seed and seedling ecology of the piñon and juniper woodlands of western North America is essential for understanding both the northward migration and expansion of the woodlands during the Holocene (< 11,500 B.P.), and the accelerated expansion of the woodlands since settlement of the West by Anglo-Americans around 200 years ago. We follow the fates of seeds and seedlings of the different piñon and juniper species within the woodlands from seed development to seedling establishment, and discuss the implications of this information for the past and present expansion of the woodlands. While seed development requires about two and one-half years in pinons, it is species-dependent in junipers and can take one, two, or even three years. Substantial seed losses can occur during seed development due to developmental constraints, and before or after seed maturation as a result of insects, pathogens, or predatory animals. In piñon pines, the primary seed dispersers are scatterhoarding birds (corvids) and rodents that harvest seeds from the trees or after seed fall and cache them in the soil. In contrast, most junipers appear to be dispersed primarily by frugivorous birds and mammals that ingest the seeds and defecate them onto the soil surface. We have recently documented that scatter-hoarding rodents also disperse juniper seeds. Disperser effectiveness, or the contribution a disperser makes to the future reproduction of a plant population, may vary among species of piñons and especially junipers. Piñon seeds are short-lived and exhibit little dormancy, and they probably only germinate the spring following dispersal. Juniper seeds are long-lived and seed dispersal can occur over one or more years. Seed germination can be delayed for several years due to impermeable seed coats, embryo dormancy, or the presence of inhibitors. Seedling establishment of piñon pines is facilitated by nurse plants but, while junipers often establish beneath nurse plants, they are capable of establishing in open environments. In the southwestern United States, higher establishment of juniper occurs in open environments due to more favorable precipitation, and competition may be more important than facilitation in determining establishment. When considering the mechanisms involved in the past and present expansion of the woodlands, short-distance dispersal, local population growth, and long-distance dispersal are all important. Different classes of dispersers, some of which appear to have coevolved with the tree species, appear to be responsible for local (short-distance) vs. long-distance dispersal in pinons and junipers. Because ecotones form the interface between the woodlands and adjacent communities, they can provide valuable information on both the seed dispersal and seedling establishment processes responsible for tree expansion. Disturbance regimes and, recently, the effects of humans on those regimes have major effects on the expansion and contraction of the woodlands. Before Anglo-American settlement, fires occurred as frequently as every 50–100 years throughout much of the woodlands. During this century, fire frequencies have been reduced due to the indirect effects of livestock grazing and the direct effects of removing Native Americans from the ecosystem and implementing active fire-prevention programs. The result has been an increase in tree-dominated successional stages at the expense of grass-dominated stages. Various management techniques, including controlled burning and chaining, have been implemented to reduce tree dominance, but their effects depend largely on the life histories of the tree species and the disturbance characteristics. Several areas relating to the seed and seedling ecology of the piñon and juniper require additional research if we are to truly understand the dynamics of the woodlands.     

WHEN MORPHOLOGY MISLEADS: INTERPOPULATION UNIFORMITY IN SEXUAL SELECTION MASKS GENETIC DIVERGENCE IN HARLEQUIN BEETLE-RIDING PSEUDOSCORPION POPULATIONS

Differences in secondary sexual characteristics of males often provide the most conspicuous means of distinguishing between closely related species. Does this therefore imply that the absence of differentiation in exaggerated male traits between allopatric populations provides evidence of a single, genetically cohesive species? We addressed this question with a comprehensive investigation of two populations (French Guiana and Panama) of the harlequin beetle-riding pseudoscorpion, Cordylochernes scorpioides. This highly sexually dimorphic pseudoscorpion is currently described as a single species, ranging throughout the Neotropics. Our morphometric analyses detected minimal differentiation between the two populations in all nine external morphological characters measured, including sexually dimorphic traits in males. Only in traits of the spermatophore was there any appreciable level of differentiation. Behavior differentiation and prezygotic reproductive isolation were also limited: 78.3% of males successfully transferred sperm to “foreign” females, and in 63.9% of these cases, females' eggs were successfully fertilized. By contrast, extensive divergence existed in two of nine electrophoretic loci, including an essentially fixed-allele difference at the Ldh locus. Most significantly, postzygotic reproductive isolation was complete, with heteropopulation zygotes invariably aborting early in development. These results strongly suggest that the two populations are, in fact, sibling species, a conclusion supported by our recently published findings on their marked divergence in minisatellite DNA. How can such interpopulation homogeneity in male sexually dimorphic traits exist in the face of strong genetic divergence? We propose that sexual selection, oscillating between favoring small and then large males, maintains such high levels of male variability within each population that it has obscured a speciation event in which genetic divergence and postzygotic incompatibility have clearly outpaced the evolution of prezygotic reproductive isolation.     

Genomic Analysis of a New Mammalian Distal-less Gene: Dlx7

We have cloned a new Dlx gene (Dlx7) from human and mouse that may represent the mammalian orthologue of the newt geneNvHBox-5.The homeodomains of these genes are highly similar to all other vertebrate Dlx genes, and regions of similarity also exist between mammalian Dlx7 and a subset of vertebrate Dlx genes downstream of the homeodomain. The sequence divergence between human and mouse Dlx7 in these regions is greater than that predicted from comparisons of other vertebrate Dlx genes, however, and there is little sequence similarity upstream of the homeodomain both between these two genes and with other Dlx genes. We present evidence for alternative splicing of mouseDlx7upstream of the homeodomain that may account for some of this divergence. We have mapped humanDLX7distal to the 5′ end of the HOXB cluster at an estimated distance of between 1 and 2 Mb by FISH. Both the human and the mouse Dlx7 are shown to be closely linked to Dlx3 in a convergently transcribed orientation. These mapping results support the possibility that vertebrate distal-less genes have been duplicated in concert with the Hox clusters.     

Fungal Catabolism of Crown Gall Opines

This study was conducted to determine the capacities of 37 fungi to utilize various crown gall opines as their sole carbon and nitrogen source. One strain of Fusarium solani, two of Cylindrocarpon destructans, and six of Cylindrocarpon heteronema catabolized octopine, mannopine, octopinic acid, succinamopine, or a combination of these opines. One C. heteronema and one Fusarium dimerum strain grew only on succinamopine. None of the fungal isolates had the ability to grow on nopaline. The catabolism of opines by fungi was confirmed by the disappearance of the opine from the growth medium and by an increase in final mycelial dry weight with rising initial concentration of test substrate. This study thus shows that the catabolism of opines is not restricted to bacteria.     

Improved agarose gel assay for quantification of growth factor-induced cell motility

Cell chemotaxis is frequently required in normal or pathological situations such as invasion, metastasis, and tumor angiogenesis and may involve many different cell types. At present, no device can simultaneously (i) make morphological observations, (ii) quantify cell migration, (iii) test multiple chemoattracting gradients, and (iv) analyze cell-cell interactions. We developed an agarose-based assay to address these questions. Two glass molds were designed, around which agarose gel could be poured to form specific well shapes. Using a vital nuclear stain (Hoechst 33258), we characterized the migration profile of adherent or suspension cells. Cells could be observed during the entire migration process. We were able to follow cells moving toward chemoattractants or being repulsed by other molecules, and we could estimate average migration speed. Using this inexpensive assay, we were able to obtain precise, reproducible results concerning the chemotactic behavior of different cell types. The resulting data differentiated between chemokinetic and chemotactic movement. Chemotactic potencies could be compared using different criteria, such as the number of attracted cells, induced speed, and morphological aspect. This improved agarose assay appears to be a reliable and inexpensive alternative to other available chemotaxis study tools.     

Plasticity in laying dates of Canada Geese in response to spring phenology

Observed phenological changes can be explained either by individual phenotypic plasticity or by evolutionary changes, but there is more evidence pointing towards phenotypic plasticity to explain the mechanism behind changes in bird phenology. However, most studies on phenology have been conducted on insectivorous bird species for which breeding is closely tied to temperature and insect emergence. In this study, we examined the consequences of climatic conditions on the nesting phenology of temperate breeding Canada Geese Branta canadensis maxima, which rely on a continuous food supply, during a 14‐year period (2003–16). We determined whether laying dates were plastically adjusted to spring environmental conditions, and whether this adjustment resulted in a laying date advancement. We further estimated the strength and shape of selection acting on breeding timing, by looking at the effect of laying date on the relative number of young successfully hatched in a nest. We found that Geese plastically adjusted their laying date to spring maximum temperature (and not to precipitation or ice break‐up), resulting in a 9‐day advancement of laying date in the population for that period. Laying date was also moderately repeatable (r = 0.23) and subject to directional selection, but stabilizing selection was negligible. We thus demonstrate how Canada Geese plastically adjust laying dates to temperature, which may further be beneficial to nesting success. Evolutionary change of laying date to selection related to climate change, however, is still possible.