Genetic and morphological evolution following a founder event in the dark-eyed junco, Junco hyemalis thurberi

An isolated population of dark-eyed juncos, Junco hyemalis, became established on the campus of the University of California at San Diego (UCSD), probably in the early 1980s. It now numbers about 70 breeding pairs. Populations across the entire natural range of the subspecies J. h. thurberi are weakly differentiated from each other at five microsatellite loci (FST = 0.01). The UCSD population is significantly different from these populations, the closest of which is 70 km away. It has 88% of the genetic heterozygosity and 63% of the allelic richness of populations in the montane range of the subspecies, consistent with a harmonic mean effective population size of 32 (but with 95% confidence limits from four to > 70) over the eight generations since founding. Results suggest a moderate bottleneck in the early establishment phase but with more than seven effective founders. Individuals in the UCSD population have shorter wings and tails than those in the nearby mountains and a common garden experiment indicates that the morphological differences are genetically based. The moderate effective population size is not sufficient for the observed morphological differences to have evolved as a consequence of genetic drift, indicating a major role for selection subsequent to the founding of the UCSD population.     

Mycorrhizal colonization mediated by species interactions in arctic tundra

The Alaskan tussock tundra is a strongly nutrient-limited ecosystem, where almost all vascular plant species are mycorrhizal. We established a long-term removal experiment to document effects of arctic plant species on ecto- and ericoid mycorrhizal fungi and to investigate whether species interactions and/or nutrient availability affect mycorrhizal colonization. The treatments applied were removal of Betula nana (Betulaceae, dominant deciduous shrub species), removal of Ledum palustre (Ericaceae, dominant evergreen shrub species), control (no removal), and each of these three treatments with the addition of fertilizer. After 3 years of Ledum removal and fertilization, we found that overall ectomycorrhizal colonization in Betula was significantly reduced. Changes in ectomycorrhizal morphotype composition in removal and fertilized treatments were also observed. These results suggest that the effect of Ledum on Betula 's mycorrhizal roots is due to sequestration of nutrients by Ledum, leading to reduced nutrient availability in the soil. In contrast, ericoid mycorrhizal colonization was not affected by fertilization, but the removal of Betula and to a lower degree of Ledum resulted in a reduction of ericoid mycorrhizal colonization suggesting a direct effect of these species on ericoid mycorrhizal colonization. Nutrient availability was only higher in fertilized treatments, but caution should be taken with the interpretation of these data as soil microbes may effectively compete with the ion exchange resins for the nutrients released by plant removal in these nutrient-limited soils.     

Adaptation of rainbow fish to lake and stream habitats

Fish occupy a range of hydrological habitats that exert different demands on locomotor performance. We examined replicate natural populations of the rainbow fishes Melanotaenia eachamensis and M. duboulayi to determine if colonization of low-velocity (lake) habitats by fish from high-velocity (stream) habitats resulted in adaptation of locomotor morphology and performance. Relative to stream conspecifics, lake fish had more posteriorly positioned first dorsal and pelvic fins, and shorter second dorsal fin bases. Habitat dimorphism observed between wild-caught fish was determined to be heritable as it was retained in M. eachamensis offspring raised in a common garden. Repeated evolution of the same heritable phenotype in independently derived populations indicated body shape divergence was a consequence of natural selection. Morphological divergence between hydrological habitats did not support a priori expectations of deeper bodies and caudal peduncles in lake fish. However, observed divergence in fin positioning was consistent with a family-wide association between habitat and morphology, and with empirical studies on other fish species. As predicted, decreased demand for sustained swimming in lakes resulted in a reduction in caudal red muscle area of lake fish relative to their stream counterparts. Melanotaenia duboulayi lake fish also had slower sustained swimming speeds (Ucrit) than stream conspecifics. In M. eachamensis, habitat affected Ucrit of males and females differently. Specifically, females exhibited the pattern observed in M. duboulayi (lake fish had faster Ucrit than stream fish), but the opposite association was observed in males (stream males had slower Ucrit than lake males). Stream M. eachamensis also exhibited a reversed pattern of sexual dimorphism in Ucrit (males slower than females) relative to all other groups (males faster than females). We suggest that M. eachamensis males from streams responded to factors other than water velocity. Although replication of muscle and Ucrit phenotypes across same habitat populations within and/or among species was suggestive of adaptation, the common garden experiment did not confirm a genetic basis to these associations. Kinematic studies should consider the effect of the position and base length of dorsal fins.     

Improved NMR spectra of a protein–DNA complex through rational mutagenesis and the application of a sensitivity optimized isotope-filtered NOESY experiment

The NMR spectra of the complex between the DNA-binding domain of the Dead ringer protein (DRI-DBD, Gly262-Gly398) and its DNA binding site (DRI-DBD:DNA, 26 kDa) have been optimized by biochemical and spectroscopic means. First, we demonstrate the utility of a modified 2D [F1,F2] ¹³C-filtered NOESY experiment that employs a ¹J_HC versus chemical shift optimized adiabatic ¹³C inversion pulse [Zwahlen, C. et al. (1997) J. Am. Chem. Soc., 119, 6711–6721]. The new sequence is shown to be more sensitive than previously published pulse schemes (up to 40% in favorable cases) and its utility is demonstrated using two protein–DNA complexes. Second, we demonstrate that the targeted replacement of an interfacial aromatic residue in the DRI-DBD:DNA complex substantially reduces line broadening within its NMR spectra. The spectral changes are dramatic, salvaging a protein–DNA complex that was originally ill suited for structural analysis by NMR. This biochemical approach is not a general method, but may prove useful in the spectral optimization of other protein complexes that suffer from interfacial line broadening caused by dynamic changes in proximal aromatic rings.     

Interchange of DNA-binding modes in the deformed and ultrabithorax homeodomains: a structural role for the N-terminal arm

The deformed (Dfd) and ultrabithorax (Ubx) homeoproteins regulate developmental gene expression in Drosophila melanogaster by binding to specific DNA sequences within its genome. DNA binding is largely accomplished via a highly conserved helix-turn-helix DNA-binding domain that is known as a homeodomain (HD). Despite nearly identical DNA recognition helices and similar target DNA sequence preferences, the in vivo functions of the two proteins are quite different. We have previously revealed differences between the two HDs in their interactions with DNA. In an effort to define the individual roles of the HD N-terminal arm and recognition helix in sequence-specific binding, we have characterized the structural details of two Dfd/Ubx chimeric HDs in complex with both the Dfd and Ubx-optimal-binding site sequences. We utilized hydroxyl radical cleavage of DNA to assess the positioning of the proteins on the binding sites. The effects of missing nucleosides and purine methylation on HD binding were also analyzed. Our results show that both the Dfd and Ubx HDs have similar DNA-binding modes when in complex with the Ubx-optimal site. There are subtle but reproducible differences in these modes that are completely interchanged when the Dfd N-terminal arm is replaced with the corresponding region of the Ubx HD. In contrast, we showed previously that the Dfd-optimal site sequence elicits a very different binding mode for the Ubx HD, while the Dfd HD maintains a mode similar to that elicited by the Ubx-optimal site. Our current methylation interference studies suggest that this alternate binding mode involves interaction of the Ubx N-terminal arm with the minor groove on the opposite face of DNA relative to the major groove that is occupied by the recognition helix. As judged by hydroxyl radical footprinting and the missing nucleoside experiment, it appears that interaction of the Ubx recognition helix with the DNA major groove is reduced. Replacing the Dfd N-terminal arm with that of Ubx does not elicit a complete interchange of the DNA-binding mode. Although the position of the chimera relative to DNA, as judged by hydroxyl radical footprinting, is similar to that of the Dfd HD, the missing nucleoside and methylation interference patterns resemble those of the Ubx HD. Repositioning of amino acid side-chains without wholesale structural alteration in the polypeptide appears to occur as a function of N-terminal arm identity and DNA-binding site sequence. Complete interchange of binding modes was achieved only by replacement of the Dfd N-terminal arm and the recognition helix plus 13 carboxyl-terminal residues with the corresponding residues of Ubx. The position of the N-terminal arm in the DNA minor groove appears to differ in a manner that depends on the two base-pair differences between the Dfd and Ubx-optimal-binding sites. Thus, N-terminal arm position dictates the binding mode and the interaction of the recognition helix with nucleosides in the major groove.     

Determination of residual dipolar couplings in homonuclear MOCCA-SIAM experiments

In solutions with partial molecular alignment, anisotropic magnetic interactions such as the chemical shift anisotropy, the electric quadrupole interaction, and the magnetic dipole-dipole interaction are no longer averaged out to zero in contrast to isotropic solutions. The resulting residual anisotropic magnetic interactions are increasingly used in biological NMR studies for the determination of 3D structures of proteins and other biomolecules. In the present paper we propose a new approach allowing the measurement of residual H^N-H dipolar couplings of non-isotope enriched proteins based on the application of the MOCCA-SIAM experiment. This experiment allows the measurement of homonuclear coupling constants with an accuracy of ca. ±0.2 Hz and is therefore particularly well suited to determine residual dipolar couplings at relatively low degrees of molecular orientation. The agreement between experimentally determined residual H^N-H couplings and calculated values is demonstrated for BPTI.     

A unified parametric regression model for recapture studies with random removals in continuous time

Conditional likelihood based on counting processes are combined with a Horvitz-Thompson estimator to yield a population size estimator that is more efficient than the existing ones. Random removals are allowed in the recapturing process. Simulation studies are shown to assess the performance of the proposed estimators. Examples on a bird banding and a small mammal recapturing study are given.     

Analysis of metabolic and physiological responses to gnd knockout in Escherichia coli by using C-13 tracer experiment and enzyme activity measurement

The physiological and metabolic responses to gnd knockout in Escherichia coli K-12 was quantitatively investigated by using the (13)C tracer experiment (GC-MS/NMR) together with the enzyme activity analysis. It was shown that the general response to the gene knockout was the local flux rerouting via Entner-Doudoroff pathway and the direction reversing via non-oxidative pentose phosphate pathway (PPP). The mutant was found to direct higher flux to phosphoglucose isomerase reaction as compared to the wild-type, but the respiratory metabolism was comparable in both strains. The anaplerotic pathway catalyzed by malic enzyme was identified in the mutant, which was accompanied with an up-regulation of phosphoenolpyruvate carboxylase and down-regulation of phosphoenolpyruvate carboxykinase. The presented results provide first evidence that compensatory mechanism existed in PPP and anaplerotic pathway in response to the gnd deletion.