Genetic analysis of sterile mutants in the dpy-5 unc-13 (I) genomic region of Caenorhabditis elegans

Essential genes were identified in the 1.5-map unit dpy-5 unc-13 region of chromosome I in the Caenorhabditis elegans genome by rescuing lethal mutations using the duplication sDp2. In this paper, we report the mapping and complementation testing of lethal mutations, 45 of which identify 18 new, essential genes. This analysis brings the number of essential genes defined by the sDp2 rescue of lethal mutants to 97; 64 of these map between dpy-5 and unc-13. 61% of these essential genes are identified by more than one allele. Positioning of the mutations was done using the breakpoints of six duplications. The mutant phenotypes of 14 loci essential for fertility were characterized by Nomarski microscopy and DAPI staining. None of the mutants were rescued by wild-type male sperm. The cytological data showed that four genes produced mutants with defects in gonadogenesis, let-395, let-603, let-605 and let-610. Mutations in seven genes, let-355, let-367, let-384, let-513, let-544, let-545 and let-606, affected germ cell proliferation or gametogenesis. Mutants for the remaining three genes, let-370, let-599 and let-604, produced eggs that failed to develop or hatch, thereby acting as maternal effect lethals. We observed a nonrandom distribution of arrest phenotypes with regard to map position. Received: 8 May 1996 / Accepted : 27 January 1997     

Structural Insights into the Mechanism of Base Excision by MBD4

DNA glycosylases remove damaged or modified nucleobases by cleaving the N-glycosyl bond and the correct nucleotide is restored through subsequent base excision repair. In addition to excising threatening lesions, DNA glycosylases contribute to epigenetic regulation by mediating DNA demethylation and perform other important functions. However, the catalytic mechanism remains poorly defined for many glycosylases, including MBD4 (methyl-CpG binding domain IV), a member of the helix-hairpin-helix (HhH) superfamily. MBD4 excises thymine from G·T mispairs, suppressing mutations caused by deamination of 5-methylcytosine, and it removes uracil and modified uracils (e.g., 5-hydroxymethyluracil) mispaired with guanine. To investigate the mechanism of MBD4 we solved high-resolution structures of enzyme-DNA complexes at three stages of catalysis. Using a non-cleavable substrate analog, 2′-deoxy-pseudouridine, we determined the first structure of an enzyme-substrate complex for wild-type MBD4, which confirms interactions that mediate lesion recognition and suggests that a catalytic Asp, highly conserved in HhH enzymes, binds the putative nucleophilic water molecule and stabilizes the transition state. Observation that mutating the Asp (to Gly) reduces activity by 2700-fold indicates an important role in catalysis, but probably not one as the nucleophile in a double-displacement reaction, as previously suggested. Consistent with direct-displacement hydrolysis, a structure of the enzyme-product complex indicates a reaction leading to inversion of configuration. A structure with DNA containing 1-azadeoxyribose models a potential oxacarbenium-ion intermediate and suggests the Asp could facilitate migration of the electrophile towards the nucleophilic water. Finally, the structures provide detailed snapshots of the HhH motif, informing how these ubiquitous metal-binding elements mediate DNA binding.     

Lipid content of terrestrial arthropods in relation to body size,phylogeny, ontogeny and sex

Energy storage in arthropods has important implications for survival and reproduction. The lipid content of 276 species of adult arthropods with wet mass in the range 0.2–6.13 g is determined to assess how lipid mass scales with body mass. The relative contribution of lipids to total body mass is investigated with respect to phylogeny, ontogeny and sex. The lipid content of adult insects, arachnids, and arthropods in general shows an isometric scaling relationship with respect to body mass (M) (M_{arthropod lipid} = ?1.09 ×M_dry^1.01 and M_{arthropod lipid} = ?1.00 ×M_lean^0.98). However, lipid allocation varies between arthropod taxa, as well as with sex and developmental stage within arthropod taxa. Female insects and arachnids generally have higher lipid contents than males, and larval holometabolous insects and juvenile arachnids have higher lipid contents than adults.     

Interacting forces of predation and fishing affect species’ maturation size

1. Fishing is a strong selective force and is supposed to select for earlier maturation at smaller body size. However, the extent to which fishing‐induced evolution is shaping ecosystems remains debated. This is in part because it is challenging to disentangle fishing from other selective forces (e.g., size‐structured predation and cannibalism) in complex ecosystems undergoing rapid change.
2. Changes in maturation size from fishing and predation have previously been explored with multi‐species physiologically structured models but assumed separation of ecological and evolutionary timescales. To assess the eco‐evolutionary impact of fishing and predation at the same timescale, we developed a stochastic physiologically size‐structured food‐web model, where new phenotypes are introduced randomly through time enabling dynamic simulation of species'' relative maturation sizes under different types of selection pressures.
3. Using the model, we carried out a fully factorial in silico experiment to assess how maturation size would change in the absence and presence of both fishing and predation (including cannibalism). We carried out ten replicate stochastic simulations exposed to all combinations of fishing and predation in a model community of nine interacting fish species ranging in their maximum sizes from 10 g to 100 kg. We visualized and statistically analyzed the results using linear models.
4. The effects of fishing on maturation size depended on whether or not predation was enabled and differed substantially across species. Fishing consistently reduced the maturation sizes of two largest species whether or not predation was enabled and this decrease was seen even at low fishing intensities (F = 0.2 per year). In contrast, the maturation sizes of the three smallest species evolved to become smaller through time but this happened regardless of the levels of predation or fishing. For the four medium‐size species, the effect of fishing was highly variable with more species showing significant and larger fishing effects in the presence of predation.
5. Ultimately our results suggest that the interactive effects of predation and fishing can have marked effects on species'' maturation sizes, but that, at least for the largest species, predation does not counterbalance the evolutionary effect of fishing. Our model also produced relative maturation sizes that are broadly consistent with empirical estimates for many fish species.

     

A comparative study of breeding seasonality and litter size in eleven taxa of captive lemurs (Lemur andVarecia)

Breeding records of 11 taxa of captive lemurs housed at the Duke University Primate Center (DUPC), North Carolina, were analyzed for differences in the timing of births, for the relationship between breeding season and photoperiod, and for differences in litter size. At DUPC there are significant differences in the timing of births among certain taxa, including differences among some subspecies of Lemur fulvus.However, changes in latitude result in changes in the timing of the breeding season. Lemurs moved to higher latitudes mate at lower light-dark ratios than on Madagascar. The data presented here are consistent with the following model: a photoperiodic cue initiating reproductive activity, presumably a light-dark threshold, precedes the actual mating season by approximately 2 months, with an intervening period of physiological and social preparation. On Madagascar, selection may have favored births that coincide with the end of dry seasons and the beginning of wet seasons, which results in lactation and weaning during times of resource abundance. Taxa from the north and east have the highest mean litter sizes; those from the west have the lowest.     

DNA ligase-AMP adducts: Identification of yeast DNA ligase polypeptides

Yeast DNA ligase is radioactively labelled in vitro by incubating a crude cell extract with [α-³²P]ATP. The product of this reaction is the stable covalent ligase-AMP adduct, which can be characterized by its reactivity with either pyrophosphate or nicked DNA and visualized by gel electrophoresis and autoradiography. The Saccharomyces cerevisiae DNA ligase was identified as an 89 kDa polypeptide by exploiting the fact that transformants with multiple copies of the plasmid-encoded DNA ligase (CDC9) gene overproduce the enzyme by two orders of magnitude. A similar strategy has been used to identify the Schizosaccharomyces pombe DNA ligase as an 87 kDa polypeptide. Both values agree well with the coding capacities of the respective cloned gene sequences. When the S. cerevisiae ligase is greatly overproduced with respect to wild-type levels, a second polypeptide of 78.5 kDa is also labelled and has the same properties as the 89 kDa adduct. We suggest that this polypeptide is generated by proteolysis.     

Sex ratios under asymmetrical local mate competition in the parasitoid wasp Nasonia vitripennis

Sex ratio theory has proved remarkably useful in testing theadaptive nature of animal behavior. A particularly productivearea in this respect is Hamilton's theory of local mate competition(LMC), which has been extended in numerous directions to includegreater biological realism, allowing more detailed tests inspecific organisms. We have presented one such extension, termedasymmetrical LMC, which occurs when egg laying by females ona patch is asynchronous, and emerging males do not disperse,resulting in the extent of LMC on a patch varying over time.Our aim here is to test whether the parasitoid wasp Nasoniavitripennis responds to variation in the degree of asymmetricalLMC. Specifically, we show that females adjust their offspringsex ratios in response to (1) variation in the amount of asynchronyin emergence between broods on a patch and (2) the number andproportion of previously parasitized hosts on the patch. Ourresults provide qualitative support for the predictions of theory,suggesting new levels of complexity in the sex ratio behaviorof this much-studied organism. However, our results do not alwaysprovide quantitative support for theory, suggesting furthercomplexities that must be clarified.     

Eye size variation reflects habitat and daily activity patterns in colubrid snakes

The functioning of the vertebrate eye depends on its absolute size, which is presumably adapted to specific needs. Eye size variation in lidless and spectacled colubrid snakes was investigated, including 839 specimens belonging to 49 genera, 66 species and subspecies. Variations of adult eye diameters (EDs) in both absolute and relative terms between species were correlated with parameters reflecting behavioral ecology. In absolute terms, eye of arboreal species was larger than in terrestrial and semiaquatic species. For diurnal species, EDs of terrestrial species do not differ from semiaquatic species; for nocturnal species the ED of terrestrial species is larger than fossorial species but not different from semiaquatic species. In relative terms, ED did not differ significantly by habitat for diurnal species. Although the ED of terrestrial species is larger than fossorial species there were no differences for nocturnal species between semiaquatic and fossorial snakes. In contrast to other vertebrates studied to date, colubrid EDs in absolute and relative terms are larger in diurnal than in nocturnal species. These observations suggest that among colubrid snakes, eye size variation reflects adaptation to specific habitats, foraging strategies and daily activities, independently of phylogeny. J. Morphol. 2012. © 2012 Wiley Periodicals, Inc.     

Dissecting the Conformational Dynamics of the Bile Acid Transporter Homologue ASBTNM

Apical sodium-dependent bile acid transporter (ASBT) catalyses uphill transport of bile acids using the electrochemical gradient of Na⁺ as the driving force. The crystal structures of two bacterial homologues ASBT_NM and ASBT_Yf have previously been determined, with the former showing an inward-facing conformation, and the latter adopting an outward-facing conformation accomplished by the substitution of the critical Na⁺-binding residue glutamate-254 with an alanine residue. While the two crystal structures suggested an elevator-like movement to afford alternating access to the substrate binding site, the mechanistic role of Na⁺ and substrate in the conformational isomerization remains unclear. In this study, we utilized site-directed alkylation monitored by in-gel fluorescence (SDAF) to probe the solvent accessibility of the residues lining the substrate permeation pathway of ASBT_NM under different Na⁺ and substrate conditions, and interpreted the conformational states inferred from the crystal structures. Unexpectedly, the crosslinking experiments demonstrated that ASBT_NM is a monomer protein, unlike the other elevator-type transporters, usually forming a homodimer or a homotrimer. The conformational dynamics observed by the biochemical experiments were further validated using DEER measuring the distance between the spin-labelled pairs. Our results revealed that Na⁺ ions shift the conformational equilibrium of ASBT_NM toward the inward-facing state thereby facilitating cytoplasmic uptake of substrate. The current findings provide a novel perspective on the conformational equilibrium of secondary active transporters.