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.     

Group versus population level demographics: An analysis of comparability using long term data on wild white‐faced capuchin monkeys (Cebus capucinus imitator)

Primates have long been used as indicator species for assessing overall ecosystem health. However, area‐wide census methods are time consuming, costly, and not always feasible under many field conditions. Therefore, it is important to establish whether monitoring a subset of a population accurately reflects demographic changes occurring in the population at large. Over the past 35 years, we have conducted 15 area‐wide censuses in Sector Santa Rosa, Costa Rica. These efforts have revealed important trends in population growth patterns of capuchin monkeys following the protection and subsequent regeneration of native forests. During this same period, we have also intensively studied a subset of the capuchin groups. Comparing these two datasets, we investigate whether the population structures of the closely monitored groups are reliable indicators of area‐wide demographic patterns. We compare the overall group size and the individual age/sex class compositions of study groups and nonstudy groups (i.e., those contacted during area‐wide censuses only). Our study groups contained more individuals overall with a larger proportion of infants, and there were indications that the proportion of adult and subadult males was lower. These differences can be ascribed either to sampling errors or real differences attributable to human presence and/or better habitat quality for the study groups. No other sex/age classes differed, and major demographic changes were simultaneously evident in both study and nonstudy groups. This study suggests that the Santa Rosa capuchin population is similarly impacted by large‐scale ecological patterns observable within our study groups.     

The position of the epistatic S locus in the halothane linkage group in pigs

The linkage of the Phi, Pgd, Po2, S, H and halothane sensitivity loci was followed in a Belgian Landrace family, heterozygous for these systems over 6 generations. Recombination next to the S locus occurred mainly in pigs belonging to this particular family. From this investigation the position of the S locus is proved to be outwith the Phi-Pgd region, next to Phi . Therefore the gene sequence S - Phi - Hal -H- Po2 -Pgd is proposed. Higher recombination rates were observed in the female parental line of the multiheterozygous family when compared to the male parental line. Additional data from animals, unrelated to this strain, confirm the evidence of close linkage of the S system to the nearest marker loci.     

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.

     

N-Terminal sequencing by mass spectrometry through specific fluorescamine labeling of α-amino groups before tryptic digestion

We present a single-step procedure for the specific mass labeling of unblocked protein N termini. We show that the dye fluorescamine, which is commonly assumed to require mildly alkaline conditions for undergoing a nonspecific reaction with α- and ε-amino groups associated with amino acids, in fact shows a specific reaction only with α-amino groups present at protein N termini when mildly acidic conditions are used. We use this finding to label, identify, and sequence the trypsinolysis-derived N-terminal peptide of lysozyme, using only mass spectrometry, to illustrate how this method could be used with other proteins.     

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.