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.     

Role of electrostatics in the binding of charged metallophthalocyanines to neutral and charged phospholipid membranes

Binding of the cationic tetra(tributylammoniomethyl)-substituted hydroxoaluminum phthalocyanine (AlPcN₄) to bilayer lipid membranes was studied by fluorescence correlation spectroscopy (FCS) and intramembrane field compensation (IFC) methods. With neutral phosphatidylcholine membranes, AlPcN₄ appeared to bind more effectively than the negatively charged tetrasulfonated aluminum phthalocyanine (AlPcS₄), which was attributed to the enhancement of the coordination interaction of aluminum with the phosphate moiety of phosphatidylcholine by the electric field created by positively charged groups of AlPcN₄. The inhibitory effect of fluoride ions on the membrane binding of both AlPcN₄ and AlPcS₄ supported the essential role of aluminum-phosphate coordination in the interaction of these phthalocyanines with phospholipids. The presence of negative or positive charges on the surface of lipid membranes modulated the binding of AlPcN₄ and AlPcS₄ in accord with the character (attraction or repulsion) of the electrostatic interaction, thus showing the significant contribution of the latter to the phthalocyanine adsorption on lipid bilayers. The data on the photodynamic activity of AlPcN₄ and AlPcS₄ as measured by sensitized photoinactivation of gramicidin channels in bilayer lipid membranes correlated well with the binding data obtained by FCS and IFC techniques. The reduced photodynamic activity of AlPcN₄ with neutral membranes violating this correlation was attributed to the concentration quenching of singlet excited states as proved by the data on the AlPcN₄ fluorescence quenching.     

Activation of superoxide production and differential exocytosis in polymorphonuclear leukocytes by cytochalasins A,B, C,D and E: Effects of various ions

All of the common cytochalasins activate superoxide anion release and exocytosis of β-N-acetylglucosaminidase and lysozyme from guinea-pig polymorphonuclear leukocytes (neutrophils) incubated in a buffered sucrose medium. Half-maximal activation of both processes is produced by approx. 2 μM cytochalasin A, C >μM cytochalasin B ? 4–5 μM cytochalasin D, E. While maximal rates of O₂^? release and extents of exocytosis require extracellular calcium (1–2 mM), replacing sucrose with monovalent cation chlorides is inhibitory to neutrophil activation by cytochalasins. Na⁺, K⁺ or choline inhibited either cytochalasin B- or E-stimulated O₂^? production with IC₅₀ values of 5–10 mM and inhibition occurs whether Cl^?, NO₃^? or SCN^? is the anion added with Na⁺ or K⁺. Release of β-N-acetylglucosaminidase in control or cytochalasin B-stimulated cells is inhibited by NaCl (IC₅₀ ≈ 10 mM), while cytochalasin E-stimulated exocytosis is reduced less and K⁺ or choline chloride are ineffective in inhibiting either cytochalasin B- or E-stimulated exocytosis. Release of β-glucuronidase, myeloperoxidase or acid phosphatase from neutrophils incubated in buffered sucrose is not stimulated by cytochalasin B. Stimulation of either O₂^? or β-N-acetylglucosaminidase release by low concentrations of cytochalasin A is followed by inhibition of each at higher concentrations. It appears that all cytochalasins can activate both NAD(P)H oxidase and selective degranulation of neutrophils incubated in salt-restricted media and that differential inhibition of these two processes by monovalent cations and/or anions is produced at some step(s) subsequent to cytochalasin interaction with the cell.     

Soil and Fertilizer Amendments and Edge Effects on the Floral Succession of Pulverized Fuel Ash

Plots of fresh pulverized fuel ash (PFA, an industrial waste) were inoculated with soils from existing PFA sites and fertilizers in a factorial design, then left unmanaged for 12 years during which time the floral development and soil chemistry were monitored annually. For the first 3 years, the site supported a sparse mix of chenopods (including the scarce Chenopodium glaucum ) and halophytes. As salinity declined, ruderals, legumes, and grasses plus the fire-site moss Funaria hygrometrica colonized, followed by Festuca arundinacea grassland (NVC community MG12) and Hippophae rhamnoides scrub. Dactylorhiza incarnata (orchidacea) appeared after 7 years, but only in plots that had received soil from existing orchid colonies. Four years later, a larger second generation of Dactylorhiza appeared, but only in the central zone of the site where vegetation was thinnest. By year 12, the site was dominated by coarse grasses and scrub, with early successional species persisting only in the sparsely vegetated center, where nitrate levels were lowest. This edge effect is interpreted as centripetal encroachment, a process of potentially wider concern for the conservation of low-fertility habitat patches. Overall, seed bank inoculation seems to have introduced few but desirable species ( D. incarnata , Pyrola rotundifolia , some halophytes, and annuals), whereas initial application of organic fertilizer had long-lasting (≥10 years) effects on cover and soil composition.     

Factors determining the reconstructive denaturation of proteins in sodium dodecyl sulfate solutions. Further circular dichroism studies on structural reorganization of seven proteins

The factors determining the onset and extent of reconstructive denaturation of proteins were considered by comparing circular dichroism (CD) data of seven proteins and previously published findings. The effects of sodium dodecyl sulfate (SDS) on the conformation of the following proteins were tested: lysozyme, the mitogens fromPhytolacca americana (fractions Pa2 and Pa4), lectin fromWistaria floribunda, ovine lutropin, a Bence Jones protein, and histone H2B. While the helix content of lysozyme was raised by SDS slightly, in the Bence Jones protein andW. floribunda lectin it increased from near zero to about 25–30%. In histone H2B the helix content was raised by SDS even to about 48%. However, no clear indication of helix formation could be observed in the mitogens and lutropin, even at low pH or 2.0–2.5. The tertiary structure of the proteins was perturbed by SDS. It was concluded that the reorganization of secondary structure of the proteins was favored by the following factors: (1) presence of helicogenic amino acid sequences in the protein, (2) availability of positively charged sites of the basic amino acids for interactions with the dodecyl ion, (3) absence of a large surplus of negatively charged sites on the surface of protein, and (4) absence of extensive disulfide cross-linking within the macromolecule. Both hydrophobic and electrostatic interactions occur in reconstructive denaturation, and the newly formed helices are stabilized by hydrophobic shielding by the alkyl chains of the alkyl sulfate.     

Detection of genetic variants affecting cattle behaviour and their impact on milk production: a genome‐wide association study

Behaviour traits of cattle have been reported to affect important production traits, such as meat quality and milk performance as well as reproduction and health. Genetic predisposition is, together with environmental stimuli, undoubtedly involved in the development of behaviour phenotypes. Underlying molecular mechanisms affecting behaviour in general and behaviour and productions traits in particular still have to be studied in detail. Therefore, we performed a genome‐wide association study in an F₂ Charolais × German Holstein cross‐breed population to identify genetic variants that affect behaviour‐related traits assessed in an open‐field and novel‐object test and analysed their putative impact on milk performance. Of 37 201 tested single nucleotide polymorphism (SNPs), four showed a genome‐wide and 37 a chromosome‐wide significant association with behaviour traits assessed in both tests. Nine of the SNPs that were associated with behaviour traits likewise showed a nominal significant association with milk performance traits. On chromosomes 14 and 29, six SNPs were identified to be associated with exploratory behaviour and inactivity during the novel‐object test as well as with milk yield traits. Least squares means for behaviour and milk performance traits for these SNPs revealed that genotypes associated with higher inactivity and less exploratory behaviour promote higher milk yields. Whether these results are due to molecular mechanisms simultaneously affecting behaviour and milk performance or due to a behaviour predisposition, which causes indirect effects on milk performance by influencing individual reactivity, needs further investigation.     

Effects of larval competition on survival and growth in Mediterranean fruit flies

1. Larval success was compared when one, two, or three egg clutches were laid in kumquat fruits (≈ 10 ml in volume) either successively on the same day or at the rate of one clutch per day. 2. Increased clutch density was associated with a significant decrease in larval survival rate and non‐significant decreases in larval growth rate and pupal mass. 3. Larval and pupal parameters showed significantly larger variance when clutches were laid on successive days than on the same day, suggesting a competitive advantage for older larvae over younger larvae. 4. The results suggest that, in small fruit, reduced fitness due to larval competition may act against possible fitness benefits due to social facilitation among adult females, hence reducing the likelihood of non‐linear population dynamics caused by processes such as the Allee effect.