Expansion of the ISWI chromatin remodeler family with new active complexes

ISWI chromatin remodelers mobilize nucleosomes to control DNA accessibility. Complexes isolated to date pair one of six regulatory subunits with one of two highly similar ATPases. However, we find that each endogenously expressed ATPase co‐purifies with every regulatory subunit, substantially increasing the diversity of ISWI complexes, and we additionally identify BAZ2B as a novel, seventh regulatory subunit. Through reconstitution of catalytically active human ISWI complexes, we demonstrate that the new interactions described here are stable and direct. Finally, we profile the nucleosome remodeling functions of the now expanded family of ISWI chromatin remodelers. By revealing the combinatorial nature of ISWI complexes, we provide a basis for better understanding ISWI function in normal settings and disease.     

Plasmodium falciparum: in vitro characterization and human infectivity of a cloned line.

The culture-adapted NF54 isolate of Plasmodium falciparum was subjected in vitro to three sequential limiting dilution titrations and the resulting clone was given the designation CVD1. DNA sequence analysis of the gene encoding the circumsporozoite (CS) protein revealed differences between CVD1 and the published NF54 CS gene. CVD1 had 1191 bp, 397 amino acids, and 42 repeat units while NF54 had 1218 bp, 405 amino acids, and 44 repeat units. The CVD1 clone was more sensitive to chloroquine than was the parental line, in vitro. Anopheles stephensi mosquitoes were infected equally by the cloned and uncloned parasites. Volunteers were readily infected by NF54 and CVD1 following infectious mosquito bites. The availability of a well-characterized, chloroquine-sensitive clone which safety infects humans should facilitate performance of experimental challenge studies to assess vaccine efficacy.     

Fine epitope mapping of anti-epidermal growth factor receptor antibodies through random mutagenesis and yeast surface display

Fine epitope mapping of therapeutically relevant monoclonal antibodies (mAbs) specific for the epidermal growth factor receptor (EGFR) was accomplished through random mutagenesis and yeast surface display. Using this method, we have identified key residues energetically important for the binding of EGFR to the mAbs 806, 225, and 13A9. A yeast-displayed library of single point mutants of an EGFR ectodomain fragment (residues 273-621) was constructed by random mutagenesis and was screened for reduced binding to EGFR mAbs. If an EGFR mutant showed loss of binding to a mAb, this suggested that the mutated residue was potentially a contact residue. The mAb 806 binding epitope was localized to one face of a loop comprised of EGFR residues Cys287-Cys302, which is constrained by a disulfide bond and two salt bridges. The mAb 806 epitope as identified here is not fully accessible in the autoinhibited EGFR monomer conformation, which is consistent with the hypothesis that mAb 806 binds to a transitional form of EGFR as it changes from an autoinhibited to extended monomer. The amino acids Lys465 and Ile467 were identified as energetic hot spot residues for mAb 225 binding to EGFR. These residues are adjacent to the EGFR ligand-binding site, which is consistent with the ability of mAb 225 to block binding of epidermal growth factor (EGF) and transforming growth factor-alpha (TGF-alpha) ligands. Ser468 and Glu472 were identified as energetically important for mAb 13A9 binding to EGFR, and the location of this epitope suggests that mAb 13A9 mediates observed TGF-alpha blocking effects through conformational perturbation of EGFR domain III. Combinatorial library screening of yeast-displayed mutagenic proteins is a novel method to identify discontinuous and heat-denaturable mAb binding epitopes with residue-level resolution.     

Horizontal transmission, vertical inactivation, and stochastic loss of mariner-like transposable elements

Horizontal transmission has been well documented as a major mechanism for the dissemination of mariner-like elements (MLEs) among species. Less well understood are mechanisms that limit vertical transmission of MLEs resulting in the "spotty" or discontinuous distribution observed in closely related species. In this article we present evidence that the genome of the common ancestor of the melanogaster species subgroup of Drosophila contained an MLE related to the mellifera (honey bee) subfamily. Horizontal transmission, approximately 3-10 MYA, is strongly suggested by the observation that the sequence of the MLE in Drosophila erecta is 97% identical in nucleotide sequence with that of an MLE in the cat flea, Ctenocephalides felis. The D. erecta MLE has a spotty distribution among species in the melanogaster subgroup. The element has a high copy number in D. erecta and D. orena, a moderate copy number in D. teissieri and D. yakuba, and was apparently lost ("stochastic loss") in the lineage leading to D. melanogaster, D. simulans, D. mauritiana, and D. sechellia. In D. erecta, most copies are concentrated in the heterochromatin. Two copies from D. erecta, denoted De12 and De19, were cloned and sequenced, and they appear to be nonfunctional ("vertical inactivation"). It therefore appears that the predominant mode of MLE evolution is vertical inactivation and stochastic loss balanced against occasional reinvasion of lineages by horizontal transmission.      

Effects on German cockroach nymphs of contact exposure to IGRs, singly and in combination

Late instar German cockroach male and female nymphs were exposed continuously for two weeks to surfaces treated with fenoxycarb, diflubenzuron, and pyriproxyfen, singly and in combination. Concentrations were determined that eliminated or nearly eliminated reproduction in matings with untreated mates, either through mortality, effects on reproduction, or a combination of mortality and sterility (no hatch). The major effect of fenoxycarb, pyriproxyfen, and pyriproxyfen plus fenoxycarb was on reproduction. The major effect of diflubenzuron was mortality. No hatch occurred in matings of females that were exposed to low concentrations of pyriproxyfen and fenoxycarb (2 ng/cm² and 6 ng/cm², respectively); sterility was incomplete when females were exposed to 600 ng/cm² of diflubenzuron. Mortality and sterility acted together to eliminate productive matings (matings that produced nymphs) when relatively high concentrations of diflubenzuron were combined with one or both of the other insect growth regulators (IGRs). In the triple combination, very small amounts of fenoxycarb and pyriproxyfen (total 1.1 ng/cm²) combined with 200 ng/cm² of diflubenzuron eliminated productive matings of treated females, but similar results with treated males were found only at higher concentrations of each IGR.     

Extended selections for pyrethroid resistance in the German cockroach (Dictyoptera: Blattellidae).

Selection experiments with a pyrethrins-susceptible and a pyrethrins-resistant strain of German cockroaches, Blattella germanica (L.), were conducted for 17 generations with either permethrin or fenvalerate as the selecting agent. Large nymphs were left on treated glass surfaces for extended periods of time each generation. Mortality was assessed at 24 h. The level of resistance was determined periodically by time-mortality testing. The VPI-susceptible strain served as the basis for comparison. The pyrethrins-susceptible strain developed resistance to pyrethrins early in the selection process; this strain ultimately became resistant to allethrin, phenothrin, permethrin, fenvalerate, cyfluthrin, and cypermethrin. Fenvalerate caused faster development of resistance than did permethrin. The pyrethrins-resistant strain, selected with fenvalerate, quickly became resistant to allethrin, permethrin, phenothrin, and fenvalerate. Ultimately, it developed resistance to all nine pyrethroids tested.     

Seasonal aspects of daily ration and diet of largemouth bass,Micropterus salmoides,with an evaluation of gastric evacuation rates

Synopsis Sample of stomach contents collected on 10 dates from May to October, 1978, were used to describe the diet and estimate the daily ration of subadult largemouth bass (primarily age-III fish) in Lake Rebecca, Minnesota. The method of Elliott & Persson (1978) was used to estimate daily ration. Data from sources in the literature were used to quantify gastric evacuation, which was found to be adequately described by an exponential decay model. The exponent of gastric evacuation increased exponentially with temperature. Seasonal changes in the diet with respect to composition, distribution of food among stomachs, and food particle size were reflected in the seasonal pattern of growth. Weight gain and the formation of scale annuli did not occur until the diet shifted from large bluegills and insects to age-0 largemouth bass and bluegills. Estimates of daily ration ranged from almost zero in mid-May and early June to over 5% in late August. The use of median weights of stomach contents was found to yield more meaningful estimates of the daily ration of individual bass than those based on means. Estimates based on medians were consistent with the observed pattern of growth and with information on maintenance rations and satiation levels. A growth-limiting lack of suitably-sized forage fish apparently occurred in the early part of the growing season.Paper Number 11,657, Scientific Journal Series, Minnesota Agricultural Experiment Station, St. Paul, MN. 55108.