P-element-induced interallelic gene conversion of insertions and deletions in Drosophila melanogaster.

We studied the process by which whd, a P-element insertion allele of the Drosophila melanogaster white locus, is replaced by its homolog in the presence of transposase. These events are interpreted as the result of double-strand gap repair following excision of the P transposon in whd. We used a series of alleles derived from whd through P-element mobility as templates for this repair. One group of alleles, referred to collectively as whd-F, carried fragments of the P element that had lost some of the sequences needed in cis for mobility. The other group, whd-D, had lost all of the P insert and had some of the flanking DNA from white deleted. The average replacement frequencies were 43% for whd-F alleles and 7% for the whd-D alleles. Some of the former were converted at frequencies exceeding 50%. Our data suggest that the high conversion frequencies for the whd-F templates can be attributed at least in part to an elevated efficiency of repair of unexpanded gaps that is possibly caused by the closer match between whd-F sequences and the unexpanded gap endpoints. In addition, we found that the gene substitutions were almost exclusively in the direction of whd being replaced by the whd-F or whd-D allele rather than the reverse. The template alleles were usually unaltered in the process. This asymmetry implies that the conversion process is unidirectional and that the P fragments are not good substrates for P-element transposase. Our results help elucidate a highly efficient double-strand gap repair mechanism in D. melanogaster that can also be used for gene replacement procedures involving insertions and deletions. They also help explain the rapid spread of P elements in populations.     

Oxide-Based Solid-State Batteries: A Perspective on Composite Cathode Architecture

The garnet-type phase Li₇La₃Zr₂O₁₂ (LLZO) attracts significant attention as an oxide solid electrolyte to enable safe and robust solid-state batteries (SSBs) with potentially high energy density. However, while significant progress has been made in demonstrating compatibility with Li metal, integrating LLZO into composite cathodes remains a challenge. The current perspective focuses on the critical issues that need to be addressed to achieve the ultimate goal of an all-solid-state LLZO-based battery that delivers safety, durability, and pack-level performance characteristics that are unobtainable with state-of-the-art Li-ion batteries. This perspective complements existing reviews of solid/solid interfaces with more emphasis on understanding numerous homo- and heteroionic interfaces in a pure oxide-based SSB and the various phenomena that accompany the evolution of the chemical, electrochemical, structural, morphological, and mechanical properties of those interfaces during processing and operation. Finally, the insights gained from a comprehensive literature survey of LLZO–cathode interfaces are used to guide efforts for the development of LLZO-based SSBs.     

Isolation and characterization of plasma membrane-associated cortical granules from sea urchin eggs

Cortical granules, which are specialized secretory organelles found in ova of many organisms, have been isolated from the eggs of the sea urchins Arbacia punctulata and Strongylocentrtus pupuratus by a simple, rapid procedure. Electron micropscope examination of cortical granules prepared by this procedure reveals that they are tightly attached to large segments of the plasma membrane and its associated vitelline layer. Further evidence that he cortical granules were associated with these cell surface layers was obtained by (125)I-labeling techniques. The cortical granule preparations were found to be rich in proteoesterase, which was purified 32-fold over that detected in a crude homogenate. Similarly, the specific radioactivity of a (125)I-labeled, surface glycoprotein was increased 40-fold. These facts, coupled with electron microscope observations, indicate the isolation procedure yields a preparation in which both the cortical granules and the plasma membrane-vitelline layer are purified to the same extent. Gel electrophoresis of the membrane-associated cortical granule preparation reveals the presence of at least eight polypeptides. The major polypeptide, which is a glycotprotein of apparent mol wt of 100,000, contains most of the radioactivity introduced by (125)I-labeling of the intact eggs. Lysis of the cortical granules is observed under hypotonic conditions, or under isotonic conditions if Ca(2+) ion is present. When lysis is under isotonic conditions is induced by addition of Ca(2+) ion, the electron-dense contents of the granules remain insoluble. In contrast, hypotonic lysis results in release of the contents of the granule in a soluble form. However, in both cases the (125)I-labeled glycoprotein remains insoluble, presumably because it is a component of either the plasma membrane or the vitelline layer. All these findings indicate that, using this purified preparation, it should be possible to carry out in vitro studies to better define some of the initial, surface-related events observed in vivo upon fertilization.     

Pleiotropic Effects on Fitness of Mutations Affecting Viability in DROSOPHILA MELANOGASTER

The relative viabilities and fitnesses of wild-type second chromosomes in heterozygous condition were determined. Joint analysis of these permitted an estimation of a parameter that relates the viability effect of a mutation to its effect on fitness as a whole. For newly arisen mutations, the estimate was slightly greater than one, indicating that the reductions in viability caused by these mutations are associated with reductions in other components of fitness. For mutations from an equilibrium population, the estimate of the parameter was near zero, implying that the deleterious viability effects of these mutations are compensated by improvements in other aspects of fitness.     

Components of Hybrid Dysgenesis in a Wild Population of DROSOPHILA MELANOGASTER

Hybrid dysgenesis is a condition found in certain interstrain hybrids of Drosophila melanogaster caused by the interaction of chromosomal and cytoplasmic factors. Germ-line abnormalities, including sterility, high mutability and male recombination, appear in the affected individuals. There are at least two distinct systems of hybrid dysgenesis. We examined a Wisconsin wild population in two consecutive years to determine the distribution of the chromosomal P factor and the extrachromosomal M cytotype that together cause one kind of hybrid dysgenic sterility. The P factor was found to be very common in the population, with all three major chromosomes being polymorphic for it. This polymorphism was strongly correlated with variability for male recombination elements, suggesting that these two traits are part of the same system of hybrid dysgenesis. There was a slight tendency for the P factor to be lost in lines taken from this population and inbred in the laboratory for many generations. A large-scale search for the M cytotype, which causes susceptibility to the P factor, showed that it is present in the population at only very low frequencies. Further evidence that the population is mostly immune to the action of the P factor was our finding of a general lack of dysgenic sterility in the wild flies themselves. However, we were able to isolate several wild strains that consistently showed the M cytotype. In some cases, the frequency of the M cytotype could be maintained in these lines, but it could not usually be increased by artificial selection. Some possible consequences of hybrid dysgenesis for the evolutionary biology of Drosophila are suggested.     

The nature of the competitive ability of spontaneous staphylocoagulase-negative mutants of Staphylococcus aureus with respect to growth of the parent strains in continuous culture

During prolonged cultivation of S. aureus strains 104 and NCTC 8178 in continuous culture, staphylocoagulase-negative mutants arose and accumulated progressively in increasing proportions. The resulting loss of production of staphylocoagulase was accompanied by a simultaneous loss of production of -haemolysin and PV-leucocidin. Characterization of the strains revealed no further differences in biotype, exoenzymes, phage pattern and plasmid content.Cultivation in batch cultures showed that the maximal specific growth rates and specific oxygen-consumption rates of the mutant strains were slightly higher than those of the parent strains, whereas the production of total extracellular protein of the mutant strains had decreased significantly.From competition experiments between parent and mutant strains in chemostat cultures at different dilution rates and cultivation temperatures, it was concluded that the underlying mechanism of accumulation of staphylocoagulase-negative mutants in the chemostat is based on differences in affinity for the limiting substrate(s) rather than on differences in the production rates of total extracellular proteins. The complete repression of three exoenzymes, a partial repression of the total extracellular protein production, and an increased affinity for the limiting substrate(s) suggested that a mutation in a regulatory gene is involved. The possible role of a transposon in this mutation is discussed.     

Human peptidylglycine alpha-amidating monooxygenase: cDNA, cloning and functional expression of a truncated form in COS cells

We have identified a cDNA encoding human peptidylglycine alpha-amidating monooxygenase (PAM; EC 1.14.17.3) with a total length of 3748 bp by screening of a human thyroid carcinoma lambda gt11 library using two heterologous oligonucleotides to conserved regions which derived from frog skin and bovine pituitary PAM sequences. Furthermore we have identified a sequence which differs in a 321 bp deletion. COS cells transfected with a truncated form of this cDNA (lacking the putative carboxyl-terminal transmembrane domain) generated a functional PAM that showed a 20-fold increase of the activity compared to the control and was visualized by immunoblotting.     

Germline hypermutability in Drosophila and its relation to hybrid dysgenesis and cytotype

In its hypermutable state, an unstable singed allele, sn^w, mutates in the germline to two other alleleic forms at a total frequency usually between 40 and 60%. In its stable state, the mutation rate of sn^w is essentially zero. Its state depends on an extrachromosomal condition indistinguishable from a property called cytotype previously studied as a component of hybrid dysgenesis. Of the two known systems of hybrid dysgenesis, denoted P-M and I-R, sn^w hypermutability is determined by the P-M system and appears to be independent of the I-R system. Cytotype, as defined by the control of sn^w mutability, is self-reproducing in the cytoplasm or nucleoplasm of the germline through at least two generations. However, it is not entirely autonomous, being ultimately determined by the chromosomes after sufficiently many generations of backcrossing. This combination of chromosomal and extrachromosomal transmission agrees well with previous studies on cytotype. Temperature differences have little effect on the mean mutation rates, but they have a pronounced effect on the intrinsic variance among individuals. The latter effect suggests that high temperatures reduce germ-cell survival during the development of dysgenic flies. Chromosomal rearrangements produce no apparent effects on the behavior of sn^w. Hypermutability is thought to be caused by the excision or other alteration of an inserted genetic element in the sn^w gene. This element might be a copy of the "P factor," which is though to be a mobile sequence capable of causing female sterility and other dysgenic traits in the P-M system.     

Aggregation-dependent turnover of flagellar adhesion molecules in chlamydomonas gametes

Previous studies on flagellar adhesion in chlamydomonas (Snell, W. and S. Roseman. 1979. J. Biol. Chem. 254:10820-10829.) have shown that as gametes adhere to flagella isolated from gametes of the opposite mating type, the adhsiveness of the added flagella but not of the gametes is lost. The studies reported here show that the addition of protein synthesis inhibitors (cycloheximide [CH] or anisomycin) to the medium of such cell- flagella mixtures causes the cells to lose their adhesiveness. This loss, however, occurs only after the cells have interacted with 4-8 flagella/cell and does not occur if the cells are kept in CH (7 h) without aggregating. The availability of an impotent (imp) mating type plus (MT(+)) mutant (provided by U.W. Goodenough), which adheres but is unable to undergo the fusion that normally follows adhesion, made it possible to determine whether a similar loss of adhesiveness occurs in mixtures of matting type minus (mt(-)) and imp mt(+) gametes. In the absence of inhibitor, mt(-) and imp mt(+) gametes adhered to each other (without fusing) for several hours; however, in the presence of CH or anisomycin, the gametes began to de-adhere 35 min after mixing, and, by 90 min, 100 percent of the cells were single again. This effect was reversible, and the rapid turnover of cells were single again. This effect was reversible, and the rapid turnover of molecules involved in adhesion occurred only during adhesion inasmuch as gametes pretreated for 4 h with CH were able to aggregate in CH for the same length of time as nonpretreated cells aggregated in CH. By the addition of CH at various times after the mt(-) and imp mt(+) gametes were mixed, measurements were made of the “pool size” of the molecules involved in adhesion. The pool reached a minimum after 25 min of aggregation, rapidly increased for the next 25 min, and then leveled off at the premixing level. These results suggest that flagellar adhesion in chlamydomonas causes modification of surface molecules (receptors, ligands), which brings about their inactivation and stimulates their replacement.