A trans-acting product needed for P factor transposition in Drosophila.

A transposable genetic element of the P family in Drosophila melanogaster was found to be unstable in the presence of other P elements but stable in their absence. A sensitive assay for P transpositional activity is provided by the snw allele, a defective P insert in the singed bristle locus which becomes hypermutable only in the presence of complete elements. This measure of activity was highly correlated with a type of female sterility normally associated with P activity. There was no cross-reactivity with transposase from another hybrid dysgenesis-causing element (the I factor).

Reversal of fortune for Drosophila geneticists?

Although Drosophila is a wonderful model organism, there is one molecular arena where it lags far behind its yeast and mouse model counterparts. Reverse genetics, whereby a piece of DNA is integrated into a target gene such that the gene is disrupted or replaced, is not easy in Drosophila. As Engels explains in his provocative Perspective, this may be set to change with the description of a new method for reverse genetics in Drosophila (Rong and Golic). This new technique should ensure that Drosophila remains the darling of geneticists for many years to come.

Site-specific X-chromosome rearrangements from hybrid dysgenesis in Drosophila melanogaster.

When the germ line of Drosophila males is destabilized by a syndrome known as hybrid dysgenesis, X-chromosome rearrangements are found in up to 10 percent of the gametes produced. Some of these aberrations are simple inversions, but many are complex multibreak rearrangements. Furthermore, most of the breakpoints fall into a few highly localized positions on the chromosome. These positions are mostly at points of intercalary heterochromatin and may vary from one strain to the next. the results suggest that they may represent points of insertion of mobile DNA sequences.

Sterility of Drosophila with mutations in the Bloom syndrome gene--complementation by Ku70.

The Drosophila Dmblm locus is a homolog of the human Bloom syndrome gene, which encodes a helicase of the RECQ family. We show that Dmblm is identical to mus309, a locus originally identified in a mutagen-sensitivity screen. One mus309 allele, which carries a stop codon between two of the helicase motifs, causes partial male sterility and complete female sterility. Mutant males produce an excess of XY sperm and nullo sperm, consistent with a high frequency of nondisjunction and/or chromosome loss. These phenotypes of mus309 suggest that Dmblm functions in DNA double-strand break repair. The mutant Dmblm phenotypes were partially rescued by an extra copy of the DNA repair gene Ku70, indicating that the two genes functionally interact in vivo.

Long-range cis preference in DNA homology search over the length of a Drosophila chromosome.

P element-induced chromosome breakage on the X chromosome of Drosophila melanogaster was repaired six times more frequently when a homologous template was located anywhere on the X chromosome rather than on an autosome. Cis-trans comparisons confirmed that recombinational repair was more frequent when the interacting sequences were physically connected. These results suggest that the search for homology between the broken ends and a matching template sequence occurs preferentially in the cis configuration. This cis advantage operates over more than 15 megabases of DNA.

Targeted gene replacement in Drosophila via P element-induced gap repair.

Transposable elements of the P family in Drosophila are thought to transpose by a cut-and-paste process that leaves a double-strand gap. The repair of such gaps resulted in the transfer of up to several kilobase pairs of information from a homologous template sequence to the site of P element excision by a process similar to gene conversion. The template was an in vitro-modified sequence that was tested at various genomic positions. Characterization of 123 conversion tracts provided a detailed description of their length and distribution. Most events were continuous conversion tracts that overlapped the P insertion site without concomitant conversion of the template. The average conversion tract was 1379 base pairs, and the distribution of tract lengths fit a simple model of gap enlargement. The conversion events occurred at sufficiently high frequencies to form the basis of an efficient means of directed gene replacement.

The first record on Brazilian stingless bees published 450 years ago by Hans Staden.

Only a few decades after 1492, when Christopher Columbus arrived on a Caribbean island and Pedro Alvares Cabral claimed Brazil for Portugal in 1500, a German mercenary gave the first description of stingless bees in 1557. He got to know them when he was imprisoned for months by an anthropophagous tribe in the coastal region of Santos, today in the State of São Paulo. This rather short but nevertheless extremely exact record on stingless bees is hidden in the first book on Brazil. Three species and important aspects of their life history were treated. This early description has been completely overlooked by bee scientists until now. My note intends to close this evident gap.

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.

Comparison of targeted-gene replacement frequencies in Drosophila melanogaster at the forked and white loci.

P element-induced gene conversion has been previously used to modify the white gene of Drosophila melanogaster in a directed fashion. The applicability of this approach of gene targeting in Drosophila melanogaster, however, has not been analyzed quantitatively for other genes. We took advantage of the P element-induced forked allele, f(hd), which was used as a target, and we constructed a vector containing a modified forked fragment for converting f(hd). Conversion frequencies were analyzed for this locus as well as for an alternative white allele, w(eh812). Combination of both P element-induced mutant genes allowed the simultaneous analysis of conversion frequencies under identical genetic, developmental, and environmental conditions. This paper demonstrates that gene conversion through P element-induced gap repair can be applied with similar success rates at the forked locus and in the white gene. The average conversion frequency at forked was 0.29%, and that at white was 0.17%. These frequencies indicate that in vivo gene targeting in Drosophila melanogaster should be applicable for other genes in this species at manageable rates. We also confirmed the homolog dependence of reversions at the forked locus, indicating that P elements transpose via a cut-and-paste mechanism. In a different experiment, we attempted conversion with a modified forked allele containing the su(Hw) binding site. Despite an increased sample size, there were no conversion events with this template. One interpretation (under investigation) is that the binding of the su(Hw) product prevents double-strand break repair.

Efficient copying of nonhomologous sequences from ectopic sites via P-element-induced gap repair.

P-element-induced gap repair was used to copy nonhomologous DNA into the Drosophila white locus. We found that nearly 8,000 bp of nonhomologous sequence could be copied from an ectopic template at essentially the same rate as a single-base substitution at the same location. An in vitro-constructed deletion was also copied into white at high frequencies. This procedure can be applied to the study of gene expression in Drosophila melanogaster, especially for genes too large to be manipulated in other ways. We also observed several types of more complex events in which the copied template sequences were rearranged such that the breakpoints occurred at direct duplications. Most of these can be explained by a model of double strand break repair in which each terminus of the break invades a template independently and serves as a primer for DNA synthesis from it, yielding two overlapping single-stranded sequences. These single strands then pair, and synthesis is completed by each using the other as a template. This synthesis-dependent strand annealing (SDSA) model as a possible general mechanism in complex organisms is discussed.

Comparative study of nest architecture and colony structure of the fungus-growing ants, Mycocepurus goeldii and M. smithii.

Nest architecture and demography of the non leaf-cutting fungus-growing ant species Mycocepurus goeldii and M. smithii (Attini: Formicidae) were studied in an agroforest habitat near Manaus, Brazil during the excavation of 13 nests. Both species built their nests in two different ways. The first type possessed a "tree-like" architecture, in which a vertical tunnel led downwards and lateral tunnels branched off at 90 degrees angles from the main tunnel, with a chamber at the end of each side branch. Alternatively, other nests displayed a "necklace-like" architecture, where the main tunnel also led down vertically, but entered each chamber from the top and exited it at the bottom, resulting in an architecture where chambers appeared like pearls on a necklace. The nest systems of M. goeldii and M. smithii consisted of 1-21 or 1-15 chambers, respectively. Of 199 excavated chambers, 57 % contained fungus-gardens. Chambers not containing fungus gardens were filled with organic matter from decaying fungus gardens or earthworm feces. Only M. smithii workers deposited loose soil in abandoned chambers during the construction of new nest chambers. Workers of M. smithii constructed significantly smaller chambers than those of M. goeldii. In both species, fungus garden-containing chambers were larger than non-garden chambers and were homogenously distributed in the soil between 17 cm and 105 cm depth. Neither fungus gardens nor abandoned chambers were encountered more frequently in deeper or shallower soil strata indicating that ants of both species did not abandon shallower versus deeper chambers, or move the colony to deeper soil layers with increasing colony age. Fungus gardens were suspended from the ceiling of the subterranean chambers and originated as small mycelial tufts. Through continual addition of organic debris, the tufts first grew vertically to strands before they expanded laterally until most of the chamber volume was filled with fungus garden curtains. New garden chambers were found at depths ranging from 17 to 83 cm, suggesting that new garden chambers were not constructed in deeper soil strata with increasing colony age. The size of M. goeldii and M. smithii colonies was dependent on their age. Worker counts varied between a few individuals in recently founded colonies and 1352 workers in an adult M. goeldii colony. The ratio of worker number per fungus garden chamber was higher in M. goeldii colonies than M. smithii colonies; the M. goeldii colonies were more populous. Both species were oligogynous with a maximum of four and three queens observed in a single chamber of M. goeldii and M. smithii, respectively. The reproductive status of each queen was unknown. In both species the ratio of brood to workers was approximately 2:3. Larvae and pupae were unequally distributed throughout the nest, but were only located in chambers containing a garden. Their location in the chamber was dependent upon the developmental state of the fungus garden.

Formation of prostanoids and hydroxy fatty acids by stimulated peritoneal mast cells: role of the dietary fat type in rat.

To study the influence of membrane fatty acid composition on the formation of prostanoids and hydroxy fatty acids by rat peritoneal mast cells (MC), animals were fed three different types of fatty acids: mackerel oil (MO), abundant in n-3 fatty acids; sunflower seed oil (SO), rich in linoleic acid; and hydrogenated coconut oil (HCO), mainly containing saturated fatty acids. The presence of n-3 fatty acids in the diet resulted in the incorporation of 20:5(n-3), 22:5(n-3) and 22:6(n-3) in MC phospholipids. A decrease of arachidonic acid, 20:4(n-6), was observed in MC-phospholipids of the MO-fed animals. Furthermore, increasing the relative amounts of 18:2(n-6) in the diet (SO group) led to an increased incorporation of linoleic acid, 18:2(n-6) in MC phospholipids when compared to both other dietary groups. The changes in MC phospholipid fatty acid composition were (partly) reflected in the formation of prostanoids and hydroxy fatty acids upon stimulation with the calcium ionophore A23187. The decrease in arachidonic acid content in MC phospholipids of MO-fed rats resulted in a decreased formation of PGD2 when compared to both other groups. Also, the increased amounts of 18:2(n-6) in MC phospholipids of SO-fed rats resulted in an increased formation of 9- and 13-HODE upon stimulation. The results show that modifications in the fatty acid composition of the diet influences MC membrane fatty acid composition which ultimately results in changes in prostanoid and hydroxy fatty acid synthesis by MC upon stimulation with the calcium ionophore A23187.

Differential release of histamine and prostaglandin D2 in rat peritoneal mast cells: roles of cytosolic calcium and protein tyrosine kinases.

We studied how the release of histamine and prostaglandin D2 (PGD2) were connected in stimulated rat peritoneal mast cells, and to what extent these processes were controlled by the cytosolic Ca2+ concentration, [Ca2+]i, and protein tyrosine kinases. In the presence of 1 mM CaCl2, the G-protein activating compound 48/80 (10 micrograms/ml) evoked a transient rise in [Ca2+]i and a relatively high secretion of histamine, but only a low release of PGD2. In contrast, 5 microM thapsigargin (an inhibitor of endomembrane Ca(2+)-ATPases) and 5 microM ionomycin evoked high and prolonged rises in [Ca2+]i, and stimulated the cells to release relatively small amounts of histamine and high amounts of PGD2. Stimulation of the cells with CaCl2 and 10 microM ATP4- gave only minor quantities of histamine and PGD2, despite of the micromolar level of [Ca2+]i reached. When CaCl2 was replaced by EGTA, rises in [Ca2+]i as well as release of histamine and PGD2 were reduced with each agonist, but the preference of agonists to release more histamine or PGD2 remained unchanged. In mast cells with depleted Ca2+ stores, the addition of CaCl2 stimulated the store-regulated Ca2+ entry resulting in a prolonged rise in [Ca2+]i. However, simultaneous addition of compound 48/80 and CaCl2 was required for release of histamine and PGD2. In cells with full stores, PGD2 release evoked by compound 48/80 was greatly reduced by genistein and methyl-2,5-dihydroxycinnamate, two structurally unrelated inhibitors of protein tyrosine kinases, whereas histamine secretion was not influenced by these inhibitors. Similarly, with thapsigargin or ionomycin as agonist, PGD2 release was more sensitive to the tyrosine kinase inhibitors than histamine secretion. We conclude that in activated rat peritoneal mast cells: (i) the influx of extracellular Ca2+ potentiates agonist-evoked rises in [Ca2+]i as well as histamine secretion and PGD2 release; (ii) the amplitude of the [Ca2+]i rise does not determine the preferential effect of agonists to release more histamine or more PGD2; (iii) the relatively high PGD2 release evoked by thapsigargin and ionomycin is probably due to their potency to evoke a prolonged rise in [Ca2+]i and to activate protein tyrosine kinases.

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.

Modified P elements that mimic the P cytotype in Drosophila melanogaster.

Activity of the P family of transposable elements in Drosophila melanogaster is regulated primarily by a cellular condition known as P cytotype. It has been hypothesized that P cytotype depends on a P element-encoded repressor of transposition and excision. We provide evidence in support of this idea by showing that two modified P elements, each with lesions affecting the fourth transposase exon, mimic most of the P cytotype effects. These elements were identified by means of two sensitive assays capable of detecting repression by a single P element. One assay makes use of cytotype-dependent gene expression of certain P element insertion mutations at the singed bristle locus. The other measures suppression of transposase activity from the unusually stable genomic P element, delta 2-3(99B), that normally produces transposase in both germinal and somatic tissues. The P cytotype-like effects include suppression of snw germline hypermutability, snw somatic mosaicism, pupal lethality, and gonadal dysgenic sterility. Unlike P cytotype, however, there was no reciprocal cross effect in the inheritance of repression.

P-element-induced male recombination and gene conversion in Drosophila.

A P-element insertion flanked by 13 restriction fragment length polymorphism (RFLP) marker sites was used to examine male recombination and gene conversion at an autosomal site. The great majority of crossovers on chromosome arm 2R occurred within the 4-kb region containing the P element and RFLP sites. Of the 128 recombinants analyzed, approximately two-thirds carried duplications or deletions flanking the P element. These rearrangements are described in more detail in the accompanying report. In a parallel experiment, we examined 91 gene conversion tracts resulting from excision of the same autosomal P element. We found the average tract length was 1463 bp, which is essentially the same as found previously at the white locus. The distribution of conversion tract endpoints was indistinguishable from the distribution of crossover points among the nonrearranged male recombinants. Most recombination events can be explained by the "hybrid element insertion" model, but, for those lacking a duplication or deletion, a second step involving double-strand gap repair must be postulated to explain the distribution of crossover points.

Formation of chromosome rearrangements by P factors in Drosophila.

We studied a collection of 746 chromosome rearrangements all induced by the activity of members of the P family of transposable elements in Drosophila melanogaster. The chromosomes ranged from simple inversions to complex rearrangements. The distribution of complex rearrangement classes was of the kind expected if each rearrangement came about from a single multibreak event followed by random rejoining of chromosome segments, as opposed to a series of two-break events. Most breakpoints occurred at or very near (within a few hundred nucleotide pairs) the sites of preexisting P elements, but these elements were often lost during the rearrangement event. There were also a few cases of apparent gain of P elements. In cases in which both breakpoints of an inversion retained P elements, that inversion was capable of reverting at high frequencies to the original sequence or something close to it. This reversion occurred with sufficient precision to restore the function of a gene, held-up-b, which had been mutated by the breakpoint. However, some of the reversions had acquired irregularities at the former breakpoints that were detectable either by standard cytology or by molecular methods. The revertants themselves retained the ability to undergo further rearrangements depending on the presence of P elements. We interpret these results to rule out the simplest hypotheses of rearrangement formation that involve cointegrate structures or homologous recombination. The data provide a general picture of the rearrangement process and its possible relationship to transposition.

Hybrid dysgenesis in Drosophila melanogaster: the biology of female and male sterility.

High levels of female and male sterility were observed among the hybrids from one of the two reciprocal crosses between a wild strain of D. melanogaster known as pi2 and laboratory strains. The sterility, which is part of a common syndrome called hybrid dysgenesis, was found to be associated with the rudimentary condition of one or both of the ovaries or testes. All other tissues, including those of the reproductive system were normal, as were longevity and mating behavior. The morphological details of the sterility closely mimic the agametic condition occurring when germ cells are destroyed by irradiation or by the maternal-effect mutation, grandchildless. We suggest that sterility in hybrid dysgenesis is also caused by failure in the early development of germ cells. There is a thermo-sensitive period beginning at approximately the time of initiation of mitosis among primordial germ cells a few hours before the egg hatches and ending during the early larval stages. Our results suggest that hybrid dysgenesis, which also includes male recombination, mutation and other traits, may be limited to the germ line, and that each of the primordial germ cells develops, or fails to develop, independently of the others. This hypothesis is consistent with the observed frequencies of unilateral and bilateral sterility, with the shape of the thermosensitivity curves and with the fact that males are less often sterile than females. The features of this intraspecific hybrid sterility are found to resemble those seen in some interspecific Drosophila hybrids, especially those from the cross D. melanogaster X D. simulans.

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 lest 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.

Evolution of the RECQ family of helicases: A drosophila homolog, Dmblm, is similar to the human bloom syndrome gene.

Several eukaryotic homologs of the Escherichia coli RecQ DNA helicase have been found. These include the human BLM gene, whose mutation results in Bloom syndrome, and the human WRN gene, whose mutation leads to Werner syndrome resembling premature aging. We cloned a Drosophila melanogaster homolog of the RECQ helicase family, Dmblm (Drosophila melanogaster Bloom), which encodes a putative 1487-amino-acid protein. Phylogenetic and dot plot analyses for the RECQ family, including 10 eukaryotic and 3 prokaryotic genes, indicate Dmblm is most closely related to the Homo sapiens BLM gene, suggesting functional similarity. Also, we found that Dmblm cDNA partially rescued the sensitivity to methyl methanesulfonate of Saccharomyces cerevisiae sgs1 mutant, demonstrating the presence of a functional similarity between Dmblm and SGS1. Our analyses identify four possible subfamilies in the RECQ family: (1) the BLM subgroup (H. sapiens Bloom, D. melanogaster Dmblm, and Caenorhabditis elegans T04A11.6); (2) the yeast RECQ subgroup (S. cerevisiae SGS1 and Schizosaccharomyces pombe rqh1/rad12); (3) the RECQL/Q1 subgroup (H. sapiens RECQL/Q1 and C. elegans K02F3.1); and (4) the WRN subgroup (H. sapiens Werner and C. elegans F18C5.2). This result may indicate that metazoans hold at least three RECQ genes, each of which may have a different function, and that multiple RECQ genes diverged with the generation of multicellular organisms. We propose that invertebrates such as nematodes and insects are useful as model systems of human genetic diseases.