A Polycomb and Gaga Dependent Silencer Adjoins the Fab-7 Boundary in the Drosophila Bithorax Complex

The homeotic genes of the Drosophila bithorax complex are controlled by a large cis-regulatory region that ensures their segmentally restricted pattern of expression. A deletion that removes the Frontabdominal-7 cis-regulatory region (Fab-7(1)) dominantly transforms parasegment 11 into parasegment 12. Previous studies suggested that removal of a domain boundary element on the proximal side of Fab-7(1) is responsible for this gain-of-function phenotype. In this article we demonstrate that the Fab-7(1) deletion also removes a silencer element, the iab-7 PRE, which maps to a different DNA segment and plays a different role in regulating parasegment-specific expression patterns of the Abd-B gene. The iab-7 PRE mediates pairing-sensitive silencing of mini-white, and can maintain the segmentally restricted expression pattern of a BXD, Ubx/lacZ reporter transgene. Both silencing activities depend upon Polycomb Group proteins. Pairing-sensitive silencing is relieved by removing the transvection protein Zeste, but is enhanced in a novel pairing-independent manner by the zeste(1) allele. The iab-7 PRE silencer is contained within a 0.8-kb fragment that spans a nuclease hypersensitive site, and silencing appears to depend on the chromatin remodeling protein, the GAGA factor.     

Enhancer Traps in the Drosophila Bithorax Complex Mark Parasegmental Domains

Eight P elements carrying a β-galactosidase (lacZ) reporter have been mapped to sites within the Drosophila bithorax complex. The bithorax complex contains three homeotic genes, and at least nine regulatory regions which control their expression in successive parasegments of the fly. The enhancer traps inserted at the promoter of one of the genes, Ultrabithorax, express lacZ in patterns which mimic the Ultrabithorax protein pattern. Enhancer traps in the regulatory regions do not mimic the endogenous genes, but express lacZ globally in the relevant parasegments. Some P elements carry large DNA fragments upstream of the lacZ promoter but internal to the P element. In cases where these internal sequences specify a lacZ pattern, that pattern is generally suppressed when the element is inserted in the bithorax complex. In embryos mutant for genes of the Polycomb group, the lacZ expression from the enhancer traps spreads to all segments. Thus, the enhancer traps reveal parasegmental domains that are maintained by Polycomb-mediated repression. Such domains may be realized by parasegmental differences in chromatin structure.     

Functional Requirements for Fab-7 Boundary Activity in the Bithorax Complex

Chromatin boundaries are architectural elements that determine the three-dimensional folding of the chromatin fiber and organize the chromosome into independent units of genetic activity. The Fab-7 boundary from the Drosophila bithorax complex (BX-C) is required for the parasegment-specific expression of the Abd-B gene. We have used a replacement strategy to identify sequences that are necessary and sufficient for Fab-7 boundary function in the BX-C. Fab-7 boundary activity is known to depend on factors that are stage specific, and we describe a novel ∼700-kDa complex, the late boundary complex (LBC), that binds to Fab-7 sequences that have insulator functions in late embryos and adults. We show that the LBC is enriched in nuclear extracts from late, but not early, embryos and that it contains three insulator proteins, GAF, Mod(mdg4), and E(y)2. Its DNA binding properties are unusual in that it requires a minimal sequence of >65 bp; however, other than a GAGA motif, the three Fab-7 LBC recognition elements display few sequence similarities. Finally, we show that mutations which abrogate LBC binding in vitro inactivate the Fab-7 boundary in the BX-C.     

The Mode of Action of the Bithorax Genes of Drosophila melanogaster

SYNOPSIS. The activity of the genes of the bithorax complex(BX-C) of Drosophila is necessary for the normal developmentof thoracic and abdominal segments. Primordia destined to formthe different segments of the fly are established in differentpositions prior to BX-C function. Different combinations ofBX-C genes are then activated in each position to specify forthe different segment types. In this process some BX-C genesact before others. I propose a temporal binary mechanism whichaccounts for the different combinations of active and inactiveBX-C genes in the thoracic segments.     

Bithorax Complex genes control alary muscle patterning along the cardiac tube of Drosophila

Cardiac specification models are widely utilized to provide insight into the expression and function of homologous genes and structures in humans. In Drosophila, contractions of the alary muscles control hemolymph inflow and support the cardiac tube, however embryonic development of these muscles remain largely understudied. We found that alary muscles in Drosophila embryos appear as segmental pairs, attaching dorsally at the seven-up (svp) expressing pericardial cells along the cardiac dorsal vessel, and laterally to the body wall. Normal patterning of alary muscles along the dorsal vessel was found to be a function of the Bithorax Complex genes abdominal-A (abd-A) and Ultrabithorax (Ubx) but not of the orphan nuclear receptor gene svp. Ectopic expression of either abd-A or Ubx resulted in an increase in the number of alary muscle pairs from seven to 10, and also produced a general elongation of the dorsal vessel. A single knockout of Ubx resulted in a reduced number of alary muscles. Double knockouts of both Ubx and abd-A prevented alary muscles from developing normally and from attaching to the dorsal vessel. These studies demonstrate an additional facet of muscle development that depends upon the Hox genes, and define for the first time mechanisms that impact development of this important subset of muscles.     

The Border Between the Ultrabithorax and abdominal-A Regulatory Domains in the Drosophila Bithorax Complex

The bithorax complex in Drosophila melanogaster includes three homeobox-containing genes—Ultrabithorax (Ubx), abdominal-A (abd-A), and Abdominal-B (Abd-B)—which are required for the proper differentiation of the posterior 10 segments of the body. Each of these genes has multiple distinct regulatory regions; there is one for each segmental unit of the body plan where the genes are expressed. One additional protein- coding gene in the bithorax complex, Glut3, a sugar-transporter homolog, can be deleted without phenotype. We focus here on the upstream regulatory region for Ubx, the bithoraxoid (bxd) domain, and its border with the adjacent infraabdominal-2 (iab-2) domain, which controls abdA. These two domains can be defined by the phenotypes of rearrangement breakpoints, and by the expression patterns of enhancer traps. In D. virilis, the homeotic cluster is split between Ubx and abd-A, and so the border can also be located by a sequence comparison between species. When the border region is deleted in melanogaster, the flies show a dominant phenotype called Front-ultraabdominal (Fub); the first abdominal segment is transformed into a copy of the second abdominal segment. Thus, the border blocks the spread of activation from the bxd domain into the iab-2 domain.     

Genetic interactions between the Polycomb locus and the Antennapedia and Bithorax complexes of Drosophila

Summary We have studied the embryonic and adult phenotypes of genetic combinations between Polycomb (Pc), Regulator of bithorax (Rg-bx) and the genes of the Bithorax complex (BX-C) and the Antennapedia complex (ANT-C). The products of Pc and Rg-bx genes act antagonistically, their mutant combinations leading to the ectopic expression of genes of the BX-C and ANT-C. The genetic analysis of the Pc locus suggests it is a complex gene. Pc⁺ products behave as members of a regulatory set that negatively control the expression of BX-C and ANT-C genes. Genetic combinations between different doses of Pc, Rg-bx and the genes of the BX-C and ANT-C have phenotypes which may be interpreted as resulting from ectopic derepression of posterior selector genes repressing selector genes of anterior segments. The transformation phenotypes of certain genetic combinations differ in embryos and adults. A model of regulation of the BX-C and the ANT-C genes during the imaginal cell proliferation is presented, in which the specification state is maintained by self-activation of a given selector gene and down modulation of other selector genes in the same cell.     

Transvection in the Iab-5,6,7 Region of the Bithorax Complex of Drosophila: Homology Independent Interactions in Trans

The Abdominal-B (Abd-B) gene of the bithorax complex (BX-C) of Drosophila controls the identities of the fifth through seventh abdominal segments and segments in the genitalia (more precisely, parasegments 10-14). Here we focus on iab-5, iab-6 and iab-7, regulatory regions of Abd-B that control expression in the fifth, sixth and seventh abdominal segments (parasegments 10-12). By analysis of partial BX-C deficiencies, we show that these regions are able to promote fifth and sixth abdominal segment identities in the absence of an Abd-B gene in cis. We establish that this ability does not result from cis-regulation of the adjacent abd-A or Ubx genes of the BX-C but rather occurs because the iab-5,6,7 region is able to interact with Abd-B in trans. We demonstrate that this interaction is proximity dependent and is, therefore, a case of what E. B. LEWIS has called transvection. Interactions of this type are presumably facilitated by the synapsis of homologues that occurs in somatic cells of Dipterans. Although transvection has been detected in a number of Drosophila genes, transvection of the iab-5,6,7 region is exceptional in two ways. First, interaction in trans with Abd-B does not require that homologues share homologous sequences within, or for some distance to either side of, the BX-C. This is the first case of transvection shown to be independent of local synapsis. A second unusual feature of iab-5,6,7 transvection is that it is remarkably difficult to disrupt by heterozygosity for chromosome rearrangements. The lack of requirement for local synapsis and the tenacity of trans-interaction argue that the iab-5,6,7 region can locate and interact with Abd-B over considerable distance. This is consistent with the normal role of iab-5,6,7, which must act over some 20-60 kb to influence its regulatory target in cis at the Abd-B promoter. Evidence is presented that trans-action of iab-5,6,7 requires, and may be mediated by, the region between distal iab-7 and Abd-B. Also, we show that iab-5,6,7 transvection is independent of the allelic state of zeste, a gene that influences several other cases of transvection. The long-range nature of interactions in trans between iab-5,6,7 and Abd-B suggests that similar interactions could operate effectively in organisms lacking extensive somatic pairing. Transvection may, therefore, be of more general significance than previously suspected.     

Probing domain swapping for the neuronal SNARE complex with electron paramagnetic resonance

Highly conserved soluble N-ethylmaleimide sensitive factor attachment protein receptor (SNARE) proteins control membrane fusion at synapses. The target plasma membrane-associated SNARE proteins and the vesicle-associated SNARE protein assemble into a parallel four-helix bundle. Using a novel EPR approach, it is found that the SNARE four-helix bundles are interconnected via domain swapping that is achieved by substituting one of the two SNAP-25 helices with the identical helix from the second four-helical bundle. Domain swapping is likely to play a role in the multimerization of the SNARE complex that is required for successful membrane fusion. The new EPR application employed here should be useful to study other polymerizing proteins.     

Seamless cloning and domain swapping of synthetic and complex DNA

The use of synthetic DNA can avoid problems that are sometimes encountered with conventional molecular biology techniques using DNA with high GC content, strong secondary structures, or repeat sequences. However, very complex DNA may still resist PCR and synthesis of DNA from oligonucleotides. In the method described here, separately synthesized DNA segments were seamlessly joined independently of the presence of restriction sites in the target DNA. This method allowed the reconstruction of complex DNA by concatenation of easily synthesized segments and permitted repeated swapping of segments, from a few nucleotides to large fragments of complex DNA for phenotypic analysis.     

DNA strand displacement, strand annealing and strand swapping by the Drosophila Bloom's syndrome helicase

Genetic analysis of the Drosophila Bloom's syndrome helicase homolog (mus309/DmBLM) indicates that DmBLM is required for the synthesis-dependent strand annealing (SDSA) pathway of homologous recombination. Here we report the first biochemical study of DmBLM. Recombinant, epitope-tagged DmBLM was expressed in Drosophila cell culture and highly purified protein was prepared from nuclear extracts. Purified DmBLM exists exclusively as a high molecular weight (~1.17 MDa) species, is a DNA-dependent ATPase, has 3′→5′ DNA helicase activity, prefers forked substrate DNAs and anneals complementary DNAs. High-affinity DNA binding is ATP-dependent and low-affinity ATP-independent interactions contribute to forked substrate DNA binding and drive strand annealing. DmBLM combines DNA strand displacement with DNA strand annealing to catalyze the displacement of one DNA strand while annealing a second complementary DNA strand.     

Drosophila bipectinata species complex

The Drosophila bipectinata species complex belongs to the ananassae subgroup of the melanogaster species group (Genus Drosophila, Subgenus Sophophora). The members of the complex are: D. bipectinata, D. parabipectinata, D. malerkotliana, and D. pseudoananassae. Of the four species, D. bipectinata is most widely distributed. Females are indistinguishable, but males are distinguishable by their sex-comb teeth number and pattern and by abdominal colouration. Chromosomal inversions have been detected in these species. In natural populations of D. bipectinata the frequency of inversions and the level of inversion heterozygosity were found to be very low but in laboratory stocks inversions persisted for more than 20 generations due to heterotic buffering. On an average 9.3 fixed interspecific inversions separate each species pair. Non-random association between linked inversions indicated epistatic interaction in natural populations of D. bipectinata. Certain spontaneous mutations were detected and mapped for the first time in D. bipectinata. Low frequency of spontaneous male recombination has also been reported in D. bipectinata. Sexual isolation study in the complex indicated strong preference for homogamic mating. The results also indicated incomplete sexual isolation among different members of this complex. The isolation estimate among six different geographic populations of D. bipectinata ranged from 0.54 - 0.92 representing positive assortative mating which is an evidence for incipient sexual isolation. Incipient sexual isolation was also found within D. malerkotliana and D. parabipectinata . Chromosomal, hybridization and allozyme studies revealed close phylogenetic relationship among the four species of the bipectinata complex. Mitochondrial DNA study revealed net nucleotide difference (delta) between these species to be very small (0.0002 +/- 0.0008) reflecting closeness. Evidence for genetic control of sexual activity and existence of sexual selection in D. bipectinata has been shown on the basis of mating propensity tests carried out on geographic strains, their hybrids and diallel crosses. Significant variation was found among the strains tested with respect to courtship time, duration of copulation and fertility. A positive correlation between duration of copulation and fertility in D. bipectinata was found. Evidence for rare-male mating advantage was also found in D. bipectinata. A positive response to selection for high and low mating activity provided evidence for polygenic control of this phenomenon in D. bipectinata. Bilateral outgrowths on thorax, a unique phenotype, reported for the first time in D. bipectinata has been shown to affect mate recognition ability. Results of the study on pupation site preference (larval behaviour) and oviposition site preference (non-sexual behaviour) have also been included.     

Flexible navigational computations in the Drosophila central complex

Insects can perform impressive feats of navigation, suggesting a sophisticated sense of direction and an ability to choose appropriate trajectories toward ethological goals. The hypothesized substrate for these navigational abilities is the central complex (CX), a midline brain structure with orderly topology. The circuit transformations performed by the CX are now being concretely described by recent advances in the study of fruit fly neural circuits. An emerging theme is dynamic representation of navigational variables (e.g. heading or travel direction) computed in a manner distributed across specific neuronal populations. These representations are shaped by multimodal inputs whose weights evolve rapidly as surroundings change. Investigation of CX circuits is revealing with precise detail how structured wiring and synaptic plasticity enable neural circuits to flexibly subsample from the currently available sensory and motor cues to build a stable and accurate map of space. Given the sensory richness of natural environments, these findings are encouraging insect neuroscientists to no longer ask which cues insects use to navigate, but instead which cues can insects use, and under which contexts.