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H W Brock

Publications and source records attributed to H W Brock.

At least 37 records · Page 2Linked to original sources

Suppressors of position-effect variegation in Drosophila melanogaster affect expression of the heterochromatic gene light in the absence of a chromosome rearrangement.

Suppressors of position-effect variegation (Su(var)s) in Drosophila melanogaster are usually studied in the presence of chromosomal rearrangements, which exhibit variegated expression of euchromatic genes moved near to, or heterochromatic genes moved away from, centromeric heterochromatin. However, the effects of Su(var) mutations on heterochromatic gene expression in the absence of a variegating re-arrangement have not yet been defined. Here we present a number of results which suggest that Su(var) gene products can interact to affect the expression of the light gene in its normal heterochromatic location. We initially observed that eye pigment was reduced in several Su(var) double mutants; the phenotype resembled that of light mutations and was more severe when only one copy of the light gene was present. This reduced pigmentation could be alleviated by a duplication for the light gene or by a reduction in the amount of cellular heterochromatin. In addition, the viability of most Su(var) double mutant combinations tested was greatly reduced in a genetic background of reduced light gene dosage, when extra heterochromatin is present. We conclude that Su(var) gene products can affect expression of the heterochromatic light gene in the absence of any chromosomal rearrangements. However, it is noteworthy that mutations in any single Su(var) gene have little effect on light expression; we observe instead that different pairings of Su(var) mutations are required to show an effect on light expression. Interestingly, we have obtained evidence that at least two of the second chromosome Su(var) mutations are gain-of-function lesions, which also suggests that there may be different modes of interaction among these genes. It may therefore be possible to use this more sensitive assay of Su(var) effects on heterochromatic genes to infer functional relationships among the products of the 50 or more known Su(var) loci.

Animals↗

The polyhomeotic locus of Drosophila melanogaster is transcriptionally and post-transcriptionally regulated during embryogenesis.

The polyhomeotic (ph) locus of Drosophila is a complex locus essential for the maintenance of segmental identity. Genetic analysis suggested that two independent units contribute to ph function. Comparison of genomic sequence shows that the ph locus has been duplicated, and that it contains proximal and distal transcription units. The proximal transcription unit encodes two embryonic mRNAs of 6.4 and 6.1 kb, and the distal unit encodes a 6.4-kb embryonic mRNA. The proximal and distal transcription units are differentially regulated at the mRNA level during development as shown by developmental Northern analysis. The distal protein is very similar to the proximal product, except for the absence of an amino terminal region, and a small region near the carboxy terminus. The long open reading frame in the distal cDNA does not begin with an ATG codon, and an internal ATG is used for a start codon. We show that the proximal protein occurs in two forms that are developmentally regulated, and that probably arise from use of two different initiator methionine codons. We find no evidence for differential binding of proximal and distal products to polytene chromosomes. Nevertheless, we show that mutations in the proximal and distal proteins have differing effects on regulation of a reporter under the control of a regulatory region from bithoraxoid, suggesting that ph proximal and distal proteins have different functions. These results show that the ph locus undergoes complex developmental regulation, and suggest that Polycomb group regulation may be more dynamic than anticipated.

Animals↗

A domain shared by the Polycomb group proteins Scm and ph mediates heterotypic and homotypic interactions.

The Sex comb on midleg (Scm) and polyhomeotic (ph) proteins are members of the Polycomb group (PcG) of transcriptional repressors. PcG proteins maintain differential patterns of homeotic gene expression during development in Drosophila flies. The Scm and ph proteins share a homology domain with 38% identity over a length of 65 amino acids, termed the SPM domain, that is located at their respective C termini. Using the yeast two-hybrid system and in vitro protein-binding assays, we show that the SPM domain mediates direct interaction between Scm and ph. Binding studies with isolated SPM domains from Scm and ph show that the domain is sufficient for these protein interactions. These studies also show that the Scm-ph and Scm-Scm domain interactions are much stronger than the ph-ph domain interaction, indicating that the isolated domain has intrinsic binding specificity determinants. Analysis of site-directed point mutations identifies residues that are important for SPM domain function. These binding properties, predicted alpha-helical secondary structure, and conservation of hydrophobic residues prompt comparisons of the SPM domain to the helix-loop-helix and leucine zipper domains used for homotypic and heterotypic protein interactions in other transcriptional regulators. In addition to in vitro studies, we show colocalization of the Scm and ph proteins at polytene chromosome sites in vivo. We discuss the possible roles of the SPM domain in the assembly or function of molecular complexes of PcG proteins.

Amino Acid Sequence↗

beta3-tubulin is directly repressed by the engrailed protein in Drosophila.

In Drosophila, Engrailed is a nuclear regulatory protein with essential roles during embryonic development. Although Engrailed is a transcription factor, little progress has been achieved in identifying its target genes. We report here the identification of an effector gene, the beta3-tubulin gene, as a direct target of Engrailed. The cytological location of beta3-tubulin, 60C, is a strong site of Engrailed binding on polytene chromosomes. Immunostaining analysis of a transgenic line containing a P[beta3-tubulin-lacZ] construct shows an additional site of Engrailed binding at the location of the transgene. Molecular analysis allowed identification of several Engrailed binding sites, both in vitro and in vivo, within the first intron of the beta3-tubulin locus. Engrailed binding sites identified in vitro are active in larvae. Furthermore, expression of beta3-tubulin is derepressed in the ectoderm of engrailed mutant embryos. Repression of beta3-tubulin by Engrailed is also obtained when Engrailed is ectopically expressed in embryonic mesoderm. Finally, two different sets of Engrailed binding sites are shown to be involved in the early and late regulation of beta3-tubulin by Engrailed during embryogenesis.

Animals↗

Locust ion transport peptide (ITP): primary structure, cDNA and expression in a baculovirus system.

Ion transport peptide (ITP) purified from locust nervous corpus cardiacum (CC) has previously been shown to stimulate salt and water reabsorption and inhibit acid secretion in the ileum of Schistocerca gregaria. We used the partial amino acid sequence of purified ITP to derive degenerate primers. These were used to amplify a cDNA from brain RNA using reverse transcription and the polymerase chain reaction (RtPCR). This sequence was extended using anchored PCR to yield a partial, 517bp cDNA clone. This cDNA encodes a putative ITP prohormone which could be cleaved at two dibasic amino acid sites to yield a 72 residue active amidated peptide. The deduced amino acid sequence from the cDNA agrees completely with the amino acid sequence and molecular mass (8564Da) derived from analysis of purified ITP. Relative to a family of crustacean hyperglycaemic hormones (CHH), all six cysteine residues and many other amino acid residues are conserved in ITP, establishing that ITP is a homologue. However, CHH, crab eyestalk and CC extracts from distantly related insects have no action, whereas CC extracts from closely related insects are active on the locust ITP assay, showing that the bioassay is selective. Insect Sf9 cells transfected with a baculovirus containing our partial cDNA secreted a potent stimulant of locust ileal transport, confirming that the peptide encoded by our ITP clone has biological activity. The mRNA for ITP is restricted to the brain and CC. Interestingly, a related mRNA is observed in other tissues which are not active on the ITP bioassay.

Amino Acid Sequence↗

Genetic interactions and dosage effects of Polycomb group genes of Drosophila.

The Polycomb (Pc) group of genes are required for maintenance of cell determination in Drosophila melanogaster. At least 11 Pc group genes have been described and there may be up to 40; all are required for normal regulation of homeotic genes, but as a group, their phenotypes are rather diverse. It has been suggested that the products of Pc group genes might be members of a heteromeric complex that acts to regulate the chromatin structure of target loci. We examined the phenotypes of adult flies heterozygous for every pairwise combination of Pc group genes in an attempt to subdivide the Pc group functionally. The results support the idea that Additional sex combs (Asx), Pc, Polycomblike (Pcl), Posterior sex combs (Psc), Sex combs on midleg (Scm), and Sex combs extra (Sce) have similar functions in some imaginal tissues. We show genetic interactions among extra sex combs (esc) and Asx, Enhancer of Pc, Pcl, Enhancer of zeste E(z), and super sex combs and reassess the idea that most Pc group genes function independently of esc. Most duplications of Pc group genes neither exhibit anterior transformations nor suppress the extra sex comb phenotype of Pc group mutations, suggesting that not all Pc group genes behave as predicted by the mass-action model. Surprisingly, duplications of E(z) enhance homeotic phenotypes of esc mutants. Flies with increasing doses of esc+ exhibit anterior transformations, but these are not enhanced by mutations in trithorax group genes. The results are discussed with respect to current models of Pc group function.

Alleles↗

polyhomeotic appears to be a target of engrailed regulation in Drosophila.

In Drosophila, Engrailed is a nuclear regulatory protein with essential roles in embryonic segmentation and in normal development of posterior compartments. One of its regulatory targets appears to be polyhomeotic (ph), a Polycomb group gene. We observed, by immunostaining, that Engrailed protein binds to the site of the polyhomeotic locus in region 2D of polytene chromosomes. The same analysis carried out on a transgenic line containing one copy of a P(ph-lacZ) construct shows an additional Engrailed-binding site at the location of the insert. In vivo, polyhomeotic depends on engrailed function in germ-band-elongated embryos, when engrailed and polyhomeotic genes are expressed in similar patterns. By in vitro immunoprecipitations and gel shift assays, we identified two classes of high affinity Engrailed-binding sites upstream of each of the two polyhomeotic transcription units. DNA fragments containing these sites were also immunoprecipitated from embryonic UV crosslinked chromatin in presence of anti-Engrailed antibody. These results suggest that polyhomeotic activation in germ-band-elongated embryos could be mediated by Engrailed-binding to these sites.

Animals↗

Mutations in some Polycomb group genes of Drosophila interfere with regulation of segmentation genes.

Mutations in several Polycomb (Pc) group genes cause maternal-effect or zygotic segmentation defects, suggesting that Pc group genes may regulate the segmentation genes of Drosophila. We show that individuals doubly heterozygous for mutations in polyhomeotic and six other Pc group genes show gap, pair rule, and segment polarity segmentation defects. We examined double heterozygous combinations of Pc group and segmentation mutations for enhancement of adult and embryonic segmentation defects. Posterior sex combs and polyhomeotic interact with Krüppel and enhance embryonic phenotypes of hunchback and knirps, and polyhomeotic enhances even-skipped. Surprisingly, flies carrying duplications of extra sex combs (esc), that were heterozygous for mutations of even-skipped (eve), were extremely subvital. Embryos and surviving adults of this genotype showed strong segmentation defects in even-numbered segments. Antibody studies confirm that expression of eve is suppressed by duplications of esc. However, esc duplications have no effect on other gap or pair rule genes tested. To our knowledge, this is only the second triplo-abnormal phenotype associated with Pc group genes. Duplications of nine other Pc group genes have no detectable effect on eve. Expression of engrailed (en) was abnormal in the central nervous systems of most Pc group mutants. These results support a role for Pc genes in regulation of some segmentation genes, and suggest that esc may act differently from other Pc group genes.

Abdomen↗

Screen for enhancers of Polycomb and Polycomblike in Drosophila melanogaster.

There are 11 Polycomb group genes known in Drosophila. These genes are negative regulators of homeotic gene expression, and may act by modifying chromatin structure. It is not clear how many members of the Polycomb group of genes exist. Many were discovered because of their homeotic phenotypes, or because they enhance homeotic mutations. Systematic screens for enhancers of Polycomb have identified previously known members of the Polycomb group. In an attempt to discover cytological locations of new Polycomb group genes, we crossed deletions uncovering about 20% of the genome to Polycomb-like and Polycomb and scored for enhancement of the extra sex combs phenotype. Haploidy for four regions, 36F7-37A, 43E18; 44B5-9, 70C2-6, and 70C6-15; 70D enhanced the extra sex comb phenotype associated with strong Polycomb group mutations. These regions have homeotic phenotypes either as homozygous embryos or heterozygous adults, or both. We also show that spalt enhances Polycomb group mutations. These results are discussed with respect to previous estimates of Polycomb group gene number.

Animals↗

Interactions of polyhomeotic with Polycomb group genes of Drosophila melanogaster.

The Polycomb (Pc) group genes of Drosophila are negative regulators of homeotic genes, but individual loci have pleiotropic phenotypes. It has been suggested that Pc group genes might form a regulatory hierarchy, or might be members of a multimeric complex that obeys the law of mass action. Recently, it was shown that polyhomeotic (ph) immunoprecipitates in a multimeric complex that includes Pc. Here, we show that duplications of ph suppress homeotic transformations of Pc and Pcl, supporting a mass-action model for Pc group function. We crossed ph alleles to all members of the Polycomb group, and to E(Pc) and Su(z)2 to look for synergistic effects. We observed extragenic noncomplementation between ph503 and Pc, Psc1 and Su(z)2(1) in females, and between ph409 and Sce1, ScmD1 and E(z)1 mutations in males, suggesting that these gene products might interact directly with ph. Males hemizygous for a temperature-sensitive allele, ph2, are lethal when heterozygous with mutants in Asx, Pc, Pcl, Psc, Sce and Scm, and with E(Pc) and Su(z)2. Mutations in trithorax group genes were not able to suppress the lethality of ph2/Y; Psc1/+ males. ph2 was not lethal with extra sex combs, E(z), super sex combs (sxc) or l(4)102EFc heterozygotes, but did cause earlier lethality in embryos homozygous for E(z), sxc and l(4)102EFc. However, ph503 did not enhance homeotic phenotypes of esc heterozygotes derived from homozygous esc- mothers. We examined the embryonic phenotypes of ph2 embryos that were lethal when heterozygous or homozygous for other mutations. Based on this phenotypic analysis, we suggest that ph may perform different functions in conjunction with differing subsets of Pc group genes.

Alleles↗

Polycomb and polyhomeotic are constituents of a multimeric protein complex in chromatin of Drosophila melanogaster.

The polycomb group (Pc-G) genes are responsible for maintaining the repressed state of homeotic genes during development. It has been suggested that the Pc-G exerts its transcriptional control by regulating higher order chromatin structure. In particular, the finding of genetic and molecular similarities to components involved in heterochromatin formation, led to the proposal that homeotic genes are permanently repressed by mechanisms similar to those responsible for heterochromatin compaction. Because of synergistic effects, Pc-G gene products are thought to act in a multimeric complex. Using immunoprecipitation we show that two members of the Pc-G, Polycomb and polyhomeotic, are constituents of a soluble multimeric protein complex. Size fractionation indicates that a large portion of the two proteins are found in a distinct complex of molecular weight 2-5 x 10(6) Da. During embryogenesis the two proteins show the same spatial distribution. In addition, by double-immunofluorescence labelling we can demonstrate that Polycomb and polyhomeotic have exactly the same binding patterns on polytene chromosomes of larval salivary glands. We propose that some Pc-G proteins act in multimeric complexes to compact the chromatin of stably repressed genes like the homeotic regulators.

Animals↗

Genetic analysis of the additional sex combs locus of Drosophila melanogaster.

Additional sex combs (Asx) is a member of the Polycomb group of genes, which are thought to be required for maintenance of chromatin structure. To better understand the function of Asx, we have isolated nine new alleles, each of which acts like a gain of function mutation. Asx is required for normal determination of segment identity. AsxP1 shows an unusual phenotype in that anterior and posterior homeotic transformations are seen in the same individuals, suggesting that AsxP1 might upset chromatin structure in a way that makes both activation and repression of homeotic genes more difficult. Analysis of embryonic and adult phenotypes of Asx alleles suggests that Asx is required zygotically for determination of segment number and polarity. The expression pattern of even-skipped is altered in Asx mutant embryos, suggesting that Asx is required for normal expression of this gene. We have transposon-tagged the Asx gene, and can thus begin molecular analysis of its function.

Alleles↗

The polyhomeotic gene of Drosophila encodes a chromatin protein that shares polytene chromosome-binding sites with Polycomb.

The Polycomb group (PcG) genes in Drosophila melanogaster are required for maintenance of correct spatial expression of homeotic genes, and their products are thought to form either a regulatory network or act as a multimeric complex. Recently, it has been suggested that because of homology between Polycomb (Pc) and Su(var)205, PcG genes encode chromatin proteins required for the maintenance of a determined state in chromatin. The polyhomeotic (ph) gene is a member of the PcG of genes. We present DNA sequence of a ph cDNA, which encodes a 169-kD protein with a single putative zinc finger, a serine/threonine-rich region, and has glutamine repeats, suggesting that ph is a DNA-binding protein. Polyclonal antisera directed against ph protein bind to approximately 80 sites on polytene chromosomes. Most of these sites appear to be the same as those recognized by antibodies to Pc protein. ph protein binds to insertion sites of constructs containing DNA from the bithoraxoid (bxd) region of the Bithorax complex, showing that ph binding to chromatin is DNA dependent. The same bxd constructs are recognized by Pc protein, strongly supporting the hypothesis that ph and Pc interact directly.

Amino Acid Sequence↗

The complex genetic locus polyhomeotic in Drosophila melanogaster potentially encodes two homologous zinc-finger proteins.

Differential expression of the homeotic gene complexes, ANT-C and BX-C, of Drosophila melanogaster is partly controlled by trans-regulating factors located outside the two complexes. The complex genetic locus, polyhomeotic (ph), is one of these trans regulators required during development for correct expression of the homeotic selector genes. The ph locus comprises two genetically independent units whose functions are largely redundant. There are two duplicated sequences arranged as a tandem repeat in the ph region, defining two molecular ph units. Sequence analysis of the 28.6 kb of DNA comprising the locus shows varying degrees of sequence conservation between these two molecular units. Long open reading frames with a high degree of conservation have been localized in each tandem repeat. Putative protein products encoded by both the proximal and the distal unit contain several identical or practically identical protein domains: a zinc-finger-forming motif, an alpha-helix motif, a domain rich in serine and threonine residues and stretches of glutamine residues. The presence of these protein domains supports the hypothesis that ph encodes a transcription factor that may function as part of a protein complex. Possible molecular mechanisms leading to the particular structure of the locus are discussed.

Amino Acid Sequence↗

A complex genetic locus, polyhomeotic, is required for segmental specification and epidermal development in D. melanogaster.

Two mutagenic events are required to make null mutations of polyhomeotic (ph), which suggests that the locus is complex. Amorphic mutations (ph degrees) die in mid-embryogenesis and completely lack ventral thoracic and abdominal epidermal derivatives, whereas single-event mutations lead to transformations similar to those of known dominant gain of function mutants in the Antennapedia and bithorax complexes. After a chromosomal walk, the ph gene was localized using deficiencies and ph mutations that result from DNA rearrangements. Hybridization analyses show that there are two large, duplicated sequences in the ph region, and DNA lesions affecting either one of these repeats alter the function of the ph locus. We propose a model that may account for this unusual functional organization.

Alleles↗

Sequence and genomic structure of ras homologues Dmras85D and Dmras64B of Drosophila melanogaster.

The ras homologues of Drosophila melanogaster located at 85D and 64B on the polytene chromosome map were cloned using the Ha-ras gene of Harvey murine sarcoma virus as a probe. The genomic sequences of Dmras85D and Dmras64B were determined and shown to differ from previously published sequences. Dmras85D is much more similar to the Ha-ras, Ki-ras, and N-ras genes than it is to either the Dmras64B gene or to the ras genes of Saccharomyces cerevisiae. Comparison of the Dmras85D genomic sequences with the previously published nucleotide sequence (Neuman-Silberberg et al., Cell 37 (1984) 1027-1033) shows that the positions of the two introns are not conserved relative to the positions of the introns in Dmras64B or in vertebrate ras genes. The Dmras64B and Dmras85D transcripts were analyzed by blot hybridization and shown to be dissimilar. The data suggest that the divergence of the Dmras genes was ancient, and that Dmras85D and Dmras64B have different functions.

Amino Acid Sequence↗

Evolution of P transposable elements: sequences of Drosophila nebulosa P elements.

P elements have been cloned and sequenced from Drosophila nebulosa. Their sequences have diverged less than 6% from P elements of Drosophila melanogaster. However D. nebulosa P elements have nucleotide changes that close all four open reading frames found in the D. melanogaster P element. Microinjection experiments show that D. nebulosa P elements cannot provide transposase function for D. melanogaster P elements, nor are D. nebulosa P elements mobilized by the transposase provided by a D. melanogaster P factor. Three D. nebulosa P elements appear to have integrated into the same position of a complex, centromeric repeated sequence. Comparison of nucleotide sequences suggests that D. nebulosa P elements have diverged upon different pathways from a common ancestor that was 99% homologous to the P elements of D. melanogaster.

Base Sequence↗

Distribution and conservation of mobile elements in the genus Drosophila.

Essentially nothing is known of the origin, mode of transmission, and evolution of mobile elements within the genus Drosophila. To better understand the evolutionary history of these mobile elements, we examined the distribution and conservation of homologues to the P, I, gypsy, copia, and F elements in 34 Drosophila species from three subgenera. Probes specific for each element were prepared from D. melanogaster and hybridized to genomic DNA. Filters were washed under conditions of increasing stringency to estimate the similarity between D. melanogaster sequences and their homologues in other species. The I element homologues show the most limited distribution of all elements tested, being restricted to the melanogaster species group. The P elements are found in many members of the subgenus Sophophora but, with the notable exception of D. nasuta, are not found in the other two subgenera. Copia-, gypsy-, and F-element homologues are widespread in the genus, but their similarity to the D. melanogaster probe differs markedly between species. The distribution of copia and P elements and the conservation of the gypsy and P elements is inconsistent with a model that postulates a single ancient origin for each type of element followed by mating-dependent transmission. The data can be explained by horizontal transmission of mobile elements between reproductively isolated species.

Animals↗