Daniel Pauli

The dose of a putative ubiquitin-specific protease affects position-effect variegation in Drosophila melanogaster.

A dominant insertional P-element mutation enhances position-effect variegation in Drosophila melanogaster. The mutation is homozygous, viable, and fertile and maps at 64E on the third chromosome. The corresponding gene was cloned by transposon tagging. Insertion of the transposon upstream of the open reading frame correlates with a strong reduction of transcript level. A transgene was constructed with the cDNA and found to have the effect opposite from that of the mutation, namely, to suppress variegation. Sequencing of the cDNA reveals a large open reading frame encoding a putative ubiquitin-specific protease (Ubp). Ubiquitin marks various proteins, frequently for proteasome-dependent degradation. Ubps can cleave the ubiquitin part from these proteins. We discuss the link established here between a deubiquitinating enzyme and epigenetic silencing processes.

Heat shock response in Drosophila.

Major alterations in genetic activity have been observed in every organism after exposure to abnormally high temperatures. This phenomenon, called the heat shock response, was discovered in the fruit flyDrosophila. Studies with this organism led to the discovery of the heat shock proteins, whose genes were among the first eukaryotic genes to be cloned. Several of the most important aspects of the regulation of the heat shock response and of the functions of the heat shock proteins have been unraveled inDrosophila.

The Drosophila Sin3 gene encodes a widely distributed transcription factor essential for embryonic viability

Abstract Expression of many mammalian genes is activated by the binding of heterodimers of the Myc and Max proteins to specific DNA sequences called the E-boxes. Transcription of the same genes is repressed upon binding to the same sequences of complexes composed of Max, Mad/Mxi1, the co-repressors Sin3 and N-CoR, and the histone deacetylase Rpd3. Max-Mad/Mxi1 heterodimers, which bind to E-boxes in absence of co-repressors, do not inhibit gene expression simply by competition with Myc-Max heterodimers, but require Sin3 and Rpd3 for efficient repression of transcription. We have cloned a Drosophila homolog of Sin3 (dSin3) and found it to be ubiquitously expressed during embryonic development. Yeast, mouse and Drosophila proteins share six blocks of strong homologies, including four potential paired amphipathic helix domains. In addition, the domain of binding to the histone deacetylase Rpd3 is strongly conserved. Null mutations cause recessive embryonic lethality.

Characterization of HSP27 and three immunologically related polypeptides during Drosophila development

The low-molecular-weight heat-shock protein HSP27 is made in the absence of heat shock during Drosophila melanogaster development. An analysis of the accumulation of HSP27 during specific stages of development is presented using an antiserum recognizing this protein. Whereas HSP27 is abundant during embryogenesis, the level of this protein begins to decrease in the 20-h old embryo and is no longer detectable in second instar larvae. A high level of HSP27 is again observed in third instar larvae and reaches a maximal level in late pupae. While still abundant in young adult flies of both sexes, a greater amount of HSP27 is found in females with the protein being highly concentrated within the ovaries. Following lysis of whole pupae, about 60% of HSP27 is found in the soluble lysate fraction in a form which sediments between 5 and 20 S. Anti-HSP27 serum also recognizes three other developmentally regulated polypeptides with apparent MW of 33, 85 and 120 kDa. The 33 kDa protein accumulates in pupae while those of 85 and 120 kDa are more abundant in third instar larvae. Unlike HSP27, these proteins are not detected in embryos or ovaries. Immunoblot analysis of V8 proteolytic fragments suggests that HSP 27 and 33 kDa are related polypeptides. Exposure of the developing insect to heat-shock treatment results in increased level of HSP27. In larvae, a small amount of the 33 kDa protein accumulates following heat shock, while in pupae and adult flies a decrease in the concentration of this protein is observed after heat shock. Finally, different cellular localizations and distributions within the pupal body have been found for these developmentally regulated polypeptides.

Transcriptional regulation in Drosophila during heat shock: a nuclear run-on analysis

We used a nuclear run-on assay as a novel approach to study the changes in transcriptional activity that take place in Drosophila melanogaster during heat shock. In response to a rapid temperature upshift, total transcriptional activity in cultured K C161 cells decreased proportionally to the severity of the shock. After extended stress at 37° C (15 min or more), transcription was severely reduced, and at 39° C most transcription was instantaneously arrested. However, strikingly different responses were observed for individual genes. Transcription of histone H1 genes was severely inhibited even under mild heat shock conditions. Transcription of the actin 5C gene decreased progressively with increasing temperature, while transcription of the core histone genes or of the heat shock cognate genes was repressed only under severe heat shock conditions. Transcriptional activation of the D. melanogaster heat shock genes was also investigated. In unshocked cells, hsp84 was moderately transcribed, while transcriptional activity at the other protein-coding heat shock genes was undetectable (less than 0.2 polymerases per gene). Engaged but paused RNA polymerase molecules were found at the hsp70 and hsp26 genes, but not at the other heat shock genes. The rates of transcription increased with increasing temperature with a peak of expression at around 35° C. At 37° C, induction was less efficient, and no induction was achieved after a rapid shift to 39° C. Increased transcription of the heat shock genes was observed within 1–2 min of heat shock, and maximal rates were reached within 2–5 min. Despite very similar profiles of response, different heat shock genes were transcribed at strikingly different rates, which varied over a 20-fold range. The noncoding heat shock locus 93D was transcribed at a very high rate under non-heat shock conditions, and showed a transcriptional response to elevated temperatures different from that of protein-coding heat shock genes. An estimation of the absolute rates of transcription at different temperatures was obtained.

A GAL4-HP1 fusion protein targeted near heterochromatin promotes gene silencing

Abstract. We have constructed a new reporter transgene, Winkelried, equipped with a synthetic binding site for the yeast GAL4 transcriptional activator. The binding site is inserted between the white and lacZ reporter genes, and is flanked by FRT sequences. These elements allow excision of the GAL4 binding site by crossing the transgenic line with an FLP recombinase producing strain. We have generated by X-ray irradiation two independent chromosomal rearrangements, Heidi and Tell, relocating Winkelried next to pericentromeric heterochromatin. These rearrangements induce variegation of both white and lacZ. Variegation of Winkelried in the rearranged transgenic lines responds to the loss and excess of doses of the dominant suppressors of position-effect variegation (PEV) Su(var)3–7 and Su(var)2–5. Winkelried therefore constitutes a unique tool to test the effect on variegation in cis of any factor fused to the GAL4 DNA binding domain. Indeed, a chimeric protein, made of the DNA binding site of GAL4 and of HP1, the modifier of PEV encoded by Su(var)2–5, is shown to enhance variegation of Heidi and Tell. Excision of the binding sites for GAL4 in the variegating rearrangements Heidi and Tell abolishes the modifier effect of the GAL4-HP1 chimera. Therefore, in the Heidi and Tell rearrangements, enhancement of position-effect variegation depends strictly both on the concentration of GAL4-HP1 and on the presence of its binding site in the vicinity of the reporter genes.

Transcriptional activation by GAGA factor is through its direct interaction with dmTAF3

The GAGA factor (GAF), encoded by the Trithorax like gene (Trl) is a multifunctional protein involved in gene activation, Polycomb-dependent repression, chromatin remodeling and is a component of chromatin domain boundaries. Although first isolated as transcriptional activator of the Drosophila homeotic gene Ultrabithorax (Ubx), the molecular basis of this GAF activity is unknown. Here we show that dmTAF3 (also known as BIP2 and dTAF(II)155), a component of TFIID, interacts directly with GAF. We generated mutations in dmTAF3 and show that, in Trl mutant background, they affect transcription of Ubx leading to enhancement of Ubx phenotype. These results reveal that the gene activation pathway involving GAF is through its direct interaction with dmTAF3.

Drosophila OVO zinc-finger protein regulates ovo and ovarian tumor target promoters

Abstract The ovo+ and ovarian tumor+ genes function in the germline sex determination pathway in Drosophila, but the hierarchical relationship between them is unknown. We found that increased ovo+ copy number resulted in increased ovarian tumor expression in the female germline and increased ovo expression in the male germline. The ovo locus encodes C2H2 zinc-finger proteins. Bacterially expressed OVO zinc-finger domain bound to multiple sites at or near the ovo and ovarian tumor promoters strongly suggesting that OVO is directly autoregulatory and that ovarian tumor is a direct downstream target of ovo in the germline sex determination hierarchy. Both positive and negative regulation by OVO proteins appears likely, depending on promoter context and on the sex of the fly. Our observation that two strong OVO-binding sites are at the initiator of the TATA-less ovo-B and ovarian tumor promoters raises the possibility that OVO proteins influence the nucleation of transcriptional pre-initiation complexes.

A Drosophila heat shock gene from locus 67B is expressed during embryogenesis and pupation.

We present a detailed characterization of the structure and expression of gene 3 from the 67B locus of Drosophila melanogaster. Northern blot analysis reveals a major poly(A)+ transcript during two stages of development: mid-embryogenesis and beginning of pupation. After heat shock, the abundance of this mRNA is increased and small amounts of larger RNAs representing alternate terminations of the major transcript appear. In Schneider 3 tissue culture cells, beside the major transcript, we also observe small amounts of the larger RNAs after a heat shock. The sequencing of cDNA and genomic clones shows an intronless transcription unit with one long open reading frame. The deduced polypeptide has 169 amino acids. It shares a strong homology with the four small heat shock proteins in the region also conserved in the mammalian alpha-crystalline B2 chain. In gene 3, this homology is restricted to the first 50 residues along the conserved 83 amino acid stretch. Two heat shock regulatory elements are localized upstream from the gene.

Suppression of distinct ovo phenotypes in the Drosophila female germline by maleless− and Sex-lethalM†

Mutations in ovo result in several different phenotypes, which we show are due to the regulation of distinct developmental pathways. Two X (female) germ cells require ovo+ activity for viability, but 1X (male) germ cells do not. In our study, we observed suppression of the ovo germline-lethality phenotype in loss-of-function maleless (mle) females indicating that ovo+ and mle+ have opposing effects in female germ cells; or that they are hierarchically related. Gain-of-function Sex-lethal (Sxl) alleles and male specific lethal-2 alleles did not suppress the ovo germline death phenotype. Many of the surviving germ cells in females mutant for both ovo and mle showed ovarian tumors. In contrast to the germline viability phenotype, we did observe suppression of the tumor phenotype in females heterozygous for gain-of-function alleles of Sxl. Further, females mutant for some hypomorphic ovo alleles were rendered fertile by Sxl gain-of-function alleles. Thus, ovo+ is required for at least two distinct functions, one involving mle+, and one mediated by Sxl+ gene products. The existence of ovo+ functions independent of mle+ and Sxl+ is likely.