Targeted mutagenesis of Drosophila RNaseZ gene by homologous recombination.

For the first time we used a homologous recombination method to obtain complete and precise deletion of Drosophila dRNaseZ gene. In the founder line of flies in which the RNaseZ sequence was replaced by attP site, the full-length sequence of the gene was reintegrated, and its functionality was shown. This approach will allow us to generate further gene mutations in different domains of dRNaseZ protein and discover a broad spectrum and uncover functions outside of tRNA processing.

Juvenile hormone signaling during oogenesis in Drosophila melanogaster.

Juvenile hormone (JH) participates both in the control of insect development and the establishment of reproductive maturity. In cultured Drosophila cells and in ovarian nurse cells, JH and its synthetic analog, methoprene, induce the expression of two related genes. These genes encode highly similar amino acid transport proteins that are homologous to transporters found in a variety of eukaryotes. JhI-21 is a novel Drosophila gene, and minidiscs (mnd) is a gene that was identified earlier. Two JH-inducible genes are regulated by different molecular mechanisms; JhI-21 behaves as a secondary JH-responsive gene, while mnd behaves as a primary responsive gene. Both JhI-21 and mnd transcripts show developmental profiles, which are consistent with JH regulation. Following eclosion, transcripts from JhI-21 and mnd are synthesized in ovarian nurse cells and subsequently sequestered in the mature egg. Their ectopic accumulation in ovaries can be induced by topical methoprene application. In apterous (ap4) mutant adults defective in JH secretion, mnd and JhI-21 RNA levels are severely reduced, but normal abundance is rescued to a high degree by topical methoprene treatment. Based on the evidence, we propose that during sexual maturation of Drosophila, JH provides a signal to the ovary that leads to the production of several maternally inherited mRNAs.

Selective binding of Drosophila BR-C isoforms to a distal regulatory element in the hsp23 promoter.

The Broad-Complex (BR-C) gene plays a key role in the ecdysone regulatory hierarchy. Together with other early ecdysone-inducible genes BR-C transmits the hormonal signal to a set of secondary response genes in a tissue-specific manner. Among its targets is the hsp23 gene. Previously we showed that expression of the hsp23 gene in late third instar is BR-C-dependent, and accompanied by the appearance of a BR-C-dependent DNase I hypersensitive site at position -1400 (DHS-1400). BR-C encodes a family of transcription factors, and we show here that at least three BR-C protein isoforms--Z1, Z2, and Z3--bind to the sequences around DHS-1400 in vitro. A DNase I footprinting assay reveals five protected regions, designated site 1 to site 5, each of which specifically associates with one or several BR-C protein isoforms. We also show that a 100 bp region overlapping site 5, which binds all three isoforms in vitro, is required for hsp23 activity in vivo. The deletion of binding site 5 in a reporter gene construct reproduced the effect of the npr class mutations, that is, hsp23 is no longer expressed in any tissue tested except brain. Thus, BR-C regulates hsp23 expression via direct interaction of the predominant isoform with the distal regulatory element.

The isolation of two juvenile hormone-inducible genes in Drosophila melanogaster.

Juvenile hormone (JH) is an important regulator of both insect development and reproductive maturation. Although the molecular mechanism of JH action is not yet known, there is growing circumstantial evidence that JH directly regulates gene expression. In the absence of a JH target gene, however, this suggestion has remained speculative. Cultured Drosophila S2 cells have been used to identify genes whose expression is regulated by JH. Employing differential display we identified several genes whose transcripts accumulate in cells treated with the JH agonist methoprene. Two of the genes-JhI-1 and JhI-26-were cloned and characterized in detail. For both genes, transcripts showed rapid and specific induction in the presence of either methoprene or JHIII, but not in the presence of other biologically inactive compounds of similar chemical structure. Accumulation of JhI-1 and JhI-26 RNAs requires continuous hormone presence. The developmental expression of the two JH-inducible genes corresponds to the abundance profile of JH in vivo. Furthermore, topical methoprene application to pupae leads to the ectopic accumulation of JhI-1 and JhI-26 transcripts.

The Broad-Complex gene is a tissue-specific modulator of the ecdysone response of the Drosophila hsp23 gene.

The steroid hormone ecdysone causes dramatic changes in the genetic programs leading to the pupariation of Drosophila melanogaster, and the Broad-Complex (BR-C) gene is known to play a key role in this process. Previously we showed that BR-C regulates developmental changes in transcription and chromatin structure of the 67B heat shock gene cluster, which contains four small hsp genes. Importantly, the downregulation of the hsp23 gene in the BR-C mutants correlates with the absence of a DNase I-hypersensitive site (DHS) at position -1400. To study the functional importance of the DHS-1400, we have introduced genomic fragments containing a modified hsp23 gene into the Drosophila germ line. Our analysis shows that the ecdysone response element is necessary but not sufficient for full developmental expression of hsp23 in the late third instar and that there is, indeed, another regulatory element, in the vicinity of DHS-1400. We also show that hsp23 developmental expression is not tissue specific. A construct lacking the ecdysone response element is unable to direct normal hsp23 expression in all tissues except the brain. Similarly, brain-specific expression is BR-C independent, although in the other tissues we find different requirements for BR-C genetic functions. The effect of the br mutations is restricted to wing imaginal discs and midgut tissue, while that of 2Bc is restricted to the fat body and Malpighian tubules, and mutations in the rbp group have no effect in any of the tissues studied. Thus, BR-C regulatory action is mediated through different genetic functions in a tissue-specific manner.

The Drosophila broad-complex regulates developmental changes in transcription and chromatin structure of the 67B heat-shock gene cluster.

At the end of the third larval instar of Drosophila, ecdysone triggers the tissue-specific activation of gene expression. In cytogenetic and molecular studies, the Broad-complex (BR-C) locus has been defined as a key element in the hierarchy of hormonal regulation of gene activity. Here we show that BR-C function is required for the regulation of the small hsp genes, which are clustered in the 67B puff and are known to be activated by heat shock and by ecdysone during development. We have found that the genes of the 67B cluster are expressed differentially in the salivary glands. While hsp23 and hsp27 transcripts accumulate at relatively high levels, those of hsp22 and hsp26 are present at low and intermediate levels, respectively. The complete BR-C deficiency as well as mutations of the npr class reduce the expression of genes hsp23 and hsp27 by 95 to 99%. The analysis of mutants representing two subfunctions of the BR-C-l(1)2Bab and l(1)2Bc, has shown that the latter is principally required for complete hsp induction. As sites of DNase I hypersensitivity in chromatin are believed to correspond to gene regulatory sequences, we have studied the changes of chromatin structure in the 67B region at different states of hsp gene activity. Upon hormonal induction, at the onset of metamorphosis, additional DNase I hypersensitive sites (DHS) appear in the 5' regions, four DHSs are associated with hsp23 and two with hsp27. We suggest that they are due to the binding of the hormone-receptor complex and/or transcription factors, related to ecdysone action. Finally, two DHSs (at -1400 of hsp23 and at -1200 of hsp27) are absent in the mutant nuclei, and thus may correspond to the target sequences for the BR-C-dependent regulatory protein(s).

GEBF-I activates the Drosophila Sgs3 gene enhancer by altering a positioned nucleosomal core particle.

The enhancer region of the Drosophila melanogaster ecdysone-regulated glue gene, Sgs3, shows dramatic modifications of chromatin structure in strict correlation with changes in gene expression during development. We show that there is a positioned nucleosomal core particle over the enhancer which is displaced or disrupted during gene activation. This transition is prevented in Drosophila larvae mutated in the ecdysone-dependent 2B5 locus, in which Sgs3 is inactive and GEBF-I, a Glue Enhancer Binding Factor, is missing. We have defined the GEBF-I binding sites in vitro and shown that mutation of these sequences abolishes the enhancer activity in vivo. This combined in vitro and in vivo approach reveals new aspects of the dynamic organization of a regulatory element during development and highlights the potential of this model for studies of the relation between chromatin structure and gene activity.

Trans-regulation of ecdysterone-induced protein synthesis in Drosophila melanogaster salivary glands.

A set of coordinately expressed genes is actively transcribed after a dramatic increase in the ecdysterone titer in late third-instar development of Drosophila melanogaster, as shown by the appearance of a number of puffs in salivary gland chromosomes and by the synthesis of a number of new proteins. Previous work has suggested that a product of the ecs gene, which is located within the 2B3-5 puff, is necessary for providing alterations in transcriptional activity at the sites of ecdysterone-dependent puffs. The experiments reported here were designed to determine whether the ecs gene's regulatory effect on puffing is confirmed by its regulatory effect on the synthesis of ecdysterone-inducible proteins (EIPs). The first series of experiments showed that in salivary glands in vivo ecdysterone induces 24 EIPs and in vitro induces 26 EIPs. The sets of polypeptides revealed are in good conformity. The second set of experiments demonstrated that mutations in the ecs locus disturb EIP synthesis: ecsl(1)t10 and ecsl(1)t143 mutations affect EIP synthesis to a lesser extent, while ecsl(1)t435 and ecsl(1)t324, as well as the 2B3-5 puff deficiency, prevent EIP synthesis completely. The experiments on dosage dependency revealed two EIPs whose rate of synthesis correlates with the dosage of the 2B3-5 X-chromosomal region. These EIPs are shown to be in fact small heat-shock proteins 23 and 28K, which are known to be encoded within the 67B puff and are under dual control--transient and developmental. The final set of experiments followed the 2B3-5 dosage dependency in vitro and showed that 15 EIPs display either an affected rate of synthesis or, mainly, a quicker induction time. Data obtained show that the ecs locus is trans-regulatory and that its product is necessary for spreading the effect of ecdysterone to other loci.