Nucleotide Sequence Comparison of the rp49 Gene Region between Drosophila subobscura and D. melanogaster.

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mRNA decay analysis in Drosophila melanogaster: Drug-induced changes in glutathione S-transferase D21 mRNA stability

We have established an in vivo system to investigate mechanisms by which pentobarbital (PB), a psychoactive drug with a sedative effect, changes the rate of decay of gstD21 mRNA (encoding a Drosophila glutathione S-transferase). Here we describe methods for the use of hsp70 promoter-based transgenes and transgenic lines to determine mRNA half-lives by RNase protection assays in Drosophila. We are able to identify and map putative decay intermediates by cRT-PCR and DNA sequencing of the resulting clones. Our results indicate that the 3′-UTR of gstD21 mRNA is responsive to PB by regulating mRNA decay and that the cis-acting element(s) responsible for the PB-mediated stabilization resides in a 59 nucleotide sequence in the 3′-UTR of the gstD21 mRNA (Akgül and Tu, 2007).National Institute of Environmental Health Sciences (NIEHS) (ES02678

Multiple transcripts from the Antennapedia gene of Drosophila melanogaster

Journal ArticleThe structures of four major transcripts from the homeotic gene Antennapedia of Drosophila melanogaster were determined. These transcripts constitute two RNA classes, each class initiating from a unique promoter but sharing 3' exons. Within the shared sequences is a major open reading frame encoding a 378-amino-acid protein as well as alternative polyadenylation sites. Although the RNA classes differ in their 5' sequences, both leaders contain many AUGs upstream of the major open reading frame. For the two RNA classes, neither gross tissue nor temporal specificity was observed. However, the second poly(A) site is preferred in neural tissue. The structural diversity of the RNAs is discussed in relation to biological functions of the Antennapedia locus

Regulated chromatin domain comprising cluster of co-expressed genes in Drosophila melanogaster

Recently, the phenomenon of clustering of co-expressed genes on chromosomes was discovered in eukaryotes. To explore the hypothesis that genes within clusters occupy shared chromatin domains, we performed a detailed analysis of transcription pattern and chromatin structure of a cluster of co-expressed genes. We found that five non-homologous genes (Crtp, Yu, CK2βtes, Pros28.1B and CG13581) are expressed exclusively in Drosophila melanogaster male germ-line and form a non-interrupted cluster in the 15 kb region of chromosome 2. The cluster is surrounded by genes with broader transcription patterns. Analysis of DNase I sensitivity revealed ‘open’ chromatin conformation in the cluster and adjacent regions in the male germ-line cells, where all studied genes are transcribed. In contrast, in somatic tissues where the cluster genes are silent, the domain of repressed chromatin encompassed four out of five cluster genes and an adjacent non-cluster gene CG13589 that is also silent in analyzed somatic tissues. The fifth cluster gene (CG13581) appears to be excluded from the chromatin domain occupied by the other four genes. Our results suggest that extensive clustering of co-expressed genes in eukaryotic genomes does in general reflect the domain organization of chromatin, although domain borders may not exactly correspond to the margins of gene clusters

Drosophila gene for antizyme requires ribosomal frameshifting for expression and contains an intronic gene for snRNP Sm D3 on the opposite strand

Journal ArticlePreviously, a Drosophila melanogaster sequence with high homology to the sequence for mammalian antizyme (ornithine decarboxylase antizyme) was reported. The present study shows that homology of this coding sequence to its mammalian antizyme counterpart also extends to a 5* open reading frame (ORF) which encodes the amino-terminal part of antizyme and overlaps the 11 frame (ORF2) that encodes the carboxy-terminal three-quarters of the protein. Ribosomes shift frame from the 5* ORF to ORF2 with an efficiency regulated by polyamines. At least in mammals, this is part of an autoregulatory circuit. The shift site and 23 of 25 of the flanking nucleotides which are likely important for efficient frameshifting are identical to their mammalian homologs. In the reverse orientation, within one of the introns of the Drosophila antizyme gene, the gene for snRNP Sm D3 is located. Previously, it was shown that two closely linked P-element transposon insertions caused the gutfeeling phenotype of embryonic lethality and aberrant neuronal and muscle cell differentiation. The present work shows that defects in either snRNP Sm D3 or antizyme, or both, are likely causes of the phenotype

Expression and function of the ultraspiracle (usp) gene locus during development in Drosophila melanogaster

The usp locus encodes a member of the nuclear hormone receptor superfamily in Drosophila melaaogaater that interacts with EcR (ecdysone receptor) to mediate ecdysteroid-induced gene expression. A 2.7-kb usp mRNA was detected at all developmental times tested, although its abundance varied. Among premetatnorphic stages, both the 2.7-kb transcript and Usp protein attained their highest levels in the late third larval instar. The 2.7-kb usp transcript was also found in adult stages and a 1.2-kb transcript was detected in the polyadenylated RNA fraction of both mature adult females and early embryos. Aneuploids carrying two asp mutant alleles and a putative variegating usp+ allele often developed deformities of the adult wing disc that apparently resulted from mutational disruption of uap activity before metamorphosis and whose frequency was affected by maternal genotype. Both of the recessive lethal uap mutations associated with this "cleft thorax" phenotype involved substitutions of conserved arginine residues in the DNA-binding domain, although the frequency of the phenotype was not the same for the two alleles. Both mutant proteins retained the ability to form heterodimers with EcR in vitro but showed reduced affinity for an ecdysone response element

The mode of action of intersex in terminal sexual differentiation in Drosophila.

The intersex gene (2-60.5) lies at the terminus of the regulatory pathway that determines sex-type in Drosophila. Its product functions with the female-specific product of doublesex, another gene in the self-determination regulatory pathway, to regulate female-specific differentiation. However, the mechanism of this regulation has not been clearly demonstrated. Using a temperature-sensitive allele at intersex to eliminate its function at time points both during development and in the adult stage, the mode by which intersex regulates female determination was addressed. When chromosomal females bearing a temperature-sensitive intersex allele are raised at a permissive temperature, they develop as phenotypic females. Animals raised at a restrictive temperature also develop as normal females. In contrast, animals kept at a restrictive temperature past the mid-pupal stage, or animals raised at a permissive temperature and then shifted to a restrictive temperature before the mid-pupal stage develop as sterile females. Therefore, the fertility to be retained, intersex function must be present at least until the mid-pupal stage. To determine if the function of intersex is also required in the adult to maintain the female differentiated state, as well as to address its mode of action, intersex function was eliminated in the adult female. To this end, diplo-X females bearing a temperature-sensitive intersex allele were place at a restrictive temperature as adults and used to analyze whether intersex exerts transcriptional control over the female-specific expression of the yolk protein gene, yp1. Even after placing animals bearing the temperature-sensitive allele at a restrictive temperature for up to twenty days, yp1 transcription persisted at levels equivalent to sibling controls having normal intersex function. These data suggest that intersex does not function to positively regulate female-specific gene expression, but does not rule out that intersex functions to repress male-type gene expression in females

Programmed cell death of primordial germ cells in Drosophila is regulated by p53 and the Outsiders monocarboxylate transporter

Primordial germ cell development uses programmed cell death to remove abnormal, misplaced or excess cells. Precise control of this process is essential to maintain the continuity and integrity of the germline, and to prevent germ cells from colonizing locations other than the gonads. Through careful analyses of primordial germ cell distribution in developing Drosophila melanogaster embryos, we show that normal germ cell development involves extensive programmed cell death during stages 10-12 of embryogenesis. This germ cell death is mediated by Drosophila p53 (p53). Mutations in p53 result in excess primordial germ cells that are ectopic to the gonads. Initial movements of the germ cells appear normal, and wild-type numbers of germ cells populate the gonads, indicating that p53 is required for germ cell death, but not migration. To our knowledge, this is the first report of a loss-of-function phenotype for Drosophila p53 in a non-sensitized background. The p53 phenotype is remarkably similar to that of outsiders (out) mutants. Here, we show that the out gene encodes a putative monocarboxylate transporter. Mutations in p53 and out show nonallelic noncomplementation. Interestingly, overexpression of p53 in primordial germ cells of out mutant embryos partially suppresses the out germ cell death phenotype, suggesting that p53 functions in germ cells either downstream of out or in a closely linked pathway. These findings inform models in which signaling between p53 and cellular metabolism are integrated to regulate programmed cell death decisions

The Ecdysone Regulatory Pathway Controls Wing Morphogenesis and Integrin Expression during Drosophila Metamorphosis

AbstractDrosophila imaginal discs are specified and patterned during embryonic and larval development, resulting in each cell acquiring a specific fate in the adult fly. Morphogenesis and differentiation of imaginal tissues, however, does not occur until metamorphosis, when pulses of the steroid hormone ecdysone direct these complex morphogenetic responses. In this paper, we focus on the role of ecdysone in regulating adult wing development during metamorphosis. We show that mutations in the EcR ecdysone receptor gene and crooked legs (crol), an ecdysone-inducible gene that encodes a family of zinc finger proteins, cause similar defects in wing morphogenesis and cell adhesion, indicating a role for ecdysone in these morphogenetic responses. We also show that crol and EcR mutations interact with mutations in genes encoding integrin subunits—a family of αβ heterodimeric cell surface receptors that mediate cell adhesion in many organisms. α-Integrin transcription is regulated by ecdysone in cultured larval organs and some changes in the temporal patterns of integrin expression correlate with the ecdysone titer profile during metamorphosis. Transcription of α- and β-integrin subunits is also altered in crol and EcR mutants, indicating that integrin expression is dependent upon crol and EcR function. Finally, we describe a new hypomorphic mutation in EcR which indicates that different EcR isoforms can direct the development of adult appendages. This study provides evidence that ecdysone controls wing morphogenesis and cell adhesion by regulating integrin expression during metamorphosis. We also propose that ecdysone modulation of integrin expression might be widely used to control multiple aspects of adult development

Two distinct pathways can control expression of the gene encoding the Drosophila antimicrobial peptide metchnikowin

Metchnikowin is a recently discovered proline-rich peptide from Drosophila with antibacterial and antifungal properties. Like most other antimicrobial peptides from insects, its expression is immune-inducible. Here we present evidence that induction of metchnikowin gene expression can be mediated either by the TOLL pathway or by the imd gene product. We show that the gene remains inducible in Toll-deficient mutants, in which the antifungal response is blocked, as well as in imd mutants, which fail to mount an antibacterial response. However, in Toll-deficient;imd double mutants, metchnikowin gene expression can no longer be detected after immune challenge. Our results suggest that expression of this peptide with dual activity can be triggered by signals generated by either bacterial or fungal infection. Cloning of the metchnikowin gene revealed the presence in the 5' flanking region of several putative cis-regulatory motifs characterized in the promoters of insect immune genes: namely, Rel sites, GATA motifs, interferon consensus response elements and NF-IL6 response elements. Establishment of transgenic fly lines in which the GFP reporter gene was placed under the control of 1.5 kb of metchnikowin gene upstream sequences indicates that this fragment is able to confer full immune inducibility and tissue specificity of expression on the transgene