Embryonic development in Drosophila is characterized by an early phase during which a cellular blastoderm is formed and gastrulation takes place, and by a later postgastrulation phase in which key morphogenetic processes such as segmentation and organogenesis occur. We have focused on this later phase in embryogenesis with the goal of obtaining a comprehensive analysis of the zygotic gene expression that occurs during development under normal and altered environmental conditions. For this, a functional genomic approach to embryogenesis has been developed that uses highdensity oligonucleotide arrays for large-scale detection and quantification of gene expression. These oligonucleotide arrays were used for quantitative transcript imaging of embryonically expressed genes under standard conditions and in response to heat shock. In embryos raised under standard conditions, transcripts were detected for 37% of the 1,519 identified genes represented on the arrays, and highly reproducible quantification of gene expression was achieved in all cases. Analysis of differential gene expression after heat shock revealed substantial expression level changes for known heat-shock genes and identified numerous heat shock-inducible genes. These results demonstrate that highdensity oligonucleotide arrays are sensitive, efficient, and quantitative instruments for the analysis of large scale gene expression in Drosophila embryos. R ecently the genome of the first multicellular eukaryote Caenorhabditis elegans was completely elucidated (1). Sequencing of the Drosophila melanogaster genome has now also been carried out, and currently the corresponding putative open reading frames are being defined and verified (2). On the basis of this complete genomic information, it will now be important to determine the complex expression of all encoded genes and to analyze physiological as well as pathological phenomena from a global genetic perspective. Large-scale transcript analysis is made possible by DNA micro-or oligonucleotide arrays (3, 4), both of which allow the simultaneous monitoring of hundreds of mRNA expression profiles (5, 6). In this study, we used Drosophila high-density oligonucleotide arrays to monitor the simultaneous expression of zygotically active genes during the later postgastrulation stages of embryonic development (7-9). We analyzed the relative abundance levels of hundreds of embryonically expressed genes under normal physiological conditions and in response to heat shock (10). In embryos raised under normal conditions, we obtained highly reproducible quantification for 563 expressed genes corresponding to different functional classes. After a 36°C heat shock, we detected increases in expression levels for known heat-shock genes and identified numerous heat-shock-inducible genes. Materials and MethodsEmbryos. D. melanogaster Oregon R stocks were kept on standard cornmeal͞yeast͞agar medium at 25°C. Embryos were collected overnight on grape-juice plates for 12 h and were kept for a further 5 h at 25°C before RNA isolation...

We analyzed the expression and function of eyeless (ey) and twin of eyeless (toy) in the embryonic central nervous system (CNS) of Drosophila. Both genes are differentially expressed in specific neuronal subsets (but not in glia) in every CNS neuromere, and in the brain, specific cell populations co-expressing both proteins define a longitudinal domain which is intercalated between broad exclusive expression domains of ey and toy. Studies of genetic null alleles and dsRNA interference did not reveal any gross neuroanatomical effects of ey, toy, or ey/toy elimination in the embryonic CNS. In contrast, targeted misexpression of ey, but not of toy, resulted in profound axonal abnormalities in the embryonic ventral nerve cord and brain.

Pre-mRNA editing involving the conversion of adenosine to inosine is mediated by adenosine deaminases that act on RNA (ADAR1 and ADAR2). ADARs contain multiple double-stranded RNA(dsRNA)-binding domains in addition to an adenosine deaminase domain. An adenosine deaminase acting on tRNAs, scTad1p (also known as scADAT1), cloned from Saccharomyces cerevisiae has a deaminase domain related to the ADARs but lacks dsRNA-binding domains. We have identified a gene homologous to scADAT1 in the region of Drosophila melanogaster Adh chromosome II. Recombinant Drosophila ADAT1 (dADAT1) has been expressed in the yeast Pichia pastoris and purified. The enzyme has no activity on dsRNA substrates but is a tRNA deaminase with specificity for adenosine 37 of insect alanine tRNA. dADAT1 shows greater similarity to vertebrate ADARs than to yeast Tad1p, supporting the hypothesis of a common evolutionary origin for ADARs and ADATs. dAdat1 transcripts are maternally supplied in the egg. Zygotic expression is widespread initially and later concentrates in the central nervous system. Adenosine and cytosine have exocyclic amino groups that participate in Watson-Crick base pairing during transcription and translation. RNA editing enzymes have been discovered that deaminate specific adenosine residues to inosine (4, 32, 38, 40) or specific cytosine residues to uridine in RNA molecules (11,39). This can result in the incorporation of different amino acids at edited positions or the formation of a smaller protein due to the generation of a stop codon. Two closely related adenosine deaminases acting on RNA (ADARs) have been identified in vertebrates (3) that catalyze the deamination of specific adenosine residues to inosine in pre-mRNAs (for a review, see reference 22). These enzymes have homologous adenosine deaminase domains (32) and also contain multiple double-stranded RNA(dsRNA)-binding domains (47). ADARs recognize and deaminate specific adenosines within exons that form duplexes with flanking intronic sequences in pre-mRNA (20, 32). ADAR activity is ubiquitous and has been found in all metazoans tested and in most tissues (52). The abundance of inosine in polyA ϩ RNA has been estimated to be 1 in 17,000 nucleotides in brain and less in other mammalian tissues, correlating with ADAR expression levels (35). Inosine in edited transcripts directs the incorporation of cytosine during firststrand synthesis of cDNA (5), and RNA editing events have usually been identified in cDNA sequences in which guanosine replaces a genomically encoded adenosine (8, 45). Pre-mRNAs encoding subunits of the glutamate-gated ion channels (for a review, see reference 42) and the G protein-coupled serotonin 2C receptor (8) undergo RNA editing of their sequences by this mechanism. Proteins encoded by edited mRNAs often have functional properties that differ from the genomically encoded versions.

Background: Mutations and gene expression alterations in brain tumors have been extensively investigated, however the causes of brain tumorigenesis are largely unknown. Animal models are necessary to correlate altered transcriptional activity and tumor phenotype and to better understand how these alterations cause malignant growth. In order to gain insights into the in vivo transcriptional activity associated with a brain tumor, we carried out genome-wide microarray expression analyses of an adult brain tumor in Drosophila caused by homozygous mutation in the tumor suppressor gene brain tumor (brat).

We report the construction of a broad-host-range expression vector based on an RSF1010-derived replicon.The vector carries the strong leftward promoter (pL) of coliphage A as well as the c1857 allele, which codes for a thermolabile repressor protein. The coding region of mature human interleukin 2, which is preceded by the ner ribosome binding site of phage Mu, was cloned downstream from the pL promoter. The plasmid was introduced into Erwinia and Serratia species by means of mobilization. Heat-inducible synthesis of interleukin 2 protein was obtained, showing that the pL promoter is functional in these genera. As in Escherichia coli, the bulk of the overproduced protein was present in an insoluble form.In recent years much attention has been paid to the development of gene cloning and expression systems in bacteria other than the widely used Escherichia coli (13,22,35). Many gram-negative bacteria, some of which are of medical or agricuiLural importance (27, 38), possess a broad diversity of metabolic activities and might therefore be promising hosts for cloning and expression studies. Indeed, some genes can only be functionally expressed in a particular physiological background, e.g., when an enzyme is integrated into a specific biochemical pathway (21, 33) or when the base composition of the foreign gene differs widely from that of the host genome. It has been suggested that the weak expression of Pseudomonas genes in E. coli may be attributed to the unusual codon preference of Pseudomonas species (28). Therefore, there is a need for expression vectors that are adapted to the host of interest. The most versatile of these are likely to be based on broad-host-range plasmids, which can be stably inherited over a wide range of gram-negative species (2).We describe the cloning of the strong leftward promoter (PL) of coliphage X, together with the cI repressor gene, onto a derivative of the broad-host-range plasmid RSF1010 (19). This plasmid was introduced into three other enteric bacteria: Serratia marcescens, Erwinia chrysanthemi, and Erwinia carotovora subsp. carotovora. By obtaining controlled expression of cloned human interleukin 2 (IL-2), we demonstrate that the PL promoter is functional in these organisms and that its activity is regulated by the cI repressor as in the natural host E. coli.MATERUILS AND METHODS Bacterial strains and plasmids. The bacterial strains and plasmids used in this study are listed in Tables 1 and 2. Unless otherwise mentioned, bacterial cultures were grown at 28°C in LB medium (1% tryptone [Difco Laboratories, Detroit, Mich.], 0.5% yeast extract [Difco], 0.5% NaCl). Antibiotics were used at the following concentrations: kanamycin, 50 ,ug/ml; carbenicillin, 100 ,ug/ml; tetracycline, 10 ,Lg/ml. * Corresponding author.Transformation and conjugation procedures. E. coli strains were transformed as described previously (31). Plasmid DNA was introduced into Serratia and Erwinia species by means of conjugational transfer by using pRK2013 (16) or pRK2073 (26) as mobilizing plasmids. Conjuga...

In Drosophila, the glial cells missing (gcm) gene encodes a transcription factor that controls the determination of glial versus neuronal fate. In gcm mutants, presumptive glial cells are transformed into neurons and, conversely, when gcm is ectopically misexpressed, presumptive neurons become glia. Although gcm is thought to initiate glial cell development through its action on downstream genes that execute the glial differentiation program, little is known about the identity of these genes. To identify gcm downstream genes in a comprehensive manner, we used genome-wide oligonucleotide arrays to analyze differential gene expression in wild-type embryos versus embryos in which gcm is misexpressed throughout the neuroectoderm. Transcripts were analyzed at two defined temporal windows during embryogenesis. During the first period of initial gcm action on determination of glial cell precursors, over 400 genes were differentially regulated. Among these are numerous genes that encode other transcription factors, which underscores the master regulatory role of gcm in gliogenesis. During a second later period, when glial cells had already differentiated, over 1200 genes were differentially regulated. Most of these genes, including many genes for chromatin remodeling factors and cell cycle regulators, were not differentially expressed at the early stage, indicating that the genetic control of glial fate determination is largely different from that involved in maintenance of differentiated cells. At both stages, glial-specific genes were upregulated and neuron-specific genes were downregulated, supporting a model whereby gcm promotes glial development by activating glial genes, while simultaneously repressing neuronal genes. In addition, at both stages, numerous genes that were not previously known to be involved in glial development were differentially regulated and, thus, identified as potential new downstream targets of gcm. For a subset of the differentially regulated genes, tissue-specific in vivo expression data were obtained that confirmed the transcript profiling results. This first genome-wide analysis of gene expression events downstream of a key developmental transcription factor presents a novel level of insight into the repertoire of genes that initiate and maintain cell fate choices in CNS development.

No abstract

Background: Homeotic genes are key developmental regulators that are highly conserved throughout evolution. Their encoded homeoproteins function as transcription factors to control a wide range of developmental processes. Although much is known about homeodomain-DNA interactions, only a small number of genes acting downstream of homeoproteins have been identified. Here we use a functional genomic approach to identify candidate target genes of the Drosophila homeodomain transcription factor Labial.