Janelia Farm: an experiment in scientific culture.
Janelia Farm, the new research campus of the Howard Hughes Medical Institute, is an ongoing experiment in the social engineering of research communities.
Academies and Institutes↗
Biomedical subjects
Gerald M Rubin
Publications and source records attributed to Gerald M Rubin.
Large-scale trends in the evolution of gene structures within 11 animal genomes.
We have used the annotations of six animal genomes (Homo sapiens, Mus musculus, Ciona intestinalis, Drosophila melanogaster, Anopheles gambiae, and Caenorhabditis elegans) together with the sequences of five unannotated Drosophila genomes to survey changes in protein sequence and gene structure over a variety of timescales--from the less than 5 million years since the divergence of D. simulans and D. melanogaster to the more than 500 million years that have elapsed since the Cambrian explosion. To do so, we have developed a new open-source software library called CGL (for "Comparative Genomics Library"). Our results demonstrate that change in intron-exon structure is gradual, clock-like, and largely independent of coding-sequence evolution. This means that genome annotations can be used in new ways to inform, corroborate, and test conclusions drawn from comparative genomics analyses that are based upon protein and nucleotide sequence similarities.
Animals↗
Drosophila microRNAs exhibit diverse spatial expression patterns during embryonic development.
MicroRNAs (miRNAs) are an extensive class of regulatory RNA whose specific functions in animals are generally unknown. Although computational methods have identified many potential targets of miRNAs, elucidating the spatial expression patterns of miRNAs is necessary to identify the sites of miRNA action. Here, we report the spatial patterns of miRNA transcription during Drosophila embryonic development, as revealed by in situ hybridization to nascent miRNA transcripts. We detect expression of 15 "stand-alone" miRNA loci and 9 intronic miRNA loci, which collectively represent 38 miRNA genes. We observe great variety in the spatial patterns of miRNA transcription, including preblastoderm stripes, in aspects of the central and peripheral nervous systems, and in cellular subsets of the mesoderm and endoderm. We also describe an intronic miRNA (miR-7) whose expression pattern is distinct from that of its host mRNA (bancal), which demonstrates that intronic miRNAs can be subject to independent cis-regulatory control. Intriguingly, the expression patterns of several fly miRNAs are analogous to those of their vertebrate counterparts, suggesting that these miRNAs may have ancient roles in animal patterning.
Animals↗
Pervasive regulation of Drosophila Notch target genes by GY-box-, Brd-box-, and K-box-class microRNAs.
Although hundreds of distinct animal microRNAs (miRNAs) are known, the specific biological functions of only a handful are understood at present. Here, we demonstrate that three different families of Drosophila miRNAs directly regulate two large families of Notch target genes, including basic helix-loop-helix (bHLH) repressor and Bearded family genes. These miRNAs regulate Notch target gene activity via GY-box (GUCUUCC), Brd-box (AGCUUUA), and K-box (cUGUGAUa) motifs. These are conserved sites in target 3'-untranslated regions (3'-UTRs) that are complementary to the 5'-ends of miRNAs, or "seed" regions. Collectively, these motifs represent >40 miRNA-binding sites in Notch target genes, and we show all three classes of motif to be necessary and sufficient for miRNA-mediated regulation in vivo. Importantly, many of the validated miRNA-binding sites have limited pairing to miRNAs outside of the "box:seed" region. Consistent with this, we find that seed-related miRNAs that are otherwise quite divergent can regulate the same target sequences. Finally, we demonstrate that ectopic expression of several Notch-regulating miRNAs induces mutant phenotypes that are characteristic of Notch pathway loss of function, including loss of wing margin, thickened wing veins, increased bristle density, and tufted bristles. Collectively, these data establish insights into miRNA target recognition and demonstrate that the Notch signaling pathway is a major target of miRNA-mediated regulation in Drosophila.
3' Untranslated Regions↗
The ubiquitin ligase Drosophila Mind bomb promotes Notch signaling by regulating the localization and activity of Serrate and Delta.
The receptor Notch and its ligands of the Delta/Serrate/LAG2 (DSL) family are the central components in the Notch pathway, a fundamental cell signaling system that regulates pattern formation during animal development. Delta is directly ubiquitinated by Drosophila and Xenopus Neuralized, and by zebrafish Mind bomb, two unrelated RING-type E3 ubiquitin ligases with common abilities to promote Delta endocytosis and signaling activity. Although orthologs of both Neuralized and Mind bomb are found in most metazoan organisms, their relative contributions to Notch signaling in any single organism have not yet been assessed. We show here that a Drosophila ortholog of Mind bomb (D-mib) is a positive component of Notch signaling that is required for multiple Neuralized-independent, Notch-dependent developmental processes. Furthermore, we show that D-mib associates physically and functionally with both Serrate and Delta. We find that D-mib uses its ubiquitin ligase activity to promote DSL ligand activity, an activity that is correlated with its ability to induce the endocytosis and degradation of both Delta and Serrate (see also Le Borgne et al., 2005). We further demonstrate that D-mib can functionally replace Neuralized in multiple cell fate decisions that absolutely require endogenous Neuralized, a testament to the highly similar activities of these two unrelated ubiquitin ligases in regulating Notch signaling. We conclude that ubiquitination of Delta and Serrate by Neuralized and D-mib is an obligate feature of DSL ligand activation throughout Drosophila development.
Animals↗
Identification of putative noncoding polyadenylated transcripts in Drosophila melanogaster.
Analysis of EST and cDNA collections from a number of metazoan species has identified genes encoding long polyadenylated transcripts that do not contain ORFs of lengths typical for protein-encoding mRNAs. Noncoding functions of such polyadenylated transcripts have been elucidated in only a few examples. The corresponding genes neither contain hallmark sequence motifs nor appear to have been conserved across phyla. Thus, it is impossible to systematically identify new members of this class of gene by using sequence homology and traditional gene-finding algorithms that depend on protein-coding potential. Consequently, even their approximate number has not been established for any metazoan genome. We curated polyadenylated transcripts with limited protein-coding capacity from intergenic regions of the Drosophila melanogaster genome. We used RT-PCR assays, hybridization to RNA blots and whole-mount embryos, and computational analyses to characterize candidate transcripts. We verify the structures and expression of 17 distinct, likely non-protein-coding polyadenylated transcripts. We show that the expression of many of these transcripts is conserved in other Drosophila species, indicating that they have important biological functions.
Animals↗
A computational and experimental approach to validating annotations and gene predictions in the Drosophila melanogaster genome.
Five years after the completion of the sequence of the Drosophila melanogaster genome, the number of protein-coding genes it contains remains a matter of debate; the number of computational gene predictions greatly exceeds the number of validated gene annotations. We have assembled a collection of >10,000 gene predictions that do not overlap existing gene annotations and have developed a process for their validation that allows us to efficiently prioritize and experimentally validate predictions from various sources by sequencing RT-PCR products to confirm gene structures. Our data provide experimental evidence for 122 protein-coding genes. Our analyses suggest that the entire collection of predictions contains only approximately 700 additional protein-coding genes. Although we cannot rule out the discovery of genes with unusual features that make them refractory to existing methods, our results suggest that the D. melanogaster genome contains approximately 14,000 protein-coding genes.
Animals↗
Computational identification of developmental enhancers: conservation and function of transcription factor binding-site clusters in Drosophila melanogaster and Drosophila pseudoobscura.
BACKGROUND: The identification of sequences that control transcription in metazoans is a major goal of genome analysis. In a previous study, we demonstrated that searching for clusters of predicted transcription factor binding sites could discover active regulatory sequences, and identified 37 regions of the Drosophila melanogaster genome with high densities of predicted binding sites for five transcription factors involved in anterior-posterior embryonic patterning. Nine of these clusters overlapped known enhancers. Here, we report the results of in vivo functional analysis of 27 remaining clusters. RESULTS: We generated transgenic flies carrying each cluster attached to a basal promoter and reporter gene, and assayed embryos for reporter gene expression. Six clusters are enhancers of adjacent genes: giant, fushi tarazu, odd-skipped, nubbin, squeeze and pdm2; three drive expression in patterns unrelated to those of neighboring genes; the remaining 18 do not appear to have enhancer activity. We used the Drosophila pseudoobscura genome to compare patterns of evolution in and around the 15 positive and 18 false-positive predictions. Although conservation of primary sequence cannot distinguish true from false positives, conservation of binding-site clustering accurately discriminates functional binding-site clusters from those with no function. We incorporated conservation of binding-site clustering into a new genome-wide enhancer screen, and predict several hundred new regulatory sequences, including 85 adjacent to genes with embryonic patterns. CONCLUSIONS: Measuring conservation of sequence features closely linked to function--such as binding-site clusterin--makes better use of comparative sequence data than commonly used methods that examine only sequence identity.
Animals↗
Nurturing interdisciplinary research.
Explore the source record for details and available documents.
Biomedical Research↗
Drosophila melanogaster MNK/Chk2 and p53 regulate multiple DNA repair and apoptotic pathways following DNA damage.
We have used genetic and microarray analysis to determine how ionizing radiation (IR) induces p53-dependent transcription and apoptosis in Drosophila melanogaster. IR induces MNK/Chk2-dependent phosphorylation of p53 without changing p53 protein levels, indicating that p53 activity can be regulated without an Mdm2-like activity. In a genome-wide analysis of IR-induced transcription in wild-type and mutant embryos, all IR-induced increases in transcript levels required both p53 and the Drosophila Chk2 homolog MNK. Proapoptotic targets of p53 include hid, reaper, sickle, and the tumor necrosis factor family member EIGER: Overexpression of Eiger is sufficient to induce apoptosis, but mutations in Eiger do not block IR-induced apoptosis. Animals heterozygous for deletions that span the reaper, sickle, and hid genes exhibited reduced IR-dependent apoptosis, indicating that this gene complex is haploinsufficient for induction of apoptosis. Among the genes in this region, hid plays a central, dosage-sensitive role in IR-induced apoptosis. p53 and MNK/Chk2 also regulate DNA repair genes, including two components of the nonhomologous end-joining repair pathway, Ku70 and Ku80. Our results indicate that MNK/Chk2-dependent modification of Drosophila p53 activates a global transcriptional response to DNA damage that induces error-prone DNA repair as well as intrinsic and extrinsic apoptosis pathways.
Animals↗
Complementary miRNA pairs suggest a regulatory role for miRNA:miRNA duplexes.
microRNAs (miRNAs) are 21-22-nucleotide noncoding RNAs that are widely believed to regulate complementary mRNA targets. However, due to the modest amount of pairing involved, only a few out of the hundreds of known animal miRNAs have thus far been connected to mRNA targets. Here, we considered the possibility that miRNAs might regulate non-mRNA targets, namely other miRNAs. To do so, we conducted a systematic assessment of the nearly complete catalogs of animal miRNAs for potential miRNA:miRNA complements. Our analysis uncovered several compelling examples that strongly suggest a function for miRNA duplexes, thus adding a potential layer of regulatory sophistication to the small RNA world. Interestingly, the most striking examples involve miRNAs complementary to members of the K-box family and Brd-box family, two classes of miRNAs previously implicated in regulation of Notch target genes. We emphasize that patterns of nucleotide constraint indicate that miRNA complementarity is not a simple consequence of miRNA:miRNA* complementarity; however, our findings do suggest that the potential regulatory consequences of the latter also deserve investigation.
Animals↗
The BDGP gene disruption project: single transposon insertions associated with 40% of Drosophila genes.
The Berkeley Drosophila Genome Project (BDGP) strives to disrupt each Drosophila gene by the insertion of a single transposable element. As part of this effort, transposons in >30,000 fly strains were localized and analyzed relative to predicted Drosophila gene structures. Approximately 6300 lines that maximize genomic coverage were selected to be sent to the Bloomington Stock Center for public distribution, bringing the size of the BDGP gene disruption collection to 7140 lines. It now includes individual lines predicted to disrupt 5362 of the 13,666 currently annotated Drosophila genes (39%). Other lines contain an insertion at least 2 kb from others in the collection and likely mutate additional incompletely annotated or uncharacterized genes and chromosomal regulatory elements. The remaining strains contain insertions likely to disrupt alternative gene promoters or to allow gene misexpression. The expanded BDGP gene disruption collection provides a public resource that will facilitate the application of Drosophila genetics to diverse biological problems. Finally, the project reveals new insight into how transposons interact with a eukaryotic genome and helps define optimal strategies for using insertional mutagenesis as a genomic tool.
Animals↗
The Drosophila synaptotagmin-like protein bitesize is required for growth and has mRNA localization sequences within its open reading frame.
The vertebrate synaptotagmin-like protein granuphilin binds to the vesicle-trafficking proteins Rab27a and Munc18 and can modulate exocytosis of insulin-containing secretory granules in pancreatic beta cell lines. Here, we report the molecular and genetic characterization of bitesize, a granuphilin homolog and the only Drosophila synaptotagmin-like protein. Mutations that affect bitesize have reduced cell size and number, resulting in smaller animals that develop slowly. We also show that at least two classes of bitesize transcripts are localized to the apical plasma membrane in polarized epithelial cells. Whereas most cis-acting mRNA localization sequences map to 3' untranslated regions, bitesize contains a 2.2-kb sequence within its ORF that is necessary and sufficient for apical localization. Thus, we have found that bitesize is a metazoan example of a transcript for which all identifiable mRNA localization sequences are contained within the protein-coding region.
3' Untranslated Regions↗
Quantitative analysis of bristle number in Drosophila mutants identifies genes involved in neural development.
BACKGROUND: The identification of the function of all genes that contribute to specific biological processes and complex traits is one of the major challenges in the postgenomic era. One approach is to employ forward genetic screens in genetically tractable model organisms. In Drosophila melanogaster, P element-mediated insertional mutagenesis is a versatile tool for the dissection of molecular pathways, and there is an ongoing effort to tag every gene with a P element insertion. However, the vast majority of P element insertion lines are viable and fertile as homozygotes and do not exhibit obvious phenotypic defects, perhaps because of the tendency for P elements to insert 5' of transcription units. Quantitative genetic analysis of subtle effects of P element mutations that have been induced in an isogenic background may be a highly efficient method for functional genome annotation. RESULTS: Here, we have tested the efficacy of this strategy by assessing the extent to which screening for quantitative effects of P elements on sensory bristle number can identify genes affecting neural development. We find that such quantitative screens uncover an unusually large number of genes that are known to function in neural development, as well as genes with yet uncharacterized effects on neural development, and novel loci. CONCLUSIONS: Our findings establish the use of quantitative trait analysis for functional genome annotation through forward genetics. Similar analyses of quantitative effects of P element insertions will facilitate our understanding of the genes affecting many other complex traits in Drosophila.
Animals↗
Computational identification of Drosophila microRNA genes.
BACKGROUND: MicroRNAs (miRNAs) are a large family of 21-22 nucleotide non-coding RNAs with presumed post-transcriptional regulatory activity. Most miRNAs were identified by direct cloning of small RNAs, an approach that favors detection of abundant miRNAs. Three observations suggested that miRNA genes might be identified using a computational approach. First, miRNAs generally derive from precursor transcripts of 70-100 nucleotides with extended stem-loop structure. Second, miRNAs are usually highly conserved between the genomes of related species. Third, miRNAs display a characteristic pattern of evolutionary divergence. RESULTS: We developed an informatic procedure called 'miRseeker', which analyzed the completed euchromatic sequences of Drosophila melanogaster and D. pseudoobscura for conserved sequences that adopt an extended stem-loop structure and display a pattern of nucleotide divergence characteristic of known miRNAs. The sensitivity of this computational procedure was demonstrated by the presence of 75% (18/24) of previously identified Drosophila miRNAs within the top 124 candidates. In total, we identified 48 novel miRNA candidates that were strongly conserved in more distant insect, nematode, or vertebrate genomes. We verified expression for a total of 24 novel miRNA genes, including 20 of 27 candidates conserved in a third species and 4 of 11 high-scoring, Drosophila-specific candidates. Our analyses lead us to estimate that drosophilid genomes contain around 110 miRNA genes. CONCLUSIONS: Our computational strategy succeeded in identifying bona fide miRNA genes and suggests that miRNAs constitute nearly 1% of predicted protein-coding genes in Drosophila, a percentage similar to the percentage of miRNAs recently attributed to other metazoan genomes.
Animals↗
Drosophila matrix metalloproteinases are required for tissue remodeling, but not embryonic development.
The matrix metalloproteinase (MMP) family is heavily implicated in many diseases, including cancer. The developmental functions of these genes are not clear, however, because the >20 mammalian MMPs can be functionally redundant. Drosophila melanogaster has only two MMPs, which are expressed in embryos in distinct patterns. We created mutations in both genes: Mmp1 mutants have defects in larval tracheal growth and pupal head eversion, and Mmp2 mutants have defects in larval tissue histolysis and epithelial fusion during metamorphosis; neither is required for embryonic development. Double mutants also complete embryogenesis, and these represent the first time, to our knowledge, that all MMPs have been disrupted in any organism. Thus, MMPs are not required for Drosophila embryonic development, but, rather, for tissue remodeling.
Animals↗
Y chromosome and other heterochromatic sequences of the Drosophila melanogaster genome: how far can we go?
Whole genome shotgun assemblies have proven remarkably successful in reconstructing the bulk of euchromatic genes, with the only limit appearing to be determined by the sequencing depth. For genes imbedded in heterochromatin, however, the low cloning efficiency of repetitive sequences, combined with the computational challenges, demand that additional clues be used to annotate the sequences. One approach that has proven very successful in identifying protein coding genes in Y-linked heterochromatin of Drosophila melanogaster has been to make a BLASTable database of the small, unmapped contigs and fragments leftover at the end of a shotgun assembly, and to attempt to capture these by blasting with an appropriate query sequence. This approach often yields a staggered alignment of contigs from the unmapped set to the query sequence, as though the disjoint contigs represent small portions of the gene. Further inspection frequently shows that the contigs are broken by very large, heterochromatic introns. Methods of this sort are being expanded to make best use of all available clues to determine which unmapped contigs are associated with genes. These include use of EST libraries, and, in the case of the Y chromosome, testing of male specific genes and reduced shotgun depth of relevant contigs. It appears much more hopeful than anyone would have imagined that whole genome shotgun assemblies can recover the great bulk of even heterochromatic genes.
Animals↗
The Drosophila melanogaster genome.
Drosophila's importance as a model organism made it an obvious choice to be among the first genomes sequenced, and the Release 1 sequence of the euchromatic portion of the genome was published in March 2000. This accomplishment demonstrated that a whole genome shotgun (WGS) strategy could produce a reliable metazoan genome sequence. Despite the attention to sequencing methods, the nucleotide sequence is just the starting point for genome-wide analyses; at a minimum, the genome sequence must be interpreted using expressed sequence tag (EST) and complementary DNA (cDNA) evidence and computational tools to identify genes and predict the structures of their RNA and protein products. The functions of these products and the manner in which their expression and activities are controlled must then be assessed-a much more challenging task with no clear endpoint that requires a wide variety of experimental and computational methods. We first review the current state of the Drosophila melanogaster genome sequence and its structural annotation and then briefly summarize some promising approaches that are being taken to achieve an initial functional annotation.
Animals↗