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Susan E Celniker

Publications and source records attributed to Susan E Celniker.

At least 19 recordsLinked to original sources
PubMed2007

Chromatin looping mediates boundary element promoter interactions.

One facet of the control of gene expression is long-range promoter regulation by distant enhancers. It is an important component of the regulation of genes that control metazoan development and has been appreciated for some time but the molecular mechanisms underlying this regulation have remained poorly understood. A recent study by Cleard and colleagues1 reports the first in vivo evidence of chromatin looping and boundary element promoter interaction. Specifically, they studied the function of a boundary element within the cis-regulatory region of the Abdominal-B (Abd-B) gene of Drosophila melanogaster.

Animals↗
PubMed2006

Improved repeat identification and masking in Dipterans.

Repetitive sequences are a major constituent of many eukaryote genomes and play roles in gene regulation, chromosome inheritance, nuclear architecture, and genome stability. The identification of repetitive elements has traditionally relied on in-depth, manual curation and computational determination of close relatives based on DNA identity. However, the rapid divergence of repetitive sequence has made identification of repeats by DNA identity difficult even in closely related species. Hence, the presence of unidentified repeats in genome sequences affects the quality of gene annotations and annotation-dependent analyses (e.g. microarray analyses). We have developed an enhanced repeat identification pipeline using two approaches. First, the de novo repeat finding program PILER-DF was used to identify interspersed repetitive elements in several recently finished Dipteran genomes. Repeats were classified, when possible, according to their similarity to known elements described in Repbase and GenBank, and also screened against annotated genes as one means of eliminating false positives. Second, we used a new program called RepeatRunner, which integrates results from both RepeatMasker nucleotide searches and protein searches using BLASTX. Using RepeatRunner with PILER-DF predictions, we masked repeats in thirteen Dipteran genomes and conclude that combining PILER-DF and RepeatRunner greatly enhances repeat identification in both well-characterized and un-annotated genomes.

Animals↗
PubMed2006

RNA editing in Drosophila melanogaster: New targets and functional consequences.

Adenosine deaminases that act on RNA [adenosine deaminase, RNA specific (ADAR)] catalyze the site-specific conversion of adenosine to inosine in primary mRNA transcripts. These re-coding events affect coding potential, splice sites, and stability of mature mRNAs. ADAR is an essential gene, and studies in mouse, Caenorhabditis elegans, and Drosophila suggest that its primary function is to modify adult behavior by altering signaling components in the nervous system. By comparing the sequence of isogenic cDNAs to genomic DNA, we have identified and experimentally verified 27 new targets of Drosophila ADAR. Our analyses led us to identify new classes of genes whose transcripts are targets of ADAR, including components of the actin cytoskeleton and genes involved in ion homeostasis and signal transduction. Our results indicate that editing in Drosophila increases the diversity of the proteome, and does so in a manner that has direct functional consequences on protein function.

Adenosine Deaminase↗
PubMed2006

Genomic organization of the Drosophila telomere retrotransposable elements.

The emerging sequence of the heterochromatic portion of the Drosophila melanogaster genome, with the most recent update of euchromatic sequence, gives the first genome-wide view of the chromosomal distribution of the telomeric retrotransposons, HeT-A, TART, and Tahre. As expected, these elements are entirely excluded from euchromatin, although sequence fragments of HeT-A and TART 3 untranslated regions are found in nontelomeric heterochromatin on the Y chromosome. The proximal ends of HeT-A/TART arrays appear to be a transition zone because only here do other transposable elements mix in the array. The sharp distinction between the distribution of telomeric elements and that of other transposable elements suggests that chromatin structure is important in telomere element localization. Measurements reported here show (1) D. melanogaster telomeres are very long, in the size range reported for inbred mouse strains (averaging 46 kb per chromosome end in Drosophila stock 2057). As in organisms with telomerase, their length varies depending on genotype. There is also slight under-replication in polytene nuclei. (2) Surprisingly, the relationship between the number of HeT-A and TART elements is not stochastic but is strongly correlated across stocks, supporting the idea that the two elements are interdependent. Although currently assembled portions of the HeT-A/TART arrays are from the most-proximal part of long arrays, approximately 61% of the total HeT-A sequence in these regions consists of intact, potentially active elements with little evidence of sequence decay, making it likely that the content of the telomere arrays turns over more extensively than has been thought.

Animals↗
PubMed2005

Rapid and efficient cDNA library screening by self-ligation of inverse PCR products (SLIP).

cDNA cloning is a central technology in molecular biology. cDNA sequences are used to determine mRNA transcript structures, including splice junctions, open reading frames (ORFs) and 5'- and 3'-untranslated regions (UTRs). cDNA clones are valuable reagents for functional studies of genes and proteins. Expressed Sequence Tag (EST) sequencing is the method of choice for recovering cDNAs representing many of the transcripts encoded in a eukaryotic genome. However, EST sequencing samples a cDNA library at random, and it recovers transcripts with low expression levels inefficiently. We describe a PCR-based method for directed screening of plasmid cDNA libraries. We demonstrate its utility in a screen of libraries used in our Drosophila EST projects for 153 transcription factor genes that were not represented by full-length cDNA clones in our Drosophila Gene Collection. We recovered high-quality, full-length cDNAs for 72 genes and variously compromised clones for an additional 32 genes. The method can be used at any scale, from the isolation of cDNA clones for a particular gene of interest, to the improvement of large gene collections in model organisms and the human. Finally, we discuss the relative merits of directed cDNA library screening and RT-PCR approaches.

Animals↗
PubMed2005

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↗
PubMed2005

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↗
PubMed2005

Identification and analysis of U5 snRNA variants in Drosophila.

Distinct isoforms of spliceosomal RNAs may be involved in regulating pre-messenger RNA splicing in eukaryotic cells. During a large-scale effort to identify small noncoding RNAs in Drosophila, we isolated a U5 snRNA-like molecule containing a 5' segment identical to that of the canonical (major) U5 snRNA but with a variant Sm binding site and a distinct 3' hairpin sequence. Based on this finding, another six similar U5 snRNA-like sequences were identified within the Drosophila genome by sequence similarity to the invariant loop in the 5' half of U5. Interestingly, although all of these variants are expressed in vivo, each shows a distinct temporal expression profile during Drosophila development, and one is expressed primarily in fly heads. The presence of these U5 snRNA variants within RNP particles suggests their role in splicing and implies a possible connection to regulation of developmental and tissue-specific gene expression.

Amino Acid Sequence↗
PubMed2004

Drosophila DNase I footprint database: a systematic genome annotation of transcription factor binding sites in the fruitfly, Drosophila melanogaster.

UNLABELLED: Despite increasing numbers of computational tools developed to predict cis-regulatory sequences, the availability of high-quality datasets of transcription factor binding sites limits advances in the bioinformatics of gene regulation. Here we present such a dataset based on a systematic literature curation and genome annotation of DNase I footprints for the fruitfly, Drosophila melanogaster. Using the experimental results of 201 primary references, we annotated 1367 binding sites from 87 transcription factors and 101 target genes in the D.melanogaster genome sequence. These data will provide a rich resource for future bioinformatics analyses of transcriptional regulation in Drosophila such as constructing motif models, training cis-regulatory module detectors, benchmarking alignment tools and continued text mining of the extensive literature on transcriptional regulation in this important model organism. AVAILABILITY: http://www.flyreg.org/ CONTACT: cbergman@gen.cam.ac.uk.

Binding Sites↗
PubMed2004

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↗
PubMed2004

Benchmarking tools for the alignment of functional noncoding DNA.

BACKGROUND: Numerous tools have been developed to align genomic sequences. However, their relative performance in specific applications remains poorly characterized. Alignments of protein-coding sequences typically have been benchmarked against "correct" alignments inferred from structural data. For noncoding sequences, where such independent validation is lacking, simulation provides an effective means to generate "correct" alignments with which to benchmark alignment tools. RESULTS: Using rates of noncoding sequence evolution estimated from the genus Drosophila, we simulated alignments over a range of divergence times under varying models incorporating point substitution, insertion/deletion events, and short blocks of constrained sequences such as those found in cis-regulatory regions. We then compared "correct" alignments generated by a modified version of the ROSE simulation platform to alignments of the simulated derived sequences produced by eight pairwise alignment tools (Avid, BlastZ, Chaos, ClustalW, DiAlign, Lagan, Needle, and WABA) to determine the off-the-shelf performance of each tool. As expected, the ability to align noncoding sequences accurately decreases with increasing divergence for all tools, and declines faster in the presence of insertion/deletion evolution. Global alignment tools (Avid, ClustalW, Lagan, and Needle) typically have higher sensitivity over entire noncoding sequences as well as in constrained sequences. Local tools (BlastZ, Chaos, and WABA) have lower overall sensitivity as a consequence of incomplete coverage, but have high specificity to detect constrained sequences as well as high sensitivity within the subset of sequences they align. Tools such as DiAlign, which generate both local and global outputs, produce alignments of constrained sequences with both high sensitivity and specificity for divergence distances in the range of 1.25-3.0 substitutions per site. CONCLUSION: For species with genomic properties similar to Drosophila, we conclude that a single pair of optimally diverged species analyzed with a high performance alignment tool can yield accurate and specific alignments of functionally constrained noncoding sequences. Further algorithm development, optimization of alignment parameters, and benchmarking studies will be necessary to extract the maximal biological information from alignments of functional noncoding DNA.

Animals↗
PubMed2004

Creation of a minimal tiling path of genomic clones for Drosophila: provision of a common resource.

On the basis of shotgun subclone libraries used in the sequencing of the Drosophila melanogaster genome, a minimal tiling path of subclones across much of the genome was determined. About 320,000 shotgun clones for chromosomes X(12-20), 2R, 2L, 3R, and 4 were available from the Berkeley Drosophila Genome Project. The clone inserts have an average length of 3.4 kb and are amenable to standard PCR amplification. The resulting tiling path covers 86.2% of chromosome X(12-20), 86.2% of chromosomal arm 2R, 79.0% of 2L, 89.6% of 3R, and 80.5% of chromosome 4. In total, the 25,135 clones represent 76.7 Mb--equivalent to about 67% of the genome--and would be suitable for producing a microarray on a single slide.

Animals↗
PubMed2003

Posterior patterning genes and the identification of a unique body region in the brine shrimp Artemia franciscana.

All arthropods share the same basic set of Hox genes, although the expression of these genes differs among divergent groups. In the brine shrimp Artemia franciscana, their expression is limited to the head, thoracic/trunk and genital segments, but is excluded from more posterior parts of the body which consist of six post-genital segments and the telson (bearing the anus). Nothing is currently known about the genes that specify the identity of these posterior structures. We examine the expression patterns of four candidate genes, Abdominal-B, caudal/Cdx, even-skipped/Evx and spalt, the homologues of which are known to play an important role in the specification of posterior structures in other animals. Abdominal-B is expressed in the genital segments of Artemia, but not in the post-genital segments at any developmental stage. The expression of caudal, even-skipped and spalt in the larval growth-zone suggests they may play a role in the generation of body segments (perhaps comparable with the role of gap and segmentation genes in insects), but not a direct role in defining the identity of post-genital segments. The expression of caudal at later stages suggests a role in the specification of anal structures. A PCR screen designed to isolate Hox genes expressed specifically in the posterior part of the body failed to identify any new Hox genes. We conclude that the post-genital segments of Artemia are not defined by any of the genes known to play a role in the specification of posterior segments in other arthropods. We argue that these segments constitute a unique body region that bears no obvious homology to previously characterised domains of Hox gene activity.

Amino Acid Sequence↗
PubMed2003

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↗
PubMed2003

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↗
PubMed2002

Annotation of the Drosophila melanogaster euchromatic genome: a systematic review.

BACKGROUND: The recent completion of the Drosophila melanogaster genomic sequence to high quality and the availability of a greatly expanded set of Drosophila cDNA sequences, aligning to 78% of the predicted euchromatic genes, afforded FlyBase the opportunity to significantly improve genomic annotations. We made the annotation process more rigorous by inspecting each gene visually, utilizing a comprehensive set of curation rules, requiring traceable evidence for each gene model, and comparing each predicted peptide to SWISS-PROT and TrEMBL sequences. RESULTS: Although the number of predicted protein-coding genes in Drosophila remains essentially unchanged, the revised annotation significantly improves gene models, resulting in structural changes to 85% of the transcripts and 45% of the predicted proteins. We annotated transposable elements and non-protein-coding RNAs as new features, and extended the annotation of untranslated (UTR) sequences and alternative transcripts to include more than 70% and 20% of genes, respectively. Finally, cDNA sequence provided evidence for dicistronic transcripts, neighboring genes with overlapping UTRs on the same DNA sequence strand, alternatively spliced genes that encode distinct, non-overlapping peptides, and numerous nested genes. CONCLUSIONS: Identification of so many unusual gene models not only suggests that some mechanisms for gene regulation are more prevalent than previously believed, but also underscores the complex challenges of eukaryotic gene prediction. At present, experimental data and human curation remain essential to generate high-quality genome annotations.

Animals↗