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PubMed1998

Relative paucity of genes causing inviability in hybrids between Drosophila melanogaster and D. simulans.

Using deficiencies from Drosophila melanogaster, we looked for genomic regions in the sister species D. simulans that could cause lethality when hemizygous on a hybrid genetic background. Such genotypes allow hemizygous genes from one species to interact with heterozygous genes from other species and may correspond to the kinds of genotypes causing Haldane's rule, the observation that if only one gender is sterile or inviable in species hybrids, it is nearly always the heterogametic sex. A survey of roughly 50% of the D. simulans genome (114 chromosome regions) revealed only four regions causing hybrid lethality and five causing severe reductions in hybrid viability. However, the viability of all of these genotypes was at least partially restored by rearing hybrids at lower temperature or using different genetic backgrounds from D. simulans. We therefore detected no D. simulans chromosome regions causing unconditional hybrid lethality, although several regions were shown to be deleterious under most tested temperatures and genetic backgrounds. The relative paucity of "inviability genes" supports the idea, suggested by work on other species, that hybrid inviability between closely related species might be caused by interactions among relatively few genes, while hybrid sterility may involve many more loci.

Animals↗

PubMed1996

Structure of the phenylalanine hydroxylase gene in Drosophila melanogaster and evidence of alternative promoter usage.

The complete Drosophila melanogaster phenylalanine hydroxylase gene isolated from a genomic library was sequenced. Gene structure consisted of five exons covering a region of around 3 kb. Position of introns in the C-terminal domain was conserved with mammalian aromatic amino acid hydroxylase genes. Putative promoter sequences in the 5'UTR and intron 1 were identified. A novel transcript was detected differing from that previously reported by the inclusion of a part of the intron 1 sequence. It could be produced using an alternative promoter. The deduced open reading frame would code a protein with a small difference at the N-terminus. Expression of the alternative transcripts was examined throughout development.

Amino Acid Sequence↗

PubMed2004

The genomic rate of adaptive amino acid substitution in Drosophila.

The proportion of amino acid substitutions driven by adaptive evolution can potentially be estimated from polymorphism and divergence data by an extension of the McDonald-Kreitman test. We have developed a maximum-likelihood method to do this and have applied our method to several data sets from three Drosophila species: D. melanogaster, D. simulans, and D. yakuba. The estimated number of adaptive substitutions per codon is not uniformly distributed among genes, but follows a leptokurtic distribution. However, the proportion of amino acid substitutions fixed by adaptive evolution seems to be remarkably constant across the genome (i.e., the proportion of amino acid substitutions that are adaptive appears to be the same in fast-evolving and slow-evolving genes; fast-evolving genes have higher numbers of both adaptive and neutral substitutions). Our estimates do not seem to be significantly biased by selection on synonymous codon use or by the assumption of independence among sites. Nevertheless, an accurate estimate is hampered by the existence of slightly deleterious mutations and variations in effective population size. The analysis of several Drosophila data sets suggests that approximately 25% +/- 20% of amino acid substitutions were driven by positive selection in the divergence between D. simulans and D. yakuba.

Adaptation, Biological↗

PubMed1996

Segregation distortion in Drosophila melanogaster: genomic organization of Responder sequences.

The heterochromatic Responder (Rsp) locus of Drosophila melanogaster is the target of the two distorter loci Sd and E(SD). Rsp is located in a specific heterochromatic region of the second chromosome and is made up of AT-rich satellite sequences whose abundance is related to its sensitivity to the distorter chromosomes. Here we report that a cluster of Rsp sequences is also located in the third chromosome. The third-chromosome cluster has the same flanking sequences as the clone originally used to identify the Rsp elements, and one of the flanking sequences is a rearranged 412 retrotransposon. The presence of a second, unlinked Rsp-sequence cluster makes re-interpretation necessary for some earlier experiments in which segregation of the third chromosome had not been followed and raises interesting possibilities for the origin of the Rsp locus.

Animals↗

PubMedDate not supplied

[Genetic structure of mobile elements of the "Penelope" family in closely related Drosophila species].

Genomic libraries were obtained from species belonging to the "virilis" group of Drosophila. Several copies of Penelope elements were isolated from these libraries by using a D. virilis Penelope clone as a probe. The elements were sequenced, and their structure was determined. The geographical distribution of this family of mobile elements in closely related species of the group was studied in detail. Cytological localization of the elements was also carried out. The high variability observed between different copies of Penelope is probably due to recombination between individual copies. The role of these elements in the evolution of closely related species is discussed.

Animals↗

PubMed2005

Target-selected mutant screen by TILLING in Drosophila.

The availability of the full Drosophila genomic DNA sequence prompts the development of a method to efficiently obtain mutations in genes of interest identified by their sequence homologies or biochemically. To date, molecularly characterized mutations have been generated in around 6000 of the approximately 15,000 annotated fly genes, of which around one-third are essential for viability. To obtain mutations in essential and nonessential genes of interest, we took a reverse genetics approach, based on the large-scale detection of point mutations by Cel-I-mediated heteroduplex cleavage. A library of genomic DNA from 2086 EMS-mutagenized lines was established. The library was screened for mutations in three genes. A total of 6.1 Mb were screened, and 44 hits were found in two different mutagenesis conditions. Optimal conditions yielded an average of one mutation every 156 kb. For an essential gene tested, five of 25 mutations turned out to cause lethality, confirming that EMS mutagenesis leads to high frequency of gene inactivation. We thereby established that Cel-I-mediated TILLING can be used to efficiently obtain mutations in genes of interest in Drosophila.

Animals↗

PubMed1999

GEM, a cluster of repetitive sequences in the Drosophila subobscura genome.

GEM is a new family of repetitive sequences detected in the D. subobscura genome. Two of the four described GEM elements encompass a heterogeneous central module, with no detectable ORF, flanked by two long inverted repeats. These elements are composed of a set of repetitive modules, which are inverted repeat (IR), direct repeat (DR), palindromic sequence (PS), long sequence (LS) and short sequence (SS). These five modules can be found either clustered or dispersed as single modules in the D. subobscura genome, in euchromatic and heterochromatic regions. In addition to the 3' region of Adh retrosequences, single IR and LS blocks were found associated with the promoter region of different genes, in particular, LS-like blocks have also been found associated with functional genes in D. melanogaster and D. virilis. Conversely, the DR block is highly similar to satellite DNAs from some other species of the obscura group. In addition, GEM elements share some structural features with IS elements described in different Drosophila species. It is likely that both GEM and IS sequences would be vestiges of an ancestral transposable element.

Alcohol Dehydrogenase↗

PubMed2002

Large clusters of co-expressed genes in the Drosophila genome.

Clustering of co-expressed, non-homologous genes on chromosomes implies their co-regulation. In lower eukaryotes, co-expressed genes are often found in pairs. Clustering of genes that share aspects of transcriptional regulation has also been reported in higher eukaryotes. To advance our understanding of the mode of coordinated gene regulation in multicellular organisms, we performed a genome-wide analysis of the chromosomal distribution of co-expressed genes in Drosophila. We identified a total of 1,661 testes-specific genes, one-third of which are clustered on chromosomes. The number of clusters of three or more genes is much higher than expected by chance. We observed a similar trend for genes upregulated in the embryo and in the adult head, although the expression pattern of individual genes cannot be predicted on the basis of chromosomal position alone. Our data suggest that the prevalent mechanism of transcriptional co-regulation in higher eukaryotes operates with extensive chromatin domains that comprise multiple genes.

Animals↗

PubMed2003

Assessment of SAGE in transcript identification.

An essential step in Serial Analysis of Gene Expression (SAGE) is tag mapping, which refers to the unambiguous determination of the gene represented by a SAGE tag. Current resources for tag mapping are incomplete, and thus do not allow assessment of the efficacy of SAGE in transcript identification. A method of tag mapping is described here and applied to the Drosophila melanogaster and Caenorhabditis elegans genomes, which permits detailed SAGE assessment and provides tag-mapping resources that were unavailable previously for these organisms. In our method, a conceptual transcriptome is constructed using genomic sequence and annotation by extending predicted coding regions to include UTRs on the basis of EST and cDNA alignments, UTR length distributions, and polyadenylation signals. Analysis of extracted tags suggests that, using the standard SAGE procedure, expression of 8% of D. melanogaster and 15% of C. elegans genes cannot be detected unambiguously by SAGE due to shared sequence or lack of NlaIII-anchoring enzyme sites. Both increasing tag length by 2-3 bp and using Sau3A instead of NlaIII as the anchoring enzyme increases potential for transcript detection. This work identifies and quantifies genes not amenable to SAGE analysis, in addition to providing tag-to-gene mappings for two model organisms.

Animals↗

PubMed2006

Does lack of recombination enhance asymmetric evolution among duplicate genes? Insights from the Drosophila melanogaster genome.

Gene duplication has different outcomes: pseudogenization (death of one of the two copies), gene amplification (both copies remain the same), sub-functionalization (both copies are required to perform the ancestral function) and neo-functionalization (one copy acquires a new function). Asymmetric evolution (one copy evolves faster than the other) is usually seen as a signature of neo-functionalization. However, it has been proposed that sub-functionalization could also generate asymmetric evolution among duplicate genes when they experience different local recombination rates. Indeed, the low recombination copy is expected to evolve faster because of Hill-Robertson effects. Here we tested this idea with about 100 pairs of young duplicates from the Drosophila melanogaster genome. Looking only at young duplicates allowed us to compare recombination rates and evolutionary rates on a similar time-scale contrary to previous work. We found that dispersed pairs tend to evolve more asymmetrically than tandem ones. Among dispersed copies, the low recombination copy tends to be the fast-evolving one. We also tested the possibility that all this was explained by a confounding factor (expression level) but found no evidence for it. In conclusion, our results do support the idea that asymmetric evolution among duplicates is enhanced by restricted recombination. However, further work is needed to clearly distinguish between sub-functionalization and neo-functionalization for the asymmetrically-evolving duplicate pairs that we found.

Animals↗

PubMed2005

A BAC-based physical map of the Drosophila buzzatii genome.

Large-insert genomic libraries facilitate cloning of large genomic regions, allow the construction of clone-based physical maps, and provide useful resources for sequencing entire genomes. Drosophila buzzatii is a representative species of the repleta group in the Drosophila subgenus, which is being widely used as a model in studies of genome evolution, ecological adaptation, and speciation. We constructed a Bacterial Artificial Chromosome (BAC) genomic library of D. buzzatii using the shuttle vector pTARBAC2.1. The library comprises 18,353 clones with an average insert size of 152 kb and an approximately 18x expected representation of the D. buzzatii euchromatic genome. We screened the entire library with six euchromatic gene probes and estimated the actual genome representation to be approximately 23x. In addition, we fingerprinted by restriction digestion and agarose gel electrophoresis a sample of 9555 clones, and assembled them using FingerPrint Contigs (FPC) software and manual editing into 345 contigs (mean of 26 clones per contig) and 670 singletons. Finally, we anchored 181 large contigs (containing 7788 clones) to the D. buzzatii salivary gland polytene chromosomes by in situ hybridization of 427 representative clones. The BAC library and a database with all the information regarding the high coverage BAC-based physical map described in this paper are available to the research community.

Animals↗

PubMed1996

Structural organization of the rae28 gene, a putative murine homologue of the Drosophila polyhomeotic gene.

A putative murine homologue of the Drosophila polyhomeotic gene, named rae28, has been isolated from a genomic library of 129/SV mouse and its structural organization has been analyzed. rae28 is a single gene of approximately 22 kb long and consists of 15 exons. Its 5'-flanking region lacks typical transcriptional regulatory sequences, such as TATA and CCAAT boxes, but contains GC-rich sequences and seven putative binding sites for a transcription factor, Sp1. One major transcription start point has been determined. The overall exon-intron organization suggested that three different Rae28 mRNAs are generated through alternative splicing. Furthermore, the rae28 gene has been located on the R-positive F3 band of mouse chromosome 6 by the direct R-banding fluorescence in situ hybridization methods.

Animals↗

PubMed1994

A family of U1 pseudogenes in Bombyx mori may be derived from an ancestral pseudogene.

Seven EMBL-4 lambda clones containing U1 small nuclear RNA sequences were isolated from a Bombyx mori genomic library. Six of the seven represent unique sequences. The six unique U1 sequences exhibit fixed point 3'-end truncation. Five out of the six clones share immediate 3'-end flanking sequences while two share 5'-end flanking sequences. Fixed point 3'-end truncation and a hierarchy of shared to unique diagnostic mutations may suggest a family of U1 pseudogenes generated from a reverse-transcribed class II pseudogene in B. mori. An ancestral 'master' U1 pseudogene capable of RNA- and/or DNA-mediated transposition may give rise to generations of U1 pseudogenes that include the original pseudogene's flanking sequences. Identical 3'-end truncation in some of these U1 sequences can be explained by RNA self-priming due to intra-strand binding prior to reverse transcription.

Animals↗

PubMed2002

Digging deep for ancient relics: a survey of protein motifs in the intergenic sequences of four eukaryotic genomes.

We have examined conserved protein motifs in the non-coding, intergenic regions ("pseudomotif patterns") and surveyed their occurrence in the fly, worm, yeast and human genomes (chromosomes 21 and 22 only). To identify these patterns, we masked out annotated genes, pseudogenes and repeat regions from the raw genomic sequence and then compared the remaining sequence, in six-frame translation, against 1319 patterns from the PROSITE database. For each pseudomotif pattern, the absolute number of occurrences is not very informative unless compared against a statistical expectation; consequently, we calculated the expected occurrence of each pattern using a Poisson model and verified this with simulations. Using a p-value cut-off of 0.01, we found 67 pseudomotif patterns over-represented in fly intergenic regions, 34 in worm, 21 in human and six in yeast. These include the zinc finger, leucine zipper, nucleotide-binding motif and EGF domain. Many of the over-represented patterns were common to two or more organisms, but there were a few that were unique to specific ones. Furthermore, we found more over-represented patterns in the fly than in the worm, although the fly has fewer pseudogenes. This puzzling observation can be explained by a higher deletion rate in the fly genome. We also surveyed under-represented patterns, finding 23 in the fly, 12 in the worm, 18 in human and two in yeast. If intergenic sequences were truly random, we would expect an equal number of over and under-represented patterns. The fact that for each organism the number of over-represented patterns is greater than the number of under-represented ones implies that a fraction of the intergenic regions consist of ancient protein fragments that, due to accumulated disablements, have become unrecognizable by conventional techniques for gene and pseudogene identification. Moreover, we find that in aggregate the over-represented pseudomotif patterns occupy a substantial fraction of the intergenic regions. Further information is available at http://pseudogene.org

Amino Acid Motifs↗

PubMed2002

Genomewide analysis of the Drosophila tetraspanins reveals a subset with similar function in the formation of the embryonic synapse.

Tetraspanins encode a large conserved family of proteins that span the membrane four times and are expressed in a variety of eukaryotic tissues. They are part of membrane complexes that are involved in such diverse processes as intracellular signaling, cellular motility, metastasis, and tumor suppression. The single fly tetraspanin characterized to date, late bloomer (lbm), is expressed on the axons, terminal arbors, and growth cones of motoneurons. In embryos lacking Lbm protein, motoneurons reach their muscle targets, but initially fail to form synaptic terminals. During larval stages, however, functional contacts are formed. The newly available genomic sequence of Drosophila melanogaster indicates the existence of 34 additional members of the tetraspanin family in the fly. To address the possibility that other tetraspanins with functions that might compensate for a lack of lbm exist, we determined the expression domains of the Drosophila tetraspanin gene family members by RNA in situ analysis. We found two other tetraspanins also expressed in motoneurons and subsequently generated a small chromosomal deletion that removes all three motoneuron-specific tetraspanins. The deletion results in a significant enhancement in the lbm phenotype, indicating that the two additional motoneuron-expressed tetraspanins can, at least in part, compensate for the absence of lbm during the formation of the embryonic synapse.

Animals↗

PubMed1993

The cDNA and deduced protein sequence of house fly NADPH-cytochrome P450 reductase.

Antisera to purified house fly NADPH-cytochrome P450 reductase were used to select cDNA clones from an expression library of abdomens of phenobarbital-treated house flies. A partial cDNA of 1841 bp containing a TAG termination codon, a consensus polyadenylation site and 269 bp of 3' untranslated sequence was obtained. Sequencing of a genomic clone coupled with mRNA sequencing yielded the complete coding sequence including the starting ATG. The resulting open reading frame of 2013 nucleotides codes for a protein of 671 residues. The native reductase apoprotein has a molecular weight of 76,366 and the deduced molecular weight of the holoenzyme (i.e. with 1 mol of FAD and FMN) is 77,608. The sequence of the house fly P450 reductase protein is highly similar to that of rabbit liver, the overall amino acid positional identity is 54.5% and the overall identity among eukaryotic P450 reductases is about 25%. The P450 reductase gene of 19-23 kb was located on chromosome III, as shown by comparison of RFLP-patterns of the P450 reductase gene in two house fly strains and their hybrids.

Amino Acid Sequence↗

PubMed1998

Systematic gain-of-function genetics in Drosophila.

A modular misexpression system was used to carry out systematic gain-of-function genetic screens in Drosophila. The system is based on inducible expression of genes tagged by insertion of a P-element vector carrying a GAL4-regulated promoter oriented to transcribe flanking genomic sequences. To identify genes involved in eye and wing development, the 2300 independent lines were screened for dominant phenotypes. Among many novel genes, the screen identified known genes, including hedgehog and decapentaplegic, implicated in these processes. A genetic interaction screen for suppressors of a cell migration defect in a hypomorphic slow border cells mutant identified known genes with likely roles in tyrosine kinase signaling and control of actin cytoskeleton, among many novel genes. These studies demonstrate the ability of the modular misexpression system to identify developmentally important genes and suggest that it will be generally useful for genetic interaction screens.

Animals↗

PubMed1994

Primary structure and functional expression of a Drosophila cyclic nucleotide-gated channel present in eyes and antennae.

Cyclic nucleotide-gated (CNG) ion channels serve as downstream targets of signalling pathways in vertebrate photoreceptors and olfactory sensory neurons. Whether CNG channels subserve similar functions in invertebrate photoreception and olfaction is unknown. We have cloned genomic DNA and cDNA encoding a cGMP-gated channel from Drosophila. The gene contains at least seven exons. Heterologous expression of cloned cDNA in both Xenopus oocytes and HEK 293 cells gives rise to functional ion channels. The Drosophila CNG channel is approximately 50-fold more sensitive to cGMP than to cAMP. The voltage dependence of blockage by divalent cations is different compared with the CNG channel of rod photoreceptors, and the Ca2+ permeability is much larger. The channel mRNA is expressed in antennae and the visual system of Drosophila. It is proposed that CNG channels are involved in transduction cascades of both invertebrate photoreceptors and olfactory sensillae.

Amino Acid Sequence↗