TheDrosophila melanogaster60A Chromosomal Division Is Extremely Dense with Functional Genes: Their Sequences, Genomic Organization, and Expression

Lukácsovich, Tamás; Asztalos, Zoltán; Juni, Naoto; Awano, Wakae; Yamamoto, Daisuke

doi:10.1006/geno.1999.5746

Cited by 25 publications

(28 citation statements)

References 39 publications

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“…Our observations are consistent with the results of the Genome Annotation Assessment Project, which evaluated different annotation tools on the well-characterized Adh region (30). Moreover, they are reminiscent of data found in the Drosophila gene trap description, whose authors also have identified a significant number of fusions with transcripts absent from the databases (21). These results suggest that the algorithms used to predict genes from genomic databases have missed a significant number of genes.…”

Section: Sensitivity Of the System And Frequency Of Protein Trap Eventssupporting

confidence: 89%

“…When it integrates near a gene enhancer sequence, the reporter is expressed in the same pattern as the endogenous gene controlled by the enhancer. Recently, a gene trap has been developed, in which the reporter gene does not contain a minimal promoter and is expressed only when it integrates within the trapped gene's expressed sequences (21). In this case, the reporter is expected to reproduce the complete transcription pattern of the trapped gene.…”

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confidence: 99%

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A protein trap strategy to detect GFP-tagged proteins expressed from their endogenous loci in Drosophila

Morin

Daneman

Zavortink

et al. 2001

Proc. Natl. Acad. Sci. U.S.A.

749

771

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In Drosophila, enhancer trap strategies allow rapid access to expression patterns, molecular data, and mutations in trapped genes. However, they do not give any information at the protein level, e.g., about the protein subcellular localization. Using the green fluorescent protein (GFP) as a mobile artificial exon carried by a transposable P-element, we have developed a protein trap system. We screened for individual flies, in which GFP tags fulllength endogenous proteins expressed from their endogenous locus, allowing us to observe their cellular and subcellular distribution. GFP fusions are targeted to virtually any compartment of the cell. In the case of insertions in previously known genes, we observe that the subcellular localization of the fusion protein corresponds to the described distribution of the endogenous protein. The artificial GFP exon does not disturb upstream and downstream splicing events. Many insertions correspond to genes not predicted by the Drosophila Genome Project. Our results show the feasibility of a protein trap in Drosophila. GFP reveals in real time the dynamics of protein's distribution in the whole, live organism and provides useful markers for a number of cellular structures and compartments.

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Section: Sensitivity Of the System And Frequency Of Protein Trap Eventssupporting

confidence: 89%

mentioning

confidence: 99%

A protein trap strategy to detect GFP-tagged proteins expressed from their endogenous loci in Drosophila

Morin

Daneman

Zavortink

et al. 2001

Proc. Natl. Acad. Sci. U.S.A.

749

771

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“…However, the significance of these homologies is limited because they are predominantly localized in the central portion of TDP-43, which contains the highly conserved RRM motifs and the glycine-rich region, two elements that TDP-43 shares with many other RNA-binding proteins. Nonetheless, the cloning of homologous proteins in Drosophila (40) and Caenorhabditis (QZ0414) shows that TDP-43 is highly conserved. Interestingly, a comparison of its amino acid sequence with yeast proteins also shows a high identity (ϳ30%) with two factors, HRP1 (Nuclear Polyadenylated RNA-binding protein, involved in pre-mRNA 3Ј-end cleavage and polyadenylation) (41,42) and NSR1 (Nucleolar Protein involved in the processing of 20 S to 18 S rRNA) (43).…”

Section: Discussionmentioning

confidence: 99%

Characterization and Functional Implications of the RNA Binding Properties of Nuclear Factor TDP-43, a Novel Splicing Regulator ofCFTR Exon 9

Buratti

Baralle

2001

Journal of Biological Chemistry

595

606

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Variations in a polymorphic (TG)m sequence near exon 9 of the human CFTR gene have been associated with variable proportions of exon skipping and occurrence of disease. We have recently identified nuclear factor TDP-43 as a novel splicing regulator capable of binding to this element in the CFTR pre-mRNA and inhibiting recognition of the neighboring exon. In this study we report the dissection of the RNA binding properties of TDP-43 and their functional implications in relationship with the splicing process. Our results show that this protein contains two fully functional RNA recognition motif (RRM) domains with distinct RNA/DNA binding characteristics. Interestingly, TDP-43 can bind a minimum number of six UG (or TG) single-stranded dinucleotide stretches, and binding affinity increases with the number of repeats. In particular, the highly conserved Phe residues in the first RRM region play a key role in nucleic acid recognition.We have recently reported the identification of TDP-43 as a splicing regulator that specifically binds the (UG)m-repeated polymorphic region near the 3Ј-splice site of CFTR exon 9 and down-regulates its recognition by the splicing machinery (1). This region, acting in concert with the adjacent (u)n element, is one of the key cis-acting sequences which regulate the proportion of exon 9 skipping in the mature CFTR mRNA transcript (1-3). Considering that exon 9 skipping produces a non-functional CFTR protein (4, 5) the study of the RNA binding properties of TDP-43 is of considerable importance to gain further insight concerning the potential disease-causing consequences of its binding in vivo. Indeed, the clinical relevance of these studies is highlighted by the existence of a clear association between certain (TG)m(T)n alleles with distinct forms of Cystic Fibrosis (1, 6 -9).In addition, the study of (UG)m elements can provide further insight concerning the mRNA splicing process in general because (UG)m sequences have been described to act as splicing regulatory sequences in different genomic contexts. In fact, in addition to the CFTR gene, the presence of simple (UG)mrepeated sequences has been described to influence the splicing process of at least two other genes: the apolipoprotein AII gene (10) and the human cardiac Na ϩ /Ca 2ϩ exchanger (11). In the Apo AII gene the UG tract was shown to be functionally equivalent to a polypyrimidine tract and required for efficient splicing of Apo AII exon 2 (10) while in the human cardiac Na ϩ /Ca 2ϩ exchanger (11) it acts as a strong intronic splicing enhancer situated in intron 2. It should be noted that in contrast with these two genes, the CFTR (UG)m element was found to possess a strong inhibitory effect on CFTR exon 9 splicing, a property that may probably be linked to its peculiar evolutionary history. In fact, sequencing of the mouse CFTR exon 9 genomic region has shown that in the flanking introns, the (TG)m(T)n regulatory elements are absent and that the intron themselves are of very different length when compared with the human introns (...

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“…A transposon carrying a promoterless cDNA accurately reflects endogenous gene expression when integrated downstream of a genomic transcription start site (11). However, flies carrying this construct have to be crossed to a GFP expressing reporter line to reveal the expression pattern in live animals.…”

Section: Resultsmentioning

confidence: 99%

“…1). Because the original construct by Lukacsovich et al (11) was shown to trap promoters, the sequences upstream of GAL4 were kept the same, including a splice acceptor site (SA) and a so-called stop-start site (one small variation was added; see below). Because GFP and GAL4 are both present in our construct, the activity of endogenous promoters should be detectable in the first generation progeny of dysgenic animals.…”

Section: Resultsmentioning

confidence: 99%

An efficient promoter trap for detection of patterned gene expression and subsequent functional analysis in Drosophila

Larsen

Franch-Marro²,

Hartenstein³

et al. 2006

Proc. Natl. Acad. Sci. U.S.A.

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Transposable elements have been used in Drosophila to detect gene expression, inactivate gene function, and induce ectopic expression or overexpression. We have combined all of these features in a single construct. A promoterless GAL4 cDNA is expressed when the construct inserts within a transcriptional unit, and GAL4 activates a GFP-encoding gene present in the same transposon. In a primary screen, patterned gene expression is detected as GFP fluorescence in the live progeny of dysgenic males. Many animals expressing GFP in distinct patterns can be recovered with relatively little effort. As expected, many insertions cause loss of function. After insertion at a genomic location, specific parts of the transposon can be excised by FLP recombinase, thus allowing it to induce conditional misexpression of the tagged gene. Therefore, both gain-and loss-of-function studies can be carried out with a single insertion in a gene identified by virtue of its expression pattern. Using this promoter trap approach, we have identified a group of cells that innervate the calyx of the mushroom body and could thus define a previously unrecognized memory circuit.etermining the function of most genes is a long-term goal in the postgenomic era. This enterprise was initiated many decades ago, much before DNA sequencing, with the numerous forward genetic screens that have been carried out in Drosophila (1) and in other model organisms (2). Such screens have attained an exquisite degree of sophistication, allowing very specific biological functions to be probed. However, forward genetic screens are unlikely to uncover the function of all genes because their activity could be masked by redundancy. Moreover, the function of many genes might be overlooked if they serve a subtle function not needed for viability but essential for fitness in the wild. This is likely to be the case for many brain functions. Homologous recombination technology has the potential to knock out every gene, although this technology is still very laborious (3). Transgenic RNAi is another reverse genetic approach that has a place in the postgenomic era (4), but it is limited by the fact that it usually causes incomplete knock down and that it is still relatively laborious because it requires the construction and validation of individual transgenic strains. As a complement to the loss-of-function assays, misexpression screens based on the GAL4 system (5) have also been very successful in uncovering the activity of many genes in specific tissues (6). Ideally, however, gain-of-function analysis should always be complemented by the loss-of-function phenotype.The pattern of expression can be an alternative starting point for a genetic screen. For example, our work on embryonic boundaries in Drosophila suggests that segmentally expressed genes involved in segmental groove formation remain to be discovered (7). Presumably, these genes have not been identified in the past because of redundancy. A screen based on expression patterns could identify these genes as long as subse...

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TheDrosophila melanogaster60A Chromosomal Division Is Extremely Dense with Functional Genes: Their Sequences, Genomic Organization, and Expression

Cited by 25 publications

References 39 publications

A protein trap strategy to detect GFP-tagged proteins expressed from their endogenous loci in Drosophila

A protein trap strategy to detect GFP-tagged proteins expressed from their endogenous loci in Drosophila

Characterization and Functional Implications of the RNA Binding Properties of Nuclear Factor TDP-43, a Novel Splicing Regulator ofCFTR Exon 9

An efficient promoter trap for detection of patterned gene expression and subsequent functional analysis in Drosophila

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