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Genes involved in Drosophila glutamate receptor expression and localization.

Liebl FL, Featherstone DE - BMC Neurosci (2005)

Bottom Line: Expression pattern analyses and complementation tests, however, suggest that any single mutant - even if a mutant gene is uniquely tagged - must be interpreted with caution until the mutation is validated genetically and phenotypically.Our study identified 57 transposon mutants with qualitative differences in glutamate receptor expression and localization.Alternatively, one can focus on the types of genes identified, rather than the identities of individual genes.

View Article: PubMed Central - HTML - PubMed

Affiliation: Dept. of Cell and Structural Biology, Univ. of Illinois at Urbana-Champaign, Urbana, USA. lieblfb@msn.com

ABSTRACT

Background: A clear picture of the mechanisms controlling glutamate receptor expression, localization, and stability remains elusive, possibly due to an incomplete understanding of the proteins involved. We screened transposon mutants generated by the ongoing Drosophila Gene Disruption Project in an effort to identify the different types of genes required for glutamate receptor cluster development.

Results: To enrich for non-silent insertions with severe disruptions in glutamate receptor clustering, we identified and focused on homozygous lethal mutants in a collection of 2185 BG and KG transposon mutants generated by the BDGP Gene Disruption Project. 202 lethal mutant lines were individually dissected to expose glutamatergic neuromuscular junctions, stained using antibodies that recognize neuronal membrane and the glutamate receptor subunit GluRIIA, and viewed using laser-scanning confocal microscopy. We identified 57 mutants with qualitative differences in GluRIIA expression and/or localization. 84% of mutants showed loss of receptors and/or clusters; 16% of mutants showed an increase in receptors. Insertion loci encode a variety of protein types, including cytoskeleton proteins and regulators, kinases, phosphatases, ubiquitin ligases, mucins, cell adhesion proteins, transporters, proteins controlling gene expression and protein translation, and proteins of unknown/novel function. Expression pattern analyses and complementation tests, however, suggest that any single mutant - even if a mutant gene is uniquely tagged - must be interpreted with caution until the mutation is validated genetically and phenotypically.

Conclusion: Our study identified 57 transposon mutants with qualitative differences in glutamate receptor expression and localization. Despite transposon tagging of every insertion locus, extensive validation is needed before one can have confidence in the role of any individual gene. Alternatively, one can focus on the types of genes identified, rather than the identities of individual genes. This genomic approach, which circumvents many technical caveats in favor of a wider perspective, suggests that glutamate receptor cluster formation involves many cellular processes, including: 1) cell adhesion and signaling, 2) extensive and relatively specific regulation of gene expression and RNA, 3) the actin and microtubule cytoskeletons, and 4) many novel/unexplored processes, such as those involving mucin/polycystin-like proteins and proteins of unknown function.

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Pie chart showing the different types of proteins encoded by the insertion loci identified in the screen. The size of the pie wedge represents the proportion of genes of each type identified. Functional categories were based on previously published studies of the Drosophila gene and/or sequence similarity to functionally annotated mouse genes. Table 1 contains a complete list of the genes represented in this chart.
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Figure 2: Pie chart showing the different types of proteins encoded by the insertion loci identified in the screen. The size of the pie wedge represents the proportion of genes of each type identified. Functional categories were based on previously published studies of the Drosophila gene and/or sequence similarity to functionally annotated mouse genes. Table 1 contains a complete list of the genes represented in this chart.

Mentions: For all of the mutants identified in our screen, the Gene Disruption Project has precisely determined the genomic insertion site of the transposon using inverse PCR. In the vast majority of cases, inverse PCR results were consistent with a unique insertion, and in most cases the flanking genomic sequence revealed that the insertion was in an annotated gene. For each of the mutants identified in our screen, we used the BDGDP P-screen database and/or BLAST searches with flanking genomic sequence from iPCR results to identify which gene was mutated by the inserted P-element. Putative functions were assigned to each of these genes based on previous publications, FlyBase annotations, and/or Genbank BLAST searches. We binned each of the insertion site genes into one of the following functional categories: 1) extracellular matrix proteins (mucins), 2) cell adhesion proteins 3) cytoskeleton proteins, cytoskeletal regulators, and adaptor proteins, 4) kinases and phosphatases, 5) ubiqitination proteins (ubiquitin ligases), 6) transporters/pumps, 7) proteins involved in gene expression and protein translation, 8) enzymes, and 9) proteins of unknown/novel function. These categories, and the relative number of proteins in each category, are illustrated by Figure 2. All categories shown in Figure 2 were represented by at least two mutant genes.


Genes involved in Drosophila glutamate receptor expression and localization.

Liebl FL, Featherstone DE - BMC Neurosci (2005)

Pie chart showing the different types of proteins encoded by the insertion loci identified in the screen. The size of the pie wedge represents the proportion of genes of each type identified. Functional categories were based on previously published studies of the Drosophila gene and/or sequence similarity to functionally annotated mouse genes. Table 1 contains a complete list of the genes represented in this chart.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC1173111&req=5

Figure 2: Pie chart showing the different types of proteins encoded by the insertion loci identified in the screen. The size of the pie wedge represents the proportion of genes of each type identified. Functional categories were based on previously published studies of the Drosophila gene and/or sequence similarity to functionally annotated mouse genes. Table 1 contains a complete list of the genes represented in this chart.
Mentions: For all of the mutants identified in our screen, the Gene Disruption Project has precisely determined the genomic insertion site of the transposon using inverse PCR. In the vast majority of cases, inverse PCR results were consistent with a unique insertion, and in most cases the flanking genomic sequence revealed that the insertion was in an annotated gene. For each of the mutants identified in our screen, we used the BDGDP P-screen database and/or BLAST searches with flanking genomic sequence from iPCR results to identify which gene was mutated by the inserted P-element. Putative functions were assigned to each of these genes based on previous publications, FlyBase annotations, and/or Genbank BLAST searches. We binned each of the insertion site genes into one of the following functional categories: 1) extracellular matrix proteins (mucins), 2) cell adhesion proteins 3) cytoskeleton proteins, cytoskeletal regulators, and adaptor proteins, 4) kinases and phosphatases, 5) ubiqitination proteins (ubiquitin ligases), 6) transporters/pumps, 7) proteins involved in gene expression and protein translation, 8) enzymes, and 9) proteins of unknown/novel function. These categories, and the relative number of proteins in each category, are illustrated by Figure 2. All categories shown in Figure 2 were represented by at least two mutant genes.

Bottom Line: Expression pattern analyses and complementation tests, however, suggest that any single mutant - even if a mutant gene is uniquely tagged - must be interpreted with caution until the mutation is validated genetically and phenotypically.Our study identified 57 transposon mutants with qualitative differences in glutamate receptor expression and localization.Alternatively, one can focus on the types of genes identified, rather than the identities of individual genes.

View Article: PubMed Central - HTML - PubMed

Affiliation: Dept. of Cell and Structural Biology, Univ. of Illinois at Urbana-Champaign, Urbana, USA. lieblfb@msn.com

ABSTRACT

Background: A clear picture of the mechanisms controlling glutamate receptor expression, localization, and stability remains elusive, possibly due to an incomplete understanding of the proteins involved. We screened transposon mutants generated by the ongoing Drosophila Gene Disruption Project in an effort to identify the different types of genes required for glutamate receptor cluster development.

Results: To enrich for non-silent insertions with severe disruptions in glutamate receptor clustering, we identified and focused on homozygous lethal mutants in a collection of 2185 BG and KG transposon mutants generated by the BDGP Gene Disruption Project. 202 lethal mutant lines were individually dissected to expose glutamatergic neuromuscular junctions, stained using antibodies that recognize neuronal membrane and the glutamate receptor subunit GluRIIA, and viewed using laser-scanning confocal microscopy. We identified 57 mutants with qualitative differences in GluRIIA expression and/or localization. 84% of mutants showed loss of receptors and/or clusters; 16% of mutants showed an increase in receptors. Insertion loci encode a variety of protein types, including cytoskeleton proteins and regulators, kinases, phosphatases, ubiquitin ligases, mucins, cell adhesion proteins, transporters, proteins controlling gene expression and protein translation, and proteins of unknown/novel function. Expression pattern analyses and complementation tests, however, suggest that any single mutant - even if a mutant gene is uniquely tagged - must be interpreted with caution until the mutation is validated genetically and phenotypically.

Conclusion: Our study identified 57 transposon mutants with qualitative differences in glutamate receptor expression and localization. Despite transposon tagging of every insertion locus, extensive validation is needed before one can have confidence in the role of any individual gene. Alternatively, one can focus on the types of genes identified, rather than the identities of individual genes. This genomic approach, which circumvents many technical caveats in favor of a wider perspective, suggests that glutamate receptor cluster formation involves many cellular processes, including: 1) cell adhesion and signaling, 2) extensive and relatively specific regulation of gene expression and RNA, 3) the actin and microtubule cytoskeletons, and 4) many novel/unexplored processes, such as those involving mucin/polycystin-like proteins and proteins of unknown function.

Show MeSH
Related in: MedlinePlus