The Gene Search System: A Method for Efficient Detection and Rapid Molecular Identification of Genes in Drosophila melanogaster

Abstract We have constructed a P-element-based gene search vector for efficient detection of genes in Drosophila melanogaster. The vector contains two copies of the upstream activating sequence (UAS) enhancer adjacent to a core promoter, one copy near the terminal inverted repeats at each end of the vector, and oriented to direct transcription outward. Genes were detected on the basis of phenotypic changes caused by GAL4-dependent forced expression of vector-flanking DNA, and the transcripts were identified with reverse transcriptase PCR (RT-PCR) using the vector-specific primer and followed by direct sequencing. The system had a greater sensitivity than those already in use for gain-of-function screening: 64% of the vector insertion lines (394/613) showed phenotypes with forced expression of vector-flanking DNA, such as lethality or defects in adult structure. Molecular analysis of 170 randomly selected insertions with forced expression phenotypes revealed that 21% matched the sequences of cloned genes, and 18% matched reported expressed sequence tags (ESTs). Of the insertions in cloned genes, 83% were upstream of the protein-coding region. We discovered two new genes that showed sequence similarity to human genes, Ras-related protein 2 and microsomal glutathione S-transferase. The system can be useful as a tool for the functional mapping of the Drosophila genome.

Download Full-text

Identification of Chromosome Inheritance Modifiers in Drosophila melanogaster

Genetics ◽

10.1093/genetics/157.4.1623 ◽

2001 ◽

Vol 157 (4) ◽

pp. 1623-1637 ◽

Cited By ~ 7

Author(s):

Kenneth W Dobie ◽

Cameron D Kennedy ◽

Vivienne M Velasco ◽

Tory L McGrath ◽

Juliani Weko ◽

...

Keyword(s):

Drosophila Melanogaster ◽

Biological Activity ◽

Cancer Progression ◽

Birth Defects ◽

Mitotic Chromosome ◽

P Element ◽

Inverse Pcr ◽

Gtpase Activating Protein ◽

New Genes ◽

Genomic Analyses

Abstract Faithful chromosome inheritance is a fundamental biological activity and errors contribute to birth defects and cancer progression. We have performed a P-element screen in Drosophila melanogaster with the aim of identifying novel candidate genes involved in inheritance. We used a “sensitized” minichromosome substrate (J21A) to screen ∼3,000 new P-element lines for dominant effects on chromosome inheritance and recovered 78 Sensitized chromosome inheritance modifiers (Scim). Of these, 69 decreased minichromosome inheritance while 9 increased minichromosome inheritance. Fourteen mutations are lethal or semilethal when homozygous and all exhibit dramatic mitotic defects. Inverse PCR combined with genomic analyses identified P insertions within or close to genes with previously described inheritance functions, including wings apart-like (wapl), centrosomin (cnn), and pavarotti (pav). Further, lethal insertions in replication factor complex 4 (rfc4) and GTPase-activating protein 1 (Gap1) exhibit specific mitotic chromosome defects, discovering previously unknown roles for these proteins in chromosome inheritance. The majority of the lines represent mutations in previously uncharacterized loci, many of which have human homologs, and we anticipate that this collection will provide a rich source of mutations in new genes required for chromosome inheritance in metazoans.

Download Full-text

Structure and tissue-specific expression of the Drosophila melanogaster organellar-type Ca2+-ATPase gene

Biochemical Journal ◽

10.1042/bj3100757 ◽

1995 ◽

Vol 310 (3) ◽

pp. 757-763 ◽

Cited By ~ 18

Author(s):

A Magyar ◽

E Bakos ◽

A Váradi

Keyword(s):

Drosophila Melanogaster ◽

Transcription Initiation ◽

Initiation Site ◽

Drosophila Genome ◽

Coding Region ◽

Specific Expression ◽

S1 Nuclease ◽

Protein Coding ◽

Atpase Gene ◽

High Level

A 14 kb genomic clone covering the organellar-type Ca(2+)-ATPase gene of Drosophila melanogaster has been isolated and characterized. The sequence of a 7132 bp region extending from 1.1 kb 5′ upstream of the initiation ATG codon over the polyadenylation signal at the 3′ end has been determined. The gene consists of nine exons including one with an exceptional size of 2172 bp representing 72% of the protein coding region. Introns are relatively small (< 100 bp) except for the 3′ intron which has a size of 2239 bp, an exceptionally large size among Drosophila introns. Five of the introns are in the same positions in Drosophila, Artemia and rabbit SERCA1 Ca(2+)-ATPase genes. There is only one organellar-type Ca(2+)-ATPase gene in the Drosophila genome, as was shown by Southern-blot analysis [Váradi, Gilmore-Hebert and Benz (1989) FEBS Lett. 258, 203-207] and by chromosomal localization [Magyar and Váradi (1990) Biochem. Biophys. Res. Commun. 173, 872-877]. Primer extension and S1-nuclease assays revealed a potential transcription initiation site 876 bp upstream of the translation initiation ATG with a TATA-box 23 bp upstream of this site. Analysis of the 5′ region of the Drosophila organellar-type Ca(2+)-ATPase gene suggests the presence of potential recognition sequences of various muscle-specific transcription factors and shows a region with remarkable similarity to that in the rabbit SERCA2 gene. The tissue distribution of expression of the organellar-type Ca(2+)-ATPase gene has been studied by in situ RNA-RNA hybridization on microscopic sections. A low mRNA abundance can be detected in each tissue of adult flies, suggesting a housekeeping function for the gene. On the other hand a pronounced tissue specificity of expression has also been found as the organellar-type Ca(2+)-ATPase is expressed at a very high level in cell bodies of the central nervous system and in various muscles.

Download Full-text

Duplicated Proteasome Subunit Genes in Drosophila melanogaster Encoding Testes-Specific Isoforms

Genetics ◽

10.1093/genetics/144.1.147 ◽

1996 ◽

Vol 144 (1) ◽

pp. 147-157 ◽

Cited By ~ 3

Author(s):

Xiaoqing Yuan ◽

Mary Miller ◽

John M Belote

Keyword(s):

Drosophila Melanogaster ◽

Genomic Library ◽

Sequence Similarity ◽

Expression Patterns ◽

Primary Spermatocyte ◽

Amino Acid Identity ◽

High Sequence Similarity ◽

New Genes ◽

Cell Type Specific ◽

Elongation Phase

Abstract Using the previously cloned proteasome α-type subunit gene Pros28.1, we screened a Drosophila melanogaster genomic library using reduced stringency conditions to identify closely related genes. Two new genes, Pros28.1A (map position 92F) and Pros28.IB (map position 60D7), showing high sequence similarity to Pros28.1, were identified and characterized. Pros28.1A encodes a protein with 74% amino acid identity to PROS28.1, while the Pros28.1B gene product is 58% identical. The Pros28.1B gene has two introns, located in exactly analogous positions as the two introns in Pros28.1, while the Pros28.IA gene lacks introns. Northern blot analysis reveals that the two new genes are expressed only in males, during the pupal and adult stages. Tissue-specific patterns of expression were examined using transgenic flies carrying Zacz-fusion reporter genes. This analysis revealed that both genes are expressed in germline cells during spermatogenesis, although their expression patterns differed. Pros28.1A expression is first detected at the primary spermatocyte stage and persists into the spermatid elongation phase of spermiogenesis, while Pros28. IB expression is prominent only during spermatid elongation. These genes represent the most striking example of cell-type-specific proteasome gene expression reported to date in any system and support the notion that there is structural and functional heterogeneity among proteasomes in metazoans.

Download Full-text

An inverse PCR screen for the detection of P element insertions in cloned genomic intervals in Drosophila melanogaster.

Genetics ◽

10.1093/genetics/139.2.757 ◽

1995 ◽

Vol 139 (2) ◽

pp. 757-766 ◽

Cited By ~ 1

Author(s):

B Dalby ◽

A J Pereira ◽

L S Goldstein

Keyword(s):

Drosophila Melanogaster ◽

Large Scale ◽

P Element ◽

Inverse Pcr ◽

Drosophila Genome ◽

Screening Approach ◽

Genes Encoding ◽

Larval Serum Protein ◽

Insertion Sites ◽

Sib Selection

Abstract We developed a screening approach that utilizes an inverse polymerase chain reaction (PCR) to detect P element insertions in or near previously cloned genes in Drosophila melanogaster. We used this approach in a large scale genetic screen in which P elements were mobilized from sites on the X chromosome to new autosomal locations. Mutagenized flies were combined in pools, and our screening approach was used to generate probes corresponding to the sequences flanking each site of insertion. These probes then were used for hybridization to cloned genomic intervals, allowing individuals carrying insertions in them to be detected. We used the same approach to perform repeated rounds of sib-selection to generate stable insertion lines. We screened 16,100 insert bearing individuals and recovered 11 insertions in five intervals containing genes encoding members of the kinesin superfamily in Drosophila melanogaster. In addition, we recovered an insertion in the region including the Larval Serum Protein-2 gene. Examination by Southern hybridization confirms that the lines we recovered represent genuine insertions in the corresponding genomic intervals. Our data indicates that this approach will be very efficient both for P element mutagenesis of new genomic regions and for detection and recovery of "local" P element transposition events. In addition, our data constitutes a survey of preferred P element insertion sites in the Drosophila genome and suggests that insertion sites that are mutable at a rate of approximately 10(-4) are distributed every 40-50 kb.

Download Full-text

Regulation of P-Element Transposase Activity in Drosophila melanogaster by hobo Transgenes That Contain KP Elements

Genetics ◽

10.1093/genetics/161.1.205 ◽

2002 ◽

Vol 161 (1) ◽

pp. 205-215 ◽

Cited By ~ 1

Author(s):

Michael J Simmons ◽

Kevin J Haley ◽

Craig D Grimes ◽

John D Raymond ◽

Joseph C L Fong

Keyword(s):

Drosophila Melanogaster ◽

P Element ◽

Drosophila Genome ◽

Maternal Transmission ◽

Alternate Splicing ◽

Hsp70 Promoter ◽

P Elements ◽

Naturally Occurring ◽

Element Activity ◽

Transformation Vectors

Abstract Fusions between the Drosophila hsp70 promoter and three different incomplete P elements, KP, SP, and BP1, were inserted into the Drosophila genome by means of hobo transformation vectors and the resulting transgenic stocks were tested for repression of P-element transposase activity. Only the H(hsp/KP) transgenes repressed transposase activity, and the degree of repression was comparable to that of a naturally occurring KP element. The KP transgenes repressed transposase activity both with and without heat-shock treatments. Both the KP element and H(hsp/KP) transgenes repressed the transposase activity encoded by the modified P element in the P(ry+, Δ2-3)99B transgene more effectively than that encoded by the complete P element in the H(hsp/CP)2 transgene even though the P(ry+, Δ2-3)99B transgene was the stronger transposase source. Repression of both transposase sources appeared to be due to a zygotic effect of the KP element or transgene. There was no evidence for repression by a strictly maternal effect; nor was there any evidence for enhancement of KP repression by the joint maternal transmission of H(hsp/KP) and H(hsp/CP) transgenes. These results are consistent with the idea that KP-mediated repression of P-element activity involves a KP-repressor polypeptide that is not maternally transmitted and that KP-mediated repression is not strengthened by the 66-kD repressor produced by complete P elements through alternate splicing of their RNA.

Download Full-text

The congested-like tracheae Gene of Drosophila melanogaster Encodes a Member of the Mitochondrial Carrier Family Required for Gas-Filling of the Tracheal System and Expansion of the Wings After Eclosion

Genetics ◽

10.1093/genetics/147.4.1755 ◽

1997 ◽

Vol 147 (4) ◽

pp. 1755-1768 ◽

Cited By ~ 1

Author(s):

Kirsten Hartenstein ◽

Pradip Sinha ◽

Arati Mishra ◽

Heide Schenkel ◽

Istvan Török ◽

...

Keyword(s):

Drosophila Melanogaster ◽

Tandem Repeats ◽

Polytene Chromosomes ◽

P Element ◽

Coding Region ◽

Tracheal System ◽

Mitochondrial Carrier ◽

Transmembrane Segments ◽

Hatching Time ◽

Mitochondrial Carrier Family

Abstract A recessive semi-lethal mutation resulting from the insertion of a P-lacW transposon at the cytological position 23A on the polytene chromosomes of Drosophila melanogaster was found to affect the unfolding and expansion of the wings resulting in a loss of venation and a marked decrease in their size. Lethality was polyphasic with numerous animals dying during early larval development and displaying apparently collapsed tracheal trees. The gene was therefore designated as congested-like tracheae, or colt. The colt mutation resulted from the insertion of a P-lacW transposon within the coding region of a 1.4kb transcript. Wild-type function was restored by inducing a precise excision of the P-lacWtransposon, while a deletion of the colt locus, produced by imprecise excision of the P element, showed a phenotype similar to that of the original P insert. The colt gene consists of a single exon and encodes a protein of 306 amino acids made of three tandem repeats, each characterized by two predicted transmembrane segments and a loop domain. The COLT protein shares extensive homology with proteins in the mitochondrial carrier family and particularly with the DIF-1 protein of Caenwhabditis ekgans, which has been shown to be maternally required for embryonic tissue differentiation. Our analysis revealed that zygotic colt function is dispensable for normal embryonic morphogenesis but is required for gas-filling of the tracheal system at hatching time of the embryo and for normal epithelial morphogenesis of the wings.

Download Full-text

Developmental expression and biophysical characterization of a Drosophila melanogaster aquaporin

AJP Cell Physiology ◽

10.1152/ajpcell.00612.2004 ◽

2005 ◽

Vol 289 (2) ◽

pp. C397-C407 ◽

Cited By ~ 92

Author(s):

Nancy Kaufmann ◽

John C. Mathai ◽

Warren G. Hill ◽

Julian A. T. Dow ◽

Mark L. Zeidel ◽

...

Keyword(s):

Drosophila Melanogaster ◽

Water Transport ◽

Molecular Mechanisms ◽

Fluid Transport ◽

Genetic Manipulation ◽

Sequence Similarity ◽

Malpighian Tubule ◽

Developmental Expression ◽

Drosophila Genome ◽

Secretory Vesicles

Aquaporins (AQPs) accelerate the movement of water and other solutes across biological membranes, yet the molecular mechanisms of each AQP's transport function and the diverse physiological roles played by AQP family members are still being defined. We therefore have characterized an AQP in a model organism, Drosophila melanogaster, which is amenable to genetic manipulation and developmental analysis. To study the mechanism of Drosophila Malpighian tubule (MT)-facilitated water transport, we identified seven putative AQPs in the Drosophila genome and found that one of these, previously named DRIP, has the greatest sequence similarity to those vertebrate AQPs that exhibit the highest rates of water transport. In situ mRNA analyses showed that DRIP is expressed in both embryonic and adult MTs, as well as in other tissues in which fluid transport is essential. In addition, the pattern of DRIP expression was dynamic. To define DRIP-mediated water transport, the protein was expressed in Xenopus oocytes and in yeast secretory vesicles, and we found that significantly elevated rates of water transport correlated with DRIP expression. Moreover, the activation energy required for water transport in DRIP-expressing secretory vesicles was 4.9 kcal/mol. This low value is characteristic of AQP-mediated water transport, whereas the value in control vesicles was 16.4 kcal/mol. In contrast, glycerol, urea, ammonia, and proton transport were unaffected by DRIP expression, suggesting that DRIP is a highly selective water-specific channel. This result is consistent with the homology between DRIP and mammalian water-specific AQPs. Together, these data establish Drosophila as a new model system with which to investigate AQP function.

Download Full-text

Sites of P element insertion and structures of P element deletions in the 5' region of Drosophila melanogaster RpII215

Molecular and Cellular Biology ◽

10.1128/mcb.6.10.3312-3319.1986 ◽

1986 ◽

Vol 6 (10) ◽

pp. 3312-3319

Author(s):

L L Searles ◽

A L Greenleaf ◽

W E Kemp ◽

R A Voelker

Keyword(s):

Drosophila Melanogaster ◽

P Element ◽

Nucleotide Sequencing ◽

Insertion Mutation ◽

Activity Levels ◽

Coding Region ◽

Element Insertion ◽

Insertion And Deletion ◽

Transcribed Sequences ◽

Specific Sequences

Several P element insertion and deletion mutations near the 5' end of Drosophila melanogaster RpII215 have been examined by nucleotide sequencing. Two different sites of P element insertion, approximately 90 nucleotides apart, have been detected in this region of the gene. Therefore, including an additional site of P element insertion within the coding region, there are at least three distinct sites of P element insertion at RpII215. Both 5' sites are within a noncoding portion of transcribed sequences. The sequences of four revertants of one P element insertion mutation (D50) indicate that the P element is either precisely deleted or internally deleted to restore RpII215 activity. Partial internal deletions of the P element result in different RpII215 activity levels, which appear to depend on the specific sequences that remain after excision.

Download Full-text

Drosophila nonsense suppressors: functional analysis in Saccharomyces cerevisiae, Drosophila tissue culture cells and Drosophila melanogaster.

Genetics ◽

10.1093/genetics/126.3.625 ◽

1990 ◽

Vol 126 (3) ◽

pp. 625-637

Author(s):

D Garza ◽

M M Medhora ◽

D L Hartl

Keyword(s):

Tissue Culture ◽

Drosophila Melanogaster ◽

P Element ◽

Dominant Male ◽

Trna Genes ◽

Drosophila Genome ◽

Culture Cells ◽

Tissue Culture Cells ◽

Nonsense Suppressors ◽

Beta Galactosidase

Abstract Amber (UAG) and opal (UGA) nonsense suppressors were constructed by oligonucleotide site-directed mutagenesis of two Drosophila melanogaster leucine-tRNA genes and tested in yeast, Drosophila tissue culture cells and transformed flies. Suppression of a variety of amber and opal alleles occurs in yeast. In Drosophila tissue culture cells, the mutant tRNAs suppress hsp70:Adh (alcohol dehydrogenase) amber and opal alleles as well as an hsp70:beta-gal (beta-galactosidase) amber allele. The mutant tRNAs were also introduced into the Drosophila genome by P element-mediated transformation. No measurable suppression was seen in histochemical assays for Adhn4 (amber), AdhnB (opal), or an amber allele of beta-galactosidase. Low levels of suppression (approximately 0.1-0.5% of wild type) were detected using an hsp70:cat (chloramphenicol acetyltransferase) amber mutation. Dominant male sterility was consistently associated with the presence of the amber suppressors.

Download Full-text

Analysis of Two Cosmid Clones from Chromosome 4 of Drosophila melanogaster Reveals Two New Genes Amid an Unusual Arrangement of Repeated Sequences

Genome Research ◽

10.1101/gr.9.2.137 ◽

1999 ◽

Vol 9 (2) ◽

pp. 137-149 ◽

Cited By ~ 1

Author(s):

John Locke ◽

Lynn Podemski ◽

Ken Roy ◽

David Pilgrim ◽

Ross Hodgetts

Keyword(s):

Drosophila Melanogaster ◽

Dna Sequences ◽

Polytene Chromosomes ◽

Mobile Element ◽

The Other ◽

P Element ◽

Matrix Analysis ◽

Predicted Amino Acid Sequence ◽

Chromosome 4 ◽

New Genes

Chromosome 4 from Drosophila melanogaster has several unusual features that distinguish it from the other chromosomes. These include a diffuse appearance in salivary gland polytene chromosomes, an absence of recombination, and the variegated expression of P-element transgenes. As part of a larger project to understand these properties, we are assembling a physical map of this chromosome. Here we report the sequence of two cosmids representing ∼5% of the polytenized region. Both cosmid clones contain numerous repeated DNA sequences, as identified by cross hybridization with labeled genomic DNA, BLAST searches, and dot matrix analysis, which are positioned between and within the transcribed sequences. The repetitive sequences include three copies of the mobile element Hoppel, one copy of the mobile element HB, and 18 DINE repeats. DINE is a novel, short repeated sequence dispersed throughout both cosmid sequences. One cosmid includes the previously described cubitus interruptus(ci) gene and two new genes: that a gene with a predicted amino acid sequence similar to ribosomal protein S3a which is consistent with the Minute(4)101 locus thought to be in the region, and a novel member of the protein family that includes plexin and met–hepatocyte growth factor receptor. The other cosmid contains only the two short 5′-most exons from thezinc-finger-homolog-2 (zfh-2) gene. This is the first extensive sequence analysis of noncoding DNA from chromosome 4. The distribution of the various repeats suggests its organization is similar to the β-heterochromatic regions near the base of the major chromosome arms. Such a pattern may account for the diffuse banding of the polytene chromosome 4 and the variegation of many P-element transgenes on the chromosome.

Download Full-text