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

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.

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Efficient Recovery of Centric Heterochromatin P-Element Insertions in Drosophila melanogaster

Genetics ◽

10.1093/genetics/161.1.217 ◽

2002 ◽

Vol 161 (1) ◽

pp. 217-229 ◽

Cited By ~ 10

Author(s):

Christopher M Yan ◽

Kenneth W Dobie ◽

Hiep D Le ◽

Alexander Y Konev ◽

Gary H Karpen

Keyword(s):

Drosophila Melanogaster ◽

Large Scale ◽

Structure And Function ◽

P Element ◽

Inverse Pcr ◽

Fish Analysis ◽

Genomic Scaffold ◽

Efficient Recovery ◽

Resolution Banding ◽

And Function

Abstract Approximately one-third of the human and Drosophila melanogaster genomes are heterochromatic, yet we know very little about the structure and function of this enigmatic component of eukaryotic genomes. To facilitate molecular and cytological analysis of heterochromatin we introduced a yellow+ (y+)-marked P element into centric heterochromatin by screening for variegated phenotypes, that is, mosaic gene inactivation. We recovered >110 P insertions with variegated yellow expression from ∼3500 total mobilization events. FISH analysis of 71 of these insertions showed that 69 (97%) were in the centric heterochromatin, rather than telomeres or euchromatin. High-resolution banding analysis showed a wide but nonuniform distribution of insertions within centric heterochromatin; variegated insertions were predominantly recovered near regions of satellite DNA. We successfully used inverse PCR to clone and sequence the flanking DNA for ∼63% of the insertions. BLAST analysis of the flanks demonstrated that either most of the variegated insertions could not be placed on the genomic scaffold, and thus may be inserted within novel DNA sequence, or that the flanking DNA hit multiple sites on the scaffold, due to insertions within different transposons. Taken together these data suggest that screening for yellow variegation is a very efficient method for recovering centric insertions and that a large-scale screen for variegated yellow P insertions will provide important tools for detailed analysis of centric heterochromatin structure and function.

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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.

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

Genetics ◽

10.1093/genetics/151.2.725 ◽

1999 ◽

Vol 151 (2) ◽

pp. 725-737 ◽

Cited By ~ 6

Author(s):

Gakuta Toba ◽

Takashi Ohsako ◽

Naomasa Miyata ◽

Tsuyoshi Ohtsuka ◽

Ki-Hyeon Seong ◽

...

Keyword(s):

Drosophila Melanogaster ◽

Core Promoter ◽

Sequence Similarity ◽

Direct Sequencing ◽

P Element ◽

Drosophila Genome ◽

Coding Region ◽

New Genes ◽

Gene Search ◽

Efficient Detection

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.

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A selective screen to recover chromosomal deletions and duplications in Drosophila melanogaster.

Genetics ◽

10.1093/genetics/119.2.377 ◽

1988 ◽

Vol 119 (2) ◽

pp. 377-390

Author(s):

D Gubb ◽

S McGill ◽

M Ashburner

Keyword(s):

Drosophila Melanogaster ◽

Chromosomal Region ◽

Molecular Level ◽

Hybrid Dysgenesis ◽

P Element ◽

Genomic Clones ◽

Element Insertion ◽

Chromosomal Deletions ◽

Complementation Groups ◽

Insertion Sites

Abstract A screen is described that will select for breakpoints within a restricted chromosomal region in Drosophila. The aberrations recovered can be used to construct chromosomes carrying synthetic duplications and deletions. Such chromosomes have applications in the mapping of complementation groups at both the genetic and molecular level. In particular, breakpoints recovered after P element hybrid dysgenesis tend to be associated with P element insertion sites. Such aberration breakpoints can be genetically mapped, as synthetic deletions, and then used as transposon-tagged sites for the recovery of genomic clones.

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An Exploration of the Sequence of a 2.9-Mb Region of the Genome of Drosophila melanogaster: The Adh Region

Genetics ◽

10.1093/genetics/153.1.179 ◽

1999 ◽

Vol 153 (1) ◽

pp. 179-219 ◽

Cited By ~ 15

Author(s):

M Ashburner ◽

S Misra ◽

J Roote ◽

S E Lewis ◽

R Blazej ◽

...

Keyword(s):

Drosophila Melanogaster ◽

Transposable Element ◽

Large Scale ◽

Chromosome Region ◽

Complete Sequence ◽

Test Methods ◽

P Element ◽

Cdna Libraries ◽

Protein Coding ◽

Protein Coding Genes

Abstract A contiguous sequence of nearly 3 Mb from the genome of Drosophila melanogaster has been sequenced from a series of overlapping P1 and BAC clones. This region covers 69 chromosome polytene bands on chromosome arm 2L, including the genetically well-characterized “Adh region.” A computational analysis of the sequence predicts 218 protein-coding genes, 11 tRNAs, and 17 transposable element sequences. At least 38 of the protein-coding genes are arranged in clusters of from 2 to 6 closely related genes, suggesting extensive tandem duplication. The gene density is one protein-coding gene every 13 kb; the transposable element density is one element every 171 kb. Of 73 genes in this region identified by genetic analysis, 49 have been located on the sequence; P-element insertions have been mapped to 43 genes. Ninety-five (44%) of the known and predicted genes match a Drosophila EST, and 144 (66%) have clear similarities to proteins in other organisms. Genes known to have mutant phenotypes are more likely to be represented in cDNA libraries, and far more likely to have products similar to proteins of other organisms, than are genes with no known mutant phenotype. Over 650 chromosome aberration breakpoints map to this chromosome region, and their nonrandom distribution on the genetic map reflects variation in gene spacing on the DNA. This is the first large-scale analysis of the genome of D. melanogaster at the sequence level. In addition to the direct results obtained, this analysis has allowed us to develop and test methods that will be needed to interpret the complete sequence of the genome of this species.

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Unusual properties of regulatory DNA from the Drosophila engrailed gene: three "pairing-sensitive" sites within a 1.6-kb region.

Genetics ◽

10.1093/genetics/136.3.1025 ◽

1994 ◽

Vol 136 (3) ◽

pp. 1025-1038 ◽

Cited By ~ 6

Author(s):

J A Kassis

Keyword(s):

Marker Gene ◽

P Element ◽

Drosophila Genome ◽

Trithorax Group ◽

Eye Color ◽

P Elements ◽

In Trans ◽

Insertion Sites ◽

Regulatory Dna ◽

In Cis

Abstract We have previously shown that a 2-kb fragment of engrailed DNA can suppress expression of a linked marker gene, white, in the P element vector CaSpeR. This suppression is dependent on the presence of two copies of engrailed DNA-containing P elements (P[en]) in proximity in the Drosophila genome (either in cis or in trans). In this study, the 2-kb fragment was dissected and found to contain three fragments of DNA which could mediate white suppression [called "pairing-sensitive sites" (PS)]. A PS site was also identified in regulatory DNA from the Drosophila escargot gene. The eye colors of six different P[en] insertions in the escargot gene suggest an interaction between P[en]-encoded and genome-encoded PS sites. I hypothesize that white gene expression from P[en] is repressed by the formation of a protein complex which is initiated at the engrailed PS sites and also requires interactions with flanking genomic DNA. Genes were sought which influence the function of PS sites. Mutations in some Polycomb and trithorax group genes were found to affect the eye color from some P[en] insertion sites. However, different mutations affected expression from different P[en] insertion sites and no one mutation was found to affect expression from all P[en] insertion sites examined. These results suggest that white expression from P[en] is not directly regulated by members of the Polycomb and trithorax group genes, but in some cases can be influenced by them. I propose that engrailed PS sites normally act to promote interactions between distantly located engrailed regulatory sites and the engrailed promoter.

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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.

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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.

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Transposable element-induced response to artificial selection in Drosophila melanogaster: molecular analysis of selection lines.

Genetics ◽

10.1093/genetics/125.4.803 ◽

1990 ◽

Vol 125 (4) ◽

pp. 803-811 ◽

Cited By ~ 1

Author(s):

A E Shrimpton ◽

T F Mackay ◽

A J Brown

Keyword(s):

Drosophila Melanogaster ◽

Artificial Selection ◽

Genetic Variance ◽

Selection Response ◽

P Element ◽

Induced Response ◽

Phenotypic Variance ◽

P Elements ◽

Insertion Sites

Abstract Artificial selection lines for abdominal bristle score of Drosophila melanogaster established from P-M hybrid dysgenic crosses showed increases in selection response, heritability and phenotypic variance compared to similar lines started from nondysgenic crosses. To determine whether this increased genetic variance could be due to enhanced transposition of P elements following the dysgenic cross, the cytological locations (sites) of P elements were determined by in situ hybridization for the whole genome of samples of 20 individuals from the parental P strain, 20 individuals from each of the eight dysgenic selection lines, and ten individuals from each of the eight nondysgenic selection lines. Variation among and within the selection lines and the parental P strain in P element insertion sites was exceptionally high. A total of 601 sites were identified, but there was no difference in total number of sites per line, mean number of sites per individual, mean copy number per individual, or site frequency between dysgenic and nondysgenic selection lines, or between lines selected for high and low bristle score. Transposition following nondysgenic crosses may explain additional observations of accelerated selection responses in nondysgenic selection lines. It was not possible to deduce which, if any, of the several hundred insertions in the dysgenic selection lines were responsible for their extreme bristle phenotypes.

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Large-Scale Identification of Virulence Genes fromStreptococcus pneumoniae

Infection and Immunity ◽

10.1128/iai.66.12.5620-5629.1998 ◽

1998 ◽

Vol 66 (12) ◽

pp. 5620-5629 ◽

Cited By ~ 308

Author(s):

Alessandra Polissi ◽

Andrea Pontiggia ◽

Georg Feger ◽

Mario Altieri ◽

Harald Mottl ◽

...

Keyword(s):

Virulence Factors ◽

Dna Sequences ◽

Large Scale ◽

Virulence Genes ◽

Dna Recombination ◽

Inverse Pcr ◽

Chloramphenicol Resistance ◽

Virulence Attenuation ◽

Genes Encoding

ABSTRACT Streptococcus pneumoniae is the major cause of bacterial pneumonia, and it is also responsible for otitis media and meningitis in children. Apart from the capsule, the virulence factors of this pathogen are not completely understood. Recent technical advances in the field of bacterial pathogenesis (in vivo expression technology and signature-tagged mutagenesis [STM]) have allowed a large-scale identification of virulence genes. We have adapted to S. pneumoniae the STM technique, originally used for the discovery of Salmonella genes involved in pathogenicity. A library of pneumococcal chromosomal fragments (400 to 600 bp) was constructed in a suicide plasmid vector carrying unique DNA sequence tags and a chloramphenicol resistance marker. The recent clinical isolate G54 was transformed with this library. Chloramphenicol-resistant mutants were obtained by homologous recombination, resulting in genes inactivated by insertion of the suicide vector carrying a unique tag. In a mouse pneumonia model, 1.250 candidate clones were screened; 200 of these were not recovered from the lungs were therefore considered virulence-attenuated mutants. The regions flanking the chloramphenicol gene of the attenuated mutants were amplified by inverse PCR and sequenced. The sequence analysis showed that the 200 mutants had insertions in 126 different genes that could be grouped in six classes: (i) known pneumococcal virulence genes; (ii) genes involved in metabolic pathways; (iii) genes encoding proteases; (iv) genes coding for ATP binding cassette transporters; (v) genes encoding proteins involved in DNA recombination/repair; and (vi) DNA sequences that showed similarity to hypothetical genes with unknown function. To evaluate the virulence attenuation for each mutant, all 126 clones were individually analyzed in a mouse septicemia model. Not all mutants selected in the pneumonia model were confirmed in septicemia, thus indicating the existence of virulence factors specific for pneumonia.

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