A P-Element Insertion Screen Identified Mutations in 455 Novel Essential Genes in Drosophila

Genetics ◽  
2003 ◽  
Vol 163 (1) ◽  
pp. 195-201 ◽  
Author(s):  
Su-Wan Oh ◽  
Tracy Kingsley ◽  
Hyun-hee Shin ◽  
Zhiyu Zheng ◽  
Hua-Wei Chen ◽  
...  
Keyword(s):  
Biological Function ◽  
P Element ◽  
Drosophila Genome ◽  
Gene Products ◽  
Induced Mutations ◽  
Element Insertion ◽  
Lethal Mutations ◽  
Chromosome Mutations ◽  
Eukaryotic Genomes ◽  
Insertion Mutations

Abstract With the completion of the nucleotide sequences of several complex eukaryotic genomes, tens of thousands of genes have been predicted. However, this information has to be correlated with the functions of those genes to enhance our understanding of biology and to improve human health care. The Drosophila transposon P-element-induced mutations are very useful for directly connecting gene products to their biological function. We designed an efficient transposon P-element-mediated gene disruption procedure and performed genetic screening for single P-element insertion mutations, enabling us to recover 2500 lethal mutations. Among these, 2355 are second chromosome mutations. Sequences flanking >2300 insertions that identify 850 different genes or ESTs (783 genes on the second chromosome and 67 genes on the third chromosome) have been determined. Among these, 455 correspond to genes for which no lethal mutation has yet been reported. The Drosophila genome is thought to contain ∼3600 vital genes; 1400 are localized on the second chromosome. Our mutation collection represents ∼56% of the second chromosome vital genes and ∼24% of the total vital Drosophila genes.

scholarly journals Zygotic Lethal Mutations With Maternal Effect Phenotypes in Drosophila melanogaster. II. Loci on the Second and Third Chromosomes Identified by P-Element-Induced Mutations

Genetics ◽  
1996 ◽  
Vol 144 (4) ◽  
pp. 1681-1692 ◽  
Author(s):  
Norbert Perrimon ◽  
Anne Lanjuin ◽  
Charles Arnold ◽  
Elizabeth Noll
Keyword(s):  
Maternal Effect ◽  
Genome Project ◽  
P Element ◽  
Berkeley Drosophila Genome Project ◽  
Drosophila Genome ◽  
Gene Products ◽  
Induced Mutations ◽  
Lethal Mutations ◽  
Egg Formation ◽  
Normal Embryonic Development

Screens for zygotic lethal mutations that are associated with specific maternal effect lethal phenotypes have only been conducted for the X chromosome. To identify loci on the autosomes, which represent four-fifths of the Drosophila genome, we have used the autosomal “FLP-DFS” technique to screen a collection of 496 P element-induced mutations established by the Berkeley Drosophila Genome Project. We have identified 64 new loci whose gene products are required for proper egg formation or normal embryonic development.

scholarly journals P-Element Insertion Alleles of Essential Genes on the Third Chromosome of Drosophila melanogaster Correlation of Physical and Cytogenetic Maps in Chromosomal Region 86E-87F

Genetics ◽  
1997 ◽  
Vol 147 (4) ◽  
pp. 1697-1722 ◽  
Author(s):  
Peter Deák ◽  
Mahmoud M Omar ◽  
Robert D C Saunders ◽  
Margit Pál ◽  
Orbán Komonyi ◽  
...  
Keyword(s):  
Drosophila Melanogaster ◽  
Genetic Maps ◽  
P Element ◽  
Drosophila Genome ◽  
Induced Mutations ◽  
Lethal Mutant ◽  
P Elements ◽  
Element Insertion ◽  
The Third ◽  
Complementation Groups

Abstract We have established a collection of 2460 lethal or semi-lethal mutant lines using a procedure thought to insert single P elements into vital genes on the third chromosome of Drosophila melanogaster. More than 1200 randomly selected lines were examined by in situ hybridization and 90% found to contain single insertions at sites that mark 89% of all lettered subdivisions of the Bridges' map. A set of chromosomal deficiencies that collectively uncover ~25% of the euchromatin of chromosome 3 reveal lethal mutations in 468 lines corresponding to 145 complementation groups. We undertook a detailed analysis of the cytogenetic interval 86E-87F and identified 87 P-element-induced mutations falling into 38 complementation groups, 16 of which correspond to previously known genes. Twenty-one of these 38 complementation groups have at least one allele that has a P-element insertion at a position consistent with the cytogenetics of the locus. We have rescued P elements and flanking chromosomal sequences from the 86E-87F region in 35 lines with either lethal or genetically silent P insertions, and used these as probes to identify cosmids and P1 clones from the Drosophila genome projects. This has tied together the physical and genetic maps and has linked 44 previously identified cosmid contigs into seven “super-contigs” that span the interval. STS data for sequences flanking one side of the P-element insertions in 49 lines has identified insertions in the αγ element at 87C, two known transposable elements, and the open reading frames of seven putative single copy genes. These correspond to five known genes in this interval, and two genes identified by the homology of their predicted products to known proteins from other organisms.

scholarly journals Molecular cloning of suppressor of sable, a Drosophila melanogaster transposon-mediated suppressor.

1986 ◽  
Vol 6 (5) ◽  
pp. 1520-1528 ◽  
Author(s):  
D Y Chang ◽  
B Wisely ◽  
S M Huang ◽  
R A Voelker
Keyword(s):  
Drosophila Melanogaster ◽  
Dna Sequences ◽  
Insertion Site ◽  
Hybrid Dysgenesis ◽  
P Element ◽  
Dna Transformation ◽  
Wild Type ◽  
Induced Mutations ◽  
X Ray ◽  
Element Insertion

A hybrid dysgenesis-induced allele [su(s)w20] associated with a P-element insertion was used to clone sequences from the su(s) region of Drosophila melanogaster by means of the transposon-tagging technique. Cloned sequences were used to probe restriction enzyme-digested DNAs from 22 other su(s) mutations. None of three X-ray-induced or six ethyl methanesulfonate-induced su(s) mutations possessed detectable variation. Seven spontaneous, four hybrid dysgenesis-induced, and two DNA transformation-induced mutations were associated with insertions within 2.0 kilobases (kb) of the su(s)w20 P-element insertion site. When the region of DNA that included the mutational insertions was used to probe poly(A)+ RNAs, a 5-kb message was detected in wild-type RNA that was present in greatly reduced amounts in two su(s) mutations. By using strand-specific probes, the direction of transcription of the 5-kb message was determined. The mutational insertions lie in DNA sequences near the 5' end of the 5-kb message. Three of the seven spontaneous su(s) mutations are associated with gypsy insertions, but they are not suppressible by su(Hw).

scholarly journals The fitness consequences of P element insertion in Drosophila melanogaster

1988 ◽  
Vol 52 (1) ◽  
pp. 17-26 ◽  
Author(s):  
Walter F. Eanes ◽  
Cedric Wesley ◽  
Jody Hey ◽  
David Houle ◽  
James W. Ajioka
Keyword(s):  
Drosophila Melanogaster ◽  
Relative Number ◽  
African Population ◽  
P Element ◽  
Insertion Event ◽  
Element Insertion ◽  
Lethal Mutations ◽  
Fitness Consequences ◽  
Per Se

SummaryIn this study we estimate the frequency at which P-element insertion events, as identified by in situ hybridization, generate lethal and mild viability mutations. The frequency of lethal mutations generated per insertion event was 0·004. Viability dropped an average of 1% per insertion event. Our results indicate that it is deletions and rearrangements resulting from the mobilization of P elements already in place and not the insertions per se that cause the drastic effects on viability and fitness observed in most studies of P–M dysgenesis-derived mutations. Elements of five other families (I, copia, 412, B104, and gypsy) were not mobilized in these crosses. Finally, we contrast the density of P elements on the X chromosome with the density on the four autosomal arms in a collection of thirty genomes from an African population. The relative number of P elements on the X chromosome is too high to be explained by either a hemizygous selection or a neutrality model. The possible reasons for the failure to detect selection are discussed.

scholarly journals Mapping and characterization of P-element-induced mutations at quantitative trait loci in Drosophila melanogaster

1993 ◽  
Vol 61 (3) ◽  
pp. 177-193 ◽  
Author(s):  
Chaoqiang Lai ◽  
Trudy F. C. Mackay
Keyword(s):  
Drosophila Melanogaster ◽  
Physical Mapping ◽  
Hybridization Analysis ◽  
P Element ◽  
Complementation Test ◽  
Induced Mutations ◽  
Element Insertion ◽  
Bristle Number ◽  
Chromosomal Bands

SummaryX chromosomes derived from crosses of inbred P and M Drosophila melanogaster strains that had extreme effects on abdominal and/or sternopleural bristle number in males, were further analyzed to determine their effects in females and to map the loci at which the mutations occurred. Seven lines that had on average 3.9 fewer sternopleural bristles than wildtype in males had average homozygous sternopleural bristle effects of −2·2. The bristle effects were partially recessive, with an average degree of dominance of −0·60. Physical mapping of the sternopleural bristle effects of these lines placed them all at approximately 24·7 cM. These mutations are apparently allelic on the basis of a complementation test, and deficiency mapping indicates they occur within chromosomal bands 8A4; 8C6. In situ hybridization analysis of the sites of P element insertions of these lines suggests that mutations probably resulted from excision of P elements at 8C on the original inbred P strain chromosome. Two additional lines, NDC(19) and DP(146), had reduced numbers of sternopleural and abdominal bristles. NDC(19) males had 9·7 fewer abdominal and 8·6 fewer sternopleural bristles than wildtype. The corresponding homozygous abdominal and sternopleural bristle number effects were −5·8 and −3·8, respectively; with the abdominal bristle effect completely recessive and the sternopleural bristle effect nearly additive. DP(146) males had 6·2 fewer abdominal and 4·1 fewer sternopleural bristles than wildtype, with homozygous abdominal bristle effects of −4·3 and sternopleural bristle effects of −2·0. Abdominal bristle effects of this line were partially recessive whereas the sternopleural bristle effects were additive. Physical mapping showed effects on both bristle traits segregated jointly in these two lines, with the NDC(19) mutation closely linked to y and the DP(146) mutation 0·17 cM from it. Complementation tests and deficiency mapping also indicate the mutations in lines NDC(19) and DP(146) are at closely linked but separate loci within chromosomal bands 1B2; 1B4–6 and 1B4–6; 1B10 respectively, with some epistatic effects. In situ hybridization analysis of sites of P element insertion suggest that the NDC(19) mutation, which may be a scute allele, was probably caused by a P element insertion in the IB region; the DP(146) mutation is also associated with an insertion at IB.

scholarly journals DEVELOPMENTAL USE OF GENE PRODUCTS IN DROSOPHILA: THE MATERNAL-ZYGOTIC TRANSITION

Genetics ◽  
1984 ◽  
Vol 108 (2) ◽  
pp. 361-375
Author(s):  
Leonard G Robbins
Keyword(s):  
X Chromosome ◽  
Developmental Stages ◽  
Large Fraction ◽  
Temperature Shift ◽  
Clonal Analysis ◽  
Drosophila Genome ◽  
Gene Products ◽  
Lethal Mutations ◽  
Wide Range

ABSTRACT Recent results suggest that activity of a large fraction of the Drosophila genome is needed at multiple developmental stages. The timing of the transition from dependence on maternally stored gene products to reliance on zygotically coded products has been examined for several zygotic-lethal mutations in the z-w region of the X chromosome. The mutants differ in zygotic sensitivity to reduced maternal activity, and they have a wide range of times of lethality. Nevertheless, both temperature shift experiments and clonal analysis indicate that all of the maternal-zygotic transitions occur around the time of blastoderm formation.

scholarly journals The Drosophila clathrin heavy chain gene: clathrin function is essential in a multicellular organism.

Genetics ◽  
1993 ◽  
Vol 134 (4) ◽  
pp. 1119-1134 ◽  
Author(s):  
C Bazinet ◽  
A L Katzen ◽  
M Morgan ◽  
A P Mahowald ◽  
S K Lemmon
Keyword(s):  
X Chromosome ◽  
Heavy Chain ◽  
P Element ◽  
Cdna Clones ◽  
Drosophila Genome ◽  
Wild Type ◽  
Degenerate Primers ◽  
Coated Pits ◽  
Lethal Mutations ◽  
Clathrin Heavy Chain

Abstract The clathrin heavy chain (HC) is the major structural polypeptide of the cytoplasmic surface lattice of clathrin-coated pits and vesicles. As a genetic approach to understanding the role of clathrin in cellular morphogenesis and developmental signal transduction, a clathrin heavy chain (Chc) gene of Drosophila melanogaster has been identified by a combination of molecular and classical genetic approaches. Using degenerate primers based on mammalian and yeast clathrin HC sequences, a small fragment of the HC gene was amplified from genomic Drosophila DNA by the polymerase chain reaction. Genomic and cDNA clones from phage libraries were isolated and analyzed using this fragment as a probe. The amino acid sequence of the Drosophila clathrin HC deduced from cDNA sequences is 80%, 57% and 49% identical, respectively, with the mammalian, Dictyostelium and yeast HCs. Hybridization in situ to larval polytene chromosomes revealed a single Chc locus at position 13F2 on the X chromosome. A 13-kb genomic Drosophila fragment including the Chc transcription unit was reintroduced into the Drosophila genome via P element-mediated germline transformation. This DNA complemented a group of EMS-induced lethal mutations mapping to the same region of the X chromosome, thus identifying the Chc complementation group. Mutant individuals homozygous or hemizygous for the Chc1, Chc2 or Chc3 alleles developed to a late stage of embryogenesis, but failed to hatch to the first larval stage. A fourth allele, Chc4, exhibited polyphasic lethality, with a significant number of homozygous and hemizygous offspring surviving to adulthood. Germline clonal analysis of Chc mutant alleles indicated that the three tight lethal alleles were autonomous cell-lethal mutations in the female germline. In contrast, Chc4 germline clones were viable at a rate comparable to wild type, giving rise to viable adult progeny. However, hemizygous Chc4 males were invariably sterile. The sterility was efficiently rescued by an autosomal copy of the wild-type Chc gene reintroduced on a P element. These findings suggest a specialized role for clathrin in spermatogenesis.

scholarly journals Somatic reversion of P transposable element insertion mutations in the singed locus of Drosophila melanogaster requiring specific P insertions and a trans- acting factor

1989 ◽  
Vol 54 (2) ◽  
pp. 101-112 ◽  
Author(s):  
John F. Y. Brookfield ◽  
Alan P. Lewis
Keyword(s):  
Drosophila Melanogaster ◽  
Transposable Element ◽  
Maternal Effect ◽  
P Element ◽  
Chromosome 2 ◽  
Restriction Maps ◽  
Element Insertion ◽  
Somatic Reversion ◽  
P Transposable Element ◽  
Insertion Mutations

SummaryDestabilization in somatic cells of P-element insertions in the X-linked singed gene of Drosophila melanogaster has been studied. We have shown that some but not all unstable P-element insertions in singed can form mosaics. The cause of this variation is not clear from studies of the restriction maps of the mutations tested. The transposable element movements occur early in development and require, in addition to an appropriate P-element insertion in singed, a trans-acting maternal effect component. Movements appear to occur preferentially in attached-X stocks. However, the maternal effect component maps to the central region of chromosome 2.

scholarly journals CYTOGENETIC ANALYSIS OF THE cSOD MICROREGION IN DROSOPHILA MELANOGASTER

Genetics ◽  
1986 ◽  
Vol 112 (2) ◽  
pp. 205-215
Author(s):  
Shelagh D Campbell ◽  
Arthur J Hilliker ◽  
John P Phillips
Keyword(s):  
Drosophila Melanogaster ◽  
Hot Spot ◽  
P Element ◽  
Single Group ◽  
Induced Mutations ◽  
Euchromatic Region ◽  
Hypomorphic Allele ◽  
Lethal Mutations ◽  
Chromosome Bands

ABSTRACT This report describes the genetic organization of a euchromatic region on the third chromosome of Drosophila melanogaster extending cytologically from 68A2 to C1, an interval comprising 10 or 11 polytene chromosome bands. The gene for cytoplasmic superoxide dismutase (cSOD) maps within this interval, as does low xanthine dehydrogenase (lxd).—Recessive lethal mutations were generated within the region by ethyl methanesulfonate mutagenesis and by hybrid dysgenesis. These lethals fall into 11 functional groups, which were partially ordered by complementation with deletions having breakpoints within the region. The distribution of dysgenesis-induced mutations in the region is highly nonrandom, the majority being within a single group. The mutability of this gene is comparable to that of singed (sn), a documented "hot-spot" for P-element insertion.—One of the EMS-induced lethals, l-108, fulfills biochemical criteria expected of a hypomorphic allele of cSOD. To our knowledge this is the first such allele recovered of this gene, and it should prove very useful in an analysis of the in vivo function of cytoplasmic SOD. Indeed, it has been demonstrated that cSOD is almost certainly a vital gene.

Molecular cloning of suppressor of sable, a Drosophila melanogaster transposon-mediated suppressor

1986 ◽  
Vol 6 (5) ◽  
pp. 1520-1528
Author(s):  
D Y Chang ◽  
B Wisely ◽  
S M Huang ◽  
R A Voelker
Keyword(s):  
Drosophila Melanogaster ◽  
Dna Sequences ◽  
Insertion Site ◽  
Hybrid Dysgenesis ◽  
P Element ◽  
Dna Transformation ◽  
Wild Type ◽  
Induced Mutations ◽  
X Ray ◽  
Element Insertion

A hybrid dysgenesis-induced allele [su(s)w20] associated with a P-element insertion was used to clone sequences from the su(s) region of Drosophila melanogaster by means of the transposon-tagging technique. Cloned sequences were used to probe restriction enzyme-digested DNAs from 22 other su(s) mutations. None of three X-ray-induced or six ethyl methanesulfonate-induced su(s) mutations possessed detectable variation. Seven spontaneous, four hybrid dysgenesis-induced, and two DNA transformation-induced mutations were associated with insertions within 2.0 kilobases (kb) of the su(s)w20 P-element insertion site. When the region of DNA that included the mutational insertions was used to probe poly(A)+ RNAs, a 5-kb message was detected in wild-type RNA that was present in greatly reduced amounts in two su(s) mutations. By using strand-specific probes, the direction of transcription of the 5-kb message was determined. The mutational insertions lie in DNA sequences near the 5' end of the 5-kb message. Three of the seven spontaneous su(s) mutations are associated with gypsy insertions, but they are not suppressible by su(Hw).

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