Skip to main content
PMC home page
Proceedings of the National Academy of Sciences of the United States of America logoLink to Proceedings of the National Academy of Sciences of the United States of America
. 1994 Jan 18;91(2):719–722. doi: 10.1073/pnas.91.2.719

Nature screen: an efficient method for screening natural populations of Drosophila for targeted P-element insertions.

A G Clark 1, S Silveria 1, W Meyers 1, C H Langley 1
PMCID: PMC43020  PMID: 8290588

Abstract

The efficiency of molecular techniques is making it increasingly necessary to rely on reverse genetics to understand the function of genes. Tissue-specific libraries allow one to identify numerous genes that can be cloned, sequenced, and mapped and whose temporal and tissue-specific pattern of expression are well characterized but whose function remains unknown. In such cases, it is desirable to generate targeted mutations to examine the phenotype of loss-of-function lesions. Here we describe a method for identifying naturally occurring variants of Drosophila melanogaster with specific genes tagged by a nearby P element. Imprecise P-element excision can then be used to generate a series of small deletions in or near the gene. In the method described here, large numbers of wild-caught males were crossed to balancer females, and inserts were identified in pooled samples by the polymerase chain reaction with one primer from each target gene and one primer from the P-element terminal repeat. We present the calculations for the probability of successfully tagging a gene and show that it is greatly improved by simultaneously screening inserts into several genes. If a large natural population is available, a nature screen is faster and easier than inducing P-element transposition in the laboratory, but the resulting lines, being genetically heterogeneous, may require more subsequent work to isolate. Using this method to screen the genomes of approximately 10,400 males, we found P-element inserts in close proximity to 3 of 10 genes that were screened.

Full text

PDF
719

Images in this article

721Fig. 3
on p.721

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Ballinger D. G., Benzer S. Targeted gene mutations in Drosophila. Proc Natl Acad Sci U S A. 1989 Dec;86(23):9402–9406. doi: 10.1073/pnas.86.23.9402. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Calvi B. R., Hong T. J., Findley S. D., Gelbart W. M. Evidence for a common evolutionary origin of inverted repeat transposons in Drosophila and plants: hobo, Activator, and Tam3. Cell. 1991 Aug 9;66(3):465–471. doi: 10.1016/0092-8674(81)90010-6. [DOI] [PubMed] [Google Scholar]
  3. Charlesworth B., Langley C. H. The population genetics of Drosophila transposable elements. Annu Rev Genet. 1989;23:251–287. doi: 10.1146/annurev.ge.23.120189.001343. [DOI] [PubMed] [Google Scholar]
  4. DiBenedetto A. J., Harada H. A., Wolfner M. F. Structure, cell-specific expression, and mating-induced regulation of a Drosophila melanogaster male accessory gland gene. Dev Biol. 1990 May;139(1):134–148. doi: 10.1016/0012-1606(90)90284-p. [DOI] [PubMed] [Google Scholar]
  5. Engels W. R., Johnson-Schlitz D. M., Eggleston W. B., Sved J. High-frequency P element loss in Drosophila is homolog dependent. Cell. 1990 Aug 10;62(3):515–525. doi: 10.1016/0092-8674(90)90016-8. [DOI] [PubMed] [Google Scholar]
  6. Falkenthal S., Parker V. P., Davidson N. Developmental variations in the splicing pattern of transcripts from the Drosophila gene encoding myosin alkali light chain result in different carboxyl-terminal amino acid sequences. Proc Natl Acad Sci U S A. 1985 Jan;82(2):449–453. doi: 10.1073/pnas.82.2.449. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Green M. M. THE DISCRIMINATION OF WILD-TYPE ISOALLELES AT THE WHITE LOCUS OF DROSOPHILA MELANOGASTER. Proc Natl Acad Sci U S A. 1959 Apr;45(4):549–553. doi: 10.1073/pnas.45.4.549. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Hamilton B. A., Palazzolo M. J., Chang J. H., VijayRaghavan K., Mayeda C. A., Whitney M. A., Meyerowitz E. M. Large scale screen for transposon insertions into cloned genes. Proc Natl Acad Sci U S A. 1991 Apr 1;88(7):2731–2735. doi: 10.1073/pnas.88.7.2731. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Kaiser K., Goodwin S. F. "Site-selected" transposon mutagenesis of Drosophila. Proc Natl Acad Sci U S A. 1990 Mar;87(5):1686–1690. doi: 10.1073/pnas.87.5.1686. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Milligan C. D., Kaiser K. 'Site-selected' mutagenesis of a Drosophila gene using the I factor retrotransposon. Nucleic Acids Res. 1993 Mar 11;21(5):1323–1324. doi: 10.1093/nar/21.5.1323. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Monsma S. A., Wolfner M. F. Structure and expression of a Drosophila male accessory gland gene whose product resembles a peptide pheromone precursor. Genes Dev. 1988 Sep;2(9):1063–1073. doi: 10.1101/gad.2.9.1063. [DOI] [PubMed] [Google Scholar]
  12. Mulligan P. K., Rasch E. M. The determination of genome size in male and female germ cells of Drosophila melanogaster by DNA-Feulgen cytophotometry. Histochemistry. 1980;66(1):11–18. doi: 10.1007/BF00493241. [DOI] [PubMed] [Google Scholar]
  13. Sandler L, Hiraizumi Y, Sandler I. Meiotic Drive in Natural Populations of Drosophila Melanogaster. I. the Cytogenetic Basis of Segregation-Distortion. Genetics. 1959 Mar;44(2):233–250. doi: 10.1093/genetics/44.2.233. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Voelker R. A., Langley C. H., Brown A. J., Ohnishi S., Dickson B., Montgomery E., Smith S. C. Enzyme null alleles in natural populations of Drosophila melanogaster: Frequencies in a North Carolina population. Proc Natl Acad Sci U S A. 1980 Feb;77(2):1091–1095. doi: 10.1073/pnas.77.2.1091. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Proceedings of the National Academy of Sciences of the United States of America are provided here courtesy of National Academy of Sciences

RESOURCES