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Genetics. May 2002; 161(1): 217–229.
PMCID: PMC1462106

Efficient recovery of centric heterochromatin P-element insertions in Drosophila melanogaster.

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 approximately 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 approximately 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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Selected References

These references are in PubMed. This may not be the complete list of references from this article.
  • Adams MD, Celniker SE, Holt RA, Evans CA, Gocayne JD, Amanatides PG, Scherer SE, Li PW, Hoskins RA, Galle RF, et al. The genome sequence of Drosophila melanogaster. Science. 2000 Mar 24;287(5461):2185–2195. [PubMed]
  • Berg CA, Spradling AC. Studies on the rate and site-specificity of P element transposition. Genetics. 1991 Mar;127(3):515–524. [PMC free article] [PubMed]
  • Copenhaver GP, Nickel K, Kuromori T, Benito MI, Kaul S, Lin X, Bevan M, Murphy G, Harris B, Parnell LD, et al. Genetic definition and sequence analysis of Arabidopsis centromeres. Science. 1999 Dec 24;286(5449):2468–2474. [PubMed]
  • Cryderman DE, Cuaycong MH, Elgin SC, Wallrath LL. Characterization of sequences associated with position-effect variegation at pericentric sites in Drosophila heterochromatin. Chromosoma. 1998 Nov;107(5):277–285. [PubMed]
  • Csink AK, Henikoff S. Genetic modification of heterochromatic association and nuclear organization in Drosophila. Nature. 1996 Jun 6;381(6582):529–531. [PubMed]
  • Dej KJ, Orr-Weaver TL. Separation anxiety at the centromere. Trends Cell Biol. 2000 Sep;10(9):392–399. [PubMed]
  • Dernburg AF, Broman KW, Fung JC, Marshall WF, Philips J, Agard DA, Sedat JW. Perturbation of nuclear architecture by long-distance chromosome interactions. Cell. 1996 May 31;85(5):745–759. [PubMed]
  • Lilly MA, Spradling AC. The Drosophila endocycle is controlled by Cyclin E and lacks a checkpoint ensuring S-phase completion. Genes Dev. 1996 Oct 1;10(19):2514–2526. [PubMed]
  • Dernburg AF, Sedat JW, Hawley RS. Direct evidence of a role for heterochromatin in meiotic chromosome segregation. Cell. 1996 Jul 12;86(1):135–146. [PubMed]
  • Lohe AR, Hilliker AJ, Roberts PA. Mapping simple repeated DNA sequences in heterochromatin of Drosophila melanogaster. Genetics. 1993 Aug;134(4):1149–1174. [PMC free article] [PubMed]
  • Devlin RH, Holm DG, Morin KR, Honda BM. Identifying a single-copy DNA sequence associated with the expression of a heterochromatic gene, the light locus of Drosophila melanogaster. Genome. 1990 Jun;33(3):405–415. [PubMed]
  • Devlin RH, Bingham B, Wakimoto BT. The organization and expression of the light gene, a heterochromatic gene of Drosophila melanogaster. Genetics. 1990 May;125(1):129–140. [PMC free article] [PubMed]
  • Mukai M, Kashikawa M, Kobayashi S. Induction of indora expression in pole cells by the mesoderm is required for female germ-line development in Drosophila melanogaster. Development. 1999 Feb;126(5):1023–1029. [PubMed]
  • Dobie KW, Kennedy CD, Velasco VM, McGrath TL, Weko J, Patterson RW, Karpen GH. Identification of chromosome inheritance modifiers in Drosophila melanogaster. Genetics. 2001 Apr;157(4):1623–1637. [PMC free article] [PubMed]
  • Murphy TD, Karpen GH. Localization of centromere function in a Drosophila minichromosome. Cell. 1995 Aug 25;82(4):599–609. [PMC free article] [PubMed]
  • Donaldson KM, Karpen GH. Trans-suppression of terminal deficiency-associated position effect variegation in a Drosophila minichromosome. Genetics. 1997 Feb;145(2):325–337. [PMC free article] [PubMed]
  • Peacock WJ, Lohe AR, Gerlach WL, Dunsmuir P, Dennis ES, Appels R. Fine structure and evolution of DNA in heterochromatin. Cold Spring Harb Symp Quant Biol. 1978;42(Pt 2):1121–1135. [PubMed]
  • Eggert H, Bergemann K, Saumweber H. Molecular screening for P-element insertions in a large genomic region of Drosophila melanogaster using polymerase chain reaction mediated by the vectorette. Genetics. 1998 Jul;149(3):1427–1434. [PMC free article] [PubMed]
  • Pimpinelli S, Berloco M, Fanti L, Dimitri P, Bonaccorsi S, Marchetti E, Caizzi R, Caggese C, Gatti M. Transposable elements are stable structural components of Drosophila melanogaster heterochromatin. Proc Natl Acad Sci U S A. 1995 Apr 25;92(9):3804–3808. [PMC free article] [PubMed]
  • Roseman RR, Johnson EA, Rodesch CK, Bjerke M, Nagoshi RN, Geyer PK. A P element containing suppressor of hairy-wing binding regions has novel properties for mutagenesis in Drosophila melanogaster. Genetics. 1995 Nov;141(3):1061–1074. [PMC free article] [PubMed]
  • Gatti M, Bonaccorsi S, Pimpinelli S. Looking at Drosophila mitotic chromosomes. Methods Cell Biol. 1994;44:371–391. [PubMed]
  • Rubin GM, Hong L, Brokstein P, Evans-Holm M, Frise E, Stapleton M, Harvey DA. A Drosophila complementary DNA resource. Science. 2000 Mar 24;287(5461):2222–2224. [PubMed]
  • Schueler MG, Higgins AW, Rudd MK, Gustashaw K, Willard HF. Genomic and genetic definition of a functional human centromere. Science. 2001 Oct 5;294(5540):109–115. [PubMed]
  • Smaragdov MG, Smirnov AF, Rodionov AV. Agregatsiia geterokhromatinovykh raionov khromosom v neiroblastakh Drosophila melanogaster. Tsitol Genet. 1980 May-Jun;14(3):37–42. [PubMed]
  • Hari KL, Cook KR, Karpen GH. The Drosophila Su(var)2-10 locus regulates chromosome structure and function and encodes a member of the PIAS protein family. Genes Dev. 2001 Jun 1;15(11):1334–1348. [PMC free article] [PubMed]
  • Spradling AC, Stern DM, Kiss I, Roote J, Laverty T, Rubin GM. Gene disruptions using P transposable elements: an integral component of the Drosophila genome project. Proc Natl Acad Sci U S A. 1995 Nov 21;92(24):10824–10830. [PMC free article] [PubMed]
  • Horvath JE, Schwartz S, Eichler EE. The mosaic structure of human pericentromeric DNA: a strategy for characterizing complex regions of the human genome. Genome Res. 2000 Jun;10(6):839–852. [PMC free article] [PubMed]
  • Spradling AC, Stern D, Beaton A, Rhem EJ, Laverty T, Mozden N, Misra S, Rubin GM. The Berkeley Drosophila Genome Project gene disruption project: Single P-element insertions mutating 25% of vital Drosophila genes. Genetics. 1999 Sep;153(1):135–177. [PMC free article] [PubMed]
  • Karpen GH, Spradling AC. Reduced DNA polytenization of a minichromosome region undergoing position-effect variegation in Drosophila. Cell. 1990 Oct 5;63(1):97–107. [PMC free article] [PubMed]
  • Karpen GH, Spradling AC. Analysis of subtelomeric heterochromatin in the Drosophila minichromosome Dp1187 by single P element insertional mutagenesis. Genetics. 1992 Nov;132(3):737–753. [PMC free article] [PubMed]
  • Sullivan BA, Blower MD, Karpen GH. Determining centromere identity: cyclical stories and forking paths. Nat Rev Genet. 2001 Aug;2(8):584–596. [PubMed]
  • Sun FL, Cuaycong MH, Craig CA, Wallrath LL, Locke J, Elgin SC. The fourth chromosome of Drosophila melanogaster: interspersed euchromatic and heterochromatic domains. Proc Natl Acad Sci U S A. 2000 May 9;97(10):5340–5345. [PMC free article] [PubMed]
  • Kurenova E, Champion L, Biessmann H, Mason JM. Directional gene silencing induced by a complex subtelomeric satellite from Drosophila. Chromosoma. 1998 Nov;107(5):311–320. [PubMed]
  • Lander ES, Linton LM, Birren B, Nusbaum C, Zody MC, Baldwin J, Devon K, Dewar K, Doyle M, FitzHugh W, et al. Initial sequencing and analysis of the human genome. Nature. 2001 Feb 15;409(6822):860–921. [PubMed]
  • Le MH, Duricka D, Karpen GH. Islands of complex DNA are widespread in Drosophila centric heterochromatin. Genetics. 1995 Sep;141(1):283–303. [PMC free article] [PubMed]
  • Sun X, Wahlstrom J, Karpen G. Molecular structure of a functional Drosophila centromere. Cell. 1997 Dec 26;91(7):1007–1019. [PMC free article] [PubMed]
  • Tolchkov EV, Kramerova IA, Lavrov SA, Rasheva VI, Bonaccorsi S, Alatortsev VE, Gvozdev VA. Position-effect variegation in Drosophila melanogaster X chromosome inversion with a breakpoint in a satellite block and its suppression in a secondary rearrangement. Chromosoma. 1997 Dec;106(8):520–525. [PubMed]
  • Liao GC, Rehm EJ, Rubin GM. Insertion site preferences of the P transposable element in Drosophila melanogaster. Proc Natl Acad Sci U S A. 2000 Mar 28;97(7):3347–3351. [PMC free article] [PubMed]
  • Tower J, Karpen GH, Craig N, Spradling AC. Preferential transposition of Drosophila P elements to nearby chromosomal sites. Genetics. 1993 Feb;133(2):347–359. [PMC free article] [PubMed]
  • Zhang P, Spradling AC. Efficient and dispersed local P element transposition from Drosophila females. Genetics. 1993 Feb;133(2):361–373. [PMC free article] [PubMed]
  • Wallrath LL, Elgin SC. Position effect variegation in Drosophila is associated with an altered chromatin structure. Genes Dev. 1995 May 15;9(10):1263–1277. [PubMed]
  • Zhang P, Spradling AC. Insertional mutagenesis of Drosophila heterochromatin with single P elements. Proc Natl Acad Sci U S A. 1994 Apr 26;91(9):3539–3543. [PMC free article] [PubMed]
  • Wallrath LL, Guntur VP, Rosman LE, Elgin SC. DNA representation of variegating heterochromatic P-element inserts in diploid and polytene tissues of Drosophila melanogaster. Chromosoma. 1996 Apr;104(7):519–527. [PubMed]
  • Zhang P, Spradling AC. The Drosophila salivary gland chromocenter contains highly polytenized subdomains of mitotic heterochromatin. Genetics. 1995 Feb;139(2):659–670. [PMC free article] [PubMed]
  • Zhang P, Stankiewicz RL. Y-Linked male sterile mutations induced by P element in Drosophila melanogaster. Genetics. 1998 Oct;150(2):735–744. [PMC free article] [PubMed]
  • Wevrick R, Willard VP, Willard HF. Structure of DNA near long tandem arrays of alpha satellite DNA at the centromere of human chromosome 7. Genomics. 1992 Dec;14(4):912–923. [PubMed]

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