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Analysis of Subtelomeric Heterochromatin in the Drosophila Minichromosome Dpll87 by Single P Element Insertional Mutagenesis

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Analysis of Subtelomeric Heterochromatin in the Drosophila Minichromosome Dpll87 by Single P Element Insertional Mutagenesis Copyright 0 1992 by the Genetics Society of America Analysis of Subtelomeric Heterochromatin in the Drosophila Minichromosome Dpll87 by Single P Element Insertional Mutagenesis Gary H. Karpen’ and Allan C. Spradling Howard Hughes Medical Institute Research Laboratories, Carnegie Institution of Washington, Department of Embryology, Baltimore, Maryland 21210 Manuscript received May 28, 1992 Accepted for publication July 28, 1992 ABSTRACT We investigatedwhether single P elementinsertional mutagenesis could beused to analyze heterochromatinwithin the Drosophila minichromosome Dpll87. Forty-fiveinsertions of the P[lacZ,rosy+] element onto Dpll87 (recovered among 7,825 transpositions) were highly clustered. None was recovered in centromeric heterochromatin, but 39 occurred about 40 kb from the distal telomere withina 4.7-kb hotspot containing tandem copies a of novel 1.8-kb repetitiveDNA sequence. The DNA within and distalto this region lacked essential genes and displayed severalother properties characteristic of heterochromatin. The rosy+ genes within the inserted transposons were inhibitedby position-effect variegation, and the subtelomeric region was underrepresented in polytene salivary gland cells. These experiments demonstrated that P elements preferentially transpose into a small subset of heterochromatic sites, providing a versatile method for studying the structure andfunction of these chromosome regions.This approach revealed thata Drosophila chromosome containsa large region of subtelomeric heterochromatinwith specific structural and genetic properties. HROMOSOMES in multicellular eukaryotes due to the priming problem (WATSON1972; CAVA- C have been studied extensively at thegenetic and LIER-SMITH1974). In Drosophila, chromosomes bear- cytogenetic levels. However,much remains to be ingterminal deletions lose 50-75 bp of DNA per learned concerning the molecular structure of chro- generation (BIESSMANNand MASON 1988; LEVIS mosomes and the mechanisms underlyingchromo- 1989), presumably for this reason (LEVIS1989; BIESS- some behavior during thecell cycleand development. MANN, CARTERand MASON 1990). The structure of The study of small natural andartificial chromosomes normal Drosophila telomeres has not been described, has greatly aided the analysis of chromosome function however, and the existence of terminal GT-rich re- in yeast (MURRAYand SZOSTAK1983; NEWLONet al. peats remains to be demonstrated. 1991). For this reason we previously initiated molec- Telomericregions frequently contain other re- ular studies of a Drosophila minichromosome, peated DNA sequences in addition to hexanucleotide Dp(l;f)1187, or Dp1187 (KARPEN and SPRADLINC or oligonucleotide repeats. For example, most yeast 1990). Dpl187 contains only 1000 kb of centromeric chromosomes contain tandem repeats of a sequence heterochromatin, and 300 kb of distal DNA, making called Y’, and an X sequence (CHANand TYE1983). it the smallest known functional chromosome in Dro- Large arrays of repetitive sequences are found at the sophila and other multicellular eukaryotes. We now telomeres of many other organisms, including Plas- report the use of P element insertional mutagenesis modium (PACE et al. 1987), Secale (BEDBROOKet al. to analyze the distal subtelomeric region of @I 187. 1980), Chironomus (SAIGAand EDSTROM1985), and Chromosomal telomeres carry out specialized func- humans (COOKE, BROWNand RAPPOLD1985; BROWN tions essential forchromosome maintenance [re- et al. 1990; DELANGEet al. 1990). A specific family of viewed by ZAKIAN (1 989) and BLACKBURN(1 991)]. repeats called HeT has been mapped at many Dro- The short GT-rich oligonucleotide repeats found at sophila telomeres (RUBIN 1978; YOUNGet al. 1983). the extreme termini of chromosomes in a wide range The functions of these“subtelomeric” repeats are of organisms serveas substrates for theenzyme telom- unclear, since they varywidely in amount between erase (GREIDERand BLACKBURN1985). The sequences chromosomes andundergo rapidevolutionary added by telomerase counteract the 5’ shortening of changes (YOUNG et al. 1983; CORCORANet al. 1988; chromosomal molecules that would otherwise occur ZAKIANand BLANTON 1988).Recently, BIESSMANNet al. (1990) reported that HeT repeats can be added The sequence data presented in this article have been submitted to the onto a broken chromosome end, possible by retro- EMBL/GenBank Data Libraries under the accession number L03284. ’ Current address: Molecular Biology and Virology Laboratory, The Salk transposition. Sequence additions, along with telom- Institute, 10010 North Torrey Pines Road, La Jolla, California 92037. ere-telomere recombination, might contribute to the Genetics 132: 737-753 (November, 1992) 738 G. H. Karpen and A. C. Spradling rapid changes observed in subtelomeric regions. females. Among an average of 100 FP progeny from each In Drosophila, telomeres have been associated with vial, y+ .ry+ Sb+ males were recognized as bearing new transpositions of P[lacZ, rosy+] (hereafter called PZ; see additional genetic and cytogenetic properties. Telom- MLODZIKand HIROMI1992) not linked to the X chromo- eresare generally thoughtto beheterochromatic some. These males were mated individually with twoy ;ryro6 (SCHULTZ 1947),and share common sequences with females, however, a maximum of only twoy+ ry+ Sb+ males centric regions and the Y chromosome (YOUNGet al. were used from any one F1 vial to avoid clusters, and males 1983). It has been suggested by cytological examina- from the same vial were recorded and kept together for further study. Approximately 8,300 such F2 crosses were tions,but not proven by molecular analyses, that established, including 950 pairs. In some F1 crosses the telomeric regions become underrepresented in poly- transpositions all segregated from Dpll87, or segregated tene DNA (ROBERTS1979). Rearrangements that jux- with the chromosome containing A2-3, hence the number taposeeuchromatic andheterochromatic chromo- of F:! vials established wasless than the total number of some regions frequently cause genes near the break- transpositions that occurred. Lines containing insertions onto Dpll87 were recognized point to display position-effect variegation (reviewed by co-segregation of they+ andry+ markers in progeny from in SPOFFORD1976; HENIKOFF 1990; SPRADLINGand the F2 outcrosses (at least some y+ ry+ progeny, but no y- KARPEN1990). The expression of a white+ gene relo- ry+). Linkage of the PZ element to the Y chromosome was cated near the 3R telomere variegated (HAZELRIGG, established by observing transmission of the ry+ marker only LEVISand RUBIN1984; LEVIS,HAZELRIGC and RUBIN from fathers to sons.Lines containing an insertion on Dp1187 and a second insertion on another chromosome 1985), however, it remains unclear if telomeric re- were recognized (despite the presence of y-ry+ animals) by gions are generally capable of causing variegated po- observing that all y+ progeny were ry+; the insertions were sition effects. then separated in a subsequent generation. A total of 42 Studies of Dpl187 telomeres, and of its centromeric independent lines with insertions on Dp1187 were eventu- region, were limited by a lack of specific molecular ally recovered (Table 2). Autosomal PZ insertions were balanced over CyO, TM? ryK,or (rarely) CxL) and recessive probes and of mutations mapping to these regions. (or rare dominant) phenotypes associated withthe insertion- We thereforeused insertional mutagenesis with single bearing chromosome classified. A small number of lines P elements (COOLEY,KELLEY and SPRADLING1988) with insertions on the 4th chromosome or lines that were to determine if genetically marked P elements bearing not balanced over either Cy0 or TM? for undetermined unique sequence tags could be recovered in reasons were discarded. In addition, ovaries were dissected Dp1187 from adults of each line, stained for &galactosidase activity, heterochromatin.Heterochromatin is generally and the expression pattern recorded. Subsequent molecular thoughtto be an infrequenttarget of P element and phenotypic study revealed that approximately 50% of insertion (ENGELS1989), but our experimentsre- the lines isolated as pairs contained identical insertions, so vealed that asignificant number of insertions occurred the total number of independent insertions recovered was nearthe distal telomere of this minichromosome. approximately 8,300 - 475 = 7,825. Some of these results will be presented in further detail elsewhere. These studies have provided new information con- Plasmid rescue: Sequences flanking an insertion were cerning the structure and function of Drosophila te- rescued by first preparing DNA from ten adult males as lomeres, and their relationship to transposable ele- described (BENDER,SPIERER and HOCNESS1983). Following ments. digestion with XbaI, or XbaI and SpeI, the reaction was diluted, ligated overnight at 15", and used to transform competent cells to kanamycin resistance, essentially as MATERIALS AND METHODS DH5 described (PIROTTA1986; COOLEY,KELLEY and SPRADLINC Drosophila stocks: Flies were grown on standard corn 1988). Approximately one colony was obtained for each fly meallagar media [see ASHBURNER(1990)], at 22". Unless equivalent of DNA used for transformation. stated otherwise, strains and mutations are as described in Restrictionmapping using pulsed-field and conven- LINDSLEYand ZIMM (1992). The original stock containing tional gel electrophoresis:The location ofthe PZ insertions Dp1187 was provided byD. LINDSLEY,and was balanced in Dpll87 was determined
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