Evidence That Intergenic Spacer Repeats of Drosophila ...viously (MCKEE and KARPEN 1990). This...

Copyright 0 1992 by the Genetics Society of America

Evidence That Intergenic Spacer Repeats of Drosophila melanogaster rRNA Genes Function as X-Y Pairing Sites in Male Meiosis, and a

General Model for Achiasmatic Pairing

Bruce D. McKee,* Ledare Habera* and Julie A. Vranat

*Department of Zoology, University of Tennessee, Knoxville, Tennessee 37996, and ?Mayo Clinic, Rochester, Minnesota 55905 Manuscript received March 23, 1992

Accepted for publication June 30, 1992

ABSTRACT In Drosophila melanogaster males, X-Y meiotic chromosome pairing is mediated by the nucleolus

organizers (NOS) which are located in the X heterochromatin (Xh) and near the Y centromere. Deficiencies for Xh disrupt X-Y meiotic pairing and cause high frequencies of X-Y nondisjunction. Insertion of cloned rRNA genes on an Xh- chromosome partially restores normal X-Y pairing and disjunction. To map the sequences within an inserted, X-linked rRNA gene responsible for stimulating X-Y pairing, partial deletions were generated by P element-mediated destabilization of the insert. Complete deletions of the rRNA transcription unit did not interfere with the ability to stimulate X-Y pairing as long as most of the intergenic spacer (IGS) remained. Within groups of deletions that lacked the entire transcription unit and differed only in length of residual IGS material, pairing ability was proportional to the dose of 240-bp intergenic spacer repeats. Deletions of the complete rRNA transcription unit or of the 28s sequences alone blocked nucleolus formation, as determined by binding of an antinucleolar antibody, yet did not interfere with pairing ability, suggesting that X-Y pairing may not be mechanistically related to nucleolus formation. A model for achiasmatic pairing in Drosophila males based upon the combined action of topoisomerase I and a strand transferase is proposed.

M EIOSIS, the type of cell division associated with sexual reproduction, accomplishes two unique

functions. One is the reduction in chromosome number necessary to produce gametes, which involves pairing and segregation of homologous chromosomes. The other is genetic recombination, which involves both independent assortment of nonhomologous chromosomes and exchange between paired homologs. Both of these outcomes depend upon homologous pairing, as shown by the consequences of pairing- defective mutations, which include both high frequencies of meiotic nondisjunction and reduced recombination (BAKER et al. 1976). Pairing is usually, although not universally, accompanied by synapsis, the formation of synaptonemal complex (SC). SC is a linear, tripartite structure that connects paired homologs all along their lengths during early prophase and is thought to play some role in meiotic recombination (VON WETTSTEIN, RASMUSSEN and HOLM 1984).

Several recent models of meiotic pairing have at- tributed major roles to processes thought to be involved in recombination, such as a DNA homology search culminating in formation of heteroduplex DNA (SMITHIES and POWERS 1986; CARPENTER 1987; ROEDER 1990; ALANI, PADMORE and KLECKNER 1990). Several recent findings support such models. First, in yeast, double strand breaks, thought to be early intermediates in meiotic recombination (SUN et al. 1989; SUN, TRECO and SZOSTAK 199 1 ; CAO, ALANI

Genetics 132: 529-544 (October, 1992)

and KLECKNER 1990), can be detected at or before the onset of synapsis (PADMORE, CAO and KLECKNER 1991). Second, yeast meiotic mutants that block homologous recombination also typically prevent synapsis (ROEDER 1990; ALANI, PADMORE and KLECKNER 1990). Third, the initiation of meiotic pairing and synapsis is accompanied by the appearance of SC- associated nodules (RASMUSSEN and HOLM 1978; CAR- PENTER 1979; ALBINI and JONES 1987; ANDERSON and STACK 1988) generally similar in appearance but smaller and more numerous than the pachytene “recombination nodules” (RNs), which are thought to be packets of recombination enzymes (CARPENTER 1975). Fourth, ectopic exchange between duplicated sequences on the same or different chromosomes in yeast occurs at frequencies comparable to those of allelic recombination (PETES and HILL 1988; HABER et al. 1991), indicating that formation of stable, full- length synaptonemal complex is not a prerequisite for recombination. Finally, prokaryotic and eukaryotic strand transferases, the central enzymes in heteroduplex formation, are also pairing enzymes in vitro (RAD- DING 1988; KMIEC and HOLLOMAN 1984).

Exchange is also thought to be required for a later step in meiotic chromosome segregation, the formation of chiasmata. SC disappears at the end of pachytene, at which time homologs that have crossed over remain linked by one or more chiasmata located at the sites of the crossovers (JONES 1987). Chiasmata

530 B. D. McKee, L. Habera and J. A. Vrana

are thought to stabilize bivalents until anaphase and to enable homologous centromeres to reliably orient to opposite poles (HAWLEY 1988).

A difficulty with these ideas is that homologous pairing and segregation are more universal than recombination. There are a number of achiasmatic organisms in which one sex has abandoned recombination altogether yet still undergoes regular pairing and segregation (WHITE 1973; CALLAN and PERRY 1977). If pairing is essentially recombinational in mechanism, it is not clear how organisms that lack recombination manage to pair their chromosomes. Also unclear is the mechanism by which the bivalents formed in nonrecombinational organisms are stabilized in the absence of chiasmata.

One of the best characterized examples of achiasmatic pairing is in Drosophila melanogaster males. Un- like their recombination-proficient sisters, Drosophila males have no regular crossing over (MORGAN 19 12) and fail to form either synaptonemal complexes (MEYER 1960) or chiasmata (COOPER 1950). When the chromosomes first become visible during male meiotic prophase, they are already paired and they remain stably paired until anaphase despite the absence of chiasmata. At prophase, autosomes appear to be paired all along their lengths while the sex chromosomes pair at discrete, heterochromatic sites known as collochores (COOPER 1950, 1964).

Recent data implicate the NOS in X-Y pairing and segregation in males. There are two NOS in D. melanogaster, one in the centric heterochromatin of the long arm of the X and the other near the base of the short arm of the Y (RITOSSA 1976). Each NO contains 200-250 tandem copies of an 8-kb transcription unit that contains the sequences for the 18S, 5.8s and 28s rRNAs as well as “external” (ETS) and “internal” (ITS) transcribed spacers (see Figure 2). Adjacent transcription units are separated by a 3-4-kb intergenic spacer (IGS) region that consists in part of several tandemly 240-bp repeats with homology to the rDNA promoter (LONG and DAWID 1980; COEN and DOVER 1982; KOHORN and RAE 1982; MILLER, HAY- WARD and GLOVER 1983). X heterochromatic deletions that encompass the NO lead to X-Y nondisjunction (GERSHENSON 1933; COOPER 1964); for the larg- est such deletions, X-Y disjunction is essentially random (MCKEE and LINDSLEY 1987). The ability of heterochromatically deleted X chromosomes to pair with and disjoin from the Y is partially restored by ectopic, X-linked insertions of cloned rRNA genes. The degree of stimulation is proportional to the number of inserted rRNA genes. X-Y homology for other repetitive sequences such as Stellate and heterochromatic satellite sequences does not promote X-Y disjunction (MCKEE and KARPEN 1990). These observations indicate that the collochores consist, at least in part, of the NOS.

This study was undertaken to gain insight into the mechanism of achiasmatic pairing involving the rDNA. The goal was to identify, by deletion mapping, rDNA sub-sequences needed for stimulation of X-Y pairing. Partial deletions within an rDNA-containing, X-linked P transposon were generated in vivo by in- duction of partial excisions by P transposase. We show that this method is an efficient way to generate a large but nonrandom group of deletions, most of which have breakpoints near or within the target sequence and all of which are at the same chromosome site, thus controlling for position effects. Tests of X-Y disjunction show that an rDNA fragment containing only several copies of the 240-bp repeats from the intergenic spacer can stimulate X-Y disjunction as effectively as a complete rRNA gene. A model for achiasmatic pairing based on formation of a nonrecombi- nogenic heteroduplex by the combined action of a strand transferase and topoisomerase I is proposed.

MATERIALS AND METHODS

Chromosomes: DJl1)X-I is a deletion that encompasses most of the X heterochromatin as well as a portion of proximal X euchromatin. Its breakpoints are proximal to the NO and distal to or within 1(1)20Cb (LINDSLEY and ZIMM 1986, 1987). Its sequence is normal otherwise. Males carrying Df(I)X-I and a normal Y are inviable because of the proximal euchromatic deletion. The duplication present in BsYy+ supplies the missing functions. DflI)X-I and BsYy+ pair irregularly in male meiosis and disjoin randomly from one another (MCKEE and LINDSLEY 1987). Both y+Y and BsYy+ also carry a duplication for 1Al-1 B1 from the tip of the X chromosome (LINDSLEY and ZIMM 1987). Both Y chromosomes behave normally in male meiosis. [r ib7]( lAl- 4) is an X chromosome containing an insertion of the p(rib,ry)7 transposon near the tip of the X (KARPEN, SCHAEF- FER and LAIRD 1988; MCKEE and KARPEN 1990). X[rib7]*i are the derivatives of [rib7](IAI-4) recovered from the P destabilization scheme. [A2,3](99B) is a third chromosome carrying a stable insertion of the p(ry+,A2,3) transposon that produces P transposase constitutively (ROBERTSON et al. 1988). FM6, y2 B and TM3 ryAK Sb are balancers for the X and third chromosomes, respectively. Markers are described in LINDSLEY and ZIMM (1985, 1990).

Determination of disjunction frequencies by progeny tests To test X[rib7]* deletions for ability to stimulate X-Y disjunction, each was recombined onto DJl1)X-I and made heterozygous with pYy+. Males were crossed singly with two ry females in shell vials containing standard cornmeal-mo- lasses-agar medium and cultured at 22”-23”. The parents were transferred to fresh medium on day 7 and discarded on day 14. The progeny in the first vial were counted on days 12, 15 and 19, and in the second vial on days 19, 22 and 26. The disjunction frequency (E‘) was calculated from the formula:

P = 1/[ 1 + (CD/AB)”]

where A , B , C and D are the observed frequencies of progeny derived from X , Y , XY and nullo-X,nullo-Y sperm, respectively (MCKEE 1984; MCKEE and LINDSLEY 1987). 95% confidence intervals (C.I.) were calculated as described previously (MCKEE and KARPEN 1990). This formula compen- sates for the skewed progeny ratios that result from sper-

rDNA Spacers and Achiasmatic Pairing 53 1

matid mortality associated with genotypes with irregular X- Y pairing.

Meiotic cytology: Meiotic chromosomes were prepared by the method of LIFSCHYTZ and HAREVEN (1 977). Briefly, testes from young (0-2-day-old) adult males were dissected in testis buffer (ASHBURNER 1989), fixed for 30 sec in 45% acetic acid, stained for 5 min in 3% orcein-60% acetic acid, transferred to a drop of 60% acetic acid on a clean microscope slide, cut into pieces with a sharp dissecting needle and squashed gently by covering with a coverslip containing a drop of 2% orcein in 1: 1 1actic:acetic acid. Slides were aged overnight, viewed under phase optics with a Zeiss Universal Axioplan photomicroscope and photographed with Kodak TMAX-100 film. Anaphase I primary spermatocytes and secondary spermatocytes were scored for the presence or absence of the X and Y chromosomes. P was calculated from the formula:

P = (2a + c + d)/(2a + 26 + c + d + e +f) where a and b are the numbers of anaphase I spermatocytes in which the X and Yare disjoining to the opposite ( a ) and same (6) poles, and c, d, e andfare the numbers of X, Y, XY and nullo-X;nullo-Y secondary spermatocytes, respectively. The 95% confidence intervals were calculated as described previously (MCKEE and KARPEN 1990).

Genomic Southern blot analysis: DNA for genomic blots was prepared from 4- 12 flies by the method of BENDER, SPIERER and HOGNESS (1983). Genomic DNA was digested 2-4 hr with the appropriate restriction enzyme (Bethesda Research Laboratories, New England Biolabs, U.S. Bio- chemical), electrophoresed on 0.5% agarose gels in Tris- borate-EDTA buffer (MANIATIS, FRITSCH and SAMBROOK 1982), and transferred to Gene Screen Plus filters (New England Nuclear) by the SOUTHERN (1975) blot method according to the manufacturer's instructions. After prehy- bridization for 15 min in 50% formamide, 1% sodium dodecyl sulfate (SDS) and 5 X SSPE, denatured probe DNA, prepared by nick translation (using a kit from Bethesda Research Laboratories) in the presence of ['*P]dCTP (New England Nuclear) was added. Hybridization was carried out overnight at 42". Filters were washed twice at room temperature in 2 X SSC, twice at 65" in 2 X SSC, 1% SDS, and twice at room temperature in 0.1 X SSC, and exposed at -70" to Kodak XAR-5 film for varying amounts of time.

Cloning: High molecular weight DNA for library con- struction was prepared by the method of BINGHAM, LEVIS and RUBIN 1981. Clones from the X[ri67]*211 and HJ+B lines were isolated from libraries made in lambda pGEMl1 (Promega Biotech). Genomic DNA was partially digested with SauSA, the fragments partially filled in with the Klenow fragment of DNA polymerase (Bethesda Research Labora- tories) and dATP and dGTP, and ligated to XhoI-digested, partially end-filled pGEMl1 arms. Libraries from X[rib7] *49A, U+ and 7B were prepared by ligating HzndIII-digested, partially end-filled, gel-purified genomic DNA into the partially end-filled SpeI site of X-ZAP (Stratagene). A library from X[ri67]*21O was prepared by digesting genomic DNA with HindIII, then with BamHI, XhoI, SstI, SalI, KpnI and X6aI (to inactivate non-rDNA fragments), gel- purifying 2.0-kb fragments, and ligating them into the Hind111 site of pBluescript I1 KS- (Stratagene). The phage libraries were screened by the plaque lift procedure (BEN- TON and DAVIS 1977) and the plasmid library by the colony lift procedure (GRUNSTEIN and HOGNESS 1975) as described in MANIATIS, FRITSCH and SAMBROOK 1982. s2P-Labeled DNA homologous to PL (prepared by nick translation or random oligonucleotide priming using kits purchased from Bethesda Research Laboratories) was used as probe. Hy- bridization was carried out at 42" overnight. Nitrocellulose

filters (Schleicher and Schuell) were washed as described in MANIATIS, FRITSCH and SAMBROOK (1 982) and exposed at -80" to Kodak XAR-5 film for various time intervals. Subclones were prepared in pBluescript I1 KS-.

DNA sequence analysis: DNA sequences were determined by the Sanger dideoxy method (SANGER, NICKLEN and COULSON 1977) using double strand template DNA and the Sequenase kit (U.S. Biochemical) following the protocol of TABOR and RICHARDSON 1987. Templates were sub- cloned in the pBluescript I1 KS- vector (Stratagene).

Mininucleolus assays: Larvae for polytene chromosome preparations were cultured at 18". Salivary glands were dissected from wandering stage third-instar larvae in 45% acetic acid. The glands were transferred to a fresh drop of 45% acetic acid on a siliconized coverslip. After incubating for one minute, the glands were squashed gently to avoid damaging the nucleolus, and examined by phase-contrast microscopy. Slides with well spread chromosomes were fro- zen in liquid nitrogen, and the coverslips removed with a razor blade. Slides were then stored in 90% ethanol at 4" for up to 3 days. The slides were dried, then rinsed twice for 5 min each in PBS. An aliquot of 100 pl of a 1: 10,000 dilution (in PBS) of Ajl primary antibody (SAUMWEBER et al. 1980) was added to the squash. Slides were incubated at 22" for 2 hours in a moist chamber, then washed twice for 5 min each in PBS. An aliquot of 100 PI of secondary antibody (biotinylated anti-mouse IgG (H + L) (Vector Lab- oratories)) diluted 1 : 150 in PBS was added and incubated at 22" for 1-2 hr. After two 5-min washes in PBS, 100 pl of 1 : 100 (PBS) fluorescein-avidin DCS (cell-sorting grade, Vector Laboratories) was added to the squash and incubated at 22" in the dark for 1 hr. Slides were rinsed twice for 5 min. each with PBS. The slides were mounted in 90% glycerol-PBS solution containing 1 mg/ml p-phenylenedi- amine (to prolong fluorescence) and 0.5 pg/ml ethidium bromide (to stain the chromosomes) (VRANA and MCKEE 1993). Slides were viewed on a Zeiss Universal Axioplan fluorescence microscope with BP450-490 exciting filter, FT510 chromatic beam splitter, LP520 barrier filter and Neofluar objectives, and photographed using Kodak Ekta- chrome 160 film. The slides retained fluorescence for up to 3 months after mounting, stored at 4".

RESULTS

Targeted deletion mutagenesis of an rDNA-containing transposon generates imperfect excisions at a very high frequency: p(rib,ry)7 is a P transposon containing a complete rRNA transcription unit with 5' and 3' intergenic spacers (KARPEN, SCHAEFFER and LAIRD 1988) inserted into the Carnegie-20 transformation vector (RUBIN and SPRADLINC 1983). [rib71 ( lA1-4) (see Figure 2) is an insertion of p(rib,ry)7 near the tip of the X , obtained by P element-mediated transformation (KARPEN, SCHAEFFER and LAIRD 1988). [rib7](1Al-4) has been shown to be transcribed, to organize a mininucleolus (KARPEN, SCHAEFFER and LAIRD 1988), and to stimulate X-Y pairing and disjunction when located on the X chromosome Dfll)X- 1 , which is deficient for the native pairing site (MCKEE and LINDSLEY 1987; MCKEE and KARPEN 1990).

To generate deletions within [rib7](lA1-4), males carrying both [A 2,3](99B) and [rib7](lAl-4) were generated by the cross-diagrammed in Figure 1. [A 2,3](99B) is a potent, genomically integrated source


X [ r i b 7 ]

p::: X [ r i b 7 ]

X [ r i b 7 ]

F1 - M

-0- Y

TM3, r y Sb

- x U

FM6, B -u "-0u

r y

1 (1)ZOCb rY

X [ r i b 7 ] * TM3, r y Sb FM6, B r y

w u -* F2 __o u x " o -

FM6, B rY Y r y

X [ r i b 7 ] * rY

F3 - - viable? -"u Y r y

FIGURE 1 .-Mating scheme for generation and recovery of partial excisions within an X-linked, rDNA-containing P transposon. For each individual, sex and third chromosome genotypes are represented. Circles represent centromeres; tilled rectangles represent [rib7](1AZ-4) (abbreviated as X[rib7]) and its destabilized derivatives (X[rib7]*); open rectangles represent [A 2,3](99B). F1 males are dysgenic, containing both a source of P transposase ([A 2,3](99B)) and a target ([rib7](IAZ-4)). The resulting destabilization of [rib7](ZAZ-4) leads to excisions, some partial and some complete, in the germ line of F1 males. The products are recovered in their Sb daughters (F2) which may be ry or ry+ depending on whether or not the excision event affected rosy sequences in [rib71 (ZAI-4). The X[rib7]* chromosomes are stable in the F2 and subsequent generations because the transposase source has been seg- regated away. The F2 cross establishes a balanced stock and provides a test for viability of X[rib7]* chromosomes.

of P transposase useful for remobilizing defective transposons (ROBERTSON et al. 1988). The products of remobilization protocols such as this typically include substantial frequencies of imperfect excisions, some of which result in partial deletions within the transposon (DANIELS et al. 1985; DREESEN, HENIKOFF and LOUCHNEY 1991). The F1 males were crossed with females carrying the X chromosome balancer, FM6. Both F1 males and females were homozygous for the recessive eye-color marker, rosy (ry) which is located on chromosome 3. The males had wild-type (ry+) eyes, however, because both [rib7](1A1-4) and [A 2,3](99B) carry ry+. The F1 males were also heterozygous for TM3, a third chromosome balancer marked with Stubble ( S b ) and ry. In the initial experiment, F2 S b ry daughters (which lack [A 2, 3](99B) and have lost the X-linked ry+ marker) were recovered. They were crossed singly to FM6; ry males to establish stocks. As shown in Table 1 ,62% of the F2 Sb females were ry, indicating an extraordinarily high excision frequency. The majority of these "treated" X chromosomes (designated X[ri67]*) proved to carry newly induced recessive lethal mutations, as indicated by the inviability of the Fs X[rib7]*/Y sons. This indicates that most of the excisions effected not only the rosy

TABLE 1

Phenotypic classification of X[rib7]* chromosomes

rosy rosy+

Expt. Nonlethal Lethal Nonlethal Lethal Total

1 8 (A) 27 (B) 2 la 56 2 1 04a 37(C) 19(D) 160

The letters in parentheses are the names of the classes. a These chromosomes were not tested for viability and were

discarded. In experiment 1, only the rosy X[rib7]* chromosomes were stocked and tested for viability. In experiment 2, only the rosy+ X[rib7]* chromosomes were recovered.

sequences internal to the transposon but also one or more essential loci external to it.

The high excision frequency suggested that deletions might also be recovered among ry+ progeny at reasonable frequencies. Therefore, the mobilization cross was repeated and Sb ry+ daughters recovered (Figure 1). 34% of the X[rib7]* chromosomes carried by these females proved to contain new recessive lethals (Table 1). The remainder (66%) of the treated x's in experiment 2 were phenotypically indistinguishable from the original X (ry+ and viable). For the subsequent analysis, the products of the deletion mutagenesis protocol were classified into four categories on the basis of eye color and viability: (A) rosy, nonlethal; (B) rosy, lethal; ( C ) rosy+, nonlethal, and (D) rosy+, lethal (Table 1).

Targeted deletion breakpoints are nonrandomly distributed within [rib7](1A1-4): The [rib7]X* chromosomes in each category were further characterized by genomic Southern blot analysis using radiolabeled probes homologous to transposon sequences. rDNA- containing probes could not be used because the large amount of DNA from the nucleolus organizers ob- scured transposon-derived bands. However, it was possible to test each of the X[rib7]* lines for homology to probes for PL and rosy sequences, and to detect rearrangements involving the rDNA by alterations in restriction fragments containing both P and rDNA sequences or both the 3' half of rosy and rDNA sequences (see Figure 2). Each line was also tested for junction fragments characteristic of the [rib7](IA1-4) insertion site using probes for PL and for the 5' half of rosy.

The results, summarized in Figure 2, showed that all of the X[rZb7]* chromosomes in classes A, B and D and five of those in class C had undergone one or more deletions. Of the class A lines, four (class AI) lacked homology to all transposon probes and thus were complete excisions of [rib7]; the remainder (class A2) involved a deletion with one breakpoint in the 5' IGS of the rDNA and the other either within or beyond PR, thus deleting all of the rDNA and rosy transcription units. All but one of the class B lines were complete excisions that also removed one or more nearby essential genes. The exception, X[rib7]

rDNA Spacers and Achiasmatic Pairing 533

A <- ............................................................................. DWOeaCe

......................................................... D/OY "'

PROBES PL

- - 3' ROSY S ROSY

C

...................................... ....

.-.... ........................................... "

.............................. .............. ........

<. ........

<. .......... , p", 7 , , I I ,,I*.,, + <""""""""". ......................

- l k b

FIGURE 2.-Map of P transposase-induced deletions ( lA1-4) . (A) Map of the terminal region of the X chromosome. The two upper lines show the extents of DfT1)Basc and Dfll)y'", respectively, relative to a genic map of the distal 1A region (third line). Rectangles represent known genes and sequences, solid lines r e p resent uncharacterized intervening sequences, and dashed lines represent deleted material. (B) Detailed map of [rib7](1AI-4). PL and PR represent the left and right ends of the P element in Carnegie-20, as described in RIJBIN and SPRADLING (1 983). V (solid lines) refers to vector sequences at the borders of the rDNA insert. IGS (open rectangles) = intergenic spacer (also known as nontran- scribed spacer); ETS and ITS (dotted rectangles) = external and internal transcribed spacers; 18S, 5.8s and 28s (left-to-right- hatched rectangles) = sequences responsible for 18S, 5.8s and 28s rRNAs. ROSY (right-to-left-hatched rectangles) = 7.2-kb Hind111 fragment containing ry+allele. Horizontal arrows indicate directions of transcription units (TUs). Restriction sites: filled triangles rep resent Hind111 sites, filled circles represent EcoRl sites, open circles represent Sstll sites. The probes used for the genomic blot analysis are indicated below. (C) Maps of X[rib7]* deletions based on genomic blot analysis. Dashed lines represent deleted material. Arrows indicate that deletions extend to left or right of mapped sequences. Class names and the numbers of X[rib7]* chromosomes in each class (in parentheses) are to the right of each map. Since breakpoints vary among members of a class and since precise breakpoints can not be determined by genomic blot analysis, the indicated breakpoints are only approximate. They are accurate to within the indicated interval ( i e . , 5' IGS, 3' IGS, 5' ROSY) with the exception of the class D2 breakpoints which could be in either the 5' IGS or V regions. See Figures 4 and 5 for more precise mapping of some of the breakpoints.

*144 (class B2), contained a deletion with one breakpoint within the 5' IGS and the other to the right of

PR, deleting one or more essential loci. The class C lines included five with transpositions to other sites on the X (analyzed elsewhere (MCKEE and KARPEN 1990)) and five with deletions internal to the rDNA. Four of the deletions involved breakpoints in the 5' and 3' IGS regions (class C1) and the fifth involved one breakpoint in the 5.8s sequence and one in the 3' IGS (class C2). The remainder of the class C lines were indistinguishable from the parental [ r ib7] ( lAl - 4 ) chromosome. All but one of the class D lines proved to be terminal deletions (evidence to be presented elsewhere). Class Dl lines involved deletion of sequences to the left of PL and did not affect the transposon. Classes D2 and D3 involved one break in either the 5' (D2) or 3' (D3) V/IGS regions. Analysis of these lines is complicated by the fact that terminal deletions are unstable (BIESSMANN, CARTER and MA- SON 1990) so that the positions of the breakpoints change over time. A more detailed analysis of these lines will be presented elsewhere. The lone class D4 line involved one break upstream of the rosy transcription unit and the other to the right of PR.

The genomic blot data, combined with the results of complementation tests of lethal X[rib7]* lines against deficiencies and lethal alleles from the 1A region (Table 2) permit a determination of the loca- tion and orientation of [rib7](lAl-4) on the X chromosome. The excisions with at least one breakpoint internal to the transposon can be classed into three broad categories: internal deletions (those with two internal breakpoints (classes C1 and C2)), leftward- extending deletions [those that delete PL and X chromosome sequences adjacent to PL (classes D2 and D3)], and rightward-extending deletions (those that delete PR and X chromosome sequences adjacent to PR (classes A2, B2 and D4)). Complementation tests (Table 2) show that all leftward-extending deletions remove the distalmost essential locus on the X (l(1)lAa) but no other essential loci and the lethal rightward-extending deletions (classes B2 and D4) remove the penultimate essential locus on the X (l(1)lAb) but neither l(1)lAa nor loci proximal to l(1)lAb. Thus, [rib7](lAI-4) is located between l(1)lAa and l ( l ) lAb, oriented with PL nearer the telomere than PR (Figure 2).

These data indicate a highly nonrandom distribution of breakpoints associated with P-induced deletions. All except one of the 30 breakpoints mapped between PL and rosy were in either the 5' or the 3' V/IGS region. Several of these have been localized more precisely (see below) and found to be within the IGS. Only one breakpoint occurred within the rDNA transcription unit and only one in rosy sequences.

Pairing/disjunctional ability depends on presence of rDNA: All of the deletions from the targeted mutagenesis protocol were tested for ability to stimulate X-Y disjunction by the progeny test method and


TABLE 2

Results from complementation tests of lethal X[rib7]* chromosomes against deficiencies and lethal alleles from the 1A

region

Tester chromosomes X[rib7]* Lethals" y'Y DJI)Basc DAI)y'" I(I)IAa 4 J ) I A b I ( I ) I A c

B1 (26) + - + - + + B2 (1) + - + Dl (3) + - + - + + D2(11) + - + - + + D3 (4) + - + + + D4 (1) + - +

- + -

- - + -

Complementation tests were carried out by crossing X[rib7]*/ FM6.3 B by FM6,f/y+Y males (y+Y column) or by crossing "Tester"/ Balancer females to X[rib7]*/y+Y males (remaining columns). y+Y carries wild-type alleles of all loci in 1A appended to the tip of YL (LINDSLEY and ZIMM 1985). In the body of the table, a "+" indicates that the relevant X[rib7]*/tester genotype was viable, a "-" indicates that it was not. There were no borderline cases. See Figure 2 for map of 1 A region.

" The first entry in this column is the class name (see Figure 2). The number in parentheses is the number of lines tested.

many (all of those in the partial deletion classes A2, C1 and C2) were also tested cytologically for X-Y disjunction frequency in orcein-stained meiotic chromosome preparations (see MATERIALS AND METHODS for details and Figure 3 for examples). T o d o this, each deletion was recombined onto DJT1)X-1 (an X chromosome deficient for the native pairing site (MCKEE and LINDSLEY 1987) and tested in conjunction with the marked Y chromosome, @Yy+. The results (Table 3) were combined within classes when all members of the class gave the same result but presented individually otherwise.

The results for lines with breakpoints external to the rDNA were as follows (Figure 2 and Table 3). (i) Complete excision of [rib7](1A1-4) resulted in loss of disjunctional ability, as expected, for 29 out of the 30 tested lines. X-Y disjunction for the 26 class B lines averaged 55.5%, and was not significantly different from that of negative control Dfl1)X-1 males; these lines did not differ from one another and their results are combined in Table 3. Three of the four class AI lines also showed disjunctional frequencies indistinguishable from that of DJT1)X-1 males. X[rib7]*207 showed a slight but statistically significant deviation, in the direction of increased disjunction, from DJT1)X- 1. This result was based on a rather small sample size and the line was lost before it could be retested so it is not clear if this difference is real. (ii) Deletions that did not remove any rDNA did not interfere with disjunctional ability, again as expected. X-Y disjunction for lines in class Dl averaged 66.9% and was not significantly different from that of the positive control [rib7](1AZ-4),DfTZ)X-l males. The single class D4 line, X[rib7]*K+, also showed unimpaired disjunctional ability.

The 28s gene is not essential for X-Y pairing: The

- 4 . 1.

!r- I

FIGURE J.-Meiotic chronlosome squashes. Acetic-orcein stained chromosomes from various meiotic stages are shown. Large arrowheads denote Y chromosomes and small arrow heads denote X chromosomes. (A) Late prophase I in a wild-type male. (B) Late prophase I in a DffI)X-I/B'Yy+ male. The X and Yare unpaired. (C) Late prophase I in X[rib7]*H~B,Dfll)X-I/BSYy+ male. Pairing between the X and Y is evident. (D) Late prophase I in a X[rib7] *21 I,DffI)X-I/B'Yy+ male. X-Y pairing involving the tip of the X is evident. (E) Anaphase I in an X(rib7]*H~B,DflI)X-l/BsYy+ male. (F) Metaphase I1 in an X[rib7]*211,DffI)X-I/BsYy+ male. Magnifi- cations: 583X.

lone representative of class C2, X[rib7]*HTB, ap- peared from genomic blot analysis to have undergone a small deletion in the 5' IGS region and a second, much larger one in the proximal region of the rDNA. T o permit a more detailed analysis, DNA from X[rib7]*HTB was cloned and the breakpoints identified by a combination of restriction mapping and DNA sequence analysis. The results confirmed that there had indeed been two deletions. Restriction analysis of the 5' end of the rDNA insert indicated that there had been an apparently exact deletion of one of the 8% copies of the 240-bp "promoter-like" repeats that make up most of the 5' IGS sequences of p(rib,ry)7, as if from a homologous crossover between adjacent repeats. The other deletion was 4.5 kb in length and encompassed all of the 28s and parts of the ITS and 5.8s sequences. DNA sequence analysis

rDNA Spacers and Achiasmatic Pairing

TABLE 3

X-Y disjunction frequency and mininucleolus formation in X[rib7]* chromosomes

535

X-Y disjunction frequency

- Class Line

Progeny test Cytological test

P+ ( C q c N P+ (C.I$ N MNJ

A1 204 0.554 (0.017)' 3,444 N T N T

205 0.565 (0.014)' 5,405 N T N T

207 0.589 (0.013)d'e 5,305 N T N T

NrB 0.542 (0.047)' 685 NT N T

A2 20 1 0.576 (0.016)d" 3,986 0.51 (0.05)' 314 N T

202 0.537 (0.018)e 6,669 0.48 (0.05)' 340 N T

210 0.566 (0.013)' 5,479 0.52 (0.05)' 368 - 211 0.691 (0.017f 2,565 0.67 (0.05)d 336

c1 7B 0.644 (0.012)d7c 6,404 0.62 (0.05)d 317

B1 Lumped (26) 0.555 (0.009)' 13,419 N T N T

B2 144 0.650 (0.029)d'e 939 N T

-

-

U+ 0.603 (0.025)d'C 3,837 0.56 (0.06)' 289 - -

49A 0.550 (0.021)' 2,7 17 0.51 (0.06)' 267 42A 0.564 (0.013)' 5,503 0.53 (0.05)' 354

-

c2 HJ+B 0.823 (0.013)d'c 3,029 0.80 (0.04)d" 372

-

Dl Lumped (3) 0.669 (0.029f 955 N T N T

D2 Lumped (1 1) 0.647 (0.013)d" 4,592 N T N T D3 Lumped (4) 0.555 (0.007)' 19,464 N T N T D4 K+ 0.726 (0.039)d" 43 1 N T N T

+ ControIa 0.684 (0.012)d 6,559 0.64 (0.05)d 318 + - Controlb 0.547 (0.018)e 3,544 0.51 (0.06)' 302 -

-

Meiotic tests were carried out by recombining each X[rib7/* onto Df(Z)X-I and testing over the @Yy+ Y chromosome. Mininucleolus formation was assayed in salivary gland squashes of homozygous females, except for X[rib7/*Z##, which was assayed in FM6 heterozygous females. NT = not tested.

+Control = [rib71 (ZAZ-#), Df(Z)X-Z. -Control = ~~1)x-l. P = the disjunction frequency; C.I. = 95% confidence interval (see MATERIALS AND METHODS for calculation formulae). Significantly different (p = 0.05) from - control. ' Significantly different ( p = 0.05) from + control. f M N = mininucleolus formation.

showed that the deletion fused the 5' end of the 5.8s sequences to the 5' end of the 3' IGS region (Figure 4). These deletions not only did not interfere with the ability of the X to disjoin from the Y, they actually improved it. Disjunction percentages for X[rib7] *HJ+B,Dfl)X-1 males were significantly higher than those for [rib7](1Al-#),Dfl)X-l controls by both the progeny count and cytological methods (Table 3). Thus, sequences in the downstream (3') region of the rDNA transcription unit (TU) are not essential for X- Y disjunction and may actually hinder it.

Pairing ability correlates with copy number of 240-bp IGS repeats: Estimates of the X-Y disjunction frequency for lines with complete deletions of the rDNA transcription unit resulting from breakpoints within the 5' or 3' IGS region (classes A2, B2 and Cl) ranged from 53.7% to 69.1% by the progeny test method and from 48% to 67% by the cytological method; several lines exhibited intermediate frequencies. The lower figures are indistinguishable from the negative control and the higher figures are indistinguishable from the positive control. As all of these deletions had breakpoints within the 5' and/or 3' IGS regions, it seemed likely that the variation in disjunc-

tional ability reflected different amounts or organization of IGS sequences. T o further investigate this relationship, DNA from several of the lines was cloned and analyzed by restriction mapping and DNA se- quencing.

DNA homologous to the PL probe was cloned from two of the class A2 lines, X[rib7]*210 and X[rib7] *211, which show weak (52% X-Y disjunction) and strong (67% X-Y disjunction) pairing ability, respectively. For X[rib7]*210, a 2.0-kb Hind111 fragment encompassing the residual rDNA from the P transposon was cloned and partially sequenced. Its deletion breakpoints proved to be within one of the 5'IGS 240-bp repeats and in X chromosome sequences flanking PR (Figure 5). It retained slightly less than two 240-bp repeats. For X[rib7]*211, it was known from genomic blot analysis that there were two P transposons inserted on the X chromosome, one at the original site and a second at a new site. Clones encompassing the two transposons (referred to as X[rib7]*21 l a and X[rib7]*2llb in Figure 5A) proved to contain approximately 7 and 1 copies of the 240-bp repeat, respectively (see Figure 5 and MATERIALS AND METH- ODS for details). No other rDNA was present in either

536 B. D. McKee. L. Habera and J. A. Vrana

A

X/rib/PHJ*B 1 kb

B HJ'B

5.8s" T T A T A T W ATOAATTATA AAACTCTAAO COGTQQATCA CTCGGCTCAT GGGT t I T S l - t S . 8 S "z Y

1900b T A T W T A A ATGGTTGCCA AACAGCTCGT CATCAATTTA GTGACGCAGG 2 0 AluI 4 0

HJ+B c

CATATGATAT TGMTCCCTA TCATATAATT TTAATATAAA Q A A T T T . . . . . . NdeI 60 8 0

FIGURE 4.-The structure of X[rib7]*HJ+B and the sequence flanking its major deletion. (A) The top line is a restriction map of [ r i b 7 ] ( l A l - 4 ) . from Figure 2 (see legend for abbreviations and shadings). Restriction sites: tilled triangles = HindIII sites; filled circles = EcoRI sites; open circles = SstII sites. The second line is a detailed map of the indicated regions of [rib']]. Horizontal arrows represent 240-bp IGS repeats. Vertical arrows represent the positions of the breakpoints that define deletion X [ r i b 7 ] * H f B . The numbered regions within the ITS represent: 1, ITSl; 2, 5.8s; 3, ITS2a; 4, 2s; and 5, ITS2. The third line is a map of the X[r ib7] * H f B double deletion. Dotted lines represent deleted sequences. (B) DNA sequences of the "5.8s and 'IGS (1900 region) DNAs flanking the breakpoints of the major deletion in X [ r i b 7 ] * H f E . Breakpoints are indicated by vertical arrows. Sequences in boldface are present in X [ r i b 7 ] * H f B ; sequences in normal typeface are deleted from X[rib7]*HJ+B and are from TAUTZ et a l . ( 1 988).

transposon or in genomic DNA flanking the transposons. DNA sequence analysis identified the same junction sequence in both transposons, resulting from fusion of a 240-bp repeat to PR-derived sequences (Figure 5B), indicating that 211b arose from 211a by replicative transposition (accompanied by a second deletion encompassing exactly six 240-bp repeats) after the initial deletion of the rDNA transcription unit. X[rib7]*201 and 202 were not cloned, but genomic restriction analysis indicates that their rDNA content is more similar to X[rib7]*210 than to X[rib7] "21 I . The pairing behavior of these lines is consistent with that conclusion.

Analysis of cloned DNA from lines X[rib7]*49A, X[rib7]*U+ and X[rib7]*7B (class C1) confirmed the preliminary conclusion from genomic blot studies that in all three lines the deletion had resulted from an apparent unequal crossover between 240-bp repeats located in the 5' and 3' IGS regions, so that the remaining rDNA consisted solely of an uninterrupted block of 240-bp repeats. The three lines differ only in the residual copy number of 240-bp repeats: 5 in 49A, 7 in U+ and 8 in 7B. These copy number differ-

- B

llcb

T C MOCTA~YXQ ITCTACOAU amamran A M C T I ~ A T aaaTnaacaa ~oorrocco~ CCTCICATAT

- AluIAluI 220 1

210 w

20

TSTXA&MC O+ISOTORC ATA?OLTI¶T W C M l T A T A ~ A O T A M l T MATCATATA UTA- 40 NdeI 60 80 NdeI 100

2 1 1 a a T $,

TTMTATXTA ~TATATOTAT ATQ.XM" " A T A T KC=- m a a A a M u 120 140 DdeI 160

C C e C A r m r O Z O M T W A T A TAOTAOTOTA A O C T I W

180 (ScaI)2OO AluIAlul

2 1 1 c

PR' . . . . .. . ATICAMCCC CACCCACAX: CTMOOORA A T C M W T C AT&- ... .. .. . 2 1 0 *

210' . . . . . . . . XIQATMOW COCmUcoO m M T C A C M T C A C I T A C . . . . . . . FIGURE 5.-Detailed maps and sequences of cloned class A2 and

C1 deletions. (A) Restriction maps of cloned deletions. The top line is a map of [ r i b 7 ] ( l A l - 4 ) drawn to emphasize intergenic spacers (untilled rectangles). Only the ends of the rDNA and rosy transcrip tion units (cross-hatched rectangles) are shown. Filled rectangles represent PL and PR, as in Figure 2. Bold lines represent vector sequences flanking the rDNA insert. Thin lines represent sequences flanking [rib7](1A1-4). Small horizontal arrows represent 240-bp "promoter-like" repeats within the IGS. Larger horizontal arrows represent the rDNA and rosy transcription units. Restriction enzyme sites: filled triangles = HindIII sites; filled circles = EcoRI sites; open circles = SstlI sites; open boxes = ScaI sites. Other lines show structures of deletions. Dotted lines represent deleted material. For X[r ib7]*211 phage clones from a partial SauSAdigest library were recovered so that the proximity of the two transposons could be determined and so that DNA flanking the transposons could be tested for presence of rearranged rDNA. Restriction and hybridization analysis of the clones indicated that the two transposons (referred to as X[r ib7]*211a and 2 1 1 b ) contain 7 and 1 240- bp repeats, respectively, that they are not immediately adjacent (separated by at least 10 kb), that 2 1 l a is present at the original site while 2 1 l b is at a new site, and that no rDNA sequences other than 240-bp repeats are present. (B) Sequences of several of the 240-bp repeats in the 5' IGS of p(rib,ry)7 and locations of the breakpoints of deletions in X[r ib7]*210 and 2 1 I repeats of p(rib,ry)7. Six of the 8% repeats in the 5' IGS were sequenced. Polymorphic sites are indicated by alternative bases above the main line. The polymerase 1 transcription initiation site is indicated by the horizontal arrow. The consensus topoisomerase I cleavage site is underlined. Vertical arrows represent the breakpoints of the deletions in line X(rib71 * 2 1 0 and X[r ib7]*211. The breakpoints in X[rib7]*21 l a and 2 1 1 b are identical. "Sequence of PR flanking the breakpoint in X[r ib7] * 2 1 1 (vertical arrow). Sequences in boldface are present in X[rib7] * 2 1 1 and were determined as described. Sequences in normal typeface are deleted from X[r ib7]*211 and are from RUBIN and SPRADLING (1 983). 'Sequence of flanking X chromosome material adjacent to breakpoint in X[r ib7]*210.


0.5 0 - c I 0 2 1 0

HJ+B

211

o + c 0 70

0 "+

0 49A

I I I / / I I I I I ( I I I [

0 1 2 3 4 5 6 7 0 9 1 0 1 1 1 2 1 3

# OF 240 BP IGS REPEATS FIGURE 6.-Summary of structural and functional analyses of

deletions in classes A2, C1 and C2. The X-Y disjunction percentage as determined by the cytological assay method (Table 3) for each line is plotted against the approximate number of 240-bp repeats. Repeat numbers were determined in cloned DNA for all lines (see Figures 4 and 5) . Open circles represent negative (-C) and positive (+C) controls (see Table 3). Lines from class A2, C1 and C2 are indicated by filled circles, open squares and filled squares, respectively.

ences correlate with the differences in X-Y disjunction frequency for the class C1 deletions (51%, 56% and 62% for 49A, U+ and 7B, respectively).

The results of the analyses of cloned DNA from lines in classes A2, C1 and C2 are summarized in Figure 6 in which X-Y disjunction frequencies are graphed as a function of the number of copies of the 240-bp IGS repeat. It is clear that there is a positive correlation between copy number of 240-bp repeats and ability to stimulate X-Y disjunction. It is also clear, however, that there are differences among classes that do not reflect 240-bp repeat copy number. First, none of the C1 chromosomes paired as effectively as the X[rib7]*2II chromosome from class A2 yet the IGS copy number in X[rib7]*211 is comparable to that of two of the class C1 chromosomes. Second, X[rib7] * H S B stimulated X-Y disjunction more effectively than the positive control despite having one less 240- bp repeat. The possible significance of these differences is addressed in the DISCUSSION.

Nucleolus formation is prevented by complete and partial deletions of the rDNA transcription unit: It has been suggested that the pairing ability of rDNA sequences might be functionally related to their ability to form a nucleolus (MCKEE and KARPEN 1990). The nucleolus could promote rDNA pairing either by providing a region of high rDNA concentration, thus facilitating the homology search process, or by providing a pool of rDNA-binding proteins, one or more of which could be modified to function in a pairing capacity during meiosis. If the nucleolus plays

an important role in pairing, then rDNA sequences needed for nucleolus formation are likely also to be important in pairing. It is possible to test rDNA sequences for nucleolus-forming ability because single, euchromatically located rRNA genes form mininucleoli at the sites of insertion on polytene chromosomes. Mininucleoli can be easily detected in salivary gland squashes by indirect immunofluorescence using the antinucleolar antibody Ajl (SAUMWEBER et al. 1980; KARPEN, SCHAEFFER and LAIRD 1988), and are also visible, although less easily so, in unstained, acid- squashed salivary gland preparations. Figure 7A shows an unstained mininucleolus and Figure 7B an anti-Aj 1 -stained mininucleolus formed by [rib7](1A 1-

Each of the partial rDNA deletions in classes A2, C1 and C2 were tested for mininucleolus formation by both the antibody and acid-squash tests. The results (Figure 7 and Table 3) were unambiguous: all of the partial deletions were found to be negative for nucleolus formation by both tests. As discussed above, the class A2 and C1 deletions are deficient for the entire rDNA T U but retain variable amounts of the 5' and 3'IGS region; their inability to form mininucleoli indicates that part or all of the rDNA T U is essential for nucleolus formation. Line X[r ib7]*HrB (class C2) is deficient for the entire 28s gene and for parts of the 5.8s gene and the ITS region; it also has a 5' IGS shortened by a single 240bp repeat. The failure of this line to form a mininucleolus indicates either that there are sequences in the proximal half of the rDNA transcription unit that are essential for nucleolus formation or that nucleolus formation depends upon having a minimum of eight 240-bp IGS repeats 5' to the transcription unit. The former conclusion seems more likely.

4).

DISCUSSION

Targeted deletion mutagenesis: The results presented here show that destabilization of a P transposon containing a target sequence by exposure to the genomically integrated transposase source [A 2,3](99B) is an effective method of producing deletions within the target. The deletion frequency proved to be high enough that useful deletions could be recovered without a phenotypic screen. Deletions were recovered in five out of 37 phenotypically parental (ry', nonlethal) X[rib7]* chromosomes screened by genomic blot analysis. In addition to the ease with which deletions can be generated, the major advantage of this method over conventional in vitro mutagenesislp element- transformation protocols is that all of the deletions can be tested at a single insertion site, thus eliminating any confounding position effects.

The chief drawback of this method is that the breakpoints may be nonrandomly distributed within the target sequence. The present results provide a


FIGURE 7.-Mininucleolus formation at sites of ectopic rRNA genes in polytene chromosome squashes from salivary glands of homozygous females. Arrowheads indicate the 1 A region of the X chromosome where [rib7](IAI-4) and its deletion derivatives are located. (A) Unstained squash of [r ib7]( lAl-4) showing a dis- tinct nucleolus adhering to the tip of the X in addition to the usual one adjacent to the chromocenter. (B) Squash of [r ib7]( lAl-4) female stained with the antinucleolar antibody Aj 1 showing mininucleolus just below the 1 A region on the micro- graph. Under the conditions used, nucleoli fluoresce green and chromosomes (which are stained with ethidium bromide) fluoresce yellow. (C and D) Aj 1-stained squashes from an X[rib7]*HJ+B female (C) and an X[rib7]*211 female (D) showing 1A region with no associated mininucleolus. Magnifications: 212X for A and 334X for B-D.

clear, although probably extreme, example of this danger. It seems likely that the high concentration of direct repeats in the IGS regions accounts for their high susceptibility to involvement in P-induced rearrangements. Previous studies have documented a strong tendency for the breakpoints of imprecise excisions to occur within direct repeats (ENGELS 1989). Targets not containing internal repeats are less likely to show such a skewed breakpoint distribution. The more random distribution of breakpoints reported by DANIELS et al. (1985) and DREESEN, HENIKOFF and LOUCHNEY (1 99 l), who used similar protocols to generate deletions in transposon-borne copies of the unique sequence rosy and brown genes, respectively, supports this interpretation.

rDNA sequences involved in X-Y pairing and nucleolus formation: Despite their nonrandom distribution, the deletion breakpoints generated in this study proved useful for functional mapping. The chief conclusions that emerge from phenotypic analysis of the deletions are as follows.

The 240-bp IGS repeats are capable of stimulating X-Y pairing in the absence of other rDNA sequences: Deletion of the entire rDNA T U as well as the rosy sequences had no effect on the ability of the remaining IGS sequences to stimulate X-Y disjunction, as evidenced by the disjunctional frequency in line X[rib7]*211. This does not imply that T U sequences do not participate in pairing. No fragments that lack spacers but retain the T U have been tested. However, the fact that deleting the 3’ half of the transcription unit including the 28s gene actually improves pairing abil-

ity suggests that at least the 3’ half of the rDNA may not be very important for pairing.

Disjunctional ability is proportional to the number of copies of the 240-bp IGS repeat: Within both the A2 and C1 classes there is a clear correlation between IGS copy number and pairing ability. Within class A2, X chromosomes with fewer than two copies of the 240- bp repeat (X[rib7]*210 and probably 201 and 202) show little or no pairing ability while one with 8 copies (X[rib7*211) pairs as effectively as X chromosomes with a complete rRNA gene. Within class C l , chromosomes with 5, 7 and 8 copies show a similar correlation between copy number of 240-bp repeats and ability to stimulate X-Y disjunction. However, none of the class Cl chromosomes is as potent as X[rib7]*21 I , despite having comparable numbers of 240-bp repeats. The only consistent difference between class A2 and Cl chromosomes is that, in the latter, IGS sequences are adjacent to a pol11 transcription unit (rosy) while in the former the rosy sequences have been deleted. It is possible that transcription of the rosy sequences exerts an inhibitory effect on IGS pairing.

The demonstration that IGS sequences alone have pairing ability provides an explanation for the otherwise puzzling observation that in Drosophila simulans, a close relative of D. melanoguster, the Y chromosome lacks a nucleolus organizer, yet pairs and disjoins regularly from the X (which has the only NO) during male meiosis. It has recently been shown that the D. simulans Y chromosome contains a very large (3000 kb) block of tandemly repeated 240-bp sequences homologous to the IGS repeats in the X-linked N O


(LOHE and ROBERTS 1990). In light of the present results, this IGS-derived block is a very strong candi- date for the pairing site of the D. simulans Y chromosome. It is possible that the requirement for accurate X-Y pairing dictated the retention of IGS sequences during evolution of the D. simulans Y .

Nucleolus formation requires sequences from the 3‘ half of the rDNA TU: The lack of visible mininucleolus formation in lines deleted either for the entire rDNA T U or for just the 3’ half indicates that autonomous nucleolus formation depends on presence of some of these sequences. Since pairing ability does not require sequences other than the 240-bp IGS repeats, this observation argues against a close mechanistic connec- tion between nucleolus formation and chromosome pairing. The apparent improvement in pairing ability accompanying deletion of 28s sequences suggests the possibility that nucleolus formation actually inhibits pairing. It is not known when pairing occurs but it must precede nucleolar dissolution because chromosomes are already paired when they first become visible during prophase; the nucleolus is still intact at this time. The enzymes and structures involved in chromosome pairing could be restricted to non-nucleolar portions of the nucleus. If some rRNA genes are also not located in the nucleolus, they may participate preferentially in pairing. Deletion of sequences necessary for nucleolar participation but not for pairing would then increase pairing efficiency.

The present results must be interpreted with cau- tion, however. They do not rule out the possibility that the initial steps in nucleolus formation, which are likely to involve initiation of transcription at the pol1 promoter, can occur in the absence of 28s sequences but that formation of a cytologically detectable nucleolus including proteins such as Ajl depends on their presence. A functional polymerase I promoter is present in each 240-bp repeat (COEN and DOVER 1982; KOHORN and RAE 1982; MILLER, HAYWARD and GLOVER 1983). These spacer promoters can direct transcription in the presence or absence of the rDNA transcription unit (KOHORN and RAE 1982; MILLER, HAYWARD and GLOVER 1983; MURTIF and RAE 1985; GRIMALDI, FIORENTINO and DI NOCERA 1990), so the possibility that pairing is mechanistically related to the initiation of polymerase I transcription can not be dismissed. Clearly it will be of considerable importance to investigate expression of the rDNA fragments in these deletion lines.

Implications for the mechanism of achiasmatic meiotic pairing: These results provide strong evidence that X-Y pairing in Drosophila males is based on underlying DNA homology. The nucleolus organizers each contain some 200 copies of the rDNA (LONG and DAWID 1980), each copy being about 12 kb in length. Thus, rDNA homology provides for at least 2000 kb of homology between the otherwise

largely nonhomologous sex chromosomes. Approxi- mately one-quarter of this homology is accounted for by the intergenic spacer regions which have been shown herein to contain strong pairing ability. Auto- somal pairing shows a similar dependence on DNA homology, but in a more dispersed fashion. Segments of second chromosome euchromatin transposed to the Y are able to pair with a normal chromosome 2 at meiotic prophase. The frequency with which they do so is proportional to the length of homology (B. D. MCKEE, S. DAS and S. LUMSDEN, submitted for publication). Thus meiotic pairing ability in Drosophila males is broadly distributed among chromosomal sequences and reflects underlying DNA homology.

However, not all sequences participate in male meiotic pairing. For the sex chromosomes, the highly repetitive satellite sequences in the vicinity of the X centromere, the moderately repetitive Stellate locus in the X euchromatin (LIVAK 1984), and transpositions of unique-sequence X markers to the Y chromosome are all ineffective at promoting X-Y disjunction when the X NO is absent, despite the presence of homologous sequences on the Y (MCKEE and KARPEN 1990). For the autosomes, centric heterochromatin appears to be excluded from participation in meiotic pairing, as shown by the failure of heterochromatic free second chromosome duplications to pair and disjoin regularly from each other or from intact second chromosomes (YAMAMOTO 1979), and by the random segregation of compound chromosomes such as C(2L) and C(2R) that share homology only in their centric heterochromatic regions (HILLIKER, HOLM and APPELS 1982). These observations suggest that many but not all sequences are active with respect to meiotic pairing. The apparent exclusion of heterochromatic sequences other than the rDNA from participation in pairing suggests that chromatin conformation is an important variable.

Similar observations have been made in organisms with “normal” (recombinational) meiosis. Widespread participation of homologous sequences in the initiation of pairing is implied by the ability of heterozy- gotes for reciprocal translocations or inversions to achieve point-for-point matching of homologous regions (VON WETTSTEIN, RASMUSSEN and HOLM 1984), and by the recovery of crossovers between largely nonhomologous chromosomes sharing short, essentially arbitrary regions of homology (PETES and HILL 1988; CRAYMER 1981). However, some sequences, especially those in heterochromatic regions (JOHN 1989), participate weakly or not at all in recombinational pairing. Conversely, there are clearly strong hotspots for the initiation of meiotic recombination (NICOLAS et a / . 1989; CAO, ALANI and KLECKNER 1990) although it is not yet clear whether these are also hotspots for the initiation of chromosome pairing.

These parallels between the rules for pairing initi-


ation in chiasmatic and achiasmatic meiosis suggest that the mechanisms of pairing may share fundamen- tal similarities. The mechanism of meiotic pairing is not known in any organism. Recent evidence from yeast that the initial events in recombination precede synapsis (PADMORE, CAO and KLECKNER 1991) and that synapsis depends upon functions needed for recombination (CAO, ALANI and KLECKNER 1990; ALANI, PADMORE and KLECKNER 1990; ENGEBRECHT, HIRSCH and ROEDER 1990; ROEDER 1990) suggests that a DNA-level homology search culminating in heteroduplex formation may be a key step in chromosome pairing. There is no evidence relevant to whether heteroduplex is formed in achiasmatic pairing, but the evidence for importance of DNA homology summarized above raises it as a possibility.

A general model for achiasmatic pairing: The 240-bp IGS repeat contains two well characterized sites of possible significance for meiotic pairing. One is a highly conserved hexadecameric sequence that functions as a high affinity cleavage site for topoisomerase I . The topoisomerase I site was first identified in the rDNA spacer of Tetrahymena and is also present in the rDNA spacers of a variety of taxonomically distant organisms (BONVEN, GOCKE and WESTER- GAARD 1985). This site is preferentially cleaved by eukaryotic topoisomerase I enzymes from a wide variety of sources including humans and Drosophila (CHRISTIANSEN, BONVEN and WESTERGAARD 1987). The other site is a copy of the 52-bp sequence that extends from -24 to +28 relative to the rDNA transcription initiation site and that contains the minimal pol1 promoter (Figure 5). These “spacer promoters” are transcribed both in vivo and in vitro (KOHORN and RAE 1982; MILLER, HAYWARD and GLOVER 1983, MURTIF and RAE 1985; GRIMALDI, FIORENTINA and DI NOCERA 1990). They function in vivo to enhance transcription of the rDNA transcription unit located immediately downstream. The level of pre-rRNA transcription is linearly related to the number of 240- bp repeats preceding the pre-rRNA promoter, but only when the 240-bp repeats are oriented toward the downstream pre-rRNA promoter, suggesting that the enhancing activity is related to transcription of the spacer promoters (GRIMALDI and DI NOCERA 1988). Deletion analysis demonstrates that this enhancing activity maps to a 70-bp region of the 240-bp repeat that includes the spacer promoter (GRIMALDI, FIOR- ENTINA and DI NOCERA 1990).

The topoisomerase I site is of interest with respect to meiotic pairing because in vitro studies indicate that topoisomerase I in conjunction with a strand transferase enzyme such as recA protein from Escherichia coli or recl protein from Ustilago maydis, can catalyze pairing and heteroduplex formation between complementary single stranded and duplex DNA molecules that lack homologous free ends (CUNNINGHAM et al.

1981; KMIEC et al. 1983; CASSUTO 1984). Heterodu- plex can be formed by strand transferases alone as long as a free end is available, but it seems unlikely that the chromosome breaks needed to generate the free ends would occur in achiasmatic meiosis since they are highly recombinogenic. In the absence of a free end (e.g. , if both molecules are circular or if the homologous region is flanked by heterologous sequences), strand transferases can carry out a “homology search” that culminates in the formation of “paranemic” joints in which the complementary regions are homologously associated but not topologically interwound (KMIEC and HOLLOMAN 1984; Cox and LEHMAN 1987; RADDING 1988). Addition of topoisomerase I permits the complementary regions to be interwound in a stable heteroduplex, known as a “hemicatenane.” No free ends are transferred during hemicatenane formation and, consequently, no op- portunities for intermolecular ligation and crossing over arise. Such structures are highly stable and could provide a substitute for chiasmata in stabilization of bivalents until anaphase. Hemicatenanes could be resolved by reversal of the process of formation using a topoisomerase again instead of an ordinary resolvase, thus preventing crossing over (Figure 8).

There is genetic evidence consistent with the idea that topoisomerase I is an inhibitor of recombination. Yeast strains doubly mutant for the topoisomerase I and topoisomerase I1 genes show elevated frequencies of recombination in the tandemly repeated rRNA genes (CHRISTMAN, DIETRICH and FINK 1988). More- over, a mutation that causes hyper-recombination between terminal repeats of T y l elements proved to be in a novel eukaryotic topoisomerase I gene homologous to the topA gene of E. coli (WALLIS et al. 1989).

The polymerase I spacer promoters are potentially of interest with respect to meiotic pairing because of evidence that pairing between homologous duplexes is strongly stimulated by transcription. Intact circular duplexes do not form homologous joints in the presence of the U. maydis recl protein, apparently because this protein, like the E. coli recA protein, requires a stretch of single stranded DNA as the initial binding site (RADDING 1988). However, the formation of stable joints can be stimulated either by adding RNA polymerase and NTPs concurrently with rec 1 protein, or by adding short, single stranded oligonucleotides homologous to the duplexes in the presence of recl (KMIEC et al. 1983). Both treatments generate D-loops in the duplex and provide binding sites for recl protein. Thus it is possible that in nonrecombinational pairing, access of strand-transferase to single-strand DNA is promoted by transcription of the DNA rather than by the action of nucleases. This suggestion is consistent with evidence that concurrent transcription promotes mitotic recombination (reviewed in THOMAS and ROTHSTEIN 199 1). A particularly relevant finding

rDNA Spacers and Achiasmatic Pairing 54 1

A

B

C

D

a I

b 1

1 a b

f i l

i I

+ +

1 a b

+ +

a I b

+ +

FIGURE 8.-A general model for achiasmatic pairing. a/+ and b/ + represent outside markers. Open circles represent strand transfer proteins, closed circles topoisomerase I molecules. (A) A site on one chromatid is “activated” for an homology search by binding of strand transfer proteins. This may require rendering the site single stranded via transcription or helicase activity or by some other means. Binding of strand transfer protein to only one strand is shown for simplicity but both strands may participate in the homology search. (B) An homology search by the activated pairing site leads to formation of a paranemic joint with an homologous site. Topoisomerase I binds to a nearby site and introduces transient nicks. (C) A hemicatenane is formed in which the homologously paired DNA strands are topologically interwound. Topoisomerase I activity is needed for interwinding. Three-stranded structure is shown but actual structure of hemicatenane is not known. Four- stranded structure or two two-stranded structures are also possible. (D) Hemicatenane is resolved by topoisomerase I-mediated unwind- ing. The resolved molecules are always noncrossovers because no free ends have been transferred and so there have been no oppor- tunities for interchromatid ligation.

is that in yeast a fragment of DNA containing an RNA polymerase I promoter stimulates recombination of an adjacent gene 25- to 100-fold (KEIL and ROEDER 1984). This stimulation apparently reflects transcription into the adjacent sequences because it depends on orientation of the promoter and can be prevented by inserting a transcription termination site between the rDNA promoter and the recombining sequences (VOELKEL-MEIMAN, KEIL and ROEDER 1987). In meiosis, some recombination hotspots have also been mapped to promoter and/or enhancer regions (NI- COLAS et d . 1989; SUN et d . 1989; SHENKAR, SHEN and ARNHEIM 1991). However, this may reflect a chromatin conformation characteristic of promoters rather than active transcription, since inhibiting transcription from the yeast argl promoter does not in- hibit its activity as a meiotic recombination hotspot

(SCHULTES and SZOSTAK 1991). Thus, the proposed model of achiasmatic pairing would seem to require some mechanism of generating single-strand DNA other than nucleolytic degradation, and transcription provides one possible mechanism, but there may be others.

Hemicatenane formation mediated by a strand transferase and topoisomerase I (and, perhaps, RNA polymerase) provides a potentially general model for achiasmatic pairing. Topoisomerase I is abundant in the nucleus, and is associated preferentially with ac- tively transcribed genes (FLEISCHMANN et al. 1984). Although topoisomerase I cleaves the hexadecameric rDNA spacer sequence with especially high affinity, it also cleaves at a large number of other sites that fit only a loose, 4-base consensus (BEEN, BURGESS and CHAMPOUX 1984). Thus, although the proposed model fits the present results on rDNA pairing particularly well, it is not restricted to rDNA pairing. It could also account for the widespread participation of autosomal sequences in meiotic pairing, since none of the proposed enzymes are sequence-specific. The model is diagrammed in Figure 8.

Aspects of this model may also apply to recombinational pairing. The homology search stage, which requires a strand transferase and which leads to formation of a paranemic joint, could be common to chiasmatic and achiasmatic pairing. Since paranemic joints are not topologically interwound, it is likely that they could be disassembled more rapidly than true heteroduplexes. This feature would make them useful intermediates in a trial-and-error homology search. Hemicatenane formation via combined action of a strand transferase and a topoisomerase, is likely to be unique to achiasmatic pairing. Double strand DNA breaks are clearly associated with recombination in yeast (SUN et al. 1989; CAO, ALANI and KLECKNER 1990), suggesting that recombinational heteroduplexes are formed by transfer of free ends generated by a simple endonuclease. Heteroduplex resolution in chiasmatic meiosis would also presumably not involve topoisomerases, but rather conventional resolvases that generate strand breaks subject to intermolecular ligation. Thus it is possible that the major difference between chiasmatic and achiasmatic pairing lies in the types of enzymes-simple endonucleases us. topoisomerases-used to make the strand breaks needed for heteroduplex formation and resolution.

This model has the virtue of being directly, though not necessarily easily testable. Potentially testable pre- dictions relevant to Drosophila male meiosis include: the presence of heteroduplex DNA during meiotic prophase persisting until anaphase; a requirement for a strand transferase and topoisomerase I for meiotic pairing of all chromosomes; a requirement for topoisomerase I for homolog segregation at anaphase I; and a requirement for a functional high-affinity to-


poisomerase I site for the strong pairing of the 240- bp IGS spacer repeat. A prediction that arises from the suggestion that hybrid DNA formation is common to both chiasmatic and achiasmatic meiosis is that male and female Drosophila should share at least one essential meiotic function, a gene for a strand transfer enzyme. Nearly all meiotic mutants that have been isolated thus far in Drosophila affect only one sex (BAKER et al. 1976), but it is unlikely that the existing mutations saturate the meiotically active genome.

We thank MARY ANN HANDEL, RANJUN GANGULY, DHRUBAJYOTI CHAKRAVARTI, AMY ROE and two anonymous reviewers for valuable suggestions on the manuscript. We are grateful to GARY KARPEN for supplying the [rib7](IAI-4) stock and for the gift of anti-Aj1 antibody. We thank W. ENGELS for the [A 2,3](99B) stock and the Mid-American Drosophila Stock Center at Bowling Green State University, Bowling Green, Ohio, for supplying most of the other Drosophila stocks used in this study. We also thank CYNTHIA MERRILL for valuable technical assistance with the complementation tests. This study was supported by U.S. Public Health Service grants R01 GM40489 and KO4 GM00522 from the National Institute of General Medical Sciences to B.D.M.

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