Fine Mapping of Satellite DNA Sequences Along the Y Chromosome ...

Copyright 0 1991 by the Genetics Society of America

Fine Mapping of Satellite DNA Sequences Along theY Chromosome of Drosophila melanogastec Relationships Between Satellite Sequences and Fertility Factors Silvia Bonaccorsi* and AllanLohet *Centro di Genetica Evolurionistica del CNR, Universita di Roma “La Sapienza,” 00185 Roma, Italy, and ?Department of Genetics, Washington University School of Medicine, St. Louis, Missouri 63130 Manuscript received February 11, 1991 Accepted for publication May 29, 1991 ABSTRACT The entirely heterochromatic Y chromosome of Drosophila melanogaster contains a series of simple sequence satellite DNAs which together account for about 80% of its length. Molecular cloning of the three simple sequence satellite DNAs of D. melanogaster (1.672, 1.686 and 1.705 g/ml) revealed that each satellite comprises several distinct repeat sequences. Together 11 related sequences were identified and 9 of them were shown to be located on the Y chromosome. In the present study we have finely mapped 8 of these sequences along the Y by in situ hybridization on mitotic chromosome preparations. The hybridization experiments were performed on a series of cytologicallydetermined rearrangements involving the Y chromosome. The breakpoints of these rearrangements provided an array of landmarks along the Y whichhave been used to localize each sequence on the various heterochromatic blocks defined by Hoechst and N-banding techniques. The results of this analysis indicate a good correlation between the N-banded regions and 1.705 repeats and between the Hoechst-bright regions and the 1.672 repeats. However, the molecular basis for banding does not appear to depend exclusivelyon DNA content, since heterochromatic blocks showingidentical banding patterns often contain different combinations of satellite repeats. The distribution of satellite repeats has also been analyzed with respect to the male fertility factors of the Y chromosome. Both loopforming (kl-5, kl-3 and ks-I) and non-loopforming (kl-2 and b - 2 ) fertility genes contain substantial amounts of satellite DNAs. Moreover, each fertility region is characterized by a specific combination of satellite sequences rather than by an homogeneous array of a single type of repeat. We discuss the possible functional role of these satellite sequences in the light of the available information on the Y chromosome fertility factors of D.melanogaster.

T

HE Y chromosome of Drosophila melanogaster is an entirely heterochromatic element which carries a limited but well defined set of genetic functions: the fertility factors, a series of loci whichare essential for male fertility (BRIDGES1916; STERN1929; BROSSEAU 1960); the bobbed locus (bb), which is allelic to the bobbed locus located inthe X heterochromatin and corresponds to the ribosomal cistrons (for review see RITOSSA1976); a pairing site(collochore) required during male meiosis for regular pairing and segregation of the sex chromosomes (COOPER1964); a locus necessary for normal male meiosis, whose deficiency causes the appearance of needle shaped crystals inthe primary spermatocytesand anabnormal chromosome distribution at meiosis (HARDYet al. 1984; LIVAK 1984, 1990).

Classical genetic and cytological analyses of these functions have beenfor a long time precluded because the Y chromosome does not undergo meiotic recombination and is included in the chromocenter of polytene chromosomes.More recently, by applying a series of banding techniques to neuroblast prometaphase chromosomes, it was possible to differentiate Genetics 1 2 9 177-189 (September, 1991)

the mitotic Y chromosome into 25 regions (GATTIand PIMPINELLI1983). Thusan extensivecytogenetic analysis could be undertaken which permitted the mapping of the functions carried by the Y chromosome and provided some insights into their genetic organization (GATTI and PIMPINELLI 1983; BONACCORSI et al. 1988). The most extensively studied Y chromosome functions are the fertility factors. Four fertility factors have been cytologically localized on the long arm of the Y chromosome and two on the short arm(KENNISON 1981; HAZELRIGG, FORNILIand KAUFMAN 1982; GATTIand PIMPINELLI 1983). Starting from the tip of the long arm they are designated as k1-5, kl-3, kl-2, kl1, ks-1 and ks-2; the existence of kl-4, postulated by BROSSEAU (1960), has not been confirmed (KENNISON 198 1; HAZELRICG,FORNILIand KAUFMAN 1982; GATTIand PIMPINELLI 1983). Moreover, it has been shown that each of k1-5, kl-3 and KS-1 fertility factors possesses extremely large physicaldimensions;they are defined by a series of noncomplementing sterile breakpoints scattered over chromosome regions containing up to 4000 kb of DNA. The cytogenetic data

178

S. Bonaccorsi and A. Lohe

on kl-2, kl-1 and ks-2 enabled a ratherprecise localization of these loci along the Y but not an estimation of their physicalsize (GATTI and PIMPINELLI1983; C . PISANO,S. BONACCORSI and M. GATTI,manuscript in preparation). More recently, it has been shown that the kl-5, kl-3 and ks-I fertility factors are responsible for the development of three giant lampbrush-like loops in primaryspermatocyte nuclei. These loops are analogous to those described in Drosophila hydei and are thought to represent the cytological manifestation of fertility factor activity during this stage of spermaet al. 1988). togenesis (BONACCORSI A number of studies performed during the past yearshaveshown that a seriesofsimplesequence satellite DNAs are located on the Y which together et al. account for about 80% of its length (PEACOCK 1977; APPELSand PEACOCK1978; STEFFENSEN, APPELS and PEACOCK1981). D. melanoguster contains four well defined satellite DNAs which amount to about 20% of the genome and can be divided into two groups based on sequencecomplexity (GALL, et al. 1973). One COHENand POLAN197 1; PEACOCK group contains tandem repeats of a simple sequence, only 5, 7 or 10 bp in length; these satellites band at 1.672, 1.686 and 1.705 in CsCI. The other group is represented by the 1.688 satellitewhichconsistsof tandem repeats of a longer sequence, 359 bp in length. Molecular cloning of D. melanogaster satellite DNA provided a more refined view of this genetic material, by showing that more than one sequence is present in each satellite (LOHEand BRUTLAC1986). Together, 11 related sequences were identified in thethree simple satelliteDNAs, all mapping to the heterochromatic portions of the genome, and 9 of them were shown to be present on the Y chromosome (LOHEand and ROBERTS,unpubRoberts 1988; LOHE,HILLIKER lished). However, the in situ hybridization experiments of LOHEand ROBERTS(1988) were performed on wild-type mitotic chromosomesand did not permit a precise localization of the satellite sequences with respect to the cytological and genetic entities previ1983; ously identified on theY (GATTIand PIMPINELLI BONACCORSI et al. 1988). Here we describe a series of in situ hybridization experiments performed on a number of cytologically determined rearrangements involving the Y chromosome. The heterochromatic breakpoints of these rearrangements provided an array of unambiguous landmarks along the Y which have been used to localizeeachsequence onthe various heterochromatic blocks defined by the banding techniques. This allowed us to relate the molecular composition of specific Y chromosome blocks to their cytochemical and functional properties. MATERIALS AND METHODS

Drosophila stocks All the Y autosome translocations used here were generated by LINDSLEY et al. (1972) using a B"Yy+.

The genetics and cytology ofthese translocations have been described by LINDSLEY et al. (1 972), GATTIand PIMPINELLI (1 983) and by BONACCORSI et al. (1 988). All the T(X;Y)s used here, except T(X;Y)Tl3, are fertile reciprocal translocations having the X breakpoint in the proximal heterochromatin; they involve an X chromosome marked withy wfand aE'Yy' chromosome (KENNISON198 1; HARDY et al. 1984). T(X;Y)Tl3 is a sterile reciprocal translocation (KENNISON198 1) having the Y breakpoint in region h5 and carrying an additional deficiency that encompasses regions h5-h6. The X and Y chromosome breakpoints of T(X;Y)FIZ, T(X;Y)F15 and T(X;Y)T13 were determined in the present study and are reported in Table 1 , which summarizes the cytological features of all the rearrangements used here. Before being used for in situ hybridization experiments, all the Y chromosome rearrangements were reexamined cytologically by Hoechst and N-banding according to GATTI and PIMPINELLI (1 983). This analysis revealed that 17 of the 18 rearrangements previously described (GATTIand PIMPINELLI 1983; HARDYet d . 1984; BONACCORSI et d . 1988) maintained their original constitution. T(Y;Z)A77 picked up an additional deficiency encompassingregions h 1-h 16. All the othermutations, special chromosomes and genetic markers used in this work are described by LINDSLEY and GRELL(1968). All stocks and crosses were maintained at 25" f 1 " . Recombinant plasmids:Isolation and cloning of D. melanogaster satellite DNA sequences are described in detail elsewhere (LOHEand BRUTLAC1986). The eight satellite DNA clones used inthe present work are reported in Table 2. The 1.672-1 plasmid clone contains a tandem array of AATAC satellite repeats adjacent to part of a mobile element DNA (LOHEand BRUTLAC1987b). AnEcoRI fragment of this plasmid contains the complete satellite array of 181 bp flanked by 7 1 bp of mobile element DNA. This fragment was subcloned intothe pSP65 vector and the plasmid was named 1.672-18 1 . ['HIRNA probes of each sequence were synthetized from pSP64/65 DNA templates. Nick-translated ['HIDNA probes were also synthesizedand used for asubset of in situ experiments with 1.672-1 8 1 , 1.705-42 and 1.705--34. Stringency of hybridization: The mean melting temperature (Tm) of the simple satellite DNAs is dependent on nucleotide composition and sequence, and varies widely among these closely related sequences (LOHEand BRUTLAG 1987a). Since different satellite repeats can show up to 80% sequence homology, stringent hybridization criteria were established to avoid cross-hybridizationamong the classes of satellite sequences. The Tm values for RNA-DNA hybrids were determined for each of the eight satellite probes and a hybridization temperature of 10-1 3" below the Tm value was chosen for the in situ hybridizations (Table 2). I n situ hybridization: Mitotic chromosome preparations were made following the proceduredescribed by GATTIand PIMPINELLI (1 983). Minor modifications to their procedure were that slides have been frozen in liquid nitrogen and, after removing the coverslip, immediately placed in 95% ethanol and air dried. The in situ hybridization procedure is similar to that used et al. (1 977). Freshly made, air driedslides were by PEACOCK placed in 0.2 M HC1 (37") for 30 min, rinsed briefly in distilled water, dehydrated in 70% ethanol (2 times), 95% ethanol (2 times) and air dried. 3H-labeled probe (1 X lo5 cpm) in 3 X SSC, 50% formamide (vol/vol) was applied to the slide, an 18 X 18 mm coverslip placedover the solution, and the coverslip was sealed with rubber cement. Prior to incubation slides were placed at 65" in an air incubator for 15 min and slides were then placed at the hybridization

Y Chromosome SatelliteDNAs

179

TABLE 1 Cytological features of rearrangements Y breakpoint"

Translocation

Other breakpoint Reference'

28B 85E 48E h30 h26 34B 38B h26 33B 72AB 36C h29 76D h26 26B 25A 24D 31EF 35CD 66A 57F

Fertilitf

1, hl/h2 TCy;2)R50 1 , 3, h2/h3 T(Y;3)G42 1, 3, 5 h3 TCy;2)D 19 2, 3, h4 T(X;y)V24 2 h5 Df(h5-h6) T(X;Y)T13 1, 3, 5 h7 ln(h1-h3) TCy; 2)G 74 1, 3, 5 h9/h 10 TCy;2)P5 7 F 2, 3, 4 hl1 T(X;Y)E 1 F 1, 3 hll TCy;2)E92 F3 1, h13 TCy;3)B223 S(R1-1) 1, 3 h14 T(Y;2)B242 2 F h15 T(X;Y)F1 2 F 1, 3 h15 TCy;3)B115 2 F h15/h16 T(X;y)Fl5 S(kZ-2,3,5) 3 1, h16 Df(h3-h13) T(Y;2)H121 h19 Df(h1-hl6) S(KL) 3 1, TCy; 2)A 77 h2 1 5 S ( h - 1 ) 3, 1, T(Y;2)PS S(ks-1) 1, 3, 5 h2 1 TCy; 2)A 162 5 S(h-1) 3, 1, h2 1 T(Y;2)J165 S(kS-1) 1 , 3, 5 h22/h23 T(Y;3)R119 5 S ( h - 1 ) 3, 1, h23/h24 T(y;2)J163 S. BONACCORSI,DIMITRI P. and M.GATTI in The nomenclature of the X chromosome breakpoints is described in detail in S. PIMPINELLI, preparation. The X heterochromatin has been subdivided into 9 regions; region h29 corresponds to the nucleolus organizer and region h33 to thecentromeric area. A slash between two Y regions means that the rearrangement is broken at thejunction of these two regions. * The fertility factors which are disrupted by the rearrangement are indicated between the brackets. 1 , LINDSLEY, et al. (1972); 2, KENNISON(1981); 3, GATTIand PIMPINELLI (1983); 4, HARDY et al. (1984); 5, BONACCORSI et al. (1988).

+

+

+ +

Mean melting temperatures of satellite RNA-DNA hybrids and hybridization temperatures

1.705-42 1.705-34 1.686-198 1.672-38 1.672-349 1.672-181 1.672-453 1.672-563

Repeating sequence

AAGAG AAGAGAG AAGAC AATAT AATAG AATAC AATAAAC AATAGAC

Tm value of RNA-DNA ("C)

Hybridization temperature ("C)

59

48 50 40 16 23 23 25 35

61

53 26 35 36 37 48

S(k1-5) 3, S(R1-5) 5 S(kZ-5) F 5 S(k1-3) S(kl-3,5) S(R1-3)

RESULTS

TABLE 2

Satellite clone

5

The melting temperatures were determined by P. A. ROBERTS and A. R. LOHE(unpublished results).

temperature (see above) for 16 hr. Following hybridization, coverslips were removed and residual probe washed away by incubation in the hybridization solution at the hybridization temperature for15 min (2 times). Slides were washed in 2 X SSC for 15 min, treated with RNAse A (2 rg/ml in 2 X SSC, 30 min at room temperature), washed again in 2 X SSC (4 times, 15 min each), placed in 70% ethanol, 95% ethanol (2 times), airdried, dipped in Ilford K2 emulsion (diluted 1:1 with water) and exposed at 4 " in a light-tight box. Exposure times were determined empirically andvaried for different probes from 3 days to several weeks. Slides were developed in Kodak Dektol D l 9 (2 min.), washed in water and fixed in Kodak Fixer for4 min. They werestained for 20 min. with a 10%solution of freshly preparedGiemsa (BDH, R66) in 8 mM KHzP04, 6 mM Na2HP04,pH 6.8, rinsed in tap water,air dried and mountedin immersion oil (Zeiss). Slides were stored indefinitely in this way.

I n situ hybridization experiments were performed using eight satellite DNA clones recently isolated by LOHEand BRUTLAG (1986). The repeating sequence contained in each clone, together with the hybridization temperatures used for thein situ experiments are reported in Table 2. Four sequences (AAGAG, AAGAGAG, AAGAC, AATAT) map to multiple sites along the Y and to other heterochromaticportions of the Drosophila genome; three sequences (AATAC, AATAAAC, AATAGAC) map only to unique sites on the Y, and one (AATAG) toa single site on the Y and to the 2nd chromosome heterochromatin (LOHE and ROBERTS1988). To finely map the 8 satellite sequences associated with the Y chromosome we used 16 Y autosome and 5 X-Y cytologically determined translocations (GATTI and PIMPINELLI 1983; BONACCORSI et al. 1988) whose breakpoints span the length of the Y chromosome. The breakpoints of these translocations are diagramatically reported in Figure 1. All these rearrangements were induced on a FYy+ which carries two X heterochromatin blocks appended at the distal ends of YL and P,respectively (GATTIand PIMPINELLI 1983). The presence of these blocks of X heterochromatin does not interfere with the satellite mapping along the Y chromosome because their principal component is the 359-bp repeats of the 1.688 satellite DNA, which are absent from the wild type Y (HILLI-


180 G74

mo

-

(AAGAG)

Lu

u

w

(AAGAGAG), (AAGAC), (AATAT),

PA

P7AO

u

U

u

U

(AATAG),

u

U

U

U

(AATAC),

U

(AATAAAC),

U

(AATAGAC),

U

1

2

35 4

A I

6 7 6

9 11 10

13 12

E

14 16 15

17

16 19

22 21

23 24 25

C

FIGURE1.-Localization of 8 satellite DNA sequences along the Y chromosome of Drosophila melanoguster. The diagram is a schematic representation of the Y chromosome stained with Hoechst 33258 (GATTI and PIMPINELLI 1983). The chromosome is subdivided into 25 regions defined by the degree of fluorescence and the presence of constrictions. Filled segments indicate bright fluorescence, hatched segments indicate dull fluorescence and open segments indicate no fluorescence. C: centromere; N: N-banded regions. The vertical lines above the diagram indicate the Y chromosome breakpoints of the 21 translocations used for the in situ hybridization experiments. G 7 4 , 8 2 4 2 and P8 were used for the gross localization of each satellite sequence, the other translocations were employed for fine mapping (see text and Table 1 for further explanation). The horizontal lines below the upper diagram indicate the localization of each sequence. The breakpoints that allowed each localization are reported as short vertical lines distributed along each horizontal line. Each sequence has been localized in chromosome regions defined by at least two breakpoints; the only exception is represented by the localization of the AAGAG repeats within region h24-h25 (see text). The thin horizontal lines below the lower diagram indicate the maximum physical size of the fertility factors and 1983; BONACCORSI et al. 1988). In the lower row, thick the thicker lines below them their minimum physical size (GATTIand PIMPINELLI lines indicate the loop forming regions (BONACCORSI et al. 1988). KER and APPELS 1982). T h e X heterochromatin also contains low amounts of AACAG and AATAT repeats but these sequences are restricted to the periand centromeric areaof the X chromosome (HILLIKER APPELS1982; LOHEand ROBERTS1988). None of the X;Y translocations used in the present work has an X breakpoint falling near the centromere(see Table 1); thus, X-Y translocations could be used to map these repeats along the Y chromosome without significant interference from the centric materialof the X . For each sequence, agross localization was obtained by using three Y autosome translocations [T(Y;2)G74, T(Y;2)B242 and T(Y;2)P8] whose breakpoints approximately divide the Y chromosome intofour equal sections (see Figure 1). Once its overall distribution was determined, each sequence was finely mapped by using a series of breakpoints that further subdivide the Y. In general, each sequence was mapped to chromosome regions defined by two breakpoints. In some cases, the examination of particularly elongated prometaphasechromosomespermittedamore precise localization of a sequence within these regions. Examples of hybridization on complete metaphases carrying Y autosome translocations are shown in Figure 2. Figure 3 shows the cytological definition of the breakpoints of three Y chromosome rearrangements

by sequential staining with Hoechst 33258 (Figure 3, a-c) and N-banding (Figure 3, d-f) and illustrates the method used for the localization of a sequence along a Y region definedby three translocation breakpoints. Localization of AAGAG repeats: T h e AAGAG sequence is the main component of the 1.705 satellite DNA; it is very abundant, accounting for about 5.6% of the haploid genome of D.melunoguster (LOHEand 1986). This sequence maps to multiple sites BRUTLAG along the Y and, in different amounts, to the heterochromatin of all the chromosomes of the complement 1981; LOHEand (STEFFENSEN, APPELSand PEACOCK ROBERTS1988; see also Figure 2). In situ hybridization on T(Y;2)G74 shows the presence of labeling on both the Y-distal and Y-proximal elements of this translocation. The Y-distal element of G74 exhibits a single cluster of silver grains near the translocation breakpoint while its Y-proximal element exhibits grainson two sites, one close but notadjacent to the breakpoint, and the other on the short arm. Silver grains are also present on both sides of the breakpoint of T(Y;2)B242 (Figure 4c). However, in this case it is the Y-distal element which exhibits two clusters of grains, one located near the breakpoint and the other in the distal part of the long arm. The Y-proximal element of B242 appears to be heavily

Y Chromosome Satellite DNAs

labeled only on the short arm, showing the chromosome segment h15-h20, comprised between the breakpoint and the distal end of the nucleolar constriction, completely devoid of grains. Accordingly, the Y-proximal element of TCy;2)P8 is labeled onlyon the long arm, while its Y-distalelement shows a heavy labeling throughout its length. Together these data indicate that the labeling is present only on three of the segments defined by G74, B242and P8;the B242P8 segment, which comprises regions h15-h20, does not appear to contain AAGAG sequences. The remaining three segments of the Y, each showing at least one main site of hybridization with this sequence, have been further dissected using a series of suitable breakpoints. The Y breakpoints used and the results of this analysisare reported below (see also Figure 1). No AAGAG repeats are present in regions hl-h2 as shown from the lack oflabeling on the Y-distal elements of both TCy;2)R50 , and T(Y;3)G42 (Figure 3g). A heavy labeling is present on the Y-distal elements of both TCy;2)D19 (Figure 3h) and T(X;y)V24 (Figure 3i), indicating that the AAGAG repeats are located in region h3. The Y-proximal element of V24 exhibits a heavy labeling site quite close to its breakpoint (Figure 3i); the Y-distal element of G74 also showsa heavy labeling site close to its breakpoint. Together these observations demonstrate the presence of the AAGAG sequences somewhere within regions h4-h6. Neither the Y-proximal element of G74 nor the Ydistal element of TCy;2)P57 exhibit labeling close to the breakpoint; this provides evidence that no AAGAG sequences are located in regions h7-h9. The Yproximal element of P57, on the other hand,appears to be heavily labeled just at its breakpoint. Labeling is alsoobservedclose to and on both sidesof the breakpoints of T(X;y)El and T(Y;2)B92 (Figure 4a). The Y-distal element of Tfy;3)B223 is labeled close to the breakpoint, but no consistent labeling is apparent near thebreakpoint of the Y-proximal element, which carries regions h14-h25. Thus, AAGAG repeats appear to be located in region h l O-h13 but no clear evidence of their presence in region h14 has been obtained. These results also indicate that theAAGAG sequence is located in region h 10,but do not permit a fine mapping of this sequence within region h l 1h13. The data are consistent with a localization ofthe AAGAG sequence in region h l 1 or h l 2 or both. As mentioned above, no AAGAG repeats are present in the Y chromosome segment comprised between the breakpoints of B242 and P8,while multiple sites of labeling are observed along the remaining portion of the shortarm (regions h21-h25). The breakpoints of T(Y;2)P8, T(Y;2)A162(Figure 2a) and T(Y;2)J165 all fall in region h21, in the proximal, middle and distal third, respectively. The Y-distal elements ofthese

181

translocations carry regions h2 1-h25 and are heavily labeled in each case. The Y-proximal element of P8 is not labeled whilethe proximal elements of both A1 62 and J165 showheavylabeling, indicating that the AAGAG sequence is localized in region h2 1. The Y-distal element of J165 shows a heavy labeling sitewhich,in particularly elongated chromosomes, appears to be separated from its breakpoint and from an additional site of labeling located more distally on the short arm. Twosites of labeling are still apparent on the Y-distal element of T(Y;3)R119 (Figure 4e), whereas onlyone site of labeling is left on the Y-distal element of TCy;2)J163 (Figure 4g). Thus, tandem repeats of the AAGAG sequence are present in region h23 and in an additional site comprised between the middle of region h24 and the end of the short arm. Since no breakpoints are available along region h24h25 a more refined localization of the AAGAG sequence within this interval was precluded. In conclusion, AAGAG repeats were mapped to five separate sites along the Y, corresponding to regions h3-h6 and h O-h 1 1 3 of the long arm and to regions h2 1, h23 and h24-h25 of the short arm. Localization of AAGAGAG repeats: The 7-bp sequence AAGAGAG is a minor component of the 1.705 satellite DNA. Repeats of this sequence, which are located on the Y chromosome and on the heterochromatin of the second andthird chromosomes, account for about 1.5% of the haploid genome of D. melunoguster (LOHE and BRUTLAC 1986; LOHE and ROBERTS1988). I n situ hybridizationanalysisusing G74, B242 (Figure 4d) and P8 showed the presence of two main sitesof hybridization of the AAGAGAG sequence along the Y chromosome. One site is located on the long arm, distal to the breakpoint of G74, and the other on the short arm, distal to the breakpoint of P8. The use of four additional breakpoints on the long arm and four on the short demonstrated arm a partial coincidence betweenthe location of this sequenceand that of the AAGAG repeats. The labeling pattern of R50, G42, Dl9 and V24 demonstrated that AAGAGAG repeats are restricted to region h3 of the long arm and do not extend into regions hl-h2 or h4-h6. In addition, none of the Y-proximal elements of P8, A162, J165, R119 (Figure 4f) and J163 showed a consistent labeling at the breakpoints, whereas all the Y-distal elements ofthese translocations are clearly labeled. The AAGAGAG cluster of the short arm appears therefore to map between the breakpoint of J16.3, at the junction between region h23 and h24, and the distal end of the short arm. Localization of AAGAC repeats: The AAGAC sequence is a component of the 1.686 satellite and comprises about 2.4% of the haploid genome. It maps to multiple sites along the Y and to the heterochromatin of the second chromosome (LOHEand BRUTLAG

182


-

FIGURE2.-Examples of in situ hybridization on metaphase plates carrying different Y autosome translocations. The numbers identify each chromosome of the complement and the arrows point to the translocation breakpoints. YP and YD indicate the Y-proximal and Y-distal elements of each translocation. a: T(Y;2)A162, translocation breakpoints in region h21 of the Y chromosome and in 2L (31EF), hybridized with the AAGAG probe. The heterochromatin of all the chromosomes appears heavily labeled. In particular, the Y-proximal element shows a heavy signal at the translocation breakpoint and multiple sites of labeling on the long arm; the Y-distal element shows a heavy labeling on


1986; LOHEand ROBERTS1988). Repeats of this sequence are present in the regions distal to the breakpoints of G74 (hl-h7) and P8 (h21-h25) and in the interval comprised between the breakpoints ofG74 and B242, (regions h7-hl5) but not in the segment defined by the breakpoints of B242 and P8, corresponding to regions h 15-h2 1. For the fine mapping of the AAGAC sequence within these regions 11 additional breakpoints were used. The hybridization patterns on R50, G42, Dl9 and V24 demonstrate that the AAGAC repeats are absent from regions hl-h2, abundantly present throughout region h3 and scarcely present within regions h4-h5. The results obtained using P57, B92 (Figure 4b) and E l clearly indicate that the AAGAC repeats present in the middleof the long arm are restricted to region hl0. Finally, the labeling patterns on P8, A162 and J165 and J163 indicate the presence of two hybridization sites on the short arm: one in region h21 and the other between the breakpoint of J163 (Figure 4h) and the terminus of the short arm, somewhere within region h24-h25. Localization of AATAT repeats: The AATAT repeats represent the major component of the 1.672 satellite DNA. They account for 3% of the haploid genome and map in the heterochromatic portions of all the chromosomes of the complement with a principal localization in the 4 and the Y (LOHEand BRUTLAG 1986; LOHE and ROBERTS1988). AATAT repeats are contained in all the four segments defined by the breakpoints ofG74,B242 and P8. Further analysis of the location of the A A T A T repeats was carried out using 10 additional breakpoints on the long arm and two on the short arm.The hybridization patterns observed on R50 (Figure 5a) and G42 strongly suggest that region hl-h2 contains the AAT A T repeats, while region h-3 is devoid of these sequences. The whole region comprised between the breakpoints of V24 and P57 appears heavily labeled with the AATATsequence. Moreover, T(X;Y)T13 and G74 (Figure 5b) both exhibit a quite strong signal on each sideof their breakpoints, indicating thatthe AATAT sequences are located in region h4 and in regions h7-h9.However,they do not prove their localization in region h5-h6, since TI3 carries a deletion of this region, and do not permit a decision on whether they map throughout the entire h7-h9 region or only to aportion of it.

183

Neither the Y-proximal element of P57 nor the Ydistal element of B242 show any labeling located close to their breakpoints (Figure 5c). Thus, region h10h14 does not appear to contain A A T A T sequences. The Y-proximal elements ofB242 (Figure 5c) and T(X;Y)F12 show a heavy labeling close to their breakpoints; the Y-proximal elements of both T(Y;3)B115 and T(X;Y)Fl5 are slightly labeledat their breakpoints, while no grains are observed near the breakpoint of the Y-proximal element ofT(Y;2)H121. Conversely, the Y-distal elements of these translocations consistently show silvergrains at their breakpoints. Together these observations strongly suggest that AATAT sequences are mainly clustered within region h15, still present in region h16 and absent from regions h 17 and h18. The Y-distal element of T(Y;2)A77 and the Y-proximal elements of P8, J165 and J163 (Figure 5d) donot exhibit any labelingnear their breakpoints, indicating that noAATAT sequences are located in regions h19-h23. On the otherhand, the Y-distal elements of P8, J165 and J163 (Figure 5d) show a rather heavy labeling suggesting that the localization of A A T A T repeats on the short armis distal to the breakpoint of J163, somewhere within region h24-h25. Localization of AATAG,AATAC,AATAAAC and AATAGAC repeats: All these sequences were clonedas minor components of the 1.672 satellite DNA (LOHEand BRUTLAG1986), each amounts to only 0.1-0.5% of the D. melunoguster genome (LOHE and BRUTLAC1986) and maps to unique sites along the Y chromosome (A. R . LOHE, A. J. HILLIKER and P. A. ROBERTS unpublished). Tandem arrays of the AATAG repeats are mainly located at a single site of the Y chromosome and on the heterochromatin of chromosome 2 (LOHE and ROBERTS 1988). The AATAG sequence hybridizes only with the Y-distal element of G74 and with the Y-proximal element of V24 and is thus restricted to region h4-h7. A more precise localization of the AATAG sequence within this region was obtained using T13. Only the Y-distal element of thistranslocation, carrying regions hl-h4, is labeledwith the AATAG probe, mappingthese repeats to region h4. However, since the Y-proximal element of T13 carries a deficiency that encompasses regions h5-h6, we cannot exclude that some AATAG sequences are also present in the deleted region. The AATAC sequence is exclusively located onthe

both the second chromosome heterochromatin and the Y short arm. b and c: T(Y;2)G74, breakpoints in region h7 of the Y chromosome and in 2L (34B), hybridized with the A A T A T (b) and the AATAC sequences (c). In b, the Y-proximal element of this translocation appears heavily labeled at the translocation breakpoint and atadditional sites on both the long and the short arm; the Y-distal element shows a strong signal extending from the translocation breakpoint to most of the long arm. Note the heavy labeling of the fourth chromosomes and of the tip of the attached X s . The pericentromeric area of the third chromosome is also labeled while the second chromosome heterochromatin appears completely devoid of grains. In c, after hybridization with the AATAC probe only the attached-XY and the Y-distal element of the translocation are labeled at a site located on the long arm, distal to the breakpoint of G74. Thus thehybridization is restricted to a single site of Y'..

184


FIGURE3.-Mapping the AAGAG sequence to region h3. The six upper panels (a-f) show the reciprocal elements of three translocations sequentially stained with Hoechst 33258 (a-c) and N-banding (d-f). The three lower panels show the same translocations after in situ hybridbation with the AAGAG sequence (g-i). T h e arrows point to thetranslocation breakpoints and the numbers alongthe Y chromosome 1983). a, d, g: T(Y;3)C42, breakpoint at the distal end of region h3 indicate cytological landmarks (cf. Figure 1 and GATTIand PIMPINELLI (a, d). The Ydistal element shows hybridization only on the third chromosome heterochromatin (g, left), while a heavy signal is observed at the translocation breakpoint and atmultiple sites of the Y-proximal element (g, right). b, e, h: T(Y;2)D19, breakpoint in the middle of region h3 @, e). The Ydistal element shows a site of hybridbation coincident with the translocation breakpoint (arrow) and an area of heavy labeling on thesecond chromosome heterochromatin (h, left). T h e Y-proximal element is labeled at thetranslocation breakpoint, in the middle of the long arm and on the short arm (h, right). c, f, i: T(X;y)V24, breakpoint between regions h3 and h4 (c, f). The Ydistal element is labeled at the translocation breakpoint (i. left). A weaker signal is observed near the translocation breakpoint of the Y-proximal element which also shows two additional sites of hybridi~ationin the middle of the long arm andon the shortarm (i, right).

Y chromosome (LOHEand ROBERTS1988).It maps to a single site located between the breakpoints of V24 and G74 (Figure 2c). None of the reciprocal elements of T13 is labeled with this sequence. Thus, the AATAC repeats map to regions h5-h6 which, as mentioned above, are deleted in T13. The AATAAAC repeats map to region h22, in the interval comprised between the breakpoints of J165 and RI 19, while the

AATAGAC sequence is localized in region h10, between the breakpoints of E l and P57. DISCUSSION

In the present study we have finely localized eight satellite DNAsequences along the Y chromosome. Several improvements in the satellite mapping strategy compared with previousstudies were essential for

Satellite Y Chromosome

185

DNAs

a

b

d

VL

w

f

VL

4

FIGURE4.-h situ hybridbation of the AAGAG (a, c, e, g), AAGAC (b, h) and AAGAGAG (d, f) sequences on different Y autosome translocations. The arrows point to the translocation breakpoints. a, b: T(Y;2)R92,breakpoint in region h 1 1 (see Fig. I for the localization of this and the following breakpoints), hybridized with the AAGAG (a) and AAGAC (b) sequences. The Y-distal element shows the same pattern of labeling after hybridization with either of these sequences (a and b, left). The Y-proximal element of this translocation is labeled at its translocation breakpoint after hybridi~ationwith the AAGAG sequence (a, right) but not after hybridization with the AAGAC sequence (b, right). c, d: T(Y;2)B242, breakpoint in region h14, hybridized with the AAGAG (c) and AAGAGAG (d) sequences. In (c, left) the Y-distal element of this translocation shows a site of heavy labeling at the translocation breakpoint and a second site of hvbridization located more distally. In (d. left) only a distal site of hybridi~ationis observed, while no grains are present at the translocation breakpoint. Note that the Yproximal element of this translocation shows a heavier labeling of the short arm with the AAGAG (c, right) than with the AAGACAG (d, right) sequence. e, f T(Y;3)R119, breakpoint at the proximal end of region t123, after hvbridi7~tionwith the AAGAG (e) and AAGAGAG (f) sequences. In(e) the Y-proximal element (left) shows a clear site of labeling at the translocation breakpoint, distal to thenucleolus organizer constriction (NO), and multiple sites of labeling on the long arm, while the Y-distal element (right) shows two close but distinct sites of labeling. In (f) only a distal region of the Y-proximal element (left) is labeled, and a single site of hybridization is present on the Y-distal element (right). g, h: T(Y;2)J163,breakpoint at the distal end of h23, afterhybridbation with the AAGAG (g) and AAGAC (h) sequences. In both cases the Y-proximal element shows a rather heavy labeling close to the translocation breakpoint and multiple sites of hybridization on the long arm (g and h, left), while the Y-distal element appears labeled at a stngle site close to the translocation breakpoint (g and h. right). Note the heavier labeling of the second chromosome heterochromatin observed after hybridization with the AAGAG sequence (g. right).

186


4

.

0

a

b

FIGURE5.-In sifuhybridization of the AATATsequence on different Y-2 translocations. The arrows point to thetranslocation breakpoints. a: T(Y;2)R5O, breakpoint in region h2. The Ydistal element (left) shows a site of labeling close to the translocation breakpoint while the Yproximal element (right) shows multiple sites of hybridization both on the long and on the shortarm. b T(Y;2)G74, breakpoint in region h7. T h e Y-distal element (left) is labeled at its translocation breakpoint and at an additional site located more distally. The Y-proximal element (right) is labeled at the translocation breakpoint, near the centromereregion and on the short arm. c: T(Y;2)B242, breakpoint in region h14. T h e Ydistal element (left) shows a heavy labeling only on its distal portion, while the Y-proximal element shows an area of heavy labeling comprised between the translocation breakpoint and thenucleolar constriction (NO) and an additional site of hybridbation on the short arm. d: T(Y;2)J163, breakpoint at the distal end of region h23. T h e Y-proximal element (left) does not show any labeling along the entire region comprised between the translocation breakpoint and the nucleolar constriction (NO), while two main sites of hybridization are evident near the centromere and in a rather distal portion of the long arm. The Ydistal element (right) is clearly labeled right at the translocation breakpoint.

the generation of a high resolution mapof the Ylocatedsatellites. First, w e usedcloned probes that contain a single sequence repeated in tandem for the entire length (LOHEand BRUTLAC1986) ratherthan probes from gradient-purified, bulk satellites.Second, different satellites wereaccurately mapped using stringent hybridization conditions that are specific for each of the satellite repeats, to avoid cross hybridization betweentheseclosely related sequences (LOHEand BRUTLAC1987a). Finally, we mapped the satellite sequences with respect to translocation breakpoints. Using 16 Y autosome and 5 X-Y cytologically determinedtranslocations(GATTI and PIMPINELLI 1983; BONACCORSI et al. 1988)a ratherfine mapping of each sequence was possibleevenin the absence of extremely elongated chromosomes, since for every translocation analyzed witha given probe we only had to determine on which side of the breakpoint the hybridization occurred. In this way most of the satellitesequencescouldbeunambiguouslyassigned to one or more heterochromatic blocks. This permits us to relate the molecular composition of the Y regions to their banding patterns and their functional properties. Relationship between satellite sequence distribu-

tion and chromosome banding: Three types of regions can be basically distinguished along the Y after staining with quinacrine, Hoechst and N-banding (6 with the diagram in Figure 1): (1) Hoechst and quinacrine brightly fluorescent and N-banding negative regions (hl, h2, h4, h6, h8, h9, h15, h17, h22 and h24); (2) Hoechst and quinacrine dully fluorescent and N-banding negative regions (h7, h l 1, h13, h19 and h20); (3) Hoechst and quinacrine nonfluorescent and N-banding positive regions (h3, h5, h10, h12, h14, h21, h23 and h25). It has been suggested that this cytochemical heterogeneity reflects the different base composition of the highly repeated DNAs contained in the three classes of regions. In particular, fluorochrome-bright blocks have been associated with the 1.672 AT-rich satellite DNA while N-banded regions have been suggested to co-map with the relatively GC-rich 1.705 satelliteDNA (GATTI,PIMPINELLI and SANTINI1978; PIMPINELLI, SANTINIand GATTI1978; APPELSand PEACOCK 1978; STEFFENSEN, APPELS and PEACOCK 1981; GATTIand PIMPINELLI 1983). The high resolution mapof the Y chromosome satellite DNA sequences reported here shows that in somecases a given sequence could be mapped to


homogeneously stained blocks defined by 2 or 3 breakpoints, while inothers, due to the lack ofsuitable breakpoints, it was mapped to asegment composed of two differently stained blocks(see Figure 1). The results obtained on the homogeneously stained regions clearly show that the AT-rich sequences from the 1.672 satellite DNA are almost exclusively localizedinHoechst bright regions while the N-banded regions always contain one or more sequences from the relatively GC-rich1.705 and 1.686 satellite DNAs. The only exception is represented by region h16, that is Hoechstnegative and appears to accommodate some A A T A T repeats. In this respectit must benoted that theresolution of our cytological method does not permit us to exclude that the breakpoint of F15 falls in the very proximal part of region h15; the Y-proximal element of this translocation could carry a very small proportion of the Hoechst bright region h15, not detectable by banding methods, which could be responsible for the labeling observed after hybridization with the AATAT sequence. Based on these results we can make someinferences about the localizationof satellite DNAsin regions which contain differently stained heterochromatic blocks. For example, the Hoechst bright region h24 andthe N-banded region h25together havebeen shown to contain the AAGAG and AAGAGAG sequences of the 1.705 satelliteDNA, the AAGAC sequence of the 1.686 satellite and the AATAT sequence of the 1.672 satellite DNA (Figure 1). An obvious suggestionis that region h24 is similar to the other Hoechst bright regions and contains the AAT A T sequence, while region h25 contains the AAGAG, AAGAGAGand AAGAC repeats like the other N-banded regions. The situation of region hlO-h13 with respect to the localization of the AAGAGsequence is much less clear. A likely possibility is that the AAGAG sequence is contained only in region h10 and h12. However, we cannot exclude that these repeats are located, perhaps in small amounts, also in regions h l l and h13. A puzzling problem is represented by region h4h6, a brightly fluorescent block which accommodates a smallN-band (h5) and contains five repeated sequences belonging to three different satellite DNAs (see Figure 1). Three of the five sequences havebeen mapped throughout region h4-h6 since the lackof breakpoints that further subdivide this interval made it quite difficult to resolve the sequence distribution within these regions (the only breakpoint available is that of T l 3 , which, however, carries an additional deficiency of region h5-h6). These three sequences are the AAGAG and AAGAC sequences typical of N bands and the AATAT sequence usually located in the Hoechst bright regions. Thus it appears likely that the first two sequences map to the N-banded region h5 while theAATAT repeats are locatedin the

187

Hoechst bright regions h4 and h6. The two minor 1.672 repeats (AATAG and AATAC) that exclusively map to these regions appear to be located on the two sides of the breakpoint of T 1 3 , in region h4 and h6, respectively (see Figure 1). However, due to the deficiency carried by T13, we cannot exclude that some of these sequences are also locatedsomewhere within region h5. The presence of AATAC repeats exclusively in region h5-h6 is rather surprising, since quantitations of these repeats showed that they comprise nearly 10% of the Y (A. R. LOHE,A. J. HILLIKER and P. A. ROBERTS, unpublishedresults).Cytologically, the entire region h4-h6 spans less then 10% of the Y chromosome and nevertheless it contains four different satellitesequences in addition to the AATAC repeats. The possibility exists therefore that the degree ofcompactionof the DNA contained in this particular region is higher than in other regions of the Y chromosome. No satellite sequences have been localized in the Hoechst bright regions h17 and h 18,in regions h l 9 and h20 and in the N-banded region h14. Region h20, which corresponds to the nucleolus organizer constriction, contains the ribosomalDNAcistrons (BONACCORSI, PIMPINELLI and GATTIunpublished). Satellite sequences which have not yet been identified could be contained in the remaining regions. Alternatively, they couldbe organized differently from the rest of the Y, accommodating long stretches of nonrepetitive or middle repetitive DNA. Together theresults reported above clearlyindicate that there is a good correlation between N-bands and 1.705 repeats and between brightly fluorescent regions and 1.672 sequences.However, heterochromatic blockscontaining different combinations of satellite repeats exhibit identical banding patterns. Thus the molecular basis for banding does not appear to depend solely on DNA content; otherfactors, like the presence of specific proteins which can interact with various repetitive DNAs in similar ways, might play a major role in determining the response to banding techniques. Satellite DNA and Y chromosome fertility factors: The Y chromosome of D. melanogaster carries six complementation groups required for male fertility (KENNISON1981; HAZELRIGG et al. 1982; GATTIand PIMPINELLI 1983). Four fertility factors havebeen mapped to the long arm (kl-5, kl-3, kl-2and k l - I ) and two to the short arm (ks-I and ks-2). Moreover, the kl-5,kl-3 and ks-I fertility factors wereshown to possess extremely large physicaldimensions. These three loci are defined by a series of noncomplementing breakpoints and deficiencies distributed over chromosomal regions containing up to 4000 kbof DNA(GATTI and PIMPINELLI 1983; BONACCORSIet al. 1988). The availablecytogenetic data permitted the mapping ofk1-I, kl-2and b - 2 but not an estimation

188


of their size. The localization of the fertility factors, different, fully fertile stocks (HALFER1981). Thus, as results from the extensive analysis performed by region h4-h6 could be a site of accumulation of closely GATTI and PIMPINELLI (1983), is reportedonthe related repetitive DNAs, which, at least in some popbottom of Figure 1. ulations, is dispensable. More recently it has been shown that primary sperThe regions that correspond to the non-loop-formmatocyte nuclei of X/Y males of D. melanogaster exing geneskl-2 and ks-2 also contain satellite sequences. hibit three giant lampbrush-like loops formed by the ks-2 co-maps with 4 different repeats (AAGAG, AAY chromosome (BONACCORSI et al. 1988). These strucGAGAG, AAGAC and AATAT), while kl-2 co-maps tures are absent from X/O males and consist of three with the AATAGAC, AAGAG and AAGAC repeats. conspicuous skeins thread-like of material which begin No evidencefor asubstantial presence of any of these to develop in young spermatocytes, grow throughout satellite sequences within kl-1 (region h14) has been spermatocyte development and reach their maximum obtained in the present analysis.In general, no specific size in mature spermatocytes; they disintegrate just association between a satellite sequence and a given before the first meiotic division. Deficiency and breakfertility factor can be observed. The same sequence point mapping studies showed that the loop-forming often maps to different fertility factors and each fersites map withinthe kl-5, kl-3and ks-1 fertility factors. tility region is characterized by a specific combination In particular, region h3, which spans about one third of satellite sequencesrather than by an homogeneous of kl-5 is responsible for the formation of the kl-5 array of a single type of repeats. Moreover, both the loop, while region h2 1, which comprises one third of loop-forming and the non-loop-forming segments of ks-1, forms the ks-1 loop (Figure 1). The loop-forming the kl-5 and ks-1 factors contain large amounts of site of the kl-3 fertility factor is coextensive withregion satellite sequences. h7-h9 which corresponds to the minimum physical It is currently very difficultto propose an hypothesis et al. sizeofthislocus (Figure 1 and BONACCORSI for the biological role of the satellite DNA contained in the non-loop-forming fertility factors and in the 1988). A comparison of the map position of the fertility non-loop-forming regions of kl-5 and ks-1. Less diffifactors with the molecular map ofthe Y chromosome cult is to envisage a possible function for the satellite repeats contained in the loop-forming regions. The Y constructed in the present workclearlyshows that chromosome loops are giant nuclear structures conthese loci correspond to large blocks of satellite resisting of a DNA axis with laterally attached growing peats(see Figure 1). The kl-5 locus contains four transcripts in turn associated with large amounts of different satellite DNA sequences; the loop-forming proteins encoded by the autosomes and/orthe X site of kl-5 (region h3) accomodates the AAGAG, chromosome (BONACCORSI et al. 1988; for the strucAAGAC and AAGAGAG sequences, while the kl-5 ture of the Y loops of D. hydei see HENNIG1985 and non-loop-forming region h 1-h2 contains AATAT reLIFSCHYTZ 1988). I n situ hybridization experiments peats. Similarly, four satellite sequences are located on testes preparations have recently shown that the within the ks-1 locus. The AAGAG and AAGAC 1.686-AAGAC sequence is actively transcribed along sequences map to the loop-forming region h2 1; the et al. 1990). The the kl-5 and ks-1 loops (BONACCORSI ks-1 non-loop-forming regions h22 and h23 contain transcripts are accumulated on the loops and do not the AATAAAC and AAGAG repeats, respectively. appear to migrate to thecytoplasm; theyare degraded Only AATAT repeats have been mapped to the klwhen the loops disintegrate prior to meioticmeta3 loop-forming region h7-h9, which corresponds to phase I. Togetherthese observations ledus to suggest the minimum physical size of this fertility factor. Acthe hypothesis that these structures play a structural tually, the physical size of kl-3 could exceed region rather than a coding role during spermatogenesis, h7-h9 and possibly include part of region h4-h6. providing the structural framework for the compartUnfortunately, there are not currently available mentalized accumulation of non-Y-encodedproteins. breakpoints within the h4-h6 interval that permit a better definition of the distal limit of this locus (GATTI The recent finding that thekl-3 loop binds a tektinS. BONACCORSI and M. GATTI, like protein (C. PISANO, and PIMPINELLI 1983; BONACCORSI et al. 1988). Thus manuscript in preparation) gives further support to the kl-3 fertility factor might also contain a non-loopthis hypothesis and indicates that some of the proteins forming portion corresponding to a region (h4-h6) bound to the loops are sperm-specific polypeptides to which appears to contain at least fivedifferent satellite be utilized later in development during sperm differsequences (see above). In this respect it is interesting entiation. An alternative hypothesis about the nature to note that region h4-h6seems to be the only Y of the Y chromosome fertility factors has been put chromosome block exhibiting a certain degree of cyforward by GOLDSTEIN, HARDY and LINDSLEY (1982) tological polymorphism. Ananalysis carried out on kl-5 and kl-3 genes who observed that mutations in the mitotic chromosomes of 16 stocks of D. melanogaster lead to the simultaneous absence of two different high demonstrated the presence of partial or complete (M, 300,000) and of molecular weight polypeptides deletions of these regions of the Y chromosome in two

Satellite Y Chromosome

the outer dynein arms of the peripheral doublets of the axoneme. They interpreted these results as indicating that the kZ-? and kl-5 fertility genes contain the coding sequences for the axonemal dyneins of Drosophila (GOLDSTEIN, HARDYand LINDSLEY 1982). In this respect, we would liketo point out thatour results do not exclude the possibility that the kt-3 and kE-5 polypeptides observed by GOLDSTEIN,HARDYand LINDSLEY are encoded either by a segment of the loop or by a region just outside the loop. Similarly, their results do not exclude the possibility that, in addition to coding for dyneins, the Y loops fulfill a protein binding function. We wish to thank M. GATTIand P. HARTE forhelpful discussion and critical reading of the manuscript. This work has been s u p ported in part by a grant from Fondazione Cenci Bolognetti, and also by the Commonwealth Scientific and Industrial Research Organization, Australia.

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in Drosophila hydei. Advan. Genet. 23: 179-234. HILLIKER,A. J., and R.APPELS, 1982 Pleiotropic effects associated with the deletion of heterochromatin surrounding rDNA on the X chromosome of Drosophila. Chromosoma 86: 469490. KENNISON, J. A., 1981 The genetical and cytological organization of the Y chromosome of Drosophila melanogaster. Genetics 9 8 259-548. LIFSCHYTZ, E.,1988 The developmental program of spermiogenesisin Drosophila: a genetic analysis. Int. Rev. Cytol. 1 0 9 21 1-258. LINDSLEY, D. L., and E. H. GRELL,1968 GeneticVariations of Drosophila melanogaster. Carnegie Inst. Wash. Publ. 627. LINDSLEY, D. L., L. SANDLER, B. S. BAKER,A. T . C. CARPENTER, R. E. DENELL, J. C. HALL,P. A.JACOBS,G . L. G . MIKLOS, B. K. DAVIS,R. C. GETHMANN, R. W. HARDY, A. HESSLER, S. M. MILLER,H. NOZAWA, D. M. PARRY andM. GOULD~OMERO, 1972 Segmental aneuploidy and the genetic gross structure of the Drosophila genome. Genetics 71: 157-184. LIVAK,K. J., 1984 Organization and mapping of a sequence on the Drosophila melanogaster X and Y chromosomes that is transcribed during spermatogenesis. Genetics 107: 61 1-634. LIVAK,K. J., 1990 Detailed structure of the Drosophilamelanogaster Stellate genes and their transcripts. Genetics 124: 303316. LOHE,A. R., and D.L. BRUTLAC,1986 Multiplicity of satellite DNA sequences in Drosophila melanogaster. Proc. Natl. Acad. Sci. USA 83: 696-700. 1987a tdentical satellite DNA LOHE,A.R., and D. L. BRUTLAG, sequences in sibling speciesof Drosophila. J. Mol. Biol. 194 161-170. 1987b Adjacent satellite DNA LOHE,A. R., and D.L. BRUTLAC, segments in Drosophila melanogaster: Structure of junctions. J. Mol. Biol. 194: 171-179. LOHE,A. R., and P. A. ROBERTS,1988 Evolution of satellite DNA sequences in Drosophila, pp 148-186 In Heterochromatin: Molecular and Structural Aspects, edited by R. VERMA. Cambridge University Press, Cambridge. MEYER,G . F., 0. H E Sand ~ W. BEERMANN, 1961 Phasenspezifische Funktionsstrukturen in Spermatocytenkernen von Drosophila melanogaster und ihre Abhangigkeit vom Y Chromosom. Chromosoma 12: 676-716. PEACOCK, W. J., D. BRUTLAG, E. GOLDRING, R. APPELS,C. W. HINTONand D. L. LINDSLEY,1973The organization of highly repeated DNA sequences in Drosophilamelanogaster chromosomes. Cold Spring Harbor Symp. Quant. Biol. 38: 405-4 16. P. DUNSMUIR, E. S. PEACOCK W. J., A. R. LOHE,W. L. GERLACH, DENNIS and R. APPELS,I977 Fine structure and evolution of DNA in heterochromatin. Cold Spring Harbor Symp. Quant. Biol. 42: 1121-1 135. RITOSSA, F., 1976 The bobbed locus, pp 801-846 in The Genetics and Biology of Drosophila, Vol. lb, edited by M.ASHBURNER and E. NOVITSKI. Academic Press, London. STEFFENSEN, D.L., R. APPELS and W.J. PEACOCK, 1981 The distribution oftwohighly repeated DNA sequences within Drosophila melanogaster chromosomes. Chromosoma 82: 525541. STERN,C., 1929 Untersuchungen uber Aberrationen des Y-chromosoms vonDrosophila melanogaster. 2. Indukt. Abstammungs. Vererbungsl. 51: 253-353, Communicating editor: V. G . FINNERTY