Chromosomal homology and molecular organization of Muller's ...

Copyright 0 1997 by the Genetics Society of America

Chromosomal Homology and Molecular Organization of Muller’s Elements D and E in the DrosophiZa repzeta Species Group Jose Maria b,* Carmen Segarrat and Alfredo Ruiz* *Departament de Gene‘tica i de Microbiologia, Uniuersitat Autonoma de Barcelona, 081 93 Bellaten-a, Barcelona, Spain and tDepartament de Gene‘tica, Uniuersitat de Barcelona, 08071 Barcelona, Spain Manuscript received April 2, 1996 Accepted for publication October 22, 1996 ABSTRACT Thirty-three DNA clones containing protein-coding genes have been used for in situ hybridization to the polytene chromosomes of two Drosophila repleta group species, D.repleta and D.buzzatii. Twenty-six clones gave positive results allowing the precise localization of 26 genes and the tentative identification of another nine. The results were fully consistent with the currently accepted chromosomal homologies and in no case was evidence for reciprocal translocations or pencentric inversions found. Most of the genes mapped to chromosomes 2 and 4 that are homologous, respectively, to chromosome arms 3R and ?L of D. melanogaster (Muller’s elements E and D).The comparison of the molecular organization of these two elements between D.melanogaster and D.+eta (two species that belong to different subgenera and diverged some 62 million years ago) showed an extensive reorganization via paracentric inversions. Using a maximum likelihoodprocedure, we estimated that 130 paracentric inversions have become fixed in element E after the divergence of the two lineages. Therefore, the evolution rate for element E is approximately one inversion per million years. This value is comparable to previous estimates of the rate of evolution of chromosome Xandyields an estimate of 4.5 inversions per million years for the whole Drosophila genome.

T

HE interspecific comparison of gene maps provides a far-reaching tool to disclose the patterns and rates of genome evolution (HARTL and LOZOVSKAYA 1994; HARTLet al. 1995). In the genus Drosophila, comparative gene mappinghas a longhistory (STURTEVANT and NOVITSKI1941; PATTERSON and STONE1952) but has become an even more powerful and reliable approach with the development of the in situ hybridization technique (PARDUE et al. 1970) that allows the localization on thepolytene chromosomes of a given species of DNA sequences complementary to those included in recombinant clones obtained from the same or a different species. A large number of genes have already been cloned in D. melanogaster and other Drosophila species (LINDSLEY and ZIMM 1992; FLYBASE1996). In addition, several projects to physically map the genome of Drosophila melanogaster have been undertaken using cosmids (SIDEN-KIAMOS et al. 1991; MALWENO et al. 1995), P1 phages and yeast artificial chromosomes (AJIOKA et al. 1991; SMOLLER et al. 1991; CAI et al. 1995; HARTLand LOZOVSKAYA 1995).Thusan increasing number of DNA clones are being made available for comparative mapping by these molecular studies. I n situ hybridization has already been used to test the chromosomal homologies proposed by MULLER(1940) in several species groups (STEINEMAN 1982; STEINEMAN Corresponding author: Alfredo Ruiz, Departament de Genktica i de Microbiologia, Facultat de Cihcies-Edifici C, Univenitat Authnoma de Barcelona, 08193 Bellaterra, Barcelona, Spain. E-mail: [email protected] Genetics 1 4 5 281 -295 (February, 1997)

et al. 1984; WHITING et al. 1989; LOZOVSKAYA et al. 1993; PAPACEIT and JUAN 1993) and, in a few cases, to comparethe molecular organization of chromosomes among Drosophila species. Using both gene-containing clones and anonymous phages, SEGARR.4 and AGUADF: (1992) and SEGARRA et al. (1995,1996) have compared the molecular organization of Muller’s elements A, D and E between D. melanogaster and six species of the obscura group also included in the subgenus Sophophora. Likewise, KRESS (1993) relying on both genetically mapped markers (GUBENKO and EVGEN’EV 1984) and in situ hybridization data (WHITING et al. 1989; LOZOVSKAYA et al. 1993) has compared the organization of chromosomal elements A and D between D. melanogaster and D. virilis, two species that belong to different subgenera, Sophophora and Drosophila, respectively. The repleta species group, comprising over 90 species, is one of the largest groups in the genus Drosophila (WASSERMAN 1982, 1992).The cactophilic species of this group provide a model system for ecological and evolutionary studies that has proved very fruitful (BARKER and STARMER 1982; BARKER et al. 1990). Many of the species have been cytologically investigated and a fairly complete inversion phylogeny has been built (WASSERMAN 1982,1992). Thekaryotype of most repleta species, including D.repkta, consists of five telocentric chromosomes ( 2 = X, 2, 3, 4 and 5 ) and a dot ( 6 ) chromosome (WASERMAN 1992) and is thus similar to the putative ancestral karyotype of the genus (MULLER 1940; CLAWONand GUEST1986). Apparently, chromo-

J. M. Ranz, C. Segarra and A. Ruiz

282

TABLE 1 Genes previously localized on the polytene chromosomes of the D. repleta group species

position'

Gene" Map

Act5C Act42A/5 7A Act 79B Act8 7E/ 88F Adh br dor JYsPl) Fum buzatii Gapdhl hJiL, Hsp 70B Hsp68 Hsp8? Hsr93D

His-C Lspla Lsplp

PP

5SRNA tra tRNA Ubi-pb3E

Speciesb D. D. D. D. D. D.

hydei hydei hydei hydei buzatii repleta D. repleta D. hydei D. D. hydei D. repleta D. hydei D. hydei D. hydei D. hydei D. hydei D. hydei D. hydei D. buzatii D. hydei D. hydei D. hydei D. hydei

Reference

X( 12B) 5(119D/121D) 4 (78B) 2 (38B/46A) ?(Gla) X(A1) X(A1) 2 (26C/D) X(D3b-e) 5(?) X(A1) 2 (32A) 2 (36A) 4(81B) 2 (48B) "5) 4 (80C3 2 (33A) 2 (33A) 4 (Ala-g) 2 (23B1-2) 4 (93A) Several 4 (90B)

LOUKASand KAFATOS (1986) LOUKASand KAFATOS (1986) LOUW and KAFATOS (1986) LOUKASand KAFATOS (1986) LABRADOR et al. (1990) KOKOZAet al. (1992) KOKOZAet al. (1992) MAIER et al. (1993) NAVEIRAet al. (1986) WOJTASet al. (1992) KOKOZAet al. (1992) PETERSet al. (1980) PETERSet al. (1980) PETERSet al. (1980) PETERSet al. (1984) FITCHet al. (1990) BROCKand ROBERTS(1983) BROCKand ROBERTS(1983) NAVEIRAet al. (1986) LONSO and BERENDES(1975) O'NEIL andBELOTE(1992) TONZETICH et al. (1990) IZQUIERDO et al. (1981) ~

~~

See reference or LINDSLEY and ZIMM (1992) for full name. Some genes were localized in other species as well. 'Notation refers to the map of BERENDES(1963) for D. hydei and to the map of WHARTON(1942) for D. repleta and D.buzzatii. soma1 elements have been conserved within the repleta group; rearrangementsinvolving heterologous chromosomes arerare (only fourcentric fusions have been observed) and pericentric inversions seem entirely absent although paracentric inversions are relatively frequent (WASSERMAN 1992). Probable homologies of the chromosomes of the repleta species with Muller's elements and D. melanogaster chromosomes have been established on the basis of linkage relationships of visible mutants and allozyme loci: X-A-X, 2-E-X, 3-B-2L, 4-DX, 5-C2R and 6-F-4 (STURTEVANT and NOVITSKI 1941; SPENCER1957;HESS 1976; ZOUROS 1976; SCHAFER et al. 1993). The genes localized so far on the polytene chromosomes of the repleta group species (Table 1) in general support these homologies yet some remarkable exceptions have been found (see DISCUSSION). The number of genes so far mapped in the repleta group species is clearly insufficient for a detailed interspecific comparison of the molecular organization of any of the chromosomal elements. As a matter of fact, aside from the work with D. virilis cited above, such comparisonhas not been carried out for any pair of species as distantly related as D. melanogasterand D. repleta, which belong to different subgenera, Sophophoraand Drosophila, respectively. Inthepresent study, 33 DNA clonescontainingprotein-coding genes previously localized (all but two) on chromosome arms 3L and X of D. melanogaster (Muller's ele-

ments D and E ) were used as probes for in situ hybridization onthechromosomes oftwo repleta group species, D. repleta and D. buzzatii. Our results: (1) allow a thorough comparison of the molecular organization of chromosomal elements D and E between two distantly related species groups, melanogaster and repleta; (2) furnish new and useful basic genetic information on the repleta group species; and (3) provide a test of the proposed cytological relationships between D. repleta and D. buzzatii. The chromosomal arrangements of D. repleta drawn by WHARTON (1942) were chosen as the reference for the cytological studies within this group (WMSERMAN 1982, 1992).Therefore, assuming thatthe inversion phylogeny is correct, the localization of a given gene on thechromosomes of D. repleta will automatically predict its map position in the 70 species already studied cytologically. Conversely, the cytogenetic relationships between D. repkta and any group species may be tested by mapping genes on the chromosomes of the two species. Any discrepancy between the predicted and the observed localization of a gene might indicate possible a cytological misjudgment (or a transposition event; see DISCUSSION).As an example, we have performed such a test withD. buzzatiiwhose cytological relationship to D. repkta has recently been revised (RUIZ andWASSERMAN 1993). Moreover, D. buzzatii is polymorphic for several inversions on chromosomes 2 and 4 whose adaptive

283

Chromosome Drosophila Evolution in

significance in natural populations is being actively investigated (RUIZet al. 1986,1991; BARBADILLAet al. 1994; BETRANet al. 1995). Our mapping study aimed also to provided molecular markers located in different positions in relation to the breakpoints of the chromosome 2 inversions that may be useful for futuremolecular studies of this polymorphism. MATERIALSAND

METHODS

Stocks: The following species and stocks were used: two stocks of D. melanogaster, Oregon R and Canton S; one stock of D. virilis from Tokyo (Japan); one stock of D. repkta from Siboney (Cuba);one stock of D. hydei from Carboneras (Spain); and four stocks of D. buzzatii, each homokaryotypic for a different arrangement on thesecond chromosome (2st, 2j, 2jz' and 2jq') and isolated by repeated sib-mating from wild flies collected in Argentina and the Canary Islands (see RUIZet al. 1984). Genes and probes: Twenty-eight recombinant DNAplasmids and four recombinantphages containing totally or partially known protein-coding genes were used as probes (Table 2). An additional recombinant phage,hDsubFC4, whose genic content has not yet been characterized (S. RAMOS~NSINS, unpublished data) was used as well. Most of the 33 clones, kindly provided by different authors, were isolated from genomic or cDNA libraries of D. melanogaster but seven of them came from other Drosophila species (Table 2). All genes but two have been reported to map on the third chromosomeof D.melanogaster (27 on 3R and four on X); Ubi-j80 maps on 2L and the localization of Xpcc is unknown. Polytenechromosomepreparationand in situ hybridization: Third instar larvae were grown at low density in a modified version of David'skilled-yeast culture medium (DAVID 1962). Salivary gland chromosomes suitablefor in situ hybridization were preparedaccordingto MONTGOMERYet al. (1987). Probes were labeled with biotin-1GdUTP by nick translation.Prehybridization, hybridization and detection were carried out as described by MONTGOMERY et al. (1987) using the ABCElite Vector Laboratories kit (SEGARRA and ACUADE1992). Hybridization temperature was in general 25". Micrographs were obtained by phase contrast with a Zeiss Ortoplan Photomicroscopeat 800X magnification using EKTAR-25 KODAK film and a blue filter. Chromosomemaps: Hybridization signals were localized on thepolytene chromosomes using the following cytological maps: D.melanoguster (LEFEVRE 1976), D.virilis (GUBENKO and EVGEN'EV 1984),D. repkta (WHARTON 1942),D.hydei (WASSERMAN 1962; BERENDES 1963) and D.buzzatii (RUIZand WASSERMAN 1993). The maps of D. buzzatii (RUIZand WASSERMAN 1993) and D. hydei (WASSERMAN 1962) are cut-and-paste reconstructions of the D. repkta map (WHARTON1942) according to the sequence of inversions proposed for their respective phylogenies. A maximum likelihood method for estimating the number of fixedinversion differences between twospecies using comparative mapping data: This method was designed to apply to species of the genus Drosophila, such as those dealt with here, in which chromosomal elements have been conserved Its logic is based on maximum likelihood (see DISCUSSION). estimation theory (KENDALL and STUART1967, Chapter 18) and can be easily extended to other types of chromosome rearrangements in a more general framework. Consider a given chromosomal element with m homologous gene markers mapped in two different species. Each pair of consecutive markers delimits a chromosomal segment. Thus we have a total of m + 1 segments if the centromere and the telomere are also considered as homologous markers.

The methodrequires that thephysical length of each segment is known (or can be estimated) in one of the two species (the reference species) and 1, denotes the length, relative to that of the entire chromosome, of segment i. Each chromosomal segment in the reference species is then scrutinized for conservation or not in the other species. Let us assume that theinversion breakpoints are distributed at random along the chromosomal element. If the number of fixed inversion differences between the two species is n, then the total number of breakpoints in the chromosomal element is 2n and the number of breakpoints in each chromosomal segment will approximate the Poisson distribution (if 1, 5 0.1 and 2n 2 30). Accordingly, the probability that a segment of length 1, has been conserved, ix., contains zero breakpoints, is e?", and the probability that it has not been conserved, i.e., contains at least one breakpoint, is 1 - e"% Therefore, the jointprobability of observing a given set of s conserved and m + 1 - s nonconserved segments (the likelihood function) is m+ I

L

=

n (e""') n 1

( 1 - e""!),

(1)

,+I

The value of n that maximizes L is found making zero the first derivative of In L

A general expression for the ML estimator of n was not obtained, but a numericalvalue can be calculated in each particular case solving the likelihood equation (2). The variance of the estimate of n may be calculated as the inverse of the amount of information, I, which is equal to

Finally, the fit of the data to the model may be tested by means of the likelihoodratio (G) test (SOKALand ROHLF 1995, Chapter 17). For a given value of n, each segment has an expected and anobserved probability of conservation and no-conservation. Thus the numberof classes equals twice the number of segments and the test has m degrees of freedom. RESULTS

Control hybridizations: All probes but six were hybridized tothe salivary gland chromosomes of the source species (Table 2) as a control to assure the identity of the genes being mapped and the specificity of hybridization under low stringency conditions. This was not done in the case of Acph-1, Cec, Hsp7OB, M(3)99D, 9 and xDsubFC4 because the controlhybridizations had been already carried out elsewhere (SEGARRA et al. 1996). The results of all control hybridizations were in good agreement with the published cytological localizations of the studied genes with the following noteworthy exceptions. Ubi-j32produced three signals in D. melanogaster on different chromosomes, X(5E), X(63F)and Z(25A).While the first two, resulting from polyubiquitin blocks, agree well with previous results (IZQUIERDO et al. 1981; IZQUIERDO 1994), the third one (Figure l a ) , which must correspond to the localization of the ubiquitin-52-amino-acid fusion-protein gene itself, differs from the reported site, X(97A) (IZQUIERDO 1994). PI-

284

J. M. Ranz, C. Segarra and A. Ruiz TABLE 2

origin of the 33 clones used as probes and reported position of the genes on the cytological mapof D. m l a m g e Clone Insert Gene”

Map position Origin

clone of nature name

Clone

size (kb)

Acr9Ma Acph-1

3R(96A) 3R (99C5-7)

D. melanogaster D. subobscura

FCD 1 XDsubAcph-la

cDNA Genomic

Act8 7E Antp bcd Cec

3R (87E9-12) 3R(84B1-2) 3R(84A1) 3R(99E45)

D. D. D. D.

pK60 H5 bcd hDsubCec6

Genomic Genomic cDNA Genomic

- 15.0

cP0 Dl dsx ems E(sPl) Est-6 fih hb Hsp 7UB kni lbe lbl M(3)99D MtnA Pp1-87B Pros35 Rb97D

3R(90D/E) ?R(92A2) ?R(84E1-2) 3R(88A1-2) JR(96F11-14) 3L (69A1-3) 3R (98D2-3) 3R (85A3-B1) 3R(87C1) 3L (77E1-2) 3R (93D9-E2) 3R(93D9-E2) 3R(99D1-9) 3R(85E10-15) 3R (87B6-12) 3R (89F/90A) ?R(97D)

D. D. D. D. D. D. D. D. D. D.

melanogaster melanogaster melanogaster melanogaster melanogaster melanogaster melanogaster melanogaster melanogaster melanogaster D.melanogaster D. melanogaster D.pseudoobscura D. melanogaster D. melanogaster D. melanogaster D. melanogaster

61.2 c3.2 B2 pE4cDNA12 dSmX PGDW-1 fkh hbDm-X26 232.1 pcEH2.9 lbe XDpseudoRp49-1 cDm51 Ppl-87B pSk-DM35 Rb97D

cDNA cDNA cDNA cDNA Genomic cDNA cDNA Genomic Genomic cDNA Genomic Genomic Genomic cDNA Genomic cDNA Genomic

2.5 4.7 2.6 2.2 1.0 1.8 1.5 2.4 1.0 2.9 4.5 5.0 -15.0 0.5 7.8 0.9 8.0

RpIIl4U

3R (88A10) 3R(87D11-12) 3R( 100A6-Bl) 3L (73A8-9) 3R (97A) 2L(31E) 3L (63F2-4) 3R (89E1-2)

D. D. D. D. D. D. D. D. D.

PGEX-Dl XDsubXdh N4 pIBI/3H pG52H-B pG808 pDM63F pcw.10 pcD1.3 XDsubFC4

cDNA Genomic cDNA Genomic Genomic Genomic Genomic Genomic cDNA Genomic

1.5 -15.0 1.8 3.0 4.1

9 t I1 tra Ubif52 Ubi-j80 Ubij163E UbX

xpcc

-

-

3R (82F)

melanogaster virilis melanogaster subobscura

melanogaster subobscura melanogaster hydei melanogaster melanogaster melanogaster melanogaster melanogaster 11. subobscura

lbl

2.3

- 15.0 7.0 5.0 -

-

5.0 1.8 4.3 14.0

Reference BOSSYet al. (1988) A. NAVARRO-SABATER (personal communication) MANSEAUet al. (1988) HOOPER et al. (1992) BERLETHet al. (1988) S. RAMOS-ONSINS (personal communication) BELLENet al. (1992) VASSIN et al. (1987) BURTISand BAKER(1989) DALTONet al. (1989) KNUST et al. (1987) et al. (1991) PROCUNIER et al. (1989) WEIGEI. TAUTZ et al. (1987) LWAKet al. (1979) NAUBERet al. (1988) et al. (1994) JAGLA JACIAet al. (1993) SECARRA and AGUADE(1993) MARONIet al. (1986) DOMBRADI et al. (1989) HAMSet al. (1989) KARSCH-MIZRACHI and HAYNES (1993) HAMILTON et al. (1993) COMERON and ACUADE(1996) PICNONI et al. (1990) O’NEILand BEIDTE (1992) CABRERA e1 al. (1992) BARRIOet al. (1994) et al. (1981) IZQUIERDO BENDERet al. (1983) HENNINC et al. (1994) S. RAMOS-ONSINS (personal communication)

and ZIMM (1992) or FLYBASE(1996) for full name of’ genes and/or additional information. “See LINDSLEY

et al. (1990) localized tll o n chromosome ?R (100A5,fkB1,2). This signal appeared in our controlas well as a weaker one on the same chromosome (85A) (Figure l b ) . Xpcc, whose localization on the chromosomes of D. rnelanogaster has not been previously reported, gave two signals on chromosome 2R of D.melanogastm, a primary one on 51F and a secondary one in 53C (Figure IC). Negative results In spite of several attempts, seven clones did not produce any detectable hybridization signal on the polytene chromosomes of D. repleta or D. buzzatii. These clones were bcd, cpo, dsx, Est-6, hb, Pros35 and Rb97D. Positive hybridizations to the chromosomes of D. repZetu and D. buzzaeii: Table 3 gives the hybridization sites on the salivary gland chromosomes of D. repleta and D. buzzatii of the 26 clones that yielded positive results. Seventeen of them produced a single and conGNONI

sistent hybridization signalthat must correspond to the site of the gene homologous to that contained in the clone (Figure 2, a-q). The other nineprobes gave rise to two or morehybridization signals, one of them being usually more intense than the rest (Figures 2, r-v, and 3). This primary signal was interpreted as pointing the position of thehomologousgeneinthe two repkta group species. In all cases the primary hybridized band was the same in D. repkta and D. buzzatii, although its relative position along the chromosome may differ due to thefixed paracentricinversions that differentiate the two species (see DISCUSSION). When multiple signals were observed, the additional signals besides the primary one were most likely due to the presence of other copies of the same gene family, pseudogenes or DNA segmentssharingacertain sequence homology with the cloned gene. In somecases a match can be made between these additional signals

Chromosome Evolution in Drosophila

285

TABLE 3 Localization on the salivary gland chromosomes of D. repleta and D. buzzatii of 26 genes mapped by in situ hybridization Gene" Acr96Aa Acph-1 Act87E

Primary signal 2 (E5a) 2(Alj) 2(B4d)

Additional signals

X(D2h-3a)(E4b)b, 2(C5e), 4 ( F l ~ ) ~ , 5(A3a)(A4b) (A5d)

P(F1c-d) Antp Cec 2 (B4a) Dl 2(C%) ems 2 (F5a) 2 (Fla) E(@) @h 2(F2b) Hsp70B 2(D5b) 2(B2c) (C6d) (E6k), 4(F4a) kni 4 (E3a) lbe 2(Alf) lbl 2(Am M(3) 99D 2 (B2a) MtnA (F4h) 2 Ppl-87B 2(F4a-b) X(F4g?)( F ~ c ? )2(E4g-5a), , 5(E4h-5a) RpI7140 (D2h) 2 ry 2 (B2c) tll 2 (C4d) 2 (F3a) tra X(centromere)', 4(A3c) 2(G5~)~ Ubi-fs2 3(Alb) X(C3a), 4(Bld) Ubi-flO 3(F4d-e) Ubi$63E 4(Bld) X(C3a), 3(Alb)' UbX 2(Flc-d) xpcc 4(F3a)" 5(A4g) W.subFC4 2 (EIf) 2 (B4a)

FIGURE1.-Control hybridizations in D. melanogaster. (a) Ubi-fs2, (b) tll, (c) Xpcc. Large and small arrowheads point to primary and additional signals, respectively.

and known genes of D. melanogaster. Four information sources were used in this tentative identification: (1) the relative intensity of the various signals, which was coincident in D.rgbleta and D.buzzatii, (2)the accepted homologies between chromosomal elements of D.melanogaster and D. rgbleta (see DISCUSSION), (3) the additional information from other species of the rgbbta group (Table l ) , and (4) the close proximity of the additional signals to other genemarkers of known localization. A brief comment on the results of the probes producing multiple signals follows. Act87E: Using as probe a clone of the A c d C gene, FYRBERG et al. (1980) localized on the polytene chromosomes of D. melanogasterfive other members of the Actin family: Act42A and Act57A on 2R, Act79B on X, and Act87E and Act88Fon X.LOUKAS and KAFATOS (1986), who used the same clone, observed the same six signals on the homologous chromosomes of severalDrosophila species, including D. hydei (Table 1).The same six genes were also apparent in our control hybridization using a clone containing the Act87E gene (MANSEAU et al.

Chromosome and site (in parenthesis) refer to the cytological map of D. repbta (WHARTON1942). See LINDSLEY and ZIMM (1992) or FLYBASE(1996) for full name. 'Detected in D. repbta only. ' Detected in D. buzzatii only.

1988). On the chromosomes of D.rgbleta and D.buzzatii this clone produced six hybridization signals common to bothspecies plus two weaker signalsdetectable inD. repleta only (Table 3). Based on signal intensity, chromosome homologies and the information from D. hydei (Table 1 ) , the signals 2(B4d), 2(C5e), X(D2h-3a) and 4(Flc) observed in D.rgbleta are probably homologous, respectively, to Act87E, Act88fi AcdC and Act79B in D. melanogaster and the two signals 5(A3a) and 5(A5d) must correspond to Act42A and Act57A. Hsp70: Six other genes sharing significant homology to Hsp 7 0 have been foundin D.melanogaster: Hsp68 in 3R(95D), Hsc70-1 in X(7OC), Hsc70-2 in 3R(87D), Hsc7G3 in X(lOE), Hsc70-4 in X(88E) and Hsc70-5 in 2R(50E) (LIVAKet al. 1978; HOLMGREM et al. 1979; CRAIGet al. 1983; LINDSLEY and ZIMM 1992; RUBINet al. 1993;FLYBASE 1996). Five hybridization signals were observed on the chromosomes of D.rgbleta and D.buzzatii: a strong one in 2(D5b), a medium-intensity one in 2(C6d) and three weaker signals in 2(B2c), 2(E6k)

286

FIGURE 2.-Positive


hybridizations to the chromosomes of (1) D. buzzatii (standard arrangements unless otherwise stated) and

(2) D. repktu. Clones producing a single hybridization signal are as follows: (a) Am96Aa, (b) Acph-1, (c) Antp, (d) Cec, (e) Dl, (f) ems, (8) E(@), (h) jkh, (i) kni (chromosome 4 s ) , (j) Zbe, (k) Ibl, (1) MtnA, (m) M(3)99D, (n) RpIIl40, (0) Ubi-PO,(p) Ubx, (9) y. Clones producing multiple signals are as follows: (r) tra, (s) Ubi-fs2, (t) Ubi-p63E, (u) Xpcc, (v) hDsubFC4. Large and small arrowheads point to primary and additional signals, respectively (some additional signals are not shown).


287

02

FIGURE2.- Continued

and 4(F4a) (Table 3). The first two sites correspond, respectively, to the cytological sites of the Hsp70 and Hsp68genes in D. hydei (Table 1).The signal at Z(B2c)

can be ascribed to Hsc70-2 due its close proximity to 9 (LIVAKet al. 1978). Finally, based on chromosome homology, the other two signals, Z(E6k) and 4(F4a),

288

FIGURE3.-Positive

J. M. Ranz, C . Segarra and A. Ruiz

hybridizations to the chromosomes of (1) D.buzzatii (standard arrangements unless otherwise stated) and

(2) D. repbta. Clones producing multiple signals are as follows: (a) Act87E, (b) Hsp70B, (c) PPI-87B (chromosome 2 j ) , (d) tll. Large and small arrowheads point to primary signals and additionalsignals, respectively (some additional signals are not shown).

Chromosome Evolution in Drosophila are likely homologous to Hsc70-4 and Hsc70-1, respectively. PPI-87B: Using a clone of this gene, DOMBRADI et al. (1989) detected four hybridization signals in D. melanogaster, two on chromosome 3R (87B and 96A) and another two on chromosome X (9C and 13C). The same four signals wereobserved in our control hybridization. On the other hand, five signals were apparent in both D. repleta and D. buzzatii (Table 3). Due to its higher intensity, the signal in 2(F4a-b) is very likely the site of PpI-87B, while the other signal on the same chromosome (E4g), which is in close proximity with Acr96Aa (see DISCUSSION), is that of Ppl-96A. The two signals on chromosome X are likely homologous to the D. melanogaster Ppl-9C and Ppl-l?C genes. tll: Two signals on chromosome 2 were observed in both D.repleta and D. buzzatii. The primary one in F3a must correspond to the site of the gene itself. The secondary and weaker one in C4d is probably homologous to the additional and previously unreported signal detected in our control hybridization (85A). tra: A strong signal on chromosome 4 (A3c) was detected in the two repkta species. This band is coincidental with that previously described in D. hydei by O’NEILand BELOTE(1992) if one takes into account the different notation in the cytological maps of WASSERMAN (1962) and BERENDES(1963). In D. repkta, the tra probe produced two additional signalsin chromosome 2 (G5c) and the centromere of chromosome X (Table 3). Ubi-f52 and Ubip65’E: Using a clone of Ubif52, three signals were detected in D.rqbleta and D. buzzatii, a primary one in chromosome ? (Alb) and two additional on chromosomes 4 and X (Table 3). This result agrees well with our control hybridization on the basis of element homologies between D.melanogasterand D. repleta. When the Ubip63E clone was used, two shared signals were observed in D. repleta and D. buzzatii: one strong in chromosome 4 (Bld) and another less intense in chromosome X (C3a). Thesetwo sites, identical to those of the additional signals observed with Ubi-f52, must correspond to UbipG?E and Ubip5E in D. melanogaster (IZQUIERDO et al. 1981; IZQUIERDO 1994) taking into account element homologies. Xpcc: A single hybridization signal on chromosome 5 (A4g) was observed in both species. In D. buzzatii an additional signal was detected in chromosome 4 (F3a). xDsubFC4: A strong signal on chromosome 2 (Elf) was apparent in both D. repleta and D.buzzatii, whereas a weak one was detected in D.repleta only (Table 3). Molecular organization of chromosome elements E and D in D. repleta and D. buzzatii: Our results allow the unambiguous localization on the polytene chromosomes of D.repleta and D.buzzatii of 26autosomal genes: 20 on chromosome 2, three on chromosome 4, two on chromosome 3 and one on chromosome 5 (Table 3). In addition, theuse ofcomplementary information and other criteria (see above) has led to the tentative identification of another nine genes. Five of these genes are

289

located on chromosome 2 (Act88E Hsp68, Hsc70-2, Hsc70-4 and Ppl-96A), two on chromosome 4 (Act79B and Hsc70-I) and two on chromosome X (Act5C and Ubip5E). If the secondary signal of tll is added, we mapped a total of 36 gene markers. Our information is most complete for chromosomes 2 and4, with 26 and five markers, respectively, whoselocalization is shown in Figure 4. Position of mapped genes in relation to the polymorphic inversions of D. buzzatii: All hybridizations to the D.buzzatii chromosomes described above were carried out using a stock homokaryotypic for the 2 standard (2st) arrangement. Seven probes (Acr96Aa,Antp, Dl, Hsp7OB, Ppl-87B, RpIIl40 and Ubx) produced signals on chromosome 2 near the breakpoints of inversions 2j, 2z3 or 24,which are polymorphic in natural populations of this species (RUIZet al. 1984; RUIZand WASSERMAN 1993). To define more accurately the position of these signals in relation to the inversion breakpoints, additional hybridizations of the seven probes were performed using D. buzzatii stocks homokaryotypic for the relevant arrangements, 2j, 2jz’ and 2j4. The results are summarized in Figure 4. The observation that Dl is located outside of inversion 2j, about eight to 10 bands from its distal breakpoint, agrees with its linkage map position (SCHAFER et al. 1993) and indicates that recombination in this segment is supressed in the 2j/st heterokaryotypes. On the other hand, the location of the signal belonging to Ppl-96A inside inversion 2j but outside inversion 22 shows that the distal breakpoints of these two inversions are not precisely identical as previously reported (RUIZ et al. 1984). DISCUSSION

Conservation of protein-coding nucleotide sequences and gene families: In our study, 26 out of the 33 clones (-79%) hybridized to the polytene chromosomes of D. repkta and D. buzzatii. Only sevenclones did not produce detectable hybridization signals. Sucha high proportion of success is remarkable because D. melanogaster and D. subobscura are only distantly related to D. repleta and D. buzzatii (belonging to different subgenera: Sophophora and Drosophila, respectively) and their divergence time has been estimated at -62 million years (BEVERLYand WILSON 1984; SPICER 1988). An inspection of the list of mapped genes (Tables 2 and 3)shows a wide functional variety of the encoded proteins: there are transcription factors (Antp or lbe) and transmembrane receptors ( D l ) that operate during development, antibacterial proteins (Cec) , ribosomal proteins (M(?)99D),structural proteins (Act87E or Ubip6?E), metal-binding proteins (MtnA) and enzymes (Acph-1 or 9). No clear-cut functional differences between the successful and unsuccessful genes are apparent. Perhaps the most remarkable case is that of the D. melanogaster MtnA probe that contains a 120bp coding sequence (MARONI et al. 1986) but still produced a detectable hybridization signal. Thisshows both

290


-

- - Pgm - - tra Hsp83 Ubi-p63E

3L

- - Ubi-p63E

4

Hsc~O-1

-1 Hsp83 " .

kni

--

Act796

--

H~c70-1

kni Act796

1 DsubFC4 Antp tll*

,MtnA Hsp7OA 1PPI-876 Hsp7OB

= > Act87E ems Rp11140

, \

L

-

2

Act88F

Dl Hsr93D !/be

.

Ibl

fkh ems MtnA Ppl-876 tll*

fkh

Ubx An@ EfSPl) Hsc7G4 Acr96Aa Ppt96A

X DsubFC4

\~

3R

t

a

. =_

Antp

=iqHsc70-4 WPl)

"

Acr96Aa Ppl-96A

- - h DsubFC4 - - lisp70

Hsp70

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Rplll40

Dl Hsp68 Act88F tll Act87E Cec HSc70-2 ry M(3)99D Acph-I Ibl Ibe Hsr93D

D. melanogaster

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D. repleta

a highly conservedcoding sequence and asensitive technique. Our results showconservation not only of the nucleotide sequence of each gene but, in many cases, of the number anddistribution of members of multigene fam-

Rp11140 - - Hsp68 Dl --

"

- - Act88F 111

-

FIGURE 4.-hdization of the 31 gene markers mapped on chromosomes 2 and 4 of D. r q b h and D. bwmtzi. A single chromosome 4 is depicted because the two species are homosequential for this chromosome. The position of three markers (Hsp83,Hr9?D and Pgm) mapped previously by other authors (see Table 1) has been also included. The graph showsalso the position of all the markers on the homologous chrome somes ?R and X of D. melanogaster for comparison. til" indicates the secondary signal of tailless (tu). The relative order of y and Hsc7@2 (and that of Antp and Ubx) in D. v+&a and D. bwzatii is not known for certain. Likewise for the gene pair h a n d Zbl in the three species. The breakpoints of the pol rphic inversions 2j, 2zF oand 2q7 are shown on the D. bmmtii map. To recover the chromosomal segmentsincluded in inversions 22 and 24 (whicharose on a 2j chromosome) segment 2j must be inverted fint.

M(3)99D ry Hsc70-2 Cec - & Act87E ACph-I IbI Ibe

--

I

Hsr93D

D. buzzatii ilies as well. This indicates that these families arose before thesplit of the lineages leading to the Sophophora and Drosophila subgenera and have been not involved in translocations or transposon-mediated movements between chromosomal elements (see below) (LOUKAS

in

Evolution Chromosome

Drosophila

29 1

genes are still together as in the ancestor of the genus and KAFATOS 1986; TONZETICH et al. 1990). In the case or split in the same way as in D. virilis. of the Actin family, for instance, we have detected the Chromosomal homologies between D. melunogaster same six copies found previously by LOUKASand KAand D. repleta: The production of a single signal or FATOS (1986) in D. hydez at the same chromosomal sites a more intense(primary) one among severalsignals (Table 1).The fourmembers of the Ppl family observed allowed the unambiguous mapping of26 autosomal by DOMBRADIet al. (1989) in D. melanogaster were also genes on the salivary gland chromosomes of D. repleta found in D. repleta and D. buzzatii. The Hsp70 gene famand D. buzzatii. In all these cases the results were fully ily seems also preciselyconserved. In D. melanogasterand consistent with the currently accepted chromosomal different species of the obscura group, several Hsp70 homologies and in no case was evidence for reciprocal gene copies are distributed between two chromosomal translocations or pericentric inversions found. Twenty sites (LNAKet al. 1978; MOLTO et al. 1993; SEGARRA et out of 21 genes reported to be located on chromosome al. 1996). In D. buzzatii and D. repleta, the Hsp70 clone 3R of D. melanogaster (Muller’s element E ) were located produced a single and intense primary signal that indicates a clustered arrangement of the several copies at a on the homologous chromosome 2 of D. repleta and D. single site. This organization is similar to that of several buzzatii (Figure 4). The only apparent exception, U b i j52, is probably a case of cytological misidentification: other Drosophila species, including D. virilis, D. hydei our controlshowed that this gene is located on chromo(Drosophila subgenus) and D. lebanonensis (Scaptodrosome 2L of D. melanogaster (Muller’s element B ) and sophiia subgenus), which show a single Hsp70 site (EVon the homologous chromosome 3 of D. repleta and D. GEN’EV et al. 1978; PETEFS et al. 1980; PAPACEIT and JUAN 1993). The most simple interpretation for these buzzatii. The only gene reported on chromosome2L of observations is that the ancestral Hsp70 gene cluster D. melanogaster, Ubi-fSO, was mapped on thehomologous split in the phylogenetic lineage leading to the melanochromosome 3 of D. repleta and D. buzzatii. Likewise, gaster and obscura groups while it still is conserved in the three genes reported on chromosome 3L of D. melaother lineages, includingthatleading to the repleta nogaster (Muller’s element D ) were mapped on the homologous chromosome 4 of D. repleta and D. buzzatii group. Four other members of the Hsp70 gene family, Hsp68, Hsc70-1, Hsc70-2and Hsc7@4,were also detected (Figure 4). Finally, Xpcc turned out to be located on in D. repleta and D. buzzatii. chromosome 2R of D. melanogaster (Muller’s element Twoblocksof tandemly repeated ubiquitin genes C ) and on thehomologous chromosome 5 of D.repleta have been found in D. melanogaster, Ubi+63E and U b i and D. buzzatii. p5E (IZQUIERDO 1994). The same two blocks were deOnly four exceptions to the chromosomal homolotected in D. repleta and D. buzzatii using the D. melanogasgies between D. melanogaster and the repleta group speter Ubip63E clone (IZQUIERDO et al. 1981) or the Ubicieshave beenreported: H i s 4 (FITCHet al. 1990), j52 clone that containsalso the ubiquitin sequence(a- 5SRNA (ALONSO and BERENDES 1975), Lspla and L s p l p BRERA et al. 1992). The site of the Ubi963E block in (BROCKand ROBERTS 1983) and Est-6 (OAKESHOTT et chromosome 4was coincident with itsprevious localizaal. 1990). TheHistone and 5SRNA genes are organized tion in D. hydei (IZQUIERDO et al. 1981). The homeobox as a blockof tandemly repeated copies (ASHBURNER gene family (GEHRING and HIROMI1986; RUDDLE et al. 1989) and this kind of gene often shows nonhomolo1994) is distributed in D. melanogasterin two separated gous locations in Drosophila species (STUART et al. 1981; gene complexes: the Antennapedia complex (ANT-C) STEINEMAN 1982; STEINEMAN et al. 1984; FELGERand at 3R(84B1-2) (KAUFMAN et al. 1990) and the Bithorax PINSKER 1987) perhaps as a result of transposon-medicomplex (BX-C) at X(89E1-2) (DUNCAN 1987).We sucated movements. The location of the Lspl genes is strikcesfully hybridized one gene clone from each complex ing and might perhaps be a case of transposition as to the chromosomes of D. repleta and D. buzzatii. The well (BROCKand ROBERTS 1983). Est-6 is located in D. chromosomal sites of Antp and Ubx were coincident or melanogaster on chromosome 3L (Muller’s element D ) adjacent in both species, a situation also found in D. (PROCUNIER et al. 1991), whereas the presumably hovirilis, another species of the Drosophila subgenus (VON mologous gene Est-1 of D. buzzatii maps to chromosome ALLMEN et al. 1996). In the latter species, the BX-C is 2 (Muller’s element E ) (SCHAFER et al. 1993). This obsplit between the Ubx and Abd-A transcription units; servation has been explained by a rearrangement inUbx is clustered with the ANT-C at one site (24E) of volving chromosomal elements D and E (OAKESHOT~ et chromosome 2, whereas Abd-A and Abd-B reside at anal. 1990). This hypothesis could not be tested directly other site (26D) of the same chromosome (VONALL here because, unfortunately, the Est-6 probe did not MEN et al. 1996). Thus, most likely, a single homeobox yield positive results. However, none of the genes that gene complex was present in the ancestor of the genus are evenly scattered along the 3R chromosome of D. Drosophila, as it is today in other insects like Tribolium melanogaster mapped on the D. repleta chromosome 4 (RUDDLE et al. 1994). This complex split in a different and none of the genes located on D. melanogaster chroway in the two lineages leading to the melanogaster and mosome ?L fell on D. repleta chromosome 2 (Figure 4). the virilis species groups. More work is needed to deterThis makes this hypothesis veryunlikely:only a very mine whether in the r@leta group the eight homeobox small rearrangement wouldhave goneundetected.

292


Thus, the D. buzzatiiEst-1 locus (SCHAFER et al. 1993) might be nonhomologous to the D. melanogasterEst-6 locus (OAKESHOTT et al. 1990). Evolution of chromosomal elements E and D: When the arrangement of the 35 available gene markers located on elements E and D is compared between D. melanogaster and D. repleta (Figure 4), anextensive reorganization within each element becomes evident. This reorganization has surely been producedby a sequence of paracentric inversions fixed in the two phylogenetic lineages leading to D. melanogaster and D.repleta since their common ancestor.Assuming independent origin, the fixation of n inversions requires 2n inversion breakpoints and generates 2n + 1 unbroken chromosomal fragments with an average relative size 1 / ( 2 n + 1).Thus theestimation of theaverage size of unbroken fragments allows an assessment of the number of fixed inversions, and vice versa. The number of paracentric inversions fixed between D. melanogaster and D. repleta in chromosomal element E, for which our information is more extensive (Figure 4), was estimated using two different methods. The comparison of all possiblepairs of contiguous gene markers on this element reveals that only three small chromosome segments have seemingly been conserved: H s r 9 D lbelbl, yHsc70-2, and Ppl-96A-Acr96Aa. The approximate size of these segments in D.'melanogaster is 82, 20 and 92 kb, respectively (BENDER et al. 1983; SPIERER et al. 1983; PETERSet al. 1984; KEITH et al. 1987; BOSSYetal. 1988; GARBE et al. 1989; DOMBRADI et al. 1990;JACLA et al. 1993, 1994). The next closest pair of genes in D. melanogmter is Acph-1 and M(?)99D, distant some 115 kb. These two markers are much further apart(-29 bands) in D. repkla (Figure 4) and, thus, have very likely been involved in chromosomal rearrangements. Applying the method of NADEAUand TAYLOR (1984) to these data, an estimate for the average sizeof conserved fragments of 68 kb was obtained. Given that the DNA content of the euchromatic portion of chromosome ?R is -23,850 kb (HEINO et al. 1994), the total number of conserved fragments is 23,850/68 = 351. This yields 175 inversions fixed in this element during the divergence of D.rqbleta and D. melancgaster. One of the assumptions of NADEAU and TAYLOR'S method is thatthedistribution of gene markers through the chromosomeis random and independent, which may not be true in our case. A more accurate estimate of the number of fixed inversions may be obtained by a maximum likelihood (ML) method(see MATERIALS AND METHODS) that (1) takes into account the actual distribution of markers along the chromosome, (2) makesuse of all the information available (not only the size of conserved segments), and (3) assumes random (Poisson) distribution of the inversion breakpoints. The lengths of the 29 segments in chromosome 3R of D. melanogaster were estimated from the cytological position of the gene markers and the information about the DNA content of each chromosomal

band provided by HEINOet al. (1994). The fit of the data to the model was very good ( G = 6.29; d.f. = 28; P > 0.05). The ML estimate of the number of fixed inversions was n ? SD = 130 ? 56, which gives a value for the average size of unbroken fragments of 23,850/ 261 = 91 kb. The stability of this estimate was checked by recalculating n after omitting from the dataone, two or three gene markers. The average estimate of n after subtracting one marker (27 cases) was 132 +- 14; after omitting two markers (50 randomly chosen pairs) 133 ? 17; and after excluding three markers (50 randomly chosen trios) 138 ? 35. Thus ourestimate seems reasonably robust. Our results allow us to calculate rates of chromosomal evolution. Taking 62 million years as the divergence time between the two subgenera (BEVERLY and WILSON1984; SPICER 1988), we obtain a rate of fixation of inversions on element E of 130/124 = 1 inversion per million years. This value is comparable to the estimate of one X-linked inversion per million years obtained by SEGARRA et al. (1995) from the comparison of D. melanogaster and D. pseudoobscura, but higher than the rateof fixation estimated by NURMINSKY et al. (1995) from a comparison of the region around Adh between D. melanogaster and D. virilis. If the rate of evolution were similar among chromosomal elements, a rate of 4.5 fixedinversions per million years for the entireDrosophila euchromatic genome would be estimated. Cytological phylogeny of D. repleta and D. buzratik D. repleta belongs to the repleta subgroup and arose from the ancestor of the species group (PRIMITIVE I) throughthe fixation of eight inversions: Xu, Xb, Xc, 2 4 2b, 2tK, 2u8 and ?b (WASSERMAN 1992; RLJIZand WASSEW 1993). D. buzzatiz belongs to the mulleri subgroup and arose from the same ancestor through the fixation of four inversions: 2m, 2n, 2 2 and 5g (RUIZ and WASSERMAN 1993). Thus the karyotypes of these two species differ by 12 fixed inversions, seven of them on chromosome 2. Some of these inversions are apparent when comparing theorder of the 28 gene markers available on this chromosome between D. repleta and D. Duzzatii (Figure 4). Thedistally located 2a and 2b inversions are independent of each other andwith respect to the remaining inversions (RUIZ andWASSERMAN 1993). Accordingly, thegenes M(?}99D, 9, Hsc70-2, Cec and Act87E, included within the 2a segment, are oriented in opposite direction in D. repleta and D. buuatii; the same happens with Hsp68and Dlincluded within the 26 inversion (Figure 4). Inversions 2m and 2n are tandemly arranged and share the middle breakpoint while inversion Zz', which occurred on a 2mn chromosome, overlaps both 2m and 2n (RUIZ and WASSERMAN 1993). The genes ems, Mtn,A, Ppl-87E, til (secondary signal) andfih are located within inversion 2n but outside inversion 22 and, as expected, show the opposite direction in the two species (Figure 4). On the other hand, (ibx, Ant$, E($}, Hsc70-4, Acr96Aa, Ppl-96A, kDsubFC4 and Hsp70 are included within both 2n and Zz7 and thus show the


same orientation in D. rqbleta and D. buzzatii (Figure 4). In conclusion, our results are consistent with the proposed cytogenetic relationships between D. repleta and D. buzatii. Nevertheless, more work is needed for a definite corroboration (or falsification) because no markers are currently available for some of the rearranged chromosomal segments. We thank very sincerely all the authors who provided flies, DNA clones and/or relevant information: M. AGUADE, J. M. COMERON, E. JUAN, A. NAVARRO-SABATER and E. RAMOS-ONSINS (Universitat de Barcelona); M.BALLIVET (University of Geneva); M. BELLARD (Institut National de la Sant6 et de la Recherche M6dicale-Centre National de la Recherche Scientifique, Stmbourg); H. J. BELLEN(Howard Hughes Medical Institute, Houston); J. M. BELOTE(Syracuse University); J. A. CAMPOS-ORTEGA(Institutfur Entwicklungphysiologie, Koln); S. CAMPUZANO (Madrid): E. A. CRAIGand W. WALTER(University of Wisconsin); E. C. FRIEDBERG (University of Texas); A. L. GREENLEAF (Duke University); S. R. HAYNES (National Institutes of Health, Bethesda); J. E. HOOPER(University of Colorado); M. IZQUIERDO (Universidad AutBnoma de Madrid); H. JACKLE and M. A. GONZALEZ (Max-Planck-Institut fur Biophysikalische Chemie, Gattingen); P. M. KLOETZEL and R.STOHWASSER(University of Heidelberg); G. MARONI (University of North Carolina); W. J. MCGUINNIS and N. MCGUINNIS (Yale University); H. MORAWIETZ (University of California, San Francisco);R. C. RICHMOND (University of South Florida); L. SANCHEZ(Madrid); and M.WASSERMAN(City University of New York). We also thank M. AGUADEfor encouraging this project and A. BARBADII.~, E. BETRAN,0. CABRE, M. ~ C E R E S ,D. LORENZO, A. NAVARRO, A. NAVARRO-SABATER, S. RAMOS-ONSINS J. V. MARANAS, and J. ROZAS for technical advice and helpful discussion of results. Work supported by grants PB93-0844 and PB91-0245 from the Direcci6nGeneral de Investigaci6n Cientifica y Tecnica(Ministerio de EducaciBn y Ciencia, Spain) awarded toA.R. and Montserrat AguadC, respectively.

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