Proc. Natl. Acad. Sci. USA Vol. 90, pp. 5737-5741, June 1993 How Y become genetically inert ( structure/ miranda/sienced Lcp /retrotransposons/ degeneration) MANFRED STEINEMANN*t, SIGRID STEINEMANN*t, AND FRIEDRICH LOTTSPEICHt *Institut fur Genetik und Mikrobiologie, Universitat Munchen, Maria-Ward-Strasse la, D-8000 Munich 19, Federal Republic of Germany; and tMax-Planck-Institut fur Biochemie, Genzentrum, Am Klopferspitz, D-8033 Martinsried, Federal Republic of Germany Communicated by M. M. Green, February 16, 1993 (receivedfor review January 11, 1993)

ABSTRACT We have investigated the mechanistic aspects inally autosomal genes, now present on a degenerating Y of inactivation of the major larval cuticle genes chromosome. (Lcpl-4) in Drosophila miranda during Y chromosome evolu- tion. The Lcp genes are located on the X2 and neo-Y chromo- MATERIALS AND METHODS somes in D. miranda but are autosomaily inherited in all other Drosophila investigated so far. In the neo-Y chromo- Cloning and DNA Sequencing of the Lcp Region of X2 and some all four Lcp loci are embedded within a dense cluster of Y Chromosomes of D. miranda. Cloning and standard DNA transposable elements. The X2Lcpl-4 loci are expressed, while techniques were carried out according to ref. 16. The strategy the Y chromosomal shows reduced and for cloning and sequencing the Lcp region from the X2 and Y Lcp3 only activity chromosomes ofD. miranda has been detailed (9, 15). DNA the Lcpl, Lcp2, and Lcp4 are completely inactive. Our results and protein sequence alignments were performed using MAC- suggest that Lcpl and Lcp3 loci on the degenerating Y chro- MOLLY (Soft , Berlin) or DNASIS and PROSIS (Pharmacia) mosome of D. miranda are silenced by neighboring transpos- alignment programs. able elements. These observations support our assumption that Isolation and Separation ofthe LCPs. LCPs from single late the first step in Y chromosome degeneration is the successive third-instar larvae were prepared according to a published silencing of Y chromosomal loci caused by trapping and protocol (17), with modifications. Several cuticles prepared accumulation of transposons. in this way were extracted simultaneously. After centrifuga- tion the supernatants were used for gel electrophoresis. The Regulated and efficient gene expression is based on a struc- gels were overlaid with 0.075 M Tris citrate, pH 8.6. Standard tured chromosome organization. Interactions between chro- nondenaturing 8.5% polyacrylamide slab gels were run in 0.3 mosome structure and gene expression are most obviously M borate buffer, adjusted to pH 8.6. exemplified in genes adapted to a euchromatic or a hetero- Protein Microsequencing of the Electrophoretically Sepa- chromatic environment. Rearrangements that result in trans- rated LCPs. LCPs separated by nondenaturing gel electro- of such genes into a novel context-e.g., from a euchro- phoresis were electroblotted onto siliconized glass-fiber matic to a heterochromatic one-can lead to a change in sheets (Glassybond, Biometra, Gottingen, F.R.G.) and expression pattern (1, 2). stained with Coomassie blue (18). Protein-containing bands The Drosophila miranda is characterized by a were excised and sequenced by using a 477A sequencer pair of heteromorphic chromosomes in the male that are equipped with an on-line 120A phenylthiohydantoin analyzer still evolving from a pair of initially homologous . (Applied Biosystems). This exceptional situation is due to the translocation of one Constructs and P Element-Mediated Germ-Line Transfor- of the autosomes to the Y chromosome, resulting in a neo-Y mation. DNA fragments of interest were inserted into the chromosome and a monosome (neo-X), designated X2 (3-6). polylinker of the pCaSpeR vector (19) by standard cloning Both chromosomes are undergoing an evolutionary process techniques. For the Y chromosomal Lcpl and Lcp3 loci we of chromosome remodeling, each along a different pathway prepared two types offragments, A and B. The Y Lcp3 locus was cloned together with the complete flanking retroelement (7, 8). TRIM and part ofLcp2 (Y3A). In Y3B we removed the TRIM The larval cuticle protein genes (Lcpl-4) are located on the element. The Nsi I fragment (Y1A) encompasses the 5' X2 and Y chromosomes in D. miranda (9, 10), while they are flanking ISY1-3 insertion sequences (see Fig. 3). For the Y1B autosomally inherited in the two sibling species Drosophila fragment we deleted the ISY1-3 sequences. pseudoobscura and Drosophila persimilis (here we follow the As an internal control for expression we designed hybrid locus designation Lcp of ref. 11). They are expressed in the constructs in which Lcpl genes were fused to the LX2/3 epidermal cells of late third-instar larvae (12, 13) and repre- construct. The Hl(YlA;X2/3) construct contains the Y1A sent a set of genes which encode the larval cuticle , fragment cloned into the Pst I site ofthe LX2/3 construct. In LCP1-LCP4 (in the original literature CP1-CP4; for consist- the H2(YlB;X2/3) construct we cloned the Pst I and Bgl II ency, we suggest LCPs). On comparing the DNA structure of sites of the Y1B fragment into the Pst I and BamHI sites of the Lcp region from the two chromosomes ofD. miranda we the LX2/3 construct. As an autosomal reference gene we observed insertions, deletions, and a large duplication asso- used the BamHI fragment (P1) containing Lcpl from D. ciated with the loci on the Y chromosome (14, 15). The pseudoobscura. The P1 fragment was cloned in the BamHI densely clustered insertions were identified as retrotrans- site of the LX2/3 construct to give H3(P1;X2/3). The inte- posons and transposon-like DNA elements (10, 15). grated fragments are indicated in Fig. 3. Constructs were We have used Lcpl-4 as test genes to address the question injected into homozygous Ore- of the impact of these DNA insertions and heterochromati- gon-R w snw embryos, together with a helper , A2-3 nization processes on the regulation and expression of orig- (20). The constructs were integrated into the Drosophila

The publication costs ofthis article were defrayed in part by page charge Abbreviation: LCP, larval cuticle protein. payment. This article must therefore be hereby marked "advertisement" tPresent address: Institut fur Molekulare Genetik, Grisebachstrasse in accordance with 18 U.S.C. §1734 solely to indicate this fact. 8, D-3400 Gottingen, F.R.G. 5737 Downloaded by guest on October 3, 2021 5738 Gehetics: Steinemaiin et al. Proc. Natl. Acad. Sci. USA 90 (1993)


LCP 1 NPPVP.. LCP2 'we PVSLCP...... 1 4 GVAHV. LCP 1 GVAHV... LCP 1 _' GVAHV..

LCP 3 NANVE.. VAPVS... LCP2 _U~ VAPVS... LCP 2 _gp0 VAPVS.. LCP 2 '4mM' LCP 4 iI*- NENPE...

NENAE.. LOP 3/4 LCP 5 " LCP 3/4 _ NENAE... LCP 3/4 _wNENAE... NENAE... NENAE NENAE..

LCP 5 41 LCP 5 qupi LCP 5 4p LCP 6 -.P LCP66 LCP 6 , FIG. 1. Identification ofthe LCPs encoded by Lcpl-4. The proteins were isolated and separated in nondenaturing 8.5% polyacrylamide gels. For illustration the photograph ofa Coomassie blue-stained gel was scanned with a laser scanner and processed for the application ofdesignations without changing the positions ofthe bands. The faint band corresponding to the LCP3 encoded by the Y chromosomal Lcp3 locus is not visible (cf. Fig. 4B). Protein designations and the sequences of the first five N-terminal amino acids (single-letter symbols) are indicated for the corresponding bands. LCP3 (X2 chromosomal) and LCP4 show the same mobility. MEL(OR), D. melanogaster Otegon-R; MIR, D. miranda; PSEU, D. pseudoobscura; f, ; m, male. by P element-mediated germ-line transformation suggesting that they originate from a duplication that oc- (21). Localization of the constructs was performed geneti- curred after the separation of the obscura and melanogaster cally. Balancer chromosomes, FM7c, CyO, and TM3 Sb Ser, groups. It is of interest that the strong D. miranda LCP band used to balance the established lines are described in ref. 11. with highest mobility, LCP6, must also be encoded on the X2/Y chromosome pair, as we consistently have observed RESULTS two LCP6 bands in males. This is obviously due to a in the Y chromosomal Lcp6 locus. The poly- Correlation of Individual LCPs with Lcpl-4 Genes. We morphic band shows the same mobility as the D. pseudoob- isolated the LCPs from single cuticles ofthird-instar male and scura LCP6 (Fig. 1; see also Fig. 4). The position ofthe locus female larvae ofD. melanogaster Oregon-R (w snw, the strain is unknown. Interestingly, the Y chromosomal Lcp6 locus is used for germ-line injections), D. pseudoobscura, and D. expressed normally (see below). miranda. The electrophoretically separated LCPs were elec- Are X2 and Y Loci Both Expressed? We sequenced the X2 troblotted onto a hydrophobic membrane and the N-terminal and Y loci of Lcpl-4. Alignment of the allelic DNA se- amino acids from each of the bands (Fig. 1) were determined quences revealed several point , some of which by microsequencing (18). Based on their sequence relation- lead to amino acid exchanges in the signal peptides and ships with the corresponding proteins from D. melanogaster mature proteins. We used the amino acid substitutions which (details of sequence comparisons will be published else- are closest to the N-terminal ends (Fig. 2) as genetic markers. where), we identified LCP1-4 from D. pseudoobscura and D. Thus, we were able to address the question of whether each miranda (male and female). The X2 chromosomal LCP3/4 of ofthe Lcpl-4 loci on the X2 and Y chromosomes is active or D. miranda and also the autosomal LCP3/4 ofD. pseudoob- inactive. With the exception of the faint band of the Y scura run together as one band and the latter show a slower chromosome-encoded LCP3 (see Fig. 4B; in Fig. 1 the faint mobility. The amino acid sequences deduced for the X2 Lcp3 band is not visible), we found in male LCPs exclusively the and Lcp4 loci reveal that they encode identical proteins, marker amino acids characteristic of the X2 loci. The Y GENE1 S I G NAL PEPTI D E IN VIVO PROTEIN 1 X2 MFKFVMVFAVLGVA AAGVAHVPeHPQVSH4_VGRSEDVHAEVKSEHSD V RADGFDADLLVSN Y MFKFVMVFAVLGLAAAGVAHVPHPQVSH CVGRSEDVHAEVKSEHSDIR A D G F D AD L L V S N 61 X2 SIQQASSGDVHGNIHGSFSWISPEGEIHIVEIKYVADENGYOPVGAVLPTPPPI PEAIARAV Y S IQQASSGDVIHGN HGSFSWI SPEGEHVE IKYVADENGYOPVGAVLPTPPP PEA IARAV 121 X2 AWLEAHPQAPEPVHHSHH Y AWLEAHPQVPEPVHHSHH GENE2 S I G NAL P EPT ID EI N V IVO P ROTEIN 1 X2 MFKFVMVFAVLGLAAAVAPVSRSDDVH1AEVKVLSSDVRADGFDTDLVjVJDNS IQQAASGDI Y LFKFVMGFAVLGLAAAVAPVSRSDDVHAEVKVLSSDVRADGFDTDLVjJIDNS IOQAASGDI FIG. 2. Alignment of the de- 61 X2 HGNAIHGSFSWI SPEGEHVDI KYVADENGYQPVGAVLPTPPP PEA VRALAWLEAHPOAP duced amino acid sequences of the Y HGNAHGSFSWI SPEGEHVDI KYVADENGYQPQGSVLPTPPP PEA IVRALAWLEAHPaAP X2 and Y Lcpl-4 loci. The deduced 121 X2 EHGAHH amino acid sequences of the coding Y EHGAHH regions from X2 and Y of the D. miranda GENE3 S IGNAL PEPTI DE IN VIVO PROTEIN Lcpl-4 genes are 1 X2 MFK LLVCALAALVAANENAEVKELVN[EVNPDGFKTVVSLSDGSASQASGDVHGN DGVF aligned. The alignments start with Y MFKI LLVCALAALVAANENAEVKELVNMJVNPDGFKTVVSLSDGSAQASSGDVHGN DG V F the methionine of the signal peptide which comprises a 16-residue motif. 61 X2 EWVSPEGVHVRVAYKADENGYQPSSDLLPVAPP P E AILKSLAWI iEAHPSKE Y EWVSPEGVHVRVAYKADENGYQPTSDLLPVAPP PEA The border between the mature pro- LKSLAWIOAHPSKE tein and the signal peptide is indi- GENE4 S I G NA l P E P T I D E I N VI VO P R O T E I N cated by a vertical bar. Amino acid 1 X2M F K LL V C A L AA LV A A N E N A EVK E LVNVNGFKTVLS SQASGOGNGF Y M F K L L V CA L A A L V AA N E NA E SRWL PGRS:GV(P RRLCLC PG NCAFRPGR IlS substitutions are shaded. The amino ECOGAGQUG acid exchanges used as markers for 61 X2 EWVSVP.EGVIHV RVA YK A D ENGY QP S S D L L PV ApP IPEAW^lK S ELAW E. PSPKE individual alleles are shaded and Y L V A A A boxed. Downloaded by guest on October 3, 2021 Genetics: Steinemann et al. Proc. Natl. Acad. Sci. USA 90 (1993) 5739 A LCP REGION FIG. 3. Organization of the Lcpl-4 re- D.pseudoobscura 1 kb gion from D. pseudoobscura and D. miranda 3 XBH e R "HI B HH H B R X a and constructs derived from it. (A) Compar- mmn . - 0 0 I ison ofthe autosomal Lcpl-4 region from D. imm. 0- pseudoobscura and the X2 and Y Lcpl-4 1 Gene 2 Gene Gene 4 Gene 3 regions of D. miranda. Double-headed ar- rows indicate unsequenced sections. For ori- D.miranda H entation some restriction sites are included. X2 xsB H HH %X H R Positions of the Lcpl-4 genes and the ori- ON .. a entation of transcription are indicated (large arrows). In the Y chromosomal Lcpl-4 re- GMene I gion the positions of the various inserts 18Y4 (ISYs) are shown. Lcpl is flanked on the 5' 1SY2 ISY_ side by the ISY1-3 insertions. Two large retrotransposons, TRIM (3.1 kb) and TRAM ISY1 N H (about 2.5 kb), have inserted between Lcp2 y XBH N H Bg Bg PH H N X H and Lcp3 and between Lcp3 and Lcp4, re- .I-*-' spectively. In addition Lcp4 is flanked on the 3' side by a large duplication (14). Small Gene 1 jGene 2 Gene G3a4 DY D Y2 DY32 DiDYt deficiencies are indicated by DY. Restriction sites: H, HindIII; R, EcoRI; B, BamHI; X, DY4 DYE B Xho I. The Nsi I (N), Psi I (P), andBgl II (Bg) sites are indicated only for the Y chromoso- CONSTRUCTS mal Lcpl region. (B) Type A and B con- R structs are designed for the analysis of the LP2/3 , , r Mr22MnCHROMOSOME 3 influence of the presence or absence of the 0 R flanking transposable elements on the ex- N7777 CHROMOSOME X2 behavior of the Lcp loci. Cloned LX2/3 R pression fragments are shown below their corre- KAF-F.F,r _C77 CHROMOSOME y LY3A (D sponding positions on the restriction maps MZ CHROMOSOME y (above) and are indicated with black bars. LY3B DNA fragments were cloned into the P ele- ment-derived vector pCaSpeR (hatched N N bars) which utilizes the white (w+) gene as LY1A CHROMOSOME Y visual marker (19). The constructs are ori- ented with the w+ gene on the right. The N N R circled EcoRI sites are artificial. The Y1A -----dzzz HzH1 (Y1 A;X2/3) fragment is inserted in the orientation shown in the restriction map. The hybrid constructs P R 9 H1-3 are based on the LX2/3 construct. The 1, .. CZ22M H2(Y1 B;X2/3) orientation of Y1A in Hl(YlA;X2/3) is the same as in LY1A. The Y1B fragment in B R B® H2(YlB;X2/3) is inverted with respect to s>Zz H3(P1; X2/3) the restriction map above.

chromosomal Lcp2 locus carries an A -- T transversion the tested independent lines containing the Y chromosomal changing the first ATG triplet (methionine) into LY3A construct inserted in different chromosomes showed TTG (leucine) (Fig. 2). Therefore we cannot decide from the an obvious (see below) expression pattern (Fig. 4A). The expression pattern whether nonexpression of the Y Lcp2 LY3A construct includes the Y Lcp3 gene together with the locus is due to an effect on translation or to an effect on complete flanking retroelement TRIM and part of Lcp2. In transcription. Comparison ofthe sequence motifTGTAATG the LY3B construct the TRIM element has been removed flanking the second methionine codon in the signal peptide from the LY3A fragment (Fig. 3). Homozygous transgenic with the Drosophila consensus sequence for translation ini- lines carrying the LY3B construct revealed an additional tiation, (C/A)AA(A/C)ATG (22), makes use of this ATG as band behind LCP4 of D. melanogaster (Fig. 4B). From the an initiation site improbable. In the case of the Y locus of expression pattern ofthe LY3B construct we realized that the Lcp4, a frameshift mutation in the first position of the valine LCP3 encoded by the Y chromosomal Lcp3 locus is charac- triplet in the mature protein (Fig. 2) should lead to a trunca- terized by a different mobility than the X2-encoded LCP3. tion of the protein. However, further sequence work made it Detailed reinspection ofthe D. miranda LCP bands revealed obvious that the Lcp4 locus on the Y chromosome is partially a faint band specifically associated with males (Fig. 4B). The deleted, supposedly due to the insertion and excision of a level of expression of the LY3A and LY3B constructs was mobile element. Northern analysis has revealed that this determined in relation to LCP4 ofD. melanogaster (Table 1). locus is not transcribed (15). Thus in D. miranda males the Analyzed pooled LY3A constructs from independent trans- Y-linked Lcp3 locus is expressed only at a reduced level and genic lines revealed an LCP3 mir/LCP4 mel ratio of <1:20, the Y chromosomal Lcpl, Lcp2, and Lcp4 loci are completely whereas the pooled LY3B lines showed an increased expres- inactive. sion of 1:4 (Table 1). The LYlA construct containing the Y Transformation ofD. melanogaster with Autosomal, X2 and Lcpl gene flanked by ISY1-3 about 1.6 kb 5' upstream (Fig. Y Lcp Genes. Both the LP2/3 construct, which contains the 3) was expressed in low amounts or not at all in different Lcp2 and Lcp3 genes from D. pseudoobscura, and the LX2/3 independent lines (Table 2). In addition we designed a series construct, which has the Lcp2 and Lcp3 genes from the X2 of hybrid constructs in which the LX2/3 DNA was fused to chromosome of D. miranda (Fig. 3), were expressed in the Y chromosomal Y1A or YlB fragment. The latter con- transgenic D. melanogaster flies (Fig. 4A). However, none of tained the Lcpl gene without flanking ISY1-3 (see Fig. 3). We Downloaded by guest on October 3, 2021 5740 Genetics: Steinemann et al. Proc. Natl. Acad. Sci. USA 90 (1993)

A MEL MIR MEL MEL MEL PSEU Table 1. Characterization of D. melanogaster lines transformed LX2/3 LY3A LP2/3 with the Y of Lcp3 from D. miranda LCP1 LCP3 LCP2 mirl -Of LCP1 LCP4 mel LCP3 LCP2 Construct* Line Chromosomet ratio$ LCP4 LCP3/4 LY3A LY3A-23 3rd LY3A-33§ 3rd <1:20 LY3A-43 3rd J <12 LY3A-793 3rd LY3B LY3B-21§ 3rd B MEL MEL MEL MEL MIR MIR LY3B-211 3rd 1:4 LX2/3 LY3A LY31 m t LY3B-34 3rd *See Fig. 3B. LCP1 tIt was determined genetically that the lines carried the D. miranda LCP2 LCP1 Lcp3 allele on their . Lines were made homozygous. LCP3 tLCPs were isolated from third-instar larval cuticles, electrophoret- LCP2 ically separated, and analyzed for the presence or absence of the LCP4 expected bands. The mobility difference between the LCP4 band of LCP3/4 D. melanogaster (met) and the Y chromosomal LCP3 band of D. miranda (mir) is only slight. Hence, we performed protein micro- sequencing after electroblotting onto siliconized glas-fiber mem- branes. Pooled LCP extracts from independent lines (two larval

C PSEU MEL MIR MEL MEL MEL MEL cuticles per line) were electrophoresed and the LCP3 mir/LCP4 mel H3 H2 LYlA Hi ratio in the band was estimated from the phenylthiohydantoin amino acid ratios of the different N-terminal sequences. LCP1 LCP1 §Lines used for Fig. 4. Ilmommosom" LCP2 A*Ai" LCP2 LCP3 LCP1 than was detected with the low LCP1-producing LYlA LCP3/4 LCP4 construct. The autosomal D. pseudoobscura LCP1 on the H3(P1;X2/3) construct showed the expected normal expres- sion, with apseu/mel ratio of 1:1 (Table 2 and Fig. 4C). With

Table 2. Characterization of D. melanogaster lines transformed FIG. 4. LCPs from transgenic D. melanogaster lines. LCPs were with Lcpl alleles from D. pseudoobscura (autosomal) and D. extracted from single cuticles of late third-instar larvae as detailed in miranda (Y chromosomal) Materials and Methods and Fig. 1. For comparison the control samples from untransformed strains are included. MEL, D. mela- Expression* nogaster Oregon-R (w snw), the strain used for embryo injections; MIR, D. miranda MPI; PSEU, D. pseudoobscura. In A and C female LCP1/ cuticles were used. (A) Lcp2 and -3 genes. LCP2 and LCP3 of D. Chromo- LCP2 LCP1 pseudoobscura and the X2-derived LCP2 and LCP3 of D. miranda Construct* Line somet ratio (pooled) were expressed. The Y-derived LCP3A band is missing at its LYlA LY1A-41 2nd 1:1 expected position (star). (B) Lcp3 gene. With the LY3B construct a LYlA-11§ 3rd 1:1 1:7 mir/mel double band was resolved. The additional band expressed from the LY1A-72 3rd 1:1 construct shows a mobility which is slightly slower than LCP4 ofD. melanogaster (arrowhead). A faint corresponding band (arrowhead) Hl(YlA;X2/3)1 H1-217 2nd >1:1 occurs specifically in D. miranda males (MIR m). (C) Lcpl gene. The H1-41§ 2nd >1:1 1:1.5 mir/mel H1-H3 constructs are hybrid constructs carrying the LX2/3 con- H1-221 3rd >1:1 struct as a positive internal reference for expression and various H2(YlB;X2/3)1 H2-11 2nd 2:1 Lcpl-containing fragments. H2-13§ 2nd 2:1 1:1 mir/mel H2-16 2nd 2:1 took the X2 Lcp2 and Lcp3 expression as an internal refer- H3(P1;X2/3)1 H3-11 2nd 2:1 ence (Fig. 4 A and C). The mobility difference between the H3-13 2nd 2:1 1:1 pseulmel LCP1 of D. melanogaster and the LCP1 of D. miranda and H3-15§ 3rd 2:1 D. pseudoobscura is very slight. LCP1 and LCP2 of D. *See Fig. 3B. melanogaster are expressed in comparable amounts (Fig. tDetermined genetically. Lines were made homozygous. 4A). Therefore we used the relative increase in the LCP1 tLCPs were isolated from third-instar larval cuticles, electrophoret- band versus LCP2 (cf. MEL H2 and H3, Fig. 4C) as an ically separated, and analyzed for the presence or absence of the indication of the expression of the introduced Lcpl genes expected bands. The LCP1 bands of D. miranda (mir), D. pseu- (Table 2). In addition we performed protein microsequencing doobscura (pseu), and D. melanogaster (mel) differ only slightly in mobility. As LCP1 and LCP2 of D. melanogaster are present in on the electroblotted LCP1 band. The amino acid ratio comparable amounts (see Fig. 4), we used the relative increase of determined for the N-terminal LCP1 sequences of D. mela- the LCP1/LCP2 ratio (see MEL H2 and H3 in Fig. 4C) as an nogaster and D. miranda (mir/met) or D. pseudoobscura indication of expression or nonexpression of the exogenous Lcpl (pseulmel) was used as additional evidence for expression or alleles. In addition, we performed protein microsequencing after nonexpression of Lcpl-containing constructs (Table 2). electroblotting onto siliconized glass-fiber membranes. Pooled LCP LCP1/LCP2 comparisons indicate expression of the Y Lcpl extracts from three independent lines (three larval cuticles per line) were electrophoresed and the mir/mel and pseulmel ratios in the gene from the Hl(YlA;X2/3) construct in a ratio >1:1. LCP1 band were estimated from the phenylthiohydantoin amino Protein microsequencing revealed a mir/mel LCP1 ratio of acid ratios of the different N-terminal sequences. 1:1.5 (Fig. 4C and Table 2). It is obvious that the Lcpl gene ¶Hybrid constructs (see Fig. 3). All hybrid constructs showed the X2 in the LYlA fragment in combination with the X2 chromo- LCP2 and LCP3 bands. somal Lcp2 and Lcp3 produced more of the Y chromosomal §Lines used for Fig. 4C. Downloaded by guest on October 3, 2021 Genetics: Steinemann et al. Proc. Natl. Acad. Sci. USA 90 (1993) 5741

removed flanking ISY1-3 elements the Y Lcpl gene in the The low-level expression illustrated by the Y Lcp3 gene could H2(YlB;X2/3) construct was as active as the autosomal D. generate a phase of adaptation for the male genome to pseudoobscura Lcpl gene in the H3(P1;X2/3) construct compensate for the ongoing changes in gene dosage. Several (Table 2 and Fig. 4C). These results demonstrate that the Y mutations associated with transposable elements have been chromosomal Lcpl and Lcp3 loci are intact and contain all identified in Drosophila (for review, see ref. 30). Silencing cis-acting sequences necessary for expression. Further, the could hide intact as well as mutated genes-e.g., the Y ISY1-3 insertions and the TRIM element repress the Y Lcpl chromosomal and Lcp3 loci, Lcp4 gene (15). Thus, an accumulation of respectively. mutations, even with deleterious consequences for the orga- nism, could be tolerated by the genome as long as the mutated DISCUSSION genes are silenced. Expression and Nonexpression of X2 and Y D. miranda Lcp We thank Eveline Seifert and Heike Taubert for their kind and Loci. Based on the protein sequence data, besides the Y skillful introduction to the technique of embryo injection, Eva chromosomal Lcp2 locus all other Y Lcp loci should be Praetzel for scanning ofthe proteingels, and Paul Hardy forcritically translatable. Northern analysis of Lcp4 expression showed reading the manuscript. We are especially indebted to Herbert Jackle that the Y chromosomal locus was not transcribed (15). and Hans-Joachim Fritz for their generous support and kind hospi- Therefore, we reasoned that cis-acting effects associated tality. Our work was supported by grants from the Deutsche Fors- with sequences in the vicinity of the Y loci influence tran- chungsgemeinschaft (Ste266/2-1 and Ste266/4-1). scription and are responsible for the silencing. Transgenic D. melanogaster lines transformed with the LX2/3 or LP2/3 1. Tartof, K. D., Hobbs, C. & Jones, M. (1984) 37, 869-878. constructs produce LCP2 and LCP3 in amounts comparable 2. Henikoff, S. (1990) Trends Genet. 6, 422-426. to those expressed from the 3. Dobzhansky, T. (1935) Genetics 20, 377-391. endogenous Lcp genes (Fig. 4A). 4. MacKnight, R. H. (1939) Genetics 24, 180-201. Therefore D. melanogaster can express, process, and cor- 5. Steinemann, M. (1982) Chromosoma 86, 59-76. rectly secrete D. miranda and D. pseudoobscura LCPs. The 6. Steinemann, M. (1984) Chromosoma 90, 1-5. interference on expression of the Y Lcpl and Lcp3 loci 7. Beermann, W. (1955) Biol. Zentralbl. 74, 525-544. exerted by the adjacent insertions cannot be explained by 8. Charlesworth, B. (1991) Science 251, 1030-1033. point mutations or by a simple disruption of the cis-acting 9. Steinemann, M. & Steinemann, S. (1990) Chromosoma 99, sequence motifs in the promoter region. We conclude that the 424-431. ISY1-3 sequences and the TRIM element are involved in 10. Steinemann, M. & Steinemann, S. (1991) Chromosoma 101, silencing the Lcpl and Lcp3 loci on the degenerating Y 169-179. chromosome ofD. miranda. The Lcp3 locus might be still in 11. Lindsley, D. L. & Zimm, G. (1990) Drosophila Inif. Serv. 68, the process 382. of silencing. 12. Snyder, M., Hirsh, J. & Davidson, N. (1981) Cell 25, 165-177. Silencing ofthe Y D. miranda Lcp Loci. We suggest that the 13. Snyder, M., Hunkapiller, M., Yuen, D., Silvert, D., Fristrom, observed silencing of the Y Lcp loci in D. miranda is a J. & Davidson, N. (1982) Cell 29, 1027-1040. consequence of changes in the chromosome structure of the 14. Steinemann, M. & Steinemann, S. (1993) Genetics, in press. degenerating Y chromosome, following the insertion oftrans- 15. Steinemann, M. & Steinemann, S. (1992) Proc. Natl. Acad. Sci. posable elements, especially retroelements. Theoretical con- USA 89, 7591-7595. siderations argue that transposable elements should accumu- 16. Sambrook, J., Fritsch, E. F. & Maniatis, T. (1989) Molecular late in regions of reduced recombination (23). Accumulation Cloning: A Laboratory Manual (Cold Spring Harbor Lab., of a 1.1-kb BamHI repeat at the Y chromosome Plainview, New York), 2nd Ed. has been 17. Fristrom, J. W., Hill, R. J. & Watt, F. (1978) Biochemistry 19, reported (24). In fact the neo-Y chromosome is inherited only 3917-3924. in the male line, which means that it is recombinationally 18. Eckerskorn, C., Mewes, W., Goretzki, H. & Lottspeich, F. isolated from the rest of the genome. (1988) Eur. J. Biochem. 176, 509-519. The observed insertions either disrupt an ordered chroma- 19. Pirrotta, V. (1988) in Vectors: A Survey ofMolecular Cloning tin structure where the domains are insulated from each other Vectors and Their Uses, eds. Rodrigues, R. L. & Denhardt, by border sequences (25) or change the chromosome struc- D. T. (Butterworth-Heinemann, Boston), pp. 437-456. ture by adopting a heterochromatic conformation thought to 20. Laski, F. A., Rio, D. C. & Rubin, G. M. (1986) Cell 44, 7-19. interfere with the expression of nearby genes. The inserted 21. Rubin, G. M. & Spradling, A. C. (1982) Science 218, 348-353. sequences could contain sequence motifs which are recog- 22. Cavener, D. R. (1987) Nucleic Acids Res. 15, 1353-1361. nized by 23. Langley, C. H., Montgomery, E., Hudson, R., Kaplan, N. & -associated proteins (26). As a Charlesworth, B. (1988) Genet. Res. 52, 223-235. consequence a new f3-heterochromatic region could be es- 24. Ganguly, R., Swanson, K. D., Kausik, R. & Krishnan, R. tablished. The chromosome structure which is most enriched (1992) Proc. Natl. Acad. Sci. USA 89, 1340-1344. in transposable elements in polytene chromosomes is in fact 25. Kellum, R. & Schedl, P. (1991) Cell 64, 941-950. the (-heterochromatic chromocenter. Similar to position- 26. James, T. C. & Elgin, S. C. R. (1986) Mol. Cell. Biol. 6, effect variegation in Drosophila (1, 2) and the silencing effect 3862-3872. at yeast HMloci (27-29), a silencing mechanism may account 27. Strathern, J. N., Klar, A. J. S., Hicks, J. B., Abraham, J. A., for the observed inactivity of Lcpl and reduced activity of Ivy, J. M., Nasmyth, K. A. & McGill, C. (1982) Cell 31, Lcp3 on the degenerating Y chromosome of D. miranda. 183-192. In the 28. Nasmyth, K. A. (1982) Cell 30, 567-578. evolutionary process of Y chromosome degenera- 29. Kostriken, R., Strathern, J. N., Klar, A. J. S., Hicks, J. B. & tion, the mechanism of silencing might be the first step and Heffron, F. (1983) Cell 35, 167-174. could even be a prerequisite for the process of degeneration. 30. Green, M. M. (1980) Annu. Rev. Genet. 14, 109-120. Downloaded by guest on October 3, 2021