The X and Y chromosomes of Drosophila melanogaster each contain a cluster ... the simulans Y chromosome, in contrast to their location among rRNA genes on ...
Copyright 0 1990 by the Genetics Society of America
An Unusual Y Chromosome of Drosophila simulansCarrying Amplified rDNA Spacer Without rRNA Genes Allan R. Lohe* and Paul A. Robertst *Department @Genetics, Case Western Reserve UniversitySchool ofhledicine, Cleveland, Ohio 44106, and TDepartment of Zoology, Oregon State University, Coruallis, Oregon 97331
Manuscript received December 23, 1989 Accepted for publication March 5 , 1990 ABSTRACT The X and Y chromosomes of Drosophila melanogaster each contain a cluster of several hundred ribosomal RNA genes (rDNA). A nontranscribed spacer region separates adjacent rRNA genes and contains tandem copies of 240 bp repeats that include the initiation site for RNApolymerase I transcription. We show here that Drosophila simulans, a sibling species of D. melanogaster, contains few, if any, rRNA genes on its Y chromosome but carries instead a large block (3,000 kb or 12,500 copies) of240 bp nontranscribed spacer repeats. The repeats are located at the tip of the long arm of the simulans Y chromosome, in contrast to their location among rRNA genes on the short arm of the Y chromosome of D. melanogaster. The bobbed mutation in homozygous females of D. melanogaster shortens and thins the bristles, owing to apartial deletion of rRNA genes on theX chromosome. The bristles of bobbed/Y males are normal owing to the presence of a full complement of rRNA genes on the Y chromosome. Peculiarly, in bobbed/Y males of D. simulans the short bristle phenotype does not return to normal but is enhanced by the presence of the Y chromosome. We propose that the 12,500 nontranscribed spacer repeats on the Y chromosome are responsible for this biological effect by competition for a protein factor(s) essential for normal levels of rDNA transcription at the X-linked locus.
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S occurs in many species, the rRNA genes of Drosophila are highly reiterated in tandem arrays. In Drosophilamelanogaster, the X and Y chromosomes each contain a cluster of rRNA genes (RITOSSA and SPIEGELMAN 1965) and functional repeat units are 11.5 kb in length (see LONGand DAWID 1980). The 28s and 18s genes in each repeat unit are separated by regions of so-called nontranscribed spacer (NTS) DNA, and rDNA repeat units share a similar arrangement of three sub-repeats in the NTS region, the 95, 330, and240-bp repeats (SIMEONE, LA VOLPEand BONCINELLI 1985). Both species-specific transcription factors and RNA polymerase I are required for the accurate initiation of rDNA transcription. The NTS sub-repeats precede the site of transcription initiation at the 18s gene and appear play to an important role in rDNA transcription. The function of the NTSin transcription of rDNA is currently under investigation in several species. The NTS region separating adjacent rRNA genes in D. melanogaster contains multiple sites identical to the initiation site for transcription of the rRNA precursor by RNA polymerase I (KOHORN and RAE et al. 1982; 1982a; COEN andDOVER1982; SIMEONE MILLER,HAYWARD and GLOVER1983). The initiation site for precursor transcription is distinct from the majorpromoterbut it can sustain a lowlevel of i n vitro (KOHORN and RAE accuratetranscription Genetics 125: 399-406 (June, 1990)
1983). The sequence of the initiation site for precursor transcription occurs once in the 240-bp repeat. Since each rRNA gene containsseven to eight tandem copies of the240-bp repeat,the promoter-like sequence is reiterated up to eight times in the region immediately precedingthestart of transcription. Transcriptsoriginatingfrom240-bprepeatshave been detected at a low level, both in vitro (KOHORN and RAE (1982a,b) and i n vivo (MILLER, HAYWARD and GLOVER1983; MURTIF and RAE 1985). Further evidence for a functional role of 240-bp repeats in RNA polymerase I transcription has come from DNA transfectionexperiments using varying numbers of 240-bp repeats.These have shown that 240-bp repeats stimulate transcription of the precursor RNA in proportion to the number of tandem copies of repeats preceding the promoter (GRIMALDI and DI NOCERA 1988). Deletions of rRNA genes produce phenotypic effects such as shorter and thinner bristles, abdominal etching andslowed development, or lethality in major deletions (bobbed mutation; RITOSSA,ATWOODand SPIEGELMAN 1966).STURTEVANT(1929) reported that the Y chromosome of Drosophila simulans intensifies the bobbed phenotype in X-linkedbobbed mutants. This enhancement is opposite in effect to that seen in the sibling species D. melanogaster, where the Y chromosome suppresses an X-linked bobbed mutant (STERN
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1927). Our examination of NTS and rDNA repeats in D. simulans shows that the Y chromosome carries an unusually large block of 240-bp NTS repeats but carries few, if any rRNA genes. These results suggest thatthe basis forthemoreextremephenotype in bobbed males of D. simulans is an excess of 240-bp NTS repeats on the Y chromosome. MATERIALS AND METHODS DNA clones and probes:The clone A3235 was obtained as described (LOHEand BRUTLAG 1987) using a purified satellite fraction of D. simulans DNA.T o enable preparation of labeled RNA for in situ hybridizations, a Hind111 fragment containing the 568-bp satellite fragment of A3235 was inserted into plasmid pSP64 (Promega) to generate plasmid A3235SP. The 28s probe, plasmid 28SSP, contained a 1.3kb BglII-Hind111 fragment from the 28s gene in pDm238 (ROIHAet al. 1981), introducedbetween the BamHI-Hind111 sites of pGEM-2 (Promega). Single-stranded RNA was synthesized usingATP, CTP, GTP and ['HIUTP (42 Ci/mmol, Amersham; 1 Ci = 37GBq)with SP6 RNApolymerase. The integrity of the labeled RNAwas verified by acrylamide gel electrophoresis. DNA analysis: The insert in A3235 was subcloned into the phagemids pTZ18U and pTZ19U using Hind111 and BamHI, and both strands were sequenced by dideoxy-chain termination (SANGER, NICKLIN and COULSON 1977). DNA was labeled by nick-translation, and Southern hybridization and quantitation of DNA sequences with embryonic DNA (D. melanogaster Oregon R Stanford, andD. simulans, ebony mutant, California Institute of Technology) were carried out as described elsewhere (LOHEand BRUTLAG1986). Quantitations and most Southern hybridizations were carried out using DNA isolated from embryos, or from heads for the comparison of 28s rDNA hybridization patterns in male and females. For the rapid preparation of DNA from heads, from 30 to 300 flies were frozen in liquid nitrogen, vortexed, and heads were purified by sieving (MIKLOS 1984). Heads were ground to a paste and extracted with 400 of extraction buffer (100 mM Tris-HC1, 50 mM EDTA, 0.5% sodium dodecyl sulfate, 0.2 M sucrose, 0.5% diethylpyrocarbonate, pH 9.0) at 65"for 30 min. Sixtymicroliters of 8 M potassium acetate were added and the solution was incubated on ice for 30-60 min. Following centrifugation at 16,000 X g for 5 min, the supernatant was removed to another tube, 2 volumes of ethanol at room temperature were added to the supernatant andnucleic acidspelleted by centrifugation (R. LIFTON,personal communication). The yield of DNA was about 20 ng per head and the DNA was used for restriction enzyme digestion without further purification. DNA was obtained from carcasses (flies lacking heads) by the same procedure. DNA quantitations: Estimation of the kilobase amount of 240-bp spacer repeats on the X and Y chromosomes was carried out by the combination of two sets ofdata. First, the genomic proportion of 240-bp repeats in embryonic DNA was measured by "dot-blot" hybridizations (LOHEand BRUTLAG 1986). This value was converted to a kilobase estimate per haploid genome, assuming a haploid DNA content of 170,000 kb in both D. melanogaster and D. simulans. Second, the proportion of 240-bp repeats on the X and Y chromosomes was determined by quantitative autoradiography. Assignment of kilobase amounts of 240-bp repeats to individual chromosomes must take into account the relative con-
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tributions of different chromosomes in the DNA preparation used for quantitation. Since DNA for genomic quantitations of 240-bp repeats was prepared from equal numbers of male and female embryos, calculations of the chromosomal values in kilobases have been made using the equation, 3/4 X chromosome 1/4 Y chromosome one each of chromosomes 2, 3 and 4 = average haploid chromosome content of the DNA preparation = 170,000 kb et al. 1977). The kilobase estimate of 240-bp (PEACOCK repeats per haploid genome (from quantitative dot-blot hybridizations) can be partitioned between the X and Y chromosomes by multiplying the proportions represented by each chromosome in the DNA preparation with the proportions of 240-bp repeats per chromosome (determined by quantitative autoradiography). In situ hybridization:This was carried out in 3 X SSCet 50% formamide (vol/vol) at 37" as described (PEACOCK al. 1977) except slides were treated with RNase A (2 rg/ml, 30 min at room temperature) before hybridization, and the probe was single-stranded RNA synthesized with SP6 polymerase. Grain counts were made before saturation of the autoradiographic emulsion. The positionsof thethree grains recorded on the Y chromosome of D. simulans using the 28s probe were not similar and these grains were probably due to background. Several grains were also recorded in euchromatic regions of both major autosomes in these same slides.
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RESULTS
240-bp satellite in D. simulans is an NTS sequence: Repeats 240-bp in length are abundant enough to band as a satellite at 1.694 g/ml in cesium chloride gradients of total D. simulans DNA. Short fragments of purified satellite were cloned into pBR322 (LOHE and BRUTLAC1986) and plasmids were isolated that contained DNA sequences representative of the satellite. The insert in one plasmid, A3235, was 568-bp in length and contained pure 240-bp repeats in tan1987). The repeats are 98% dem (LOHEand BRUTLAC homologous to each other. We noticed that these repeats are 89% similar to the 240-bp AluI repeats in the NTS of the rDNA of D. melanogaster, a sibling species. Repeats between the two species differ primarily by single base changes, including both contiguous AluI sites in the D.melanogaster repeat, but the distribution of changes is nonrandom. When changes common to all D. simulans repeats are considered, in one half of the 240-bp sequence there are 20 nucleotide changes betweenthe sequence of both species but the otherhalf shows only three differences (Figure 1). In addition, the 568-bp fragment of A3235 migrates with 800-bp marker fragment in a 5% acrylamide gel, suggesting an unusual conformation of the DNA. Southern hybridizations to total D. simulans DNA probed with 240-bp satellite repeats of A3235 showed labeling of bandscontaining therRNAgenes,as would be expected from hybridization to the spacer sequences flanking these genes. There was also significant hybridization to DNA migrating at limiting mobility (Figure 2A). The hybridization to this region of
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D.melunogusrcr 240-bp spacer CTGTTCTACG ACAGAGGGTT CAAAAACTAC TATAGGTAGG CAGTGGTTGC CGACCTCTCA TATTGTTCAA AACGTATGTG D. simuluns 240-bp satellite A 1 DS A2 -(+AAT"-DS A 3
