Chromosoma (Berl) (1990) 99:118-124 C H R O M O S O M A © Springer-Verlag 1990

On the origins of tandemly repeated genes: Does histone gene copy number in Drosophila reflect chromosomal location ? David H.A. Fitch 1, , , Linda D. Strausbaugh 1, and Victoria Barrett 2' **

1 Department of Molecular and Cell Biology, The University of Connecticut, Storrs, CT 06269, USA 2 Department of Biology, The University of Utah, Salt Lake City, Utah, USA

Received June 1, 1989 / in revised form November 1, 1989 Accepted November I, 1989 by M.L. Pardue

Abstract. Widely regarded beliefs about Drosophila histone gene copy numbers and developmental require- ments have been generalized from fairly limited data since studies on histone gene arrangements and copy numbers have been largely confined to a single species, D. melanogaster. Histone gene copy numbers and chro- mosomal locations were examined in three species: D. melanogaster, D. hydei and D. hawaiiensis. Quantitative whole genome blot analysis of D N A from diploid tissues revealed a tenfold variability in histone gene copy numbers for these three species. In situ hybridization to polytene chromosomes showed that the histone D N A (hDNA) chromosomal location is different in all three species. These observations lead us to propose a relation- ship between histone gene reiteration and chromosomal position.

Introduction

The fact that genes which are tandemly repeated often encode important products that cells use in large amounts (such as rRNA or histones) has suggested to some the possibility that high copy numbers of identical genes are required to meet a high demand for these prod- ucts (Jacob et al. 1976; Kunkel and Weinberg 1978; En- gel and Dodgson 1981; Old and Woodland 1984; van Dongen et al. 1984; Kedes 1979). The assumption has been that increased transcription from single gene copies would still be inadequate to meet this demand, therefore higher reiteration levels are required.

The approximate one-to-one mass ratio of histone proteins to D N A required for the formation of chroma- tin (Kornberg 1974) suggests that the demand for

* Present address: Department of Molecular Genetics, Albert Ein- stein College of Medicine, Bronx, NY 10461, USA ** Present address': Department of Botany, Auburn University, Auburn, AL 36830, USA

Offprint requests to : L.D. Strausbaugh

histones is proportional to the size of the genome and the rate at which replication proceeds. Among different urodele species, histone gene reiteration levels are ap- proximately proportional to genome size (Stephenson 1984), but this relationship between histone DNA (hDNA) copy number and genome size does not hold up when additional species are analyzed (Old and Wood- land 1984). It is generally believed, however, that histone gene reiteration levels are in part determined by the high- est rate of D N A replication in the developmental sched- ule of an organism, usually during cleavage stages (Kun- kel and Weinberg 1978; Engel and Dodgson 1981; Old and Woodland 1984; Anderson and Lengyel 1980).

At the low end of histone gene reiteration levels, the genomes in early mammalian embryos generally have small numbers of non-tandemly repeated histone genes ( I ~ 4 0 copies per haploid genome: Yu et al. 1978; Marz- luff and Graves 1984; Stein et al. 1984) and low rates of cell division (one division every 8 12 h: Graves et al. 1985; van Dongen et al. 1984). Even this copy number seems high since theoretical calculations indicate that the highest mammalian cell division rates might require only one to two histone gene copies (Old and Woodland 1984). This relatively high copy number is probably due to the existence of functionally different histone subtypes (Stein et al. 1984; Kim et al. 1987; LaBella et al. 1988). At the high end of the scale, sea urchins contain a large number of tandem histone genes (300-800), presumably to maintain the rapid rate of one division every hour (Kedes and Birnstiel 1971; Hinegardner 1967; Kunkel and Weinberg 1978; Newrock et al. 1978). The identical members of the tandem gene family comprise the early histone gene families; other lower copy number genes encode functionally different subtypes (Lieber et al. 1986; Halsell et al. 1987).

The nuclear division rate in D. melanogaster early embryos is among the highest known: there are 13 divi- sions in 2.5 h and the first 9 divisions occur in 81 min (Anderson and Lengyel 1984). The 100 tandemly ar- ranged histone gene copies (Lifton et al. 1978) are pre-

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sumably needed to mainta in the high rates o f histone gene t ranscr ipt ion k n o w n to occur dur ing embryogenesis or during oogenesis (Ruddell and Jacobs -Lorena 1985). A l though no histone proteins are stored, the oocytes contain a large store o f histone m R N A which is sup- posed to suppor t development up until the time zygotic histone genes are expressed (Anderson and Lengyel 1984). N o tandemly ar ranged genes that encode func- t ionally different histone proteins have yet been de- scribed in Drosophila, a l though an unl inked single copy variant has been identified in D. melanogaster (van Daal et al. 1988).

A l though teleologically satisfying, the not ion that histone gene copy numbers are directly related to devel- opmenta l demands in a simple fashion is difficult to demons t ra te experimentally or theoretically. First, calcu- lations for bo th sea urchins (Kedes 1979; Weinberg et al. 1983) and fruit flies (Anderson and Lengyel 1980) indi- cate tha t actual gene numbers far surpass the theoretical requirements for maximal rates o f t ranscript ion at known developmental stages. Second, it is difficult, if no t impossible, to quant i fy "deve lopmenta l d e m a n d s " . Third, the kinds o f organisms providing the correlat ion between copy numbers and development are so diver- gent (mammals , sea urchins, fruit flies) tha t their physio- logical and developmental demands are likely to be com- pletely different. Wi th these latter points in mind, histone gene reiteration was investigated in species repre- senting three different subradiat ions in the genus Dro- sophila: D. melanogaster (subgenus Sophophora): D. hy- dei (subgenus Drosophila, subradia t ion virilis-repleta); and D. hawaiiensis (subgenus Drosophila, subradia t ion immigrans-hirtodrosophila ( T h r o c k m o r t o n 1975). Sur- prisingly, we found variability in rei teration levels and cytogenetic locations in these relatively closely related species, suggesting a probable role for c h r o m o s o m a l lo- cat ion in high reiteration levels.

Materials and methods

Genomic blot analysis. Genomic DNA from D. melanogaster (cn bw strain obtained from Dr. T. Wright, University of Virginia). D. hydei (a strain obtained from Dr, Hallie Krider, University of Connecticut) and D. hawaiiensis (J14B8 strain from the collec- tion of Dr. J. Dickinsen, University of Utah) whole adult flies or primarily diploid cells from adult heads was extracted by a method described elsewhere (Daniels and Strausbaugh 1986). Initial DNA concentrations were measured using a Perkin-Elmer 554 spectrophotometer with quartz cuvettes. Absorbance scans were taken over a 22~300 nm range. Only samples with sharp peaks near 260 nm were used. A DNA-specific assay (3,5-diaminobenzoic acid fluorimetry) was used for a more quantitative measurement of DNA concentration (Kissane and Robbins 1958). Purified DNA was digested with restriction endonucleases as recommended by the manufacturer of the enzyme (Bethesda Research Laboratories, New England Biolabs or Boehringer-Mannheim). Fragments were separated in 0.8% agarose gels, stained with ethidium bromide, transferred to nitrocellulose (Southern 1975) or Gene Screen Plus, and hybridized to radiolabeled probes prepared by a second strand synthesis using random primers (Feinberg and Vogelstein 1983). Prewashes, hybridizations, and post washes were in 4 x SSC, 0.1% SDS, 1 x Denhardt's solution (Denhardt 1966) and 40% (unbiased

conditions) or 50% (biased conditions) formamide at 37 ° C (Over- ton and Weinberg 1978). Additional washes were at 2 x SSC, 0.1% SDS, and 1 x SSC, 0.1% SDS, at room temperature for 45 min each. (1 x SSC is 0.15 M NaC1, 0.015 M sodium citrate.) Autora- diographs of the filters were made by exposure at - 7 0 ° C with intensifying screens.

In situ hybridization. Polytene chromosomes from larval salivary glands were prepared essentially by the method described by Pardue and Gall (1975) as modified by Hyashi et al. (1978). Probes were nick-translated using Escherichia coli DNA polymerase and all four 3H-labeled deoxynucleotides as precursors (Maniatis et al. 1975); conditions for hybridization and subsequent processing of preparations were as described by Pardue and Gall (1975).

Results

The first objective o f this s tudy was to compare histone gene copy levels in the three species. In order to use quanti tat ive blots to estimate relative histone gene numbers , it was necessary to have a histone gene p robe

A Eco R1

I H1 '~ / H3 H4 H2a H2b H1

A v a l A v a l

I I

B

5 . 0 k b "-~

h m

!iii~iiii

C

h m

~!!,i~iiii,~iiiiiiilili!iiiiiiiiiiiil !!i,! ̧ i~ii~iii!ii!i!i;iiiiiiiii

i !iii, iiiii!iiiiiiiiiiiiiiiiiiii!i ili i ! !!!!ii!iiiiiiiiiiiiiiiiiiiiii

' ;ii!iiiiiiiiiiiiiiiiiiiiiiiiil

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Fig. 1 A-C. Selection of an unbiased probe. The molecular map for a typical histone gene tandem repeat from Drosophila melano- gaster (A), adapted from Lifton et al. (1978). The 5.0 kb major size class in D. melanogaster differs from the 4.8 kb size class by a substitution in the large A - T rich spacer region. The boxes show coding regions with directions of transcription indicated by arrows. AvaI digestion of a 4.8 kb repeat cloned in the plasmid pBR322 was used to produce the desired probe fragment. Approxi- mately equivalent numbers of cloned histone repeat were obtained by digestion of recombinants isolated from bacteriophage libraries of D. hydei (h) or D. melanogaster (m) with BamHI or SstI, respec- tively. Fragments were separated in 0.8% agarose gels, stained with ethidium bromide (B), transferred onto nitrocellulose, and hybridized (C) to the probe as described in Materials and methods

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which hybridized equally well with histone sequences of rather distant species. A probe consisting largely of H4 and H2A coding sequences was chosen since this segment hybridizes quantitatively to equal amounts of cloned histone repeats of D. melanogaster and D. hydei (Fig. 1) under specific conditions of reduced stringency (40% formamide, 37 ° C). It is important to note that this probe, like all others we utilized, is more species specific under conditions of higher stringency. The re- sults obtained under the lower stringency conditions demonstrate that the selected probe will not introduce any bias in comparisons between D. melanogaster and D. hydei; we assume no bias will occur with D. hawaiien- sis. Since D. hydei and D. hawaiiensis are members of the same radiation, they are more closely related to each other than to D. melanogaster. These relationships in- crease the probabili ty that the unbiased conditions will extend to D. hawaiiensis.

This probe was used under unbiased hybridization conditions to whole genome blots f rom the three species under study (Fig. 2A). Equal amounts of whole adult genomic D N A from each species were digested with a restriction endonuclease such that the major repeats of approximately 5 kb resulted in each case. Hybridization to the D N A of D. melanogaster was much greater than to the DNAs of the other two species, suggesting the possibility of more histone repeats in the former. Similar results are obtained when different enzymes are used to digest the D N A of D. hydei into unit repeats (Fitch 1986), indicating that the depicted results accurately re- flect the totality of tandem repeats in these genomes.

An alternative explanation for this result could be that equal numbers of histone repeats in the diploid ge- nomes of the three species are replicated to different extents in non-diploid D N A (Spear and Gall 1973 ; Spear 1977). To test this possibility, equal amounts of genomic D N A prepared f rom adult heads f rom the three species were examined (Fig. 2B). We assume adult head D N A to be essentially diploid - adult heads were not reported to be polyploid in an exhaustive survey of morphological studies of Drosophila tissues (Ashburner 1970). In all cases, the results confirm the interpretation that the D. melanogaster diploid genome contains more tandemly arranged histone genes than those of D. hydei or D. hawaiiensis; for all species, there are greater levels of hybridization to the largely diploid head D N A than to the whole adult DNA, consistent with an under-repre- sentation of histone sequences in total adult D N A which may have a significant component of D N A from non- diploid cell types.

Relative histone gene numbers may be estimated more accurately using a reconstruction with differing amounts of D. melanogaster diploid D N A (Fig. 3). Based on this technique, and assuming 100 copies of histone repeat per haploid genome in D. melanogaster, we estimate that D. hydei contains about 5, and D. haw- aiiensis contains about 20 tandem histone repeat copies per haploid genome. Since the amount of hybridization to 0.40 gg o fD. hydei D N A is the same as that to 0.02 gg of D. melanogaster, we may estimate that there are 5

tandem copies of histone repeat per D. hydei genome (100 copies x 0.02 lag/0.40 gg = 5 copies). Using the same logic, we estimate 20-25 tandem copies per haploid ge- home of D. hawaiiensis (100 copies x 0.08 gg/0.40 lag= 20 copies or 100 copies x 0.05 lag/0.2 g g = 2 5 copies).

Since we have cloned histone genes f rom D. hydei, it is possible to confirm some of these estimates with D. hydei probes. In this set of experiments, conditions of higher stringency for hybridization (50% formamide, 37 ° C) were utilized with a probe fragment f rom the

A B

me hy ha me hy ha

5 . 0 k b ~ -~- 5 . 0 k b

Fig. 2A, B. Different degrees of hybridization of the unbiased probe to equal amounts (1.0 ~tg) of genomic DNA from Drosophila melanogaster (me), D. hydei (hy), and D. hawaiiensis (ha) whole adult flies (A), or primarily diploid cells from adult heads (B). Genomic DNA was digested with either BamHI (me, by), or Hin- dIII (ha) and the resulting fragments separated by electrophoresis in a 0.8% agarose gel and blotted onto nitrocellulose filters. The filters were hybridized and rinsed using the conditions described in Materials and methods. DNA concentrations were determined using 3,5-diaminobenzoic acid fluorimetry. Under the conditions of electrophoresis used, the 4.8 kb-sized repeat normally found in D. melanogaster comigrated with the 5.0 kb repeat

ug DNA

me hy ha i i

6 6 , 5 5 6 6 5 0

5.0 k b ~

Fig. 3. Quantitative reconstruction for copy number estimation. Specific amounts of primarily diploid genomic DNA from adult heads of Drosophila melanogaster (me), D. hydei (hy), or D. haw- aiiensis (ha) were digested with either BamHI (me, by) or HindIII (ha). The fragments were separated by electrophoresis in a 0.8% agarose gel, blotted onto nitrocellulose, and hybridized using the unbiased conditions described in Materials and methods. Due to distortion at the right-hand edge of the gel, the major D. hawaiiensis repeat artifactually migrates much more slowly than the major D. melanogaster and D. hydei repeats

121

A

me hy

B

me hy

C m h m h

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0.10 0.20

Fig. 4A-C. Similar degrees of hybridization of a biased probe to equal amounts of genomic DNA from Drosophila melanogaster or D. hydei. Approximately equivalent numbers of cloned histone repeats were obtained by digestion of DNA isolated from plasmid clones of D. melanogaster (me) in pBR322 or D. hydei (hy) in pUC13 with BamH[. Fragments were separated in 0.8% agarose gels, stained with ethidium bromide (A), transferred to nitrocellu- lose, and hybridized to the SstI-EcoRI fragment of the cloned D. hydei repeat under biased hybridization conditions (B). Under these conditions, the D. hydei probe hybridization signal is 15- to 20-fold greater for its homologous DNA than to the D. melanogaster DNA. C Equal amounts (0.1 or 0.2 ~tg) of genomic DNA from adult heads of D. melanogaster (m) or D. hydei (h) were digested with BamHi and analyzed as described in Materials and methods using biased hybridization conditions

D. hydei repeat (SstI-EcoRI) partly analogous to the probe from D. melanogaster. This fragment is about 1 kb in size and contains all of H4, the 5' end of H3, and adjacent intergenic regions (Fitch 1986). Under these conditions, the probe is biased for its homologous D. hydei genomic repeat, resulting in a 15 to a 20-fold stronger signal to equivalent amounts of cloned repeat (Fig. 4A, B). When this probe is hybridized to equal amounts of digested genomic DNA from heads of D. melanogaster or D. hydei (Fig. 4C), the hybridization signals are equivalent. These results suggest that D. hydei contains 6-7 tandem histone repeats, consistent with the estimates based on hybridizations with the D. melano- gaster probe.

The validity of these conclusions about relative copy numbers is predicated on a number of additional consid- erations. First, comparisons of quantitative blots be- tween species are only valid if equal genome equivalents of the three species have been used. The haploid D. mela- nogaster genome is 1.7 x 108 bp (Rasch et al. 1971); that of D. hydei is 1.8 x 108 bp (Dickson et al. 1971), and that of a very close relative of D. hawaiiensis, D. silvaren- tis is 1 .7x108 bp (Triantaphyllidis and Richardson 1980). Second, the amount of DNA per lane for each species must be exact. DNA in the present experiments was measured using a highly specific and sensitive fluori- metric procedure (Kissane and Robbins 1958). Further- more, ethidium bromide staining and hybridization to single copy sequences confirm the equivalency of D N A amounts (Fitch 1986). Third, digestions must be com- plete for valid comparisons of unit-sized repeats. Since multicopy, tandemly repeated genes yield distinctive par- tial digests (Strausbaugh and Weinberg 1979) and none of these were observed, partial digestions that would erroneously underestimate numbers of tandem genes may be eliminated as possibility. Although our results do not preclude the possibility that non-tandem arrange- ments of histone genes may occur, such arrangements clearly do not represent a major portion of the total number of histone genes. Fourth, there is no reason to suspect that DNA from D. hydei or D. hawaiiensis has any unusual properties that would result in inappropri- ate signal strengths in hybridization experiments. Results of experiments with both the single copy hsrco locus of D. hydei (J. Garbe, personal communication) and the Adh locus of D. hawaiiensis (V. Barrett, personal com- munication) support this notion.

Given these differences in numbers of tandemly re- peated histone genes, it was of interest to determine if there were any differences in the chromosomal locations of histone genes in the species. The result of in situ hy- bridization studies are shown in Figure 5. Squashes of polytene chromosomes from salivary glands from third instar larvae of D. melanogaster (A), D. hydei (B), or D. hawaiiensis (C) were hybridized to the [3H]-labeled, nick-translated, 4.8 kb D. melanogaster histone repeat (large arrowheads). In situ hybridization shows that histone D N A is clearly located adjacent to the quasi- heterochromatic base of chromosome 2L (Spofford 1976) at polytene chromosome position 39DE in D. mel- anogaster (Fig. 5A) in agreement with Pardue et al. (1977). In D. hydei, histone genes are located exclusively in the middle of the euchromatic arm of chromosome 4, at cytogenetic location 80C3-5 (Fig. 5B; see Berendes 1963, for standard D. hydei map). In D. hawaiiensis, the histone genes are also located in a euchromatic inter- val (Fig. 5 C).

These in situ results not only demonstrate the differ- ent chromosomal locations for histone genes in the three species, but also lend support to the conclusions about variable copy numbers. The D. melanogaster and D. hy- dei chromosomes were hybridized with the same probe and had equivalent exposure times. The number of grains over the D. melanogaster histone regions is much

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greater than that over D. hydei, supporting further the contention that fewer h D N A copies are present per hap- loid genome in D. hydei than in D. melanogaster. This supportive evidence must, however, be qualified since we do not know the relative representation of histone D N A following polytenization in the two species.

Fig. 5A-C. Chromosomal localization of histone sequences. Squashes of polytene chromosomes from salivary glands from the third instar larvae of Drosophila melanogaster (A), D. hydei (B), or D. hawaiiensis (C) were hybridized in situ to 3H-labeled, nick- translated, 4.8 kb D. melanogaster repeat (large arrowheads). In addition, the D. hawaiiensis chromosomes shown in the insert to

Discussion

This investigation has shown that while D. melanogaster contains 100-110 tandem histone repeats per haploid ge- nome, D. hawaiiensis has about 20, and D. hydei only about 5 10 copies per haploid genome. In addition, the more highly repeated D. melanogaster histone genes are located adjacent to quasi-heterochromatin while both D. hawaiiensis and D. hydei histone DNAs are located in the midst of clearly euchromatic intervals. In another species, D. virilis, there is also a lower copy number of total histone genes (50) than in D. melanogaster (Domier et al. 1986) and two separate chromosomal lo- cations (Anderson and Lengyel 1984). These observa- tions suggest that some of the widely held ideas about histone reiteration generalized from studies on D. melanogaster are unsupported when data are considered from a broader phylogeny.

It has been part of the dogma about histone gene reiteration that the abundance of histone gene copies is related to the developmental demands of the organism. The argument is rendered less convincing given our ob- servations of variations among copy numbers in these three Drosophila species which have similar developmen- tal times and mechanisms. If not simply related to devel- opmental demands, how then might one explain the striking differences in tandem repeat numbers among the three Drosophila species analyzed here? One possibil- ity is that each of the tandemly arranged histone genes is equivalently active in the three species but that wide ranges in the levels of histone gene expression are tolerat- ed. Alternatively, individual genes in the three species may not be equally active, perhaps due to position ef- fects (Spofford 1976). For example, the close proximity to heterochromatin of the genes in D. melanogaster may render them less active than their euchromatic counter- parts in D. hydei. To achieve similar levels of expression, over evolutionary times, D. melanogaster histone genes may have come to exist in higher copy numbers. Finally, it is possible that the copy number of tandemly arranged histone genes has no relationship to functional demands, but may be a consequence of chromosomal location. That is, copy number increases may be facilitated by genetic mechanisms that maintain highly repetitive se- quences in centromeric regions.

In both of the latter two alternatives, tandem gene families with high copy numbers are viewed as a conse-

C were hybridized to the plasmid 328A (small arrowhead), which contains the 1.6 kb Eco-RI fragment from the D. affinidisjuncta alcohol dehydrogenase (ADH) gene. Bars represent 10 gm

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quence of chromosomal location near classical hetero- chromatic regions. By these models we would predict that such gene families would be associated either cyto- logically with heterochromatic regions or, in cases where cytological localization is not possible, by the identifica- tion of recombinant D N A molecules with adjacent high- ly repeated sequences. In the case of D. melanogaster, r D N A and histone genes are both cytogenetically local- ized adjacent to heterochromatin. In this respect, it is noteworthy that one urodele genome characterized by high tandem reiteration of histone repeats contains histone D N A interspersed with centromeric satellite se- quences (Stephenson et al. 1981). The suggestion that this interspersion is directly related to both high repeti- tion and sequence homogeneity (Stephenson 1984) is strengthened by the observed correlation between tan- dem repetition levels and chromosomal location in Dro- sophila. In another highly repeated, tandemly arranged gene family, the 5S oocyte-type r R N A genes of Xenopus laevis, the genes are located at another chromosomal site associated with highly repeated sequences, the telo- mere (Pardue et al. 1973; Narayanswami and Hamka lo 1986). In tomato, tandemly repeated 5S genes are also located adjacent to heterochromatin at the centromere of chromosome I (N. Lapitan, M. Ganal , and S. Tanks- ley, personal communication). One exception to the het- erochromatic association of highly repeated tandem genes that immediately comes to mind is the 5S gene family of D. melanogaster; however, one cannot rule out the possibility that these genes were associated with heterochromatic sequences at some point in their evolu- t ionary history.

Although a great body of evidence exists that demon- strates altered activities of euchromatic genes placed next to heterochromatin, the possibility that selection for high copy numbers is a consequence of lowered activity of individual genes due to position effects is difficult to prove. Circumstantial evidence for this notion may be found in one example relevant to the specific chromo- somal location of the histones: a white t ransposon in- serted at the base of 2L shows a dramatic position effect (Hazelrigg et al. 1985).

The difficulty of associating gene copy numbers with developmental demands for the products of highly re- peated tandemly arranged genes is further illustrated by recent experiments addressing the function of single r R N A genes inserted by t ransformation into euchromat- ic locations of the chromosome (Karpen et al. 1988). In these experiments, a single r R N A gene in euchroma- tin is sufficient to partially rescue the bobbed phenotypes of delayed development and etched abdomen.

Based on a comparison of histone repeat reiteration levels and chromosomal locations in three species f rom the genus Drosophila, we propose that the relatively high copy numbers in D. melanogaster are largely a conse- quence of their current association with classical hetero- chromatic regions of the chromosome. A number of other examples of heterochromatic locations for tan- demly repeated gene families exist as well. The fact that such a correlation occurs across a wide phylogenetic

range suggests that the association of genes encoding histones and r ibosomal R N A products with regions that facilitate increases in copy numbers has probably oc- curred several times over evolutionary time. We envision that the exact number of repeats is not directly related in a simple way to functional demands on the genes but primarily reflects some proper ty of these regions such as frequent unequal exchange. That is, frequent unequal exchange among simple sequences within het- erochromatic regions may extend to adjacent genes, re- sulting in an increased number of tandemly arranged gene copies. Furthermore, we imagine that genetic mech- anisms such as inversions or translocations that remove tandemly repeated genes f rom heterochromatic environ-

m e n t s might ultimately result in a decreased copy number of those genes. Following relocation, it is still possible that selective forces act to maintain at least the minimum number of tandem copies required to meet functional demands. Tandemly repeated genes located in euchromatin would thus be viewed as having a history of close association with heterochromatic regions. This model can be tested by examining species with inversions that relocate linkage groups containing tandemly repeat- ed gene sequences.

Acknowledgements. The authors are grateful to James Garbe and Steven Tanksley for sharing unpublished results. We thank Dr. W. Joseph Dickinson for his support in the D. hawaiiensis work, Dr. Subhash Lakotia for confirmation of the histone cytogenetic locus in D. hydei, and Rachel Sage and Kelley Rochefort for assis- tance in preparing this manuscript. This research was supported by NIH grant GM28680 to L.D.S. and grants from the University of Connecticut Research Foundation.

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