CopyTight 0 1997 by the Genetics Society of America Perspectives

Anecdotal, Historical And Critical Commentaries on Genetics Edited by James F. Crow and William F. Dove

Chromosome Changes in Differentiation

Orlando J. Miller

Center for Molecular Medicine and Genetics, Wayne State University, Detroit, Michigan 48201

N a recent “Perspectives” article, EEVA THEW the H19 . The maternal H19 allele is expressed, I (1995) called attention to a variety of alterations and its cis-acting, nontranslatable RNA product inhibits in chromosomes that occur regularly in differentiating the expression of the maternal alleles of the other three cells, have been known for many years, and are still , mash-2, Ins-2, and I@. The paternal H19 allele poorly understood. Theseincludedfacultatiue heterochro- is methylated and notexpressed, so the paternal alleles matinization, polyploidization by endoreduplication, under- of the other three genes are expressed. Imprinting of replication ofsome sequences inpolytene chromosomes, the Inns-2 and I@ genes is disrupted by maternal inheri- and gene amplijication. The related programmed DNA tance of a targeted deletion of the H19 gene and its loss phenomena called chromatin diminutionand chromo- flanking sequence, while paternal inheritance has no some eliminationalso belong tothis group of highly regu- effect, reflecting the normally silent state of the pater- lated developmental chromosomechanges. Here Ishall nal HI 9 allele (LEIGHTONet al. 1995). There is also a briefly review these changes, with particular emphasis cluster of several genes on human chromosome 15 that on thecell and molecular genetic approaches thathave are expressed exclusively on thepaternal chromosome; provided, or could provide, insights into the signaling these may play a role in thePrader-Willi syndrome. One pathways and molecular mechanisms involved. of these genes has only an RNA product (WEVRICKet Facultative heterochromatinization during differenti- al. 1994). It remainsto be seen whether such &acting, ation is widespread in metazoans and is functionally nontranslatable RNAs play a more general role in im- equivalent to programmed DNA loss. The best known printing and facultative heterochromatinization in ver- examples are mammalian X chromosome inactivation tebrates, coccids, or other taxons. (LYON1961) and the inactivation of the paternally de- DNA methylation plays a role in imprinting as well rived set of chromosomes incoccids destined to become as in gene and X chromosome inactivation (CHAILLET males (HUGHES-SCHRADER1948; BROWNand NELSON- et al. 1995). Histone underacetylation may be an even REES 1961). Both involve imprinting,the epigenetic more general mechanism in imprinting and facultative process that leads to differential expression of the two heterochromatinization UEPPESEN1997). While hypera- parental alleles at a locus. When the fragmented chro- cetylation of histones is characteristic of the DNA in mosomes produced by massive doses of ionizing radia- active genes, underacetylation of histones is characteris- tion are transmitted from fatherto son in the mealybug, tic of the inactive micronucleus of Tetrahymena, the each fragment undergoes heterochromatinization,sug- inactive X chromosome of eutherian or metatherian gesting there are multiple cis-acting centers of inactiva- , and the inactive spermatocyte X chromo- tion in these holocentricchromosomes (BROWNand some of the desert locust, Schistocerca gregaria (WOLF NELSON-REES1961). These may contain nuclease-resis- and TURNER1996); surprisingly, this may not be the tant, matrix-associated AT-rich fragments (KHOSW et case for the inactive Xchromosome in the male germ- al. 1996). In contrast,there is a single centerof X- line of the mouse (ARMSTRONG et al. 1997). inactivation in mammals, and inactivation is mediated Programmed DNA loss is a common event in meta- by the cis-acting RNA product of the XISTgene (BROWN zoan differentiation. It mayinvolve whole chromo- et al. 1.991). somes, large or small segments of chromosomes, or Centers of inactivation and cis-acting RNA products precisely defined short sequences. The earliest exam- appear to be involved in at least one other type of im- ples of programmed DNA loss were chromatin diminu- printing. The imprinting of a cluster of four genes on tion in the Ascaris (BOVERI 1887) and chro- mouse chromosome 7 is mediated by one of the four, mosome elimination in the dipteran Sciaridae (METZ 1938). Cell and molecular genetic approacheshave pro- Author mail: [email protected] vided exciting insights into themechanisms responsible

Gmc~tir.;146 1-X (May, 1997) 2 0.J. Miller for each of these, as well as those involved in theV(D)J whose ends are then cappedby telomeric GGGGTTTT recombination that assembles diverse functional immu- repeats. Additional rounds of DNA replication produce noglobulin and T-cell receptor genes from segments the mature macronucleus (PRESCO~1994). In Tetrahy- that are separated in the germline . This is di- mena thermophila, the degree of is only about rected by evolutionarily conserved cisacting heptamer 45C, and only some 15% of the micronuclear genome and nonomer recombination signal sequences flanking is eliminated during macronuclear development. The the segments (TONEGAWA1983). Recognition and deletion process is very precise and involves specific cis- cutting at V(D)J recombination signal sequences re- acting sequences(YAO 1996). InParamecium tetraaurelia, quires the B and T lymphocyte-specific expression of developmental genomicrearrangements also affect two recombination-activating genes, RAG1 and RAG2 mating type determination (MEYERand KELLER 1996). (MCBLANEet al. 1995), but repairof the resultant dou- Chromosome elimination shares a number of fea- ble-strand breaks (DSBs) uses the same enzyme that all tures with chromatin diminution. Both occur at a pre- cells use for repairing DSBs, a DNAdependent cise time in early development, bothlead to the elimina- (BLUNTet al. 1995). tion of a large fraction of the germline genome, both Chromatin diminution in Ascaris and Parascaris spe- can be involved in sex determination, and both can cies involvesfragmentation at the third tofifth cleavage occur in thesame organism, as in several primitive agna- divisions of the very large chromosomes then present than hagfish . In oneof these, Eptatretus okinosea- and elimination of most of the chromatin. The frag- nus, some of the restricted sequences are highly repeti- mentation occurs at specific chromosome breakage re- tive, and there is variation in the number of germline- gions (CBRs) and is followed by the addition of 2-4 kb restricted chromosomes, leading to the suggestion that of telomeric TTAGGT repeats (MULLERet al. 1991). some supernumerary (B) chromosomes are germline- Chromatin diminution takes place at a specific stage in restricted chromosomes that have escaped their pro- the early and occurs in all somatic precursor grammed elimination (KUBOTA et al. 1992, 1993). So- cells. It is prevented in germline cells by cytoplasmic matic elimination of supernumerary chromosomes has factors close to the vegetal pole and can be induced by been described in both and animals (DARLING chemical treatment of eggs, suggesting that the inhibi- TON and THOMAS1941; MELANDER1950). In the myr- tory cytoplasmic factors are already present in the zy- micine ant, Leptothoraxspinosior, B chromosomes are gote (ESTEBANet al. 1995). Chromatin diminutionleads usually restricted to the germline of the haploid males, to the eliminationnot only of alldetectable heterochro- although they are rarely seen in the germline of the matin but also of some euchromatic genes. Three sin- diploid females (IMAI1974). Chromosome elimination gle-copy genes were identified inAscaris suum that were is common in several genera of gall midges (Diptera: eliminated from somatic cells by chromatin diminution; Cecidomyidae), such as Miastor, in which there is weak each is clearly related to a gene thatis retained, leading but suggestive evidence linking chromosome elimina- to the suggestion that chromatin diminution is linked tion to partial genome duplication (BREGMAN1975). to partial genome duplication (MULLERet al. 1996). This point could be clarified with chromosome-specific Chromatindiminution has been most extensively libraries (painting probes) for in situ suppression hy- characterized in ciliates and shows considerable varia- bridization (LICHTERet al. 1988; PINKELet al. 1988). tion among the various taxons (PREscOTT 1994; YAO Chromatin diminutionand chromosome elimination 1996; PREER1997). After conjugation and completion have repeatedly led to the evolution of organisms in of the sexual phase of its life cycle,hypotrichous ciliates which the germline contains DNA sequences not pres- such asStylonychia or Euplotes transform a mitotic ent in somatic cells. Since germline-restricted DNA se- copy of the transcriptionally silent micronucleus into a quences are subject only to selective forces operating macronucleus by a process that takes about 4days. The on the germline and gametes, in them will first half of this period is taken up by endoreduplication inactivate any genes not expressed in the germline and and produces polytene chromosomes. An extreme form will drive the evolution of separate germline-specific of precise chromatin diminution then occurs, resulting and soma-specific , whether interspersed on in lossof more than 90% of the DNA. Early in the the same chromosomes or carried by different chromo- process of endoreduplication, abundant long transpo- somes. As a result, the germline may transcribe a par- son-like elements (Tecl and Tec2 families in Euplotes tially or completely different set of genes than does crassus) are excised; a bit later, short unique DNA se- the soma. Some genes essential in both compartments quences called internal eliminated sequences (IES) are might be present in two copies, one germline-specific, excised. Both Tec and IES are bounded by a direct the other soma-specific. These might arise by partial repeat of the TA dinucleotide, and excision is precise, genomeduplication, as suggested by MULLER et al. leaving one TA. Excision, presumably by nuclease-medi- (1996) for some genes in Ascaris, or by polyploidy, with ated staggered cuts, is associated with rejoining of the elimination of unwanted chromatin or chromosomes flanking sequences. After Tec and IES have been elimi- only from somatic cells and capture of these by the nated and polytenization is completed,the chromo- germline to serve as its own set of genes. somes undergo fragmentation to form linear molecules Chromosomeelimination in thedipteran fungus Perspectives 3 gnat Sciara coprophila is particularly interesting. All zy- complex (APC), which also degrades (KING et gotes contain three X chromosomes and several large al. 1996). L (limited) chromosomes. The L chromosomes are lost Endoreduplication: Polyploidization during somatic from all somatic cells by anaphase lag at the fifth or cell differentiation is extremely widespread in both sixth cleavage division.Shortly thereafter, asingle pater- and animal kingdoms. I shall limit my discussion nal X chromosome is lost to produce X/X or X/X' fe- to polyploidy arising by endoreduplication rather than males, or two paternal X chromosomes are successively by endomitosis, failed cytokinesis after telophase, or cell lost to giverise to X/O males. In male meiosis the fusion. Endoreduplication is highly regulated. It occurs maternal chromosome set plus the paternal L chromo- only at certain stages of differentiation in specific cell somes proceed to the single pole of the monopolar lineages and reaches levels that are specific to spindle; the rest of the paternal set is lost. The two the tissue, the species, or the inbred strain within a chromatids of the Xchromosome fail to separate at the species. In addition, one or more genes or segments of second meiotic division. Thus, each primary spermato- the genome often show greater or lesser degrees of cyte yields only one sperm, and it contains all the L amplification than expected from the ploidy level. The chromosomes and two X chromosomes as a result of formation of polytene chromosomes by endoreduplica- nondisjunction (METZ 1938). HELENCROUSE (1960) tion in plants and animals involves repeated rounds of found that a &acting controlling element near the DNA replication without intervening mitotic cell divi- centromere of the X chromosome was responsible for sions. This requires bypassing or suppressing X nondisjunction. When this element was translocated mechanisms that block further replication until to an autosome, thetranslocation chromosome was lost has occurred. during male meiosis. Cell cycle progression depends upon a coordinated system in which unstable regulatory subunits, called How does such regular, or programmed, nondisjunc- cyclins because of their changing abundance through- tion of both limited and Xchromosomes occur? Confo- outthe cellcycle, activate specific CDKs (NASMYTH cal laser scanning microscopy combined with fluores- 1996). For example, in animal cells, is induced cence in situ hybridization (FISH) with an X-specific by CDK2 complexed with S phase cyclins ofE or A type, paintingprobe has provided some insight into this, and M phase, mitosis, is induced by CDKl complexed showing that the long armsof the sister chromatids fail with M phase cyclins of A or B type. The maturation- to separate completely, although the centromeres are or M-phase promoting factor, MPF, is an activated M attached to the spindle and progress towards the poles. phase CDK It is found in late G2 or M phase cells and The chromosome remains at the metaphase plate and is capable of inducing nuclear disruption and chromo- is lost. The sister chromatids of the X-autosome translo- some condensation in G1, s, or G2 cells. cation chromosome also fail to separate,and L chromo- In , the switch from mitotic cycles to en- somes are lost by the same mechanism (DE SAINT doreduplication (endocycles) is associated with the loss PHALLEand 1996). SULLIVAN of the mitosis-promoting cyclins A andB and the contin- The molecular mechanisms involved inanaphase sep- ued periodic expression of the S phase-promoting aration of sister chromatids are beginningto be worked E (SAUERet al. 1995; LILLYand SPRADLINC1996). out. In general, two typesof enzyme activity are re- Endocycles are composed of alternating S and Gap quired: topoisomerase 11, to separate interlocked DNA phases and do notrequire the product of the cd~25"""~ strands, and . The role of topoisomerase I1 gene, a key regulator of normal mitotic cycles (SMITH in sister chromatid separation is best understood in and ORR-WEAVER1991). Endoreduplication inmaize (HOLMet al. 1989),but in mammals chemical involves changes similar to those seen in inhibition of topoisomerase I1 causes prolonged mitosis Drosophila. The onset of mitosis is blocked by the in- and anaphase separation is completely prevented, al- duction of an inhibitor of MPF, and S phase-promoting though the cells still attempt cleavage. With lowercon- protein are induced (GRAFIand LARKINS 1995). centrations of the inhibitor, abnormalities of chromo- A changealso occurs in the level and state of phosphor- some segregation are seen (DOWNESet al. 1991). In ylation of the Rbl05-like protein ZmRb, a negative regu- Drosophila, chromatid segregation atanaphase re- lator of cell cycleprogression. ZmRb can be phosphory- quires the product of the bawen gene, a protein that lated in vitro by an S phase protein kinase from endore- associates withtopoisomerase I1 throughout mitosis and duplicating endosperm cells (GRAFIet al. 1996). activates topoisomerase activity. In homozygous bawen DNA replication in eukaryotic nuclear genomes gen- mutants, centromeres move apart as anaphase bepns, erally begins at specific origins and proceeds bidirec- but sister chromatids fail to separate (BHATet al. 1996). tionally. Origins in yeast are very short, wellcharacter- Separation of sister chromatids requires specific prote- ized sequences, while those in metazoans are less well olysis of gene products such as PDSl in budding yeast, defined; e.g., that of a salivary secretory protein gene CUT2 in fission yeast, pimples in Drosophila, and CENP- in S. copophila is contained within a 2-kb region of the E in mammals. Proteolysis of the first two, and possibly gene ( GEREHet al. 1993), and thatof the Chinese ham- theothers, is mediated by the anaphase-promoting ster dihydrofolate reductase gene within a 0.45-kb re- 4 0.J. Miller gion (BURHANSet al. 1990). Initiation of replication parent, suggesting a role for imprinting (MCDONALD requires the assembly at origins of a prereplication (pre- and JACKSON 1994). The genes influencing endore- RC) complex composed of a fairly stable origin replica- duplication in this system might be mapped to short tion complex (ORC), a Cdc6p-type protein, and a min- regions within the genome by the quantitative trait loci ichromosome maintenance (Mcm) complex. ORCs are (QTL) approach (LANDER and SCHORK1994) with a bound to origins for most of the cell cycle. The other large number of the murinemicrosatellite markers now bind at origins in the , when Cdc6p available. Any genes known to be involvedin signal is synthesized. The binding of Cdc6p is essential for the transduction or cell cycle controlthat have been binding of the Mcm complex and the formation of a mapped to one or more of these locations would be pre-RC. likely candidates as regulators of poly- Activated S phase, cyclindependent kinases (CDKs) ploidy, but additional genes in the regulatory pathway initiate replication only from origins with pre-RCs, and might also be identified by this positional cloning ap replication is followed by dissociation of pre-RCs. Both proach. S phase and M phase CDKs phosphorylate a component Identification of the genes involved in megakaryocyte of the Mcm complex, thus inhibiting thede novo assem- differentiation would permit a detailed molecular anal- bly of pre-RCs and preventing any new pre-RCs from ysis of the signaling pathways for polyploidization. At- forming between late G1 and anaphase, when M phase tention might be directed first to the genes for recep cyclins are degraded by the proteolytic anaphase-pro- tors or ligands known to be involved in megakaryocyte moting complex (APC). This prevents origins from fir- differentiation. , the ligand of the lym- ing more than once during normal cell cycles (KING et phokine receptor c-mpl, is a good candidate. It acts syn- al. 1996; NASM~TH1996). In fissionyeast, overex- ergisticallywith erythropoietin, stem cell factor, and pression of the Cdcl8 gene, the homologue of Cdc6 in interleukin-1 1 to increase murine megakaryocyte col- budding yeast, leads to multiple rounds of replication ony growth and ploidy level in mouse cell cultures, as and the formation of giant nuclei (NISHITANIand shown by flow cytometric analysis (BROUDYet al. 1995). NURSE 1995). In Drosophila, a member of the Mcm Erythropoietin alone can yield ploidy levels up to 16C, family of replication factors, the disc proliferation ab- but with thrombopoietin 30% of the cells achievea 64C normal (dpa)gene product,is essential for mitotic repli- level. The rho gene product, a small molecular weight cation but is not required for endoreduplication (FEGER GTP-binding protein, is also involved in polyploidiza- et al. 1995). Perhaps an as yetunrecognized Mcm family tion in a megakaryocyte cell line (TAKADAet al. 1996). member is required for pre-RC formation or stabiliza- The differentiation of polyploid can tion in endoreduplicating chromosomes. be mimicked in human erythroleukemia cells by expo- Studies in mammalian cells have added to our under- sure to a phorbol ester such as TPA. Mitosis is arrested standing of the signaling pathways in endoreduplica- because cdkl protein level falls markedly and cyclin B1 tion. One ofmy former associates, MENASHE MARCUS, is modified so it cannot physically associate with cdkl. produced atemperature-sensitive mutant Chinese ham- Thus, even though the expression of cyclin B1 is ele- ster cell line, ts41. At the nonpermissive temperature, vated and sustained, there is a lack of cdkl/cyclin B1- the DNA content of these cells rose from 2C to 16C associated Hl-histone kinaseactivity, ie., a failure to ( HIRSCHBERGand MARCUS 1982). These cells replicate form MPF, so the completion of S phase does not trig- normally, but after completing S phase they skip G2, ger mitosis (DATTA et al. 1996). This is similar to the M, and G1 phases and go directly into S again (HANDELI elevation of Sphase-related protein kinases and inhibi- and WEINTRAUB1992). Thus,the ts41 geneproduct tion of MPF activity seen during endoreduplication in appears to couple S phase to M phase, blocking entry maize endosperm (GRAFIand LARKINS 1995). into S and fostering entry into G2 and mitosis. Is this Trophoblast cells ofthe rodentplacenta attain ploidy gene turned off during the normal induction of poly- levels of 512C or higher. Chromosomes are not easily ploidy in megakaryocytesor placental trophoblasts, and visualized in these giant cells, leaving some question as does a homologous gene play a similar role in endore- to whether they have arisen by endoreduplication or duplication in other animals or plants? endomitosis (THERMAN 1995).However, in the rat,poly- The differentiation of bone marrow precursor cells ploid trophoblast cells from females always contain a into -producing giant megakaryocytes is associ- single giant sex chromatin body, whereas those from ated with a low degree of polyploidization. The DNA males have none (NAGL1972). Multiple inactiveXchre content, or ploidy level, in megakaryocytes is slightly mosomes in a cell do not fuse together (THORLEYet higher in mice of the C3H inbred strain than in those al. 1967), so a giant sex chromatin body is unlikely to of the C57BL6 strain, reaching atmost 128C.The DNA represent fusion of endomitotic inactive X chromo- content is intermediate in F, mice and shows a continu- somes. It is much more likely that endoreduplication ous rather than bimodal distribution in backcrosses to is the process involved and that all the polytene sister either parental strain, suggesting that several genes in- chromatids of the inactive X chromosome have re- fluence ploidy level. Furthermore, the female parent mained tightly apposed. has a greater influence on ploidy level than the male Cell and molecular genetic approaches generally sup Perspectives 5

port the occurrenceof tight bundling of sister chroma- or over-represented regions on a chromosome (KALLIO- tids during endoreduplication in trophoblast cells. In NIEMI et al. 1992). situ hybridization with probes fortransgene integration The process leading to under-representation of some sites on two different chromosomes showed only one sequences in polytene chromosomes is unclear. Chro- site of hybridization for each probe in 16G512C nuclei matin diminution, analogous to that seen in from mice heterozygous fora tandemly repetitive (BOVERI 1887), was suggested by PAINTER(1933) and transgene inserted at a single site and two sites of hy- more recently by KARPEN and SPRADLING(1990), who bridization in comparable nuclei from homozygotes. suggested it as one cause of position-effect variegation. A class I MHC probe gave comparable results. Thus, While there is no convincing cytological or molecular endoreduplication appears to beinvolved in the forma- evidence for chromatin diminution in Drosophila, its tion of polyploid trophoblastic cells, with all the newly occurrence early in polytenization in the macronucleus replicated strands of both chromatids of a chromosome of hypotrichous ciliates such as Euplotes crassus (PRES remaining closely apposed, but no pairing of the ho- COTT 1994) provides an attractive model. An alternative mologous chromosomes (VARMUZAet al. 1988). In situ mechanism, under-replication of heterochromatin, was hybridization to the endogenous alpha-1 antitrypsin lo- suggested by HEITZ (1934), and this term is the one cus gave similar results, but with less strand clustering generally used when referring to under-representation in this chromosome region (BOWER1987). of particular sequences. Recently, LILLYand SPRADLING Four of the five loci tested with molecular probes (1996) have provided evidence that appears to favor showed tight clustering of the amplified copies. Is this the under-replication model. They identified a hypo- representative of the entire genome? Themost efficient morphic mutantof the gene. Mutant polyploid approach to answering this question would require visu- ovarian nurse cells in Drosophila had a reduced level alizing all the chromosomes in polyploid megakaryo- of Cyclin E, with altered cyclical oscillation in this level. The mutant polyploidcells failed to show the usual cytes. VARMUZA et al. (1988) suggested treating the cells under-representation of satellite DNA, and, in contrast with Xenopus egg extract, which induces premature to wild-type cells,had alate S pattern of bromodeoxyuri- chromosomecondensation (PCC) in diploid in- dine incorporation similar to that in mitotic cells, pre- terphase cells. Amuch more informative approach sumably reflecting the presence of abundant hetero- would be touse in situ hybridization with wholechromo- chromatin in both cases. Their results support the idea some painting probes (PINKELet al. 1988) to evaluate that oscillating levels of CyclinE control the endocycle the looseness or compaction of each chromosome at Sphase and may indicate the absence of a checkpoint every point throughout its length during polyploidiza- ensuring Sphase completion. This would permit in- tion. In seeking the mechanism of polyploidization, one complete replication of late-replicating sequences such might first determine whether the phorbol ester TPA as satellite DNA during endocycles, but this could occur will induce polyploidy in trophoblasts, it does in meg- as only if the Sphase in at least some of the endocycles is akaryocyte precursors and whether the same changes too short forlate-S sequences to complete their replica- in cdkl and MPF occur during polyploidization of tro- tion. Does the in the Cyclin E gene prolong phoblasts as occur in mammalian megakaryocytes and the Sphase?Do histone and rRNA genes replicate late maize endosperm. enough in endocycle Sphases for this mechanism to Under-replication: An eclectic range of transcribed account for their under-representation?Do the regions as well as nontranscribed DNA sequences are under- of theunder-represented histone and ultrabithorax represented in Drosophila cells that have undergone genes that are closest to an origin of replication show endoreduplication. EMIL HEITZ (1934) noted that cyto- the least under-representation? Alternatively, the firing logically visible heterochromatin is markedly reduced of late-replicating origins may be suppressed in endo- in polytene chromosomes and used this as a basis for cycles, leading to a shorter S phase and more efficient distinguishing a-heterochromatin (under-represented) increase in copy number of expressed genes. from 0-heterochromatin (still present). Thevarious sat- Whatever the mechanism of under-representation of ellite DNAs of a-heterochromatin in Drosophila mlano- some sequences in Drosophila, it acts not long after gaster and D. virilis are virtually totally unreplicated in endoreduplication begins. In the first two rounds of salivary polytene DNA (GALLet al. 1971). A surprising endoreduplication in ovarian follicle cells, DNA con- number and type of genes are also under-represented tent doubles each time, but in the next few rounds the in Drosophila polytene DNA, e.g., 18S+28S rRNA genes DNA content falls below that expected (~HOWALDet (rDNA, HENNIGand MEER 1971), histone genes, and al. 1979). The DNA content at stages corresponding the 3’ end of the 100-kb Ultrabithorax ([fix) gene, but to ploidylevels of 16C to 1024C increases at a rate not its distant 5’ end (LAMBand LAIRD 1987).Are other comparable to that expected if 25% of the genomic sequences also under-represented in polytene chromo- DNA were unreplicated and 75% of the DNA were un- somes? Comparative genomic hybridization (CGH) dergoingendoreduplication. Satellite 1.703 DNA, provides a general method thatmight answer this ques- rDNA, and histone DNA all replicate normally during tion, as it enables the visualization of minimallyunder- the last one or two rounds of salivaryendoreduplication 6 0.J. Miller

(HAMMOND and LAIRD 1985).After one roundof endor- tion fire once, andonly once, duringeach S phase. The eduplication, Drosophila hindgut cellshave a ploidy first example of such amplification was the DNA puffs level of only 3.4C rather than 4C, representing an un- seen insalivary glands of Sciarid in thelast (fourth) der-replication of about 30% of the genome (SMITH larval instar ( CROUSEand KEYL 1968). Thelevel of am- and ORR-WEAVER1991). If chromatin diminution is in- plification in DNA puffs in S. copophila is 16-fold higher volved, rather than under-replication of particular se- than that of the polytene chromosomes in which they quences, it may be restricted to a narrow window, say occur. Puff II/9A contains two amplified thethird to fifth rounds of replication (the relative units with quite similar sequences; they are separated copy number would provide an estimate of the time from each other by a 2.5-kb spacer (GERBIet al. 1993). of chromatin diminution). Diminution would have to What induces DNA puffing? HELENCROUSE (1968) spare some copies of any under-represented but still showed it was the steroid molting hormone, ecdysone. amplified sequence; for example, it might act only on The molecular mechanisms involved in this signal trans- the paternal set of chromosomes, owing to imprinting. duction pathway might be clarified by cloning the genes Alternatively, imprinting of these sequencesin the whose expression is activated by ecdysone. Some will germline of one parent might modify their origins of have steroid response elements in the promoter region, replication and prevent their firing duringendore- as GERBIet al. (1993) found in aDNA puff II/9A gene. duplication. However, genes whose products are used for pupation Under-representation involves primarily theinter- are unlikely to be the ones that initiate further rounds spersed, introncontaining, generally nonfunctional of DNA replication. Novel members of gene families copies of rDNA in polytene nuclei of Drosophila (EN- involved in cell cycle regulation would be more likely DOW and GLOVER1979). This is also true in the highly candidates, and might be identified by this approach. polyploid nurse cellsof Calliphora qthrocqbhala; in The two pairs of chorion genes inDrosophila ovarian which there is no under-replication of intronless rDNA follicle cells provide another example of intrachromo- interestingly, most of the amplified rDNA is in extra- soma1 DNA amplification. During the gene chromosomal micronuclei ( BUCKINGHAMand THOMP- pair on the X chromosome is amplified about 16-fold, SON 1982). There is no under-representation of rDNA and the pair on chromosome 3 about 60-fold. The two in the polytene salivary cells of the dipteran Rhyncho- genes of each pair, and the 1-kb spacer between them, sciara, which, like the fungus gnat, S. coprophila, has a are amplified equally, while the flanking sequences much smaller amount of rDNA, all intron-free. Again, show a gradient of decreasing amplification extending there are abundant rDNA-containing micronucleoli in for 40-50 kb in each direction.This suggests that addi- these cells, suggesting that there may be under-replica- tional rounds of replication are specifically initiated tion of rDNA in thepolytene chromosomes themselves, within the central, gene-containing region and are fol- and compensatory extrachromosomal rDNA replica- lowed by bidirectional replication inthe absence of tion (GERBI 1971).The amplification of rDNA is appar- discrete termination sites (SPRADLING1981). Two-di- ently unique in another way: usually only one of the mensional (2-D) gel studies have demonstrated the ex- two rDNA clusters participates inendoreduplication pected replication intermediates, and in theproper and there is a dominance hierarchy, controlled by a abundance (HECK andSPRADLING 1990). Direct visual- factor associated with the rDNA itself, or even the num- ization of amplifying chorion genes supportsthis onion- ber of rRNA genes in the cluster (ENDOW1983). skin model of DNA amplification from nested replica- The massive endoreduplication of silk gland DNA in tion forks (OSHEIMet al. 1988). Several ckacting DNA Bombyx mor& which exceeds a 500,OOOCDNA level, is sequence elements have been identified that regulate not associated with under-representation of repetitive chorion gene amplification. An important one, ACE3 sequences, rRNA genes or tRNA genes, on the basis of (amplification control element from the third chromo- analysis of reassociation kinetics (GAGE1974). Compar- some ), is only 440 bp long, and amplification begins ative genomic hybridization might reveal sites ofunder- within it (CARMINATI et al. 1992). represented sequences missed by the less sensitive ear- GERBI’S group mapped the origin of replication of lier method.It could alsoanswer thequestion of the DNA puff II/9A gene in S. coprophila, using two whether under-replicationor chromatin diminutionoc- different 2-D gel methods, and showed that here, too, curs during endoreduplication in vertebrates. All that replication proceeds bidirectionally from the origin is known so far is that satellite sequences arenot under- (GERBIet al. 1993). Thus, intrachromosomal amplifica- representedin the giant cells of mouse trophoblast tion of DNA puff and chorion genes does not occur by (LEJEUNEet al. 1982). Thus, the apparent scarcity of the rolling circle type of replication responsible for the visible heterochromatinin large trophoblast cells extrachromosomal amplification of ribosomal genes in (THEW 1995) must have another cause. Drosophila (HOURCADEet al. 1973). Itis interesting that Amplification: In some organisms, endoreduplica- both the DNA puff II/9A genes and the chorion genes tion is accompanied by even greater amplification of a that undergo endoreduplication consist of gene pairs few specific genes. DNA amplification involves escape with quite similar sequences. Perhaps this facilitates the from theusual requirement that each originof replica- formation of easily amplifiable structures. Is a specific Perspectives 7 sequence involved, such that substitution of an origin ony growth and increases megakaryocyte ploidy in vitro. Blood 85: 1719-1726. from a chorion or DNA puff gene could lead to ampli- BROWN,S. W., and W. A. NEISON-REES,1961 Radiation analysis of fication of a different gene? a lecanoid genetic system. Genetics 46: 983-1007. The degree of amplification of chorion and DNA BROWN,C. J., A. BALIABIO,J. L. RUPERT,R. G. LEFRENIERE,M. GROMPEet al., 1991 A gene from the region of the human X puff genes is fairly extreme. Do equal or lesser degrees inactivation centre is expressed exclusively from the inactive X of intrachromosomal amplification occur at other sites chromosome. Nature 349 38-44. in these or othergenomes, and is polytenization a nec- BUCKINGHAM,K, and N. THOMPSON,1982 Under-replication of intron+ rDNA cistrons in polyploid nurse cell nuclei of Calliphora essary precondition? These questions might be an- erythrocephala. Chromosoma 87: 177-196. swered with techniques such as comparative genomic BUKHANS,W. C., L. T. VASSIIJEV,M. S. CADDI.E,N. H. HEINTZand hybridization (KALLIONIEMI et al. 1992), acomputer- M. L. DE PAMPHILIS,1990 Identification of an origin of bidirec- assisted fluorescence situ hybridization technique tional DNA replication in mammalianchromosomes. Cell 62: in 955-965. that can produce a map of DNA sequences with in- CARMINATI,J. L., C. G. JOHNSTON and T. L. ORR-WEAVER,1992 The creased (or decreased) copy number as a function of Drosophila ACE3 chorion elementautonomously induces ampli- chromosomal location throughout the genome. fication. Mol. Cell. Biol. 12: 2444-2453. CHAILLET,J. R., D. S. BADERand P. LEDER,1995 Regulation of geno- Endoreduplication in dipteran insects is associated mic imprinting by gametic and embryonic processes. Genes Dev. with a very high level ofproduction of a limited number 9: 1177-1187. of proteins, those encoded by the transcriptionally hy- GROUSE,H. V., 1960 The controlling element in sex chromosome behavior in Sciara. Genetics 45: 1429-1443. peractive genes in the RNA puffs found in virtually all GROUSE,H. V., 1968 The role of ecdysone in DNA-puff formation polytene chromosomes and the DNA puffs found in and DNA synthesis inthe polytenechromosomes of Sciara those of Sciarid flies. The overproduction of poly (A)+ coprophila. Proc. Natl. Acad. Sci. USA 61: 971-978. GROUSE,H. V., and H. G. KEYL, 1968 Extra replications inthe RNA by DNA puffs in S. coprophilu enabled DIBARTO- “DNA-puffs” of Sciara coprophila. Chromosoma 25: 357-364. LOMEIS and GERBI (1989) to clone two tandem tran- DARLINGTON,C. D., and P. T. THOMAS,1941 Morbid mitosis and scription units for salivary secretory proteins from DNA the activity of inert chromosomes in sorghum. Proc. Roy. SOC. puff II/9A from a fourth instar cDNA Lond. B 159: 127-150. DATTA,N. S., J. L. WIILIAMS,J. C~LDWELI.,A. M. CURRY,E. K. ASH- library. Differential displayof mRNAs (LIANG and CRAFT et al., 1996 Novel alterations in CDKl/cyclin B1 kinase PARDEE1992) by a PCR-based method to detect and complex formation occur during the acquisition of a polyploid clone overproduced (orunderproduced) messages DNA content. Mol. Biol. Cell 7: 209-223. DE SAINTPHALLE, B., and W. SUL.I.IVAN,1996 Incomplete sister chro- might be particularly useful for identifying additional matid separation is the mechanism of programmed chromosome overexpressed genes in the various tissues that undergo elimination during early Sciara coprophila embryogenesis. Devel- endoreduplication in species of interest. opment 122: 3775-3784. DIBARTOL.OMEIS,S. M., and S. A. GERBI,1989 Molecular character- Studies incorporating modernconcepts and methods ization of DNA puff II/9A gene in Sciara coprophilu. J. Mol. Biol. of cell and molecular have greatlyenriched our 210: 531 -540. understanding of some of the chromosome changes DOWNES,C. S., A. M. MULLINCERand R. T. JOHNSON,1991 Inhibi- tors of DNA topoisomerase I1 prevent chromatid separation in that occur regularly in cell differentiation. The many mammalian cells but do not prevent exit from mitosis. Proc. unsolved problems of classical genetics and cytogenetics Natl. Acad. Sci. USA 88: 8895-8899. will continue to present a challenge for investigators, ENDOW,S. A., 1983 Nucleolar dominance in polytene cells of Dro- and a source of interesting avenues of research. sophila. Proc. Natl. Acad. Sci. USA 80: 4427-4431. ENDOW,S. A., and D. M. GLOVER,1979 Differential replication of ribosomal gene repeats in polytene nuclei of Drosophila. Cell 17: 597-605. LITERATURE CITED ESTEBAN,M. R., G. GIOVINAZZOand C. GODAY,1995 Chromatin dim- ARMSTRONG,S. J., M. A. HULTEN,A. M. KEOHANE and B. M. TURNER, inution is strictly correlated to somatic behaviorin early develop- 1997 Different strategies of X-inactivation in germinal and so- ment of the nematode Purascari,y univalens.J. Cell Sci. 108: 2393- matic cells: histone H4 underacetylation does not mark the inac- 2404. tive Xchromosome in the mouse male germline. Exp. Cell Res. FEGER,G., H. VAESSIN,T. T. SU,E. WOLFF, L.Y. JANet al., 1995 dpa, 230: 399-402. a member of the MCM family, is required for mitotic DNA repli- BHAT,M.A.,A. V. PHILIP,D.M. GI.OVERandH. J. BELLEN,1996 Chro- cation butnot endoreduplication in Drosophila. EMBO J. 14: matidsegregation atanaphase requires the barren product, a 5387-5398. novel chromosome-associated protein that interacts with topc- GAGE,L. P., 1974 Polyploidization of the silk gland of Bombyx mori. isomerase 11. Cell 87: 1103-1114. J. Mol. Biol. 86: 97-108. BLUNT,T., N. J. FINNIE,G. E. TACCIOIJ,G. C. M. SMITH,J. DEMENCEOT GALL,J. G., E. COHENand M. L. POIAN,1971 Repetitive sequences et al., 1995 Defective DNA-dependent protein kinase activity is in Drosophila. Chromosoma 33 319-344. linked to V(D)J recombination and DNA repair defects associ- GERBI,S. A., 1971 Localization and characterization of the ribo- ated with the murine SCID mutation. Cell 80: 813-823. somal RNA cistrons in Sciara coprophila. J. Mol.Biol. 58: 499- BOVERI, T., 1887 irber Differenzierung der Zellkerne wahrend der 511. Furchung des Eies von Ascaris megalocephala. hat. Anz. 2: 688- GERBI, S. A., C. LWVG,N. Wu, S. M. DIBARTOLOMEIS,B. BIENZ- 693. TADMORet al., 1993 DNA amplification in DNA puff II/9A of BOWER,D. J., 1987 Chromosome organization in polyploid mouse Sciara copraphila. Cold Spring HarborSymp. Quant. Biol. 58: 487- trophoblast nuclei. Chromosoma 95: 76-80. 494. BREGMAN,A,, 1975 Q, C, and Gbanding patterns in the germ-line GWI, G., and B. A. LARKINS,1995 Endoreduplication in maize en- and somatic chromosomes of Miactor sp. (Diptera: Cecidomyi- dosperm: involvement of M-phase promoting factor inhibition dae). Chromosoma 53: 119- 130. and induction of S phase-related kinases. Science 269 1262- BROUDY,V. C., N. L. LINand K. KAUSHANSKY, 1995 Thrombopoietin 1264. (c-mpl ligand) acts synergistically with erythropoietin, stem cell GWI, G., R. J. BURNETT,T. HELENTJARIS,B. A. LARKINS,J. A. DECA. factor, and interleukin-1 1 to enhance murinemegakaryocyte col- PRIO et aL, 1996 A maize cDNA encodes a member of the retino- 8 0.J. Miller

blastoma protein family: involvement inendoreduplication. 1979 Loss of centrioles and polyploidization in follicle cells of Proc. Natl. Acad. Sci. USA 93: 8962-8967. Drosophila. Exp. Cell Res. 118: 404-410. HAMMONI),M. P., and C.D. hm,1985 Chromosome structure MCBIANE,J. F., D.C. VAN GENT,D. A. RAMSDEN, C. ROMEO,C. A. and DNA replication in nurse and follicle cells of Drosophila mela- CUOMOet ul., 1995 Cleavage at a V(D)J recombination signal nogastm. Chromosoma 91: 267-278. requires only RAG1 and RAG2 proteins and occurs in two steps. HANDEM,S., and H.WEINTRAUB, 19Y2 The ts41 mutation in Chinese Cell 83: 387-395. hamster cells leads to successive S phases in the absence of in- MCDONALD,T. P., and C. W. JACKSON,1994 Mode of inheritance of tervening G2, M, and GI. Cell 71: 599-611. the higher degree of megakaryocyte polyploidization in C3H HECK,M. M. S., and A. C. SPRADIJNG,1990 Multiple replication ori- mice. I. Evidence for a role of genomic imprinting in megakaryo- gins are used during Drosophila chorion gene amplification. J. cyte polyploidy determination. Blood 83: 1493- 1498. Cell Biol. 110: 903-914. MEWNIIER,Y., 19.50 Accessory chromosomes in animals, especially HEIIL,E., 1934 Uber a- und0-heterochromatin sowie Konstanz in Polycelis tenuis. Hereditas 36: 19-38. und Bau der Chromosomerenbei Drosophila. Biol. Zbl. 54588- MET%,C. W., 1938 Chromosome behavior, inheritance and sex de- 609. termination in Sciara. Am. Nat. 72: 485-520. HENNIG,W., and B. MEEK,1971 Reduced polyteny of ribosomal MF,w,R, E., and A,". KEILER,1996 A Mendelian mutation affecting mating type determination also affects developmental genomic RNA cistrons in giant chromosomes of Drosophila hydei. Nat. New rearrangementsin Paramecium tetraaurelia. Genetics 143: 191- Biol. 233 70-72. 202. HIRSCHBERG,J., and M. ~CUS,1982 Isolation by a replica-plating MUILER, F., C. WICKY,A. SPICIER andH. TOBLER, 1991 New te- technique of Chinese hamster temperaturesensitive cell cycle lomere formation after developmentally regulated chromosome mutants. J. Cell. Physiol. 115: 159-166. breakage during the process of chromatin diminution in Ascaris HOI.M,C., T. STEARNSand D. BOTSTEIN,1989 DNA topoisomerase lumbricoides. Cell 67: 815-822. I1 must actat mitosis to prevent nondisjunctionand chromosome MUILER,F., V. BERNARDand H. TOBIER, 1996 Chromatin diminu- breakage. Mol. Cell. Biol. 9: 159-168. tion in nematodes. BioEssays 18: 133-138. HOURCAIIE,D., D. DRESSIXRand J. WOLFSON,1973 Amplification NAGI.,W., 197'2 Giant sex chromatin in endopolyploid trophoblast of ribosomal genes involves a rolling circle intermediate. Proc. nuclei in the rat. Experientia 28: 217-218. Natl. Acad. Sci. USA 70: 2926-2930. NMMYTH, R, 1996 Viewpoint: putting the cell cycle in order. Sci- Hc~~II~.s-S~:I~~IIER,S., 1948 Cytology of coccids (Coccoidea-Homw ence 274 1643-1645. ptera). Adv. Genet. 2: 127-203. NISEIIrI\NI, H.,and P. NURSE,1995 ~65'~''~plays amajor role in IMAI, H. T.,1974 B-chromosomes in the myrmicine ant, Lrptothorax controlling the initiation of DNA replication in fission yeast. Crll spinmior. Chromosoma 45: 431 -444. 83: 397-405. JEPPESEN,P., 1997 Histone acetylation: a possible mechanism for OSHEIM,Y. N., 0. L. MILLER,JR. and A. L. BEYER,1988 Visualization the inheritance of cell memory at mitosis. BioEssays 19: 67-74. of Zhosophila melanogaster chorion genes undergoing amplifica- ~4I.l.IONl~MI,A,, 0.-P. ~M.I.IONIEM1, D. SUDAR, D. RUTOVITZ,J. W. tion. Mol. Cell. Bid. 8: 2811-2821. GRAYrt al., 1992 Comparative genomic hybridization for molec- PAINTER, T.S., 1933 A new method for the study of chromosome ular cytogenetic analysis of solid tumors. Science 258: 818-821. rearrangements, and theplotting of chromosome maps. Science KARPEN, G. H., andA. C. SPRAIII.IN(;, 1990 Reduced DNApolyteniza- 78: 58.5-586. tion of aminichromosome region undergoing position-effect PINKEI.,D., J. LAANDEGENT, C. COLLIKS,J. Fuscoe, R. SEAGRAW.~rl al., variegation in Drosophila. Cell 63: 97-107. 1988 Fluorescence in situ hybridization with humanchromo- some-specific libraries: detection of trisomy 21 and translocation Kllosl.A, S., P. KAN'IEIKII, V. BRAHMKHARIand H. s. CHANDRA, 1996 of chromosome 4. Proc. Natl. Acad. Sci. USA 85 9138-9142. A male-specific nucleax-resistantchromatin fraction in the PREER,J. R.,,JR.,1997 Whatever happened to Paramecium genetics? mealybug Planococrus lilacinus. Chromosoma 104: 386-392. Genetics 145: 217-225. KING, R. W., R. J. DES(:~IAIES,J.-M. PETERS and M. W. KIRSCHNER. 1996 PRESCO~,D. M., 1994 The DNA of ciliated protozoa. Microbiol. How proteolysis drives the cell cycle. Science 274: 1652-1659. Rev. 58: 233-267. KUBOTA,S., Y. NAKAI,M. Kc~Ro-Oand S. KOHNO,1992 Germ line- SAUER,K., J. A. KMBIKH, H. RICHARDSON and C. F. LEHNER,1995 restricted supernumerary (B) chromosomes in Eptatrtrus okinosm- Distinct modes of cyclin E/cdc2c kinase regulation and Sphase nus. Cytogenet. Cell Genet. 60: 224-228. control in mitotic andendoreduplication cyclesof Drosophila KUBOTA,S., K. M.&w, S. MIZUNOand S. KOHNO, 1993 Germ line- embryogenesis. Genes Dev. 9: 1327-1339. restricted, highly repeated DNA sequences and their chromo- SMITH,A. V., and T. L. ORR-WEAVER,1991 The regulation of the somal localization in Japanese hagfish (Eptatetrus okinosranus). cell cycle during Drosophila embryogenesis. Development 112: Chromosoma 102: 163-173. 997-1008. LAMB, M.M., and C. 1). LAIRI), 1987 Three euchromatic DNA se- SPRADI.ING,A., 1981 The organization and amplification of two quences under-replicated in polytene chromosomes of Drosophila chromosomaldomains containing Drosophila chorion genes. are localized in constrictions and ectopic fibers. Chromosoma Cell 27: 193-201. 95: 227-235. TAKADA,M., N. MORII,S. KUMAGAIand R. RYO, 1996 The involve- LANDER, E. S., and N. J. SCHORK,I994 Genetic dissection of com- ment of the rho gene product, a small molecular weight GTP- plex traits. Science 265: 2037-2048. binding protein, in polyploidization of a human megakaryocyte LEIGHTON,P. A., R. S. INGRAM,J. EGGENSCHWII.ER,A. EFSI'RATIADIS cell line, CMK. Exp. Hematol. 24: 524-530. and S. M. Trl.GHMAN, 1995 Disruption of imprinting caused by THERMAN,E., 1995 Chromosome behavior in cell differentiation: a deletion of the Hl9 gene region in mice. Nature 375 34-39. field ripe for exploration? Genetics 141: 799-804. THORLEY,J., D. WARBURTONand 0. J. MILLER,1967 Absence of l.qEIINF., B.,,J.VAN HCIECKand F. LEROY,1982 Satellite versus total DNA rcplication in relation to endopolyploidy of decidual cells somatic pairing of sex chromatin masses (inactivated X chromo- somes) in cultured cells from a human XXXXY male. Exp. Cell in the mouse. Chrornosoma 84: 511-516. Res. 47: 663-665. LIANG,P., and A. B. PARDEE,1992 Differential display of eukaryotic TONEGAWA,S., 1983 Somatic generation of antibody diversity.Na- messenger RNA by means of the polymerase chain reaction. Sci- ture 302: 575.581. ence 257: 967-971. VARMUZA,S., V. PRIDWUX,R. KOTHARYand J. ROSSANI, 1988 Poly- ~.I(:€lTLR, P., T. CREMER,J. BOKI)EN, L. M.4NCJl;.I.lDIS and D. C. Wm, tene chromosomes in mouse trophoblast giant cells. Develop- 1988 Delineation of individual human chromosomes in meta- ment 102: 127-134. phase and interphase cells by in situ suppression hybridization WEVRICK,R. J. A. KERNS and U. FRAN(:I(E,1994 Identification of a using recombinant DNA libraries. Hum. Genet. 80 224-234. novel paternally expressed gene in the Prader-Willi syndrome LIILY,M. A,, and A. C. SPW)I.ING,1996 The Drosophila endocycle region. Hum. Mol. Genet. 3: 1877-1882. is controlled by Cyclin E and lacks a checkpoint ensuringS-phase WOLF,K. W., and B.M. TURNER,1996 The pattern of histone H4 completion. Genes Dev. 10: 2514-2526. acetylation on the X chromosome during spermatogenesis of LYON,M. F., 1961 Gene action in the Xchrornosome of the mouse the desert locust Schistocerca gregaria. Genome 39: 854-865. (Mus musculus I,.). Nature 190: 372-373. YAO, M.-C., 1996 Programmed DNA deletions in tetrahymena: MU~OWAI.D,A. P., J. H. (:A~TTON, M. K. EDWARDSand A. D. FLOYD, mechanisms and implications. Trends Genet. 12 26-30.