Chromosome Changes in Cell Differentiation

(1)

Perspectives

Anecdotal, Historical And Critical Commentaries on Genetics

Edited

by

James F.

Crow

and William F. Dove

Chromosome Changes

in

Cell Differentiation

Orlando

J. Miller

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

I

N a recent “Perspectives” article, EEVA T H E W

(1995) called attention to a variety of alterations in chromosomes that occur regularly in differentiating cells, have been known for many years, and are still poorly understood. These includedfacultatiue heterochromatinization, polyploidization by endoreduplication, under-

replication of some sequences in polytene chromosomes,

and gene amplijication. The related programmed DNA loss phenomena called chromatin diminution and chromosome elimination also belong to this group of highly regulated developmental chromosome changes. Here I shall briefly review these changes, with particular emphasis

on the cell and molecular genetic approaches that have

provided, or could provide, insights into the signaling pathways and molecular mechanisms involved.

Facultative heterochromatinization during differentiation is widespread in metazoans and is functionally equivalent to programmed DNA loss. The best known

examples are mammalian X chromosome inactivation

(LYON 1961) and the inactivation of the paternally de-

rived set of chromosomes in coccids destined to become

males (HUGHES-SCHRADER 1948; BROWN and NELSON-

REES 1961). Both involve imprinting, the epigenetic process that leads to differential expression of the two parental alleles at a locus. When the fragmented chromosomes produced by massive doses of ionizing radia-

tion are transmitted from father to son in the mealybug,

each fragment undergoes heterochromatinization, sug-

gesting there are multiple cis-acting centers of inactiva-

tion in these holocentric chromosomes (BROWN and

NELSON-REES 1961). These may contain nuclease-resis-

tant, matrix-associated AT-rich fragments (KHOSW et

al. 1996). In contrast, there is a single center of X-

inactivation in mammals, and inactivation is mediated by the cis-acting RNA product of the XISTgene (BROWN

et al. 1.991).

Centers of inactivation and cis-acting RNA products appear to be involved in at least one other type of imprinting. The imprinting of a cluster of four genes on

mouse chromosome

7

is mediated by one of the four,

Author mail: [email protected]

Gmc~tir.; 1 4 6 1-X (May, 1997)

the H19 gene. The maternal H19 allele is expressed,

and its cis-acting, nontranslatable RNA product inhibits the expression of the maternal alleles of the other three

genes, mash-2, Ins-2, and I@. The paternal H19 allele

is methylated and not expressed, so the paternal alleles of the other three genes are expressed. Imprinting of

the Inns-2 and I@ genes is disrupted by maternal inheri-

tance of a targeted deletion of the H19 gene and its

flanking sequence, while paternal inheritance has no effect, reflecting the normally silent state of the paternal HI 9 allele (LEIGHTON et al. 1995). There is also a

cluster of several genes on human chromosome 15 that

are expressed exclusively on the paternal chromosome; these may play a role in the Prader-Willi syndrome. One

of these genes has only an RNA product (WEVRICK et

al. 1994). It remains to be seen whether such &acting,

nontranslatable RNAs play a more general role in imprinting and facultative heterochromatinization in vertebrates, coccids, or other taxons.

DNA methylation plays a role in imprinting as well

as in gene and X chromosome inactivation (CHAILLET

et al. 1995). Histone underacetylation may be an even

more general mechanism in imprinting and facultative

heterochromatinization

UEPPESEN

1997). While hypera-

cetylation of histones is characteristic of the DNA in active genes, underacetylation of histones is characteristic of the inactive micronucleus of Tetrahymena, the

inactive X chromosome of eutherian or metatherian

mammals, and the inactive spermatocyte X chromo-

some of the desert locust, Schistocerca gregaria (WOLF

and TURNER 1996); surprisingly, this may not be the case for the inactive Xchromosome in the male germ-

line of the mouse (ARMSTRONG et al. 1997).

Programmed DNA loss is a common event in meta-

zoan differentiation. It may involve whole chromo-

somes, large or small segments of chromosomes, or precisely defined short sequences. The earliest examples of programmed DNA loss were chromatin diminu-

tion in the nematode Ascaris (BOVERI 1887) and chro-

mosome elimination in the dipteran Sciaridae (METZ

(2)

2 J.

for each of these, as well as those involved in the V(D)J

recombination that assembles diverse functional immu- noglobulin and T-cell receptor genes from segments that are separated in the germline genome. This is directed by evolutionarily conserved cisacting heptamer and nonomer recombination signal sequences flanking

the segments (TONEGAWA 1983). Recognition and

cutting at V(D)J recombination signal sequences re-

quires the B and T lymphocyte-specific expression of

two recombination-activating genes, RAG1 and RAG2

(MCBLANE et al. 1995), but repair of the resultant dou- ble-strand breaks (DSBs) uses the same enzyme that all cells use for repairing DSBs, a DNAdependent protein

kinase (BLUNT et al. 1995).

Chromatin diminution in Ascaris and Parascaris spe-

cies involves fragmentation at the third to fifth cleavage

divisions of the very large chromosomes then present and elimination of most of the chromatin. The fragmentation occurs at specific chromosome breakage re-

gions (CBRs) and is followed by the addition of 2-4 kb

of telomeric TTAGGT repeats (MULLER et al. 1991).

Chromatin diminution takes place at a specific stage in the early embryo and occurs in all somatic precursor

cells. It is prevented in germline cells by cytoplasmic

factors close to the vegetal pole and can be induced by chemical treatment of eggs, suggesting that the inhibi-

tory cytoplasmic factors are already present in the zy-

gote (ESTEBAN et al. 1995). Chromatin diminution leads

to the elimination not only of all detectable heterochromatin but also of some euchromatic genes. Three sin-

gle-copy genes were identified in Ascaris suum that were

eliminated from somatic cells by chromatin diminution;

each is clearly related to a gene that is retained, leading

to the suggestion that chromatin diminution is linked

to partial genome duplication (MULLER et al. 1996).

Chromatin diminution has been most extensively

characterized in ciliates and shows considerable varia-

tion among the various taxons (PREscOTT 1994; YAO

1996; PREER 1997). After conjugation and completion

of the sexual phase of its life cycle, hypotrichous ciliates

such as Stylonychia or Euplotes transform a mitotic

copy of the transcriptionally silent micronucleus into a macronucleus by a process that takes about 4 days. The first half of this period is taken up by endoreduplication

and produces polytene chromosomes. An extreme form

of precise chromatin diminution then occurs, resulting

in loss of more than 90% of the DNA. Early in the

process of endoreduplication, abundant long transpo-

son-like elements (Tecl and Tec2 families in Euplotes

crassus) are excised; a bit later, short unique DNA sequences called internal eliminated sequences (IES) are excised. Both Tec and IES are bounded by a direct repeat of the TA dinucleotide, and excision is precise, leaving one TA. Excision, presumably by nuclease-mediated staggered cuts, is associated with rejoining of the flanking sequences. After Tec and IES have been eliminated and polytenization is completed, the chromosomes undergo fragmentation to form linear molecules

whose ends are then capped by telomeric GGGGTTTT repeats. Additional rounds of DNA replication produce

the mature macronucleus ( P R E S C O ~ 1994). In Tetrahy-

mena thermophila, the degree of polyploidy is only about 45C, and only some 15% of the micronuclear genome is eliminated during macronuclear development. The

deletion process is very precise and involves specific cis-

acting sequences (YAO 1996). In Paramecium tetraaurelia,

developmental genomic rearrangements also affect

mating type determination (MEYER and KELLER 1996).

Chromosome elimination shares a number of fea-

tures with chromatin diminution. Both occur at a precise time in early development, both lead to the elimination of a large fraction of the germline genome, both can be involved in sex determination, and both can

occur in the same organism, as in several primitive agna-

than hagfish species. In one of these, Eptatretus okinosea-

nus, some of the restricted sequences are highly repetitive, and there is variation in the number of germline- restricted chromosomes, leading to the suggestion that

some supernumerary (B) chromosomes are germline-

restricted chromosomes that have escaped their pro-

grammed elimination (KUBOTA et al. 1992, 1993). So-

matic elimination of supernumerary chromosomes has

been described in both plants and animals (DARLING

TON and THOMAS 1941; MELANDER 1950). In the myr-

micine ant, Leptothorax spinosior, B chromosomes are

usually restricted to the germline of the haploid males, although they are rarely seen in the germline of the

diploid females (IMAI 1974). Chromosome elimination

is common in several genera of gall midges (Diptera: Cecidomyidae), such as Miastor, in which there is weak but suggestive evidence linking chromosome elimina-

tion to partial genome duplication (BREGMAN 1975).

This point could be clarified with chromosome-specific

libraries (painting probes) for in situ suppression hy-

bridization (LICHTER et al. 1988; PINKEL et al. 1988).

Chromatin diminution and chromosome elimination have repeatedly led to the evolution of organisms in which the germline contains DNA sequences not pres-

ent in somatic cells. Since germline-restricted DNA se-

quences are subject only to selective forces operating on the germline and gametes, mutations in them will inactivate any genes not expressed in the germline and will drive the evolution of separate germline-specific

and soma-specific genomes, whether interspersed on

the same chromosomes or carried by different chromo-

somes. As a result, the germline may transcribe a par-

tially or completely different set of genes than does the soma. Some genes essential in both compartments

might be present in two copies, one germline-specific,

the other soma-specific. These might arise by partial

genome duplication, as suggested by MULLER et al.

(1996) for some genes in Ascaris, or by polyploidy, with elimination of unwanted chromatin or chromosomes only from somatic cells and capture of these by the germline to serve as its own set of genes.

(3)

gnat Sciara coprophila is particularly interesting. All

zy-

gotes contain three X chromosomes and several large

L (limited) chromosomes. The L chromosomes are lost

from all somatic cells by anaphase lag at the fifth or

sixth cleavage division. Shortly thereafter, a single pater-

nal X chromosome is lost to produce X / X or X/X' fe-

males, or two paternal X chromosomes are successively

lost to give rise to X/O males. In male meiosis the

maternal chromosome set plus the paternal L chromo-

somes proceed to the single pole of the monopolar

spindle; the rest of the paternal set is lost. The two

chromatids of the Xchromosome fail to separate at the second meiotic division. Thus, each primary spermato-

cyte yields only one sperm, and it contains all the L

chromosomes and two

X

chromosomes as a result of

nondisjunction (METZ 1938). HELEN CROUSE (1960)

found that a &acting controlling element near the

centromere of the X chromosome was responsible for

X nondisjunction. When this element was translocated

to an autosome, the translocation chromosome was lost during male meiosis.

How does such regular, or programmed, nondisjunction of both limited and Xchromosomes occur? Confo- cal laser scanning microscopy combined with fluorescence in situ hybridization (FISH) with an X-specific

painting probe has provided some insight into this,

showing that the long arms of the sister chromatids fail

to separate completely, although the centromeres are attached to the spindle and progress towards the poles. The chromosome remains at the metaphase plate and is lost. The sister chromatids of the X-autosome translo-

cation chromosome also fail to separate, and L chromo-

somes are lost by the same mechanism (DE SAINT

PHALLE and SULLIVAN 1996).

The molecular mechanisms involved in anaphase sep-

aration of sister chromatids are beginning to be worked

out. In general, two types of enzyme activity are re-

quired: topoisomerase 11, to separate interlocked DNA strands, and proteolysis. The role of topoisomerase I1

in sister chromatid separation is best understood in

yeast (HOLM et al. 1989), but in mammals chemical

inhibition of topoisomerase I1 causes prolonged mitosis and anaphase separation is completely prevented, although the cells still attempt cleavage. With lower con- centrations of the inhibitor, abnormalities of chromo-

some segregation are seen (DOWNES et al. 1991). In

Drosophila, chromatid segregation at anaphase re-

quires the product of the bawen gene, a protein that

associates with topoisomerase I1 throughout mitosis and

activates topoisomerase activity. In homozygous bawen

mutants, centromeres move apart as anaphase bepns,

but sister chromatids fail to separate (BHAT et al. 1996).

Separation of sister chromatids requires specific prote-

olysis of gene products such as PDSl in budding yeast,

CUT2 in fission yeast, pimples in Drosophila, and CENP-

E in mammals. Proteolysis of the first two, and possibly

the others, is mediated by the anaphase-promoting

complex (APC), which also degrades cyclins (KING et

al. 1996).

Endoreduplication: Polyploidization during somatic cell differentiation is extremely widespread in both plant and animal kingdoms. I shall limit my discussion to polyploidy arising by endoreduplication rather than by endomitosis, failed cytokinesis after telophase, or cell fusion. Endoreduplication is highly regulated. It occurs only at certain stages of differentiation in specific cell lineages and reaches ploidy levels that are specific to the tissue, the species, or the inbred strain within a species. In addition, one or more genes or segments of

the genome often show greater or lesser degrees of

amplification than expected from the ploidy level. The formation of polytene chromosomes by endoreduplication in plants and animals involves repeated rounds of DNA replication without intervening mitotic cell divisions. This requires bypassing or suppressing cell cycle mechanisms that block further replication until mitosis has occurred.

Cell cycle progression depends upon a coordinated system in which unstable regulatory subunits, called cyclins because of their changing abundance through-

out the cell cycle, activate specific CDKs (NASMYTH

1996). For example, in animal cells, S phase is induced

by CDK2 complexed with S phase cyclins of E or A type,

and M phase, mitosis, is induced by CDKl complexed

with M phase cyclins of A or B type. The maturation-

or M-phase promoting factor, MPF, is an activated M

phase CDK It is found in late G2 or M phase cells and

is capable of inducing nuclear disruption and chromo-

some condensation in G1,

s,

or G2 cells.

In Drosophila, the switch from mitotic cycles to endoreduplication (endocycles) is associated with the loss

of the mitosis-promoting cyclins A and B and the contin-

ued periodic expression of the S phase-promoting

cyclin E (SAUER et al. 1995; LILLY and SPRADLINC 1996).

Endocycles are composed of alternating S and Gap

phases and do not require the product of the cd~25"""~

gene, a key regulator of normal mitotic cycles (SMITH

and ORR-WEAVER 1991). Endoreduplication in maize

endosperm involves changes similar to those seen in Drosophila. The onset of mitosis is blocked by the in-

duction of an inhibitor of MPF, and S phase-promoting

protein kinases are induced (GRAFI and LARKINS 1995).

A change also occurs in the level and state of phosphor-

ylation of the Rbl05-like protein ZmRb, a negative regu-

lator of cell cycle progression. ZmRb can be phosphory-

lated in vitro by an S phase protein kinase from endore-

duplicating endosperm cells (GRAFI et al. 1996).

DNA replication in eukaryotic nuclear genomes generally begins at specific origins and proceeds bidirectionally. Origins in yeast are very short, wellcharacter- ized sequences, while those in metazoans are less well

defined; e.g., that of a salivary secretory protein gene

in S. copophila is contained within a 2-kb region of the

gene ( GEREH et al. 1993), and that of the Chinese ham-

(4)

4 0. J. Miller

gion (BURHANS et al. 1990). Initiation of replication

requires the assembly at origins of a prereplication (pre- RC) complex composed of a fairly stable origin replica-

tion complex (ORC)

,

a Cdc6p-type protein, and a min-

ichromosome maintenance (Mcm) complex. ORCs are bound to origins for most of the cell cycle. The other proteins bind at origins in the G1 phase, when Cdc6p is synthesized. The binding of Cdc6p is essential for the binding of the Mcm complex and the formation of a pre-RC.

Activated S phase, cyclindependent kinases (CDKs)

initiate replication only from origins with pre-RCs, and replication is followed by dissociation of pre-RCs. Both

S phase and M phase CDKs phosphorylate a component

of the Mcm complex, thus inhibiting the de novo assem-

bly of pre-RCs and preventing any new pre-RCs from

forming between late G1 and anaphase, when M phase

cyclins are degraded by the proteolytic anaphase-promoting complex (APC). This prevents origins from fir-

ing more than once during normal cell cycles (KING et

al. 1996; NASM~TH 1996). In fission yeast, overex-

pression of the Cdcl8 gene, the homologue of Cdc6 in

budding yeast, leads to multiple rounds of replication

and the formation of giant nuclei (NISHITANI and

NURSE 1995). In Drosophila, a member of the Mcm family of replication factors, the disc proliferation ab-

normal (dpa) gene product, is essential for mitotic repli-

cation but is not required for endoreduplication (FEGER

et al. 1995). Perhaps an as yet unrecognized Mcm family

member is required for pre-RC formation or stabiliza- tion in endoreduplicating chromosomes.

Studies in mammalian cells have added to our understanding of the signaling pathways in endoreduplica-

tion. One of my former associates, MENASHE MARCUS,

produced a temperature-sensitive mutant Chinese hamster cell line, ts41. At the nonpermissive temperature, the DNA content of these cells rose from 2C to 16C

( HIRSCHBERG and MARCUS 1982). These cells replicate

normally, but after completing S phase they skip G2,

M, and G1 phases and go directly into S again (HANDELI

and WEINTRAUB 1992). Thus, the ts41 gene product

appears to couple S phase to M phase, blocking entry

into S and fostering entry into G2 and mitosis. Is this

gene turned off during the normal induction of poly-

ploidy in megakaryocytes or placental trophoblasts, and

does a homologous gene play a similar role in endore-

duplication in other animals or plants?

The differentiation of bone marrow precursor cells into platelet-producing giant megakaryocytes is associated with a low degree of polyploidization. The DNA content, or ploidy level, in megakaryocytes is slightly higher in mice of the C3H inbred strain than in those

of the C57BL6 strain, reaching at most 128C. The DNA

content is intermediate in

F,

mice and shows a continu-

ous rather than bimodal distribution in backcrosses to

either parental strain, suggesting that several genes influence ploidy level. Furthermore, the female parent has a greater influence on ploidy level than the male

parent, suggesting a role for imprinting (MCDONALD and JACKSON 1994). The genes influencing endore-

duplication in this system might be mapped to short regions within the genome by the quantitative trait loci

(QTL) approach (LANDER and SCHORK 1994) with a

large number of the murine microsatellite markers now

available. Any genes known to be involved in signal

transduction or cell cycle control that have been

mapped to one or more of these locations would be likely candidates as regulators of megakaryocyte polyploidy, but additional genes in the regulatory pathway might also be identified by this positional cloning a p proach.

Identification of the genes involved in megakaryocyte differentiation would permit a detailed molecular analysis of the signaling pathways for polyploidization. At- tention might be directed first to the genes for recep tors or ligands known to be involved in megakaryocyte differentiation. Thrombopoietin, the ligand of the lym-

phokine receptor c-mpl, is a good candidate. It acts syn-

ergistically with erythropoietin, stem cell factor, and

interleukin-1 1 to increase murine megakaryocyte col-

ony growth and ploidy level in mouse cell cultures, as

shown by flow cytometric analysis (BROUDY et al. 1995).

Erythropoietin alone can yield ploidy levels up to 16C, but with thrombopoietin 30% of the cells achieve a 64C

level. The rho gene product, a small molecular weight

GTP-binding protein, is also involved in polyploidiza-

tion in a megakaryocyte cell line (TAKADA et al. 1996).

The differentiation of polyploid megakaryocytes can be mimicked in human erythroleukemia cells by expo-

sure to a phorbol ester such as TPA. Mitosis is arrested

because cdkl protein level falls markedly and cyclin B1

is modified so it cannot physically associate with cdkl.

Thus, even though the expression of cyclin B1 is ele-

vated and sustained, there is a lack of cdkl/cyclin B1-

associated Hl-histone kinase activity, i e . , a failure to

form MPF, so the completion of S phase does not trig-

ger mitosis (DATTA et al. 1996). This is similar to the

elevation of Sphase-related protein kinases and inhibition of MPF activity seen during endoreduplication in

maize endosperm (GRAFI and LARKINS 1995).

Trophoblast cells of the rodent placenta attain ploidy levels of 512C or higher. Chromosomes are not easily visualized in these giant cells, leaving some question as

to whether they have arisen by endoreduplication or

endomitosis (THERMAN 1995). However, in the rat, poly-

ploid trophoblast cells from females always contain a single giant sex chromatin body, whereas those from

males have none (NAGL 1972). Multiple inactive X c h r e

mosomes in a cell do not fuse together (THORLEY et

al. 1967), so a giant sex chromatin body is unlikely to

represent fusion of endomitotic inactive X chromo-

somes. It is much more likely that endoreduplication is the process involved and that all the polytene sister chromatids of the inactive X chromosome have re- mained tightly apposed.

(5)

port the occurrence of tight bundling of sister chroma-

tids during endoreduplication in trophoblast cells. I n

situ hybridization with probes for transgene integration

sites on two different chromosomes showed only one

site of hybridization for each probe in 16G512C nuclei

from mice heterozygous for a tandemly repetitive

transgene inserted at a single site and two sites of hybridization in comparable nuclei from homozygotes. A class I MHC probe gave comparable results. Thus,

endoreduplication appears to be involved in the forma-

tion of polyploid trophoblastic cells, with all the newly replicated strands of both chromatids of a chromosome remaining closely apposed, but no pairing of the ho-

mologous chromosomes (VARMUZA et al. 1988). In situ

hybridization to the endogenous alpha-1 antitrypsin lo-

cus gave similar results, but with less strand clustering

in this chromosome region (BOWER 1987).

Four of the five loci tested with molecular probes

showed tight clustering of the amplified copies. Is this

representative of the entire genome? The most efficient

approach to answering this question would require visu- alizing all the chromosomes in polyploid megakaryo-

cytes. VARMUZA et al. (1988) suggested treating the cells

with Xenopus egg extract, which induces premature

chromosome condensation (PCC) in diploid in-

terphase cells. A much more informative approach

would be to use in situ hybridization with whole chromo-

some painting probes (PINKEL et al. 1988) to evaluate

the looseness or compaction of each chromosome at every point throughout its length during polyploidization. In seeking the mechanism of polyploidization, one might first determine whether the phorbol ester TPA

will induce polyploidy in trophoblasts, as it does in meg-

akaryocyte precursors and whether the same changes in cdkl and MPF occur during polyploidization of trophoblasts as occur in mammalian megakaryocytes and maize endosperm.

Under-replication: An eclectic range of transcribed

as well as nontranscribed DNA sequences are under-

represented in Drosophila cells that have undergone

endoreduplication. EMIL HEITZ (1934) noted that cyto-

logically visible heterochromatin is markedly reduced in polytene chromosomes and used this as a basis for distinguishing a-heterochromatin (under-represented) from 0-heterochromatin (still present). The various sat-

ellite DNAs of a-heterochromatin in Drosophila mlano-

gaster and D. virilis are virtually totally unreplicated in

salivary polytene DNA (GALL et al. 1971). A surprising

number and type of genes are also under-represented

in Drosophila polytene DNA, e.g., 18S+28S rRNA genes

(rDNA, HENNIG and MEER 1971), histone genes, and

the 3’ end of the 100-kb Ultrabithorax ( [ f i x ) gene, but

not its distant 5’ end ( L A M B and LAIRD 1987). Are other

sequences also under-represented in polytene chromosomes? Comparative genomic hybridization (CGH)

provides a general method that might answer this ques-

tion, as it enables the visualization of minimally under-

or over-represented regions on a chromosome (KALLIO-

The process leading to under-representation of some sequences in polytene chromosomes is unclear. Chro- matin diminution, analogous to that seen in nematodes

(BOVERI 1887), was suggested by PAINTER (1933) and

more recently by KARPEN and SPRADLING (1990), who

suggested it as one cause of position-effect variegation. While there is no convincing cytological or molecular

evidence for chromatin diminution in Drosophila, its

occurrence early in polytenization in the macronucleus

of hypotrichous ciliates such as Euplotes crassus (PRES

COTT 1994) provides an attractive model. An alternative

mechanism, under-replication of heterochromatin, was suggested by HEITZ (1934), and this term is the one generally used when referring to under-representation

of particular sequences. Recently, LILLY and SPRADLING

(1996) have provided evidence that appears to favor

the under-replication model. They identified a hypo-

morphic mutant of the Cyclin E gene. Mutant polyploid

ovarian nurse cells in Drosophila had a reduced level

of Cyclin E, with altered cyclical oscillation in this level.

The mutant polyploid cells failed to show the usual

under-representation of satellite DNA, and, in contrast

to wild-type cells, had a late S pattern of bromodeoxyuri-

dine incorporation similar to that in mitotic cells, presumably reflecting the presence of abundant heterochromatin in both cases. Their results support the idea

that oscillating levels of Cyclin E control the endocycle

Sphase and may indicate the absence of a checkpoint ensuring Sphase completion. This would permit incomplete replication of late-replicating sequences such as satellite DNA during endocycles, but this could occur only if the Sphase in at least some of the endocycles is too short for late-S sequences to complete their replica-

tion. Does the mutation in the Cyclin E gene prolong

the Sphase? Do histone and rRNA genes replicate late enough in endocycle Sphases for this mechanism to

account for their under-representation? Do the regions

of the under-represented histone and ultrabithorax

genes that are closest to an origin of replication show the least under-representation? Alternatively, the firing of late-replicating origins may be suppressed in endo-

cycles, leading to a shorter S phase and more efficient

increase in copy number of expressed genes.

Whatever the mechanism of under-representation of some sequences in Drosophila, it acts not long after

endoreduplication begins. In the first two rounds of

endoreduplication in ovarian follicle cells, DNA content doubles each time, but in the next few rounds the

DNA content falls below that expected ( ~ H O W A L D et

al. 1979). The DNA content at stages corresponding

to ploidy levels of 16C to 1024C increases at a rate

comparable to that expected if 25% of the genomic DNA were unreplicated and 75% of the DNA were un-

dergoing endoreduplication. Satellite 1.703 DNA,

rDNA, and histone DNA all replicate normally during

the last one or two rounds of salivary endoreduplication

(6)

6 0. J. Miller

(HAMMOND and LAIRD 1985). After one round of endor-

eduplication, Drosophila hindgut cells have a ploidy level of only 3.4C rather than 4C, representing an under-replication of about 30% of the genome (SMITH

and ORR-WEAVER 1991). If chromatin diminution is in-

volved, rather than under-replication of particular sequences, it may be restricted to a narrow window, say

the third to fifth rounds of replication (the relative

copy number would provide an estimate of the time of chromatin diminution). Diminution would have to spare some copies of any under-represented but still amplified sequence; for example, it might act only on the paternal set of chromosomes, owing to imprinting. Alternatively, imprinting of these sequences in the germline of one parent might modify their origins of replication and prevent their firing during endoreduplication.

Under-representation involves primarily the interspersed, introncontaining, generally nonfunctional

copies of rDNA in polytene nuclei of Drosophila (EN-

DOW and GLOVER 1979). This is also true in the highly

polyploid nurse cells of Calliphora qthrocqbhala; in

which there is no under-replication of intronless rDNA interestingly, most of the amplified rDNA is in extra-

chromosomal micronuclei ( BUCKINGHAM and THOMP-

SON 1982). There is no under-representation of rDNA

in the polytene salivary cells of the dipteran Rhyncho-

sciara, which, like the fungus gnat, S. coprophila, has a

much smaller amount of rDNA, all intron-free. Again, there are abundant rDNA-containing micronucleoli in these cells, suggesting that there may be under-replication of rDNA in the polytene chromosomes themselves, and compensatory extrachromosomal rDNA replica-

tion (GERBI 1971). The amplification of rDNA is appar-

ently unique in another way: usually only one of the

two rDNA clusters participates in endoreduplication

and there is a dominance hierarchy, controlled by a factor associated with the rDNA itself, or even the num-

ber of rRNA genes in the cluster (ENDOW 1983).

The massive endoreduplication of silk gland DNA in Bombyx mor& which exceeds a 500,OOOC DNA level, is not associated with under-representation of repetitive sequences, rRNA genes or tRNA genes, on the basis of

analysis of reassociation kinetics (GAGE 1974). Compar-

ative genomic hybridization might reveal sites of under-

represented sequences missed by the less sensitive ear-

lier method. It could also answer the question of

whether under-replication or chromatin diminution occurs during endoreduplication in vertebrates. All that

is known so far is that satellite sequences are not under-

represented in the giant cells of mouse trophoblast

(LEJEUNE et al. 1982). Thus, the apparent scarcity of

visible heterochromatin in large trophoblast cells

( T H E W 1995) must have another cause.

Amplification: In some organisms, endoreduplication is accompanied by even greater amplification of a few specific genes. DNA amplification involves escape

from the usual requirement that each origin of replica-

tion fire once, and only once, during each S phase. The

first example of such amplification was the DNA puffs seen in salivary glands of Sciarid flies in the last (fourth)

larval instar ( CROUSE and

KEYL

1968). The level of am-

plification in DNA puffs in S. copophila is 16-fold higher

than that of the polytene chromosomes in which they

occur. Puff II/9A contains two amplified transcription

units with quite similar sequences; they are separated

from each other by a 2.5-kb spacer (GERBI et al. 1993).

What induces DNA puffing? HELEN CROUSE (1968)

showed it was the steroid molting hormone, ecdysone. The molecular mechanisms involved in this signal transduction pathway might be clarified by cloning the genes whose expression is activated by ecdysone. Some will have steroid response elements in the promoter region,

as GERBI et al. (1993) found in a DNA puff II/9A gene.

However, genes whose products are used for pupation are unlikely to be the ones that initiate further rounds of DNA replication. Novel members of gene families involved in cell cycle regulation would be more likely candidates, and might be identified by this approach.

The two pairs of chorion genes in Drosophila ovarian

follicle cells provide another example of intrachromo- soma1 DNA amplification. During oogenesis the gene

pair on the X chromosome is amplified about 16-fold,

and the pair on chromosome 3 about 60-fold. The two

genes of each pair, and the 1-kb spacer between them, are amplified equally, while the flanking sequences show a gradient of decreasing amplification extending for 40-50 kb in each direction. This suggests that additional rounds of replication are specifically initiated within the central, gene-containing region and are fol-

lowed by bidirectional replication in the absence of

discrete termination sites (SPRADLING 1981). Two-di-

mensional (2-D) gel studies have demonstrated the expected replication intermediates, and in the proper

abundance (HECK and SPRADLING 1990). Direct visual-

ization of amplifying chorion genes supports this onion-

skin model of DNA amplification from nested replica-

tion forks (OSHEIM et al. 1988). Several ckacting DNA

sequence elements have been identified that regulate

chorion gene amplification. An important one, ACE3

(amplification control element from the third chromo-

some ), is only 440 bp long, and amplification begins

within it (CARMINATI et al. 1992).

GERBI’S group mapped the origin of replication of

the DNA puff II/9A gene in S. coprophila, using two

different 2-D gel methods, and showed that here, too, replication proceeds bidirectionally from the origin

(GERBI et al. 1993). Thus, intrachromosomal amplifica-

tion of DNA puff and chorion genes does not occur by the rolling circle type of replication responsible for the extrachromosomal amplification of ribosomal genes in

Drosophila (HOURCADE et al. 1973). It is interesting that

both the DNA puff II/9A genes and the chorion genes that undergo endoreduplication consist of gene pairs with quite similar sequences. Perhaps this facilitates the

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sequence involved, such that substitution of an origin

from a chorion or DNA puff gene could lead to ampli-

fication of a different gene?

The degree of amplification of chorion and DNA

puff genes is fairly extreme. Do equal or lesser degrees of intrachromosomal amplification occur at other sites in these or other genomes, and is polytenization a nec- essary precondition? These questions might be an- swered with techniques such as comparative genomic

hybridization (KALLIONIEMI et al. 1992), a computer-

assisted fluorescence in situ hybridization technique

that can produce a map of DNA sequences with in-

creased (or decreased) copy number as a function of

chromosomal location throughout the genome. Endoreduplication in dipteran insects is associated with a very high level of production of a limited number of proteins, those encoded by the transcriptionally hy-

peractive genes in the RNA puffs found in virtually all

polytene chromosomes and the DNA puffs found in

those of Sciarid flies. The overproduction of poly (A)+

RNA by DNA puffs in S. coprophilu enabled DIBARTO-

LOMEIS and GERBI (1989) to clone two tandem tran-

scription units for salivary secretory proteins from DNA

puff II/9A from a fourth instar salivary gland cDNA

library. Differential display of mRNAs (LIANG and

PARDEE 1992) by a PCR-based method to detect and

clone overproduced (or underproduced) messages

might be particularly useful for identifying additional overexpressed genes in the various tissues that undergo endoreduplication in species of interest.

Studies incorporating modern concepts and methods

of cell and molecular biology have greatly enriched our

understanding of some of the chromosome changes that occur regularly in cell differentiation. The many unsolved problems of classical genetics and cytogenetics

will continue to present a challenge for investigators,

and a source of interesting avenues of research.

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