lZqJrinlell [rom til<' JIOW')/£I[ of Scientijic (~. Indllstriat Resc{/nil. \'o\' 32. :'\0. l), September ] Y. C. SHAH & S. C. LAKHOTIA Depa.rlment o( Zoolog~-. Cniversity School of Sciences, Gujarat l:niver,il~', Ahmedabad 9 & S. R V. R,\O DC'partment of Zoology, lTnivcrsity of Delhi, Delhi 7 I. Introduction Y-chromosome, the flies were 'IOrma lly viable and of male phenotypel'. ECENT developments in mOlcc. ular cytogenetics The original concc:pt of total inactivity of hetero have revivccl intercst in the nature and fUlle chromatin could not be main tained for long, though R tion of heterochromatin. The concept of the idea of absenc" of major genes on heterochro heteroc romatin. from its very inception, has been matin is held to date. Gradually, a confusing array one of uncertainty ;lI1cl vagueness. The data of 'functions' of hc,terochromatin were proposed. obtained from various cytological and genetical MatherlG suggested the localization of 'polygenes' sourceS ha.vL: not in any way helped in developing for quantitative traits in the heterochromatic a unified cOl1cept of heterochromatin. The litera regions. Position-effect variegation was also founel tun:: is exhaustive, both with respect to direct to be associated with hetero chromatin 17-~O ()b~('rva speculation~. tions and theoretical In A new dimension to tlw study of heterochromatin 1950's, though the importance of heterochromatin was added by tritium-autorac1iography~J. Lima-cle was reali71:d, there was, in general, a resigned Faria22 first showed that in grasshopper and rye, approach to the illusiveness of heterochromatin l-:l, 4 the heterochromatin synthesizes its DXA later and this was summed up by Pontecorvo in the than euchromatin. Tavlor2:l confirmed this result following words: ., the study of heterochromatin in mammals, and ~inc(; -then, exhaustive studies on is at a prescientific level. .. .. We have no alter rE'plication in a wiele; variety of materials have been native but t ignore it." However, heterochro made, and the reslllts have str"ngthened the corr matin was never ignored, and the 60's saw a renewed lation of ' late-replication' with heterochromatin21 '26. interest, and the various aspects of heterochromatin The confusion about heterochromatin was clarified have been analysed by ever-increasing number of to some exten t by Hrown~7, when he proposed workers with new techniques. These studies have the terms '!acultati'1c' and 'constitutive' hetero now provided a new insight in to thE' nature and chromatin for two basically different kinds of function of het~rochromatil1. The functional aspects condensed or inactiv 1 SH.\H cI al.: :'\.\TCRE OF HETEROCHRO:\-I.\TI:'\ In some cases, there is negative heteropycnosis41 > ., i. e. some chromosome regions appear more diffuse and light-stained at metaphase. Lima-de-Faria26 has emphasized that all such negatively-heteropyc _-f_' notic regions should not be considered heterochro . matic. However, some heterochromatic regions do show negative heteropycnosis at metaphase, especially after some treatmen ts like cold, colchicine, I etc. 42-45 (Fig. 1). It is, of course, true that such non-staining gaps arc not due to the so-called nucleic A B C o E acid starvation46 , but are probably due to some differential coiling of the chromatin. In an elegant study on the heterochromatin of the Indian l\Iunt Fig. I· l'ropc'.rti"s of hdcrochromatin ~egn1l'nt in :\I-chromo 45 ~orn" of Vic;a fa./Ja (A) Loc,dizati"n of heterochromatic seg jac, Comings has clearly shown that the differential ments; K, kinetochore, l\C, l1uclwJlar con'''triction; (D) effect response of heterochromatin to colc(~mid is due to of c 1<1 (6 C C tor 72 hr), heterochromatic region, less condcnsed; differential contraction. In this case, euchromatin (C) quinacrine mustard bin cli ng "egmen ts; Il)l la te-rerlicating contracts more than heterochromatin. It has also segrncnt~; (E) chromatid break in :\1 , heterochromatic segment inollccd hI' mitomycin C. :'\ot" thl: coincidence' in the ehro- been shown that this phenomenon is unrelated to 92 4 mos(llnal segment :\01, ("rrow) in all thc ca<;e5 ] DNA syntl1Csis .,. (2) Genetic inac#vz:ty and l:nertl1ess - It is widely believed that heterochromatin is genetically inactive. At times, it has also been implied that heterochro matin, being gendically inc,rt and 'empty', may be dispensable47 ,48. Studies 0'1 Drosophila Jnelanogastel' have shown tha t in this specil's, the en tire Y and the proximal one-thrid of the X-chromosome are entirelv hetero chromatic and devoirl. of the so-callr:d ' :VI~ndelian ' genes as wdl as the s~x-cldermi1]i'1g[actors\J·10.14,49,50, In all the other systems analysed, it has been observed that heterochromatinization is associated wi th either the abSence or tlw [(,pression of genetic Fig. 2 ... Ileterocillomatic chroIllocentres in li"cr interphasc nud,'i ot Nalllls b/an fordi (Cal'hol-fuchsin stain) Fig. 4 - Absence of "H-uridine incorporation in the sex chromatin in FUl1amhulus peu1I!IIlti'" , '" Fig. 3 - Heterochromatic segments in mdaphase cbromo somcs (bonc marrow) of male Rattus blond fordi [l\'otc thc 2 SH.\l-1 vi al.: X-\TliRE CW HETEJWCHIWM\TI~ activity5,26"2!1,:1l,51,52. This is also manifested at a nueIeus will be finishing their DNA synthesis when the molecular It;ve! by absence of transcription G1 ,53-55 others have already completed. It is not imperative (Fig. 4). Genetic inactivity and inertness of hetero that all such late-finishing replicating units be chromatin is discussed in later sections; it may be heterochromatic, though the latter would be repre mentioned here that heterochromatin or condensed sen ted in such late-replicating units. chrom;ltin is believed to be not manifestly activc Late-replication ancl condensed state of hetero in trallscription. chromatin are probably in terdependen1. Several (3) 'Late' replicatioll-The first repo.rt on the asyn studies have indicated that the heterochromatin chronous replication in heterochromatJll and euchro or the condensed chromatin undergo decondensation matin was by Lima-lle-I'aria22 . The heterochromatic during DNA synthesis for a brief periocJil-i3. This X-chromosomc in the germ cells of lHelalloplus was decondensation may be a prerequisite for replication. found to complete DNA synthesis latcr than Kuroiwa74 has also shown by electron microscopic cuchromatin22 . Since then this property of hetero autoradiognphy that the late-replicating or the chromatin has bel~n analysed in very diffen:nt sys heterochromatic regions condenSe faster than tems, and barring a fe,~' exceptions, in all cases, euchromatin during prophase. Thus, it seems that h .tcrochromatin has becn observed to be 'late' cellular regulatory systems control the condensation replicating (Figs. 1 and 5). Recently, the relation cycle of different chromatin materials in such a way ship b tween condensed chromatin and 'late' that heterochromatin is decondensed for only a brief replication has been analysed in clctail by Lima-de interval during the S-period, and this may also be Faria25 .26 , and it could be concluded that hetero related to the genetic inactivity or the repressed chromatin completes replication later than euchro state of heterochromatin. 2H matin. As :llso stressed by Lima-cIe-Faria , the (4) Nonhomologous or ectopic pairing -- A feature DNA replication is affected by differentiation, and of heterochromatin frequently refc:rred to is the as such, a change from heterochromatin to euchro , stickinf'ss' or the association of nonhomologous matin or vice versa may also be associatcLl with a regions at interphase:. The common chromocentres 56 change in replication timings . formed at interphase are the results of such associa i5 ii Another characteristic of heterochromatin re tions - . This is best seen in polytene nuclei. In 26 plication is the late beginning of D~A synthesis ; Drosophila, the Cen trom~ric heterochromatin of hO\\'e,"er, the asynchrony in the beginning may not different chromosomes fuse to form one common be as pronounced a~ in the terminal period anrl in chromocentre, while the in tercalary heterochromatin, some cases no asynchrony coulL! be rletected. Thus, distributed througholl t the length of the chromo in Drosophila polytene chromosomes. the (:ucluo somes. sho\\'s ' ectopic' pairing, or thread-like DNA matic and heterochromatic regions start DXA syn con taining in tcrconn~ctions between two nonhomo th sis eLt the san1l' time, but thl: hdCfOchromatic logous loci. Sl:veral studiesi8-81 Itave revealeclthat 5i 5n region" complete it later than euchromatin - . In the ectopic pairil~g sites show all the characteri tic CliiroJlo1Jl.1Is polytene chromosomes, hO\\'ever, the features of heterochromatin, namely a condensed heterochromatic bands both start ~l1ld finish D~A state, high susceptibility to induced breaks, intense S)'11tl1 sis later than cuchromation60 . fluorescence and late-replication5i, 79-83. Ectopic The rate of replication is alsu differc'ntial in pairings may occur by accidental sticking of tl e hetl:rochromatin and euchromatin. Several studies projecting loops in to nearby chromatin, and this in mammalian chromosomes have shown that may be an indication of redundancy of DNA heterochromatin takes lesser time to complete DKA or cl::rtain othe:r common properties of Lleoxynudeo ynth-sis than the correspomling euchromatin4-i.Gl-64. protein in heterochromatin. It is interestinO' t In a recent study on the hetcrochromatin replication note that facultative hcterochromatin2i often fails in hcdg hogs, it"has been suggested that heterochro to form such common chromocentrl:s 77 -84• G4 matin is replica inO' twice as fast as euchromatin . (5) Susceptibility to illdtlced brealwges - Various More r 'pli 011S are believcli to be replicating in factors arc known to induce chromosom,: hreaks85 . 43 synchrony in hcterochromatin ,6:;.G4. However, this Heterochromatic regions have been found to be replicati e behaviour may not be universally true especially susceptible to such induced aberrations for all the heterochromatic regions. Obviously, in both animal and plant ccllsii,85·94. in cases wher. both heterochromatin and euchro Mitomycin C has been used frequen tly to induc matin initiate DN synthesis at the same time, but chromosome aberrations and as recombinogen in the former finishes late, the rate of DXA synthesis higher organisms85-98 . In a study on the action in heterochromatin may not he hiO'hcr than ill of mitomycin C on the chromosome aberrations in euchromatin. However, in such cases, the amount Vicia Jaba, a correlation between breaks and the of Dl\' anLl its organ ization ill the t\l'O kinels of la te replication of heterochromatin has been demon chromatin have also to be take;. into consideration. strated92 . After in vivo synchronization of the c lIs Furth'r studil:s on tlw kinetics of replication in in the root tip of Vicia Jaba, the aberrations induced euchromatin and heterochromatin in different by mitomycin C were maximum during the late S, system are nleded for a clear unclclstanding ?f and of all the scrorable breaks, 93·5% were in the replicative organization of the various chromatlll heteroChromatin, indicating that the breaks are fractions. induced when the chromosomes are I' ·plicating92 ,93. It must be cmpha ized. however, that all 'late' A similar explanation has been put forward by replicating chromosome regions ne d not necessarily Haegle94 for FUdR induced breaks in the polytene be het rochromatic. In a nucleus, there are many chromosomes of Ch-irollom·us. Heterochromatic re replicating unitsS!I,6~.63-59, and these different units gions also show a hig'h frequency of induced somatic complete their DNA synthesis independently in the crossing over90, 91,95, 99-102. It is possible that these S-periorl, depellding on the amOlln t and organization two aspects of heterochromatin are manifestations of DN 32,59,69,70. Thus, some replicating units in of the same basic organization. SH.\H cl at.' ~.\TCRE 010 HETEROCHR():\r.\.TJ~ During interphase, the heterochromatic regions chromosome heterochromatin, show a position-effect remain condensed, and often different, such regions variegation, when placed in the euchromatic regions, associate into larger blocks of chromoccntres (cf due to a break between these cistrons and the Section III, 1 and 3), and this provides a basis for centromere103. This also suggests that there is a . misr pair' of the induced scissions in the DN P specific organization and regulatory action within fibrils, leading to chromosomal aberrations or cross the normally heterochromatic regions, and this may ing over. Late-replication may also favour' misre be disturbed when such genes are removed from pair '77. their influence. It is interesting to note that (6) Positiou-ejfect variegation - A variegated ex heterochromatin shows other types of gradient pression of a gene due to its new position ·near effects also, e.g. in the differential contraction heterochromatin is now regarded as a generality of following exposure to cold or colcemid. Thus, in vari gation and heterochromatin20. Position-effect a study on the heterochromatin in Indian Muntjac, variegation is seen when genes normally located in Comings45 observed that .. there is a gradient of euchromatin are brought within or in close proximity chromosome contraction with the least contraction of a broken heterochromatic region; a cis-arrange occurring in heterochromatic centromere regions ment is essential for variegation20. :\Iost of the and the greatest contraction occurring in segments earlier studies WerL; on n. melanogastcr19 and later on that are farthest removed from the centromere". extensive studies have been made on D. ~'irilis and The similarity in the gradient eHects in position mouse20 ,33. Studies in mouse on the position-effect effect variegation and differential contraction is variegation have been particularly rewarding, since striking, but it is not known whether these two in mammals, as discussed later, one of the X's aspects are related. in female is heterochromatinized in somatic cells. \Vhen there is an autosome-X translocation or inser IV. Facultative and Constitutive tion, the autosomal genes often show variegation33 . Heterochromatin The basis of variegation in these instances lies in Heterochromatin is novv usually grouped into two the inactivation of the transposed and normally broad categories: facultative and constitutive. This active gene due to its new proximity to the hetero concept; introduced originally by Brown27 , has pro chromatin. The' spreading-effect' associated with vided a basis for reasonable grouping of a wide, and position-effect variegation is also very interesting often confusing, array of instances of inactive or and important. There is a polarized gradient of condensed chromatin. Earlier, all such cases were inactivity emanating from the broken heterochro labelled simply as heterochromatin, and perhaps, matic region, and this affects the newly transposed this is one reason why so much ambiguity about the euchr matic regions. It is the variablity of the nature of heterochromatin has come to stay. . spreading-effect' that produces variegation. The Initially, Heitz104 classified Drosophila polytene inactivity or lack of realization of gene products of chromosome heterochromatin into rJ.- and ~-hetero these newly transposed regions is still explained in chromatin on the basis of their abilitv to uncoil. terms of heterochromatinization or compaction. Though this classification is still meiningful (see This, as pointed out by Baker20 , " in reality, exposes Section IV 2.C), this grouping does not provide easily our ignorance rather than our understanding". operative criteria and has not been much popular. Recently, Eicher:J:I has proposed an attractive On the other hand, the concept of constitutive and mechanism for heterochromatinization, and varie facultative heterochromatin has found wide accep gation in particular; this model can also be extended tance and usage. to g~ne regulation in general. On the basis of this Brown27 proposed that chromosomal regions which model, each gene is believed to have a starter and a show heterochromatinization in one homologue, but terminator, and heterochromatic regions are bdieved remain euchromatic in the other, should be termed to be a group of polarized starters and terminators , facultative' or . functional' heterochromatin. The working as a unit under the influence of a common regions showing heterochromatinization in both the receptor site. Thus, a new gene, brought near homologues in the same cell are termed . constitu heterochromatin due to breaks and rearrangement, tive' or 'structural' heterochromatin. too may become inactivated under the polarized influence of the ' receptor-starter-terminator ' 1. Ftlcultative Heterochromatin complex33. Best known examples of facultative heterochro Whatever be the mechanism of inactivation, the matin are the 'inactive' or the 'Lyonizecl-X' in phenomenon of V-type po~ition effect is a clec:r in.di female mammalsl05-107 and the paternal set of cation of the supragemc kvd of orgallIzatIon chromosomes in diploid male mealy bugs27 ,28,l08. of chromosomal activity and a hierarchy of levels (A) J1!lammalian X-chromosome - In all female of regulation. It is to be realized that the concept mammals, one of the two X's in somatic cells becomes of supragenic levels of control is fundamental to an inactivated and heterochromatinized and continues understanding of heterochromatin. The various to remain so in subsequent cell generatiolls105 ,106,109, properties of hderochromatin, like condl~nsatio.n, and this expresses itself in interphase as the sex 'late '-replication, 'inactivity', position-effect vane chromatin or ' Barr' bodyllo. At metaphase, this gation, etc., are not independent re~ponses of the inactive-X can be seen as the 'late'-replicating or individual units located in these regIOns, but they , hot'-X in female cells2:l,2'1,2(;,:l2,5:J,1l1-1l5 (Fig. 5). probably are collectively controlled by a ' master' This condensed and 'late'-labelled X-chromosome regulator (see also Section VI). in female mammals has been elegantly and exten Recently, Baker103 has provided evidence. for a sively correlated to genetic inactivity at morpholo revers\; type of position-effect on the r-DNA Clstrons gical, biochemical and molecular levels20- 34 . It has .in D. melanogaster. He could demonstrate that the been shown that the sex-chromatin is inactive in r-DNA cistrons, normally located in the X- and Y- RNA synthesis53 ,54; recently, some workers116,117 4- SH.\H et (/{.' X.\TCRE OF HETEROCHrW:YI.\TlX IHl.vL\ on the other hand, suggested that the 'inactive '-X synthesizes a small but significant TABLE 1 - HETEROCHROMATI1 5 SHAH (:( at.: ~.\.TlJRE OF HETEROCHl\O:\L\TJ:N matin. What should be realized is the dynamicity of cellular and chromosomaI activitv and also the cellular economy. If a particula~ chromosome or part of chromosome were always inactive A and functionless, why should the organism retain this unnecessary burden? But it seems natural that when a part of the genome is not required to function in a given cell or tissue, this may be hetero chromatinized and thereby made unavailable to the trallscribing systems of the cell. This is analo- ous to the repression of indiviclua I genes for the normal differentiation of an organism; only in the case of heterochromatin, groups of genes arc simul taneously repressed by processes which are likely to be different from the individual gene regulatory systems (see also Section VI). 1. Constitutive Heterocllrol1wtin (A) General aspects - Gnlike facultative hetero chromatin, constitutive heterochromatin is believed to be a 'perma'wntly' inactive chromatin, devoid of functional genes and is believed to be unable to support transcription in vivO154-156. f\.ccen t studies, howevL:r, suggest positive functions for constitutive heterochromatin, mainly related to structural organization of chromosomes. Yunis ami Yasmineh5 have also discussed the nature and function of o constitutive heterochromatin. Typically, constitutive heterochromatin is localized near centromeres (pericentric heterochromatin), Fig. 6 - Sex-chromo~omal hctcrochromatin ill ~omatic cells nucleolar organizer rl,gions (nucleolar heterochro of male and female somatic cells [Mall'S on the left and fema.les matin) and in some cases whole or parts of chromo on the right ,-ide. Euchromatic alcas On the X arc outlincd] "omes are of this type. Somdimes, constitutive [(A) The original 5%-type X; male with heterochromatic minute Y; female with. onc 'IYonized '-X; scen in mo,t heterochromatin is also located as small to very small mammals; (B) duplicate-type X;~ male with one-hal! X and segments along the length of chromosomes (inter entire Y heterochromatic (constitnti\-c): fcmale with one and calory hderochromatin). Association of cen tromeres a half X heterochlomatic (onc half in each X is constituti\e 'Nith constitutive heterochromatin now seems to be hetcrochromatin, in one X another half is facultatiYc hetero almost universal. All the l~xamined species have ch.romatin); seen in seyeraJ rodents and somc othcr mammals; (C) triplicatc-tvpe X: male with two-thirds X and entire shown the presence of constitutive heterochromatin o Y heterochromatic (constitntivc); f"male is normally XO, and repeated D~A sequencc:s •37 ,76,157-l66. The yet two-thirds of X is constitutive heterochromatin; seen in amount of pericentric heterochromatin varies with ]'vlicro(lIs oYegoIl'i; (D) quadruplicatc-typc X; r'lalo 'dth threc species from very small amounts to large blocks. fourths of X 7 SIl.\1·1 et al.:\.\TlJH.E (IF 1-1ETEH,()CIJH,();\!.\TJN An overall consideration of the various observa Brown27 ) contain repetitive DNAI58,I"O,161,162,195,2.l0. tions on chromosome banding, its relation to It has been shown that the DNA available for heterochromatin and repetitive j)NA would greatly complementary bindiug with isolated repetitive DNA help in our und(~rstancling of chromosome organi in the chromocentre of polytene nuclei remains nearly zatiou. I n every Iikel ihood, the bands ubserved the same as in non-pOlytene nuclci1"U,I!)", and it was on metaphase ciiromo~onws indicate local enrich condulled that while IX-heterochromatin does re ment of repetitive ~elllH;nces and constitutive plicate to some extent, f3-heterochromatin fails to heterochromatin. It is possible that the chromo replicate during polytenization1"u (Fig. 8). Althougll :-,omal proteins too have a linear differentiation along CI.-heterodlromatin docs not replicate as a whole, the 111(; kngth. of chn)mo~oml~s, and treatmen t with nucleolar organizer or the r-DNA located within 4 j rypsin and other proteolytic agenIs can thus permit this region:J· !1 docs replicate in polytene nuclei. visnalization of chromosomal bands due to differen This inllicates that within the heterochromatic re t ial anll specifIc rcsponsl~ of differen t chromosomal gions, some replicas may have their independent protein moil~ties to tlw treatmenls20H Further regulation I"iI. A similar situation presumably exists ~itudies are needed along th(~se lines. in other nrosophila species with respect to hetero (C) COn:;titUtilJC hetcrochromatin in Drosophila chromatin organization. Cousti tnti Vl~ heterochromatin in Jhosophila provides The under-replication of heterochromatin in a different aspect. As pointed out earlier, iu n. polytene nuclei is another example of differential IIICIIl'llO[;({stcr, proximal one-third of the X and entire replication of euchromatin and heterochromatin. Yare heterochromatic: besides, autosomal centro However, this may he an entirely different kind of meric regions also have hetl,rochromatie regions2Jo . regulatory system than tlw usual late-replication Jn polytene chromosomes, all heterochrolllatic regions of heterochromatin. Nevertheless, it is to be noted fuse together to form a COlllmon chromoccntre. that ill mitotic nnclei, the constitutive heterochro Heitzl04 charactl,rized the heterochromatin in n. matin of X and Y in J)rosophila are also latc-repli 215 tll-claJl.ogastcr as (J,- and f3-heterochromatin, and he cating . The Y-chromosome ]wterochromatin in sugge ted that hderocltromatic regions fail to multi J)rosoph£la is in tcresting in other respects as well. ply during polytcnic growth, resulting in an under It is wieldy belicved. tllat heterochromatin in general replication of the,;c regions in the polytene nuclei. and constitutive heterochromatin in particular is .Measurements of the DNA content in polytene genetically inert and does not transcribe in vivo. J.nd non-polytene nucleiaR.211-21:J have confirmed that However, Drosophila Y-chromosomal heterochro tlw polytene nuclei do not have DNA in exact matin is one instance where speciftc functions multiples of the non-polytene diploid nuclei. Studies at a specific stage of development could be attri with molecular in sit·u hybridization and specific buted, namely transcription of specific mRNA's to (;iemsa-staining lLave shown that both '7.- and [3 con trol spermiogenesis' ;;ol"a. Differen t regions of hdl,rochromatic regions of Heitz (both these can the Y-chromosome have been shown to have specific be groupetl as constitutive heterochromatin of functiollS in regulating spermiogellesis150-.l 5:J. While X y c A " , , , ,- :c-':g""'--." , I • " ' ~ I r n u ~ \ .. , j • " I I ~. : \ , B o .I,'i g. X .:\.- and Y -chromos(lIn,~1 hderochroma,Lin in Drosophila. i. (\) i\Iitotic chromosomes o( n. lJIc!allogasfer; entire Y and proximal one-third of X hetcrocllTOmatic (sh 9 SH.\H 01 al.: K.\Tl'RE OF HETEIWCHRO;\I.\TI2\" qualification. At metaphase, all the chron:oson:es herently inactive with respect to the known mecha arc maximally condensed. and. are almost mactlve nisms of gene function, i.e. constitutive hetero in RIA syn thesis. This certainly does not mean chromatin does not carry the so-called 'Mendelian that all metaphase chromosomes are heterochro genes' and cannot transcribe mRNA\'jG. Consistent matic! Heterochromatin is a relative tenn. When with this notion is the association of constitutive some part of chrol1lati!l behaves diff~rently fr~m heterochromatin with simple repetitive DNA, which 2J5 the rest, the concept ot heterochromat1l1 comes 111. is unlikely to code for any protein : . However, The condition in pdytene chromosomes illustrates several aspects need to be reconsidered before a different aspcct of heterochromatin characteriza concluding that constitutive heterochromatin IS tion. The nuclei are in permanen t in terphase230 inert in gene activity. and ar linearlY differentiated into bands and Genetic activity of DNA is usually studied in terbands. The· bands are the condensed regions in terms of transcription of specific RNA molecules, and are relatively inactive in I~NA synthesis23o.231. viz. mRNA, rRITA, tRNA. Is it necessary that At times, some bands decondense and begin active DNA exert its influence only through these RNA transcription. It may be asked then: should th~se molecules? Probably not. Products of regulatory banus also be considered as heterochromatic? genes rna y not be translated in to prot ins, but the These conden,;ed and inactive bands may be Bm~lo regulatory action is still achieved. Besides, there gizGCl to the heterochromatic chromosomes in are indications that much of the nuclear RNA never coccids, for example. In concknscd state they are reaches cytoplasm129 ,236-242. These observations inactive, but \vhen decondClIsed, they become active indicate that some DNA in the gl:'nomc may function in transcription. However, some vcry important in ways other than the classically established path differences between thl' two systems exist. ways of transcription and translation. Constitutive In the case of bands, both the homologucs are heterochromatin DNA may bl~ an example in this condensed or decondenscd, while in coccids, only category. one haploid set is inactivated. Besides. the chromo The transcriptive inactivity or inability of con someS of coccids are condensed aJ'd inactiva ted stitutive heterochromatin or repetitious DNA is also en masse, i.e. a un ified sigr al probably dicits not universally acccpted196. Flamm et al. H3 could hcterochromatinization in all the target chromosomes not find any Rl\'A complementary to the isolated ba~ds (supragenic regulatory system). .In the case of satellite DNA of mouse. Similarly, Sieger et of the polytene nuclcl. the activIty and condens~tlOn al.l55.156 did not find evidence for transcription of of Iifferent loci arc con trolled not by a sll1g1e constitutive heterochromatin in vivo in i11iC1'otztS stimulus, but bv individual respective regulatory 233 af[l'cstes. On the other hanel. some evidence for the gcnes . In non-polytene cells too, a similar t~anscription of the satellite sequl;nces 'in vitro has mechanism exi,;t~. In any given cdl, only a small been presentcd24l,245. In this context, the observa fraction of cr ne;; are active iii transcription, th-.: rcst tions on the centromeric heterochromatin in D. condCI~.secl~2.2~1.234. being inactive and Such ina.cti melanogastel' and the satellite DNA of mouse are vation of specific genes 111 a given cell type IS a very interesting19G.245. Evidences have been presented diffe[(;~ltiation equt:l to tile process of and deve1?p to indicate that the rcpeated sequenct's in DrosopJu:la. n1l'nt. Do we consider thes ll1actIve genes. which constitutive heterochromatin and the mouse satellite fonn the bulk of tIll' chromatin of a cell, as hetero DNA arc not simple tandem repeats196. Inter chromatin? Till'Y share \vith heterochromatin the spersed between the simple repeated sequences are prop rties .of trans.criptivl' inacti.vity, conden.sed more complex sequencl.:s, which have been provi stat\;, and In some lI1stances. pos,nbly latl; rephca sionally called the' spacer' DNA. These' spacer' tion also. However, it is important to note that sequences could bear important genetic information, the mechanisms bv which such inactivation is and the satellite DNA may be the true spacers for brought about in lleterochromatin and repressed these sequences. \Valker181 suggested that the genes arc different. satellite sequences might have originated by dupli The term heterochromatin may. with advantage, cation of ribosomal spacer sequences. Thus, it be restricted to those instances wl!er,; a snpragenic may be that while the satellite or repetitiv. levd of control is involved in bringing about con sequences in constitutive heterochromatin of centro densation, the apparent lack of activity and other meres are nonsense, as far as coding for proteins correlated manifestations. With this limitation, is concerned, the newly discovered interspersed the genes that are individually inactivated in sequcncesEJ6 may have vital information. This development \\"Quld not be considered hcterochro model for centromeric heterochromatin may be mati. This criterion may apply equ211y \\'ell to compared to the arrangement of ribosomal cistrons th large blocks of hete:ochromatin at centromer~s in the nucleolar-organizer region heterochromaf , and other locations, the 111 tercalary hcterochromatll1 where the consecutive ribosoma.l sequences are and the facultative heterochromatin. spaced by small sequences, which apparently do not I i implicit that heterochromatinization is also transCl ibe. Transcription of these interspersed the product of the regulatory processes operating complex sequences in the centromeric heterochro in the cell and, therefore. all kinds of heterochro matin of Drosophila and mouse has been indicated196. matin have the potentiality to revert to the The parallel bet\\'l;en the arrangement of functional so-called euchromatic state or to be genetically sequences in the nucleolar-organizer region and the active. Facultative heterochromatin, of course. is centromere is sign ifican t. since both the regions are well known (sec Section IV.l) to be either inactive classically considered as constitutive heterochro or active and euchromatic in different cell types of matin. It may be that all constitutive heterochro an individual. \Vith regard to the constitutive matic regions have their functional cistrons buried heterochromatin, the opinions vary. It is usually in repetitive or satellite sequences. The model believed that constitutive heterochromatin is in- o f constitutive heterochromatin with repetitive 10 SH.-\H d at.: X.\TURE OF HETEROCl-IRO:\L\TIX sequences serving as spacers for some other sequen tutive heterochromatins, though similar in many ces pro ides a basis for the proposed specialized of their responses, should be considered separately chromosomal 6hollsekeeping' fur ctions3,180, 181,196 of with regard to their roles in cell function, since it constitutive heterochromatin. is clear now that the two arc fuudamentally different Britten and Davidson238 have presented a vcry in their organization and functions. interesting model of gene regulation ill higher Facultative heterochromatin probably does not rganisms. It is postulated that every cistroll has have positive roles in chromosome activity. It is s me repetitive sequence at the initiation point and the absence of genetic activity in the facultatively that the variability and other aspects of these heterochromatinized chromosome regions that is provide a plasticity for gene regulation and moclu important in cell differentiation. For example, the lation (see also Georgiev for a similar concept239). , inactivity' of one X in somatic cells of female Recently, evidence has heen presented that mammals serves for dosage compensation. Simi Drosophila genome contains, on an average, a short, larly, other examples of facultative heterochromatin about 150 base pair'> long, middle-repetitive equence may be playing their role in differentiation and per approximately 750 bas(~ pairs of thl: singll: copy development through inactivity. However, it is DNA242; this observat;on is strikingly similar to the questionable whether the facultative heterochro postu.lation of Brittl:n and Davidson2;l8. If this matin is completely inactive. Then; are suggestions distribution of repetitive sequences holds true in that the' Lyonized-X' in mammals may be only gcn ral, which seems likely, we have a gl:noml: 01' partially inactivt;:Ja·34,127. Besides, it is also possible go..ni ion, in which the rl:petitive sequences play that the heterochromatic state itsdf may have its very ortant roles in thE regulation of ' l:uchro- own n:gulatory or other kincl of action. l11atic' es. \Ve have also seen that in consti- Constitutive heterochromatin has been implicated tutive heterochromatic regions too, there are ill a variety of functions. ~Vlajority of these can be , spac rs' of repetitive nature which may separate grouped together as chromosomal 'housekeeping' • more important (probably unique) sequences_ function.5,181. The almost universal association of Eicher33 has suggested a similar model with constitutive heterochromatin with centromere sug , starters' an,l 'terminators' for each gene ancl a gests, a pr£ol'i, that it plays important roles in unified COli trol of all these for inactivation in (i) chromosomal movements in mitosis and meiosis; facultati e heterochromatin. Thus, it may be that (ii) pairing of homologous chromosomes; and the whole genome of the eukaryotes is organized (iii) maintenance of chromosome in tcgrity, etc. 'jth 'sp cers' (which probably are of repetitive Chromosomal movements during karyokinesis are nature) in between the important functional genes. very precise and coordinated. Obviously, these are These spacers may serve to control the initiation, under a strict control. Cmtromere is indispensable tenninati n and modulation of transcription of the for these events, since acentric fragments often fail as ociated fu.nctional cistro~ls. The nature of to manoeuvre themselves for orderly segregation, spacers may vary in hetero- and euchromatin. In Constitutive heterochromatin, with its repetitive heterochromatic regions, especially thl: constitutive and other kinds of sequences (e.g. the 'spacers' 196 heterochromatin, the spacers may be of a different of Kram et al. , discussed above) may provide the dcgre of repeatedness, so that the \:'ntire region is attachment sites for the spindle fibres. Ultrastruc switched ff by a single stimulus. tural studies of centromeric regions have shown a very specialized structural organization of the In on instance of the constitutive heterochro 2 matin, transcriptive activity for 'specific functions kinetochore '11;-248. The relationship between these is known. This is the Y-chromosome of Drosophila, structural features, constitutive heterochromatin and repeated sequences, remains to be understood. which has already been discussed in Section IV.2.C. 246 This again shows that the constitutive heterochro Brinkley and Stubblefield suggested that the matin is not inherently inert or incapable of trans kinetochore DNA may transcribe special RNA's cription. The information in the constitutive at specific moments in cell cycle, which may code heterochromatin may be for some very specialized for the spindle proteins or other structural fea tures func.tions and events, as for example, the spermio in volved in chromosome movements during cell gen si in the case of Drosophila Y-heterochromatin. division. It is interesting to speculate that the so Th specialized nature of information requires that called 'spacers' of Kram et nl. 196 may have the tl e constitutive heterochromatin be active for only necessary information for some or all of these events. VdY brief periods and possibly only in some cell It is worth noting that polytene nuclei, where the types, depending on the nature of information chromosomes multiply but do not segregate, have carried in its different segments. It may be con almost dispensed with centromelic heterochromatin ceptual aud technical limitations only, which do not (see Section IV.2.C). Is it possible that since these permit us to understand and 'sec' the activity cells do not require the information for spindle p ttems of constitutive heterochromatin, when organization and associated events, the DNA con in l' ·ality they are there. Like the faculta tive hete trolling these functions is not replicatecl for the sake rochromatin, the constitutive hetcrochromcltin may of cell economy? It w:Il be interesting to examine also b con idered as repressed chromatin, the action the status of centromeric DNA in those polytene of which can be realized upon withdrawal of nuclei which can also transform into polyploid the r pres ive factors. nuclei; such nuclei would be expected to retain the • ccepting that heterochromatin cloes have func cen tromcric D~A in the same proportion as the r8st tion 1 roles in the cell, some possibilities may be of it. explored. Different workers have suggested various In addition to the involvement in centromeric 'functions' for heterochromatin. However, only activities, constitutive heterochromatin also serves rece11 tly the basps for these supposed functions are to prevent recombination in some very important being understood. The facultative and consti- and vital sequences, like ribosomal cistrons. No 11 SH.\H el al.: NATCl(E OF HETEROCHl{u"L\TI:\ chiasmata arc formed in constitutive hetcrochro 32. i.\IILL:R. U. J., A dv. 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