203

DIRECTIONAL EVOLUTION: A NEW IIYPOTIIESIS

Valent in A. Krassi lovInst i tute of Biology & PedoloryFar-Eastern Scient i f ic CentreUSSR Academy of Sciences690022, Vladivostok, USSR

Recelved June 29, L9783 l" lay 20, L979; January 4, l -980

ABSTMCT: Tf ie genome is considered as a teleological system capable ofadaptive response to environmental stimuli which induce differential under-repl icat ion or ampl l f icat ion of genes. Ttre underrepl icat ion of repet l t iveDNA is functional in controlling the onset of cel-1 differentiation and thet i rning of gene act iv i t ies. Ageing is conceived as an outcome of underrepl i -cat ion of repet i t ive genes interfer ing with the chromosome behavior, competi-t ive exculsion of recessive al leles reducing homeostasis, and heterochromatiza-t ion affect ing chromosome repl icat ion pattern and pair ing. TLre l imitat ion ofreproduct ive age is related to an age-associated decrease of recombinat ionalrepair and recEif icat ion of repet i t ive genes and also to distort ion of the sexrat io in the offspr ing. At least intermit tent sexual reproduct ion is neededto el iminate the destabi l iz lng affect of al le l ic competi t , ion in ageing clones.The ampl i f icat ion of repet i t ive genes nay promote autonomous prol i ferat ion oftumor cel ls. The ampl i f ied repeats induce breakage, provide insert ion si tesfor viruses and act as episome-like elements when excised. Environmentallyinduced changes of the repetitive DNA content result ln het,erochronies whichgive r lse to najor evol-ut ionary novelt ies. Ttr is process affects also thedevelopmental succession of isozymes, causing retent ion of juveniJ-e isozymepatterns in adul- ts. Ampl i f icat ion of preferent ial ly act ive genes explalnslast ing nodif icat ions and orthogenesis.

*

INTRODUCTION

In the current evolut ionary paradign, a populat ion of organisms isrecognized as a te leological system ( i .e. , a system capable of adapt ive responseto environmental s ignals). Among strbordinate systens, an organism has transientindividual adaptabi l i ty (modif icat ion) which is not t ransmit ted to i ts of fspr ing,whi le the genome possesses no teleological qual i t ies at al l and mutates occasion-al ly. However there are no phi losophical grounds for the bel lef that teleologyshould ar ise at the populat ion level on1y. In individual development, the geno-type behaves as a teleologieal system. Histor ical ly, the molecules of geneEicmater ial rnight have or iginated as protoorganismic aggregates, independent oftheir current funct ion. They have subsequent ly developed intr icate superstruc-tures - phenotypes - to increase their adaptabi l i ty in unpredictable environments.Davis (1978) has shown that Fisherrs theorem of natural select ion is appl icableto heterogenous populaLions of repl icat ing polymers. I t is reasonable to assumeat least residual adaptabi l i ty at the genotype level, manifested through indivi-dual development as wel l as from generat ion to generat ion. This assumption isnot Lamarckian, because Lamarckists claimed external, not internal teleology(there was also a most odous power-seeking group using pseudo-Lamarckian languageand therefore labelled neo-Lamarkian, though they had nothing in common withei ther Larmarck or science in general) . In fact, the concept of the genotype asa teleological system, propounded by Weismann and lately by Wil l ians (L966), canbe viewed as an extension of the Darwinian pr inciple.****t(******

EvoLutlonary Theory 4: 203-220 (Aprll' 1980)The editors thank J.L.King, G.A.Sacher, and another referee for help in evaluatingthis paper.

G) 1980, the author.

204V. A. Krassi lov

The genotype can be envisaged as a population of various cistrons performingtheir metabol ic and reproduct ive funct ions - t ranscr lpt ion and repl icat ion. Theenvironmental st imul i af fect ing transcr ipt ion cause adapt ive shi f ts in the di f feren-t ial reproduct ion of genes. Because survival of genes depends on the f i tness oftheir carriers, the genornic adaptations are usually advantageous for the phenotypes.SomeEimes rhey lnterfere with phenotypic viability or fitness. There is whereconvent ional natural select ion intervenes.

The unorthodox concept of direct ional evolut ion of the genome, supported bysome recent achievements of molecul-ar biology, encourages the causal analysis of manyhitherto largely unexplained phenomena, such as the regulation of gene activity indevelopment, ageing, carcinogenesis, and or igin of the evolut ionary novelty. Thisconcept al lows also a revaluat ion of nuny morphological , embryological , and paleonto-logical observat ions l inking developmental and evolut ionary processes. These obser-vations have found no place in the synthetic evolutlonary theory and have been putaside without much indicat ion of future study.

GENETIC CHANGES DURING DEVELOPMENT

According to the text-book not ion, every somatic cel l carr ies the same ful lcompl-ement of chromosomes and genes. The cloning technique based on this assumptionls part ial l -y successful in producing embryos from transplanted nuclei of somaticcel ls. I lowever the genome is largely inact lve at the onset of embryogenesis. Thegenes control l ing early di f ferent iat ion are most essent ial and evol-ut ionary conser-vat ive. Tfreir change during development is unl ikely, especial ly in somatic cel lgenomes which have passed through only a few nitoLic cycles. The notion that a full-grown individual can be produced from a nucl-eus of each somatic cel l is implausible,because i t is wel l known that cel ls become l-ess plur lpotent \^/ i th age (see Clowesr196l) .The organs which are shed and establ ished anel^7 from the same meristemaLic t issues,such as leaves, ant lers, or the exoskeletons of crustaceans, show direct ional morpho-logical changes (Severtsov, 1939) .

There is a growing body of studf-es suggest ing directed genet ic changes duringdevelopment. The genome of higher organisms consists of unique and repeated sequen-ces. The lat ter form more or less highly repet i t ive DNA consist ing of the genesfor r ibosomal RNA, transfer RNA, 55 RNA, and histones, as wel l as the essent ial lynon-transcr ibed redundant or satel l i te sequences local ized in the pericentromeric,telomeric, and intercalary heterochromatin regf-ons, and the intermediate repet i t iveDNA, which is most ly interspersed betr^/een uniqtre sequences. The repet i t ive DNA,rich in adenine and thyrnine, is rnainly slow-replicating. In the human genome thereis about 6 per cent of the fast-repl icat ing intermediate DNA and 30 per cent of s lowrepeated sequences f inely interspersed with non-repeated trni ts (Cinel l i and Corneo,L976). The slow-repl icat ing DNA is potent ial ly l iable to under-repl icat ion. Themost evident ehanges during development affect the rei terated sequences. Hetero-chromatin is drast ical ly under-repl icated in polytene nuclei (Rudkin, 1965). Thereare t$/o types of chromocentr ic heterochromatin - alpha and beta, dist inguished asrelat ively dense and di f fuse areas. Whi le the alpha type, forming centromeric andpericentr ic heterochromatic regions, is t ranscr ipt ional ly and repl icat ively inact ive,the beta type is transcr ibed and repl icated to the same extent as the euchromatin(takhot ia, L974). I t is suggested that the polytene nuclei do not require a kineto-chore because they never re-enter the mototic phase. The intercalary heterochromatinis also under-repl icated during pol-ytenizat ion (see Barr and El l ison, L972). Accor-ding to Leibovitch (L977), even the unique sequences which are transcr ipt ional lyinactive in a given cell Eype or tissue are underrepresented in the polytene chromo-somes. This si tuat ion shows the interdependence of t ranscr ipt ion and repl icat ion.

The r ibosomal cistrons (rDNA) located within the alpha heterochromatin behavedif ferent ly from i t . They repl icate during polytenizat ion, though to a lesserextent than euchromatin. Underreplication of rDNA is a widespread phenonenon,

205Direct ional evolut ion

resulting in delayed development and an inviable phenotype (reduced bristle orbobbed phenotype in Drosophi la).

Detectable changes during development occur in the innnunoglobulin genes.The llglrt and heavy chains of iuununoglobulin mol-ecules consist of variabfe (V)and constant (C) regions. In ernbryonic cel ls, the V and C genes are separatedby intercalary sequences while in lymphocytes they are joined. Hozumi andTonegawa (L976) have supported the excision model of DNA change during differenti-at ion of lymphocytes. Al ternat ively, the underrepl l -cat ion of intercaLary repet i-tive DNA can be responsible for the joining of V and C genes and simultaneousact ivat ion of V. Only one al lele is expressed in any given lymphocyte. Hozumiand Tonegawa have suggested the loss of one chromosome followed by reduplicationof i ts homologue. The segregat ion of al le les in lymphocytes can be related alsoto the converslon or competi t ive exclusion of al le les (see below).

The relative amount of repetitive DNA undergoes nore or less evident changesin ernbryogenesis of many organisms (e.g., in Cycl-ops: Beernan, L977; for reviewssee also Yunis and Yasmineh, 1971; Tobler et al- . , L972). Heterochronat ic chromo-somes or segments can be lost, as in Ascaris megacephala. EuchronatLzatLon is acharacter ist ic feature of ear ly embryogenesis. According to Nur (L967 and subse-quent studies), euchromatizat ion is best manifested in male mealy bugs (Homoptera).

Woodcock and Sabatani (1975) have found underrepl lcat i -on of intermediaterepeats and possibly also adJacent unique sequences in larval Drosqphila. DNA andsupported earl ier suggest ion of Kaufmann (1946) that the i . t t . . "al"ry t t" t . rochro-matin represents regions of underrepl icat ion in the chromosome arms. They ci tedmany cases of DNA loss during development ( in chironornids, Ascaris, etc) as wel las of DNA increase in specific chrornosome bands in Sciara and Rhynchosciara. Atthe same time they acknowledged only one wel-l--establ-ished case of specific geneampli f icat ion, the rDNA magnif icat ion associated with the bobbed phenotype, whi leno amplifi-cation of the structural genes for globin or ovalbumin has been shown inthe t issues producing large amount of these proteins. However, the experimentsof Strom et al- . ( f978) have shown t issue-specif ic ampl i f icat ion of regulatory andstructural genes during di f ferent iat ion of chicken cart i lage and neural ret ina.Al t et a l - . (1978, c i ted in Strom et al . , L978) have revealed ampl i f icat ion ofstructural genes in cul tured mouse cel ls. Perlman et al . (1976) showed di f fer-ences in the DNA sequences of var ious cel ls in Xenopus. These studies are consis-tent with earl ier cytological observat ions of var iable amount and appearance ofheterochromatin in di f ferent adult t issues.

The funct ional role of underrepl icat ion of repet i t ive DNA during developmentcan be rnany-sided. The deletions of the ribosornal RNA and transfer RNA cistronscause a decreased number of cel ls per organ, delayed development, and retent ionof fetal features (see Yunis and Yasmineh, 1971). I t is conceivable that under-repl icat ion of these cistrons affects the repression of mitot ic spindle formationand the onset of cel l di f ferent iat ion.

The underrepl icat ion of the histone genes may be essent ial in regulat ingtranscr ipt ion rates at nany loci . The redundant sequences may funct ion in (1)

chromosome behaviour during cel l cycles, and (2) t inr ing of successional gene

act iv i t ies.Barr and El l ison (L972) reviewed earl ier works on ectopic pair ing ( that is,

pairing between non-homologous positions along the chromosomes) and confirmedthat AT-rich bands were involved. Conventional pairing of mi-totic and meiotic

chromosomes depends on both the large blocks of heterochromatin and the inter-

spersed repet i t ive sequences. Heterochromatin seElments can cause aggregat ion of

"hro*o"o*." , forming chromocentres. According to Yunis and Yasmineh (1971), the

pair ing through heterochromatin is an important in i t ia l step in synapsis. I t

was suggested by Stern and Hotta (f969) and other authors reviewed in Burnhamet al .-( fg72) that synapt ic pair ing, instead of fol lowing the classical "zipper"model, occurs at selected si tes specif ied as the short late-repl icat ing sequencesdistr ibuted along the chromosome. Elast ic l igatures between homologous regions,

206 V. A. Krassi lov

suggested by Maguire (1974), may also be related to the interspersed repeats.Chiasmic pairing is rare or absent in the heterochromatic regions (John and

Lewis, L965; see also Macgregor and Andrews, L977; Jones, L97B). This is conside-red to be a device for protect ing the rDNA and other rei terated genes from crossingover (Yr:nis and Yasmineh, L97I). However, the differences between replication ratesnray cause breaks and pair ing of non-sister chromatids at the junct ions of fast andslow repl icat ing segments (see Subrahmanyam, L977; Doyle, 1978). Yamamoto andMiklos (1978) have shown that the amount and posit ion of recombinat ion on each hete-rochromatical ly deleted X chromosome i-s substant ial ly di f ferent from that of anormal X. Further evidence of the effect of heterochromatin on recornbination camefrom the studies of supernumerary chromosomes, or B chroupsomes, consisting mainlyof heterochromatin. I t is known that Brs enhance recombinat ion of norrnal or A,chromosomes in rnaize, rye, wheat, etc. there is also a dosage effect, for threeBrs cause more enhancenent than one. Ward (1973) has conf irmed experimental ly thatonly heterochromatic segments of Brs affect recombinat ion. Rtroades and Dempsey(L972) have suggested that B's cause Longer melot ic cycles wlth therefore more t imebeing avai lable for recornbinatf-on. I lowever, the enhancing effect of Bf s is c lear lyrelated to their interact ion wlth heterochrornat ic regions of A chromosomes, whichoften lose their heterochromatic knobs by the lat ter st icking to the heterochromatinof Brs. An abnormal A chromosome 10 (Kl-0), l t l th addit lonal heterochrornat ic segmentin the long arm, can also enhance recombinatlon by interactlon wlth a knob on chro-nosome 9 (Nel , L973).

To sumrnarize, the process of rec.ombinatlon depends on the alternation of seg-ments with di f ferent repl- icat ion rates along the chromosome. I t ls af fected bycentromeric heterochromatin ei ther through interact ion with interspersed repet i t ivesequences or through interst i t ia l t ranslocat ions of the kind described by l loehnand Mart in ( l -973). Changes of chiasma frequencies wouLd then be associated withpara1Le1 changes in the amount of satel l i te DNA. An age-associated decrease ofchiasma frequencles in mice was f i rst descr ibed by Crew and Kol ler (L932) and con-f i rmed by subsequent observat lons reviewed in Luthardt et al . (1973), wal lace andMacSwl-ney (L976), and King and Hayman (1978). Henderson and Edwards (1968) haveshown that the ehanges in chiasma frequency are due not to terminal izat ion ( i .e. ,

loss of once-formed chiasmata) but rather to genuine decrease a1-ong the | tproduct ion

l ine" of oocytes. Speed (L977) has suggested that chiasma frequencies are respon-sive to nutr ient gradients. King and Hayman (1978) observed seasonal var iat ion ofchiasma frequency in rept i les. Ihey argued that environmental factors, especial lytemperature, were more important than putat ive internal control . However, the tem-perature and other environmental factors nny act through thelr effect on inLernalcontrol , that is, on repet i t ive DNA. Rees and Naylor (1960) and Couzin and Fox(L974) revealed developmental variation of chiasma frequency in anthers of rye andtul ip. Meiosis in the pol len-mother cel1s proceeds in a wave away from the vascularbundle, and chiasma frequencies increase in the same direction. In the developmentof tul ip anthers, this process is associated with an increase of total chromosomelengths. I t appears that in this case the chiasma frequencies are direct ly relatedto ampl i f icat ion of (presurnably repet i t ive) DNA.

The role of interspersed repet i t ive DNA in regulat ion of t ranscr ipt ion has

been discussed by Bri t ten and Davidson (1969), Georgiev et al . (L974), Hol l iday andPugh (1975), Korochkin (1977), and other authors. I t was suggested that regulat ionis achieved by sirnul taneous formation of loops containing ident ical repeats or by

a suppressive effect of the short- l ived RNA transcr ibed from intermediate repeats.Kaufmann and lddles (1963) and later Zucketkandl (1976) have postulated a control-

l ing funct ion of AT-r ich bands in gene act iv i ty, expressed cytological l -y by puff ing(see also Dickson et al . , L975). I4y suggest ion is that the interspersed repeats

suppress cranscript ion of adJacent structural genes or batter ies of structuralgenes (depending on the organizat ion of genome), possible by attract ing histonepart ic les. Because these interspersed units are slow-repl icat ing' they are systena-

i ical ly underrepl icated in the course of successive mitoses and their ef fect on

adJacent structural genes decreases (presurnably due to release of histones and repat-

teining of nucleosomal structure). After a number of mitoses, at a certain stage of

Directional evolution 207

underrepl icat ion of repeats, a structural gene becomes accessible to a transcr i-bing enzyme. In this way the interspersed repeats might regulate the successionof gene action by counting the preceding mitoses. In the ease of irnrnunoglobulin,the underreplication of repeats between V and C genes night turn on V. Anotherconsequence of underreplication is the creation of a new gene by jolning tvro non-repet i t ive units.

Posit ion effect in relat ion to larger blocks of heterochromatin is expressedin sequent ia l gene act iv i ty (e.g. , in Saccharomyces: Tauro et a l . ,1968; Mort imerand Hawthorne, L973) and the modified activity of genes causing variegation (seereview by Quir6s, L976). The effect of heterochromatin is fel t also in the dia-chronous DNA synthesis in heteromorphic chromosome pairs observed by Latt (L975),Lau et a1. (L977), and other authors.

According to the chromosome fleld theory (lima-de-Faria, L976 and earlierwork), the posiEions of genes are specif ied with respect to the pr ime organizers -kinetochore and telomere. AmpS-ifieation of telomere heterochromatin causes dis-placement of genes maintainl-ng constant relation to the prine organizers. Thekinetochore serves as a reference point in the chromomere-size and replication-gradient f ie lds. The increase of satel l - i te DNA is usual l -y conceived as occasional(ampl i f icat ion ln a single chromosome during meiosis and subsequent spread toothers). However, in the l ight of the chromosome f ield theory, paral leL ampl i f i -cat ion in both homologues appears more probable, the more so in that reconbinat ionin heterochromatic regions ls arrested.

Directed genetic change during development comprises also such processes asgene conversion, magnif icat ion, dosage compensat ion, and segregat ion of al le les.Gene conversion, or the ehange of a recessive al lele to i ts dominant al ternat iveduring development, is regulated by transposible elements. The models of conver-s ion are reviewed in Br ink ( f973), Birky and SkavarLL (L976), and Ki tani (1978).Ttre regulat,ory elements have been compared to bacter ial episomes, but there areimportant di f ferences between them (see Doerschug, L976). These elements appearrather l ike excised segments of repet i t ive DNA affect ing gene expression and ur i to-t ic recombinat ion at the points of their reintegrat ion into Ehe chromosome.

In the case of DNA def ic iency, normal- synthesis of rRNA can be restored bythe process cal led rnagnif icat ion and ascr ibed to unequal s ister-chromatid exchange(Ritossa, 1968, L973; GrazLani et a l - . , L973i Tartof , 1975). Because in Drosophi lamelanogaster sister-chromatid exchanges do not occur in the heterochromatlc regions(Dolf in i , 1978), i t seems more plausible that magnif icat ion is due to paral lelampl i f icat ion of r ibosomal cistrons in both sister chrornat ids (see Meyer and Hennlg,L974; Tartof and Dawld, L976).

A regulaEory compensat ion of rRNA synthesis also occurs, evident ly on thesame basis as the dosage compensat lon of sex- l inked genes in males. Ktresin andLeibovitch (1974) have used the single X chromosome from rnale larvae of Drosophi-lamelanogaster as a template for RNA synthesis by Escherichia col i polymerase. Theeffect of dosage conpensat ion in this heterologous system is the sane as in theintact cells. Thus regulatory genes in the autosomes and Y chromosome are notinvolved in the dosage-compensat ion increase of RNA synthesis, which can rather berelated to competi t ion of genes for t ranscr ibing enzyme. The competi t ion of homo-logous genes in the two X chromosomes of females might result in a decreased levelof RNA synthesis in each of them and also in more exact tuning of gene act iv i t iesin general - increased developmental homeostasis of females. Schwartz (1971) hasdeveloped a model for regulat ion of gene act iv i t ies by cornpet i t ion of al le les. Anumber of st .udies (reviewed in Eanes, L97B) conf irmed an effect of heterozygosityon developmental homeostasi"s. Competition in a heterozygous system would resultin less act ive transcr ipt ion and underrepl icat ion of a recessive al lele and thedosage-compensatory act ivat lon of a dominant al le le. One can assume competi t iveexclusion of recessive al leles through a number of mit .oses.

In the case of sequent ia l segregat ion of a l le les (e.g. , dur ing di f ferent ia-t ion of lymphocytes) and isozyme transi t ion the outcome of competi t ive interact ionbetween alleles evidently depends on intercellular environments. The ontogeny of

208V. A. Krassi . lov

lactate dehydrogenase is a paradigm example of d i rect ional shi f t in the isozymepattern induced by the oxygen supply, the state of cel l d i f ferent iat ion, and thephysiological funct ion of muscular eontr ict ion (Market, L965; Morr is et a l . , L9763Kolombet , L977; Frenkel and Hart , L977).

The segregat ion of a l le les and the homogenizing processes of conversion and

compet i t ive exclusion of recessive al le les may be responsible for d isrupt i -on of

developmental homeostasis in ageing organisms and the fading of heEerosis in the

course of vegetat ive propagat ion. Dar l ington (1958) has observed var iat ion anong

vegetat ive propagules persist ing over subsequent cycles of vegetat ive propagat ion.

Breese et a l . ( f965) as wel l as Shimamoto and Hayward (1975) have shown that her i t -

able var i .at ion among vegat ive propagules is due to genet ic segregat ion which is

age-dependent: only young clones segregated, whi le the old ones, maintained by

vegetat i -ve propagat ion, d id not. The decl ine of synthet ic var iet ies was relat .ed

to segregat ion. These studies show actual s igni f icance of sex in maintaining

developmental homeostasis .

AGEING AND CARCINOGENESIS

Senescence is of ten at t r ibuted to an accumulat ion of defects (somat ic muta-

t lons) disrupt ing the genet ic program. The accumulat ion of defects, instead of

being errat ic, can be related to the direct ional developmental DNA ehanges out l ined

above, i .e. , the underrepl icat ion of repet i t ive DNA, heterochromat izat ion of in-

act ivated structural genes, and compet i t ive interact ion of a l le les. Indiv idual

l i fe histor ies can be conceived as cycl ical , beginning with euchromatLzat ion due to

heterochromat in losses and act ivat ion of structural genes and ending in heterochroma-

t izat ion associated with decreased act iv i ty.The occurrence of s imi lar ageing of paramecia and human cel ls having di f ferent

genome organizat ions (Roderrnel and Smith-Sonneborn, 1977) suggests general regular i -

t ies of senescence. Though ageing in mult icel lu lar organisms nay not be reducible

to local fa i lures (Rosen, 1978), i t is undoubtedly associated with an increaslng

number of non-funct ional ce1ls. I t was shown by Hayf l ick (1965) , Hol l iday and Tar-

rant (1972) and Orgel (L972) that human somatic cel ls have a l imi ted in v i t ro

l i fespan, inversely related to the age of the donor. Schneider and Mitsui (L976)

reported a stat ist ical ly s igni f icant decrease in the onset of cel l -cul ture sene-

scence, in v i t ro l i fespan, and cel l populat ion repl icat ion rates of cul tures der ived

from old donors.A theory of ageing as a progranrned process has been expounded by Krooth ( I974),

Berdyshev (1977), and other authors. The appearance of enzymes wj- th al tered proper-

t ies in ageing cel ls both in v i t ro and in v ivo (reviewed in Krooth, L974) has led

to assumption that ageing is related to isozymic shi f ts dur ing development. As sug-

gested above, developmental homeostasis can be disrupted by the homogenizing ef fect

of a l le l ic compet i t ion. The isozymic shi f ts can also be related to al le l ic compet i -

t ion in the changing genet ic environment. I t is af fected by t ransiEion from prol i -

ferat ion to di f ferent iat ion of cel ls, as exempl i f ied by the LDH isozymes (Frenkel

and Hart , L977). According to wada (1974), the RNA released from nucleol i has a

cr i t ical i rnportance for spindle format ion and ce1l d iv is ion. He pointed to degener-

at j -on of nucleol i in ageing cel ls. Nucleolar volumes are known to be proport ional

to the number of rDNA copies per nucleolar-organlzer region (Mi l ler and Knowland,

1970). I t is conceivable that the underrepl icat ion of rDNA dur ing successive rnl to-

ses can suppress prol i ferat ion of ce1ls. Vi lenchik (1970) postulated a decrease of

rRNA synthesis wi th age, and MuradLar. (L97 7) observed 3-or 4-fold decrease of the

rRNA-to-mRNA rat io in post-natal rat l iver cel ls. However in old rats th is rat io

suddenly increased (due to mal ignant t ransformat ion of cel ls?) Lezhava (L977 ) repor-

ted a decrease of RNA synLhesis in the centromeri-c region of A1 in hunans over

eighty. In th is age category he establ ished also considerable change of intercalary

heterochromat in due to heterochromat izat ion. The age-associated heterochromat izat ion

and increase of h istone Hl content was shown also by Khi lobok et a l . (L977). Indirect

evidence of rhe funct ional ro le of h ighly repet i t ive DNA in promot ing cel l prol i fera-

209Directional evolutlon

tion is provided by a significant correlatlon between longevity and Y-chromosomelengths (see Cherkasskaya, L977),

Cel l senescence is associated with aneuploldy (Luthard et al . , 1973; Jacobset a1., L976), s ingl-e-strand breaks (Pr ice et a l . , L97L; Chetsanga et aL., L977)and decreasing repair capacity (Little, L976). ALl these phenomena can be explalnedby satelJ- i te-DNA losses and/or heterochromatlzat lon. Satel l i te DNA ensures properbehaviour of chromosomes in mltosis and meiosis. The spindl-e-fiber proteins areattached at the centromere and at heterochromatic knobs. Ttre firmness of attachmentand the centromere strength depends on the anount of satellite DNA (Walker, L97L).Ttrerefore underrepl lcat ion of satel- l i te DNA would affect centromere potency andthe separat ion of homol-ogues, causing non-disJunct ion and aneuploidy. Other sour-ces of aneuploidy are related to the role of the centromeric heterochrornatin andlnterspersed repet i t ive sequences in aggregat ion of chrornosomes, synapsis, andchiasmic palr ing (see above). Underrepl icat ion interferes with these processes,predisposing chrornosome losses. Heterochromatizat ion can also add to aneuploidythrough i ts effect on the repl icat ion rates. A mechanism of chromosome losses inthls case can be perceived by anal-ogy with aneuploidy in TriticaLe. In this hybirdplant (wheat x rye), the rye chromosomes often form univalents and contribute toaneuploldy because they have more fel-omerLc heterochromatin than the wheat chromo-somes and fai l - to accomplish repl icat lon before the prophase of melosis (Thomasand Kal- ts lkes, L974).

Luthard et, al. (L973) have discussed the rel-ation of aneupl-oldy to the age-associated decrease of chiasma freguency. The decrease of chlasmata ls accompaniedby a signlficant increase of unlvalents, preferentiall-y among small chromosomes,which have fewer chlasma. Though aneuploldy does not inevitabl-y follow, at least'some of the univalents may be lost. The effect of maternal- and paternal age inchromosome anomalies is discussed by Penrose (f933), PoJ-ani (1969), Zeuten et al .(L973 and other authors. The increase of aneupl-oidy ln women 45-64 years old dueto sex-chromosome loss is associated with decreased sexual act iv i ty (Jacobs et al . ,L976). These observat lons probably ref l -ect more general assoclat lon of aneuploidywith inact ivat ion and concomitant heterochromatlzat ion.

Other genetlc events associated wlth senescence - chromosome breaks and de-creaslng repair capacl- ty - can also be related to heterochronat lzat ion. Since hete-rochromation affects repl- lcat lon rates, heterochromatizat ion would dlsrupt the repl i -cat l -on patterns, causing breaks. This suggest ion ls supported by assoclatLon ofexrra heterochromatin wlth mult iple breaks ln Drosophi l -a (galurat , L975, L977). Hatchet al . (L976) related the hlgh proport ion of metacentr ics in kangaroo rats to extraheterochromatin. They concluded that chromosome rearrangements were controlled bysatel l iLe DNA. Recent ly Yoon and Richardson (1978) have observed clusters of breaksat intercaLaty heterochromatin sites, producing chromosome rearrangements, in Hawai-i -an specles of Drosophi la

The decrease of repair aclivity in ageing cells lndicates that DNA danagebecomes less accessible to repair enzymes. The access of repair nucleases to darnagedsites depends on the nucleosomal structure of chromatin: excision repair is rapldin the l inker DNA regions whi le ln the core part lc les, or nucleosomes, i t is hamperedby histones. Repair is most act ive ln prol i ferat ing ce1ls (Cleaver, 1978). The

heterochromatizat ion process conceivably affects repair by al ter ing the repl lcat ionrates and/or because of the DNA condensat ion in the l inker f i laments. The effect ofrepet i t ive DNA interspersion on repair was discussed by Kram et al . (L972).

Ageing affects the genotype of progeny prirnarily by increasing chromosome ano-

mal ies. Decreased recodbinat ion diminishes genet ic var iabl l i ty. Less obvious effects

are related to recornbinat ional repair and rect i f lcat ion of repet i t ive genes. Bern-

stein (L977) has suggested that metot ic recombinat ion is essent ial ly a repair process

conserving the germ 1ine. Analdi et a1. (1973) have postulated that al l rei teratedgene famil-ies are establlshed anew at each meiosis: each of the repetitlve segments

is excised in the form of a circular nonogenic DNA molecule, which repllcates and

then reintegrates into the chromosome. It is known also that ageing has some effect

2L0 V. A. Krassi lov

on the sex rat io of of fspr ing. The fol lowing mechanism can be suggested. A spermwith more heterochrorDatin in i ts genome is npre f i t in the sense that i t preferen-t ial ly fert i l izes the egg. In maize, a sperm carrying supernumerary B chromosomesusual ly fert i l izes the egg, whi le i ts s ister sperm without Bfs unites with thepolar nuclei (Roman, L947, 1948; Car lson, L969, L973>. In nan, a sperm carry ing aY chromosome is evident ly more f i t but as the underrepl ieat ion of heterochromatinprogresses, i ts f i tness decreases. I t fol lor^rs that l imitat ion of reproduct ive agemay have an even more important stabi l iz ing effect than is usual ly conceived.

Carcinogenesis contrasts with ageing in i ts v igorous cel l pol i ferat ion, butthere are also many points of resemblance. Both are associated with aneuploidy,chromosome breakage, rearrangements, and decreased repair act iv i ty (Tiepolo andZuffardL, L973; Berghe et al . , L977; Idarx, L978). These simi lar i t ies suggest simi-lar causes, that is, changes of repet l t ive DNA. According to the above hypothesls,the dtf ferent classes of repet i tLve DNA control the onset of cel l di f ferent iat ionand the t iming of structural gene act iv i t ies. l lada (1974) has argued that v irusesand other exogenous agents causing cel1 lesions are responsible for precondit ionsof cancer only, whi le carclnogenesis i tsel f depends on cont inuous ni tot ic spindleformat, ion as a prerequisi te of cel l dlv is ion. He postulated two convent ional cate-gorles of genes - the spindLe-fornat ion inducers (SFI) and repressors (SfR). SFRdefic iency in stem cel ls causes unrestr icted prol i ferat ion wLthout enter ing thedif ferent iated state. I t seems l ikely that spindle-f iber formatlon is induced byrRNA, and possibly also by other non-translated RNArs transcr ibed from the repet i-t ive DNA, which can funct ion as SFI or SFR depending on the number of repeated copies.

Renaturat ion kinet ic studies show that repet i t ive DNA is sensit ive to var lousagencies, such as growth media, temperature, v iruses and increased act iv i ty (seeRomanov and Vanyushin, L977). Sudden changes of DNA redundancy in certain celll ineages may disrupt the t iming of cel l - di f ferent lat ion and give r ise to autonomcustumcr growth. The anpl icat ion of interspersed repeats would al ter nucleosomalstructure, resul t ing in a restr ict ion of both t ranscr ipt ion (see Fl ickenger et a l . ,L973) and repair (Marx, L978; Cayama et al . , 1978). l t re arnpl i f ied slow-repl icat lngDNA would affect the pattern of chromosome repl lcat ion, causing breaks and alsoproviding the insert lon si tes for v iruses which induce addit ional breaks (see Yunisand Yasmineh, 1971). I f excised, the ampl i f led repeats may act as episome-l iketransposible elements involved in mal ignizat ion.

At lease some character ist ics of carcinogenesis support these suggest ions.Mutagenic and paramutagenie effects of var iat ions in the redundancy of repet i t iveDNA have been demonstrated in plants and animals (see Brink, L973; Phi l l ips et al . ,L974). Atkin and Baker (L977) have related the pericentr ic inversions associatedwith cancer to heterochromatin changes.

Sachs (f978) ascr ibed reversions of mal ignancy of leukaemic cel ls to trans-posible elemenLs. Anderses (1978) has shown that neoblastomas in f ishes arecaused by a gene (Tu) present on di f ferent chromosomes in several copies. Tu islocated terminal ly on sex chromosomes and can be dose-eompensated by the autosomalTu when deleted. These features are suggest ive of ampl i f ied repet i t ive-DNA segments.Tu possesses also some virus- l ike propert ies. According to Gatef f (1978), the mu-tant genes causing tumors in Drosophila are distributed over the entire genome andinvolve developmental genes which control crucial events during di f ferent iat ion ofcel l types or t issues. Many virus- l ike part ic les occur in the nuclei of al l neo-plast ic cel ls. She concludes that ' r tumor genes in Drosophi la represent nothing more

than fundamental- developmental genes which, during normal development, affect theprol i ferat ion rates and the di f ferent ion of cel- lsrr (1.c. ' p.1458).

These considerations suggest the normaLizing of repetitive DNA content as aprerequisite of cancer therapy. It is known that in maize the B chromosomes are

more effective when in odd nurnbers, while they neuttalLze each other when in pafrs.

The ampl i f icat ion of satel l i te DNA may also be neutraLized by inject ions of comple-

mentary amounts of the repetitive DNA fractions.

zLLDirectional evolution

EVOLUTION

Developmental and phylogenetic aspects were intimately related in earlyevolut ionary concepts such as the t t lanstt of K. von Baer, E. I {aeckel , and E. Cope.It was assumed that the heterochronies - accel-eration, retardation, tel-escoping,and repatterning of developmental stages - are responsible for evolutionary novel-t ies. According to the orthogenesis concept of Th. Eirner (1898) the di f ferencesbetween closely related species were analogoust to those between successive develop-mental stages. A theory of evolut ion affect ing al lometr ic growth rates (with paedo-genic mutat ions as an extreme case) as developed by Garstang (1928), Bolk (L926) ,Goldschrnidt (1940), De Beer (1951), and later by Romer (L972), TakhtaJan (L972),

Gould (L977), and other authors.It was hypotheslzed above that changes in the repetitive DNA content affect

the t i rning of cel- l di f ferent iat ion, causing heterochronies. Increase or decreaseof the intermediate repet i t ive units seens to be related also to honceot ic mutat ions,the shift of certain cell-s lnto different developmental pathways (see Ouweneel,L976). The analogous environmental-ly induced shlfts - homoeotic phenocopies mayhave a simi lar cause.

These suggest ions are supported by (1") experimental induct lon of repet l t iveDNA changes, (2) variability of DNA redundancy in natural- populations, associatedwith variation in developmental rates and morphology and (3) differences in amountof repet i- t ive DNA between races and closely rel-ated species.

Durrant (L962) dlscovered environmental induction of heritable changes inf lax (Linum usitat issimum), var lety Stramont Cirrus. Ttre appl icat ion of ni t rogenand phosphorus to pl-ants grown in a heated greenhouse caused 3- to 6-fo1d changesin the plant weight. The large (L) and small (S) forrns have been maintained fornpny generat ions, showing stable inheri tance irrespect ive of the subsequent lyappl ied soi l nutr ients. I t was found also that af ter 5 weeks of induct ion L and Sdiverged in nuclear DNA content, L having 16 per eent more DNA than S (Evans et al . ,L966). Under low temperatures the DNA content gradual-l-y reverted toward that of theoriginal form. This process could be stopped at any selected level- of the DNA con-tent by heat ing in a greenhouse (Joarder et al , , L975). Cal l is (L975) and Tirmnisand Ingle 0973, L974) have shown that the intermediate repeats were preferent ial lyaffected, though the ampl i f icat ion of some unique sequences was also suspected.

A mechanlsm comparable to the envlronmental- lnduction in flax can be assumedin the case of t,emperature-induced modiflcations in Drosophila melanogaster. Cavic-chi et al . , (1978) have descrlbed modif icat ions of wings due to rearrangements ofdevelopmental patterns. These rnodif icat ions ate heri table and progressive overseveral generatlons. Svetlov and Korsakova (L974) have shown heritable adaptiveresponse of the temperature-sensit ive forked mutants to repeated heat ing.

Processes simi lar to the sal tat ional creat ion of genet ical ly dist inct forms

of f lax, though possibly longer than in f ive weeks, may occur in nature. Consider-

able variation in the number of rRNA genes and other repetitive gene families hasgeen observed in plants (Flavell and Mohan, L973; Maher and Fox, L973),. Drosophila(U.y"rs and Henning, 1974), amphibians ( t" t i l ler and Brown, 1969), mice (Henderson

et al . , L976), and man (Evans et al . , L97L). Forej t (1973) descr ibed a polymorphisn

of centromeric heterochromatin in demes of the house nrouse. He suggested that in

this case the polymorphisrn was maintained either because of neutrality of occasional

repet i t ive DNA arnpl i f icat ions or because of a high frequency of ampl i f icat ion events.The constancy of centromeric heterochromatin in laboratory inbred strains is rather

against the hypothesis of occasional mutations. There is ample evidence of environ-

mental induction of repetltive DNA changes, which are by no means neutral' though

the phenotypic expression var ies. Schroeter and l lewit t (L974) dlscussed the adap-

t ive effect of addit ional heterochromatin causing earl ier euergenee in grasshoppers

and faster see germination in Allir.rm. Considerable divergence in the emergence

time, growth rates, metamorphosis, germination, anthesis, and other developmental

events has been observed among loca1 populations of various organisms. Such varia-

t ion associated with changes in repet i t ive DNA can be seen as incipient speciat ion.

2L2V. A. Krassi lov

No al lozyme di f ferences were found among is land populat ions of Anol is l izards

diverging in body size and colorat ion (Gorman and Kim, L975). These and simi lar

observat ions agree with the conclusions of Bernardi (L976), Mukai and Cockerham(L977 ) , and other authors that fast adapt ive shi f ts are due to repatterning of the

repetit ive DNA. Differences in DNA redundancy have been described between races of

grasshoppers, rodents, and man (John and King, L977; Baverstock et a l . , L976; Lubs

et al . , L977) and among closely related species in plants ( t" taher and Fox, L973),

Drosophi la (Laird and McCarthy, 1969), f ishes (Narayan and Rees, L977; Vladychenskaya

and Kedrova, L977), Xenopus (Buongiorno-Nardel l i et a l . , L977), salamanders (Macgregor

and Jones , L977), and rodents (Ginatul in et a l . , L977).Ttre relative amount of repetit ive DNA diminishes in polyploids and unisexual

forms (Maher and Fox, L973, Cimino, L974). In t ransspeci f ic evolut ion, decrease of

DNA redundancy in both plants and animals is associated with special izat ion t rends

(see El-Lakany and Dugle, L972; Pathak and Worster- I l i l l , L977) and with a t ransi t ion

from long-per iod to short-per iod interspersion (Crain et a l . , 1976), which can be

relaEed to more elaborate regulat ion of developmental Processes.The accelerat ion or retardat ion of ontogenic processes caused by the changes

of repet i t ive DNA may af fect a lso the developmental suecession of isozymes' resul t ing

in retent ion of Juveni le isozyme patterns in adul ts. At least some genet j -c polymor-

phisms revealed by the electrophoret ic technique may be due to paedogenic f ixat ion of

one or another successional isozyme in di f ferent populat ions (see Nair et a l . , L977).

Isozyme spectra can be af fected by compet l t ive exclusion of recessive a1l-e1es dur ing

indiv idual development (see above). Gene conversion is another hi therto underest imated

factor in populat ion geneLics (Gutz and Lesl ie, L976). These homogenizing processes

oppose heterot ic select ion.Ihe increased act iv i ty of a structural gene due to ecological shi f ts may have

important evolut ionary consequences. The exper iments wi th behavioral ly t 'enr iched" rats

(see l , t ra] |ace, L974; Kazakhashvi l i , L974) have shown that t ranscr ipt ional act iv i ty of

certain genes, as measured by the RNA-to-DNA rat io in the brain cel1s, is sensi t ive to

environmental factors and behaviour. Because repl icat ion is related to t ranscr ipt ion'

one can assume that less act ively t ranscr ibed genes are systemat ical ly underrepl icated

and eventual ly become ext inct , whi le the repl icat ion of more act ive genes is accelerated.

The environmental st i rnul i af fect ing t ranscr ipt ion of certain genes would Lnduce also

adapt ive changes in repl icat ion and amount of genet ic mater ia l . In microorganisms,

the use of new substrates is usual ly achieved by const i tut ive synthesis of inducible

enzymes, which requires jo ined mutat ions of structural and regulatory loci (see review

in Clarke, 1974). The frequent ly observed coincidence of such mutat ions shows that

they are related to al tered act iv i ty of these 1oci . Inder l ied and Mort lock (1977) have

shown that in Klebsiel la aerogenes xylotol st imulates the select ive dupl icat ion of a

gene codi-ng for r ib i to l dehydrogenase, which promotes faster growth on the substrate.per lman and Str ickgold (L977) have descr ibed the induct ion of select ive ampl i f icat ion

of genes for sesistance to ant ib iot ics on Proteus mirabi l is . These and other analo-

goui observat ions suggest that genet ic const i tut ion can be al tered not only by sto-

chast ic events and select ion of phenotypes, but also by the direct ional evolut ion of

genes.In the case of polygenic characters, the ef fect of each pair of genes can easi ly

be modif ied by environment. I f urodi f icat ion is maintained by constant appl icat ion

of cergain environmental agents, the preferentially acEivated genes would undergo

ampl i f icat ion, making the modif icat ion i r reversible or at least last ing a number of

generat ions. This mechanism would explain some of the t tDauermodif icat ionenr ' , f i rst

Jescr ibed by Jol los (1913), as wel l as the genet ie change in the direct ion of environ-

mental ly induced modif icat ion ( the myster ious "Baldwin ef fect ' r of ear ly genet ic

wri t ings). Orthogenet ic t rends descr ibed by paleontologists (e.g. , Plate, L920)

possibiy belong in the same category of evolutionary phenomena. They can also be

related to orthogenet ic changes of chrorncsome morphologies (Lirna-de-Far ia, 1976;

Imai, L976) and paral le l evolut ion of farni l ies of repet i t ive gene (Tartof and Dawid,

1976; Buongi-orno-Nardel l i et a l . , L977) .

2L3Directional evolution

CONCLUSIONS

The genome is envisaged as a population of nucletlde sequences capable of

direct adaptive response to environmental stiuruli in the form of differential-reproduction - underreplication or arnplification - of genes. The genomic adapta-

tions are usualJ.y advantageous not only for the genes, but, also for their carriers,

and play an important role both ln ontogeny and evolution.Underrepllcation and arnplification of both regul-atory and structural genes

has been observed during polytenization and for ribosornal- and irununoglobulin genest

enbryogenic DNA losses, and ce1l di f ferent iat ion in vivo and in vi t ro.The underreplication of repeated cistrons rnay be functional- in controlling

the onset of cel l di f ferent iat ion and the rates of t ranscr ipt ion at many loci .Redundant DNA is essential for the forrration of chromocentres and for ectoplc,synaptic, and chiasmic pairing, whlch depends on the alt,ernation of segments with

di f ferent repl icat lon rates along the chromosorp. The centromeric heterochromatin

enhances breakage and chiasmic pairlng by interaction with lnterspersed repetitlve

sequences. The decrease in chiasma frequency with age may reflect progressive

underrepl icat ion of satel l i te DNA.Ttre interspersed repetitive aequences nEIy suppress transcription of adjacent

genes, possibly by attract lng the hf-stone part lcLes. These sequences are systemati-

cal1y underrepl icated in the course of successive mitoses unt i l the structural genes

are turned on. Ttrus the timlng of gene activities is regulated by counting the

preceding mltoses.The gnderrepl icat ion of interspersed repeats nay result also in the creat ion

of a new gene by Joining the u:nique sequences lnitial-ly separated by these rePeats.

The excised repetitive segments may act as transposibl-e elements and cause gene

conversion.lhe dosage compensat ion of underrepl icated cistrons is related to competi t ive

lnteract ion of aLLel-es. The competl t ion of al l -eles for t ranscr ibing enzyme is

essent iaL for adJust lng gene act iv i t les. I t is assumed that the comPetl t ive exclu-

sion of a recessive allele and the compensatory activation of a dominant one inter-

fere with developmental homeostasls in ageing organisms and clones. At least inter-

mittent sexual reproduction is needed for malntaining developmental homeostasis.

Ageing is conceived as an outcome of (1) the underrepl icat ion of repet i t ive

DNA repr-ssing cel- l div is ion and weakening centromere potency, (2) the competi t ive

exclusion of recesslve al leles interfer ing with homeostasis, and (3) heterochronat i-

zaEion of inactive genes affecting chromosome replication pattern and pairing. Tleese

processes result in aneuploidy and increased repair fai lures and breakage. The

i."r.""u of chiasma frequency with age affects recortbinational repair and the recti-

f icat ion of rei terated genes. The sex rat io in che Progeny of ageing parents can be

distorted by decreasing f i tness of spernE carrying heterochromatic chrornosomes. The

l imitat ion of reproduct ive age has an important stabi1-Lzing effect.

Carcinogenesis resembles ageing in important ways. The arnpl i f ied repet i t ive

genes n6y act as spindle-forrnation inducers, encouraging autononous proliferation of

i , r*or cel ls. The arnpl i f icat ion of interspersed repeats affects both transcr ipt ion

and repl- icat ion, causing breaks arrdfor providing the insert ion si tes for v iruses.

The excised amplified repeats act as episome-like elements. Norrnalizing of the repe-

t i t ive DNA content is suggested as a prerequisi te of cancer therapy.

Major evolutionary novelties are due to heterochronies caused by changes of

the repetit,ive DNA content. The latter are induced by environmental facEors both

experimental ly and in nature. The saltat ional creat ion of morphological ly and genet i-

""1ty distinct forms of flax can be seen as incipient speciation, and unny natural

popuiations and species differing in developmental rat,es and adaptive norphological

traits show high polynorphism of the redundant DNA'

2L4V. A. Krassi lov

Heterochronies affect developmental succession of isozymes. The retent ionof juveni- le isozyme paEterns in adults adds to the isozyme polyrnorphisrns, whichare modified also by gene conversion and the competitive exclusi.on of recessiveal leles. Because repl icat ion is related to transcr ipt ion, the increased transcr ip-t ional act iv i ty of certain genes may induce their ampl i f icat ion. l t r is mechanismexplains Dauermodif ikat ionen, the'rBaldwin effectt t and orthogenesis.

These suggest ions, admit tedly speculat ive and oversimpl i f ied, are intendedto trace the approaches to predict i -ve evolut ionary theory.

ACKNOI^ILEDGMENIS

I am grateful to Dr. Leigh Van Valen for comments.

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