Мутации – это что?

Анализируются представления о мутациях. Приведены литературные данные, не соответствующие существующим концепциям о мутациях. Сформулировано положение о функциональной неоднозначности мутаций и их биологическом значении. Согласно этому положению, мутации в соме выполняют регуляторные функции, являяс...

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Дата:	2007
Автор:	Кордюм, В.А.
Формат:	Стаття
Мова:	Russian
Опубліковано:	Інститут молекулярної біології і генетики НАН України 2007
Назва видання:	Біополімери і клітина
Теми:	Матеріали семінару
Онлайн доступ:	https://nasplib.isofts.kiev.ua/handle/123456789/157531
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Назва журналу:	Digital Library of Periodicals of National Academy of Sciences of Ukraine
Цитувати:	Мутации – это что? / В.А. Кордюм // Біополімери і клітина. — 2007. — Т. 23, № 3. — С. 215-242. — Бібліогр.: 52 назв. — рос., англ.

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Digital Library of Periodicals of National Academy of Sciences of Ukraine

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spelling	nasplib_isofts_kiev_ua-123456789-1575312025-02-09T13:12:18Z Мутации – это что? Мутації – це що? Mutations: what are they? Кордюм, В.А. Матеріали семінару Анализируются представления о мутациях. Приведены литературные данные, не соответствующие существующим концепциям о мутациях. Сформулировано положение о функциональной неоднозначности мутаций и их биологическом значении. Согласно этому положению, мутации в соме выполняют регуляторные функции, являясь, таким образом, нормальной, контролируемой организмом, составляющей биологических процессов. А выходя из-под контроля, мутации в соме приводят к онкогенезу. В зародышевом же пути мутации через каскадные интегральные процессы обеспечивают элиминацию их носителей, выполняя очистительную функцию. А при выходе из-под контроля, не приводя к элиминации их носителей, реализуются в наследственную патологию во всем ее диапазоне – от скрытой формы («мутационный груз») до яркой манифестации. Проналізовано уявлення про мутації. Наведено літературні дані, які не відповідають існуючим концепціям мутацій. Сформульовано положення стосовно функціональної неоднозначності мутацій та їхнього біологічного значення. Згідно з цим положенням, мутації у сомі виконують регуляторні функції, являючи собою, таким чином, нормальну, контрольовану організмом, складову біологічних процесів. А при виході з-під контролю мутації в сомі призводять до онкогенезу. У зародковому ж шляху мутації через каскадні інтегральні процеси забезпечують елімінацію їхніх носіїв, виконуючи очищувальну функцію. А при виході з-під контролю, не спричинюючи элімінації їхніх носіїв, реалізуються у спадкову патологію в усьому її діапазоні – від прихованої форми («мутаційний вантаж») до ярскравої маніфестації. The conceptions of mutations are analyzed. The literature data that are not consistent with the existent ideas about mutations are presented. The statement about physiological ambiguity of mutations and their biological role is formulated, according to which the mutations in soma perform a regulatory role, therefore, they are a normal component of biological processes, controlled by an organism. Once uncontrolled, mutations in soma lead to oncogenesis. As for a germ route, mutations provide the elimination of their carriers through cascade integral processes, thus realizing the function of purification. Being out of control and not resulting in elimination of their carriers, the mutations are realized into the whole range of hereditary pathologies – from a latency form («mutational load») to a bright manifestation. 2007 Article Мутации – это что? / В.А. Кордюм // Біополімери і клітина. — 2007. — Т. 23, № 3. — С. 215-242. — Бібліогр.: 52 назв. — рос., англ. 0233-7657 DOI: http://dx.doi.org/10.7124/bc.000767 https://nasplib.isofts.kiev.ua/handle/123456789/157531 577.21+575.857 ru Біополімери і клітина application/pdf application/pdf Інститут молекулярної біології і генетики НАН України
institution	Digital Library of Periodicals of National Academy of Sciences of Ukraine
collection	DSpace DC
language	Russian
topic	Матеріали семінару Матеріали семінару
spellingShingle	Матеріали семінару Матеріали семінару Кордюм, В.А. Мутации – это что? Біополімери і клітина
description	Анализируются представления о мутациях. Приведены литературные данные, не соответствующие существующим концепциям о мутациях. Сформулировано положение о функциональной неоднозначности мутаций и их биологическом значении. Согласно этому положению, мутации в соме выполняют регуляторные функции, являясь, таким образом, нормальной, контролируемой организмом, составляющей биологических процессов. А выходя из-под контроля, мутации в соме приводят к онкогенезу. В зародышевом же пути мутации через каскадные интегральные процессы обеспечивают элиминацию их носителей, выполняя очистительную функцию. А при выходе из-под контроля, не приводя к элиминации их носителей, реализуются в наследственную патологию во всем ее диапазоне – от скрытой формы («мутационный груз») до яркой манифестации.
format	Article
author	Кордюм, В.А.
author_facet	Кордюм, В.А.
author_sort	Кордюм, В.А.
title	Мутации – это что?
title_short	Мутации – это что?
title_full	Мутации – это что?
title_fullStr	Мутации – это что?
title_full_unstemmed	Мутации – это что?
title_sort	мутации – это что?
publisher	Інститут молекулярної біології і генетики НАН України
publishDate	2007
topic_facet	Матеріали семінару
url	https://nasplib.isofts.kiev.ua/handle/123456789/157531
citation_txt	Мутации – это что? / В.А. Кордюм // Біополімери і клітина. — 2007. — Т. 23, № 3. — С. 215-242. — Бібліогр.: 52 назв. — рос., англ.
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first_indexed	2025-11-26T01:43:13Z
last_indexed	2025-11-26T01:43:13Z
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fulltext	Mu ta tions: What Are They? V. A. Kordium The In sti tute of Mo lec u lar Bi ol ogy and Ge net ics 150 Zabolotny Str., Kyiv 03143, Ukraine kordium@ukr.net The con cep tions of mu ta tions are an a lyzed. The lit er a ture data that are not con sis tent with the ex is tent ideas about mu ta tions are pre sented. The state ment about phys i o log i cal am bi gu ity of mu ta tions and their bi o log i cal role is for mu lated, ac cord ing to which the mu ta tions in soma per form a reg u la tory role, there - fore, they are a nor mal com po nent of bi o log i cal pro cesses, con trolled by an or gan ism. Once un con trolled, mu ta tions in soma lead to oncogenesis. As for a germ route, mu ta tions pro vide the elim i na tion of their car - ri ers through cas cade in te gral pro cesses, thus re al iz ing the func tion of pu ri fi ca tion. Be ing out of con trol and not re sult ing in elim i na tion of their car ri ers, the mu ta tions are re al ized into the whole range of he red i - tary pa thol o gies – from a la tency form (“mutational load”) to a bright man i fes ta tion. Key words: mu ta tions, reg u la tion, soma, germ route The term ‘mu ta tion’ was in tro duced by Hugo De Vries in 1901. Now a days this term be longs to the list of those, which are com monly known to ev ery one. Gen - eral and fun da men tal con cept of what the mu ta tions are is pretty close to the clas si cal and com monly ac cepted view, i.e. mu ta tions are sud denly oc cur ring sta ble changes in ge netic ap pa ra tus, which in clude both the tran si tion of genes from one al lele state into the other, and var i ous changes in num ber and struc ture of chro - mo somes [1], and clas si cal he red i tary dis eases are at - tached as ob vi ous and ab so lutely com pre hen si ble ev i - dence to the given def i ni tion (Ta ble 1). All the rest are the is sues of spec i fi ca tion, cor rec - tion, and clas si fi ca tion, like “re ces sive al lele in flu ences phe no type, in case the ge no type is ho mo zy gous”. Oth - er wise, it seems like it does not, and so forth and so on… Such a sim ple log i cal causal-con se quen tial con nec - tion, i.e. dis or ders in ge nome > changes in phe no type, to gether with count less ex per i men tal ev i dences, their reproducibility, and, in some cases, trace abil ity of all stages, all links of the chain – from quan tum lev els to ex ter nal man i fes ta tion of the ob ject (phe no type), brought us to the sit u a tion, where not only doubts but even the signs of lack of un der stand ing were ap pre - hended with sin cere amaze ment, as the man i fes ta tions of nat u ral dull ness, la zi ness, pre vent ing from con sult - ing the text-books etc. Re gard less of the fact that re li - able, not fit ting into gen er ally ac cepted con cept, ex per i - men tal ma te rial was ac cu mu lated from the very be gin - ning of the pro cess of study ing mu ta tions, only grad u ally this ma te rial be came suf fi cient for new com - pre hen sion of this prob lem. Let us per form a sort of anal y sis, shall we. Let us be gin with phe nom en ol ogy. Penetrance and ex pres sive ness are con sid ered to be “an tiq uity leg - 215 ISSN 0233-7657. Biopolymers and cell. 2007. Vol. 23. ISS 3. Translated from Ukrainian. ã V. A. KORDIUM, 2007 ends”. This is the case when the fea ture is ei ther pres ent or ab sent, or man i fested more or man i fested less. Though the changes in ge nome are pres ent in all of such cases. Sup pres sor mu ta tions be long to the same cat e - gory. Changes in ge nome have a cor re spond ing fea - ture, but when one more mu ta tion oc curs in an other gene, even though the first mu ta tion did not dis ap pear, phe no type re turned to its nor mal con di tion. Again – com pletely or in com pletely. Or it is due to the ‘po si tion ef fect’, when the fea ture de pends on the lo ca tion of one and the same gene in some ge nome re gions. And def i - nitely we can not talk about “ei ther yes or no” at re ces - sive ness. Of course, the prin ci pal mech a nisms of such in di - rect non-lin ear cor re la tion “gene > fea ture” (or in a more gen er al ized form “ge no type > phe no type”) at the time of their in tro duc tion were put into sci en tific prac - tise as no tions, and later on stud ied in de tails, in cluded into text-books, and ac quired com mon ac cep tance. And this com mon ac cep tance put ‘what re ally mat ters’ in mu ta gen e sis on the back ground, i.e. the fact that the changes in an in di vid ual gene, as well as big genomic ones (the sets of all in for ma tion), even iden ti cal, on the one hand (“in some cases”), may not only lead to, but ac tu ally and ubiq ui tously re sult in iden ti cal changes, and, on the other hand (“in other cases”), they may re - sult in iden ti cal phe no type changes so rig or ously, and may as well be come the rea son of other changes, not iden ti cal ones at all. With all the ob vi ous ness of why it hap pens so, all these whys serve to the concretisation of the no tion – lo cal, iden ti cal, close, sim i lar changes in ge no type may in flu ence phe no type dif fer ently or do not have any in flu ence at all. Mean while, when this no - tion is pat tered, than all this, is com pletely and choicelessly added up to ac ci dents, pos si bil i ties, indefinitenesses. Af ter all, they are mu ta tions, are they not? Grad u ally, the data re quir ing new as sump tions be - gan ac cu mu lat ing. As it turned out, av er age (i.e. vi tally op ti mal) mu ta tion rates in dif fer ent or gan isms dif fer more than 6000 (!) times, and the bound ary mu ta tion rates are more than 10 (!!) times dif fer ent [3]. It is im - pos si ble to ex plain such dif fer ence with what ever as - sump tion in volved. There af ter, a very im por tant fun da - KORDIUM V. A. 216 Ta ble 1 Ex am ples of dis eases, as so ci ated with the re place ments of nu cleo tides in en cod ing re gions of genes [2] Gene Nucleotide replacement Amino acid replacement Defect Reference Adenosine aminase G for A Arg-Gln Inactive Bonthron, 1985 Adenosine deaminase T for G Leu-Arg Labial Valerio, 1986 Antithrombin III Pro-Leu Inactive Bock, 1985 ?1-Anti-tripsin G for A Glu-Lys Inactive Kidd, 1983 Insulin T for C Phe-Ser Receptor binding site (dominant) Haneda,1983 Insulin receptor G for T Arg-Ser Processing of receptor predecessor Yoshimasa, 1988 Factor VIII C for T Pro-Arg Inactive Levinson, 1987 Factor IX Arg-Gln Pre-peptide processing Bentley, 1986 Factor IX G for A Arg-His Activation Noyes, 1983 b-Globin T for C Leu-Pro Tetramer formation (dominant) Kobayashi, 1987 Triosephosphate isomerase G for C Glu-Asp Labial in all tissues Daar, 1986 Uroporphyrinogen decarboxylase G for A Gly-Glu Labial in all tissues de Verneuili, 1986 Aldolase Â G for C Ala-Pro Substrate binding Cross, 1988 a2(I) Collagen C for G Gly-Arg Helix formation (dominant) Wenstrup, 1988 HPRT G for A Phe-Leu Stable Davidson, 1988 men tal con clu sion has been made, which is well-proven at all of the stages of the pro cess – the mu ta tion rate is de ter mined not by spon ta ne ous mu ta gen e sis as a re sult of ex ter nal in flu ence with all of its chances, but by intracellular pro cesses. How ever, this con clu sion was fol lowed by a strict lim i ta tion by ef fi ciency, per fec tion, etc of rep a ra tion. And then, fi nally, it was re duced to the chance. Let it not be pure but via rep a ra tion ef fi - ciency (evo lu tion al ly con di tioned, as a re sult of mu ta - tions, spon ta ne ous ones, of course, of these or those genes of rep a ra tion sys tem, etc). The fol low ing load of ex per i men tal data, which did not fit the con cept, even tak ing into ac count all of its mod i fi ca tions, was deal ing with not only ma te rial ac cu - mu la tion, but to a greater ex tent, with the ap pear ance of new meth ods of in ves ti ga tion. The use of trans gen ic mice with spe cial ex per i men tal genes for the sake of in - ves ti ga tion of mu ta gen e sis al lowed mak ing the fol low - ing gen eral con clu sions: i) mu ta tion rate within the in - di vid ual is tis sue-spe cific; ii) such spec i fic ity is not cor - re lated with pro lif er a tion rate; iii) each type of tis sue is spe cific for its type of mu ta tions [4]. And it is not mice only, the gen eral per cent age of hu man chro mo somal ab nor mal i ties (mi cro scope-vis i ble!) in oocytes, ac cord - ing to the data of dif fer ent au thors, var ies from 4.5 to 47.7% [5]. More over, the data are not pre sented in mean val ues, and not in com par i son of sick vs. healthy (Ta ble 2). And in dif fer ent genes in var i ous cells (nor mally), and not in liv ing in gen eral, but in hu man be ing the fluc - tu a tions of the level of mu ta tions reach 104. Di rect de - tec tion of mu ta tions in hu man cells, ac cord ing to sev eral au thors’ data, are as fol lows: Glo bin (eryth ro cytes) 10-8 Lym pho cytes HLA-A3 (2–8)·10-5 Lym pho cytes HLA-A2 (2–8)·10-6 Kid ney tu bu lar ep i the lium (HPRT) 2·10-5 – 2.5·10-4 For dif fer ent in di vid u als, the dif fer ences in mu ta - tion rates of one and the same gene are not just mul ti ple, but those of the 95-year-old may not ex ceed those of the MU TA TIONS: WHAT ARE THEY? 217 Ta ble 2 Fre quency of chro mo somal anom a lies in hu man oocytes [5] Number of oocytes Normal chromosomal complex Abnormal chromosomal complex Total % of anomalies Reference Hypoploidy Hyperploidy Diploidy Structural abnormalities 44 42 – 2 – 1 4,5 [8] 50 34 14 1 – 1 32 [9] 17 9 6 2 – – 47 [10] 251 192 33 20 5 1 23,5 [11] 188 153 10 16 – – 18,6 [12] 316 234 76 6 – 28 [13] 139 124 12 3 – – 8 [6] 65 31 10 7 14 – 47,7 [7] 100 % 76.8 % 13.3 % 7.3 % 2.3 % 0.3 % 23.2 % – Fig.1 Fre quency of mu ta tions in gene lo cus of the main histocompatibility com plex in nor mal hu man lym pho cytes of dif - fer ent age (r = 0.69; p<0.0001) [6] new-born one (Fig.1). At the same time hu mans, mice, and other liv ing crea tures still live, and live not badly at all. The con nec tion be tween phe no type and ge no type is rather vague. The fact of its ex is tence is na ked-eye vis i ble, yet it is rather crooked one, is n’t it? In these kinds of cases, the pro cesses, which de ter mine ev ery - thing at the mo lec u lar level and are sub se quently re al - ized into the phe no type, af ter ex haus tive ex am i na tion, will put all trans for ma tions and chains of events into their places, and the pic ture will be ab so lutely clear, whereas to day’s un cer tain ties will be cast out into the His tory text books. Let’s see what is al ready known at the mo lec u lar level, shall we? Clas si cal ge net ics is the way from phe no type to ge no type and mo lec u lar ge net - ics (in its vari ants of de ter mi na tion of genes func tions), study ing this way ex per i men tally – by means of in tro - duc tion or “re di rec tion” of genes, i.e. from ge no type to phe no type, ac quired a spe cific (com monly ac knowl - edged) name of In verse Ge net ics. The lat ter has also pro vided the ma te rial of prin ci pal im por tance. The as - sess ment of the ac cu mu lated ma te rial (ac cept ing it gen - er ally and not sep a rately) re sults in pro nounced in tel - lec tual stress in the at tempts of co or di nat ing the ob - tained with clas si cal the ory. Gen er ally, the mu ta tions proper, as fun da men tal prin ci ple of all fur ther events is some sort of change in the se quence of grounds of one DNA chain, which, for some rea son (which is al ways re duced to chance), did not re store to its pre vi ous state, but re sulted in the for - ma tion of com ple men tary base in the sec ond chain, which cor re sponds to the change oc curred. As a re sult, such change is pres ent al ready in both chains. The vari - ants for such event are nu mer ous, but they are al ways fol lowed by some sort of “se cur ing of changes”. While the change (any change) is pres ent in one DNA chain it is not a mu ta tion yet. It ac ti vates the cor re spond ing rep - a ra tion sys tem. In case when the change is re paired ac - cord ing to the complementarity of the sec ond chain, then ev ery thing will re sume to its nor mal course. No mu ta tion ac quired. But if the base of the sec ond chain “cor rected” it self to the com ple men tary changed sec - ond chain, the mu ta tion is re al ized. And from now on, it is no lon ger the mu ta tion but some thing of the or gan - ism’s proper, its own, which has to be pro tected, se - cured (change-ap pro pri ately), re paired in the case of dam age (even if the dam age is di rected to wards the res - to ra tion of the ini tial state), and so forth and so on. The change has been se cured, trans fer ring from the cat e gory of dis or ders into the cat e gory of mu ta tions, as on mo - lec u lar, ge netic, in for ma tional level, the se cured change does not dif fer es sen tially from the rest of base se quence. Even ter mi no log i cally, the re turn to the norm is called the re verse mu ta tion. It is the re al iza tion of the in for ma tion on all sub se quent stages of the pro - cess from the DNA se quence to the dis tinc tive fea ture that de ter mines “what is what”. The am bi gu ity of the is sue of mu ta tion starts at this point – as “what is what” is some sort of ab so lute. And this “what is what” is com pletely de pend ent on where, at what mo ment, and in what com bi na tion the re al iza tion takes place. All al - ter na tive pro cesses in the cell, i.e. al ter na tive tran scrip - tion, polyadenilation, splic ing, etc can ev i dence to that. One and the same DNA se quence, de pend ing on its re - al iza tion, may be phenotypically cor rect (if ev ery thing in the cell cor re sponds to its con di tion) or phenotypically in cor rect, not dif fer ent from mu ta tions (if there is no cor re spon dence to the con di tion). Al - though it does not take place at the level of DNA changes, but at the level of its main te nance, yet the phe - nom e non is dem on strated very dis tinctly. Now let us see what hap pens at the level of DNA. What se cured changes, pres ent in it, are those to be re garded as mu ta - tions? The bright est, the clos est to each one of us, and the most stud ied ex am ple will be the hu man. Some time ago there was a sort of “genre cri sis” in med i cal ge net ics. For many cen tu ries ev ery thing was ac cu rate and “ab so lutely clear” – there are some he red i - tary dis eases and mass pa thol o gies. The for mer in - cluded the dis eases which were clearly in her ited and had vivid man i fes ta tion – thalassemia, drepancytic anae mia, haemo philia, chil dren progeria – Hutch in - son-Gilford syn drome, Du chenne’s myodystrophy etc. Mean while, even at that time there were some un cer - tain ties due to dif fer ent lo cal isa tion of reali sa tion of pa - thol o gies (e.g. lungs are pre dom i nantly in jured dur ing mucoviscidosis, yet pan creas can be in jured greatly too) or dif fer ent de gree of dam age in the cases of mu ta - tions in the same gene. How ever, it all had a clas si cal ex pla na tion – dif fer ent ex pres sive ness etc. Grad u ally, the num ber of he red i tary dis eases in creased and equalled thou sands and the pos si bil ity of their ap pear - ance (the same penetrance) dif fered more from firmly KORDIUM V. A. 218 de ter mined (or strictly de ter mined pos si bil ity as in the cases of clas si cal re ces sives). Now the no tion of pre - dis po si tion oc curred. Then the sci en tists be gan dis - cuss ing the prob lem of he red i tary pre dis po si tion and the things re lated to it. As it had hap pened ear lier, ex - treme cases again made it all “clear” and these ex treme cases were given as ex am ples. Yet at that pe riod of time the tech nique of DNA se quenc ing shifted from the ge - nome of hu man to the ge nome of man kind. And the un - cer tainty reached its ab so lute. As it turned out two un - re lated hu mans dif fer from each other in three mil lion of mononucleotide mis matches, so called sin gle nu cle - o tide polymorphisms – SNPs (Fig.2). Hu man - kind-wise dif fer ences in SNPs reach 10 mil lion. The SNPs also in clude not mononucleotide dif fer ences only, but also those, the per cent age of which is not less than 1% in the in ves ti gated pop u la tion. If all the dif fer - ences were in ves ti gated, than one could find more than three mil lion of dif fer ences be tween two in di vid u als, and they would count bil lions in the whole hu man pop - u la tion. All chro mo somes are filled with them (Ta ble 3). How ever, SNPs pres ent some thing, which has been firmly se cured by ge nome of hu man, com pris ing the pop u la tion, as he red i tary changes. In or der to con sider them as mu ta tions in ac cor dance with the clas si cal def i - ni tion, the only thing re quired is the in flu ence on phe - no type. Stat ing on the ba sic con sid er ations, it is ev i - dent that as ba sic he red i tary pa thol o gies and pre dis po - si tions (viewed by med i cal ge net i cists as those of “their own” in re gards to pro cess and the ob ject) are re sulted in the ma jor ity of cases by the changes in one gene and of one nu cle o tide only, then, all this can be re garded as SNP. And then, med i cal ge net ics is a par tic u lar case of genomic poly mor phism. Yet at the time be ing (if we es ti mate sta tis ti cally) the ques tion is put in a slightly dif fer ent way – is there a pos si bil ity of no ef fect what - so ever on the or gan ism if any change in any base oc - curs? Ear lier this is sue was nar rowed down to synonymic and non-synonymic re place ments. And, of course, non-synonymic re place ments were of greater in flu ence on the phe no type. How ever, now a days, it is ev i dent that func tion of ge nome is de ter mined, to a great ex tent, by the spe cial struc ture of DNA, which is im por tant is sue for rec og ni tion of ser vic ing pro teins, RNA-DNA in ter ac tion, etc. Stat ing on these ba sic con - sid er ations we can sup pose that mononucleotide re - MU TA TIONS: WHAT ARE THEY? 219 Fig.2 Il lus tra tion of what mononucleic poly mor phism is [7] Ta ble 3 Dis tri bu tion of SNPs in the chro mo somes [8] Chromo somes Length, b.p. SNPs total TSC SNPs SNPs b.p.·103 on SNP SNPs b.p.·103 on SNP 1 214,066,000 129,931 1.65 75,166 2.85 2 222,889,000 103,664 2.15 76,985 2.90 3 186,938,000 93,140 2.01 63,669 2.94 4 169,035,000 84,426 2.00 65,719 2.57 5 170,954,000 117,882 1.45 63,545 2.69 6 165,022,000 96,317 1.71 53,797 3.07 7 149,414,000 71,752 2.08 42,327 3.53 8 125,148,000 57,834 2.16 42,653 2.93 9 107,440,000 62,013 1.73 43,020 2.50 10 127,894,000 61,298 2.09 42,466 3.01 11 129,193,000 84,663 1.53 47,621 2.71 12 125,198,000 59,245 2.11 38,136 3.28 13 93,711,000 53,093 1.77 35,754 2.62 14 89,344,000 44,112 2.03 29,746 3.00 15 73,467,000 37,814 1.94 26,524 2.77 16 74,037,000 38,735 1.91 23,328 3.17 17 73,367,000 34,621 2.12 19,396 3.78 18 73,078,000 45,135 1.62 27,028 2.70 19 56,044,000 25,676 2.18 11,185 5.01 20 63,317,000 29,478 2.15 17,051 3.71 21 33,824,000 20,916 1.62 9,103 3.72 22 33,786,000 28,410 1.19 11,056 3.06 X 131,245,000 34,842 3.77 20,400 6.43 Y 21,753,000 4,193 5.19 1,784 12.19 RefSeq 15,696,674 14,534 1.08 Total 2,710,164,000 1,419,190 1.91 887,450 3,05 Nota Bene: The data on length (b.p.) were ob tained from the se ries of works on ge net ics, dated Sep tem ber 5, 2001. The den sity level of SNPs on each chro mo some is de ter mined by the num ber of ac ces si - ble genomic se quences, in cluded into genomic se lec tion, the depth of over lap ping was ob tained from TSC of read and cloned overlappings, which are known to be het ero zy gotes. place ments may cause the whole range of ef fects on phe no type – from le thal cases to a small value. That is the way it went down af ter concretizing these “gen eral con sid er ations”. To day al most all mass pa thol o gies are spec i fied to some SNPs (Ta ble 4). Why is it so viv idly seen on the ex am ples of pro - teins stud ied in de tail? Par tic u larly, BRCA1 is one of the key sig nal-reg u la tory intercellular pro teins. BRCA1 mol e cule con tains app. 30 sites, in ter act ing with al most 30 dif fer ent pro teins, many of which be - long to reg u la tory pro teins (Fig.3). De tailed study of both gene and its en cod ing pro tein, is de fined by the con nec tion (to be more pre cise, the con nec tion be tween mu ta tions in it) and very high pre dis po si tion to the breast can cer. The con nec tion of BRCA1 SNPs and other dis or ders in the or gan ism has not been stud ied enough as lit er ally the whole pro tein (!) (with all amino ac ids), en coded by BRCA1, in ter acts with other pro - teins, then the poly mor phism will af fect this sort of in - ter ac tions. How ever, BRCA1 is noth ing more than just an ex am ple. Yet the in flu ence of SNPs is uni ver sal and may af fect all pro teins – there is not a sin gle thing in the or gan ism that would func tion on “its own”. All macromolecules in ter act, mu tu ally in ter fere, in ter - change conformationally and per form all other inter-kinds of in ter ac tions… Even the de tailed stud ies of struc ture of en zymes and genes that en code them re - vealed that SNPs, which have no in flu ence on cat a lytic ac tiv ity, may de ter mine the in ter ac tion of the en zyme with some other pro teins [10]; many synonymic re - place ments in exons are not com pat i ble with nor mal RNA pro gress ing [11] etc. Now a very in ter est ing and un usual sit u a tion oc - curs, this sit u a tion is full of un cer tainty of any (ab stract and the o retic) uni ver sal, com pre hen sive, and in wardly con tra dic tory not even a def i ni tion, but a no tion of what is con sid ered to be a mu ta tion and what is not. In or der for pro teins to pro vide the pos si bil ity of ex is tence to the cells, where they are, in fact, lo cated, in com pound and highly-dy namic com plexes, the co or di na tion of in ter - act ing do mains, conformational tran si tions, en ergy lev - els of their in ter ac tions, etc. they have to be of ex treme ac cu racy. Spa tial de vi a tions from the ideal (max i mally en ergy-wise con ve nient for a par tic u lar pro cess) should not ex ceed the tenth part of ang strom and en ergy-wise – ten kilocalories per mole. Oth er wise, the com plex sys - tems would fail to func tion prop erly pretty soon, which would be the end to ev ery thing. To pres ent a vivid il - lus tra tion of this type of in ter ac tion it is pos si ble to pro - vide the ex am ple from an other area (which has no con - nec tion with the area un der in ves ti ga tion, yet is a very vi sual one). Let us take two gear units, pro duced by two dif fer ent com pa nies, with iden ti cal ex ter nal pa ram - e ters (en trance and exit ve loc ity, sizes, weight, etc.); if we dis man tle (i.e. in side it self) the gear unit of one com pany and then put it back again, hav ing switched the gears, then ev ery thing would work fine. But if we take a set of gear of one man u fac turer and switch them for that of the other man u fac turer, then these gear units would not work or would work badly and get bro ken soon. Ev ery man u fac turer pro jects things in a slightly KORDIUM V. A. 220 Ta ble 4 Par tial list of SNP-re lated dis or ders Disorder Gene Reference Asthma EDN 1 and NOS 1 Immervoll et al., 2001 ÐÎÀÑ Microcillin Colomb et al., 2001 Systemic sclerosis Fibrillin 1 Tan et al., 2001 Lung cancer MMP-1 Zhu et al., 2001 Arrhythmias KCNQ1 Kubota et al., 2001 Idiopathic arthritis MIF Donn et al., 2001 Blood pressure TAF1 Koschinsky et al., 2001 Gallbladder cirrhosis MBL Matsushita et al., 2001 Diabetes type II Syntaxin 1À Tsunoba et al., 2001 Systemic lupus erythematosus Prolactin Stevens et al., 2001 Indigestion disorders Melanocortin Adan and Vink 2001 Migraine Insulin receptor Mc Carthy et al., 2001 Ossification Npps Koshizuka et al., 2002 Lung cancer p53 Biros et al., 2001 Late ÐD tau Martin et al., 2001 Nota Bene: SNPs – sin gle nu cle o tide polymorphisms; POAc – pri - mary open-an gle glau coma; MMP-1 – metalloproteinase 1 ma trix; EDN 1 – endothelin 1; NOS 1 – neu tron synthetase 1 of ni tro gen ox ide; MBL – mannose-bind ing pro tein; Npps – nu cle o tide pyrophosphatase; TAF1 – fibrinolysin in hib i tor, ac ti vated by thrombin; KCNQ1 – pro tein of po tas sium chan nels; MIF – macrophage in hi bi tion fac tor; PD – Par kin son’s dis ease. dif fer ent way than the other (dec i mal part of the milli - metre, axes-wise, gear con fig u ra tion etc). Of course, mol e cules are not gears. Spa tial and en ergy in ter ac - tions be tween them have got to be many times more pre cise, so that ev ery thing would func tion prop erly in the or gan ism. And if any site in any multi-pro tein com - plex is changed, then the chain of pro cesses will lead to the ex ter nally vis i ble changes in the phe no type. We may now talk about mu ta tions. In case when changes are nu mer ous and all of them are “inter-ad justed”, then the sys tem would func tion suc cess fully. But if we cross breed these or gan isms, with all in ter nally com pen - sated changes (but sep a rately for each in di vid ual), then the prog eny will ac quire pro tein-en cod ing genes in the changed vari ant in ter act ing with high-pre ci sion. It is pretty of ten seen in the ev ery day life, when the chil dren of healthy par ents are very un healthy and it can not be ex plained by any “re ces sive” of par ents as the par ents do not have these “re ces sives”. Sim ply be cause their mol e cules are ad justed in a dif fer ent way. Yet there may be some harder con se quences. Ev ery thing in the cell is con trolled, in clud ing the sta bil ity of mo bile ge - netic el e ments of ge nome, which are nu mer ous in all eukaryotic spec i men (hu mans in cluded). At dis or der of pre ci sion of in ter ac tion pro teins, con trol ling the ac tiv - ity of mo bile el e ments, the lat ter are ac ti vated, re sult ing in mass mu ta gen e sis. The fol low ing was dem on strated by a se ries of ex per i ments. Cross breed ing of two sta - ble lab o ra tory lines of drosophilae of the sec ond and the third gen er a tion (not the first one!!!) re vealed nu mer - ous mu ta tions and ge nome in sta bil ity [12]. Thus, mu ta tions are the changes, that may be as - sessed in the re la tion with some ini tial ob ject, which the phe no types can be com pared to, or within the con - trolled short re pro duc tion line. In a short suc ces sion of gen er a tions at least some thing more pre cise can be MU TA TIONS: WHAT ARE THEY? 221 Fig.3 In ter ac tions of BRCA1 pro teins. Scheme, sum ma ris ing BRCA1 pro tein-pro tein in ter ac tions. Ab bre vi a tions: ATF-1 – ac ti vat ing tran - scrip tion fac tor 1; BAP1 – BRCA1-bind ing pro tein 1; BARD1 – BRCA1-bound pro tein 1 of cir cu lar do main; BRCT – C-ter mi nal re peat BRCA1; CBP – p265 CREB-bind ing pro tein; CtIP – C-ter mi nal bind ing pro tein; ER-a – oes tro gen a-re cep tor; HDAC1/2 – histone deacetylase 1/2; LCXCE – con sen sus of bind ing mo tif of RB fam ily; NLS1 – pri mary sig nal of nu clear lo cal iza tion; RB1 – retinoblastoma 1; RbAp46/48 – ret i no blas toma-as so ci ated pro tein, m.w. 46/48 kDa; RING – cir cu lar do main (zinc fin ger); TAD – tran scrip tion ac ti va tion do - main [9]. traced. But with some thing which is cor re lated with the cat e gory of epigenetics, quite of ten the out side ob - server can be un dis tin guished from com plete chaos. The clear est (and the most stud ied) no ti fi ca tion is re - vealed at the level of co va lent mod i fi ca tion of cy to sine, i.e. bind ing of the methyl group in the fifth po si tion. The ex act quan ti ta tive de ter mi na tion of methylcytosine con tent in the gen eral pool of cell DNA (and, thus, the metC to to tal cy to sine ra tio) is rather a com plex pro ce - dure me thod i cally, which has be come avail able in the re cent years only. How ever, once avail able, this pro ce - dure al lowed ob tain ing some very in ter est ing re sults. Av er agely, if we ac cept some young healthy in di vid ual for the av er age value, then hu man ge nome metC con - tents is av er agely 16–17% of cy to sine to tal [13]. Yet if we take the ex treme val ues, then in nor mal healthy peo ple (de pend ing on ge no type, me tab o lism of folates, di ets, etc) the fluc tu a tions in metC con tents in dif fer ent in di vid u als may be ex ceeded seven-fold (Ta - ble 5). There fore, some fan tas tic changes take place in ge nome, but out wardly it is not re flected at all (the phe - no type is the same). There it is, that no mat ter what mu - ta tion re gion one se lects, one has to state on some sep a - rate, con stantly given, ex am ples, where ev ery thing is “as it should be”, how fast ev ery thing be comes as “it should not be”, i.e. in def i nite, con tro ver sial and so on. There is some thing wrong in all this, is n’t it? All this should-not-be in all of its fea tures tes ti fies about one sim ple thing – dif fer ent events have been piled into a num ber of no tions and views on mu ta tions, all of them be ing placed into Pro crus tean bed. The sit u a tion should meet its end, which is ob vi ous. The ques tion is how to force it. In or der to un der stand “how”, to deal with all con tro ver sies, in con sis ten cies, am bi gu ities, etc, i.e. all the no tions the term mu ta tion in cludes, we are to start from the be gin ning… from the very be gin ning. Let us take the hu man kind as the sam ple to be com - pared (it is both most stud ied one and, ap par ently, clos - est to all of us). This “very be gin ning” is the “start ing point” of the in di vid ual-to-be (any) and the whole bulk of pro cesses and events fol low ing it. Let us start with our selves. Over 100 years ago Weissmann in tro duced the con cept of the germ plasma. It was noth ing but the idea in the be gin ning. Grad u ally ex per i men tal data were ac cu mu lated and to day, suc - cinctly, it looks as fol lows (Fig ure 4). Once fer ti lised, zy gote starts di vid ing. Ini tially (2-3 days) there are no sig nif i cant dif fer ences be tween these di vid ing cells. Later on, they are dif fer en ti ated, and on day 5-6, only small amount of cells (so called, in ter nal cell mass of blastocyst) pre serves its po ten tial of turn ing into any - thing – any dif fer en ti ated or not dif fer en ti ated cells. In the course of sub se quent 3-4 days this part of cells (keep ing on mul ti ply ing) is di vided into two groups. One of them mi grates into the in ter nal cav ity of blastocyst and takes part in the for ma tion of a yolk bag. The sec ond group forms the em bryo. These pro cesses re main ac tive on day 20-21: the group of cells, which ini ti ated the for ma tion of em bryo, fin ishes this pro cess with first mor pho log i cally formed el e ments, while ini - tial germ cells are cre ated out of the group, which par - tic i pated in the for ma tion of ini tial yolk bag. There fore, the pre de ces sors of hu man germ cells find their be gin - ning not from this hu man. Both the lat ter and his germ cells are formed in de pend ently from the last group of totipotent cells (api cal part of in ter nal cell mass of blastocyst) which still pos sessed com plete dif fer en ti a - tion po ten tial. To be more pre cise, our kids are not re - ally ours. Be ing si mul ta neously dif fer en ti ated cells of fu ture (!) soma (“us”), and sub se quent germ route KORDIUM V. A. 222 Ta ble 5 Dis tri bu tion of folates in the eryth ro cytes and DNA methylation sta tus ac cord ing to MTHFRC677T ge no type and low and high con tent of folates in the eryth ro cytes [13] Index Low folate erythrocytes High folate erythrocytes Ñ/Ñ Ò/Ò Ð value Ñ/Ñ Ò/Ò Ð value Folate total content, nmole /g Íb 0.81±0.20 0.68±0.27 0.003 1.69±0.70 1.48±0.27 N. S. Methyltetrahydrofolate of erythrocytes, % from total 98.8±5.7 67.3±29.0 <0.0001 99.4±1.1 69.6±30.9 0.002 Methylation status of genomic DNA, ng metS/mg of DNA 64.07 (49.89-81.45) 21.93 (14.73-32.45) <0.0001 57.97 (45.60-73.55) 57.39 (29.96-109.94) N. S. (“germ cells pre de ces sors”) our kids and we orig i nate from (prior to dif fer en ti a tion) in ter nal cell mass of blastocyst (which in its turn is zy gote de riv a tive), i.e. from our par ents. The mat ter is that our chil dren and we are cous ins (!!!) ge net i cally. Hav ing been formed in yolk bag on day 20-21, ini tial germ cells colo nise the em bryo (soma, ac cord ing to Weismann). And all this goes on for count less mil lions of years. Germ route al - ways fol lows its pat tern, with no in ter rup tions, soma be ing noth ing but its tem po rary car rier and dis trib u tor. Con sis tency of germ plasma has got to be max i mal (oth er wise, the life will go off its track). The des tiny and the func tions of soma are quite dif - fer ent. Soma has got to ad just, con form, re act, adapt etc. to the en vi ron men tal con di tions – dif fer ent, vary - ing, dif fi cult, un fa vour able, and so forth and so on. And the wages for all this is in ev i ta ble end, in a while. The pe riod of this “while” is spec i fied by the bi o log i cal time, nec es sary for de vel op ment and sub se quent trans - fer round of germ plasma. Hav ing all these dif fer ences (life task, goal of ex is tence, time of ex is tence, con di - tions of ex is tence in the tur bu lent world or in se cured soma, etc) all pro cesses, mu ta tions in cluded, in soma and germ plasma can no wise be the same, nei ther in es - sence nor in func tion al ity, nor in tasks, put for ward by life as phe nom e non. Mean while, due to his tor i cally de - vel oped ideas, mu ta tions are eval u ated by the events, oc cur ring (spon ta ne ously or in duced) in the germ route, more over, the eval u a tion takes place not in the lat ter di - rectly, but via the reali sa tion in soma. Even when the mu ta tions are de ter mined in so matic cells, gen er ally, they are de ter mined by mul ti pli ca tion of such cells in se lec tive con di tions, which have hardly any thing in com mon with the things that take place in the or gan ism. Soma is us in the course of de cades in the con di tions which are im pos si ble to de scribe, per ceive, es ti mate or even imag ine. So, what is go ing on in soma with the thing, in di vid u ally per ceived dif fer ently, but de fined as MU TA TIONS: WHAT ARE THEY? 223 Fig.4 Early stages of hu man embryogenesis (com prised by [14]) mu ta tions? To un der stand what takes place in soma (i.e. in you and me), let us start with dam ages in DNA, i.e. some thing pre ced ing the mu ta tions, some thing which is nei ther norm any more, nor the mu ta tion yet, in com mon opin ion. But it con cerns com mon opin ion only, as in the es sence of the pro cess as well as its reali - sa tion (un til this pro cess is brought ei ther to norm by rep a ra tion or to changed and fixed state, i.e. “com mon mu ta tion”) the dam age is the ac tual mu ta tion in its ut - most pre sen ta tion. The dam age of the base blocks the prog ress of RNA poly mer ase, and the gene is not read off any more. It is dys func tional un til the dam age is re - paired. In the case of cell it is phenotypically (but only at the level of cell) in dis tin guish able from mu ta tion, com pletely switch ing off the man i fes ta tion of this gene. Cer tainly it goes on only for the time of dam age ex is - tence, but rather rad i cally. What is the av er age num ber of dam ages in ev ery hu man cell? The fig ure is de ter - mined by nu mer ous fac tors, the first of which (in the chain of the lat est events) is the re ac tion-ac tive prod - ucts. The most mass de struc tive ones are ox y gen and its de riv a tives. This is the very ox y gen, the ab sence of which makes one die in sev eral min utes. Av er age ox y - gen con sump tion by one in di vid ual per day is 640 g (420 l) [15]. Av er age num ber of ox y gen rad i cals formed daily in ev ery cell due to breath ing is ~6.5·1010 [16]. Quan tity-wise (i.e. item-wise) it is man i fold more than the to tal num ber of all bases of nu cleic ac ids and pro tein amino ac ids in a cell, and weight-wise it con sti - tutes 0.1% of the av er age cell weight. To tal weight of all re ac tion-ag gres sive prod ucts, formed in the cell dur - ing its ab so lutely nor mal and vi tal ac tiv ity daily, ex - ceeds the to tal weight of all macro- and non-macro mol - e cules of the cell [17]. And no mat ter what ul - tra-super-new per fect pro tec tion sys tems (i.e. at the level of pre vent ing and avoid ing) from these agents ex - ist, some part of these agents will over come the pro tec - tion and re sult in dam ag ing ev ery thing, in clud ing ge - nome. In the course of time there have been many as - sump tions on the is sue. Grad u ally the meth ods have im proved and cor rect quan ti ta tive data have been ac cu - mu lated. Ox i da tive dam ages of DNA in the cell are the most stud ied ones. They are di verse enough and their to tal num ber is 0.5–2·106 of dam ages per ge nome of ev - ery cell per day [18]. The level of dam ages is sig nif i - cantly higher (up to 2-fold) in mitochondrias [19]. But ox i da tive dam ages con sti tute only a part of the whole pool of DNA dam ages in the cell. The other types of dam ages are stud ied less. How ever, their con tri bu tion proves to be rather valu able in the course of stud ies. For in stance, depurinisation amounts to 104 per nu clear DNA of ev ery cell daily [20]. Great is the con tri bu tion of other dam ages as well. Ex trap o lat ing all the abovementioned, one has all the grounds for ac cept ing the level of dam ages of nu clear DNA of ev ery cell as app.107 daily. And it con cerns the whole life. Mu ta - tions (as com monly per ceived) oc cur, even if we do not con sider the whole time but their di vi sion pe ri ods only, and take fast-di vid ing cells (e.g. blood cells, on the way of their for ma tion from ini tial stem cell to the dif fer en ti - ated one, which en ters the blood flow), with the fre - quency (av er aged a lot) ~10–7 per gene, i.e. (tak ing into ac count the num ber of genes in hu man body is ~35 000 – 40 000) it is at least 10-times (!) less fre quent. Fac tor, de ter min ing the quan ti ties of dam ages, is the dam age pro tec tion sys tem. Ef fi ciency of the pro tec - tion sys tem is con trolled both evolutionarily and func - tion ally. The com par i son of these sys tems re veals sig - nif i cant dif fer ences de pend ing on the kinds of mam - mals. Gen er ally, it could be ex pected – dif fer ent evolutionarily dis tant taxons can not be the same. How ever, un ex pected is the fact that (and con tra dict ing early re search) the ac tiv ity of sys tems, pre vent ing DNA dam ages, does not change through out the life time (Ta - ble 6). It can tes tify in fa vour of the ex is tence of some pro - grammed level as the level of sys tems, pre vent ing DNA dam ages, de ter mines the level of damageability proper. It is note wor thy that the num ber of dam ages and the level of damageability are not the same. If we speak not about the num ber of dam ages per time unit, but about the level, i.e. about cer tain con stant num ber of DNA dam ages, it is go ing to be quite a dif fer ent no tion. Dam - ages are re paired in some time. They are re paired by the other sys tem – not the sys tem of pre vent ing DNA dam ages but the rep a ra tion sys tem. The level of dam - ages will look like some bal anced value be tween the damageability rate and the rep a ra tion rate. Fun da men tal is the ques tion of what ex actly bal - anced value will be pro vided by these op po site pro - cesses. Will this bal anced value be spot-timely, i.e. bal - KORDIUM V. A. 224 anced only for short pe ri ods of time, within which both ex ter nal and intercellular pa ram e ters are ac tu ally the same, or will it change due to their changes (which takes place con tin u ously in life)? Will it be bal anced in the wide range of ex ter nal con di tions? Di rect ex per i - ments re vealed rapid in crease in spot-level of damageability (achieved by in tense g-ir ra di a tion) to re - store back to the ini tial val ues fast (in sev eral min utes) in a sharp straight line (Fig.5). Re store back to the ini - tial val ues! The pro cess is the same in both old and young an i - mals. The sit u a tion is ab so lutely mar vel lous – the rep a - ra tion sys tems are so pow er ful as to re store the dam - ages, oc cur ring due to ex ter nal dis tur bance, in a sharp straight line. How ever, later on, hav ing achieved cer - tain pri mary level, the rep a ra tion slows down the res to - ra tion and is “main tain ing the same level”. What if one tried to help the cell to de crease the level of dam ages (i.e. their bal anced num ber in ge nome)? Seem ingly, the cell should profit from it. In re al ity it takes place “quite the con trary”. The in tro duc tion of ad di tional genes, en - cod ing the rep a ra tion en zymes, into the nor mal cell and the in crease in their quan ti ties re sults in neg a tive con se - quences. For ex am ple, over-ex pres sion of rep a ra tion en zymes of alkyl-N-pu rine glycosilase or DNA-poly - mer ase b re sulted in ge nome in sta bil ity, and, con se - quently, in the oc cur rence of instable phe no type [21]. It could hap pen in one case only, the cells re quire a cer - tain level of dam ages of their ge netic ma te rial to func - tion prop erly. Though seem ingly im pos si ble to be con - sid ered, this as sump tion can be proved by di rect ex per i - ments. The first step for ward was made af ter the daily num ber of dam ages oc cur ring had be come known. This first step was ac tual ad mis sion of the fact that ox i - da tive DNA dam ages are “nor mal cell me tab o lism” [18]. It turned out to be harder on the as sump tion on the role of ge nome dam ages dur ing nor mal cell me tab o - lism. To be strictly con sis tent, the role of dam ages de - rives from their di rect ef fect on the ex pres sion. Once the gene is dam aged, the read ing-off it stops at the dam - aged base. That is, the dam age switches the gene off, tem po rarily, un til the rep a ra tion deals with the dam age. In its phe nom en ol ogy, switch ing off the gene – in this case due to the dam age of its base – and its sub se quent (in some time) switch ing on (by rep a ra tion, res to ra tion af ter the dam age) is noth ing but reg u la tion. There fore, it is pos si ble to sup pose that the dam age of the bases is of some reg u la tory mean ing. Its es sence and na ture arise from the anal y sis of some ex per i ments on hu man cells. Age-de pend ent pro teins are pres ent in brain cells (the work was per formed on au topsy ma te rial). This de pend ence re lies on the fact that the num ber of one type of pro teins de creases with time, that of the sec ond type re mains the same, and that of the third one in - creases. The study on the level of their dam ages re - vealed the pro mot ers of these pro teins to be dam aged and to be re paired in strict ac cor dance to what “should be” dur ing age ing (Fig.6). In the es sence of the whole chain of pro cesses it is the reg u la tion, but very un usual to us. De tailed in ves ti ga tion on this is sue re vealed it to be con di tioned by the cor re spond ing lev els of DNA dam ages in the pro moter re gions and the rep a ra tion, match ing them (Fig.7). As in this case the pro mot ers were treated with the agent, caus ing the dam age of the MU TA TIONS: WHAT ARE THEY? 225 Ta ble 6 Fre quency of gene am pli fi ca tion in con trol or mICAD-ex - pres sion of HCT116 and L929 cells [27] Tissues Anti-oxidative enzymes Enzymatic activity, relative units per 1 mg of protein Young mice Old mice Liver Catalase 4.65±0.17 4.56±0.09 Gluthationperoxidase 7.16±0.60 8.36±0.28 Mn-superoxide-dismut ase 11.52±1.32 12.12±0.31 CuZn-superoxide-dism utase 192.53±20.23 174.28±16.19 Heart Catalase 0.42±0.07 0.51±0.02 Gluthationperoxidase 0.40±0.06 0.61±0.02 Mn-superoxide-dismut ase 63.08±1.95 68.04±3.69 CuZn-superoxide-dism utase 66.92±0.33 73.20±3.91 Brain Catalase 0.29±0.03 0.34±0.06 Gluthationperoxidase 0.86±0.12 0.90±0.07 Mn-superoxide-dismut ase 13.76±1.02 18.01±1.34 CuZn-superoxide-dism utase 43.31±1.69 46.53±4.75 Nota Bene: The ac tiv ity of dif fer ent anti-ox i da tive en zymes was mea sured by ex trac tion of tis sues from young (6–7-month) and old (26–28-month old) fe male mice of C56B1/6 cell line. bases out side the cell (i.e. in strictly equiv a lent, con - trolled con di tions) and then (again the same) were in - tro duced into the iden ti cal cells, dif fer ent de gree of rep - a ra tion (and con se quently dif fer ent bal anced level of dam age) de pended on the specificities of pro mot ers (pri mary se quence, formed spa tial struc tures etc). Ab - so lutely new and very in ter est ing sys tem of the reg u la - tion of gene ac tiv ity emerges. The ma jor ity of genes in the cell are con sti tu tively ex pressed. They do not have the sites for in ter ac tions with reg u la tory pro teins. But ge net i cally pro grammed nu cle o tide se quence pre de ter - mines damageability level of these se quences. Con sti - tu tive genes turn out to be reg u lated, though via ab so - lutely dif fer ent mech a nism. This is the way for new con cepts to oc cur: damageability of ge nome as nat u ral mass mech a nism of its ac tiv ity reg u la tion; reg u la tions via dif fer ent damageability due to dif fer ent pri mary se - quence. Let us take a look at what is go ing on at the level of real ised mu ta tions, i.e. changes fixed in ge - nome of so matic cells. The changes in pri mary DNA se quence, i.e. at the role of mu ta tions in soma – in you and me. The first thing to dwell upon is the fact that the spe cial phe nom e non – dy na mism of mu ta tions – ex ists in soma. The es sence of dy na mism is that the mu ta tions (fixed, clas si cal ones) not only oc cur but also dis ap pear, elim i nate. There are two ways for this elim i na tion: the first one is com monly known and is de ter mined by the elim i na tion of cells with mu ta tions. For this pur pose, the mu ta tion should ac ti vate the pro cesses, caus ing the elim i na tion of cell, bear ing it. To per form the elim i na - tion there is apoptosis, im mune sur veil lance, autophagy etc. Ev ery thing seems to be clear here. Though the quan ti ta tive side of this self-pu ri fi ca tion from mu ta - tions is so high that de serves to be the topic for sep a rate dis cus sion. Through out hu man life-time to tal weight of cells, elim i nat ing in his body, ex ceeds the “sta ble” (i.e. av er age 70 kg – av er age body weight), but ac tu ally some bal anced body weight 100-fold [17]. But this is KORDIUM V. A. 226 Fig.5 In flu ence of age on elim i na tion of oxo-8dG from nu clear DNA af ter acute g-ir ra di a tion of the whole or gan ism. Young (1) and old (2) fe male mice of C57BL/6 were sub jected to g-ir ra di a tion, 2 Gy, im me di ately af ter 7, 5, 15, and 30 min af ter ir ra di a tion; nu clear DNA was iso - lated from liver, brain, heart. Ev ery value rep re sents the level of oxo-8-dG, caused by g-ir ra di a tion, i.e. oxo-8-dG level in tis sues, not sub - jected to g-ir ra di a tion, was sub tracted from the level of oxo-8-dG, ob tained af ter ir ra di a tion. Ev ery value is the av er age value ob tained for six mice ±SEM. Ve loc ity of oxo-8-dG elim i na tion from nu clear DNA was cal cu lated us ing Microsoft Ex cel, which al lows ob tain ing the straight line, com ing out of the point of the big gest dam age (time 0), com ing through the tem po ral points till it reaches oxo-8-dG/105dG of base line. Then Microsoft Ex cel makes the in cli na tion of the line and these lines are pre sented in Fig. Elim i na tion ve loc ity of oxo-8-dG/105dG was 0.113±0.049 against 0.109±0.025 oxo-8-dG/105dG per 1 min for liv ers of young and old mice re spec tively; 0.134±0.033 against 0.086±0.009 of oxo-8-dG/105dG per 1 min for brain of young and old mice re spec tively; 0.118 young and old mice re - spec tively 0.100±0.029 of oxo-8-dG/105dG per 1 min for heart of young and old mice re spec tively; ve loc ity of elim i na tion of oxo-8-dG/105dG for young and old mice was com pared sta tis ti cally us ing Stu dent’s test. No sta tis ti cally re li able dif fer ences were ob tained for all three tis sues. How ever, the lev els of oxo-8-dG/105dG were higher (p<0.001) in old mice than in young mice, in all tem po ral points, ex cept for 30-min point of all tis sues, and the point, cor re spond ing to 7.5 min for liver [18] quite an other topic. “Gen er ally speak ing” ev ery thing is “clear”, mainly. The sec ond way to elim i nate the mu ta tions is un - usual. This is the way to elim i nate the mu ta tions from the cells, pre serv ing the lat ter, which is ob vi ous from the fol low ing ex am ple [23]. The con tents of dif fer ent types of mu ta tions were stud ied in com pletely char ac - ter ised se quence in dif fer ent tis sues of trans gen ic mice. Young mice, 3.5-month-old, (by this time all their tis - sues and or gans were formed) and old (de crepit) mice, 32-month-old, (spe cific mice life time is 3 years) were used in this ex per i ment. The de tailed study on the mu - ta tion spec tre re vealed that 2 mu ta tions of 4 dis cov ered in the brain (i.e. where cell sub sti tu tion is min i mal, and with no dam ages is even in sig nif i cant) of young mice dis ap peared com pletely by 32-month-old age. One type of mu ta tions dis ap peared in the spleen (cell re - newal in it takes place reg u larly) (Fig.8). This sort of pu ri fi ca tion from mu ta tions re sulted in ei ther no in - crease in mu ta tions with age or its be ing in sig nif i cant in some tis sues (Fig.9). There fore, both the level of dam ages and the level of fixed mu ta tions (i.e. mu ta tions, clas si cal ac cord ing to all the views) are main tained in the or gan ism (soma) at the level, re quired by it (or gan ism). Re quired level! For the misbalance to wards any side (i.e. to any, ei ther higher or lower level) turns out to be harm ful and leads to neg a tive con se quences. As it has been men tioned above, it has been proven by the ex per i ments. Al most the ma jor part of ge nome is in volved into the rep a ra - tion, i.e. in the ag gre ga tion of pro cesses, de ter min ing the level of mu ta tions, to some ex tent. Thus, in the case of bac te ria, their con tri bu tion is 30% of ge nome to tal, ex ceed ing 1 000 of genes in the ab so lute num ber [25]. MU TA TIONS: WHAT ARE THEY? 227 Fig.6 “In creased sen si tiv ity to ox i da tive dam ages of DNA pro mot ers of genes, ac tiv ity is age-down-reg u lated. Hu man cul ture of cor ti cal neu rons was in cu bated in the pres ence or ab sence of 100 mM H2O2/20 mM FeCl2 in the course of 12 hours, and then dam age of DNA pro - moter was de ter mined. The val ues are the av er age val ues of ±s.d., n=3. Stars in di cate p<0.05 re gard ing un treated sam ples; p<0.001 for the groups of genes with pro mot ers, ac tiv ity of which de creases with age in re gards to the pro mot ers of genes, ac tiv ity of which does not de pend on the age, or genes, ac tiv ity of which in creases with age” [22] Fig.7 “Dam age and rep a ra tion of DNA pro mot ers of b-tubulin and calmodulin 1. Re porter plasmids, dam aged in vi tro with H2O2, were transfected, then in each pro moter dam aged DNA se quence was de - ter mined. The val ues are in di cated in re gards to un dam aged transfected re porter DNA, and are the av er age value of three changes (n=3) ±s.d. (stan dard fluc tu a tion). Stars in di cate p<0.05 in re gards to b-tubulin” [22] In mam mals it is more pre cise (and, as one can sup pose, much more di verse). In the case of spot mu ta tions (e.g. nu cle o tide sub sti - tu tions) their reg u la tory func tion re mains un known (and for the sake of jus tice, it has to be said that it has never been stud ied). Only some thing spe cial can be dis tin guished – tau to meric pas sages. Tau to meric changes in bases can not be re paired. By no means and in no way. They are reg u lated by the con di tion of DNA: spa tial struc ture, de pend ent on the pri mary se - quence, DNA in ter ac tions with pro teins, small mol e - cules, etc. For some rea son no at ten tion is paid to the fact that the form of mu ta gen e sis, reg u lated by the cell, ex ists in the lat ter con tin u ously. Lots of ex per i men tal ma te rial have been ac cu mu lated at the level of larger changes and their reg u la tory sig nif i cance is man i fested viv idly. Viv idly enough not to be ig nored. How ever, be ing de scribed, it is masked by other ter mi nol ogy (like it is valu able enough to change the mean ing). Chromatin dim i nu tion in Ascaridae was de - scribed the first in this sense (i.e. reg u la tory role of mu - ta tions), be ing strictly func tional and pre ci sion elim i na - tion of a part of ge netic ma te rial in soma af ter di vi sion of the germ route into its own new con tin u a tion (germ route) and dead end – soma. Later on, dim i nu tion was dis cov ered in other or gan isms as well. More over, it KORDIUM V. A. 228 × à ñò î ò à ì óò à ö è é ,· 1 0 –5 × à ñò î ò à ì óò àö è é, ·1 0 –5 4,0 3,0 2,0 1,0 0,0 4,0 3,0 2,0 1,0 0,0 Ìîçã Ñåðäöå Ïå÷åíü Ñåëåçåíêà Òîíêèé êèøå÷íèê G -C ä î A -T G -C ä î T -A G -C ä î C -G A -T ä î n -n D el (- 1 ) G -C ä î A -T G -C ä î T -A G -C ä î C -G A -T ä î n -n D el (- 1 ) G -C ä î A -T G -C ä î T -A G -C ä î C -G A -T ä î n -n D el (- 1 ) G -C ä î A -T G -C ä î T -A G -C ä î C -G A -T ä î n -n D el (- 1 ) G -C ä î A -T G -C ä î T -A G -C ä î C -G A -T ä î n -n D el (- 1 ) 3,5 ì å ñ ÿ ö à 3 2 ì å ñ ÿ ö à Fig.8 “Point mu ta tion spec tra for re porter gene lacZ in brain, heart, liver, spleen, and small in tes tines in young (3.5 month) and old (32 months) mice. The col umns rep re sent the fre quency for each type of men tioned point mu ta tions. Black part of the col umn of G:CA:T shows the part of mu ta tions, oc cur ring in CpG-sites, grey part – in Del(-l)-col umns in di cate the fre quency of each mu ta tion, tak ing place in the re - peated sites, e.g. se quence of three or more nu cleo tides. Cor rec tions have been made for the re peated mu ta tions” [23] was shown to be in larger amounts, com pared to Ascaridae. Thus, in cil i ates in some cases, elim i na tion reaches up to 95% of ge nome, com pared to the one pre - served in germ route [26]. Dim i nu tion is the most re li able form of reg u la tion. What is ab sent can not be re vealed, ac ti vated, real ised il le gally, etc. There is no need for spe cial sys tems of reg u la tion (com plete switch ing off and keep ing up to this state), which is ob vi ously prof it able for com plex sig nal ling path ways. How ever, it is ab so lute in mam - mals only (hu mans in cluded). 100% elim i na tion of ge nome is their spe cific fea ture solely, which takes place dur ing erythropoiesis. At cer tain stages of dif - fer en ti a tion, the nu cleus is re moved com pletely from the cell in eryth ro cytes pre de ces sors. On the one hand, this is the way to save the space, nec es sary for the ma te rial, pro vid ing ox y gen trans fer. On the other hand, mas sive and the larg est cell pop u la tion, con - stantly formed and dis ap pear ing, po ten tially max i - mally dam aged (ox y gen trans fer, i.e. its con tin u ous con sump tion, stor ing, and de liv ery) would be of ex - treme threat of mas sive con tin u ous over all malignisation, if it had the nu cleus. No pow er ful im - mune sur veil lance would be enough to stop it. In this re spect the reg u la tion is also anti-oncogenic (and thus ab so lute). Puffs of diptera were the next dis cov ery. The puffs are ba si cally formed on the chro mo somes in the sialaden cells. They are me di ated with the sec tors of nor mal size and con sti tute man i fold (more than 1 000-fold in their ex tremes) am pli fied genes, whose func tion is to be strength ened. In these cases, the reg u - la tion of gene ac tiv ity is achieved by their cor re spond - ing mul ti pli ca tion. The for ma tion of puffs as a type of reg u la tion trig gered new re search. Heat shock pro teins (pre ced ing chap er ons) were dis cov ered via the oc cur - rence of the new puff in drosophila at rap idly in creased tem per a tures. The am pli fi ca tion in mam ma lian cells was dis cov ered un der the in flu ence of toxic sub stances (start ing with methotrexate) as the re sponse, lead ing to the in crease in pro duc ing de fen sive pro teins. Yet “spon ta ne ous” am pli fi ca tion of dif fer ent genes is nat u - ral for the mam ma lian cells. Ac tu ally, the term spon ta - ne ous is nei ther syn on y mous to cha otic nor all of a sud - den. Spon ta ne ous is when the rea son is un known… un - known, but not ab sent. So far, the sci en tists do not know how to study the am pli fi ca tion in mam ma lian or - gan isms di rectly. They can only de ter mine it in cell cul ture. It can nei ther be ne glected nor sta tis ti cally the same for dif fer ent genes. Thus, for two hu man genes, back ground dif fer ences of the lev els of am pli fi ca tion be tween dif fer ent cell lines were over 170 times one gene-wise and 3 times higher an other gene-wise, as for these genes within one and the same cell line – 120 times higher for one pair and app. 4.5 times for the other one (Ta ble 7). Am pli fi ca tion is typ i cal “large change in ge nome with ap par ent phenotypic man i fes ta tion”. Ac cord ing to clas si cal def i ni tions these are the mu ta tions with reg u - la tory func tions. Spe cial class of mu ta tions is dis tin guished on the ba sis of combinatory am pli fi ca tion-re com bi na tion mech a nism. The first one to be men tioned (de tec tion time-wise) is mag ni fi ca tion. Any re peats are in clined (due to their iden tity ac cord ing to the pri mary se - quence) to recombinational chip ping off and, as one of the con se quences, to losses. Ri bo somal genes in ge - nome are multi-copy ones. Due to this fact their num - ber may de crease in gen er a tions. In such cases, as it has been ini tially dis cov ered in drosophila, the mech a nism of their res to ra tion is switched on in the germ route cells. At a cer tain stage, re main ing part of genes of ri - MU TA TIONS: WHAT ARE THEY? 229 Fig.9 Fre quency of re porter gene lacZ in brain, heart, liver, spleen, and small in tes tines in trans gen ic mice of 60 line (Dolly et al., 2000, Mar tin et al., 2001; Geise et al., 2002). Lines are pre sented as “man - u ally drawn” curves, cor re spond ing to the av er age mu ta tion fre - quen cies in dif fer ent age groups. Re gions marked in grey rep re sent the curve of mice sur viv abil ity, cor re spond ing to 100% at birth and fall ing down to 50% at 26.5 months” [24] bo somal DNA is chipped off le gally from the chro mo - some, am pli fied in this au ton o mous con di tion, and then in te grated back into the chro mo some (where it has got to be, homology-wise). Thus, the num ber of nec es sary genes is re stored in the gen er a tive line. Sim i lar mech a nism is the ground for a qual i ta tively new phe nom e non – the for ma tion of new genes (not pres ent in the germ route, and, there fore, in germ cells as well) and “on-de mand genes”. A well-known ex am ple of the for ma tion of new genes in soma (not pres ent in the germ route and non-in cor po rat ing into it af ter their for ma tion in soma) is the genes of an ti bod ies. Due to the spe cial mech a nism, i.e. so called, V(D)j-re com bi na tion in the line of B-lym pho cytes there may be (and ac tu ally takes place) the for ma tion (al most end less in its di ver sity) of new genes, en cod ing an ti bod ies to var i ous an ti gens. These genes are not pres ent in the germ route, they oc cur in soma de novo. If nec es sary, one more mech a nism gets in volved – clone se lec tion, and the cells, hav ing this new gene, are mul ti plied in ten sively. This is a good sign of the fact that in suf fi cient fre quency of mu ta tions in the or gan ism can not be put for ward as the ob jec tion to their func - tional load. Genes of an ti bod ies can serve a good ex - am ple of the for ma tion of any amount of new nec es sary genes in soma (not any where but where it is re ally needed), which are not pres ent (and could not be pres - ent) in germ route ge nome. Yet an ti bod ies are far from be ing just the el e ment of im mune de fence. The dis cov - ery of abzymes – cat a lytic an ti bod ies – re veals that their func tions may be much broader. At the same time these cat a lytic an ti bod ies may be as com plex as pos si ble. For in stance, DNA-hy dro lys ing an ti bod ies to na tive DNA (they were dis cov ered in blood se rum of pa tients with sys temic lupus erythematosus) are metal-de pend ent (i.e. com plex func tion ing) endonucleases [28]. As V(D)j-re com bi na tion is known to spread on the events, oc cur ring in not im mune sys tem solely, and there are some other re com bi na tion sys tems, then the dis cov ery of the for ma tion of new genes in soma is log i cal to be the mat ter of time. The other ex am ple (not yet de scribed in mam mals) is the oc cur rence of “on-de mand genes”, i.e. the phe - nom e non un der dis cus sion since Lamark’s times as the prob lem of “in her i tance of the ac quired fea tures”. In 1988–1991, the au thors of [29, 30] de scribed the first trust wor thily re pro duc ible ex am ple of the oc cur rence of the fea ture (char ac ter is tic) “needed” with sub se quent in her i tance. These ex per i ments re vealed that re verse mu ta tions in mu tant gene b-galactosidase take place more of ten at the con tent of cells with these mu ta tions in the me dium, where the only nu tri tion source was lac - tose, than at av er age ge nome-sta tis tic con tent. This fre - quency is also higher than in the cur rent gene (i.e. in the same mu tant gene b-galactosidase with re turn to the nor mal state) com pared to the fre quency of mu ta tions on rich nu tri tion sub strates. De tailed study on this is sue re vealed that its ef fi ciency is de pend ent on the de gree of com plete ness of am pli fi ca tion and re com bi na tion mech a nisms (Fig.10). To achieve the max i mal ef fi ciency of such events, the gene has got to be flanked by the re peats (ide ally – to be on the trans po si tion el e ment), and the cell has got to con tain the pro teins, pro vid ing both DNA am pli fi ca - tion and its re com bi na tion [31]. In such com bi na tion the prob a bil ity of “nec es sary” mu ta tion in creases 1 000-fold. Not the gen eral level of mu ta tions, but the level of mu ta tions of a cur rent cell. The mech a nism of such pref er ence is not clear yet. Ba si cally, it co mes down to the ac ti va tion of am pli fi ca tion and re com bi na - tion. So far, this phe nom e non is most stud ied in bac te - ria. How ever, all nec es sary el e ments for this pro ce dure are pres ent in eukaryotes gen er ally, and hu mans in par - tic u lar, in com pa ra bly better, more of ten, abun dant, than in bac te ria. Eukaryotic ge nome con sists of the re - peats and var i ous po ten tially (and at some con di tions ac tu ally) mo bile el e ments. Prin ci pally im por tant for this con cept is the fact that vari ants of re peats in the ge - nome are way too nu mer ous (in the num ber of el e ments of re peats, their to tal num ber, i.e. lengths) and all of them (all of them!) dif fer from ran dom dis tri bu tion man i folds [32]. They are sup ported by the se lec tion in the cur rent form, they should be like that in or der to per - form their func tion. The bright est, the most ev i dent, the most wide-spread func tion of the re peats is the pro vi - sion of the re com bi na tion events as well as par tic i pa - tion in them. More over, al most all hu man genes are flanked with re peats (Alu mainly). The reg u la tory role of ge nome re or gani sa tion (though very lim ited yet) is al ready ad mit ted prac ti cally for all the liv ing be ings (Ta ble 8). KORDIUM V. A. 230 Yet men tion ing this role, the most ap par ent thing is not noted – all this takes place at the level of soma and not in the germ route. The unique mech a nism (al most not stud ied yet) which can both per form reg u la tory func tions and cre ate qual i ta tively new in for ma tion, is the mu ta tions with the shift of the read ing frame (+1, +2, i.e. the in ser tion of one or two ad di tional nu cleo tides, or –1, –2, i.e. de let - ing one or two nu cleo tides). At RNA level, the for ma - tion of new in for ma tion is well known and stud ied. It is due to the fact that at the stage, suit able to per form the func tion (at least in eukaryotes), the syn the sised RNA is in ad e quate in pri mary se quence to its ma trix – gene where tran scrip tion was ini ti ated. Be sides fa mil iar splic ing, dur ing which the in for ma tion, read off the gene, “is gath ered” by frag ments and pro vides the syn - the sis of dif fer ent pro teins at dif fer ent al ter na tive vari - ants, there is also the shift of read ing frame (due to RNA-poly mer ase slip ping) and ed it ing. All this is suit - able for RNA. But for DNA it is not ac cepted at all. Only reg u la tory role of mu ta tions with the shift of read - ing frame is slowly in tro duced (and rather care fully) at the level of ge nome. Thus, ad he sion an ti gen, which switches back and forth with the fre quency of 10–3–10–4 ac cord ing to the phase-vari able prin ci ple (i.e. both ways: ex pressed - not ex pressed) has been dis cov ered in mycoplasma. Such switch ing back and forth is real - ised due to the shift of the read ing frame. It may re sult in the for ma tion of stop-codon or the oc cur rence of a new read ing frame, i.e. RNA, which has to be trans lated into a new pro tein, is read off [34, 35]. Both the over com ing of mu ta tion with the shift of read ing frames, and the for ma tion of such mu ta tions can be reg u lated. Thus, spe cial 8-mem ber se quence (CGCGCGCG), which is a hot-spot of mu ta tion – 2 shifts of read ing frame – is de scribed in ge nome of eubacteria. Such dou ble shift is pro vided (by skip ping 8th gua nine of the hot-spot) by spe cial holoenzyme of DNA-poly mer ase III, i.e. the pro tein, spe cif i cally en - coded in ge nome (and reg u lated) for this pur pose [36]. In eukaryotes, this sys tem of reg u la tion (i.e. with the shift of the read ing frame and its over com ing) ac - quires more com pli cated char ac ter. In yeasts the com - plex sys tem of reg u la tion of met a bolic pro cesses, with the par tic i pa tion of SVF-group genes at the ex pense of cor rect ing the mu ta tion with the shift of read ing frame has been de scribed. SVF13 gene is en coded by the tran - scrip tion fac tor Mbx1p. But mu ta tion +1 of the read ing frame is re vealed in this gene. Syn the sis of sup pres sor, pro vid ing the skip ping of +1 base, takes place si mul ta - neously. As a re sult the level of tran scrip tion, de ter - mined by Mbx1p fac tor, de pends on the level of syn the - MU TA TIONS: WHAT ARE THEY? 231 Ta ble 7 Fre quency of gene am pli fi ca tion in con trol or mICAD-ex pres sion of HCT116 and L929 cells [27] Type of cells Irradiation dose, Gy PALA selection (amplification of cad gene) MTX selection (amplification of dhfr gene) PE, % LD50, mM Total quantity of selected cells, ·106 PALAr, frequency• 10–5 PE, % LD50, mM Total quantity of selected cells, ·106 MTXr, frequency• 10–5 HCT116/control – 45.3 31.4 4.5 5.54 42.0 17.5 15.6 0.046 HCT116/mICAD – 64.7 15.0 1.5 27.4 41.0 13.3 15.6 0.25 HCT116/control 3 59.3 31.4 2.4 8.50 13.4 17.5 14.3 0.21 HCT116/mICAD 3 66.0 15.0 0.6 41.7 18.0 13.3 11.5 1.01 L929/control – 47.3 21.3 20 0.032 47.3 31.0 10 0.15 L929/mICAD – 55.5 9.9 3 21.3 55.5 16.7 12 0.81 L929/control 4.5 22.8 21.3 4.8 3.66 22.8 31.0 4.8 10.5 L929/mICAD 4.5 11.8 9.9 2.4 81.6 11.8 16.7 3 37.4 Nota Bene: PE – clon ing ef fec tive ness; LD50 – the dose of 50 % growth cells in the pres ence of the in hib i tor; PALAr – PALA-sta ble clones; MTXr – MTX-sta ble clones. sis of sup pres sor of mu ta tion with the shift of read ing frame [37]. The pro cess of reg u la tion can be com plex and com pli cated. Thus, one of the key reg u la tors of such pro cess in yeasts is the MMR-pro teins. The in - crease in mu ta tions with the shift of read ing frame takes place at any kind of switch ing off their ac tiv ity (func - tional or mutational). It takes place on 8-mem ber hot-spots. Al most 25% of all genes in these or gan isms con tain 8-mem ber poly-A or poly-T tracks. And these tracks are highly-sta ble in ge nome (i.e. are not chipped off). How ever, there are highly-la bile poly-G or poly-C tracks, on which the mu ta tions take place es pe - cially ac tively. The in ten si ties of these pro cesses have been dem on strated on the re verse of lys– into lys+. The fre quency of re stor ing lys+ in creases 100 000 times at the pres ence of such tracks in lys– gene and the con tent of in ac ti vat ing Rsh2 mu ta tion in ge nome [38]. Con trary to spot-mu ta tions with the sub sti tu tion of the bases, the reg u la tory role of mu ta tions with the shift of read ing frame may have rather log i cal ex pla na tion by the fact that the shift oc curs pref er a bly (or even solely) on some hot-spots, i.e. on cer tain se quences (not just any where), and due to this it may be per formed in the gene spot, spec i fied for this pur pose. Over com ing of the shift of the read ing frame is con di tioned by spe - cial pro teins, also en coded in ge nome. There fore, both KORDIUM V. A. 232 Fig.10 Re ver sion of lac operon at the in ser tion of MudCF in sev eral dif fer ent po si tions: a – typ i cal in ser tion of MudCF class I (shaded squares TT18302 (rec+/F’lac); empty squares – TT20853 (rec+, oadB10::MudCF)); b – A mutS177::MudCF in ser tion (shaded squares TT18302 (rec+/F’lac); empty squares – TT18306 (rec–, oadB10::MudCF)); shaded tri an gles – TT20864 rec+, mutS::MudCF)); c – typ i cal in - ser tion class II (shaded squares TT18302 (rec+/F’lac); TT18306 (rec–/F’lac)); shaded tri an gles – TT20009 (rec+/pSLT MudCF); empty tri - an gles – TT23011 (rec– pSLT MudCF)); d – typ i cal in ser tion class III (empty tri an gles TT22996 (rec+, dbpA::MudCF); shaded tri an gles – TT23014 (rec+, dbpA::MudCF)) [31]. shifts of the frames and their over com ing, and the pos - si bil ity of the lat ter, are con trolled pro cesses. The shift of the frame in these pro cesses is just one of the el e - ments of the reg u la tion. Con sid er ing the mu ta tions in soma cells, as el e - ments of reg u la tion, pos si ble ob jec tion is the rel a tive rar ity of such events. Let it be 10–5, or even 10–3, but this is in a sin gle cell per 100 000 or per 1 000. And the reg u la tion of all tar gets by the pro teins is 100%. It is true ar ith met i cally. But it is im pos si ble to limit the reg - u la tion by arith me tic. Pro tein reg u la tion, which is both di verse and quan ti ta tively dif fer ent for some types of cells (in clud ing closely lo cated ones), is not the same (Fig.11). Neigh bour ing cells are func tion ally not the same! There fore, the cells of one and the same tis sue are het er o ge neous in their reg u la tion. But even if we come out of the fre quency of mu ta tions only, 10–5–10–3 is the enor mous value for soma. Ev ery hu man “con - sists” of ~5·1013 of cells. Strictly quan ti ta tive data show that the mu tant cell may pro duce the clone of any size at any prob a bil ity of its oc cur rence, which is ev i - denced by the clone se lec tion of lym pho cytes, car ry ing the an ti body, nec es sary for the or gan ism. And it con - cerns all tis sues and or gans. The at ten tion to this phe - nom e non is paid only when it is hard not “to see” it. E.g. Dif fer ent types of mu ta tions by dif fer ent genes in soma re sult in the oc cur rence of spots in swine (Fig.12). Ev ery spot is the mu ta tion in one gene of one cell. Then, the mul ti pli ca tion to the size of the spot oc curs. The re sult can not be ne glected – the spots are of dif fer - ent colours. These spots were sub dued to the de tailed anal y sis. How ever, the mu ta tions are not lim ited by the oc cur rence of the spots. Cor re spond ing mu ta tions will also take place in all the genes, where it is nec es sary to the or gan ism. And this can be omit ted… and it is omit - ted. Fi nally, let us take a look at the methylation. It seems like the works, car ried out on monozygotic twins, are the most prom i nent in this re spect. Monozygotic twins are clones both ac tu ally and ac - cord ing to the mech a nism of orig i na tion. As in any clones, their genomes are iden ti cal. Iden ti cal in ev ery - thing, in clud ing the methylation. How ever, when the amaz ing method, al low ing the iden ti fi ca tion of methylation of ge nome in each sep a rate twin, i.e. dis tin - guish ing the dif fer ences be tween them, was elab o rated, the re sult was as ton ish ing. In the young age, the con di - tions of life of twins were “twin-like” and the pat tern of methylation re vealed the iden tity ex per i men tally. But, later on, the con di tions of their lives were sep a rated and the methylation of genomes, iden ti cal since birth, ac - quired some sig nif i cant dif fer ences [41]. It may oc cur only in the case when the methylation is the reg u la tion of fine ad just ment of the whole ge nome. The ad just - ment to ac tual life and ac tual en vi ron ment. Only a part (and very lim ited part) of the whole ge nome is sub ject to the clas si cal reg u la tion – via sig nal ling path ways and pro tein reg u la tors. The other part is as sumed to work in the con sti tu tive mode. It is true in re gards of ex per i - men tal de ter mi na tion of ex pres sion range at dif fer ent ef fects in the real time of reg u lar ex per i ment. Con sti tu - tively work ing genes do not change their ex pres sion at once, and, in fact, they should not. But gen eral fine re-ad just ment can not but take place at long-term changes in liv ing con di tions. Gen eral and gen er - ally-cor re lated! Af ter that, con sti tu tive genes will work again in the nar row range of changes in the lev els of their ex pres sion (or gan ism is way too com pli cated to change ev ery thing, ev ery where, and at once in re spect of each, usu ally tran sient, change in some ex ter nal fac - tor). As for new sta tion ary level of liv ing, re-ad just - ment is nec es sary. “Mutational cri sis” i.e. un der stand ing of the fact that the as sump tions of mu ta tions re quire se ri ous re - view ing, leads to the prob lem of re con sid er ing ba sic bi - o log i cal con cepts. In ves ti ga tions of cer tain pro cesses and events pre vent us from see ing the life as phe nom e - MU TA TIONS: WHAT ARE THEY? 233 Ta ble 8 Three types of de vel oped reg u la tory genomic re or gani sa - tions and taxons, where the re or gani sa tions oc curred Category Type of reorganisation Taxon 1. Wide range of reorganisations Animals: Nematodes, Copepods, Hagfish, Foraminifera, Ciliates 2. Target reorganisation Immune system of vertebrates Tripanosomes antigenes Type of yeast coupling 3. rNDA – special case Animals, Enthamoeba, Euglenids, Dictyostelids and myxomycetes Ciliates non! Mean while, life is a unique phe nom e non. It is based (based, ex actly so) on ab so lutely out ra geous de - struc tive pro cesses. The lat ter are so in ex press ibly pow er ful and rapid, that no non-liv ing mat ter would sur vive them. Whereas they are foun da tions of life, nec es sary for the ex is tence: breath ing (all types of ox y - gen trans for ma tion), per ox ides and rad i cals, high-en - ergy in ter me di ary prod ucts of en zy matic chains, en ergy trans fer be tween macromolecules and in side them, en - zy matic (i.e. high-ca tal y sis) re ac tions, func tion ally di - rected to wards the de struc tion, deg ra da tion of ev ery - thing, in clud ing cell ma te rial of its own (pro teas es, nu - cleases, li pases etc) and it goes like that ev ery where. From the stand point of the oc cur ring events, life is a con tin u ous self-di rected met a bolic in for ma tional ex - plo sion. These pro cesses proper sup ply the en ergy, suf - fi cient for liv ing, for all con struc tive me tab o lism and all forms of pro tec tion from any thing, pres ent on the Earth (and reach ing the Earth from the Space). In or der for all this con stantly and per ma nently work ing, ex plo - sion-like de struc tive stuff not to tear the cell apart into tiny mo lec u lar pieces, it is or gan ised prop erly in space and time, with strict sub or di na tion to the most ef fec tive con trol. In its struc tural and func tional or gani sa tion the sim plest or gan ised cell is the dream of all nanotechnologies, ab so lutely im pos si ble in the near est fu ture. Mu ta tions are to be viewed in the con text of all these pro cesses and not as just oc ca sions. The first stage on the way to such new con cepts on mu ta tions is the eval u a tion of role, con tri bu tion, mean ing, etc of mu ta tions in var i ous life con tinua, where mu ta tions are sim i lar in their role, con tri bu tion, mean ing, etc (some “con tinua of mutational sim i lar ity”). Con tin uum of mutational sim i lar ity of germ plasma (germ route, germ line) and con tin uum of mutational sim i lar ity of vertebrata soma are the first in the con text of the afore - men tioned ma te ri als (i.e. the most stud ied ones, and the most cur rent for us). Germ route is the clas sics of con cepts on mu ta tions. All ex ist ing con cepts on the laws of in her i tance, namely men de lian in her i tance, re ces sives and dominants, penetrance and expressivity etc, were cre - ated on the ba sis of mu ta tions on germ route (through the reali sa tion in soma, but in all of its cells as de riv a - tives of germ route). Later on, all this was ex trap o lated, trans ferred, gen er al ised etc onto soma in its pure form ac cord ing to ready made, well-es tab lished and com - monly ac cepted pro vi sions (per fectly proven in prac - tice), well stud ied and jus ti fied in gen er a tive lines only, de pend ing on the ap pear ance of meth od olog i cal pos si - bil i ties of soma anal y sis. The sit u a tion of such ab so - KORDIUM V. A. 234 Fig.11 In creased vari abil ity from cell to cell of gene ex pres sion in cardiomyocytes of old mice com pared to hose of the young mice: a – ex am ples, re veal ing sta tis ti cally trust wor thy dif fer ences from cell to cell in the ex pres sion of four genes (Actb, B2m, Lpl, and Actc1, stand ard ised by Gapdh) b – ex pres sion of mi to chon drial genes of cytochrome c oxidase (stand ard ised by COX1) in the same cells did not re veal sta tis ti cally sig nif i cant in crease at ran dom se lec tion (10 repaets for old and young cells from one etalon were ana lysed for de ter min ing RT-PCR er ror); c – rep re sen ta tive cards of one young and one old mouse re vealed sig nif i cant dif fer ences in cell sam ples (es ti mated for each gene), which tes ti fies on ran dom ness of the dif - fer ences ob served from cell to cell [39]. lutely in ad e quate trans fer was com pli cated by the fact that, though mu ta tions oc curred and se cured in the germ route, they were real ised in soma! Even so the gen er ali sa tion seemed so ob vi ous that there was no room for doubt left. Re al ity made its way through with enor mous ef forts – mu ta tion in the germ route re sults in sub se quent oblig a tory re pro duc tion, mul ti pli ca tion in all cells, de scen dants of such ini tial mu tated cell of germ route. If the mu ta tion of the germ cell ini ti ated the soma, it will pro vide “its” mu ta tion in all soma cells. All of them! And germ route ge no type (!!) is real ised in soma phe no type (!!!). Ev ery thing is pre des tined by it – mu ta tion takes place in the germ route, but later on, once it is se cured, it is to be trans ferred in “its” germ route and real ised in soma. Trans ferred in the germ route and real ised in soma. At the same time, func tional task of the germ route is the max i mal (the o ret i cally ap - proach ing the ab so lute) in for ma tional con ser va tism. Func tion-wise, there should be no mu ta tions in the germ plasma what so ever. Oth er wise, the lat ter does not ful fil its func tions and is to be elim i nated as a line. It oc curs due to in con sis tency of soma, which oc curred from such germ route, car ry ing it, to the ex ter nal con di - tions. Due to in con sis tency of the germ route to soma, which it con tains, i.e. to the in ter nal con di tions. In con - sis tency of com pet i tive ness of “re veal ing agent” of its func tional ap pli ca bil ity in re gards to other germ routes in other soma etc. There fore, ev ery thing in the germ route is di rected to wards the elim i na tion of mu ta tions by any means – both “in ter nal” (us ing sys tems of pro - tec tion, rep a ra tion, max i mal de crease of bio chem i cal ac tiv ity while hav ing the max i mum of ev ery thing re - quired from sur round ing soma cells, etc) and “ex ter nal” (by elim i na tion in side, in gen er a tive sphere of cells – MU TA TIONS: WHAT ARE THEY? 235 Fig.12 Dem on stra tion of dif fer ent types of col our phe no types, con nected with al lele of Ep lo cus Ex ten sion/MC1R: a – F2 gen er a tion – hy - brid of wild sore and big white soar (pigs (left to right): 1 –(Ep/Ep), red col our with black spots, phenotypically close to Lingirod swine (not pre sented); 2 – white spots, oc cur due to the pres ence of white al lele; 3 – heterozygote (E+/Ep),has got white type colour ing on bot tom right side, which shows so matic re verse; 4 – (Ep/Ep), white with black spots, colour ing is sim i lar to Pietrain (not pre sented)); b – Tam worth; c – Glou ces ter Old Spot; d – Berk shire (photo A. Chris tian and M. Rothschild, Lowa State Uni ver sity) [40] mu ta tion car ri ers). For this pur pose, in some cur rently un known way, mu ta tions are absolutized in cas cades into dis or ders of germ route cells, which are in com pat i - ble with fur ther ex is tence (ful fill ing func tions) of those germ route cells, which have the mu ta tions (ini tial, re - sult ing in chain ac cel er a tive re ac tion). This is the only way to ex plain in con ceiv able re al ity. The fi nal stage of each sin gle func tional cy cle of germ plasma is re sulted in the way from ma ture germ cells through the en tire sub se quent embryogenesis back to ma ture germ cells. Ev ery end of this way is char ac ter ised by both enor - mous ab so lute value and per cent age of ma ture germ cells with large chro mo somal dis or ders, in com pat i ble with vi tal ity and/or ful fil ment of func tions. Thus, in case of hu man oocytes, the data of dif fer ent lab o ra to ries re gard ing chro mo somal ab nor mal i ties in healthy women in dif fer ent pop u la tions were shown to fluc tu - ate from 4.5 to 47.7% (Ta ble 2), and the av er age value de ter mined by the karyo types of 1 120 oocytes was 35% [42]. In case of sper ma to zoids these val ues amounted from one to sev eral doz ens per cent in the most fa vour able se lec tions [43–45]. These val ues grew in ten sively in un fa vour able pop u la tions reach ing 100%. This sit u a tion is the mas sive elim i na tion of some thing which is the cri te rion for the germ route, and is con tin u ously ob served at all of the germ route stages. The num ber of germ cells in fe male germ route in 7-month-old fe tus in creases to (6–7)•106 as a re sult of mul ti pli ca tion. Then the elim i na tion takes place and not more than 400 re mains till ovu la tion, i.e. app. 99.994% is elim i nated [46, 47]. Elim i na tion is even higher in male germ route tak ing into ac count post-na - tal pro cesses. There it is – if we as sess the num ber of mu ta tions ac cord ing to com monly ac cepted meth ods, it is higher in germ route cells than in soma, and their elim i na tion is so high that if it were the same for soma, there would be noth ing left of soma what so ever (sev - eral thou sandths per cent would be suf fi cient for one ex fo li ated furfur). How ever, due to such un com pro - mis ing char ac ter, the life on the Earth has been go ing on for 4 bil lion years, and the spe cies (in its rather sta ble con di tion) ex ists on av er age for sev eral mil lions years. Soma has whole way dif fer ent tasks (and ev ery - thing, in clud ing mu ta tions, is sub mit ted to these dif fer - ences). Con trary to the germ plasma, whose line is po - ten tially end less, the line of soma is al ways the dead end, rep re sented by an or gan ism, spec i men, in di vid ual, i.e. ter mi nal some thing, po ten tially lim ited by spe cies life time, func tional task of which is the pres er va tion and the trans fer of the germ route only (only!). This is the “na ture”-pre de ter mined way. And no mat ter who, how, and how much would dis agree with it, it means noth ing for life as phe nom e non. Some body dis agrees, so what? Does this some body live lon ger af ter that? No, he does not. Has he not per formed his func tion, pre scribed to him by life as phe nom e non? Then germ plasma line will elim i nate him, def i nitely and ex clu - sively him (which is also fore seen by life), along with him and in him. Only now the work starts on turn ing “dis agree ments” into some thing tan gi ble. One of the el e ments of such “turn ing into” is un der stand ing of the specificities, dif fer ences, mech a nisms of ev ery thing, which takes place in soma and the germ route. Mu ta - tions com prise one of the el e ments of such un der stand - ing. Mu ta tions in soma are rad i cally, con cep tu ally, prin - ci pally etc dif fer ent from what is spe cific for germ route, both ac cord ing to the tasks of soma and in its or i - gin, reali sa tion, func tions. In the case of soma these are dif fer ent vari ants of reg u la tion (of ev ery thing!!), in - clud ing in for ma tional va ri ety. Re com bi na tion and read ing frame shift are the ge nius so lu tions of life as phe nom e non. In its po ten tial pos si bil i ties, this is al - most end less in crease in soma in for ma tional bulk with no change in germ plasma in for ma tional bulk. As of date there are no lit er a ture data. Still the at ten tion has been paid to a nem a tode, hav ing a larger gene num - ber-wise ge nome than an in sect (Drosophila). As for hu man it is only 2–3 times larger than in a fly. How is that pos si ble? One of the vari ants is the oc cur rence of new genes in soma. In the case of germ plasma, the less num ber of genes is, the better (eas ier to pre serve their sta bil ity). Al most all of its genes, ex cept of house hold genes, are quiet. Soma is sup posed to re act to ev ery - thing ex ter nal and in ter nal. The pos si bil ity of in creas - ing the in for ma tional bulk is en coded in ge nome. This means that not the whole in for ma tion is en coded, but rather the pos si bil ity of its in crease and cre ation of new one. Be sides po ten tial pos si bil i ties of recombinations and mu ta tions with the shift of read ing frame, all al ter - na tive vari ants of gene ex pres sion (in clud ing al ter na - KORDIUM V. A. 236 tive splic ing, transcriptional frame shift and ed it ing) may re sult into genes via re verse tran scrip tion (func tion of RNA-de pend ent DNA-poly mer ase). They re sult in sep a rate (and dif fer ent) cells of dif fer ent tis sues and or - gans. On com ple tion of the re quired func tions, the cells with such new genes are elim i nated in loads as it takes place in embryogenesis. Elim i na tion of cells in soma is the con tin u ous and mas sive pro cess. There are more than enough pos si bil i ties for the re verse syn the sis in soma. Only L1-el e ments, en cod ing the pro tein with the se quence, which is con sen sus-like to known re - verse-transcriptases, are abun dant in all mam mals [48, 49], their num ber in hu man is close to the num ber of his struc tural genes [50]. Ac tu ally (in case of the pres ence of nec es sary se quences) hom ing-endonucleases may as sist the same (i.e. trans fer not only introns) [51]. In di - rect ev i dence to the fact that genes formed anew oc cur in fact, is the block ing of em bry onic de vel op ment through the in tro duc tion of an ti bod ies, in ac ti vat ing revertase [52]. Fine ad just ment of the whole ge nome via its methylation “pur su ant to re quire ment” pro vides some thing which can not be en coded as a ready made prod uct in ge nome “for all life oc ca sions” by any means. Some thing like this can ei ther not be pres ent in the germ route (the for ma tion of new genes due to re - com bi na tion, re-syn the sis or read ing frame shift, dim i - nu tion, reg u la tory mu ta tions), or it has com pletely dif - fer ent func tional load (mag ni fi ca tion, pu ri fy ing chain mutational re ac tion, switch ing the de vel op ment programmes by methylation). Ac cord ing to clas si cal ideas, mu ta tions are de ter mined by their se cur ing in germ route (and gen er ally called “ge no type”) via the reali sa tion in soma (al ready men tioned “phe no type”). Later on it goes to com mon way. It is ei ther he red i tary dis eases (if we are talk ing about hu mans) or “evo lu tion - ary mean ing”. And what ever is de fined as changes in ge nome of so matic cells (which are not trans mit ted via sex ual re pro duc tion) is epigenetics. What ever, the main thing is not to step aside from can ons. There is a need to change the mutational par a digm. Change it de novo, to re view what the mu ta tion is, tak - ing into ac count the whole bulk of data and knowl edge. Hugo De Vries was an out stand ing nat u ral ist. How - ever, it has been more than 100 years since his in tro duc - tion of the no tion of mu ta tion. In the last 100 years sci - ence in gen eral and bi ol ogy in par tic u lar ac cu mu lated enor mous amounts of ex per i men tal data, cre ated new meth ods and tech nol o gies, de vel oped con cepts and the - o ries and so forth and so on, many times more, than through out pre vi ous his tory of the man kind. And as for now a days, it is pos si ble to speak about mu ta tions, al though they are con sid ered to be the con - se quences of nat u ral pro cesses (as well as other things in the liv ing), yet they are de signed by life to per form nec es sary tasks and to bear func tional loads. The kinds of tasks vary on the groups of the liv ing. More over, they are to be viewed not gen er ally but sub stan tially. And not like the “sto chas tic oc ca sion” as ev ery thing in the liv ing is highly or ga nized. Three lines can be dis tin guished in ver te brates (one of the most stud ied mutational con tinua), three con tinua of mutational com mu nity, i.e. 1) mu ta tions and mu ta - gen e sis in the germ plasma; 2) mu ta tions and mu ta gen - e sis in soma; 3) mu ta tions and mu ta gen e sis in ar ti fi cial, un nat u ral sys tems (cell cul tures, cell lines, cell pop u la - tions, etc). Each of them should have its own ideas, the o ries, meth ods of the re search, and tech nol ogy of ap pli ca tion. Mod ern “clas sics” does look weird, does n’t it? For in - stance, the cul ture of im mor tal ized cells is dropped upon by dif fer ent sub stances, and ac cord ing to the oc - cur ring mu ta tions “pro tec tive ef fect” or “mutational spec trum” of dif fer ent prep a ra tion for the hu man be ing is de ter mined. In the cell cul tures the re ac tions and the be hav ior of cells is one, in soma (where the per spec tive prep a ra tion will be in tro duced to) the func tion of mu ta - tions is dif fer ent (and their “in hi bi tion” in or gans and tis sues may re sult in any thing), whereas in the germ line ev ery thing is quite op po site at all (if there would be any ob sta cles on the way of chain mutational pu ri fy ing pro cesses, it may pro duce the op po site ef fect, i.e. some classes of mu ta tions will be en riched, but they do not re sult in le thal ends or bright phenotypical man i fes ta - tions, which will not be ob served by the ex per i menter im me di ately). And it in cludes ev ery thing. Let us make some ab so lutely non-ca non i cal re - sume. Mu ta tions are co va lent changes in ge nome, sup - ple ment ing tra di tional reg u la tion (based on pro tein-nu - cleic in ter ac tions) – highly la bial one with more con ser - va tive one. Ex facte five lev els of reg u la tion by co va lent changes in ge nome (mu ta tions) can be dis tin - guished as fol lows: 1) dam age of bases; 2) methylation MU TA TIONS: WHAT ARE THEY? 237 of bases; 3) point mu ta tions (of all types); 4) re or gani - sa tion of ge nome; 5) for ma tion of new genes. Com mon views on mu ta tions are based on the events, tak ing place in the germ route, via re al iza tion in soma. But in both soma and the germ route the ways of reg u la tion and re al iza tion are quite dif fer ent. And there fore, the no tion of “mu ta tion”, ap plied to them can not be sim i - lar. All cell cul tures out side of the or gan ism live their own spe cial extraorganismal life, noth ing sim i lar with the nat u ral one (and it has not been ana lysed at all, what it re ally is). Mu ta tions in sev eral gen er a tions in germ plasma are the sub ject of ge net ics (the sci ence on he red ity and in her i tance). Whereas mu ta tions in soma are sub ject to reg u la tions. The at tempts to bring it down to he red ity and in her i tance, i.e. to ge net ics, hav ing added epi- pre - fix is noth ing but trib ute to tra di tion. Of course there is epigenetics – in her i tance is thus com bined, con tacted, and in ter acted etc. with it. Yet to limit the reg u la tion (even if it is not com mon to us and is de ter mined by co - va lent changes of ge nome in soma) to ge net ics (in its mod ern, but yet tra di tional un der stand ing) is im pos si - ble, i.e. pos si ble no more. For germ plasma (con tin u ous, “in di vid ual-through” line of life), mu ta tion, in flu enc ing some thing is not only the change in gene. It is also po ten tial dan ger for the weak, which changes not the func tions of changes “al most” at the level of mononucleotide poly mor phism of mu ta tions. Some sig nal, in di cat ing that the sys tem of rep a ra tion in this ex act cell of the germ line did not cope with, missed it (while in other cases, coped with, there are no mu ta tions, yet the level of damageability is the same ev ery where sta tis tic-wise). If it did not cope with that it is pos si bly dan ger ous as it may not cope with other mu ta tions. And this cell has got to be elim i nated (in ac cor dance to the func tions of germ line) from the germ line. The cas cade of changes, re sult ing in sub se - quent large, in com pat i ble with fur ther ex is tence, chro - mo somal dis or ders. Rep a ra tion in its reg u la tion and reali sa tion is very large-scale. One third of all genes is in volved in rep a ra tion even in bac te ria, and al most all of them are in volved in cytogenesis. There fore, there is some thing to be in cluded for check ing. And thus, nor - mal hu man germ cells (!) from one to tens per cent of ga - metes are aneuploid. These are the authorised clean ing mu ta tions, ini ti at ing the clean ing cas cade. Whereas those that for some rea son did not cause the lat ter and went through into the re pro duc tion are the un author ised mu ta tions. They are the source of poly mor phism, he - red i tary dis eases in cluded. Mu ta tions per form reg u la tory func tions in ge nome, di rected to wards the pro vi sion of soma ex is tence. These are the authorised mu ta tions. Be ing out of con - trol, mu ta tions in soma cause pa thol ogy, i.e. oncogenesis. These are un author ised mu ta tions. In germ route mu ta tions per form “clean ing” func tions, di - rected to wards pro vid ing con sis tency of germ plasma in the con tin u ous line of life. Once un con trolled, mu ta - tions in germ plasma re sult in patho log i cal in fe ri or ity, in com pat i ble with con tin u ous ex is tence of life, i.e. he - red i tary dis eases. Mu ta tions in soma and the germ route are dif fer ent in their or i gin, con tents, tasks, sig nif i cance, func tions, and con se quences. Com mon thing for all of them is the fact that mu ta tions are as nec es sary at trib ute of life as breath ing, en er getic me tab o lism, biosynthesis. There - fore, it is con cep tu ally hope less to fight mu ta tions “in gen eral”. Mu ta tions are to be di rected, as ev ery thing else in the liv ing. Not to fight them but to con trol them. We can not stand the fact that we are the ter mi nal state in the form of self-con trolled com mu nity of cell pop u - la tions, car ry ing, pro tect ing, and feed ing, etc con tin u - ous line of germ plasma. But we will have to, of course, if we want to un der stand how to step out of the ter mi nal. Â. À. Êîðäþì Ìóòàöèè – ýòî ÷òî? Ðåçþìå Àíàëèçèðóþòñÿ ïðåäñòàâëåíèÿ î ìóòàöèÿõ. Ïðèâåäåíû ëèòåðàòóðíûå äàííûå, íå ñîîòâåòñòâóþùèå ñóùåñòâóþùèì êîíöåïöèÿì î ìóòàöèÿõ. Ñôîðìóëèðîâàíî ïîëîæåíèå î ôóíêöèîíàëüíîé íåîäíîçíà÷íîñòè ìóòàöèé è èõ áèîëîãè÷åñêîì çíà÷åíèè. Ñîãëàñíî ýòîìó ïîëîæåíèþ, ìóòàöèè â ñîìå âûïîëíÿþò ðåãóëÿòîðíûå ôóíêöèè, ÿâëÿÿñü, òàêèì îáðàçîì, íîðìàëüíîé, êîíòðîëèðóåìîé îðãàíèçìîì, ñîñòàâëÿþùåé áèîëîãè÷åñêèõ ïðîöåññîâ. À âûõîäÿ èç-ïîä êîíòðîëÿ, ìóòàöèè â ñîìå ïðèâîäÿò ê îíêîãåíåçó. Â çàðîäûøåâîì æå ïóòè ìóòàöèè ÷åðåç êàñêàäíûå èíòåãðàëüíûå ïðîöåññû îáåñïå÷èâàþò ýëèìèíàöèþ èõ KORDIUM V. A. 238 íîñèòåëåé, âûïîëíÿÿ î÷èñòèòåëüíóþ ôóíêöèþ. À ïðè âûõîäå èç-ïîä êîíòðîëÿ, íå ïðèâîäÿ ê ýëèìèíàöèè èõ íîñèòåëåé, ðåàëèçóþòñÿ â íàñëåäñòâåííóþ ïàòîëîãèþ âî âñåì åå äèàïàçîíå – îò ñêðûòîé ôîðìû («ìóòàöèîííûé ãðóç») äî ÿðêîé ìàíèôåñòàöèè. Êëþ÷åâûå ñëîâà: ìóòàöèè, ðåãóëÿöèÿ, ñîìà, çàðîäûøåâûé ïóòü. REFERENCES 1. Ãåðøåíçîí Ñ. Ì. Îñíîâû ñîâðåìåííîé ãåíåòèêè.–Êèåâ: Íàóê. äóìêà, 1979.– 508 ñ. 2. Mohrenweiser H. W., Jones I. M. Re view at the mo lec u lar char ac ter is tics at gene mu ta tions at the germline and so matic cells at the hu man // Mutat. Res.–1990.–P. 87–108. 3. Drake J. W. Mu ta tion: ma jor evo lu tion ary trends // 18th Symp. Nucl. Ac ids Chem. (Sendai, Oct. 29–31, 1991).–Ox - ford etc., 1991.– Ð. 159–160. 4.Ono T., Lehara Y., Saito Y., Ikehata H. Mu ta tion the ory of ag - ing, as sessed in trans gen ic mice and knock-out mice // Mech. Age ing and De velop.–2002.–123.–P. 1543–1552. 5. Ñëîçèíà Í. Ì., Íåðîíîâà Å. Ã. Õðîìîñîìíûå àíîìàëèè ãàìåò ÷åëîâåêà è âíóòðèóòðîáíûé îòáîð. Èññëåäîâàíèÿ æåíñêèõ ãàìåò // Öèòîëîãèÿ è ãåíåòèêà.–1992.–26, ¹ 6.–C. 58–63. 6. Morley A. A., Turner D. R. The con tri bu tion at ex og e nous and en dog e nous muta gens to in vivo mu ta tions // Mutat. Res.–1998.–428.–P. 11–15. 7. Shastry B. S. SNP al leles in hu man dis ease and evo lu tion // J. Hum. Genet.–2002.–47.–P. 561–566. 8. The In ter na tional SNP Map Work ing Group. A map at hu man ge nome se quence vari a tion con tain ing 1.42 mil lion sin gle nu - cle o tide polymorphisms // Na ture.–2001.–40.–P. 928–933. 9. Rosen E. M., Fan S., Pestell R. G., Goldberg I. D. BRCA1 gen in breast can cer // J. Cell. Physiol.–2003.–196.–P. 19–41. 10. Hasin Y., Avidan N., Bercovich D., Korczyn A., Silman I., Beck mann J. S., Sussman J. J. A par a digm for sin gle nu cle o - tide poly mor phism anal y sis: The case of the acetylcholinesterase gene // Hum. Mutat.–2004.–24.– P. 408–416. 11. Pagani F., Raponi M., Baralle F. B. Syn on y mous mu ta tions in CFTR exon 12 af fect splic ing and are not nat u ral in evo lu - tion // Proc. Nat. Acad. Sci. USA.–2005.–102.– P. 6368–6372. 12. Õàòûíîâà Ì. À., Áóôô Å. Ì., Ìîãèëà Â. À., Íàáèðî÷êèí Ñ. Ä., Ñèìîíîâà Î. Á., Ãåðàñèìîâà Ò. È. Èíäóêöèÿ ìóòàãåíåçà â ðåçóëüòàòå íåêîòîðûõ ãåíåòè÷åñêèõ ñêðåùèâàíèé // Ãåíåòèêà.–1992.–28, ¹ 3.–Ñ. 56–67. 13. Eriso S., Choi S.-W., Girelli D., Ma son I., Dolnikowski G. G., Bagley P. J., Olivieri O., Jacques P. F., Rosenberg I. H., Carrocher R., Selhub J. A com mon mu ta tion in the 5, 10-methylenetetrahydrofolate reductase gene af fects genomic DNA methylation through an in ter ac tion with fo late sta tus // Proc. Nat. Acad. Sci. USA.– 2002.–99.– P. 5606–5611. 14. Ñàäëåð Ò. Â. Ìåäè÷íà åìáð³îëîã³ÿ çà Ëàíãìàíîì.–Ëüâ³â: Íàóò³ëóñ, 2002.–550 c. 15. Ìèøèí Â. Ï., Áîãíàðåâ Â. Â., ×óðþêàíîâ Â. Â. Êèñëîðîä // Áîëüøàÿ ìåä. Ýíöèêëîïåäèÿ.–M.: Ñîâ. Ýíöèêëîïåäèÿ, 1979.–T. 10.– C. 325–326. 16. Fraga C. G., Shigenaga M. K., Park J.-W., Degan P., Ames B. N. Ox i da tive dam age to DNA dur ing ag ing: 8-Hydroxy-2¢-deoxy- guanosine in rat or gan DNA and urine // Proc. Nat. Acad. Sci. USA. – 1990.–87.–P. 4533–4537. 17. Êîðäþì Â. À.. Íàøà «øàãðåíåâàÿ êîæà» – ýòî íàøà ïðîáëåìà. Íàì åå è ðåøàòü.–Êèåâ: Ëîãîñ, 2006.–245 ñ. 18. Ham il ton M. S., Remmen H. V., Drake J. A.,Kang H., Guo Z.M., Kewitt K., WalFrter C. A., Rich ard son A. Does ox i da - tive dam age to DNA in crease with age? // Proc. Nat. Acad. Sci. USA –2001.–98.– P. 10469–10474. 19. Rich ter Ch., Park J.-W., Ames B. N. Nor mal ox i da tive dam - age to mi to chon drial and nu clear DNA is ex ten sive // Proc. Nat. Acad. Sci. USA.–1988.–85.–P. 6465–6467. 20. Loeb L. A., Cheng K. C. Er rors in DNA syn the sis: A source of spon ta ne ous mu ta tions // Mutat. Res. Rev. Genet. Toxi- col.–1990.– P. 297–304. 21.Frosina G. Overexpression of en zymes that re pair en dog e - nous dam age to DNA // Eur. J. Biochem.–2000.–267.–P. 2135–2149. 22. Lu T., Pan Y., Kao S.-Y., Li C., Kohane I., Chan J., Yankner B. A. Gene reg u la tion and DNA dam age in the age ing hu man brain // Na ture.–2004.–429.–P. 883–891. 23. Dolle M. E., Snyder W. K., Dunson D. B., Vijg J. Mutational fin ger prints of ag ing // Nucl. Ac ids Res.–2002.–30– P. 545–549. 24. Vijg J., Dolle M. E. T. Large ge nome rearrgements as a pri - mary cause at ag ing // Mech. Ñaging and De - velop.–2002.–213.– P. 907–915. 25. Khil P. P., Camerino-Otero R. D. Over 1000 genes are in - volved in the DNA dam age re sponse of Esch e richia coli // Mol. Microbiol.–44.– P. 89–105. 26. Jahn C. L., Klobutcher L. A. Ge nome re mod el ing in cil i ated pro to zoa.–Palo Alto, 2002.–520 ð. 27. Yan B., Wang H., Peng Y., Hu Y., Zhang X., Chen Q., Bedtard J. S., Dewhirst M. W., Li C.-Y. A unique role at the DNA frag men ta tion fac tor in main tain ing genomic sta bil ity // Proc. Nat. Acad. Sci. USA.–2006.–103.– P. 1504–1509. 28. Íåâçîðîâà Ò. À., Âèíòåð Â. Ã. Èññëåäîâàíèå ÄÍÊ-ãèäðîëèçóþùåé àêòèâíîñòè àíòèòåë ê ÄÍÊ // Ó÷. çàï. Êàçàí. ãîñ. óí-òà.–2005.–Ò. 147, ÷. 2.–Ñ. 136–148. 29. Cairns J., Overbough J., Miller S. The or i gin of mu tants // Na ture.–1988.–335.–P. 142–145. 30. Cairns J., Fos ter P. L. Adap tive re ver sion of a frameshift mu - ta tion in Esch e richia coli // Ge net ics.–1991.–128.– P. 695–701. 31. Slechta E. S., Har old J., Andersson D. I., Roth J. R. The ef fect at genomic po si tion on re ver sion at a lac frameshift mu ta tion (lac LZ33) dur ing non-le thal se lec tion (adap tive mu ta tion) // Mol. Microbiol.–2002.–44.–P. 1017–1032. 32. Metzgar D., Bytot J., Wills C. Se lec tion against frameshift mu ta tions lim its microsatellite ex pan sion in cod ing DNA // Ge nome Res.–2000.–10.–P. 72–80. 33. Zufall R. A., Rob in son T., Katz L. A. Evo lu tion of de vel op - men tally reg u lated ge nome re ar range ments in eukaryotes // J. Exp. Zool. B.–2005.–304.–P. 448–455. 34. Zhang Q., Wise K. S. Lo cal ized re vers ible frameshift mu ta - tion in an ad he sion gene con fers a phase-vari able ad her ence phe no type in mycoplasma // Mol. Microbiol.–1997.–25.–P. 859–869. 35. Theiss P., Wise K. S. Lo cal ized frameshift mu ta tion gen er ates se lec tive, high-frequensy phase vari a tion at a sur face li po pro - tein en coded by a mycoplasma ABC trans porter operon // J. Bacteriol.–1997.–179.– P. 4013–4022. 239 MU TA TIONS: WHAT ARE THEY? 36. Matsui K., Yamada M., Imai M., Yamamoto K., Nohmi T. Spec i fic ity of replicative and SOS-in duc ible DNA polymerases in frameshift mu ta gen e sis: Mu ta bil ity at Sal mo - nella typhymurium strains overexpressing SOS-incducible DNA polymerases to 30 chem i cal muta gens // DNA Re - pair.–2006.–5.–P. 465–478. 37. Hendrick J. L., Wil son P. J., Edelman I. I., Sandbaken M.G., Ursic D., Culbertson M. R. Yeast frameshift sup pres sor mu ta - tions is the genes cod ing for tran scrip tion fac tor MbH1p and ri bo somal pro tein S3: Ev i dence for autoregulation at S3 syn - the sis // Ge net ics.–2001.–157.– P. 1141–1158. 38. Harfe B. D., Jinks-Rob ert son S. Se quence com po si tion and con text ef fects on the gen er a tion and re pair at frameshieft in - ter me di ates in mononucleotide runs in Saccharomyces cerevisine // Ge net ics.–2000.–156.– P. 571–578. 39. Bahar R., Hartmann C. H., Ro dri guez K.A., Denny A., Busuttil B.A., Dolle M. E. T.,Cal der R. M., Chis holm G. B., Pol lack B. H., Klein C. A., Vijg J. In creased cell-to-cell vari a - tion in gene ex pres sion in age ing mouse heart // Na - ture.–2006.–441.–P. 1011–1014. 40. Kijas J., M. H., Moller M., Plastow G., Andersson L. A Frameshift mu ta tion in MC1R and a high fre quency at so - matic re ver sions cause black spot ting in pigs // Ge net - ics.–2001.–158.–P. 779–785. 41. Froga M. F., Ballestar E., Paz M. F. Epigenetic dif fer ences arise dur ing the life time at monozygotic twins // Proc. Nat. Acad. Sci. USA.–2005.–102.– P. 10604–10609. 42. Zenzes M. T., Cas per R. F. Cytogenetics of hu man oocytes, zy gotes, and em bryos af ter in vi tro fer til iza tion // Hum. Genet.–1992.–88.– P. 367–375. 43. Tempiado C., Benet J., Genesca A., Navarro J., Cabalin M. R., Miro R., Egozcue J. Hu man sperm chro mo somes // Yum. Re- prod.–1988.–3.–P. 133–138. 44. Pellestor F., Jel B. Étude cytogénétique du sperme humain // M/S: Med. Sci.–1989.–5.–P. 244–251. 45. Benkhalifa M., Malet P. Menezo Y., Janny L., Boucher D. Cytogenetic anal y sis of hu man ga metes to em bryos: Abstr. 6 Int. Conf. Early Prenat. Diagn. Genet. Dis eases from Ga - metes to Em bryo // Prenat. Diagn.–1992.–12, Suppl.–P. 50. 46. Òåïïåðìåí Äæ., Òåïïåðìåí Õ. Ôèçèîëîãèÿ îáìåíà âåùåñòâ è ýíäîêðèííîé ñèñòåìû.–Ì.: Ìèð, 1989.–656 ñ. 47. Ô³ëü÷åíêîâ Î. Î., Ñòîéêà Ð. Ñ. Àïîíòîç ³ ðàê. Â³ä òåîð³¿ äî ïðàêòèêè.–Òåðíîï³ëü: Óêðìåäêíèãà, 2005.–524 c. 48. Swergold G. D. Indentification char ac ter iza tion, and cell spec i fic ity of a hu man LINE-1 pro moter // Mol. and Cell. Biol.–1990.–10.– P. 6718–6729. 49. Mathias S. L., Scott A. F., Kazazian (Jr.) H. H., Boeke I. J. D., Ga briel A. Re verse tran scrip tase en coded by a hu man transposable el e ment // Sci. Mag.–1991.–254.– P. 1808–1810. 50. Woods-Samuels P., Wong C., Mathias S. L., Scott A. F., Kazazian H. H., Antonarakis S. E. Char ac ter iza tion of a nondeleterious L1 in ser tion in an intron of the hu man fac tor VIII gene and fur ther ev i dence of open read ing frames in func tional L1 el e ments // Genomics.–1989.–4.–P. 290–296. 51. Haugen P., Bhattacharya D. The spread of LAGLIDADG hom ing endonuclease genes in rDNA // Nucl. Ac ids Res.–2004.–32.– P. 2049–2059. 52. Pittoggi C., Sciamanna I., Mattei E., Beraldi R., Lobascio A. M., Mai A., Quaglia M. G., Lorenzini R., Spadafora C. Role of en dog e nous re verse tran scrip tase in murine early em bryo de vel op ment // Mol. Reprod. and De velop.–2003.–66.– P. 225–236. ÓÄÊ 577.21+575.857 Íàä³éøëà äî ðåäàêö³¿ 18.04.07 240 KORDIUM V. A.

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