•   v
2008-09 1. PREDNÁŠKA MOL. BIOL.
Nucleic acids
Historical view
Emil Paleček
Institute of Biohysics, Acad. Sei. CR v.v.i., 612 65 Brno
Czech Republic
The Road to DNA started in Brno
G.J. Mendel 1866
« Back to previous results list
«..        D       -                            Paleček Emil
Citation KepOľt  Author (Paleček E)
Timespan=AII Years. Databases=SCl-EXPANDED, SSCI, A&HCI, IC, CCR-EXPANDED [back to 1340]
This report reflects citations to source items indexed within Web of Sciertce. Perform a Cited Reference Search to include citations to items not indexed within Web of Science.
Chemická reaktivita a struktura nukleových kyselin. Lokální struktury DNA stabilizované superhelikálním vinutím; Interakce DNA a bílkovin s povrchy; Interakce ĎNA-protein;
Published Items in Each Year
16 14
i:
10
í e
4
j 0
Ct>  <?t  <Ji   i3"p  >íTi  ťŕl  ťT.  <T1   CT.  CT,  <T.  O  <=>   O  O  Ol   O  O  *=>  -Z>
CT, Cr, CT, O". T« T". CT« ffi   O". O CFp .3 O  O O, O,  O O, O O
rt-.rt  ««^^.^-i^-iPirlfiřKlCiririri
rears
The latest 20 years are displayed View a graph with all years.
SŮ0 500 400 300
JdC i 100
0
Citations in Each Year
iiiin.il
ffi o- H^ri^i/níh » CT, o —i r j rvi Tfl- u-L irp r--, to
«HTiďiUiaifflCmj^mffiůŮOOlíOOOO U'ffiíilJVJiClClfllJl^lTiOOOOOOOOO
--i --i --i --i «-i-i^-t-i«rjrjrjr,|NMrMí\]ru
Years
The latest 20 years are displayed. View a graph with all years.
Results found:   246
Sum of the Times Cited [?] ;   7,524
View Citing Articles View without self-citations
248 7 524
Average
Citations per
Item [?] :
h-index [?] :
30.34 50
50
Elektrochemie nukleových kyselin a bílkovin;
Nádorové supresory, zejména protein p53; Agregace bílkovin
v neurodegenerativních chorobách (zejména agregace a-synucleinu
v Parkinsonově chorobě)
Results   248
Page    1
of 25      C'O
► H
Sort by:     Times Cited
Use the checkboxes to remove individual items from this Citation Report or restrict to items processed between
1945-1954 J_ and   2008              T'     Go
Title: Peptide nucleic acid probes for sequence-specific DNA biosensors
Author(s): Wang J, Paleček E, Nielsen PE, et al. Source: JOURNAL OF THE AMERICAN CHEMICAL SOCIETY Volume: 116   issue: 33   Pages: 7667-7670   Published: AUG 21 1996
Titas: From polarography of DMA to microanalysis with nucleic acid-modified electrodes Author(s): Paleček E
Source: ELECTROANALYSIS   Volume: 8   Issue: 1   Pages: 7-14 Published: JAN 1996
Title: Detecting DNA hybridization and damage
Author(s): Paleček E, Fojta M
Source: ANALYTICAL CHEMISTRY   Volume: 73   Issue: 3   Pages:
74A-83A   Published: FEB 1 2001
Title: LOCAL SUPERCOIL-STABILIZED DNA STRUCTURES
2004   2005   2006   2007   2008   Total    Average A                                                                         Citations
per Year
531       325
IB
21
33
14
13
428
11
447       582     7,524
28
11
26
209
16       206
20
190
147.53
16.08
15.85
23.75
Aulhorts): PALECEKE
Court»: CRITICAL REVIEWS I« BIOCHEMISTRY AND MOLECULAR
BIOLOGY   Volume.26   Issue: 2   Pages- 1S1-SZE   Published: 1991
Titte: DNA electrochemical hiosensors for environment monitoring A review AuMionsj Wana J Rnbb G. Ca X, e: al Sni« ANALYTICA CHIMICA ACTA   Volume: MT  rau: 1-S Page». 1 -G   P jb iah od J U L 10 19 9 7
Title: Electrochemical biosensors tuf DNA hybrirJizatiun and DNA carnage
Authors). Paleček E. Fojta M. Tomsohík M. er al Source BIOSENSORS A BIOELECTRONICS   Volume: 13   I «u* S Pages: 621-62!   Published. SEP 15 1991
TrrJe: ťlfc SUPERCOIL -STABILIZED CRUCIFORM OF COLE1 IS HYPER-REACTIVE TO OSMIUM-TETROXIDE Authors). LILLEY DMJ. PALEČEK E Source EMBO JOURNAL   Volume: 3   Issue: G   Pages: 11S7.1192
■LS   5-0=   19M
Tl»ľ SUPERHELICAL TORSION IM CELLULAR DMA RESPONDS DIRECTLY TO ENVIRONMENTAL AND GENETIC. FACTORS
Autt»r{s}. MCCLELLAN JA, BQUBLIKQVA P, PALECÉKE. e< SI. Source PROCEEDINGS OF THE NATIONAL ACADEMY OF 5CIE NC E 5 OF T H E UNIT ED STATE 8 OF A M ERICA   Volume- Í7 Issue. 31    Pages Í373-B377   Published- NOV 1990
Tilt*: Past present and future of nucleic acids electrochemistry Author* sr Palace K E
Source: TALANTA   Volume. GÍ   Issue. G   Paces' 109-119 Published. APR t 2002
Title: OSCILLOGRAPHIC POLAROGRAPHY OF HIGHLY POLYMERIZED DEOXYRIBONUCLEIC ACID Author(a)- PAL EC Eh E
Source: NATURE Volume: 1«   Issue: 4TG1   Pages an-es? Put* shed. I960
esjns   24Ä
Page   1
Sort by:     Times Cked          t
88
Chemical nature and spatial organization STRUCTURE
F. MIESCHER, TÜBINGEN 1871
\CIDS
Biological function
G. J. MENDEL, BRNO 1866
Timeline of DNA
1865: Gregor Mendel discovers through breeding experiments with peas that traits are inherited based on specific laws (later to be termed "Mendel's laws"). By mentioning Elements of Heredity he predicts DNA and genes (published 1866) 1866: Ernst Haeckel proposes that the nucleus contains the factors responsible for the transmission of hereditary traits. 1869: Friedrich Miescher isolates DNA/NUCLEIN for the first time. 1871: The first publications describing DNA (nuclein) by F Miescher, Felix Hoppe-Seyler, and P. Plosz are printed.
1882: Walther Flemming describes chromosomes and examines their behavior during cell division.
1884-1885: Oscar Hertwig, Albrecht von Kölliker, Eduard Strasburger, and August Weismann independently provide evidence
that the cell's nucleus contains the basis for inheritance.
1889: Richard Altmann renames nuclein to nucleic acid.
1900: Carl Correns, Hugo de Vries, and Erich von Tschermak rediscover Mendel's Laws.
1902: T Boveri and W Sutton postulate that the heredity units (called genes as of 1909) are located on chromosomes. 1902-1909: A Garrod proposes that genetic defects result in the loss of enzymes and hereditary metabolic diseases. 1909: Wilhelm Johannsen uses the word gene to describe units of heredity.
1910: T H Morgan uses fruit flies (Drosophila) as a model to study heredity and finds the first mutant with white eyes. 1913: Alfred Sturtevant and Thomas Hunt Morgan produce the first genetic linkage map (for the fruit fly Drosophila).
1928: Frederick Griffith postulates that a transforming principle permits properties from one type of bacteria (heat-inactivated virulent Streptococcus pneumoniae) to be transferred to another (live nonvirulent Streptococcus pneumoniae). 1929: P Levene identifies the building blocks of DNA, incl. four bases adenine (A), cytosine (C), guanine (G), thymine (T). 1941: George Beadle and Edward Tatum demonstrate that every gene is responsible for the production of an enzyme. 1944: Oswald T. Avery, Colin MacLeod, and Maclyn McCarty demonstrate that Griffith's transforming principle is not a protein, but rather DNA, suggesting that DNA may function as the genetic material
1949: Colette and Roger Vendrely and A Boivin discover that the nuclei of germ cells contain half the amount of DNA
that is found in somatic cells. This parallels the reduction in the number of chromosomes during gametogenesis and provides further evidence for the fact that DNA is the genetic material.
1949-1950: Erwin Chargaff finds that the DNA base composition varies between species but determines that the bases in DNA are always present in fixed ratios: the same number of A's as T's and the same number of C's as G's. 1952: Alfred Hershey and Martha Chase use viruses (bacteriophage T2) to confirm DNA as the genetic material by demonstrating that during infection viral DNA enters the bacteria while the viral proteins do not and that this DNA can be found in progeny virus particles.
1953: Rosalind Franklin and Maurice Wilkins use X-ray analyses to demonstrate that DNA has a regularly repeating
helical structure.
1953: James Watson and Francis Crick discover the molecular structure of DNA: a double helix in which A always pairs
with T, and C always with G.
1956: Arthur Kornberg discovers DNA polymerase, an enzyme that replicates DNA.
1957: Francis Crick proposes the central dogma (information in the DNA is translated into proteins through RNA) 1958:
Matthew Meselson and Franklin Stahl describe how DNA replicates (semiconservative replication).
1960-63: Julius Marmur and Paul Doty show separation of DNA strands and reformation of DNA double-helical structure
- DNA renaturation/hybridization
1961-1966: Robert W. Holley, Har Gobind Khorana, Heinrich Matthaei, Marshall W. Nirenberg, and colleagues crack the
genetic code.
1968-1970: Werner Arber, Hamilton Smith, and Daniel Nathans use restriction enzymes to cut DNA in specific places
for the first time.
1972: Paul Berg uses restriction enzymes to create the first piece of recombinant DNA.
1977: Frederick Sanger, Allan Maxam, and Walter Gilbert develop methods to sequence DNA.
1982: The first drug (human insulin), based on recombinant DNA, on the market.
1983: Kary Mullis invents PCR as a method for amplifying DNA in vitro.
1990: Sequencing of the human genome begins.
1995: First complete sequence of the genome of a free-living organism (the
bacterium Haemophilus influenzae) is published.
1996: The complete genome sequence of the first eukaryotic organism—the yeast
S. cerevisiae—is published.
1998: Complete genome sequence of the first multicellular organism—the
nematode worm Caenorhabditis elegans—is published.
1999: Sequence of the first human chromosome (22) is published.
2000: The complete sequences of the genomes of the fruit fly Drosophila and the
first plant—Arabidopsis—are published.
2001: The complete sequence of the human genome is published.
2002: The complete genome sequence of the first mammalian model organism—the
mouse—is published.

•9 75
1950
T 925
1000
1875
:ßSO
-ess
■-
1700 —
íeoo —
Perulz determmes structure first protein by sc-ray dillraction
Sanger and Tuapy eteiermine Ihe firsl protein primary sequence
Avery. MacLeod, and McCarty sJiow DNA io be ihe ageni ol genelic Lrsnsfarinaiion
Krebs elucidates the etile acid cycle ■
Sved berg develops the ullracentrifuga —
Embden and Meyerhoi describe — the giycoiylic oairiway
Sumner crystallizes urease -
Buchnsr öemortSlrates —. fermentation wflti    i ceil extracts m
MOLECULAR BIOLOGY
Davflopmsnt ol DNA cloning
GeneliC code
Walson and CricJi propose double helix icn DMA
Heishey and Chase establish DNA as me genetic material
Claude isolates first mitochondrial tractions
MIESCHER?
Pasleur links -Iwing organisms
to specific a'cc esse5
Wohles synthesizes -urea i*i me laboratory
inverHion of the — microtome
Development ol — dyes and stains
Vtrchow every cell — Comes from a ceil
SchleirJen and Schwann — formulate cell theory
Brown describes ■ nuclei
BIOCHEMISTRY
van LeeLíWBíihosk -improves lenses
HoOke describes -cellular'
invention c-1 electron microscope
-Levene postulates DNA as a repealing, tetranbcleatide structure
t-—Morgan and colleagues
devetop genetics ol Dfosopfňů
Sutton formulates! chromosomal theory of heredity
Rediscovery ot Mendel's laws by Correns, von Tschermak, and de Vries
RoiiK and weissman ctirorriosomos carry genetic information
------------1------------------
Flemming identifies
chromosomes
GENETICS
Miesctier discovers DNA Mendel discovers firs fundamental laws ol geneiics
MENDEL
CELL BIOLOGV r /Jll-L 1.2
Interweaving of the historical tradition of biochemistry, cell biology, and genetici. These three disciplines, which originally were considered tu be quite separate, have become intertwined tn yield a true molecular biology, the subject matter of present-day biochemistry.
C K MATHEWS , K E van HOLDE, BIOCHEMISTRY, 1990
Darwin C. 1858*. Book - On the Origin of Species by Means of Natural Selection
Mendel G. 1866 Miescher F. 1871  papers
Charles Darwin - Important claims:
A.  Universal Common Descent - Tree of Life - the first one-celled organism, representing the root or trunk of the Tree, gradually developed and changed over many generations into new and more complex forms, representing the branches
B.  Natural Selection as a mechanism responsible for the branching pattern Variations in living forms arise at random
Nature selects the adaptive ones Adaptive organism survive and reproduce Inherited adaptations may cause population changes
Darwin understand neither how genetic traits were passed to the progeny nor how the variations arose. He is a founder of Evolution Biology
At present: - Natural Selection as a mechanism for relatively simple processes is fully confirmed
- Universal Common Descent - Tree of Life and the role of natural selection in the origin of species are questioned
On the evolution of celts
Ort A. Wo*»*
lJitur^ltUtl-JW
noM n hinri on ih» (4*u HrWOnttfi tt^WWl ltl*T sh* 4f AM4 *T h4r44HVÜ 0#a# LumwIw (Htf) n p*4m*»lly 4r1*™-'*»4 by ÜH *q*ň.(*i-5'i of lb« ™;>h ip« Abp^AH uÉ 4+boiH » l*M« Hú tM n-*ip4* wi4 y»«*]f «^nnd «nwf)i l*M Ml (•«ulV (OaipOM^vrtin ba Miľtd n4 h-dhi<i4K*a tiViwjgjb **^1. *#*■ ■tg HQ1 If** pflrripal OVtfrľq Kmt m «trip ti Im If noluUon
Flblrilh* 4 **1 44 Mri IMfTf É llM** fatf*rll*iTril j|F lllpQ  UJ IMC *
iwvlly 'tqwvd Uh ¥¥**** ■.*■ «Tl-fwi 4 * pf o^Mrt oP cMwwid IWWlMl ol In* unkwwlMar Ml, n« ifHřtfi'li|l MmW q H A* Mtnt^^ir «4 -i tBMMh tť* HOrtyilHn. «huh M4HK Tht imJhrtdu+l «w *n*g«< t*»#i *»o<'rt « i*«* *■** ■" iii*H*Bfcji*
■r* icifJJ*Ťi|T 4n4«fHii Ai 4 í»H dwf)i fa****** *w ccmb^i And lnl*Moi»Mtptf i (**■*«■ HWil n r**H»ťl «rtm i me*« Él6H¥*l*df*j[* Pf^f^rjtf^H- +*■#> frü **i ■**« E*V ■ ■*«"■*.■■'
H-CiiU rn* ~|>*f*1ra*rt Fw-nhaM-- frw lhi i ■**.! M f/i in
TbtrroJrllM l*l FWricrPfrQ» n..ur.iiftr-11-lhr ŕľV.*l iľh«ÍVr.l>r-|
ľi la Drťwúi * dar ihr ^i+4rm Ofrkf bwtftj h" ti^pwJ Fw IWtcfe «f Ifcr L»« íCnrtyrT ■■ »u -MUliHf la MT* CM. lb* pKMcalqrtiutwlUi jKí*íb iB mht iMifUiíríliit-—*b»?ít bcCHt* M n * bw4u«K-«t men ■ (üi)cfanMnl priMr*. M ™ rlfccibvfj iliWKPii 5cé««>Uk- lakrrol la uiHalii- en"".P<a« ■4MUJ {4 p«cL up *T*er bW aafranal ffcjlofitir kre- U*í haoc^il »x h** ■***. nw proMna lud tobe ■JJf— ad, »u ilrLcirannJlpCti » MhJ i. Fit 11. ÍMtf "t *» W* "-tí «MOohri esnntBia .winTd « iturvcac ihM hu^vitfA cubU •CTuH) 40 nhwTft. jltami iV (tfíMcn iit MFÜwlir f".'•Infill
IbiUI MMfllpU In lume Ihr Bdoe hnr lipu aJti hťrfl ifi tbc L'liM>d*J Dmii*ai4^k «*Oť. jftJ ibŕ bVbL M JJ*1 hu hevn iJnuW
f        lUMHL M mrw, ň {fc-jf-ibc jppejl ul ib£ ■■JjiiiibTiIbé finrcpi Hf^jBwri"itiÄyirifMr>^liH«»t»LíBi»4idř(k*<ltfca^(il«w]
HhI qM'^N^HlrkUB, hlul rb AtwU il BJVf ÜDCH m thf flfcJCC PriHC4t JM»!1 fl*-ki(*ei lu(«!iH(lmct] »ilh ift rtlA**>!!*»«*C (éw i-fllul-í hmnal mif>H (m I he fiA-umo: «fcliu), hkI mn Ih ÜK «AtifT cuk u yntoľ ľ-rB 11 -1^). ľhOŕ ťtnuc-ri rcpuiiuĽLin hr i im er i^urxlcnritt* tfv> {0 n«ii ttlh iLm «ic h^nlh 5* rti*ťd. 111 ť*vhí 4bc flWPti^ ™v-ív.nif «Jl »ťl jfrr ,n ^Ľtuŕdi *nJ hu-icii«! ciniarerpini fi* hM J*jw b^a ti «o bp3hJ m ibc hm '»i>t»r«nr  l, uhI éu| fcp<v. «tte«H>a -m
r^Lpr^vtW ŕŕllalit MlUEiihA. Mta'h rfnpkA thiL 1hc omIuIh iftt gf lbe~fiu4u(yťM;-nUlvpn. ihr ^nhK^F *0(vkpcnil, jtt j'l ■ Jjffwtf Ľiiarjcar-—«inijw-J. *öJ. it *t*íM »fill*. h=M mií*jt»: Lnc, Wr iľauAul ŕl^fl to CiplMQ <rllul« ťrtrfuli» J <*t MJ* Li, tíO iDlii the rtAiťal fíwf^Hiin itůit <i| LMwiiBf}
r*DfatK*iaadoH*iiui Darwinh»i ^,J  'IdHikviIIi^
»hen »e ilkďlwc^ťnlkiihinteerarikipcidurn^rcAíh
[řrji ljopLTJ H>f b*í*tc" (lt|  A ŕrtfar^ Uiŕr ihr jn«^f«i
pvúúiki JU |Wfťii*h; lhjt iStd irfM ihr ÍUlbn^ ľ'PJÍ úťp ■t"
un .í* »I I , J I tM: f.řr Jl Í vn jiiin*."' «tu iVie ufif k -ŕOfVf' 1 J h pK'f*aír*l vufiiltjň f"-"**Jhr,ic wn^erMl twT»T*t o*i"»«i
■m-«ii^     #w» ■  hMtn^iaU  I  ^ •*     •*-'?
I »I OmJ •« LiH ViPnt Uh>it«T.
<tacplfoífw^r]rifrti«^d^*twtai_tWfa«t*JnJ Jamu hh: HctcioliCMbtAwiUHbfgirttTrfTWiivJ.Ll Knom»* r.vpcI■cfiťt [í*c4e* "h^i «he í«"|ilfi Icwfc I" J»"* fnwi thr umpk aAJ hiu4ifitit hint WMUDCtl n m* in lP>í ;j^ ul f>* f n ťrlh Hutlhii ÉMHpfilflStl vmíjTN *Vi«piirjftl tw jabber ftu* tu ■*■!!■ Ľ-nicm *wr luifta-h 1 Tu I Ihr ^(dfifiA tTAf ««ŕlhHil tN 1ÍK nx* iM UK WmTnAJ Irr* •» nfl*J«»:iiä ri^t J»i*fiJlt -rtd
inir4-n*H4it»Lfili*fMbK«nro«wfnf t« *«wíc*fl **■■»* «*<rt1
ha ■ ■ mmlciti e rl. S*k*i ifi mvn p n * nwlm itw rvM ncHiiun ■ti rrhNlriii lcUi hoi-1 *nhi «ti- fUbrj «ŕi "!mh Oiio LV fin44ťm m-4 nim .ílu ^iAn^JŕAf Ihn^ji Errambťt T~*mt h h4 ■ YK04llVplh' *,,rpt J^le tiunpl^ iPeJn» irf «Mil f*rt* JtLl M« OÜKI^v«. I hi p-Mffahet qiwJ hf ffifn Mpcd ilut kmr |hR vlAlbJaf 4TwirialKjf»4iMiUhflif orcvflttJiSnnf 4hc íkthnI
Thľrr  i i noyur. ■■■«] nilcui. El» «|f:^  lhal I h* tü*4V
tJifJfl lefmnwflifJ^ lií moííiJlhc iDnfmJ Iitv IW h>l n( ihá mdHtt rúca«, ffoei Ihr Uhct sm «rtHiu mtutisiŔiio in in Mil. qrutoL Tn-tkUtum n* hifhf« Jrtfhqird k>i ibii
«jjt iPN\*,íKS\i Mfelthc ľljj|(rNcU'n^ľP-,i ľ»L.-fi.**iiŕ !-■ iíwii m» hdUL-jITv m w nmlnii h^rn. hrivV. Ibciä «Annul dotrA^iHMn-  A|rai*yi Jl trf Ihf I K% A iti*tpbf •M.lŕďn »it* u
iHWdHmleirnmwrlIJľl (Im »hirr-uhrrupfih LJr#».^Tiii pn^rt« m- kjoATntJ -P JpinKriaL«. a hibihIM lií larn ■ m* A rrtaiAťtt lamí -nrirf u *pKi&: lo ihc htevna. i *«jb»»**uí
Urpcŕ Hl ťurti/fW* lákl ŕUCJkafd MI DC fetťtwc* HhJ niarjftrtC*. ml j Ir« uCbm iiŕ uAMjnŕty ŕ«trf*UI*r
Aku>ri all 4 lbe BAIWrUl If ifiilhTtf Hf piMilM í H «rf « Ibow h ■■ u*raplH*l Utovj «hu ■ orilni dm rumnáfW ptw««. U« Ibc akuiuI  HtJ wdkvjl wutt* id ihr dahmb ur
iMMÉ JifMŕrJ kam ■" JM>^t*r. -1 Hat M lV»l <K--EhlIŕJCU« h Jnufeŕ>tth«d »W Ol -■HR'41 llí- f-WTp* kv thr ■muKunl i ANA UhltM Ibc rwipyifMllBi PttlQiiilf nf
hnu m íBindh jll »f úm TchjMl ttfi t LIX Ww» nm íle»
p«(lŕrnnbykB j da^ ufWMrrrd ifMalp« I H Ia *f +»*í*i| H wwvwU fc«ra Thttl ini-wlilm*- iM«>«fli IbMbvr Jt%ctnpcd M ihf iKViullbc uftftHulliT*. tftutiyih uDJrr-rnl »thMixrMk
m>3*Pi4 .irhii» ia f ih 'H «hí iVíV n^n r^H I^Tin. TľÉntai^^hfi wTini K>a>«rbcnr«ibri k»oinr^nn3 •■ Ihr
iih-l--l"*ir iirn-ifř-LJlrTC Y*X c*" IíPrC« i i^í -T** A'»: foJ-uaih
pí ibeDKA-JrpíAurai RKA (K»Kmťi a>ť. J 4*1 ď u tacicrôl OMlŕaClltMTT, MT WuirnjJ Jt ialtibBlK«. Bm thr H11«líli|: hwlrruJ iMbWiil I -t O- fmK funulh ua HWlr/iil * «*«■» M >*-u Ľ4>pt» i* llH hfrZkful p»*raťi«í U« ílíh-rW Mif"^ piHiBUriHŕl ťcmfmMs ™t' Jl^Kni pajiiafiaA. ť-*rh prcKirt «k hnilr   t«p  U   lie   «LPUSí   and   IpWl^w   of|   r^h ifu*i3(
bofDotofv iii jwnc«h^i Jiltti-cľni pmin^h .ií h*wrui « <é4 kwY im* 111) A -tfnKti#r J liainťmr n4 lau njfBillMJt «u KprCM« M L74MI umf Eurk^hm^ JuJiwInv Tbt J.-h*<J UdfnmptKíti ipifWM b*^> í*s« **eni Wki^kiwulmlr«! •■<> ■dul ume «I -birti MV h»»] vi KWfii Nm JI.jí «Wr>.,.\*i ia nJuHnvnic» MU (A* i* dv enc «rf iraauVi« ibx MIm-hj «tkjryatk irrŤiiiir.tfrii i^iréi mmmíbh>< ruLM^i-fcfĺpcrJÉf ■ul wMMi! Bmtktím tnvmriM *i*iM.|rt jua n« k
-^^^.•«'»'.'IMU»«
Horizontal gene transfer - cell conglomerate instead of single cell ancestor
REVISED *TRE£" OF [ JFF Jíťj_r:f j 0ÉkI ^jmííu.'í ac oVl Mf n? É1 ■:uL^->n^ ^v
■AKPhuhL All ■ mihMmatalilHTtMMii of MaCnaiH iHHWIOl W"*»Io-*A Tbowr Ibbd u ■* Bern ykirnca iqbivwbbI randoatr)1 n> tTrjBwfcPT Dbr emji^mmc npir* •J [tFK truttfer oftta^t « itwIti(4* «.mti Jmhji aJttj^ HXCMf<ŕd t^r^Wri i»rij<dTuj nur J^":'"' Thiľ"ErM~ jJuQ ll^Ll J linfl« í e II K ihr rr>ai; Cfif Lhfif m4|Cr Jf-m j. n
of life probo^ <j^h ŕVwn j otmhJm^
MOtftlA OflWbJťtfrt*       CTvfUbhŕlrfH
Pl^PTOtMCtlfil
EUKARVOTES
W, K»D DOOLTITIE. *tw boli it- ,Thi Uwvuut. AwľHTOt CíH »1»™ m du Phundufi u/ikr MuAsnJ Audimrat iiíKí», W. »f, N» 12, pas» Ik« (4-il (*, Job, », I W«.
t You Ail WiMr Yeti Ejir A Cliví TWunru Rji/hiiT Could Atcovmt ich buuľnMUL Cum m Euujtnnu: Nucuui Gunun. W, Fscd Dubfllf n Trtmtí m CmAci. VU. N. M». I. Mfn »T-J1 li Ayfiw IV».
«ľKinJciMtic ri.viiini.Mrnj;> .v o IH1 Ľ^iviiML Titi.W Fotd Powlinfc n Srmu.
vot. wt, mm ím-iiiitHra, twn
DCV ■ fnénMlf £3/ tu^tmalrf juj jhukc-
■hl häkv 11 DiIuh Uhwiw) a Kift-bm Kxu 9DXM, ad dmluf ci n* Pki^am
lUrir hr .Uwd hand
tumjm*«Dt!bi   iggi
I   U
Biology's next revolution
ThecTWfUna ?ln.ie ff ricrrimn jgeiG-s'-w spina. tilecívK dmiarsa b rwliloi of lütt coneiimar ricnliTi, Bptd»crd»nlbdsn RiiH.
CtlXtl
■   r-,'11 ľ »' tri**"
NiviOalMWiidGariffiräK
(k« uTua naá fuaanarlil aaJm ■( MFJnilladha*Br>-h Ihvmáiliun h
Ůr\;4i ti,:
law,
latÉiiMiinabi
lliufirfMlqur.žkita
BHanlnarif ■raitikr
rinnw^iTrvi
r i.. J titňt L kifTi ifiŕ-s «í i ü i f * i i." nÉbnbf iif prockik* hAirí *:» ŕ Éiwii Audm^WtM^I JI. ■nlhUrtapadhiHiMifiťu.i.JTV
hu -t*—T1" -J —±ji—
ruarlnaMHmMün, aal why w b*w 1». LaaajHarJalaaanlkaFjaihial
rad^k* ur ix.lrilrlir \íi waJ. J..
UAiartriWlhaallaviut trilka iiy ibaurar* mé ilaaa* TI* hanaa Fj.krFjaajIfc^ap^auja.iirt.sa Fa tfcki HriMt I A« jii*_»*j.hiuKf T.PtaJnl r» täifnvwiuJ lilhaam
11.. i J« JMflMiif fe rilná ikri
;i7, křikl, k- ifrfn.i intaiJMi to i» iň^jivJťaMaBtaaaaBL
lamriÜMi arita? u iMaum itawjpaaa^lai br rfJnmaai-
Mhrinwabf
liui^JtaňuuÉabinHiMJJc     ".m.;, rii..;ttlii. rixut-ita^.i-JiJJt» ,j*;^!^iu    iUTIiHi^daiibaihiywki*riir tmpijJuiiMhfÜATbciiubilfel-   nqoJiarťHíciiltari Inderin
Ja,ftu juji ikaujih aat «bii.! Ji)
iHMUFJ
■mhu
íl.ř J.i ■* folii JMl'd briLih.ttH»
nlw4ft i M r h pruh h Mij^» wM
■laad^PkibMliiliiufllitoilaiBi     ihaaraNai i«|iajménbalHlbf UibriJudiaa-    biimuíM
nL~.T.'~j.>. 11, h.. 'Jali
l^jTl.lV. Ulf
irii
ai J iftmrd aan ■ ihmIbL b ibwwm II IM ■rkVHNlL UitiÜuJiatt piHta. aiaai iMauuna^fBMai HťwCflJir. di.-di driMkťi m Ük *T J1L7 ■'■' Píiuih^f ■','.-■ ifikat1 1 Jai T*L W,l I ŕilh L JiF M l*f i. I -a ,r« krMřjr-JMifWfíhrtinwib üumfr.r*h-h.^no|Hiur/ii b hMih H (hiHim numiuJ Ihai NJdnJ 1M «jafaahf JbbI ailhn lir aaiaVUa ifJUiliklf haaa Fťrnái|iÉipmhi KaMoCiBki^rHiiiri ni rutina
MHtlIp
Hujif iinU^H ■ um laJbMtH Ihn iJi.iK..ujir«iiHnMáaHhÉiau-HBMMfhibulhh
iffaMdaaiMJaiiauuarlLVfcujua
p 111■»!■ iwpi iku hitlautb»
FjJarlMnaf H h ihraiH^ÜBHihnv ni ili h hr» . 1 jhTi'ĽTi h» hr fc fI1 ynr;q .«-i.kri-.i^vr fJť-.T jin«, i^Mrlhn un :íp f i^i.rJ/ih ti .z. ... ir^hiVia.ahijnJfln!^ iJYi n I^n iňíTT ja hnpaf im ■ Lnnubuni Aui i^ ja HkaiM i Jyi; bm'.n 1 nft U— Wurf ■Amititaalhf ■um ■■u hr ilkwihn iirimÉ aft n "if In worthy dan alFjiwrilkii a «ukMd ad i^aaLTla ,1.1a-a nil ufwiulMa«. nanHllaJ li jvlkiJw hj- '.*. .fiiť.Ä u«4 ŕ an» 1 ,i Jh J«hx jJuHJKlcf-i^Lnl;.^
iruHIUBkbBBBBljElhlBVftTIir*
Ufa B^ihilkfiliUihaHM«kllwi ikil
jjfr^-nUBaVhnwaílKrT.            /
llaTaairtlfci^jhlh    nrrni t ■«i/pnamujHnam»«uilhHlifci-    ■ nťpiaiaMalituJ
[jglgjjj
ľ ÜanUfllar mim i nml inUdinriillkni '[hh,ma|iiáaii Uimli
láBBII
.AAňWH
■bahbalua/irilliih hlh-> rifr;!.F«9irdhiaila»Naa rfPíí^ FfiA irikř bprwc'uil alli i|ianľn 'Jwillnii parr ^ da IPHik inm^ liúiMMMi iii
■nniimhiiBahii laMakahaial juAjMVupatwauUunwtiMahJ MMklhal mJBEfl nu dpautcil ■/!' laaFJlhan JnUaa,UBHinail htumé i«;>«ffe*:iHiMlaiwl*i qlyubr
■FF«; iitw ajalF^iHipauaia«
laF^HitnaiulUa, nvnaHitn
■-----------^ ^'-f1]--------1------
r«F?Jxat luBjajaaBFjnM^aaHaa 11 Éa a—ilnai ihJan ihillaai iťlhi TSŠ"in*IT«íllWl l^flnaMhj iki imhaakahuJ aaarkWhlHaH mm ijwť«iaa lal lagiwtfw " llltlrflaaMlaari jfaiiiihffBbHhM
AijvIa jrrier vtJi'Mhiii hJctJL ■■faviJaiaFrihřAi
IAĽ
■diBCjn «ejh baJioland.bapin uflBMvIlaM BBPMliilhrhiFjiap-iii ATHibw «Iéé hanaiH Laa^u
■ HlMIIlMBBlJlJlj?              I
■W OiaBnaP ahUJa thartn^it Htlřai i«l BriÉMi a-ffaarl-. ütei UF/wthTTfl iJiál>iwOqiJr, lWIHaaHnilaafcUwMFa,  "vt^ iWn.UM.Ca1 Vaaatoaia 3aaa aref ť-jfj. iéb »aaii rfliai "r CarTa bmtfl arJh Cnéia Am» Urar» aWk * ». LVb
■k iiitAr.
u*r\ VirrwÁ -J-^ii, Hum- •**•
'l IIP J ■■!' >lll 4l l^^lfl
■•* 11" »tIp^Ji 4* ilJITI
■kif p.F* .'«■■ "i !«■-»■■ \ŕiwir*ľ «.■■Maii^ir^ín-TPi
Z
O
f-
o
Thus we regard as regrettable the conventional concatenation of Darwin's name with evolution, because other modalities must also be considered
11
JOHANN 6RE60R MENDEL
* 1822                          in Hynčice (Moravia, Austro-Hungarian Empire)
+ 1884                          in Brno (buried at Central Cemetery in Brno)
discovered through breeding experiments with peas that traits are inherited based on specific laws (later to be termed "Mendel's laws"). By mentioning Elements of Heredity he predicted DNA and genes (published 1866, lecture in Brno 1965)
In the 1950's Mendelism declared to be a reactionary teaching (LYSENKO, LEPESHINSKAYA)
Mendel statue removed and its destruction ordered
Brno geneticist J. Kříženecký jailed
His pupil V. Orel forced to work manually in industry
1964 attempts to rehabilitate Mendel
Academicians B. Němec (biologist) and F. SORM (biochemist, President of the Czechoslovak Academy of Sciences) backed by Soviet Academicians. Dealing between N. Khrushtchov, A. Novotný (President of Czechoslovakia), F. Sorm and biologist J. Pospíšil (later the Party Secretary) resulted in the decision to organize an international conference in 1968 (100 anniversary of publication of Mendeľ s paper) in Brno (F. Sorm warned by Novotný that his attempts may result in the end of his career if the action will get out of control). Beginning of Mendeľs Museum in Brno
A milestone not only in the approach of Party and State to Mendel but also a beginning of rehabilitation of SCIENCE against the COMMUNIST IDEOLOGY
Brno Augustinians 1860-62          Abbot C. Napp
Mendel's Medal, Moravian Museum, Brno
Abbot G. Mendel
& J MENDEL, priest, teacher, scientist and abbot in BRNO
Teachers of Brno gymnasium (High School)
THE STATUE STORY
In 1906 Dr. Hugo litis, the gymnasium professor in Brno organized an international collection to build the Mendel's Statue in Brno. Created by a French sculpturer T. Charlemont the Statue was errected at the Mendel Square in 1910
In 1956 Mendel's Statue was ordered by the Regional Authorities to be destroyed. The workers who were supposed to the job decided not to do it because they believed that the statue was nice. Moreover it would be difficult to destroy it.
After February 1948 Soviet „Lysenkism" (T. D. Lysenko 1896-1974) strongly
affected biology in Czechoslovakia. After Stalin death (1953) attempts were made
by soviet scientists (particularly by physists and chemists) to substitute Lysenko's
„materialistic biology" for normal science and by the end of 1950's plans were made to organize in Brno International Mendel
Memorial Symposium. In 1962 Lysenko's work was criticized by the Soviet Academy but
still in September 1964 N.S. Khrushtchov raised objections against the Mendel Symposium in 1965 in Brno. During his visit
in Prague he dealt with the President A. Novotný who finally agreed with the meeting organization after the President of the
Academy F. Sorm personally guaranteed that the Symposium will not be politically misused. (F. Sorm was well informed about
the activities of the influential Soviet scientist to rehabilitate fully the genetics - Soon after his visit of this country N.S.
Khrushtchov was removed from his position).


n * • *-
Ä?    J?»"**
Before the Smposium the Director of the Institute of Biophysics prof. F. Hercik was entrusted by the Academy to help with the organization of the Mendel International Meeting in Brno. To fulfill his duties he turned to the City Authorities asking to move the Mendel's Statue to the Abbey garden. As his request was ignored he asked his graduate students J. Koudelka and B. Janík to move the Statue from the Abbey yard to the garden. Both fellows were quite strong young men but they found the marble Statue too heavy.
1844 -1895 Friedrich MIESCHER
1. sdělení v r. 1871
Žák Hoppe-Seylera v Tübingen se zabýval izolací jaderných komponent (z hnisajících buněk, které získával z tamnější chirurgie). Buňky hydrolyzoval pepsinen>HCI a po třepáni s éterem izoloval jádra jako separovanou vrstvu na dně nádoby. Z tohoto materiálu „nukíeín" -
řp3 rTfu/ü I   LrwCala    n/rhia  c?a  m?nAi ičtal \/j3  -rra.r\    IruiHu   o  nhcahAwal wůrLtís
množství P.
Vysoký obsah P byl považován za velmi pozoruhodný - jediná tehdy známá organická látka obsažená v tkáni - lecitin. Když F.M. předložil práci k pubL shledá! ji H.S. tak překvapující, že ji odmítl uveřejnit dokud ji sám neprověřil.
. i V r.   OU   L>ui\   viulii   vju   uuuviUi   r \ ii> v,,   t iuiV-1    ¥ iiwiiivjvi   iiiuibi  iu i   n   iĺuiuui
nukleinu v i n5 víCKciCi i SpGľiuii i0S0S3 - Z niCii mUKi6ím O Vy 5 OK 8 r u.V.
a zásadiiý materiál bílkovinné povahy, který nazval protamin; obsah P vnukieinu 9,59%.
Purinovébase (A,G) objevili Piccard a Kossel (1874-85) U 1885, Altman nazva! nukiein poprvé nukleová kyselina, NK (nukteinsäure) (1889); koncem 19. století identifikován T a vzápětí C
Kolem roku 1930 již známy DNA (thymus) a RNA (kvasnice) i jejich základní složení. Ve čtyřicátých letech - DNA v jádře, RNA v cytoplazmě a jádře.
Fig. 1. Friedrich Miescher and his mentors. (A) Friedrich Miescher (1844-1895) as a young man. (B) Wilhelm His (1831-1904), Miescher's uncle. His still is famous for his work on the fate of cells and tissues during embryonic development and for his insights into neuroembryology. He, for example, discovered neuroblasts and coined the term bdendriteQ (Finger, 1994; Shepherd, 1991). (C) Felix Hoppe-Seyler (1825-1895), one of the pioneers of physiological chemistry (now biochemistry). Hoppe-Seyler performed seminal work on the properties of proteins, most notably hemoglobin (which he named), introduced the term bproteidQ (which later became bproteinQ), and worked extensively on fermentation and oxidation processes as well as lipid metabolism (Perutz, 1995). He was instrumental in founding Germany's first independent institute for physiological chemistry (in 1884) and in 1877 founded and edited the first journal of biochemistry, the Zeitschrift fu--r Physiologische Chemie, which still exists today as Biological Chemistry. (Ď) Adolf Strecker (1822-1871), a leading figure in chemistry in the mid-19th century and professor at the University of Tubingen from 1860 to 1870. Among other achievements, he was the first to synthesize aamino acid (alanine from acetaldehyde via its condensation product with ammonia and hydrogen cyanide) in a reaction known today as Strecker synthesis (Strecker, 1850). (E) Carl Ludwig (1816-1895), a protagonist in the field of physiology in the second half of the 19th century. His focus was the physiology of the nervous system and its sensory organs. In 1869, he founded Leipzig's Physiological Institute.
Hoppe-Seyler's laboratory around 1879
t-'Jg. Z. rkrtojintf orŕillítilrŤPC ľifilrŕii Irtanftiy w?u± ÜW.PrwifVibrcrami: 'JkciKm,-.;,. l±NLttiy aľ L'ümhzcü Uronuy il Lfttt. Ali nm w* HUriuga «tic i Jmmflry- Hex. Etowc-hoflcr hid dbíc arttiLL-brcak-M ducoraic* jmsrdhtn dB prowL» or tcmnáobir. "Jh* ubcrancti vu s ■tair.flcut itsp hr Ism invcw jptuiu into tbc irntmlEi ud ťjraimiä -r. iľ.> »ni tfder priKou» Fhntwaphy try Ľfeíl Kiccu; TütiLgai.
F. Miescher's laboratory
1 if. 4.1ha Idncibcy n ůk krme Lfrüzii uff it anih t TDHrijpii h r ra t: ťí*. í: «i i. ún* :vľi ihr MjhrHie- Pud tf aítoraí i:03A 13 >an iirux TP it HTihcjoir ľ'J :\*f rŕ. im-zMe u fetiaüor u Qk ihn: wauä ivu mn no lirriK. uKh n kus dtf illi.i;«i vocnfiu t. ', jz ft ivrjjLT fiI" Uie iwt/. ',o -jfwliu.1 •ifeiili.'ü wfcj nJ MM^d grille u.ijik «l+i i> cto* iIehihl^ ud c ilftt dňlilkufcc. »tonn in Üie skL :kki1 EjElajnnhy \y \<zn\
Tübingen castle
A, in Miescher's time
B, at present
FIRST PROTOCOL
Before attempting the isolation of cells from the pus on surgical bandages, Miescher took great care to ensure that his
source material was fresh and not contaminated. He painstakingly examined it and discarded everything that showed signs
of decomposition, either in terms of smell, appearance under the microscope, or by having turned acidic. A great deal of the
material he could obtain did not meet these strict requirements (Miescher, 1871d). Those samples that did were
subsequently used to isolate leucocytes.
In a first step, Miescher separated the leucocytes from the bandaging material and the serum (Miescher, 1869a,
1871d). This separation posed a problem for Miescher. Solutions of NaCI or a variety of alkaline or alkaline earth salt
solutions used to wash the pus resulted in a "slimy swelling" of the cells, which was impossible to process further
(His, 1897b). (This "slimy swelling" of the cells was presumably due to high-molecular-weight DNA, which had been
extracted from cells that had been damaged.) Only when Miescher tried a dilute solution of sodium sulfate [a mixture
of one part cold saturated Glauber's salt (Na2S04d 10 H20) solution and nine parts water] to wash the bandages did he
manage to successfully isolate distinct leucocytes, which could be filtered out through a sheet to remove the cotton
fibers of the bandaging. Miescher subsequently let the washing solution stand for 1-2 h to allow the cells to sediment
and inspected the leucocytes microscopically to confirm that they did not show any signs of damage.
Having isolated the cells, Miescher next had to separate the nuclei from the cytoplasm. This had never been
achieved before and Miescher had to develop new protocols. He washed the cells by rinsing them several (6-10) times
with fresh solutions of diluted (1:1000) hydrochloric acid over a period of several weeks at "wintry temperatures"
(which were important to avoid degradation). This procedure removed most of the cells' bprotoplasm,Q leaving behind
the nuclei. The residue from this treatment consisted in part of isolated nuclei and of nuclei with only little fragments of
cytoplasm left attached. Miescher showed that these nuclei could no longer be stained yellow by iodine solutions, a
method commonly used at the time for detecting cytoplasm (Arnold, 1898; Kiernan, 2001).
He then vigorously shook the nuclei for an extended period of time with a mixture of water and ether. This caused
the lipids to dissolve in the ether while those nuclei, still attached to cytoplasm, collected at the water/ether interface.
By contrast, the clean nuclei without contaminating cytoplasm were retained in the water phase. Miescher filtered these
nuclei and examined them under a microscope. He noticed that in this way he could obtain completely pure nuclei
with a smooth contour, homogeneous content, sharply defined nucleolus, somewhat smaller in comparison to their
original volumes (Miescher, 1871d).
Miescher subsequently extracted the isolated nuclei with alkaline solutions. When adding highly diluted (1:100,000)
sodium carbonate to the nuclei, he noticed that they would swell significantly and become translucent. Miescher then
isolated a yellow solution of a substance from these nuclei. By adding acetic acid or hydrochloric acid in excess, he
could obtain an insoluble, flocculent precipitate (DNA). Miescher noted that he could dissolve the precipitate again by
adding alkaline solutions.
Although this protocol allowed Miescher for the first time to isolate nuclein in appreciable purity and quantities, it was
still too little and not pure enough for his subsequent analyses. He consequently improved on this protocol until he
established the protocol detailed in Box 2, which enabled him to purify sufficient amounts of nuclein for his first set of
experiments on its elementary composition.
Box 1
M. SECOND PROTOCOL TO ISOLATE DNA
A key concern of Miescher's was to get rid of contaminating proteins, which would have skewed his analyses of the
novel substance. "I therefore turned to an agent that was already being used in chemistry with albumin molecules on
account of its strong protein-dissolving action, namely, pepsin solutions (Miescher, 1871d). Pepsin is a proteolytic
enzyme present in the stomach for digesting proteins. Miescher used it to separate the DNA from the proteins of the cells'
cytoplasm. He extracted the pepsin for his experiments from pig stomachs by washing the stomachs with a mixture of 10
cc of fuming hydrochloric acid and one liter of water and filtering the resulting solution until it was clear.
In contrast to his earlier protocol, Miescher first washed the pus cells (leucocytes) three or four times with warm
alcohol to remove lipids. He then let the residual material digest with the pepsin solution between 18 and 24 h at 37-
458C. After only a few hours, a fine gray powdery sediment of isolated nuclei separated from a yellow liquid. Miescher
continued the digestion process, changing the pepsin solution twice. After this procedure, a precipitate of nuclei without
any attached cytoplasm formed. He shook the sediment several times with ether in order to remove the remaining lipids.
Afterwards, he filtered the nuclei and washed them with water until there was no longer any trace of proteins.
He described the nuclei isolated in this way as naked (...). The contours were smooth in some cases or
slightly eaten away in others (Miescher, 1871d). Miescher washed the nuclei again several times with warm alcohol and
noted that the bnuclear mass cleaned in this way exhibited the same chemical behavior as the nuclei isolated with
hydrochloric acid.
Miescher subsequently extracted the isolated nuclei using the same alkaline extraction protocol he had previously employed on the intact cells (see Box 1) and, when adding an excess of acetic acid or hydrochloric acid to the solution, again obtained a precipitate of nuclein.
Fig. 5. Glass vial containing nuclein isolated from salmon sperm by Friedrich Miescher while working at the University of Basel. The faded label reads Nuclein aus Lachssperma, F. Miescher (Nuclein from salmon sperm, F. Miescher). Possession of the InterfakuIt-res Institut fqr Biochemie (Interfaců I tary Institute for Biochemistry), University of
Tubingen, Germany; photography by Alfons Renz, University of Tubingen, Germany.
Fig. 6. This picture of Friedrich Miescher in his later years is the frontispiece on the inside cover of the two volume collection of Miescher's scientific publications, his letters, lecture manuscripts, and papers published posthumously by Wilhelm His and others (His et al., 1897a,b).
Pathogenic
S (smooth) cell
Bacterial cell
Bacterial DNA
Heal lo kill; S Iragment
released
Nonpaihogenic
H (rough) cell
Eniry ot chromosome liagmem dealing
S gene mto fl call
RecomĎinaiion
and cell division
Some daughter cells are S-iype
S cell
W
Figure 4 ..s
Crucial experiments thai demonstrated DNA as the genetic substance, (a) The experiment of Avery et a!, showing that nonpathogenic pncumococci could be made pathogenic by transfer oi DNA from a parhngenic strain, (b) "t"he experiment of Hers hey and Chase showing thai it is transfer of the DNA from a bacteriophage to a bacterium that gives rise to new bacteriophage*.
^■labeled
viral DMA
^-labeled protein coat
Muting c-1 vkus
wiih host cells results in
flílšO'Oľ or i Of
virus to host cell
9
Protein "ghost" labeled wiih »S
Violent satiation m a mixer
Viral DNA labeled with "P
Multiplication of viral DMA m the absence ol Ihe ., original protein coal
©4sS
(a) 1944: Oswald T. Avery, Colin MacLeod, and Maclyn McCarty demonstrate that Griffith's transforming principle is not a protein, but rather DNA, suggesting that DNA may function as the genetic material
(b) 1952: Alfred Hershey and Martha Chase use viruses (bacteriophage T2) to confirm DNA as the genetic material by demonstrating that during infection viral DNA enters the bacteria while the viral proteins do not and that this DNA can be found in progeny virus particles.
Helease of ne* progeny viruses, some of which contain KP in their DNA and none of which contains MS in its coat
<b)
-^4
A
1»M

■■ ■ i	■	■ ■ ■'"-"■ ■-"■ sr '^
		B»^y;''<£iKy
«á*$S**	^■dfc^^^R^^	□
		
ICLIJ1E   6,9
ťnmpariMni of ihe A, B, and Z Tornu ůf DNA. ŕart ŕal «hra* »ídr «b
A, B and left-handed Z-ĎNA
as we know them now
How did we arrive to them ?
Double helical conformations of DNA: (left) A-DNA, (center) B-DNA, (right) Z-DNA.
21st Anniversary: The DNA Double Helix Comes of Age
27
April 25, 1953               NATURE
MOLECULAR STRUCTURE OF NUCLEIC ACIDS
A Structure for Deoxyribote Nucleic Acid
WE wiah to suggest a structure for tho salt ijf deoxyribos.. nucleic acid (D.N.A.). This structure hi» novn! features which am of considerable biological interest,
A structure for nucleic acid lias already boon proposed by Pauling anil Corey1. Thny kindly made Ihoir manuscript available To ua in advance of puhtiaation. Their model consists of three intertwined chains, with the phosphates near the libro •Kali and the bôŕtos on the outside. In our opinion, this structure is unsatisfactory fůr two reasons r 111 We Iwliove that the material which gives tho X-ray diagrams isihs salt, not the free acid. Without the acidic hydrogen atoms it is it»! clear what form« would hold the structure togotlier, especially as the negatively chargi>d phoaplistos near tho axis will wpel pach other. (Í) Rome of tho van dor Waals ilistaneey apfMtar to be tot» umalí.
Anolher throo chain structure has also' boon sug-
lil  by Frasor (in the proas).     In  In« model  the
phosphates are on the outside and I hi
Xniclure n» described is rather ill-derlnWlj
The novel feature of tho structure is the manner in which the two chains are held together by th« purine and pjTimidine hasea. The planes of the bases are perpendicular to r,hu fibre axis. They are joined togsllier in pairs, a single bow from one chain being hydrogeň-bondod to a «ingle base from the other ohain, so that the two lie side by side with identical i-oo-orttinatos. Ona of the pair must be a purine and the other a pyr.midino for bonding to occur. The hydrogen bond« are made a« follows ■ purine position 1 to pyrimidine position I ; purine position Ů to pyrimidine position 8.
If it is assumed that the bases only ooour^ structure in the most plausible tautomeric (that is. with the keto rather thf figurations) it is found thai onlySspňcjJu^JLirf of bases can bond together. Thoe*TS*ife are : adenine {purine) with thymine (torirrMrJejT and guanine (purine) with oytosine iawanidine).
In other words, i^CUp forms uno member of a pair, on e:tlie£fma\^fnnn on these assumptions the other m*mť\ rVm. bo thymine : aimilarly for guanine mdL-yJiHírno. The sequence of banc* on a singlu, oliyjn^iTnin mit tijjiKxir to b« owrictod in any HowTWer,irVjftj- specific pairs of base« can U-, it follndg Ithat if tho sequence of bases on ii'lM? W™. rhftrL the sequence on the other |Hj ihvlultfmatioally determined.
liases on ' '■*v' iiwul...   linked   together   h\   liydmiri'i]   bond»,    \' V-1 ' ed is rather ill-defuTed,/7nW\í5r t hi« reason we ah, IUA Wnml      JÄT*^™" fotmd exper.meritAlly.. that the ratio an jt                 x/^iX.^^       |t  *pj V«* «mount« or adenine to thymine, and tho ratio
We   wttu£«   \j/forward   ■nrjf.*!?^!a?ff^B_*!L*ř'^* *** °l0*t *° ""^
radii" "
w
f*jllY"Vi\i'>'»' K1nii;iiVeM\>r l«Vt ^ariEiixyTnti^LWujcleio VJ™Sl   strtuMwr has   two
'liains each, coiled round In same axis [afta diagram). We have made tho usual chemical assumptions, tiAmely. that oaoh chain consists of phosphate dieter group« joining ß-u-donxy-ríboŕnranose residues with }',5' linkages. The two chains ibut mil their bases) aru related by a dyad [xrpendioular to tho fibre axis. Both nlinint follow righi-haniled* helices,   hut   owing   to
il„.   .[\,i.|    Id..   H-qii,.)!...«   ..f   H,,.
atoms in tho two chair» run ill opposite directions. Each chain loosely resembles Fur-het^'fl" model No. 1 ; that is, 1 ho bases are on t lie inside of the helix and the phosphates on the outside. The configuration of the sugar and the atoms near it is close to Furberg's 'standard configuration*, the sugar being roughly perpendicular to the ftttach.id buxe. There
i liil   tlmiFL-    \i    i.Lin- li-
iuKriEiiii.nl iľ. Tat i vij : bboiH H>iiLbuLize ll|9 1 r$   phcwphkte—MIRM
llftllU,    dh-L    l].|.    hi i .-r | -
omul ntů* the pal« ůf 'UMllulillUttlif rliilii, ■ Ktclh«.    Trit VcrtLc»!
1 ní iDSTkj, ll.r flLhr« n)d«
in a roaidue on each chain every J-4 A. in the i-direc-tian. We have asaumed an angle of 36" belweei adjacent residues in the same chain, so that the Itructure repeats after 10 residues on each chain, that is. after 34 A. The diatftnee of a phosphorus atom from the fibre axis is 10 A. Aa the phosphates are oil the outside, cations have easy access to thorn.
The structure is an open one, and it« water content il rather high. At lower water contents we would expect the bases to tilt so that the structure could become more compact.
for dooxyriboae nucleic acid.
It ia probably impossible to build thin atruoture with ft riboae sugar in plane of tho deoiyribose, as the extra oxygen atom would mako too close a van der Waala contact.
The previously published X-ray data»-" on deoxy-ribose nucleic acid are insufficient for a rigorous test of our otructura. So far aa we can tell, it is roughly compatible with the experimental data, but it must lie regarded as unproved until it has boen checked against more exact results. Some of those are given iti the following communications. Wo were not aware of Ihe details of the nosulta presented there when we devised our structure, which rest* mainly though not entirely on published experimental data and stereochemical argumenta,
It has not oscapod our notice that the specific [iniring wo have postulated immediately suggests a possible copying mechanism for the gontitio material.
Full details of the atruoture. including the conditions iiH-uimed in building jt, together with a set of co-ordinates for the atoms, will bo published elsewtiero.
We are much indebted to Dr. Jeny Donnhue for constant advice and criticism, especially on interatomic distances. We liftvo also been stimulated bv a knowledge of Ihr genunil nature of the impuhliuheď iixporimental results and ideas of Ľr. M, H. F, Wilkin«. Dr. U. E. Franklin and their co-workers at. Klug« ('ollere. Lumbal, i )m> uf u« t.j. 11. \\ i imn in,.. aided liv a li-llownlii]i limn llio \aiii.iuil Kiniiiiiaiion
ľoľ   [iiliintilľ   J "iir.ilvM'..
J. 1). Watson F. H. C. (.RICK Medicol líemjarrli Coiincil Unit for the Htudy of the Molecular Stmotun "ľ
Iliologioat Sy-:-i-in-,
t'avendish laboratory, Cambridge.
April 2.
28
1953
A paragraph dealing with nucleic acids from a text book of Organic Chemistry (in Czech) is shown. Briefly, it says nucleic acids (NA's) form complexes with proteins which are the building blocks of plant and animal viruses and of cell nucleus. Total hydrolysis of NA's proceeds according to the following scheme:
alkaline hydrolysis        enzym, digestion
Polynucleotide     ^mononucleotide     ^uracil or purine bases
lyto ltjr*o!.iny  J new lÄtfty'HloiitfpT^SOiCiJBfilekül**-  ■ ní, Jot va apojntit "o toll*vrlnou tTtíi  M* ETBňť nuKlíoprotPÍ-iy rl^jaa^Ť" >*°ajM  fMBlljmŕoh i  tlvůBJ.iii*gh tIt* « put-na8n*n& j*dra^_
7nt(iy*T»ií « í .tt*flBJ.e»klíHJců ť&QítÍnníútiŤB brBlüu, srí1-fcraatu,» *tfaioti »jHíroťUnHJÍ ,m »vol*  a' tc n »ŕÄBe-bonín ****?" wA a -rejtulkovoho aeaano • eí vaklíSíiirřW hraůtců, St4>í DO go_ljniamdr n» fcjaaliml rosfírsÍTKrti a^ laukloliiů * Ja-iJ' je taAy BlOuSantnoů alilaafijioi  »e a oďkru ^aitaldim «b« Borlntt*Trto.i*ucl*oalŮř obn-huji BrartS š Osobní rifraro imTjP ťlbediaoHA p  ú^etoj» Ailíaw tíl ä» ' PlMal^r, «nUtft Jí OÍ i"J (L típnul mJtlítiHjyí Hjiflllny t Twaiinio o ťťbaaanpaiar ,   .
""**»•' ■ R*^!ie-ľiÍBft«*lÍ o L J Www hyujgj.ľŕľ. ::>;"
\
1A7HQ11    r>o%a mri:»T^ sAosaj
lutnu.
:T    lOCO   ««r
___j tra*
5-MtČ    («St
Dnunulc le o ti äj 2* Pol^iutleůtliy
BRSANÍCWÍCHF|l!|;
Pťf Dodnfi porno putl*infvá 3cyes ü f^ a /j lí 1 s Uta íít"". eukru' na-r                                                                                 "' ■
Considering that uracil and adenine were discovered in 1885 and G in 1844 while C in 1894 and T in 1900, our lectures on NA's were up-todate in 1885 but not in 1894
In courses of Marxism-Leninism (obligatory to all students)
we were tought that G. Mendel was a burgeous reactionary pseudoscientist.
Interestingly there was not a single chemist among us who believed it.
To my surprise there were some biologists who took this nonsenses seriously
Chargaffs Rules
Tetranucleotide hypothesis originated in 1906: DNA is a "statistical tetranucleotide". During the 1950's E. Chargaff showed a number of ĎNAs, which differ in their base content. Chargaffs rules: 1. 6-amino residues = 6-keto-residues; in another expression A+C - G+T; . py = pu; C+T = G+A   3. A/T = G/C - 1 (consequence of combining equations 1 and 2)
Watson and Crick (1953) proposed their famous double-helical structure of B-form of DNA on the ground of          Chargaff' s rules
X-ray diffraction of DNA fibers obtained by Maurice Wilkins and Rosalind Franklin
Construction of molecular models This structure consists of two antiparallel helical strands. One turn contains 10 residues in every strand, the distance between bases is 3.4 A, the bases are almost perpendicular to the axis, the phosphate group is 9 A from the axis. Bases are specifically paired through hydrogen bonds - AT and GC. The strands are complementary - hydrogen bonds between two strands, the bases are inside the structure. Difference from a-helix in polypeptides. Further forms A and C (besides B): dependence on humidity. The differences are principally in the tilt of bases and in the number of residues per turn, strands are commonly antiparallel, bases are stacked and base pairs located in one plane. It seems that the B-form is the prevalent one in solution as well as in cells and viral particles.
Crick, Watson and Wilkins: Nobel Prize 1962
"The structure is produced like a rabbit out of a hat, with no indication as to how we arrived at it"
F. Crick, NATURE 248(1974) 766- on the occasion of the 21st anniversary of the discovery (commenting their first paper in NATURE). What experimental evidence was available to W+C in 1953?
X-ßAV FIBER ANALYSIS OF DNA
represented the main evidence for the Watson-Crick double helix model
This method enabled analysis of high-molecular OUA, but orovided onív few basic oarameters of the helix
such as
distance between base pairs
number of base residues per turn
Further data were derived from model building considering the laws of structural chemistry
Base pairing from physical-chemical measurements Text Sugar configuration (PUCKER)   \
Angles of the glycosidic bonds were fixed within certain limits
Handedness of the helix The direction of rotation was guessed and then subjected to testing

6. .jto
tí
:«
'Ol

a.+ rrn
W
^nar u>oov
Nitrogenous
Bases
RNA
Ribonucleic acid
fClCytw
I
.....
■"v
L
N'        O
I H
m
Canine
H /yV"
yvVH
H                          H
|  A  | f"i<tn«
NM?

[ T   | Thymine

Ca
Ä
DNA
Deoxyribonucleic acid
Witrogenous Bases
DNA is a polyanionic biomacromolecule with bases in its interior and sugar-phosphate backbone on the surface. At neutral pH it carries one negative charge per nucleotide. Below pH 5 and and above pH 9 ionization of bases become important
Parameters of DNA structures
TABLE 1					
Comparison of A-, B-	, arid Z-DNA				
		A-ONA*	Ö-DNA-	B -ONA"	Z-DNA
Helix sense		right-handed	right-handed	right-handed	lefl-hande
Base pairs per uirn		11	10	10	12 (6dimei
Helix twist 0		32.7	36.0	34.1. 36.8	- 10. -50
Rise per base pair (A)		2.9	34	3.5. 3.3	3.7
Helix pitch (A)		32	34	34	45
Base pair lilt O		13	0	0	-7
P distance from helix axis (A)		9.5	93	9.1	5.9, 8-0
Glycoside orientation		anti	anti	anti	anti, syn
Sugar conformation		CZ'-enüo	Wide range	C2-endo	C2-endo, C2 end<y
Numerical values lor each form were oblamed by averaging ihe global parameters of
corresponding double-helix fragments
ß DNA values are for a double helix backbone conlormation alternating between confori
nonai stales I and II
The two values given correspond to CpG and GpC steps for the twist and P distance valu
to cytosme and g nanosme for the oihers.
Two values correspond to the two conformational slales From Kennard, O and Hunter. W.
Q. Rev. Biophys. 22. 3427, 19Q9 Wilh permission
TA8LE 2
Average Helical Parameters (or Selected Right-Handed Structures
A
B
Double helical conformations of DNA: (left) A-DNA, (center) B-DNA, (right) Z-DNA.
DNA structures from X-ray crystal analysis
DNA double helix is polymorphic depending on the nucleotide sequence
		Rise per			Groove width		
	Helix twist ! )	base pair (A)	Base pair tu« n	Propeller twist n	(A)		Displacement
					Minor	Ma|or	Da {A)
A-form							
d(GGTATACC)	32	29	13	10	102	6.3	4.0
d(GGGCGCCC)	32	3.3	7	12	9.5	10.1	3.7
d(CTCTAGAG)	32	3.1	10	11	87	8.0	3.6
r(GCGjd(TATACGC)	33	2.5	19	12	10,2	3.2	4.5
r(UUAUAUAUALIAUAA)	33	2.8	17	19	10.2	3.7	3.6
Fiber A-DNA	33	2 8	22	S	11.0	2.4	4.4
B-form							
d(CGCGAATTCGCG)	36	33	2	13	5.3	11.7	-0.2
d(CGCGAATTBrCGCG)	36	34	2	18	4.6	12.2	-0.2
Fiber B-DNA	36	34	Z	13	60	11.4	-0.6
BrC - S-bronecytosimo.
Adapted from Kennard. O- and Hunier. W N., O Rev. 8iophys.r 22. 327, 1989 With permission.
33
CRUCIFORM Negative SUPERCOILING Stabilizes     inverted repeat
local DNA structures
*i


TRIPLEX structure homopuhomopy\
SUPERC&lL^r
! LEFT-HANDED Z-DNA Y alternating pu-py
,.    --^CURVATURE ,££? ■-* 4-6 A's in phase with ~^% 'the helix turns
HAIRPIN
SINGLE-STRANDED region AT-rich
Physical methods such as NMR and X-ray analysis indispensable in the research of linear DNA structures are of limited use in studies of local structures stabilized by supercoiling
34
Problems of life origin
What was first - DNA, RNA or protein?
Well-known Oxford zoologist Professor Richard Dawkins (who declares himself to be passionate fighter for the truth) writes in his book River out of Eden:
"At the beginning of Life Explosion there was no mind, no creativity, no intent, there was only chemistry"
Let us try to summarize what chemistry it was
New York/Tune*
June 13,2000, Tneidiy SCIENCE DESK
Life's Origins Get Murkier and Messier; Genetic Analysis Yields Intimations of a Primordial Commune
By By NICHOLAS WADE (NYT) 2179 wordi
The surface of the earth is molten rock. The oceans are steam or superheated water, Fivery so often a wandering asteroid slams in with such energy that any incipient crust of hardened rock is melted again and the oceans are reboiled to an incandescent mist. Welcome to Hades, or at least to what geologists call the 1 ladcan interval ofeartlťg history. It is reckoned to have lasted ftom the planet's formation 4.6 billion years ago until 3.8 billion
years »go, wlirti the rain of ocean-boiling asteroids ended. Tlic Ism grccnjtonc belt of western Green and. one of the olden known rockt, was funned m lne Hadem interval ended. And amazingly, tu judu» by unemicil trauni in the Iiuan rocki. life on earih was already old.
Everything about the origin of life on earth Is a mystery, and It seems the more that Is known, the more acule the puzzles gel.
The dates have become increasingly awkward. Instead of there being a billion or so years for the first cells to emerge from a warm broth of chemicals, life seems to pop up almost Instantly after the last of the titanic asteroid impacts that routinely sterilized the infant planet. Last week, researchers reported discovering microbes that lived near volcanic vents formed 32 billion years ago, confirming that heat-loving organisms were among earth's earliest inhabitants.
The chemistry of the first life is a nightmare to explain. No one has yet devised a plausible explanation to show how the earliest chemicals of life — thought to be RNA, or ribonucleic acid, a close relative of DNA - might have constructed themselves from the inorganic chemicals likely to have been around on the curly earth. The spontaneous assembly of small RNA molecules on the primitive earth "would have been a near miracle," two experts in the subject helpfully declared last year.
A third line of inquiry Into the beginnings of life has now also hit an unexpected roadblock. This is phytogeny, or the drawing of family trees of the various genes found in present-day forms of life. The idea is to run each gene tree backward to the ancestral gene at the root o r the tree. The collection of all these ancestral genes should define the nature of the assumed universal ancestor, the living cell from which all the planet's life is descended. The universal ancestor would lie some distance away from life's origin from chemicals, but might at least give clues to how that process started.
"It is not so preposterous anymore to think of the common ancestor as a sort of Noah's ark, where pretty much every protein domain has been
represented," Dr. Koonin said. The proteins ofliving organisms arc composed of mix-and-match functional units known as domains.
Still, this idea is a disturbing concept. Evolutionists are accustomed to portraying the evolutionary process in terms of neatly branching trees, not Noah's arks............
BIOLOGICKÉ LISTY 68 (I): 3-14, 2003
Problémy vzniku života na Zemi
Emil Paleček
Biofyzikálni'ústav Akademie véd České republiky, Královopolská 135,61265 Brno Přijato do tisku
L   Úvod........................................................................................................................    3
2.   Původ prebiotických molekul (biomonomerů)........................................................   5
2.1. Abiotická syntéza v redukční atmosfére...........................................................   5
2.2. Import biomonomerů z vesmíru.......................................................................    6
2.3. Podmorská syntéza na površích minerálů........................................................    6
3.    Organizované systémy schopné replikace...............................................................   7
3.1.  Svět RNA..........................................................................................................   8
3.2.  „Světy" před světem RNA?.............................................................................   8
3.3. Metabolismus před přenosem genetické informace?.....................•.................   9
4.    Problém společného předka a konstrukce genealogického stromu.........................   10
4.1. Nový strom života bez společného předka?.....................................................   10
4.2. Horizontální přenos genů a organizace primitivních buněk.............................  11
5.    Závěr...................................................................................................................       [i
1. Úvod
V úterý 13. června 2000 vyšel v New York Times článek „Life's Origins Get Murkier and Messier; Genetic Analysis Yields Intimations of a Primordial Commune" („Původ života se stává mlhavější a zmatenější; genetická analýza naznačuje prvotní (buněčnou) komunu" , překlad EP) (Wade 2000). Vzhledem k tomu, že nemám vždy úplnou důvěru k novinovým článkům zabývajícím se vedeckými problémy, rozhodl jsem se trochu podívat, co se o otázce vzniku života na Zemi píše ve vědecké literatuře. Nakonec jsem článku v New York Times musel dát za pravdu.
Mám v živé paměti přednášku, kterou přednesl před mnoha lety v Liblicích Harold Urey o vzniku aminokyselin v laboratorních podmínkách, napodobujících podmínky předpokládané na Zemi v době, kdy pravděpodobně vznikl život. Přednáška byla jednoduchá a elegantní a dávala tušit, že během několika málo desetiletí budou problémy vzniku života vědecky zcela objasněny. Experimenty Ureyho studenta Stanley Millera vycházely z předpokladu, že v době vzniku života existovala na Zemi silně redukční atmosféra (Miller 1953, Ring et al. 1972, Wolman et al. 1972), Literatura z pozdější doby však nasvědčuje tomu, že prebiotická atmosféra nebyla silně redukční, jak vyžadují experimenty zaměřené na prebiotickou syntézu stavebních kamenů bílkovin a nukleových kyselin, a že obsahovala kyslík (Florkin 1975, Lumsden a Hall 1975, To we 1978, 1996, Carver 1981,
E. PALEČEK
Wocsc, CR. 2002. - Proc. Natl. Acad. Sei.                  Wolman, Y„ Haverland, W.J., Miller, Si.
USA 99: 8742.                                                                1972. - Proc. Nat. Acad. Sei. USA 69:
809.
E. Paleček (Institute of Biophysics, Academy of Sciences of the Czech Republic, Brno, Czech Republic) Problems of life origin on the Karth
There are three popular hypotheses attempting to explain the origin of prcbiotic nucleic acid building blocks, i.e. (a) synthesis in a reducing atmosphere, (b) input in meteorites and (c) synthesis on surfaces of metal sulfides in deep sea vents. At present it is hard to say whether any of these hypotheses is correct. It is particularly difficult to imagine the prebiotic synthesis of cytosinc based on the known chemistry; similarly the prcbiotic synthesis of pyrimidine nucleosides and nucleotides represent unsolved problems. The progress in RNA chemistry and elucidation of their catalytic functions offer an interesting system that might play an important role in the origin of life but it appears highly impro-bable that such a complicated molecule as RNA could have appeared de novo on the primitive Earth. Unfortunately, it is unclear whether the RNA world was preceded by some simpler world. Darwin's idea that all living species have a single cell common ancestor is questionable. Recently Woese has suggested that the universal ancestor was probably not a single-celled organism but a commune - a loosely built conglomerate of diverse cells in which the horizontal transfer of genes played a critical role. New important discoveries are necessary for better understanding of the origin of life on Earth.
(i)=<0)==
Abiotic synthesis of small organic molecules. Miller, a graduate student who was working with Harold Urey, began the modern era in the study of the origin of life at a time when most people believed that the atmosphere of the early earth was strongly reducing. Miller6 subjected a mixture of methane, ammonia and hydrogen to an electric discharge and led the products into liquid water. He showed that a substantia! percentage of the carbon in the gas mixture was incorporated into a relatively small group of simple organic molecules and that several of the naturally occurring amino acids were prominent among these products. This was a surprising result; organic chemists would have expected a much-less-tractable product mixture. The Urey-Miller experiments were widely accepted as a model of prebiotic synthesis of amino acids by the action of lightning.

PROBLEMS OF LIFE ORIGINS
S. Miller and H. Urey subjected mixture of methane, ammonia and hydrogen to an electric discharge and led the product into water ...
The Miller-Urey experiment attempted to recreate the chemical conditions of the primitive Earth in the laboratory, and synthesized some of the building blocks of life
but geologists showed that prebiotic atmosphere was not strongly reducing and not oxygen-free, dif f erring from that expected by Miller and Urey
f ň*«-. Ntitl> A'Vtí. Sťi. USA V.iĽ*. pp. 4í9fi-4-MH. April IWÍ IMtKhŕnwiry
Prebiotic cytosine synthesis: A critical analysis and implications for the origin of life
Runt- k t Shapiro*
Dtp-rime m uf (Twmmrir, Sam Y*h1l Uarwuty, J«) W«»hiiijfon S*nuie Ecu. Ne* Twrh. NY MAM
Owwmufiiiriir«/ 6y *.«*«■ i>S*t J*# &» InttituU for Bioioffral Studio. Jton /Wrgui Q«. /flnmťy 331 1999 (n<eir«l for reritw November í 9. í9Wí>
ABSTRACT A Dumber of theories propos« ihm RNA, or an KNA-llkc snbitnnc*, played a rak In «he origin of life.
Usually, smh hvpmhcMs prtiunt lhal lbe Watson-Crick hiisťs were readily available an prtrblotlc t-.arth. for spon In-Drous Incorporation Into a replicator, tľy Losině, homrvtr, hu> mil bi-cn reported In analyse* of mil mr ito n»r is it among tbe products or electric spark dl-wharfr; experiments. The reported prebiotic syntheses of cytosine involve [be reaction of cyanoacclyltnc <or its hydrolysis product, cynoonceialde-hjdv>. *llh cyanale, cyanogen, or ar*u- these substance* undergo side reactions with common nucleophllrs that appear 10 proceed more rapidly than cytosine formation. To favor cyloslne formation, reactani concent rations are required that ore Implausible In a natural setting, Hirlhcrmore, c> losine is consumed by deaminalion (lb* bulMifc far dramlnoltou at 25"t b -J40 yr) and olher reactions No reactions have been described thus far thai would produce cytosine, even in a specialized local Ntfjng, at a rate sufficient to compensate Tor its decomposition. On the basis oí this evidence, it appears quite unlikely that cytosine played a role In the origin of lift-. Theories that involve replicators that function without lbe Watson-Crkk pair», or no replicator at all, remain as viable ■ He mat Ives.
Cytosine synthesis would not be possible even strongly in reducing prebiotic atmosphere.
Similar problems arise with the abiotic m synthesis of nucleotides
Abiotic synthesis of a complicated molecule such as RNA is highly improbabli
BREAKING NETWORK LOGJAMS  ■  TRULY 3-D IMAGES
SCIENTIFIC AMERICAN
, .   E 270?
WWW.SCIAM.CI3M
\
Bring
'
Back   \ America's Prehistoric Beasts


h?/
f/fri
A m ¥ #
Did thi
is moiecu e
FORGET DNA AND RNA, MAYBE IT ALL BEGAN WITH SOMETHING
MUCH SIMPLER
BY ROBERT SHAPIRO
The sudden appearance of a large self-copying molecule such
as RNA was exceedingly improbable. Energy-driven networks
of small molecules afford better odds as the initiators of life
NOBEL lareate Christian de Ďuve has called for "a rejection of improbablities so incomensurably high that they only can be called miracles, phenomena that fall outside the scope of scientific inquiry". DNA, RNA and PROTEINS must then be set aside as participants in the origin of life.
f n ir\  ŕ\ f I
viwvv/i^fiuiff Lil   i_/Yr?

■ Theories of how iife first originated from nonliving matter fall into two broad Classes^replicatorfirst, in which a large molecule capable of replicating (such as RNA] formed by chance, and metabolism first, in which small molecules formed an evolving network of reactions driven by an energy source.
■  Replicator-first theorists must explain how such a complicated molecule could have formed before the process of evolution was underway.
m Metabolism-first proponents must show that reaction networks capable of growing and evolving could have formed when the earth was young.
REPLICATOR VS. METABOLISM
Scientific theories of the origin of life largely fall into two rival camps: replicators and metabo ,sm first. Both models must štart from molecules formed bu nonbiólogi-cal chemical processes, represented here by balls labeled with symbols [í]
In the rephcator-first model, some of these compounds join together in a chain S Srvľrm'n,8 3 m0,ec,Jle-PerhaPS seme kind of RNA-capable of reproducing itself [2] The molecule makes many copies of itself [3], sometimes forming mutant versions that are also capable of replicating [4). Mutant replicators that are better
adaptedtothecondrtionss.pplantearlierversionslSJ.Eventuallythis evolutionary process mustlead to the development of compartments (like cells] and metabolism m which smallermolecules use energy to perform useful processes [S]
Metabolism first starts.offwith the spontaneous formation of compartments \?\ Some compartments contain mixtures of the starting compounds that undergo cycles of reactions (S], which overtime become more complicated (9] Finally the system must make the leap to storing information in polymers [JO]
REPLICATOR FIRST
Replicator
molecule          ť^SUii-
2 Chance /
METABOLISM FIRST
Compartments
r Chance
«       I
3 Replication
4 Mutation I
Reaction cycles   8   Selection
1                   V,-.
5 Selection
9 More complex
b Compartments Form, and metabolism develops
N
10 Information is stored ^------in polymers
Metabolism
FIVE REQUIREMENTS FOR METABOLISM FIRST
At least five processes must occurforsmall molecules« achieve a kind of life—here defined asthe creation of greater order in localized regions by chemical cycles driven by an energy flow. First, something must create a boundary to separate the living region from the nonliving environment [1). A source of energy must be available, here depicted as a mineral [blue] undergoing a heat-producing reaction [2], The released energy
must drive a chemical reaction [3].A network of chemical reactions must form and increase in complexity to permit adaptation and evolution (4j. Finally, the network of reactions must draw material into itself fasterthan it loses material, and the compartments must reproduce [5]. Wo information-storing molecule [such as RNAor DN A] is required; heredity is stored in the identity and concentration of the compounds in the network.
'•-•>
•    •
42
What Readers Want to Know
In Scientific American's Hog, Robert Shapiro answered questions raised by readers of the Web version of this article. An edited selection follows.
Does the metabolism-first hypothesis point to a single origin or multiple independent origins of life?-jr
A: Multiple origins seem more viable with the metabolism-first scenario Gerald Feinbergand I discussedthe possibility of alien life (life notbased on RNA DNAand other biochemistry familiar to us} in our 1980 book, Life beyond Earth. Researchers at a conference hosted by Paul Davies at Arizona State University in December 20Q6 concluded that alien life may even exist, undetected, on this planet. The great majority of microorganisms that can be observed under a microscope cannot be grown in conventional culture media and remain uncharacterized. Alien microbes may also exist in habitats on the earth that are too extreme for even the hardiest forms of our familiar fife.
Why do we have to demonstrate metabolism first in a reaction vessel? Can't we simulate it in software ? -Dave Evanoff
A: Stuart Kauffman, Doron Lancet and others have used computersimulations to illustrate the feasibility of self-sustainingreaction cycles. Such simulations have not specified the exact chemical mixtures and reaction conditions needed to establish self-sustaining chemical networks. We do not yet knowall the reaction pathways open to mixtures of simple organic compounds, let alonetheir thermodynamic constants. Ever, if such data were available, most chemists would not be convinced bu a computer simulation but would demand an experimental demonstration.
The fact that all biological molecules are of one handedness needs some explanation. -John Holt
A: If the m íneraí transformation that powered the reaction cycle I discuss in my article were selective foronly one mirror-image form of chemical A, then the product B and other members of the cycle might also occur in only one mirror-image form Control of handedness, orchirality, becomes crucial when small chiral molecules are linked togetherto form larger ones. A modern enzyme may contain 100 linked amino acids, all of the same handedness (so-called L-amino acids), if a D-amino acid were substituted for its mirror-image L-form at a sensitive site within the enzyme then the enzyme's shape would change and its function might be lost.
An RNA-First Researcher Replies
Steven A. Benner of the Westheimer Institute for Science and Technology in Gainesville, Fla., argues thüt RNA-first models are alive and well.
Even as some declare that the RNA-first model of life's origin is dead because RNA arising spontaneously is fantastically improbable, research is lendingsupport to the model.
Let me first acknowledge that most organic molecules when hit with energy (such as lightning or heat from volcanoes) become something resembling asphalt, more suitable for paving roads than sparking life. But metabolism-first models, to the extentthatthey have been supported with arty real chemicals, must also deal with this paradox: molecules reactive enough to participate in metabolism are also reactive enough to decompose. There are no easy solutions.
Like many others, my research group has returned to the scientific imperative; actually do laboratory research to learn about how RNAmight have arisen on the earth.
The sugar ribose, the "R" in RNA, provides an object lesson in how a problem declared "unsolvabie" may instead merely be"not yet solved." Ribose long remained "impossible" to make by prebiotic synthesis (reactions among mixtures of molecules that could plausibly have existed ona prebiotic earth) because it contains a carbonyl group—a carbon atom twice bonded to an oxygen atom. The carbonyl group confers both good reactivity (the ability to participate in metabolism] and bad reactivity [the ability to form asphalt). AdecadeagoStanley L. Millerconciudedthatthe instability of ribose stemming from its carbony] group "preclude(s) the use of ribose and other sugars as prebiotic reagents.,.. It follows that ribose and other sugars were not components of the first genetic material."
But prebiotic soups need soup bowls made of appropriate minerals, not Pyrex beakers. One attractive "bowl" is found today in Death Valley. In a primordial Death Valley, the environment was alternately wet and dry, rich in organic molecules from planetary accretion and (most important) full of minerals containing boron. Why care about boron? Because boron stabilizes carbohydrates such as ribose. Further, if borate (an oxide of boron) and organic compounds abundant in meteorites are mixed and hit with lightning, good quantities of ribose are formed from formaldehyde and the ribose does not decompose.
The fact that such a simple solution can be found for a problem declared"unsolv-able" does not mean that the first form of life definitely used RNA to do genetics. Sut it should give us pause when advised to discard avenues of research simply because some of their problematic pieces have not yet been solved.
EVOLUTION OFCHEMICAL NETWORKS
The metabolism-first hypothesis requires the formation of a network of chemical reactions that increases in complexity and adapts to changes in the environment.
CYCLE FORMATION: An energy source (heretheso-called redox reaction converting mineral X to mineral Y] couplesto a reaction that convertsthe organic molecule Ato molecule B. FurtherreactionsfBtoC, Cto D....)form a cycle back to A. Reactions involving molecular species outside the cycle (E) will tend to draw more material intothe cycle.
INCREASING COMPLEXITY: If a change in conditions inhibits a reaction in the cycle (for example, C to D], then other pathscan be explored. Herea bypass has been found by which C is converted to Othrough intermediates F, G and H. Anothersolution would be the incorporation into the reaction network of a catalyst [I) whose action [dot ted tine] unblocks the C to 0 transformation. Tosurvive,the evolvingnetworkmustdrawin carbon-containing materials from the environment more rapidly than it loses them by diffusion and side reactions.such as the formation of tars that settle out ofthe solution.
CYCLE FORMATION
INCREASING COMPLEXITY
Redox reaction
Driver reaction
Redox       ^	.*_/	
reaction JĚ	"^Hea	t
;>^		
Driver	(7)	
reaction		^          Catalyst !"■-•.   action
Mineral
Organic molecule
Chemical
Carbon supply
in thfi fWrrWimff PVarnnlŕl their -arr" r-ŕin-       rrpotp  in   nninfitv   Tňi»  i-pirt-zír umní
Or did life come from another world?
The hypothesis of F. Crick is discussed in November issue of Scientific American 2005.
It is concluded that microorganism could have survived a journey from Mars to Earth
44
DNA ĎENATURATION and RENATURATION/HYBRIĎIZATION
Native DNA AR
melti
A260
D r
f
premelting   /
-A-------------B--------
^
quick cooling
melted DNA D
melti
%
slow cooling renaturation
quick cooling
denatured DNA     RENATURED DNA
Temperature
STRAND SEPARATION AND SPECIFIC RECOMBINATION IN DEOXYRIBONUCLEIC ACIDS: BIOLOGICAL STUDIES
By J. Marmur and D. Lane
CONANT LABORATORY,  DEPARTMENT C1F CHEMISTRY,  HARVARD  INIVERSITY
Communicated by Paul Doty, February 8$, 1960
It is clear that the correlation between the structure of deoxyribonucleic acid (DNA) and its function as a genetic determinant could be greatly increased if a means could be found of separating and reforming the two complementary strand«. In this and the succeeding paper1 some success along these lines is reported. This paper will deal with the evidence provided by employing the transforming activity of DNA from Diplococcus pneumoniae while the succeeding paper1 will summarize physical chemical evidence for strand separation and reunion.
158 results found (Set #1} Records 1 -- 10    show ío wont J_'
Go to Page:   i
of 16    so
[ J I 2 13 I á I 5 I & I Z lfl I S I 111]^   ►► ►
Lise the checkboxes to select records for output. See the stdebar for options
n  i
MARMURJ
PROCEDURE FOR ISOLATION OF DEOXYRIBONUCLFIC
ACID FROM MICRO-ORGANISMS
JOURNAL OF MOLECULAR BIOLOGY 3 (2): 208& 1961
Times Cited: q?34
2.     MARMURJ, DOTY P
DETERMINATION OF BASE COMPOSITION OF DEOXYRIBONUCLEIC ACID FROM [TS THERMAL DENATURATIQN TEMPERATURE
JOURNAL OF MOLECULAR BIOLOGY 5 fl): 109& 1962 Times Cited: 3210
Q   3,     SCHILDKRAUT CL, DOTY P, MARMUR J
DETĚRMINATION OF BASE COMPOSITION OF DEOXYRIBONUCLEIC ACID FROM ITS BUOYANT DFNSÍTY IN CSCl
JOURNAL OF MOLECULAR BIOLOGY 4 (5): 430& 1962 Times Cited: 1619
_   4.      MARMUR J, DOTY P
HFTFROGFNF1TY IN DEOXYRIBONUCLEIC ACID?; .1
DEPENDENCE ON COMPOSITION OF THE
CONFIGURATIONAL STABILITY OF DEOXYRIBONUCLEIC
ACIDS
NATURE 183 {4673); 1427-1429 1959
Times Cited: 4J>Z
WÍW Hill TFřT
9.     MARMURJ, LANE D
STRAND SEPARATION AND SPECIFIC RECOMBINATION IN DEOXYRIB-QNUClFrC ACIDS - BIOLOGICAL
STUDIES
PROCEEDINGS OF THE NATIONAL ACADEMY OF
SCIENCES OF THE UNITED STATES OF AMERICA 46
(4): 453-461 1960
Times Cited: 246
, , r -, .-...JdniluWltÍLilJT'. lulu, |fUI III.lvliW?)ltKM*nV»l Will —IHMtt
Sort by:
1 imri Cited
50HT
■
Analyze Results:
=   ANALYZE
,s of the authors, journals, etc. for these rect
Citation Report:
JU   ;ITATK)H RÍPCITT
Vrew detailed citation CC-
and Erte ti-inde* value for tne -e-s^cir.
Output Records; •  Selected records an page Q *M records on paqc i.j Records
K»
BiblioqTjptiir Fifldl
1
& PRINT        H J MAU      Q Í*Vt ĹXPÚRT 10 REfERÍNCL SOFTWARE.
iAvt to Mľ E ndN nt P: Web      i,
I Sinn m tú ar.r.fiss FndNote Webl
Or add them to che Marked List
for later output and mgre potions.
ADD TO M AS It f Dl IM
[0 articles marked]
Pifl* 1 of 2
Microbiologist, biochemist and molecular biologist
Julius Marmur- discovered renaturation of DNA
22 March, 1926 Bialystok (Poland) - 20 May, 1996 New York, NY
Oswald   Avery    1944 - DNA is a genetic material
(Rockefeller Institute, New York, NY)
Rollin Ď. Hotchkiss
Julius Marmur
1993
The double helix: a personal view
Francis Crick
Medical IWarrh Counril Laboratory for Molecular Biology.  Hula Rond, Cambridge, UK
Nothing was said about the possibility that the two chains might be melted apart and then annealed together again, correctly lined up. The discovery of this by Marmur and Doty has provided one of the essential tools of molecular biology. I can still remember the excitement I felt when Paul Doty told me about it at breakfast one day in New York in  a hotel  overlooking Central  Park. ^^£ÉĎ^^9
DNA electrochemistry
DNA and RNA are Electroactive Species
producing faradaic and other signals on interaction with electrodes
Cytosine (C)
Adenine (A)    A, C, & are reduced at MERCURY electrodes
Guanine {&)   reduction product of guanine is oxidized back to &
All bases {A, C, G, T, U) yield sparingly soluble compounds with the mercury and can be
determined at concentration down to 10_11M.
Solid amalgam electrodes can be used instead of the mercury drop electrodes.
A and G as well as C and T are oxidized at CARBON electrodes
PEPTIDE NUCLEIC ACID (PNA) BEHAVES SIMILARLY TO DNA AND RNA Microliter volumes of the analyte are sufficient for analysis
Electroactive Labels can be Introduced in DNA
Progress in genomics affects electroanalysis
Many areas of science are influenced by the fast development of the genomics and by the success of the Human Genome Project.
Classical sequencing of individual human genomes with 3xl09 base pairs is too difficult.
Sequencing by DNA hybridization is gaining importance
Relatively expensive DNA hybridization ARRAYS with optical detection are currently applied in research-krbs It is believed that electrochemistry can complement the optical detection providing new LESS EXPENSIVE hybridization detection for decentralized DNA analysis in many areas of practical life
LOW   DENSITY CHIPS
DNA isolation	PCR	electrochemical detection
LAB-ON-A-CHIP
600
i
50 years of nucleic acid electrochemistry
5twiiHir>HirK At;*
~ 1958: Nucleic acid bases, DNA and RNA are electroactive
500-
Mr«      ...part of the guanine ring important for the anodic signal is near to the surface whereas the the analogous part of cytosine is hidden inside the DNA double helix
nnrtirinntinn in thť». hvrlrnnp.n hnnrlinri        (showing a Cathodic Signal in SSĎNA
m
c o
s
400-
i um
""  '     ■ ■ !l ■ . I iliifm I-4JJ Ihre
"ľ     ......I"    !'■■    - II
MUM.    J17.*,
*J* 11-M II ,„,l X gvn-huNh*! i r,,[,(,.(, i, | r, Mtl. ■Í     *fff*rllÍFFUPTm|      fir.....I
■klliulji.....trhmrJll i I,.
""■«r II     Wh  I.,i......  -
wUmJKlin,      i-.l ....„..., -,
Kbm   ScfUan ..n.lk-.lnh
Wb   I...!, J*U   m.ni  ^||t  r|1|,r   |Umi]i
JHTlllnjv.LtHL^ifJtlKd Jlirľl ľ™      ^""1.      W*«J      Bl^r,
VVI  IVI   VU^      I  I  IV     I  I  IV    Ml IMIVSy4VSU*J     LSMI     I      VS  I      WT   I
[participating in the hydrogen bonding. %but not in dsDNA) E. Paleček, Nature 188 (1960) 656-657
300-
0)
E
200-
100-
«■i™,,, ,]..„vi.y,i,],li,. ,„.„]  ,.....llf]  hi „. ,|lrl|(.,:u|i.
is inactive. TliiH phonumrnon. nmv tjo perhaps interpreted in the following mannen   one part   nf the
£»«""■'■'"■.....ring, which isofgr.......nportanoe fbi
t Me l,.rinnt um nf the anodic indentation (apparently an .immo.Srci„p ,„ position 1 ie involved'), is found m the molecule neur thf> surf«», tt]„,reas the „naJoc-oua pari »f the pyrimidine rint; of cviosino í «»par-anthr the O.am,no-gro.,|.>) is hidden inside the iiinlcCTlL. where it participate» in the Formation of "»hyfogen bond, l„ aparmic „,id. where the i nuble.fiehcaJ etructurc is vomplelelv destroyed, (leoxycytulyJie acid ú not blocked itericallj in «re way so [hut   its oacfflc-polsrnfcrnphi^ activity  irm-v
muoift-r .t,.,.!!.   F».....leonyadenylic acid and deoxy.
"'■..... ■■......."'    '"'>    ""I    »how    the    ehimicterinli,.
Mentation in ammonium formate as medium, and conseniif-nl ly no indentations due to these nucleotides »n be -,|,„-,„] if [lK.y ,in, ,,„,„„, ;„ |hr. |n„|     , ol deoxynln)iiiii'lL-k- ncid.
t hav,,  aba  -r,r.....ti  deoxyribonucleic acid   and
Si!"""','........l -M Slldi,,m chloride aa medium
roth suhetanoa. yMJd oscillogram* that differ only
-li^hi K.   In 1 .1/ iodiam hydroxide, tl.....«rill.....ami
or deoxynbonuoleio   ncid   differ  eonaiderably   from Uu» ol apurmic acid.
|" '■"''"li nti.i nirtiri wiluti,,,«', proteine aive an »dentation even at .1 vory low concentration'. Since tne deoxyribonucleic „cid indentation is produced this medium nt a different potent isj, 1 vra, abk to use (he oseillopoliiroin-nphic reaction For iMectin« 'itswyribrnnielei.: aeid and proteins in the presence ľf I ™n other. In this manner, protein« can be detected J»«n fa the presenoe of an abundance ofdeoxvribo nucleic acid (Fip, 4).
Emu. Pasbokx        .1—
J
in—r~if—tr—ip-^r^i^-in
1960
1970
1980
1990
2000
year
E. Paleček, Fifty years of nucleic acid electrochemistry, Electroanalysis 2009, in press
The results of the DNA electrochemistry studies and development of the electrochemical DNA hybridization sensors in the last decade suggest that these sensors can complement DNA sensors with optical detection
How and when the DNA electrochemistry begun?
OSCILLOGRAPHIC POLAROGRAPHY
At controlled alternating current (constant current chronopotentiometry)
dE/dt
dsDNA
ssDNA cathodic                CA
paringly soluble :ompounds with Hg
anodic
H-E
LITERATURE in 1958: Adenine is polarographically reducible at strongly acid pH
while other NA bases as well as DNA are inactive
J.N.Davidson and E.Chargraff: The Nucleic Acids, Vol. 1, Academic Press, New York 1955
Paleček E.: Oszillographiche Polarographie der Nucleinsauren und ihrer Bestandteile; Naturwiss. 45 (1958), 186 Paleček E.: Oscillographic polarography of highly polymerized deoxyribonucleic acid; Nature 188 (1960), 656
Firsts in Electrochemistry of Nucleic Acids during the initial three decades
1958 DNA and RNA and all free bases are electrotractive
1960-61 assignment of DNA electrochemical signals to bases, relation between the DNA structure
and electrochemical responses
1961 adsorption (ac impedance) studies of ĎNA (IR Miller, Rehovot)
1962-66 DNA premelting, denaturation, renaturation/hybridization detected electrochemically,
traces of single stranded DNA determined in native dsĎNA. Nucleotide sequence affects dsĎNA
responses
1965  Association of bases at the electrode surface (V. Vetterl)
1966 application of pulse polarography to DNA studies
1967 detection of DNA damage
1967-68 Weak interactions of low m.w. compounds with DNA (P.J. Hilsson, M.J. Simons, Harrow, UK
and H. Berg, Jena)
1974 DNA is unwound at the electrode surface under certain conditions (EP and H.W. Nürnberg,
Jülich, independently)
1976 Evidence for polymorphy of the DNA double-helical structure
For two decades only mercury electrodes were used in NA electrochemistry
1978 Solid (carbon) electrodes introduced in nucleic acid research (V. Brabec and G. Ďryhurst,
Norman)
1980 Determination of bases at nanomolar concentrations by cathodic stripping
1981-83 Electroactive markers covalently bound to DNA
1986-88 ĎNA-modif ied electrodes
Results obtained at: IBP, Brno or elsewhere (author's name is given); the results which have been utilized in the DNA sensor development are in blue
Electrochemical sensors/detectors for DNA hybridization
Single-Surface Technologies:
PROBE
CCTTATCCTCCAAT
TARGET  :      CC.AaTACCACCTTA (COM PLE M E ISTÁ R Y)
CCTTATCCTCCAAT
CCTTATCCTCCAAT
DUPLEX FORMATION
PROBE : TARGET
■*■   NO   DUPLEX    {HYBRID NÍXT FORMED}
ACAATACCATTCC { NON-COM PLE M E NT A R Y >
DNA IIVBRIDIZATION  AS DELECTRDľllĽMItľAL DETECTTOrt ATTHE SAME SIRTACĽ
ľ i; ■ K
ttfget DNA
REDCX INDICATCR
COVALENTLY BOUND LABEL ENZYME, etc.
LUIIIKIM   HkCJl't.kJ'lt.SUl l>YV Ifl I'l.lAhv SlSiil.K M'kAMl

Au
J K Barton =1
Surface-attached molecular beacons
liluim-
0|HkiJ
H-
AiJ______j    .i
í
\
-.£*
™%
i  \
J V
\
JjF
A Heeger
In the last decade nucleic acid electrochemistry was oriented predominantly to DNA sensors for (a) DNA hybridization and (b) DNA damage.
This trend has been accompanied not only by interesting discoveries but also by a number of poor papers lacking the necessary control experiments,claiming sequence detection without PCR amplification but using synthetic oligos as target DNA, etc.
Electrochemical sensors for DNA hybridization
At present both single- and double-surface techniques can be used for DNA sequencing of longer oligonucleotides and PCR products.
Electrochemical detection of point mutations is also possible.
Optimization of the procedures are now necessary to develop commercially successful devices.
Challenges:
1) Sequencing eukaryotic DNA without amplification (by PCR). Great sensitivity and specificity of the analysis is required
2)   Development of electrochemical sensors for DNA-protein protein-protein interactions for proteomics and biomedicine
Science in Czechoslovakia after the Und World War
After February 1948 life in Czechoslovakia was increasingly affected by the Stalinist ideology and heavily controlled by the Party and Government.
Many scientists and scholars were fired from Universities but some of them got employment in the Institutes of the Czechoslovak Academy of Sciences established in 1952. This was possible particularly at the Institutes whose Directors were influential Party members but serious scientists.
PRAHA/PRAGUE
institute of Organic Chemistry and Biochemistry/
Director: F. Sorm
Chemistry and Biochemistry of Proteins and Nucleic Acids
B. Keil, B. Meloun, O. Mikes, J. Doskočil, D. Grunberger, A. Holy, I. Rychlík, J. Riman, J. Sponar, V. Paces, Z. Sormová, S. Zadrazil
For many years Czech scientists were efficiently isolated from the West In this respect the situation in Brno was much worse than in Prague
Institute of Biophysics, Brno
Director: F. Hercik
Founded in 1955 for radiobiological research it gradually turned into an institute devoted mainly to DNA
For a long time we received 50 -100 US $ for materials/chemicals per year and Department. The orders of materials from the West had to be planned 1-2 years ahead
Taking part in meetings in western countries was difficult not only because of currency problems
OSCILLOGRAPHIC POLAROGRAPHY
At controlled alternating current (constant current chronopotentiometry)
dE/dt
dsDNA
ssDNA cathodic                CA
paringly soluble :ompounds with Hg
anodic
H-E
LITERATURE in 1958: Adenine is polarographically reducible at strongly acid pH
while other NA bases as well as DNA are inactive
J.N.Davidson and E.Chargraff: The Nucleic Acids, Vol. 1, Academic Press, New York 1955
Paleček E.: Oszillographiche Polarographie der Nucleinsauren und ihrer Bestandteile; Naturwiss. 45 (1958), 186 Paleček E.: Oscillographic polarography of highly polymerized deoxyribonucleic acid; Nature 188 (1960), 656
J. Heyrovsky   invented
POLAROGRAPHY in 1922. After 37 years he was awarded a Nobel Prize
J Heyrovsky S Ochoa   A Kornberg
In difference to most of the electrochemists I met in the 1960's and 1970's, J Heyrovsky was interested in nucleic acids and he greatly stimulated my Polarographie studies of DNA
res 1959
-   .   i—                              • ■
-. -u-----------------
"vr" ■ i
.      :
■

I
-
-■

5
i ig. 1. de polarogiams of native and denatured calf thymus DNA" (a) native DNA at a concentratmn of 500 „g/ml in 0.5.1/ ammonium formate with 0.1,1/ sodium phosphate (pH 7.0)j 6) denatured DNA at a concentration of 500 «/ml in 0.5A/ ammonium formate with 0.0/ sodium phosphate CpH 7.0).    DNA was denatured by heat at the
Tn nlrl™ °Lf6 "g/ml ÍU ()M7M NaCI with °-7 mM citrftte.   Both curves start at U.U V, 100 mV/scale umt, capillary I, saturated calomel electrode.
DNA molecules
A. GENOMIC (chromosomal)
molecularis  pglyjiisnerse. nucleotide sequence unknown
B.    PLASMID OR VIRAL monodisperse, nucleotide seauencft known ret                sc                           oc
Nn
ds
ss
0
t
usually 3-4 kb mw ca2xi06    *
PCR VRo^i/cTs
C.   BIOSYNTHETIC   POLYNUCLEOTIDES
polydisperse, simple repeated sequence motifs or homopolymers
ATATATATATATATATATATAT                     AAAAAAAAAAAAAAAAAAAAA
dS  TATATATATATATATATATATA                    I I I I H I I H M I ITTIII I I I Ml
ss    CCCCCCCCCCCCCCCCCCCCCCCC    average mw 10 5 10 6
D.   SYNTHETIC   OLIGONUCLEOTIDES monodisperse,    programmed nucleotide sequence chemically modified bases and back bona possible
GCGCATTTCCGG ss
CGCGATATCGCG
CGCGTATAGCGC and                 ds
usual lengths 10-20 nucleotides
In 1960 when I published my NATURE paper
on electrochemistry of DNA I obtained invitations
from 3 emminent US scientists:
J. Marmur - Harvard Univ.
L. Grossman - Brandeis Univ.
J. Fresco - Princeton Univ.
To work in their laboratories as a postdoc
In 1960 new techniques were sought to study DNA Denaturation and Renaturation. To those working with DNA Oscillographic Polarography (OP) appeared as a very attractive tool. Invented by J. Heyrovsky, it was fast and simple, showing large differences between the signals of native and denatured DNA. The instrument for OP was produced only in Czechoslovakia.
I accepted the invitation by Julius Marmur but for more than two years I was not allowed to leave Czechoslovakia. In the meantime JM moved from Harvard to Brandeis Univ. By the end of November 1962 I finally got my exit visa and with Heyrovsky Letter of Reccommendation in my pocket I went to the plane just 24 hours before expiration of my US visa. Before my departure I sent my OP instrument by air to Boston. It arrived after 9 months completely broken. I nstead of OP I had to use ultracentrifuges and microbiological methods.
Julius Marmur discovered DNA Renaturation/Hybridization and proposed (in JMB) a new method of DNA isolation which was widely applied. His paper was quoted > 9000x.
J M at the 40th Anniversary of the Discovery of the DNA Double Helix
Rrprinlrii Trom Caiv rtrmrfn Hjiuoit Sr**wcA un QrA^nTATTVi BioLoor
Vokna XXVIII. IMS
/Vim«* im VJIJt.
Specificity of the Complementary RIVA Formed by Bacillus sub t His Infected with Bacteriophage SP8
At the end of my stay at Brandeis I did some OP experiments which I finished in Brno nd published in J. Mol. Biol, in 1965 and 1966.
J. Mabklr", C. If. GASCxarAN, E. Paucckk, F. M, Kahaj*+h J. LfcYisB. ind M. Hammel*
On+lwtt  fbp>trtmr*t vf fíiurhpm.i*ryt Bmwt*Í* Ľiifrrrntf,  IVnttiuim. Wt*~ŕŕÁu*ŕíl*
INCREASING peak II   CI-2,
ÍNCREASINÔ\ peak II and III
CI-2    .
only peak III CI-2
peak II no CI-2
Native DNA A, B
C
melted DNA D
H   melti
r^
quick cooling
quick cooling
slow cooling renaturation
denatured DNA     RENATURED DNA
Temperature
peak II no CI-2
only peak III CI-2
peak II no CI-2
DNA Premelting and Polymorphy of the DNA Double Helix
Li Í976
Reprinted trom;
FTOCtESS I« hUCUlC ACID  HfiEAJKH
■*?-G  «OLÍCULAI  B.C!.:: jv    v'J:    1|
■*ŕ IÍÍ4
*OÚÉMJC F««, |Mf
Before my departure to the US I observed Changes in the Polarographie behavior of DNA far below the denaturation temperature. These changes were later called DNA Premelting
J. Mol. Biol.
20 (1966) 263-281
ľ.      ľli.Kl   Y   Y,
B. siiblilis and B. brevis DNAs have the same G+C content and different nucleotide sequence
B. subtilis
Premefring Changes in DNA C<?n form oř ion
"■i    ]'M ľ.'l-v;
h	i
ľ'	B. brevis               ffl
1	^sjf\/
1l0 !	\^J
"	
Tcitpe'Otufe [*C)
FlQ. 12. TVtfrm*] tmuitiim of DN'A'i i»]atDl from b»ctwi» of Um gwiu» Bazill-u. DNA At k eonc«itr*Li™ cí IM ji^ffJn ín Í-Sí if-unjttüiiUiiu farení* pliu fl^íll ii-KidJiim pkr*phdLi+ (pH
— g-----g—, S- íubrili* lí*; — X^—M^ Jt- «níto; — C^—O-r 5. tubfüw m »iff:
—ů-----£—. B. «AtiTii v*r. BhTTimw; —D-----D—■ B- **wŕŕ (ATOC Í*H>.
F íí-í j>íiLáhix.'opri, dropping mercury aloclroa poliriied with repute] cj-tL** at A.C. Th* n'.«uu»m«il4 «rcn r-nri^l i^iii i= rl:> Ln!^.it*rorj'ůf Prof. J. Slirrňiir, D*p*rtriLtr.i t-ľ läiucliMüijiry. rEHt.dŕii Vniwríily. TCilÜism, J]*«., V.S.A.
POLAROGRAPHIC BEHAVIOR OF dsDNA At roomand premeltig temperaturse depended on DNA nucleotide SEQUENCE
A
6. PoLVMDBi'iEY of DNA Seco.sdahy Structufe
On the basis of the preceding discussion, a schematic picture of the structure of natural linear D\A in solution under physiological conditions (e.gr, ní 36 ZC. moderate ionic strength* and n H 7) can be down. We cm assume that the double-helical structure of the very long f A + T)-rich regions differs; from the structure- of thr mftjor part at the molecute and ťhat some of the (A -f-T)-rich segments are open (Fig. 20). An open ds-structure con be assumed in the region ní chain termini and/or in the vicinity of ss-breaks und other anomalies in the DNA primary structure. The eiract changes in the open ds-regions \vi\l depend on the nucleotide
sequence as well as on ihe chemicaE nature of the atiomnlv. Most of the
molecule will exhibit an "Y[j"°E Willfflirfifflli fi^J"? ""ft llfíl dťviatiúiu yivúu by UiĽ iiuckuLiuY- iuuucucfc. ETe^'atmg tiie- temperature in tlie premelting region í Fig. 20) is likelľ to lenid to the opening of other regions, and. eventualit, to expansion of the existing distorted ds-xegions and to further structural changes. Thus the course of the con^ formational changes as a function of temperature (premelting) will be determined by the distribution of the nucleotide sequences and anomalies in the primary structure, and mav have an almost continuous oh iractfir.
Consequently, even if we do not consider ''breathing," not only the architecture of a DMA double-helical molecule, but also its^ mechanics or dynamics can be taken into account.
To determine whether, e.g.., only the (A + T)'rich molecule ends will be open at a certain temperature Or also long A + T regions in the center of the molecule, further experimental research with better-defined samples of vinil and siynthetic nueleic aeids will be necessary. Further wort will undoubtedly provide new information on the details of the local arrangement of nucleotide residues in the double helix, as well us Qn DNA conformational motility. Thus a more accurate picture of DNA structure will emerge, whose characteristic feature will be poly-můrphy of the double helix, in contrast to the classical, highly regular DNA structure models.
Meeting F. Crick in Copenhagen and Arhus, 1977 (B. Clark)
December   Jj    197a
polydiA-'VMiA-T)
What the	people	said	
Sefore 1980			
No doubt that this electrochemistry must produce artifacts because we know well that the DNA double helix has i unique structure INDEPENDENT		After 1980 Is not it strange that such an obscure can recognize POLYMORPHY OF THE DNA DOUBLE HELIX?	technique
if the nucleotide SEQUENCE			
Professor   lU.il   Paltet); InAtLtute Of   biophytici CtfrchOtlOvak Acadeay of   Sci*nc*f Brno   It,   Kralovopotska   LIS
iľzť-hns Lov a It la
Dear   Professor   Patectk*
I do apologise   for   tftkinq   (0   Lonq   to   reply   to your letter   or   Septtwbtr   29   and   the   very   interesting   tvViaw ycu   lant with   it-      Mil for t und tel y   I   myself   will   not be aolt   CO attvnd   th* Symposiu* yon plan   for September,   197 7 And my   Cambridge COlLed?ua Aůťůri Klug   tells; bi«   that he too   is   unAbl*   to be present.      Had you COAtldtrtd   th* possibility of asking or.  Hank  Soball.?    H« has ju*t pub-:■...::■.-'.   In   iJ ::.■■-:   an   account of   (.hu   uí'híi    CtaM - pi i rtd)   kink and has   ideas   about presetting   eonformationa.      I   have no Uu whether   he vould b* able   to cone but should you with to   invitn hist his   address   Lti     Depaetotnt of   Ch*4niltryT Th* University of   Rochtstttn   ftivtŕ £tatiOnr   Rochastar, Ua-j York   I -; -.. J 7.
Voura   sincarely.
f. Mr C. Cťick
Pvrkauf Poundacion Visiting Professor
AĎSORPTIVE STRIPPING         ADSORPTIVE TRANSFER STRIPPING
NA is in the electrolytic cell and accumulates                 NA is attached to the electrode   NA is at the electrode but the
at the electrode surface during waiting                           from a small drop of solution      electrolytic cell contains only blank
(3-10 fl)                                   electrolyte
In 1986 we proposed Adsorptive Transfer Stripping Voltammetry (AdTSV) based on easy preparation of
DNA-modified electrodes
AdTSV has many advatages over conventional voltammetry of NAs:
1) Volumes of the analyte can be reduced to few microliters
2) NAs can be immobilized at the electrode surface from media not suitable for the voltammetric analysis
3) Low m.w. compounds (interfering with conventional electrochemical analysis of NAs) can be washed away
4) Interactions of NAs immobilized at the surface with proteins and other substances in solution and influence of the surface charge on NA properties and interactions can be studied, etc.
^ oi- o__rLi/   ^
^
H
V*
Bb
■1.5      -1.0      -0.5
DPP DUL
SWY HMDL
C.7SA
2ftnA

3 jiA
G™
I   ■
ň
!
í
2 s/V
-1.5      -1.0
1.4      -0.9       0.9       1-1J E (V)
-1.5      -1.0
1.4      -0.9 E (V)
0.9         1.1
z:
g
40
g     20
LU
d SRNA, SSC, 55 C d&RNA, SSC, 85 ^C dsRNA, 2.SxSSC, 85"C
peak MIR peak HR
-   T
20
—i— 40
60               80
TIME (MINUTES)
100
RENATURATION OF RNA AS DETECTED BY DPP Time dependence

120
Fig. 10, Time-course of renaturation of phage \1 dsRNA. (A) Thermally denatured ssRNA was incubated (•- *) at 85°C in 2.5 x sodium saline citrate {SSC) or (o o) at 85JC in SSC, and (x x) at 55X\ Samples were withdrawn in time intervals given in the graph and quickly cooled. DPP measurements were performed at room temperature at a RNA concentration of 3.2ug/mL in (UM ammonium formate with 0.2M sodium acetate, pH 5.6; PAR 174. (B) (o—o) peak IIR, (•—*) peak IHR. ssRNA (108 ug/mL) in 0.01 x SSC was heated for 6 min at 100~C, Then it was placed into a thcrniostated Polarographie vessel with the same volume of 0,6M ammonium formate with 0.2 M sodium phosphate, pH 7, preheated to 58ÜC. The pulse polarograms were measured at 5^C in times given in the graph. Southern-Harwell A 3100, amplifier sensitivity 1/8. Adapted from Paleček and Doskočil (1974). Copyright 1974, with permission from Academic Press,
IFFY stories
On this day 50 years ago, Watson and Crick published their double-helix theory. But, what  if...
By Steve Mirsky (2003)
"I am now astonished that I began work on the triple helix structure, rather than on the double helix," wrote Linus Pauling in the April 26,1974 issue of Nature.
In February 1953, Pauling proposed a triple helix structure for DNA in the Proceedings of the National Academy of Sciences (PNA5). He had been working with only a few blurry X-ray crystallographic images from the 1930s and one from 1947.
If history's helix had turned slightly differently, however, perhaps the following timeline might be more than mere musing...
August 15,1952: Linus Pauling (finally allowed to travel to England by a US State Department that thinks the words "chemist" and "communist" are too close for comfort) visits King's College London and sees Rosalind Franklin's X-ray crystallographs. He immediately rules out a triple helical structure for DNA and concentrates on determining the nature of what is undoubtedly a double helix.
February 1953: Pauling and Corey describes the DNA double helix structure in PNAS.....
Ä-l                             CIIEiíĽirStľ: FAľijyC- A AW LV&&Ľ          F'EC*. N. A
a rmixyShv ítuí-Ci ex k Km m k ?ti-ct^.sCACit)S
U* lílM'i F.'.Ľi.C^G 4NL> Hi^ikici  11, 0>ifi:v <'..'. t err, a<-i Ü-fcm.r.j^ I.A.nriR.AT\iRniH mi CcirutrsTnv,'1 Cjtuniitľix Ljecrmrju í y
TĽĽnSdľ.dliV
CroiöiDni=L[cd DcKmbcríi;, líl.K
liä                           UI&UISTRk': PA.SUKG ASW í-OREi'         ľjuOC. N. A. S.
VřlíLdi itc iijvůlvtd ill efltŕŕ IĹnĽi#ts. This distiMtuíiD of the úJiosphat* RTiütip ftoiti ti» fsffutir tíŕtraledral coDĚKuratiaD ia Jtot supported br direct estperiitisütal evidence; imfojtiuiatelr no precise structure djctmniimtijons have beai jiiuíů of anr phosphate di-estcra. Toe diEtoiTion, wbJŕti cor-reapojids to a larger amount of double bond cbarac-tcT for the inner oxygen atoniEttüm for the onrgic-n atoms involved in the c&tcr iuitages, is a reason-
HIhii nr lliŕ .»iiitHľ-l» sŕťl Miiijŕihir, *1iß»liig sí*wrnl niiŕ-fcľiiMŕ--Tť\-iť1i»í*>j.
Triple helix
with bases on the outside and sugar-phosphate backbone in the interior of the molecule
My IFFY story:
If L. PAULINO had in his lab an oscillopolarograph in 1952 he would never proposed this structure. Polarography clearly showed that bases must be hidden in the interior of native DNA molecule and become accessible when DNA is denatured
lUbCTlMX lEIlMK AL MI m H) D S RISftXiNI»! KMALLCIIANCiliSIX DXASIW I II *. AND DimWMIML TRACKS OK IMPĽUmiLH IK DNA SAMPULS

*^r
MERCľl'RY ELirTRODENAREPARTTfl'LARLY9HXMT1YE
»
/M\
ULTLHM1NA110N (>ľ TVACKK  (< H41 or
i
llLJXA
*\^IXA             ťHIJLIJhb
ťHIJLIJhS IX liMAKiJLMSOk- ill U*A
IKTÜMALATDILH    /   ťOVAľ i N T MOMHIW CiRIX)VILniNDI!RS
Probing of DNA structure with osmium tetroxide complexes
fco.
^SSSUHB****
£>£T£tTi0M tifillTS i
frfftppwc
/"V
fag/*»/

OsC> H g míÍh »f f U* DM rtMBhb
In the beginning of the 1980's Os,L complexes were the first electroactive labels covalently bound to DNA. These complexes produced catalytic signals at Hg electrodes allowing determination of DNA at subnanomolar concentrations
We developed methods of chemical probing of the DNA structure based on osmium tetroxide complexes (Os,L). Some of the Os,L complexes react with single-stranded DNA but not with the double-stranded B-DNA.
Critical ftrviěvs in Eiochtmiitry andMvlecular Biology, 2ů(í):]5l—2!S (1091)
Local Supercoll-Stabilized DNA Structures
E Pafŕčeŕí
MH-Phndi HM für Kafaiyakslsef» Chemie, GBKínflon, BHD vnt Instate o! Hopfiysia, Czechoslovak Aiadarny ol Sosnm, 6i ?65 aino, C5FH
[17] Probing of DNA Structure in Cells with Osmium Tetroxide—2,2'-Bipyridine
By Emil Paleček
These methods yielded information about the distorted and single-stranded regions in the DNA double helix at single-nucleotide resolution. DNA probed both in vitro and directly in cells.
Hľ-Tiinn^rN i;n7,vmolovy, vín   31:
CcvyiVú D IW2 bf Acadenfc i*«*, lne
All rif hi^ vi reiKVkliiťih*» in mnf ŕínn nc^nťJ
AĎSORPTIVE STRIPPING         ADSORPTIVE TRANSFER STRIPPING
NA is in the electrolytic cell and accumulates                 NA is attached to the electrode   NA is at the electrode but the
at the electrode surface during waiting                           from a small drop of solution      electrolytic cell contains only blank
(3-10 fl)                                   electrolyte
In 1986 we proposed Adsorptive Transfer Stripping Voltammetry (AdTSV) based on easy preparation of
DNA-modified electrodes
AdTSV has many advatages over conventional voltammetry of NAs:
1) Volumes of the analyte can be reduced to few microliters
2) NAs can be immobilized at the electrode surface from media not suitable for the voltammetric analysis
3) Low m.w. compounds (interfering with conventional electrochemical analysis of NAs) can be washed away
4) Interactions of NAs immobilized at the surface with proteins and other substances in solution and influence of the surface charge on NA properties and interactions can be studied, etc.
Foundations of nucleic acid electrochemistry
were laid down in 1960-1980's using   mercury and cmton electrodes After the discovery of ttw DM electroactMty I   t was shown that:
Signals of ds and £S DNA and RNA greatly differ   , This mode it possible to follow the course of : DNA denaturation/melting, renaturation/hybridization to detect: traces of ssDNA in dsDNA samples,   DNA damage, single-strand breaks, chem. modification, depurination...
Important findings :
DNA premehKng : beginning of the 3960's
DNA unwinding at the electrode surface : middle of 1970's
Polymorphy of the DNA double helix   i middle of 1970's
New approaches later utilized in DNA sensors:
First oovdentřy bound  deetrocetřve 3NA labels : beginning of th« 1980's First QNA-madfiec elfcttrattes ; middle oJ the 1980's
Electroactivity of nucleic acids was discovered about 50 years ago Reduction of bases at Hg electrodes is particularly sensitive to changes in DNA structure. The course of DNA and RNA denaturation and renaturation can easily traced by electrochemical methods
At present electrochemistry of nucleic acids is a booming field, particularly because it is expected that sensors for DNA hybridization and for DNA damage will become important tools in biomedicine and other regions of practical life in the 21st century
DNA-modif ied electrodes can be easily prepared; microL volumes of DNA are sufficient of its analysis but miniaturization of electrodes decreases these volumes to nL. Sensitivity of the analysis has greatly increased in recent years.
76