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`R ETIJR r~ TO
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`The 420A S~em is the first multi(cid:173)
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`

`469
`
`470
`
`47S
`
`47S
`
`476
`
`476
`
`476
`
`477
`
`477
`
`Protein structure determination
`by nuclear magnetic
`resonance
`Wolfgan g Kabsch and Paul Rosch
`Luminous phenomena and
`relationship to rock fracture
`JohnS Derr
`Segmentation genes and distributions
`of transcripts
`Douglas Co ulter and Eric Wiesch aus 472
`Mathematics: The class number
`problem
`Ian Stewart
`474
`- SCIENTIFlC CORRESPONDENCE-
`Evolution- the struggle continues
`C D Millar, N R Phillips
`& D M Lambert ;
`H Nakahara, T Sagawa & T Fuke
`Kenyan finds not early
`Miocene Sivapithecus
`J Kelley & D Pilbeam
`Quantum behaviour of
`superconducting rings
`T P Spiller & T D Clark
`The stability of zoological
`nomenclature
`PK Tubbs
`In the eye of the beholder
`G Westheimer
`Error rates in prenatal
`cystic fibrosis diagnosis
`D J H Brock & V van Heyningen
`Homology of trichosanthin
`and ricin A chain
`ZXuejun & WYiahuai
`Putting a charge on a quark
`LMotz
`478
`- -BOOKREVIEWS--
`Nationat Styles of Regulation:
`Environmental Policy in Great Britain
`and the United States
`by Da vid Vogel
`Eric Ashby
`Leviathan and the Air-Pump:
`Hobbes, Boyle, and the
`Experimental Life
`by Steven Shapin and Sim on Schaffer
`WDHackmann
`Normal Aging, Alzheimer's Disease
`and Senile Dementia: Aspects on
`Etiology, Pathogenesis,
`Diagnosis and Treatment
`C G Gottries, ed.
`T J Crow
`The Caledonide Orogen -
`Scandinavia and
`Related Areas
`D G Gee and B A Sturt, eds
`Chris Stillm an
`
`479
`
`480
`
`481
`
`482
`
`--COMMENTARY--
`Ecotogicat consequences of
`nuclear war
`S J. McNaughton, R W Ruess
`& M 8 Coughenour
`483
`- -ARTICLES--
`Laboratory in vestigation of the
`electrodynamics of rock fracture
`B TBrady & G A Rowe ll
`
`488
`
`Isolation of the paired gene of
`Drosophila and its spatial
`expression during early
`embryogenesis
`F Kilchherr , S Baumga rtner,
`D Bopp, E Fre i & M Noll
`493
`-
`LETTERS TO NATURE-
`Latitude and depth variation
`of solar rotation
`T L Duvall Jr . J W Harvey
`& M A Pomerantz
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`soo
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`Superparamagnetic component in the
`Orgueil meteorite and Mossbauer
`spectroscopy studies in applied
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`M B Madse n, S M0rup ,
`T V V Costa, J M Knudsen
`& MOisen
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`H Ziereis & F Arnold
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`Acetone measurements in the upper
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`implications for hydroxyl
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`F A rnold , G Knop & H Ziereis
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`Precipitation scavenging of aerosol
`particles over remote marine regions
`P Buat-Menard & R A Duce
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`Calcium-free pumpellyite, a new
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`formed at high pressures
`W Schreyer, W V Maresch.
`0 Medenbach & T Baller
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`A new 36 Cl hydrological model and
`36 CI systematics in the Jordan
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`M Paul, A Kaufman, M Magaritz.
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`SOl
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`503
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`sos
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`S08
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`SIO
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`SIS
`Contents continued overleaf
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`Ex pressio n o f the Drosophila pa ir-rule ge ne paired in
`blastode rm ( lowe r) a nd gastrul atio n (uppe r ) stage
`em bryos. As expla ined o n page 493 o f this issue. a l(cid:173)
`though paired has bee n cl assed a s a pair-rule ge ne o n
`the basis of the two-segmen t pe riodici ty in the patt e rn(cid:173)
`e leme nts de leted in paired m uta nts, in its fin a l form
`the striped p atte rn o f paired ge ne ex pressio n has twice
`th is pe riodicity. T his a nd re la te d work is reviewed in
`News a nd Views on page 472.
`
`--OPINION---
`-
`The changing of the old guard
`4S7
`What yellow rain?
`---NEWS-- - -
`Genetic regulations
`4S8
`Chernobyl
`British universities
`Yellow rain
`US research control
`Ethiopia
`Japan
`Synchrotron sources
`French reactors
`Biotechnology companies
`East European nuclear power
`Chernobyl
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`461
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`462
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`463
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`4S9
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`460
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`--cORRESPONDENCE-
`SERC failing astronomers? I Ageism/
`Paranormal theories/Nuclear risks/
`Sexist ads/etc.
`
`464
`
`-NEWS AND VIEWS-
`A child's guide to radiobiology
`467
`The flight of the dipteran fly
`H C Bennet-Clark
`
`468
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`Mechanosensitivity of mammalian
`auditory hair cells in vitro
`I J Russell , G P Richardson
`&ARCody
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`517
`
`NMDA-receptor activation increases
`cytoplasmic calcium concentration
`in cultured spinal cord neurones
`A B MacDermott, M L Mayer,
`G L Westbrook , S J Smith
`&1LBarker
`
`Replacing the complementarity(cid:173)
`determining regions in a human
`antibody with those from a mouse
`P T Jones , PH Dear , J Foote,
`M S Neuberger & G Winter
`
`Regulation of human insulin gene
`expression in transgenic mice
`R F Selden, M J Skoskiewicz,
`K B Howie , P S Russell
`& H M Good mal)
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`Genetic recombination between
`RNA components of
`a multipartite plant virus
`1 J Bujarski & P Kaesberg
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`519
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`522
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`525
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`528
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`A tobacco mosaic virus-induced
`tobacco protein is homologous
`to the sweet-tasting protein
`thaumatin
`B J C Cornelissen ,
`RAM Hooft van Huijsduijnen
`&JFBol
`531
`-MATTERS ARISING-
`Was there 26-Myr periodicity of
`extinctions?
`J J Sepkoski Jr & D M Raup;
`N L Gilinsky;
`1 A Kitchell & G Estabrook
`Reply: A Hoffman
`
`533
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`Endocast morphology of Hadar
`hominid AL 162-28
`R L Holloway & W H Kimbel
`Reply: D Falk
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`Maternal investment in mammals
`S I Zeveloff & M S Boyce
`Reply : R D Martin
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`PFIZER EX. 1033
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`

`NATUR E VOL. 321 29 MAY 1986
`s~zz:.____------------ LETIERSTONATURE ----------"-==-.:--"-'='-==-:"--'=:_:_:_::=
`
`differed between NMDA and KA (Fig. 3c, d), and in individual
`spinal cord neurones KA-evoked increases in (Ca2+]; were
`always much smaller than those evoked by NMDA. These
`experiments suggest that Na + is a poor trigger for inducing an
`increase in (Ca2+];, since in several neurones the inward (Na+)
`current activated by KA produced no detectable arsenazo III
`signal. However, our results do not exclude the possibility that
`Ca2+ influx through ion channels activated by NMDA triggers
`release of Ca2+ from intracellular stores27
`, contributing further
`to the NMDA-evoked arsenazo Ill signals reported here.
`Although the present results suggest a high Ca2+ permeability
`of NMDA-receptor-activated channels (Fig. 3), the net flux of
`monovalent cations (that is, conductance) decreases in the pres(cid:173)
`ence of Ca2+. This reflects interactions between permeant ions
`within the channel with Ca2+ acting as both a permeant ion and
`26
`28
`as a bl~cker of monovalent cation flux 25
`•
`•
`.
`The experiments reported here provide evidence for an
`agonist-triggered increase in (Ca2+]; in mammalian spinal cord
`neurones. Previously, ion-sensitive microelectrodes were used
`to measure changes in intracellular ionic activity triggered by
`excitatory amino acids in frog motoneurones9
`. The latter experi(cid:173)
`ments suggested an increase in both [Na+]; and [Ca2+]; during
`perfusion with L-glutamate but the results were difficult to
`interpret clearly as (1) neurones were not voltage-clamped and
`thus it is difficult to separate the relative contributions of Ca2+
`influx via voltage-dependent calcium channels and agonist-acti(cid:173)
`vated channels, and (2) L-glutamate is a mixed agonist that acts
`7
`at m·ultiple subtypes of excitatory amino-acid receptor2
`6
`•
`•
`.
`The response to NMDA-receptor activation thus provides a
`second source of calcium flux, distinct from that resulting from
`conventional voltage-dependent calcium channels, which may
`have important long-term effects on excitability. Our finding
`that the ion channels linked to the NMDA receptor subtype are
`more permeable to Ca2+ than those linked to KA receptors, has
`implications for the role of excitatory amino-acid receptors in
`CNS function. It is possible that Ca2+ influx activated by NMDA
`receptors underlies the synaptic plasticity generating long-term
`potentiation, as the latter is prevented by intracellular injection
`of EGTA to chelate Ca2+ (ref. 29), or by blocking NMDA
`receptors with selective antagonists30
`• For example, Ca2+ influx
`localized at transmitter-operated ion channels could have a role
`in organizing and regulating postsynaptic structures in an
`appropriate spatial relation to transmitter-releasing presynaptic
`terminal boutons, and it is important to consider that Ca2+ influx
`occurring at NMDA receptors located on dendritic spines might
`produce an especially large but localized elevation in · intracel(cid:173)
`lular Ca2+ concentration, due to restriction of Ca2+ diffusion
`along the narrow shaft of the spine. In addition, our results
`have some bearing on the mechanisms of desensitization of
`NMDA receptors, as the link that has been demonstrated
`between [ Ca2+]; and desensitization of nicotinic receptors at the
`3 2 may occur also for other receptor(cid:173)
`neuromuscular junction3 1
`•
`ionophore complexes. Thus our results may help to explain the
`· similar desensitization evoked by either large doses of NMDA
`or depolarizing voltage jumps 7
`, which trigger Ca2+ entry through
`NMDA channels and voltage-dependent calcium channels,
`respectively.
`
`Received 3 Jan uary; accepted I April 1986.
`
`1. K.rogsgaard-Larsen, P., Honore, T .• Hansen, J. J., Cunis, D. R. & Lodge~ D. Nature 284,
`64-66 ( 1980).
`2. Watkins, J. C. & Evans, R. H. A. R ev. Pharmac. Tox. 21 , 165-205 ( 1981).
`3. Mclennan, H . Ptog. Neurobiol. 20, 251 - 271 (1983).
`4. Nowak, L., Bregestovski, P., Ascher, P., Herbet, A. & Prochiantz, A. Nature 307, 462-465
`( 1984).
`5. Mayer, M. L., Westbrook, G . L. & Guthrie, P. B. Nature 309,261-263 (1984).
`6. Maye r, M. L. & Westbrook, G. L. J. Physiol., Lond. 354,29-53 (1984).
`7. Mayer, M. L. & Westbrook, G. L. J. Physiol., Lond. 361, 65-90 (1985).
`8. Dingledine, R. J. Physiol., Lond. 343, 385-405 (1983 ).
`1
`9. Biihrle, C. P. & Sonnhof, U. Pjliigers Arch. ges. Physiol. 396, 154- 162 (1983).
`10. Zanotto, L. & Heinemann, U. Neurosci. Lett. 3!:, 79-84 (1983).
`II. Pumai n, R. & Heinemann, U. J. Neurophysiol. 53, l - 16 (1985).
`12. Lansman, J. B., Hess, P. & Tsien, R. W. J. gen. Physiol. (in the press).
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`13. Ault. B., Evans, R. H., Francis, A. S., Oakes, D. J. & Watkins, J. C. J. Pllysiol., Lond. 307,
`413- 428 (1980).
`14. Crunelli, V. & Mayer, M. L. Brain Res. 311, 392-396 ( 1984).
`15. Hamill, 0. P., Many, A., Neher, E., Sakmann, B. & Sigwonh, F. Pj/iigers Arch. ges. Physiol.
`391 ,85- 100 (198 1).
`16. Cull-Cand y, S. G. & Ogden, D. C. Ptoc. R. Soc. B224, 367 - 373 ( 1985).
`17. Hagi wara, S. & Bylerl y, L. A Rev. Neurosci. 4, 69- 125 (1981).
`18. Smith, S. J., MacDermott, A. B. & Weight, F. F. Nature 304,350-352 ( 1983) .
`19. Gorman, A. L. F. & Thomas, M. V. J Physiol., Lond. 308,259-285 ( 1980).
`20. Berridge, M. J. & Irvine, R. F. Nature 312,315-321 (1984).
`21. Sladeczek, F., Pin, J. P., Recasens, M., Bockaen, J. & Weiss, S. Nature 317,7 17-7 19 (198>).
`22. Schoffelmeer, A.M. N. & Mulder, A. H. J. Neurochem. 40, 615-621 (1983).
`23. Evans, R. H. & Watkins, J. C. J Physiol., Lond. 277, 57P (1977) .
`24. Nowak, L. M. & Ascher, P. Soc. Neurosci. Abstr. 10, 23 ( 1984).
`25. Mayer, M. L. & Westbrook, G. L. Soc. Neurosci. Abstr. ll , 785 (1985).
`26. Ascher, P. & Nowak, L. J. Physiol., Lond. Proc. (i n the press) .
`27. Fabiato, A. & Fabiato, F. Ann. N.Y. Acad. Sci.,307, 491 - 522 ( 1978.).
`28. Nowak, L. M. & Ascher, P. Soc. Neurosci. Abstr. ll , 953 (1985).
`29. Lynch, G ., Larson, J., Kelso, S., Barrinuevo, G. & Schottler, F. Na ture 305, 719-721 ( 1983 I.
`30. Collingridge, G. L., Kehl , S. J. & McLennan, H. J. Physiol., Lond. 334, 33-46 ( 1983).
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`1978) .
`32. Miledi, R. Ptoc. R. Soc. B209, 447-452 ( 1980).
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`34. Ed wa rds, C. Neuroscience 7, 1335-1366 (1982 ).
`
`Replacing the complementarity(cid:173)
`determining regions in a human
`antibody with those from a mouse
`
`Peter T. Jones, Paul H. Dear, Jefferson Foote,
`MichaelS. Neuberger & Greg Winter
`
`Laboratory of Molecular Biology, Medical Research Council,
`Hills Road, Cambridge CB2 2QH, UK
`
`The variable domains of an antibody consist of a Jl-sheet
`framework with hypervariable regions (or complementarity-deter(cid:173)
`mining regions-CDRs) which fashion the antigen-binding site.
`Here we attempted to determine whether the antigen-binding site
`could be transplanted from one framework to another by grafting
`the CDRs. We substituted the CDRs from the heavy-chain variable
`region of mouse antibody 81-8, which binds the hapten NP-cap
`(4-hydroxy-3-nitrophenacetyl caproic acid; KNP-cap = 1.2 ._..M), for
`the corresponding CDRs of a human myeloma protein. We report
`that in combination with the 81-8 mouse light chain, the new
`antibody has acquired the hapten affinity of tile 81-8 antibody
`(KNP-cap = 1.9 ._..M). Such 'CDR replacement' may offer a means
`of constructing human monoclonal antibodies from the correspond(cid:173)
`ing mouse monoclonal antibodies.
`The three-dimensional structures of several immunoglobu lins
`show that the variable domains consist of two {:!-sheets pinned
`together by a disulphide bridge, with their hydrophobic faces
`packed together' -3
`. The individual {:!-strands are linked by loops
`which at one tip of the {:!-sheet may fashion a binding pocket
`2
`for small haptens 1
`• Sequence comparisons among heavy- and
`•
`light-chain variable domains (VH and VL respectively) reveal
`that each domain has three CDRs flanked by four rel atively
`conserved regions (framework regions-FRs) 4
`• As seen in the
`structure of the human myeloma protein NEWM (Fig. I), the
`CDRs include each of the three main loops. Often the CDRs
`also include the ends of the {:!-strands, suggesting that side
`chains at the ends of the {:!-strands may help to fix the conforma(cid:173)
`tion or orientation of the loops. The framework regions form
`· the bulk of the {:!-sheet, although for example in the VH domain
`of NEWM, FRI includes part of the loop between the two
`{:!-sheets and CDR2 not only forms a loop but a comple~e
`{:!-strand (Fig. 1). The structure of the {:!-sheet framewor~ IS
`similar in different antibodies, as the packing of different s1de
`chains is accommodated by slight shifts between the two f3-
`strands5. Furthermore, the packing together of V L and VH FRs
`is conserved6, therefore the orientation of V L with respect to VJ-1
`is fixed . We wondered whether the FRs represent a simple
`{:!-sheet scaffold on which new binding sites may be built, and
`
`PFIZER EX. 1033
`Page 5
`
`

`

`NATURE VOL 32 1 29 MAY 1986
`
`'-----'-'-'-=--=-=----o=--'-'-'-'-'----.:..:=------ LETTERSTONATVRE - - - - - - - - - - - -=
`
`513
`
`Fig. I Stereo pairs of the VH (right)
`and VL (left) domains of the human
`myeloma protein NEWM'·8 gener(cid:173)
`ated using the computer graphics
`program FROD0 25
`. The tracings
`indicate the backbone of co atoms
`for the framework regions. a, The co
`atoms of the C DRs (e ) cluster at the
`tip of the variable domain. b, A view
`into the hapten binding pocket with
`the CDRs in the ord~r (clockwise
`from noon) : VH CpR3, CDR! and
`CDR2, and VL CDR3, CDR! and
`CDR2. The side chains lining the
`binding pocket (V LA 28, N 30, Y 90,
`s 93, R 95; VH w 47, y SO, F 52,
`I 100, A 101 ) lie almost entirely in
`the CDRs. c, The ca atoms in the
`NEWM VH domain are marked (e )
`where side chains in the mouse B 1-8
`VH domain are different. The side
`chains (VL Y35, Q37, A42, P43,
`y 86, F 99; VH v 37, Q 39, L 45, y 94,
`W 107) involved in packing VH and
`VL framework regions are traced . In
`the V H domain all these side chains
`are conserved in mouse Bl-8.
`
`whether the structure of the CDRs (and ~ntigen binding) is
`therefore independent of the FR context. To answer these ques(cid:173)
`tions experimentally, we have grafted the CDRs from one anti(cid:173)
`body to another, to determine whether antigen binding transfers
`with the CDRs.
`We grafted the CDRs from the VH domain of the mouse
`monoclonal antibody B 1-8 (ref. 7) into the V H domain of the
`human myeloma protein NEWM, whose crystallographic struc(cid:173)
`ture is known' ·8
`• The VH domain of the CDR donor ( B1-8) is
`attached to a J.L constant region and associated with a mouse
`A 1 ligh~ chain, and the antibody is directed against the hapten
`NP-cap. Both the VH and VL domains seem to have a role in
`determining the affinity of the antibody for NP-cap as the
`substitution of either domain by other, often highly related
`variable domains can destroy hapten binding (refs 7, 9 and
`M.S.N., unpublished results). In the VH domain, each of the
`CDRs has been implicated in NP-cap binding 10, but the class
`of constant domains attached to V H does not seem to affect
`12
`binding of hapten 11
`. The CDRs from the VH domain· of anti(cid:173)
`·
`body B1-8 (ref. 13) are longer than the CDRs which they replace
`in NEWM 4 and this may give rise to a deeper binding pocket.
`Most of the residues conserved between the V H domains of
`Bl-8 and NEWM are located in FR2, FR4 and the carboxy(cid:173)
`terminal third of FR3 (Fig. 2a) and largely form the region of
`,13-sheet which is packed against the light chain. Therefore, it
`might be expected that the VH domain of Bl-8 (hereafter
`abbreviated to MVNP) and the hybrid B1-8/ NEWM domain
`(HuVNP) would dock in a similar manner with the mouse VL
`domain to form the antigen-combining site6
`• The more variable
`
`FRl and N-terminal two-thirds of FR3 form the other ,8-sheet
`which i$ exposed to solvent (Fig. lc).
`The gene encoding the HuVNP domain was constructed by
`gene synthesis (Fig. 2b ). We then constructed a plasmid, pSV(cid:173)
`HuVNpHe, in which the HuVNP domain is lin~ed to a ht,~man e
`constant region, and cloned into a _ pSV~gp~-deri~ec;i ·vector 14
`•
`The plasmid DNA was introduced into cells oftlie J558L mouse
`myeloma by spheroplast fusion . J558L s.,e6r~ie~' A lolfght chains
`which have been shown to associate wit)t'bea'vy ch\iin~ contain(cid:173)
`ing a MVNP variable domain, to create a binding si~e for NP-cap
`or the related hapten NIP-cap (3-iodo-4-hydroxy-5-nitrophenyl(cid:173)
`acetyl caproic acidf . As the plasmid pSV-HuVNPHe 'contains
`the gpt marker (encoding guanine phosphoribosyltransferase),
`stably transfected myeloma cells could be selected in medium
`containing mycophenolic acid 14
`transfectants wo,uld be ex,
`;
`pe<:ted to secrete an antibody (HuVNp-lgE) with a heavy chain
`composed of a HuVNP variable doinain and human e constant
`regions, and the A 1 light chain of the ·J558L myeloma. The
`culture supernatants of several gpt+ clones were assayed by
`radioimmunoassay and found to contain NIP-cap-binding anti(cid:173)
`body. The antibody secreted by one such clone was' purified
`from the culture supernatant by affinity chromatography on
`NIP-cap-Sepharose, and by SQS, polyacrylamide'· gel electro(cid:173)
`phoresis the protein was indistinguishable from the mouse
`chimaeric MVNp-lgE (ref. 12) (results not shown). The HuVNP(cid:173)
`IgE antibody competes effectiv'ely' with MVNp-lgE for binding
`to both anti-humane (Fig. 3q)'and NIP-cap coupled to bovine
`serum albumin (NIP-BSA) (Fig. 3b ).
`The affinities of HuVNP:IgE for NP-cap and NIP-cap were
`
`PFIZER EX. 1033
`Page 6
`
`

`

`NATURE VOL. 321 29 MAY 1986
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`
`Fig. 2 a, Amino-acid sequence of the V H domain of the human myeloma
`protein NEWM compared with that of the mouse Bl -8 (anti-NP) antibody,
`and divided into FRs and CDRs according to Kabat er a/4
`. Conserved
`residues are marked with a line above and below the residue. b, Amino-acid
`and nucleotide sequence of the HuVNP gene, in which t~e C DRs from the
`VH do!Jlain of the mouse Bl-8 antibody alternate with the FRs of human
`NEWM protein. The gene was constructed by replacing a section of the
`MVNP gene i!l the v~ctor pSY-VNP (ref. !2)' with a synthetic fragment
`encoding the HuVNr domain . Thus the 5' and 3' noncoding sequences, the
`leader sequence, the leader- variable region intron, five N-terminal and four
`C-te1111inal amino acids are derived from the ly!VNP gene; the rest of the
`coding sequence is derived from the synthetic HuVNP fragment. The oligonu(cid:173)
`cleotides ·are aligned with th~ corresponding portions of the l:juVNP gene.
`For convenience in cloning, the ends of oligonucleotides 25 and 26b form
`a Hindli site followed by a Hindll! site, and the se!juences of the 25/ 26b
`oligonucleotides therefore differ from the HuVNP gene.
`Methods. The synthetic gene for HuVNr was constructed as a Psri-Hindlll
`fragment. The nucleo!ide sequence was derived from the protein sequence
`using the computer program ANAL YSEQ 26 with optimal codon usage taken
`from the sequences of mouse constant-region genes. The oligonucleotides used in synthesis ( l-26b:! 28 in total ) vary in size from 14 to 59 bases and were made
`
`on a Biosearch SAM or an Applied Biosystems machine, and purified on 8 M urea/ polyacrylamide gels 7 •. The oligonucleotides were assembled in eight single-stranded
`blocks (A-D and A'- D') containing oligonucleotides I, 3, 5 and 7 (block A), 2, 4, 6 and 8 (block A'), 9, 11 , 13a and 13b (block B), lOa, lOb and 12/ 14 (block
`B'), 15 and 17 (block C ), 16 and 18 (block C'), 19, 21, 23 and 25 (block D), and 20, 22/24, 26a and 26b (block D'). In a typical assembly, for example of block
`A, 50 pmol of oligonucleotides I, 3, 5 and 7 were phosphorylated at the 5' end with T4 pol1,nucleotide kinase and mixed with 5 pmol of the terminal oligonucleotide
`which had been phosphorylated with 5 ILCi of [ ')'-32P]ATP (Amersham; 3,000 Ci mmol - ). These oligonucleotides were annealed by heating to 80 •c and cooling
`to room temperature over 30 min with unkinased oligonucleotides 2, 4 and 6 as splints in ISO ILl of 50 mM Tris- HCl pH 7.5, 10 mM MgC12. For the ligation, ATP
`(I mM ) and dithiothreitol (10 mM ) were added, together with 50 units of T4 DNA ligase (Anglian Biotechnology Ltd), and the mixture was incubated for 30 min
`at room temperature. EDTA was added to 10 mM, the sample extracted with phenol, precipitated from ethanol, dissolved in 20 ILl of water and boiled for I min
`with an equal volume of forrnamide dyes . Then the sample was loaded onto a thin (0.3 mm ) 8 M urea/ 10% polyacrylamide gel" and a band of the expected size
`detected by autoradiography and eluted by soaking. The two full -length single strands were assembled from A- D and A'- D' using splint oligonucleotides; thus,
`A-D were annealed and ligat ed in 30 ILl as above with iOO pmol each of oligonucleotides lOa, 16 and 20 as splints, then incubated overnight (A'- D' were co