LASL Health Division January Through Decomber 1972

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7(I €3 49 2

LA-5227-PR Progress Report uc-48 ISSUED: kch1973

Annual Report of the Biological and Medical Research Group (H-4) of the LASL Health Division January through Decomber 1972

Compiled by

C. R. Richmond G. L. Vwlz - Contributions from the staff are indicated by section. r"? u1 6" i J=:

u ExparimtaI rnimrlr used in work prmted in this rcpwt WOW maintained in animal care facilities that are fully rcarditd by the American Association for Accreditation of Laboratory Animal Care.

I i I LA-34324s L4-3610.MS In tho intuw of prompt distribution, this pfogros rspwt was not ditd by the Technical Information ruff.

Rintd in the Unitd htmof Anmiu. Awrilrbb from National Tlchniul Information Sawice u. s. orpn-t of clmfnarce 5285 Part Roy81 Rod Springfield, Virginia 22151 Price: Printed Copy $3.00:Microfiche $0.95 1 1 1 3 4 5 9 11

12 13 13 l.5 11 111 19 19 20 21 23 13 25 26 27 28

28 32 35 u 45 45 66

46 52 57

!UMW,IM RlrDI(SIOL0CY SECTIQS

1NTTIu)DCJcrION 61 l4AXmLxAN RADIoBIou)<;p 62

Biological Effects of G--l.y Dae Protraction during Cantinuoru ExPmure With Fixed md Chra&ing Dose btea 62 Coqarative 8iological Effects of Rdietioa Doae Protractioa in Dop and Wrt8 65 Radiobiological Pilot Ia~estiptiorufor the Negative Pioa Radiotherapy Progru 68 ?wpuLIAN WTABOLISI 71 Ratention of Tin-113 In Mu after Oral, Intraperitoned, and Intravenoku InfUCtiOa 71 Cesi-137 Activity in a NodNev lhxico Population 71

PUYSICAL RADIOBI~OCYSECTION

INTRDDUCTION 75 monArIoN OF DATA CoLLmxon 75 Y8I (TI) Spectrorter ad Ttuuuraniru Element Chest Counter Interface With PDP-a/I Coqurcr 75 Huwco 11 Interface with PDP-B/F Computer 76 Craf-Pen Interface to PDP-(I/E Caputar 16 ANALYTICAL sUPm1 76 Cheat and Uhole-Body Counting Data hd.ysi8 76 Flau Mcrofluorowtric (mP)D8ta AnUysis 77 9WCO 11 Data Analyaia 78 KARY0LOc;Y 78 Sami-4utastic Chro- AnJysir ?a ELECT-WNICS 3EVF.LW.W 19 -5i:., Samrrtor and Counting Logics 79 v-1 anerator 79 ‘,mllficr vith Bur-Line ReatoratiOtt 80 PION 1E;W CHI?;RCTERISTICS 80

:V 105b41-l 81 62 Wfanotirl Lfaht-Suttariq S-=.. of W* Call. a2 06 88 91 91 92

94 96 98

102 1d3 104 u4 119 119 111

125 125 US 126 126 126

128 128 118 U9 131 131 132 13b 134 135 136 137 137 139 141 fOSb418 V

U h 1

b i 8 I

e I. ASSOCLMD UBSm MmITImp

’Lh La AI- Sdamtffic L.baratory, am a Paftidpaat in tha k.0ciu.d Vote- Uai*.nitiu, be., 8.-a the iatmlrrl.d purpor of the 0. S. Atomic Ea8ru Carb.rian of proridlag .upport for educatioa cd tr-6 of advmacmd attadmats and faculty participatim appointma. The function of Awu i8 to promote .nd coordinate orfentation d trriniat o project. of a re&& =tun that re- quin iPter-rmivmaity er#).ratioa. m i. putic- AUU Graduate ?all- ulrrly concerned vltb orwutioa ad A. Bmting, ?c.S.,\-B proaru facillttu that cwt undertaken of ad be Departmat of Pbyaiu. Univemig of W.r Ilufco, with equal 8ffecti1m.rwa b7 a miwe lnatitutim. AlbuqwlQw, Neu I1.rico 8uch area of laterut $8 the promtion Of pos’zDocToRu WEAIPQI Ppocuw orientation md trdnlng in recent and specific trends in the biol.0Gc.l acieoces. A 1iJt.d nubar of r..aareh u8i~n-t. arm It Is felt that increaming opporttatitiu for available €or young indiddd with a Pb.D. &we* collaboration would aemm the beat intermats of the at the taborator).. a-8 WSL felbu8hip. are unI~r8iti-~LASL. .ad the biolofid mdencu. warded on L competitivr b8.i. for one ~8ruith Uaivwrsity personnel benefit by facreased oppor- the paalbiUty for reaw8.l for a wcod VU. -8 tmiti- for trdniag .ad teachias, by access to follaring i8 a lfat of p0mtdoccor.l appointwr, uh0 unique or spccirllted LASL facilities, .ad by rue participated in the LASL biaadical rerearch proof La Alva u a foam, or curlgat, for dti- gr.r during part or all of 1972, vith thdr pmVhUS iMtitUtid cooparati- efforta. Loa AM academic affiliatlo~.rrd ujor rmaearch intamst.* blolowu beamfit fra mater cmt8ct aminr- with H. A. Criaarm. P~.D.,C-~ aity facu1ti.r and graduate rtudrau. All biolop D i8t8 participatini io this progrr benefit fror Application of cvtology urd hi8tocbemiatq sltrul collaborativm effortr , croor-fertilfratiaa t8Chaqua to higb-.ped. flW-Sf8tU C.11 of ideu, and by rharlns apeclaliud erpertlae, .nalysfa instnrmtatloa unique capabilitim, and cmtly rquiprrnt. C. Pi&iebr.nd, €%.De*\- A truly functld rdatioruhip beNe80 L*sL E. .ad other ruN member8 la of gnat potentid value Syntheaia of prote3.a to ruearch, coatiawd proferaid d8welo~aC. cell-free and graduate training. Follaving is a list of -c. F. Stmiate, P~.D.,C-J participants in the Ayu-LASL biordical research pro8r.n for 1972. -Identification of molecular effects of varioua ionizing radiatiom uparmfully characterized mdel rymtrr

LOS ALAHOS SCLMXFIC MORATORY

i rarher spectacular aerial phorograph shws !UWF) on the righc of the prominent Loa Al.1108 the brldge over Lor hlaooe curyon vhich links the canvon. k'ithin the area shown are reaccora, ac- mjor Laboratory sites and facilitiu sitn chc aio- celerarors, iophisticarcd computing frciliricr. medical Research Laboratory-Los Alrmos ?!edical -chine shows, and all tire related equipment and - Center complex ana town site. To che east (to?) of technologv associared wich a large, vell-rsrao- the bridge are located the airstrip on rne left and lished laboratory concerned Gith researcn i: :he tne Clinron P. .:-laereon ?Leon Physics Facili?.' ?hyricnl sc-ences.

Y Two urial viua of the th.lch Ruaatch Lab- thater. ldrtolstered by the Lutheran lbpitd and oratory, which houses the Biomedical Ueaurch Group Hoa Society of Fargo, North Dakota, vhich 8ervea (H-4) of the Lo8 Alu# .Sdentific Laboratory's Loa Akaand surrounding c-1ti.S. 'he close Health Divirion, are ahom above. The left photo- physical tahtion between the Lo8 hl- &did 8raph ahow the do& proximity of the Health Center and the Health Rerearch Laboratory fo8term Reourch Laboratoty to the bridge whlch spuu Los the exchange of Ideas and intere8ta mag re8p.c- Alnoo canyon. The domsltirtorled building w11 tlvc staffs. Sham in the ribt photog?apb ia the constructed in 1952, and the single-level annex Health bse8rch Laboratory and Los AIM Kedfcd wed to house experiPcntal animals vu added in Centrr complex and its spatial rehtionrhip to the 1960. Lmdiately above the Health Rcsaarch Lab- main centralized Laboratory complex acrois Loa .. oratory is sham che 84-bed Lon Alamoa *dfcal hlollo8 canyon. The upper photograph shws the Biordical height, io buried in the side of a hill and is Ruearch Croup’s satellite B.diatioa Expomure Facil- accessible through a %-foot loa8 tunnel. Studiu ity located in a remote area of the Laboratory about of the biological effects of radiation delivered in 10 miles from che plla Hoalth Research Laboratory. dif ferent total doses, dose ratem, or fractionation# Built in 1962, the 1800-square Coot facility houses are conducted in this building. cobalt-60 sources of 10, 100, urd lo00 Ci encased The luver photograph shows the holding cage in steel capsules which can be raised or lwered arrangement within the Radiation Exposure Facility into a led shield beneath the floor from another whlch pennits simultaneous exposure oC dogs sad building about 50 yards away. The octagonal con- mnkevs to a wide range of cobalt-# gam rays. crete igloo, 40 ioet in diameter and 20 feet in LASL hu uwxcellod facilttiaa for tbe produc- ti- of lup quaatitlu of hi$tly enriched atable Irot0p.r: nitrogen-U, oay~ea-17, md urboe13. It io anticipatd that oulfur-33 productim vi11 begin in rbe noar futuro. Siau these arm all uln.tically =ti*. nuclei, they c.ll be detectd by uaatic maoomce t.choiquea Y mll w by cop vaationrl maapactromtry. A pro8rm of bio- chd4studlea utilidaa rhue isotop.. .nd orgamic mad biological sptheur. in ajmction vith .opbi.tiutod nuclear mapatic maoIIlDc1 spec- traoters rd other drurced tcchniqus of physical bioehalatrf, la jut begloaing to @in -am. MsullcIl uAcm18

Two ruruch rwctora louted MU the tkalth Reuarch Laboratory are avdlable Y radiation SOU~CU: the 'water Boiler," a haoroou reactor cap&& of operatioa at all pouer h~lafm 0 to 25 kilaratta, .ad the brguatt Or* *at reactor.

AccELtlUTOAs Smveral elactramtic .caleratoro . inelding INTPODUCTION

A aignificant hirtoricrl event vhich altered fie writer of this memorandum vu Dr. Clem T. the courae of mmdclad occurrod QO F.bwry 23, 1941, Senborg, and the date vu J.ntl.y 1944. *a Dr. C. T. Seaborg .ad collmdiaconred The biomedical resoarch program at Lo8 AI- the alennt plutdrr in room 301 of Cilmsn Rdl at hu evoAved from ita conception in 1943 as a amdl Berkeley. Thc i.otopa of mum-238 and not the more Health Croup estrbliahed to protect the health of familiar -a-239 vu first diacmred at that ti=; the workers, to develop safe working procedures, the plutdu isotope of rus-239 vu not isolated and to establish tolerance levels for exposure to until the spring of 1941, rod elernt 94 remained radioactivity, plutoniuo. and other rad1onuclld.a. uanud until Much of the follaring year. On In 1944, once sigoificant amounts of pluconim brch 28, 1941, 0.5 Yg Of plUtOd-239 VU fb- began to accumulate at Lor Xlamw, the Laboratoq aloned by them1 MU~~OD~,and the enoxaoua effort Director. 3~.J. Wbert Oppanheirr, at the reqwst to produce plutoni~m-239 in qwntlv for dlitarp of the Health Croup, authorized the tempor.- purposes vas begun. The potential toxicity of establishment of a group of four people to initiate plutonium vu recognized aooa after its discowv a research program designed to develop testa for aud availability in quaatltiu for biomedical re- setting exposure limits for plutonium. Several search. wths later, this saull group una absorbed by the It is worth recalling that anly extramoly amdl Health Group UI a Biochemistry Section, and the quantitiu of this precious material were available Laboratory's biomedical research program vas born. for experimentation during the early 1940's. As an In 1945, the Section moved into a small building example, the follaring Irwrmdm Written to the of It. own, and Its members established the urine Director of the Health Diviaion of the Metallurgical -say procedure for diagnosing exposure of Lab- Laboratory is indicative of the early concern about oratory personnel to plutonium. Experiments vare the potential toxicity of plutopiu. The unmorandum conducted which led to the first successful labal- statu in part: "It ham occurred to ot that the ing of a biologically important canpound (nlcotintc physiological hazard. of vorting with plutonium and acid) with reactor-produced carbon-14. The firat its coqouads my be very great. Due to its alpha meuureaent of carbon-14 by scintillation counting radiation and long life it m8y be that the permanent procedures was accomplished here and formed the location in the body of even very s~llamounts, say basis for chc present generation of commercially 1 milllgrr or leas, ayy be very harrful. The available liquid scintillation counting systcu. ingestion of such extraordinarily sa811 .~untsaa By 1948. the Health Group vas a Division in aom few teas of dcr0gr.r might be unpleuant, if the Laboratory, and the Biochemistry Soctioa beca it locates itaelf in a permanent position. In the Croup H-4. the Biomedical Research Croup. In handling of the nlativdy large amounts soon to begin here and at site Y (Loa AI-), there are many conceivable athods bv which amounts of thia order might be taken in unless the greatest care la exercised. In addition to helping set up meuurem in handling so aa to prmvmnt the occurrence of such accidents, I would like to suggeat that a program to trace the courae of plutonium in the body be Fig. 1. Wtahift building used for biomedical research activities duriw the war years initiated am soon u possible. In y opinion, such (photographed in 1946 sharing three ddi- a program should ham the vary highest priority." tiona to the original structure).

^,, Ocrobet 1952, the qou) wvad fra twruy woodeo ncPrPifoa dam di.tributial of 8lph-a sttocturv (Hg. 1) into ita ptw.st but- ad particdata in the l-. Intermtiwly, thfr m~ by the ed of that dad&a uubliaba itself in difficult problr hr mceivad cani&r&t 8CUr both nuidmd inaddcirclw Y - t&oa but little nrolutlan since the dd-WO's. uthority QO the effecu of dtia fror nuclear mi. puticulu prablr f. DQI QID. of th. hi)rt we~m,vorl&i& fabat, Oa the pbyriolom .Oa priority be- of the *Ut potmatial -Or taco- of tdtiu rl plutOPf-. of tha eItplutarrirr u regardm bdrrem- Ttm 1iaDdtd L.& hwp pi4mmml in rd tom, apace 00Cl.a parrr ryrt- (rdioiaoc&c bo- a lnco@Ua authority oo llquid ~dotilh- rhrrorl.ctric guwratom)* ndiul wllutkr tiar mm-, eynthrir ot i.otopiullJ l.bohd such.u the hurt pacer and artificial hmut, Y O+C coqotpdr, w of ~outiv-t-= in cnll Y productlor, tramp~rtation, sad biolom 4ddp., & uhole-body cwtinf tech- of this mtrrirl for oatiomal defelue. ft f. piqrw .nd appucatiacr to biodiJ Iu.u~.b Intermati- in a smme tbat part of tbe ~raq,Is utiliziag the drvelopuat of luy-wl- liqdd aw actively worklog on the probla vhich relata scinti1latSom detectors, the pup caatributed si#- to plutopiu efforts ducted SOI 2Q ywprr 0ific~8tlyto the field of antbr0p-t- thm& VbWly at UL. its millty to wmtotal- p0tu.i- by Tho 1970's ha*. vitourod the .rrpnca,of I qruntit8tily the natural Iml of potusi*l<) vith- interwt in the stab& isotopa prom- des- to in the htrrn body. By eqloit- the whole-body exploit the w of stable eleuotr in the # COultlog 8yst- dui~edfor botb ruearch db field of biordical resurch. In uti, .II IsoCope adYO .Id making we of the L.boratow's .i@f- Appliutiolu sectiocl Yam fonrd vithin the group to icat -uter Up.biUtiu, the liardied Wurch concaatrate m the bloordid aspects of tbe 1- Croup contributed sipifiuntly to the field of (gotopes of gubocl, --sen, gitrow, ad gulfur) radhtion protection by cdrtiog studiu OD the progrm wbicb involves smril groups withi0 the rptake, di.tributioa, .Ira utcntim of radloisotopu La ~luomScientific Laboratory. by mhds and 110. Tbe intorast in rt.bollc The Ibleculu bdiobiolow Sacciarr f. rarrrrd kimtics WY .to applied to tho err&ng field of in a rarfaty of ormc sy~thui. procedure# to nuclur wdiche durlnt the late 1930'r. usdle polynucleotides harial knam bum n- Shortly after the discorrzy in 19SS of the qrwnces. Certain en-r that cetdyu polymrl.0- prumce of caiurU7 in ma from uorl&ide nuclear tide 8y1Ithui8 arm not ody baing isohtd md prr fallout, muu-ow vet. be- 011 a controlled rifird but 4re rho be- studfed u biohctid population of sdjects residing in the Lor Ahms proteinm participating in ioforntion trmufrr rua- aru. Thue rtudiw hm cootinwd to the presmt tioar. The structure, fmction, urd uUb0li.r of tia d reprerat perhaps the mt =.ningful both acidic and bulc nuchar protoinm, belimd to in readout of specifiutioom docrp.ntatioo of the tqord ch.nt.r in lyll of a be involved genetic radioactive uterid n-d to the enriroarnt. and thus differentiation, are being investigated. Be~innintin the urly 1960's, .ore aqhuis Bioloslatr uut biochdsu in the Cellulu vu placed a~the fundrrotrl reuuch upecu of Radiobiology Section are inventigating the twral the biordiul nsearch program. Althow fnmr- sequence of a vuiety of cellular proeessw irr -10- tigations corrtinwd in the WUM mtabous. tioa to specific phases of the cell lffs cycle wing and -1.0 Wobiolow Soctiom mlated to the synchronized cultures of diMcells. A rthod msponae of hi#mr Org8nisU to iouirln~rdisciolu hu been developed in this Laboratory for producing .nd ruiloactive materialo, a =jot rqhui. vu relatively large quaotitiu of cultured ululiao W directed touard research in the fields of molecular cell. rynchronizod vith reepect to period in the and cellular biology. life cycle. Usin8 these cultures, mch8nism con- +- trolling synthetic processes, rubollsr, The late 19960'8 1Lo urked the start of a energy -3 recovery from ionizing radiation, cell-surface pht 03 rcllearch progrr related to the qwrtion of the probable biologicd effects resulting from nama. and uthep.ticrl mmthod. of cell groutla are

r- .d ur* 60 rr(ulr pu -up. Hr tL. duo t..c of kt.rrt I8 -* tha. 8mnP.-k oprwto w a(ulr, d addiaoml- uill bo intarpoktd. This hu wt pt bY-111, mhu the mtedl7 fnqvd hw of bi-4 r8ap-o obsorrrd thw fu8- that up.ctuioo my bo in orror in tho d "uomt dad' or in tho uximm prob.bffls* Or bo** nefOt0, it 8dBO- 8-d to 0- a. ra@8 of tha praihinuy urpurivt Vitb 8 n\rkr of rdto pmtpoao full-.& -t- mt-til 10- paitivm re~ultsU. ob-& 'Lbm, in March 1972, 00. yopp of 130 animal8 Vu irrj~tod via 6ooo apberes, oach uairrg b-1 2 WtAdW (0.22 pCi/8ph.I.). The total 1-8 burda for rhro qiYL is 1.3 aCi (0iril.r group. Ida4 ad 6 sphrru prorlculy h.d bo88 atArtd fr, bor 1971). C-red with tho origid tho 2.5-fold iacnro in animal ntmbar and 3-fold b- crow. in mphore odor gan a total irr~~of 7.5-fold in m.olitivity for low-lanl -#a. kbro imdiate lmg dry. and hwor twr incfdaco rote8 than thmo ve have obrorrd ham been nportd for other animala oxpoaod to dl0 total dona of plutaaiu. Tho Mia diffmuu appeur to bo that uroool won inhaled to tho othor oxporiweu, thw giving mxposurro to uch hrer n&rs of suller (and .or. .obila) par- ticlu. Thim uauld rosult in a IDTO nurly \miform irradiation of md -0 to a uch lupr fraction of tho lung. Irr UI attet eo approach tbuo coodi- ti-, we bmincrmwd #roatly tho ador of (E. c. &&~ou.c. A. rw.~.. L. w. RM, J. E. spherea ahiniotorrd win( our lanr spedfic- Loadom, J. 0. Porriaga, ad J. S. Witom) activity sphores. Although this action dohats our tho coavede~~oof tho madarb ve reput ?or original intootion of koopiw tho ndor of rphuu here tho protocol preliminary oxperirata for u *lull OI~OU#I to minimize overlapping of ruliaticm 1 (pp. 26-27). civen in lut year'm ~u.1rrport field., it prwideo a caparisaa of our tut systa TAlo lip.. tho plutonium content batchor 1 of l0 vith thooo wed 0lo.vb.n and will offor a direct of ZrOz dcr~phoru pnpand, uhicb ha*. bo- ad c-ti~a~ of localized verom diffuoo irradiation 2 rtrudtu the 8 upooure lml~which uero Table .nd give a direct indication of tho iaefflciancy of Iajectod lut you. Ihue oxporirnto uorr plm4 concentrated m8urcea uhich should bo of vdum in exploratory rurrray cmrlng a wid. dynamic u an 80locrion of ddftiaml e.po8ur* coodirioru. oaca to513430 1 --

TABU 1, PLmolIpl CmmMr OI mMsmBLs Batch SpUifiC actilig puoz -*c exact io0 Isotom Level qrrbe? pWsuhem i/dala&en 239% 1 Zr24 0.07 0.16 0.09 4.3 x lo-4 2 Zr25 0.22 0.49 0.13 1.4 x lo-3 2A 2r27 0.02 0.92 0.16 2.9 IO-’ 3 Zr22 0.91 2 .o 0.21 3.8 1.1 x lo-2 u Zr28 1.6 3.6 0.26 4.8 x 10’’ 23%u Y zr29 2.1 b.7 0.28 1.0 x 4 Zr31 4 -3 9.5 0.36 u 2r30 8.9 19.5 0.b6 2.0 5 Zr32 13.3 29 -3 0.52 3.3 x x 6 Zr33 59.4 131.0 0.86 1.3

TABLE 2.

Level Isotm nhr nCil&lnl

1 239P” 1 0.14 13 4.2 x 10 6.8 x 10-1 2 2 2 0.U 42 1.2 x 10 2.2 x loo lo 0 2A 0.84 81 2.5 x 10’ 0.1 x 10 60 1 3 1.82 175 5.5 x lo2 1.0 x 10 60 3 1 3A 3.2 310 1.0 1 10 1.7 x 10 co 23Bh 3 1 4 8.6 an 2.5 x 10 4.2 x 10 10 3 2 5 26.6 2,710 8.4 x lo 1.3 x 10 0 4 6 119 -0 U,lOO 3.6 x 10 5.8 x lo2 0 .. %si%MUS atmctut. lung, p - 0.19.‘ bA.sudag 1 g of lIQ8 irrdhtwl.

conditlolu &e fdvhich tuult in tullDr pmduc burden ruulted in survival time of only 1 pear tion. the att.qt to produce effects vith isolated vith rata. sources vi11 be rumed. Ih\u, on July 2 additional To ilmstipte pos8ibl. differences in €on*- group. of 30 auiuls each vet. injected with total body rrspome to particles oa opposite sides of the lung burdm of .bout 100 nCi. One group ructived alveolar-capillary vall, w have introduced spheres from 1,~.~to 2.000.000 8#¶8W/-M Of directly into the alveoli to aupplerprnt praviour Level 1 sphema (0.07 pCi/sphem), md the other jugular lnjectlotm. No plutooiu h8s been urd ruceiwd 200,000 Iph8NS/Mlia Of LIWl ZA pending a deterdrurtioa of the cant.dn&tiw probh (0.42 pCi/sphem). These 2 poups, dagvith the poeed by excretion; hwever, in June 1972. 20 ani- original hvel 6 2000-lphelrl.aiY1 -up. vi11 br uls received 57Co-hbeled spheres by lnaatr8eh.d cogared vith the Obse~ati~ll’that 0.1 llci 1WS i1uuffl8tion to study this problem.

2 I OSbU3 i destructive Testing) of ehr Lor Al- Sclaciflc Laboratory. :uag ciuuo is placod dinctly oa tho eulsion of a Koddt high-resolution spactropqblc plate and placed in a Luclte chaber sealed to the X-ray tube nead (Fig. 1). Tho ch-er is o..autrl ad then filled vith helium. T~rgot-to-pluo distance io 36 in. (0.91 Yter), and tho oxpoecoe is odc at 15 kg, 20 mas for lo dnutas. The entia radiographic procedure is porforwd in a dutd. safe-light cnvirolrwnt as the eulsioa of t& plate is cxpoaed. Emulsion Is on one sido only to rnduce uarhrrpnus due to parallu. Parallax rad vibra- tion dudnu the long expoouras uo Fqortat factoa

1 spheres within the lung vu pmrlouly nported (pp. 28-30). Ulrifordty of mram caaf.ntratloa wuest8blIohed by -ray cacmtiw of tho sub- divlslona (dam to 1/64the) of a laft lag. Dlm- crlbutioa of ruanst-neI&bor dfruDur uu sham to bo essentially ruda by vb-1 muurunts 00 thin smctiona. Thir rthod is tedlw and, bauuu few sphens can bo detectad in my 000 SectloD. it is very difficult to determine sphere loutioll In relation to ujor lung mutoy. &a olewnt rthod of virlulirlng both tho mlcmpherem urd lmg acre- structuro in excisod dole ltme hu been &valoprd using ultrasoft X-radiograph.. In thim prOudure, a previously injocted .rrirul is ruriflced ad ex- ruapinated. The trachea is expos4 aud an intra- tracheal cu~nuhfixed in pliu. Th. lmxv arm then rtrovld from the thorax, .nd the spocfmn is ea- closod in a plutlc bag and sumpended from 0 rioj stand. Dry helium PS im dlamd to fla, throw the cannula at rate djwtod to all# the 1-u to inflate to approdutoly chefr mrul si20 (bout 16 liten/hour or oaough eo th8t litera pus through the spachn io 18 hours). This procos drirr and fins the luna in up inflated confiwra- tion. While contlauowly nintrining the helium amsphere, the lune or lobo of cholce (lulully the entire left lung) is dissoctod fro the spmcirn. In SOPL casos. OM rldo of the 1-8 my have to be trfird slilhtly to achieve a flat surfsce for stable poritioalag. Mcroradiopaphy Is dmo by Crwp n-1 (Nan- Fig. 1. Soft X-ray erpoeure rystu.

3 .^_-.- linitfng rcmolutioa. Heliu ir used to reduce in a nodair .t10phem. ?hi8 techdew, pap 3rmospheric abrorptioa of the aoft X-ray beu. The ticdarly the ahortor taryt-to-plate di8-u. developed plate is placed on .D enlarging shad- create8 more peodra od, hma, a lW8 8h.w -0 Sraph .nd urnifid up to 2oQ X. Photovapha of A furthor .d..lrt.p of the hdirr reeonm of Intereat arm then taken for peruneat techlllque is that, after rdiography, the 1-8 W record. Exlglaa are Liven in Flu. 2. .ad 2b, in be infiltrated vitb celloidin ad studied hiate- vhich one can identify the Lo-m ZrOz rpherer md l*ully vftb aCcrpt3le pn8a~tiaoof coofloltc their locuiom relative to both ucmatructure and tim md ~IC~OUL~. kcawe th. 1-8 F. i0flW.l nicroatructure of the lung ainay~. to u approrlrtioa of itm now8- rod the p&- If desired, the entin radlolfrph can be photo- topaphic eduvtfactor. are 'kaara. interrph- graphed OD the ahdw&r.ph, prodwhg a mt8t. di8trpc.r c.~lba ruurad accurately either am QW viaualiting the complete lag. In dditim, w jectiau or. with mtersopdra, in thne-dirluiopj nave deStelrOrdiOw4ph8 Which, when photo- Sp8-q prrphicdly enlared and vi-d with a stereoviwr, Patention and Excretion afford 8 chree-diwmioarl of the dcrosphemr (E. C. Anderson. P. N. Dean, C. A. Drake, L. I4. --in situ. It is poulble to obtain similar resulcm Bollmd. J. E. Lmdom, .nd J. S. Wilmod frat thick (0.25-m) sectioaa of parrffin-.rbedd.d spccictem. A shorter targlt-twlate di8tancc (a. We haw eatiutd prevlwly the excntioo rue 12 in.) adlowar emere- (a.8 kVp) can be wed over the first 1~0th'~port injection to comapad to a biologicrl hdf-t- of .bout 7000 d.p# for plutonium rrrd about 400 dwm for 57C0. bteation studiu over an ll-mnth period with "CO ow indi- cue an initial rapid loom of about 5 percent of tbe Activity (PrUuUbly by SUrf8U lUChbs), fond by 8 sli&t decliae correspondins to a half-ti- of about 4600 day. (13 yurs) , u aha# in ?i&.3 (upper curve). The rawstill retain over 90 p.rceat of initial 57~eactivit)r at 1 par, corrfirr in# thee theme spherea are essentially perunently trapped in tho capillaty bed. 00 the other had, animal. &van 57C0--t.bded Sph.rm bg httrcrtr8chd fNUffhCiOn 8hw 8 highor excretion race (Fig. 3, lwer curve). commpording to biologicrl hllf-tiM of .bout 270 dagr. Thi. is C4qAtAble with the ran* of half-tins reported * for oxides of plutoaiur md uraniu in rata (13s to

I I I I I 1 .d, 50 ~ISO200uO300uO Tkrw (dorr) Fiq. :. Soft X-radiotraphe of (a) luag with mlcro- Fig. 3. htmtioo of dcroaphoree in harter lra# -:pnerea (200 X), ad (b) luas parenchm follavins IV injectioo (uppar CUM) and ::ith microaphene (SO X). 'IT iwufflation (lwrr cum). 305b433 record. 011 -.tie two tho optical droritla of the .quuo picturs el.nltm ("pbeb") 1.t~ tho -8. d0C-d- Domintry Our objective is to idontify puwtrm .I the [E. c. AoblllaL, ?. w. Iha, ad D. n. I(.uJi., 1-8 vhich datmmlne tho radial iaforrtia -100 Vtth omaontlal coatributiaar fra II. J. T~ul so the thio function C.D bo dd~odfm S- (C-5) md from Crorp H-21 storoolo~iulI.uur.uotn. Thorefom, um boeo TO purtitato ovmrall biolo@crl ru-w intmrutd in unipuhtlag the diglt1.J to to- of &.pi.- at tho colluhr Lvd. it t. v8- sua parortOr8 u doMity .rd U i.brr?C duidla to knau the distributiaa fmctiaa sivlng hgths in air and timu in a controlled tho ndot of eeh which meetwe a givm rdlotioo This approach rl.0 hu tho adpaat- of b-ug doe0 Y a function of dirt- from tho dcr~pboro. the problu of obt.inio(; roctioar which bmkt BO.UW of hi*ly localid &position of proportiom of tho in vivo lung (at soa pbrw of aloag rr aph8 track 8nd the possibility of sub- the respiration cycle). Coqutor dificuia of collulu tumts. it is not obvious in what tern the data wing iMy-proussing codes p.dU cor- thF. "dodB .bould bo rxprossed (e.8.. by amr4ng rection for I- of the effects of ahriduy dvriu tho mnrw dopooitioa over s- critical velu or preparatiaa of tho luog SOCtiOM . by SOY €0- of hit thoory). In either evoat, The fiat probla In di&titatico of a-0- it f8 n.cusary fiat to kam the fuad8mntd effect micrograph of a lung roction is that of field *It.

of lung structure 0111 radial distribution of en- and resolutioa. Tho former should be largo owugh counters botumon alpha tradu md tissue. This to be representative (of at leut a local rodon of distributiw YI will call the "radial interaction" the lung parenchyu) urd to permit terdnatioo of function. Invane-aqrure effocts are not included the alpha track. (rango bo urn in tisrcu of dt and uould omtor in tho calculatim of such dwi- donsity). In a typical lag soctioa. dons:- aftor metric coneopts u tho probdility of ultiple hit# fixation ad e.bddiag may bo 0.22 der3; thorefore, to .ingle t8ryts. intoractions betueen hits to the averaw alpha raga is 179 UI. Pavor thaa neigbborla8 ~011s~or local dose in rads. Tho pur- 1 percent of the alpha tracks are found to survi*. peso of tho radial interaction function is to iso- beyond Loo urn: thorefom. an ire 600 La sqrun rat0 th. contribution of lung SOrtry in a form will contain most of che alpha tracks. If tho

usoful for later calculation of "dome .'I Variation points of origin of the tracks aro coafiud to the of dE/dx .loo8 the alpha path is not included in central portion of tho imago (for ox.qlo, to om- tho prosont cJcul8tiona. half the area), then about 20 percent of tho trrlu hrrlxu tha put year, we have devo1Op.d a ver- will reach the edge of the iuy and CUI be ro- satile rysta for nurrical evaluation of the flecced. Became of che thinness o€ many alveolar radial interaction function by a Mate Carlo tech- walls, a resolution of better than 1 Um is dtsir- nique based on 8 hi*-resolution photodigitirer and &le. Using 0.6 Uo for the sire of a pixel 8fvonr 3 6 a llbnry of ip.ge-procusia8 corputor pro8r.r a 10 square array of 10 pixels to reproduor: the dovelopod elsuhorm at the Los Al~aosScientific image. Uith an original photomicrograph negative Laboatoy. UI are indobted to H. J. Trussel of ;It 85 X diameter., this requires a 50-Lm squn Croup C-5 (Statistical Services) for progrdng apercure to scan a 50- square srea of tho Des- and to D. I!. J.nny and R. C. Bagley of Croup H-2 ative. A scanning cirof &out 20 minutes is (PHERHu() €or photodigititing. required to digitize and record on magnetic tape The procedure is first to prep8re conventional the Optical density (OD) Of 1,OOO.OW pixels. photomicrograph nogativer of thin-sections of lung kcause of tho requirernt for frequnt md tissue taken at M@fiCetiOIU of 50 X to 200 X. rapid random access to any elornt of tho nillion- Thia hu been done using both normal gray-scale word arrav, it is necessary to condenae tho info- and very high contrast films. These film are then tion so that it can be stored in the central rmory scmnod by a high-speed digital dcnsitorter which of the computer. This is accomplished by replacing

5 .f

each &-bit OD word Vith sin& binary iateir (0 - air, 1 - ci~m~m),depending 00 whether or not the OD exceeds a &van velue. As dl.cwnd etc where, interesting .Id weful rodiflcatloru of lrng structure can be accoq1i.h.d by varying th OD cutoff. ~fogl. blu Of info-ti- am thm packed 60 per word into a uch s-f of word., which an \mp.ck.d for bit rmtriemi 88 needad. The coadrmad imp I8 thrn dyted by 8 lbrrte Carlo proaru (AWLWC, writtm by H. J. Trrusel of Group C-5) which mekcta a rdapoint of origin (lirlted to tiarm for most of out rorlysm) and projects m alpha-particle path in a random direc- of lmg sectioa played back at tion. The length of thfm path throu@ each pin1 Fig. 4. In* drrr- sity = 0.231. is crlculatad, and the comtemts of the plul (dr or Cimmw) are detedwd. A record is hpt of encounters with tismru u a fvactloll of distance fra origin, rad the path is tednated after bo urn in tissue (reflecting at the edge of the hnp, if necessary). The process is rspeatod until a ruf- ficiently large ader of paths hu been scanned to give the drelmd mtatimtical accuracy. For dis- 3 fMC88 to .bout urn, 10 path. are usually Ad.- 4 quate: 10 paths are required for 400 urn. Running ti-s on a CDC-7600 ctmputet an about 10 and 100 seconds, respectively. ?he image of the luna resulting from selection of a given OD cutoff CUI be reconstructed bv plot- tint the condmued bit array. Varying the cutoff changes the physical demity of the section by Fis. 5. [maw of iunq section plaved back at den- fra thdr poinu of orwn. Tbe mULTIvor8 ue thm aur4 hity(be- of wlectlolr .J col- those &OM patha WLI d1rmct.d almf the airuaym, -ti-) . Thpr, tha Hantr -10 cod. mt ed-e and a collivtloo of dilrcticm ruultr in addition the rUinUrrstl00 fuactlOa by t.b-4 tb@ to a nammifoa louliutlaa. Ihim affect im ll- wtd rate of acamtu of tlmnw by alpha par- lumtrated in Hm. 7 ad 8. Hgure 7 &arm the ti& Y a fuotioa of Utance fra orilp. d point. addimctiolu of origin for 1o.OOO alpha thon aurying Q1.r uoy r.Oaa source paftioo. track8 cbaa at rrda but vlth the rutrictioo A typical wultm hietopa baud OQ u),OOO puh. that the origin lir b thew and in tho central im mbua in lis. 9. The oUte(I..., frrtiul half of the ivm, ruultiog ln a Und of "auto- tiurr intercept lenith) ia the fractioo of e graph" of 1-8 mtructurm. Figure 8 rhous the locr ind alpha path. Utu8lly trmmtring tiui u ti- .nd directitnu of 1ooo alpha tract which giina dlsace frathe mource. Became d1 od- murvive fro8 the &on after tramling 215 Um. Tbe gins are placd in tboue, the ordinace rrtu is offecte of collhtioo am clearly vimible, urd the urrity at .em di.t.nca and fa- rapidly at .baa mtnicturem ro8pomible can be identlfiod by caqar- dimtacu (as mat of tho timmue ir in thin aln0l.r boo of the lo fimrer. vallo) .nd lenb aut at a v.lu oqul to the amr- 3 Because of thin melectioo effect. the fraction ~8 1- dearity (here 0.223 g/a 1. Inaid. 10 YI, of alpha trbrurvfvlng at a even dimtmu frcm the alpha r.ly., there C.D be no tormlnattiool. of EtlUCCUm. their origin im not a IIU\U. of the inter- path.. ond all effrctm are dm to lung action prob.billty bemom alpha track and tlmmue. Reyood 00 m, terrinrtiom bogin ad the obqe of Alpha tracL. do not pur through a mdlw of COD- the curve 1s the remult of three factors: (1) 1-0 stmt denrity; they begin in nglionr of higher thon of alpbr tracks; (2) collfution of tho mUrPIvorm; 8Ver.ge deoaity (their orlginr mst be in tlmmur), .rd (3) local lung structure. .rd at lare dlmtmcu thq are in redom of lawer Colla clooe to the alpha rource doubtally will be killed ud mterilird by the exrraordinully high dome rateo. Thus we are concerned 01y with the evaluation of dooe at larger dlmtancem. may beyond 40 u. Therefore, in *at follarm we vill coasider only that portion of the curve. We ab11 ala0 noruliro the varlablem by average denrity of

Pig. 7. Poiatm rad directlaw of origin (limited to tirmuc .ad to center of image) for 10,OOO randor alphe cracks.

I 90I TsI 100I InI. *¶a InI 4-1 Fig. 8. Loc~tlonoand dlrectian of those lo00 - alpha particlea murviving at 245 IJ~ Fig. 9. Himtogru of the radial intaraction fmc- fra their originr. tion for alpha cracks in a lung mectioa.

7 the lung soctian mdor lnvaatl~tfon. The ordinate divided by dnulty hcaa %latIve path In ClBSUI." and a value 0: unlq comapading to the phtuu in Fig. 9 iopllea that tt.0~8is klq trawra8d at a rat. euqrl to that of uuatteaucltd aourco alpha paths in a diu of conmtmt averar density. The absclr8a atltlplld by dmmity Ua-a the 3 usual rus/area dirarioa. Because pg/m3 - da , 2 a coavedent unit is pe/m ; dividing by dcrulty ncovalr the dIBtanc8 in unit. of 'm. ILtl Fiwre 10 rhara a fuily of unoorrlired cuma for 4 lung iruma (the flrst and last of which -re shown in Flga. 4 urd 5) obtained from a single photomicrograph by vrrying the OD cutoff. The density of the huge, aa indicated, ranged from 0.23 to 0.34. and the curves are clearly qulte different. Fig- ure 11 shwa the same data u normalized. Yote that -Et of the differences are elinhared, che mrximum :oar , ! residual varlabllfty being about 2 15 porcmt. Thw. for a given atructural pattern (i.e., for a given Oat distribution of 81vcohr sires and shapes), effects of varying alveolar wall thickness. to a first 2 c 0201 c approxinmtlon, are elimin8ted bv appropriate dcMity P normalization. The curves shwn in Fip. 10 and 11 5 OIOI are smoothed through the actual collputer results which, Of cwrae, hrve the staristical flUCtuPCiOM characterlstlc of a11 Hmtc Carl0 calculations.

Typical sets of primary data are sham in Fig. 12 OW! to illustrate the scatistical variations for cal- 3 G culaciona of and 10 paths, respectively. 001; 10 20 .o co .Another iaportant parrrter ai a lung section oirtoncr ~oo~z, is the "scale factor." the rac:o of mean intcrcepr Fig. il. Curves from Fig. 10 norulized by densitv. length to alpha rmge. Phis ?ar.luter varies considerably during lung inflation as vel1 is between species and, of course, also during the process of lun3 removal, fixation, and emotdding. Therefore, it is rmportant to understand the effects of these cf changer on the radial interaction function. rhiu c 5 variation can be simulated eaailv bv churgiag cht fom- assumed magnification factor for the digitized lmpe s while keeping the alpha range and density co~itant. 3esults of such calculations for a magnificataon ranue ot about ?00-501d for a :.:?ical lung section

.ire shown in Fig. 13. The ''frse" aagnificarion LS 35 X, ano the number on each '-;we gives the -8- -iiEicac ion assumed. The rect anqular dis cribut 1Ons

ldbbelrd and 1. are the lirnitrng functions expected Fig. i2. Udial interaction functions sh ing , .:c tl~egeextremes. .it vem 1- Xgnitications. the statistical fluctuacionr for 10 5" and 10. paths.

.) a pro~laingfurrctloa.’ It is not mchaniotic but doer igly an intensting mixture of strucrure ad chance (the tvo poaolble outc~oof Bernoulli trials corrrpondina to encountor of air or tisad. me nd.r and identity of the propertiu to be deterdnad to charmctarlte the lua8 sectloo are not yet est.bli.hed but probably will be the pare eter(o) charutstiring the distributioo of ma6 intercept length. in air ad in tirow. ‘Iheaa can be determined cuily, of COU~OC, by automatic ehc- tronic or optical scanlng devices, a far s2ql.r tuk than -re Carlo analyoio of digitird pbote

graphs *

Pis. 13. Radial istermtiom fuoctiaao a fmction u BIOLOCIUL RESULTS of ouk factor. Nmbor~rn tho CUI’Y.~ indicate the uourd upificatlon factor for the iuy. (S. C. Carpenter. G. A. >rake. i. !<.Bolld, J. E. London, J. R. Prine, J. 3. Miloon, 4 R. H. :bod) la18 otructum kcoms extmrly coarse compard Last year we reported on the inerrnu8 of ZrO: with tho alpha ran*. In our model. dl alpha cracks microspheres. At that ti- oaly animals in the two begin in tiosue so that. uhen the alveolar walls be- hi&est dose groupo shwed MV biological raspanae COY W- thick, the alpha paths nenr lea* tissue to the presence of the spheres, in the form of sad1 ud &Up rmp cutoff is obrmd at Go dis- accuwlatioru of macrophagto in the proxirity of tance froll the source. The sbociasa hu been nor- individual spheres. We continue to see such aC- uli2.d by mJltiplyfn# by the mrae density of the cwlations of phagocytes but have not obsed my section thfs cue, therefore. the cutoff (in 0.23); of 2 capsules fibrous connective tissue like ehae appuro at 9 pg/um At very hi& m~ificatiau, . seen in our earlier vork with larger, more radio- the otructure becoms fine coqared with the alpha active spheres.6 Yany of the animals in our earlier range, rrd the lung io effectively homganeotm. experiPwnta1 groups are approaching the and of their Again, alpha paths have the range dl suc with anticipated life span. and ve can expect to see -re uniform interaction probability alms their paths, ? spontaneous deaths in the next feu nonths. and the oharp cutoff I8 nay at pg/um’. Prelim- 40 tn January :972. ue injected 105 rats [CRL:CD- inary evidence suggests chat lung sections which (SWSPF] with about 6000 microspheres of activity are quite different in preparation and appearance Level G. These animals have been kept separated may produce the SPII~ family of curves as assumed under harrier conditions from other rodcnta. 3ur- Ilugnlfication varied. is inp the subsequent 10 reonths, vc have sacrificed The problem io not to identify an appropriate ?r of these enimals and hmvr observed no pu1.0~- uthwtical expreooion to reprerent at leut the disease of any kind. rhcre nave been no sponta- isportant arllbeto of thio fdly of curves and to neous deaths or anv signs of clinical diseue in relate the adjuotable parmten of the function to this group of animals. ruily ruurcd prapertier a given lung section. of Blood samples raken from all animals (hamsters One cwld then predict the radial interoction func- and rats) imve not revealed anv effect upon the :Ion for any giwn (or modification thereof) lung formed rlemncs of blood even after long exposure. the complexities of digitization and 3onte without ‘iariictr in the experissnts. we anticipated chat we Carlo The mathematical expression cwld analysis. %wit see an increasing lymphopenia from constant be chosen either archaistic heuristic on or exposure of the blood as it circulated through the ground., the former being more rewarding but per- lungs. This has not been the case. !n addition, haps more complicated. We presently incline to the we have not observed anv effects on re~ionallymph view that the generalized binodal dlrtriburion Fs nodes. Anid -Ndor 617 Control lS.5 Puwrutic carcinoma with abdodnd exten- 8iw; 00 PWMn ItUtU.. 330 Lave1 u 9.5 Kmo-endothelial ..?CW of lung 69 4 Love1 u 11.5 Undif forentiated sar- CQ, abdodd vis- cor.; no luna 1edOM 705 Love1 u 12 .O Uadiffonntiatod sar- e- of 1- a70 kvel 4 u .O Uadiffor.ntioted sar- coy, rrltiple Ytm- Fig. 16. Hcwgio-sarcoma in hmtcr lrng at tosos, no lung le8i0cu; &vel 2x. prhy not datemiand 1368 Lon1 6 7.5 Iribrcrarcoms. abdominal viscora; pulmonary nmt- Ut88.a

lhua fu, J laiuls from the cxperilratal graups and 1 fra a control pup have diad of aeoplutic disouo. Of them, only 2 have had a privry 1-8 lubn. Tdl. 3 Uru the nwplum found, dou lovela inwlvad. md duration of the erporuro in oach cue. Oam of the primary lung IC- si- wu a houn8io-sarcou (Fi& 14) in an animal fra one of the lover dwe groups (Level U) with au exposure ti- of 9.5 roaths (287 days). This tumor roplacd hrtentire17 the left lung and severely eapressed the adjacent nooaal lung tissue. There vas no evidenu of ncaatases to tho rest of tho lung or to lay othor orwn. Fig. 15. Undiffcrcntiaccd sdtr.com in hammer lung Th. othor primmy 1- lesion wu an undiffer- at Level 2’1. entiatod aarcou (Fig. 15) in an urfvl from the 8- dose group ad wu fouad 8fter about 1 year (354 &ye) of exporun ti-. In this CUI. there were multiple nodulu in both lungs. .Xicrosphcraa were fwod in adjacent normal lung tissue around each tuaor site. No rtutatic lesions were found in othor organs. A0 .niml Ora a control group rhwd tho only cuo of a wll-differentiated carcinou (Fig. 16). This particular t-r had originated in the pmcreu uith mctuuioa to several othw or~uuwith ao pulmonary mtutases. Bccauso moat of tho nroplur found have beon sarcacp.. ori8i~tin8in the abdorinal viscera, the quostion arlsu u to whether a rignificmt nmbcr of ricroaphcms my ha- passed through the lmg and beon picked up in the mticulo-endothalid Fig. 16. Pancreaeic carcinoma In a control hantcr.

.-_-__ 1. C. R. Richmad md C. L. Voelt, e&., And Uaport of the Biologkd .nd bdid Crovp (U-2) of the WSL Health Dfvi8a. Jawth- D8ed.r 1971, Lm UOr Sd- eatific tabor8toq npo- U-4923-?1: (U72). 2. J. R. Co- ad L. J. Perex, Jr., Coo- sid8r8tioo of a Tmor Ptob&ilig Ib.CtiO0 & Ucro-Oairt~for the Deep tag, Pm11 of Dutinl eh. c-8 year, the pri.y objectimm lind kpott OE &diologk8l Safety StuU.. for the Su1 ?-a for Saf8tY Brd, I-, aeI UPIIC, hrlrooPraul Safewnlr DiVi.tO0, llDs Corporatiom, Uochille, hry1-d (1969) , (1) To comtimm the mphen krj.ction prop- -port WS-596. d to e-4 iOtOn8t- *-- pup. u ladi- 3. 8. 0. Stulrt. Y. J. Y. J. Clb, .Id cat4 by forcbcodn( remultr. Edr, E. 1. iiarud. Acute Toxicity of Inbald (2) To inrutlyte additload lnmulu to pro- Plutonium Oxide-238 and -239 in kts. Battelle Memrlal Ia8tltute, Air Fora Vq~ar mote the inci&c1~8of 1-g dum. Laboratory tmhniul report ANI.-TR-68-49 (3) TO art8d thOOmtiCd Stdim 00 effect8 (1972). of 1mg strueturn QI dae diatribution, especially 4. Tuk Croup a, LUUE Dpdcs. Health PhyS. 011 the correWioo of structural par-ters and the L12. 173-207 (1966). doeo-distributioa fuxction. 5. U. Feller, h Introduction to Probability (4) To continu hlmtol~icil.ad pathological Theory and Its Application8. Vol. 1. John Uilcy and SOM , Inc., New York (19K)), rtudlu of expond lunp. pp. 104-110. (5) To mtudy the effects of a wre tmifom dis- 6. C. R. Richrood* J. LUt#tU, rod B. S. StOO., tribution of rmdiuioo do8e to the lung (e.g., by Biologic81 krpow to Srll Dimcrete ai@ly-. we of beta radioactivity instead of alpha lrrdia- Radioactive Sources. 11. .Yorphomoir of HiC~l~SiOMin bt Lungs from Intrmmuowly Cion) . Injected 239PuO2 nlcrospheres. Wadtb Ph78. -18, 6001-608 (1970). ?NTROWcTION to the corrclu8iorr chat the ioitl8tlm rtep is the OD.~sensitive to X-irradiation. putthemor% Efforts In molecular rdWalo&w an divided we found chat X-irruli8t.d RNA polymrua hu Lnto three tnJor ueu, all of uhich Involve both grr8tW tadency to ln8ort the bue radiation studirr adthe errntid hdrratd "wrong" into MA than doe8 mirradiated en-. In mother invertig8tloru that yinld inforrtioa rehtlve to study, ve h.*. evidence that ultraviolet irrdia- which rdiatiw data becou r&&ful. The three tioa produces cwdeut bond. betvren RNA polymerua areu of rrrearch are r0d.l WA. genetic Inform- and UNA; the utility this che isoh- tion transfer, .ad chraatln structure urd the of reaction in tion of DNA binding sites ir being investigated. In iitotic apparatw. The objectim of theoe reudies another direction, we have investigated p8ruten is to understand the rdlobiolog%cdrchanirn for of the initiation of RNA synthesir by deoxyribooyl inception, progress, and repair of rdiati~ndme. ollgovrr These oligaerr are covdently incor- Synthetic wrk, utillzln~ptocedurer both from . porated into the RlyA chain. ?remace of the deoxy- organic chedstry and enaymbw, hu 8upplicd :ibavl olfgowrr in the reaction, by clrcumenting oligo- and ?olydooxJrribonucleotbhr to replmish :he norarlly rate-limiting scep of IniitlatlO~, stocka of arch needed materials md to provide new greatly stimlotes the race of RNA svntheslir. The base sequencer. Nuocrical analysis of pl pen- aunt of stimulation is a function of o1fgo.r car- ation chromatogram hu proved to be a valuable centration. substrate concentration. and metal ion procedure In all upecta of the preparation .ad use cofactor used. 3f dlDNA. The rsjox requlrerntr this y8K for the arua of chromatin structure, experimnts radi8tion ad truucrlption studie6 h8ve been homo- In conducted during the put year have clearly de- polyucleotldeo vith a variety of established aver- strated chat scructural alter8tioar; of hlrtonu by age mlecu18r weights. Reaction of poly d(t-T) acetylation, methylation. and phosphorylation occur with osmium tetroxide has convcrted part oi the at defined times vlthin :he cell cycle of cultured thymine t 9.1. to the 5,6-dihydro-5.6-dihvdrO~ mamallan cells. The effects ionizing radiation structure chat is one of the products resulting of upon these reactions cultured cells show th8t, frola tne action of ionizing r8dhtIOn upon iJSA in in vith the decline of SXA and histone blorynthesir maerobic solution. Primav attention nas been sever81 hours mst-irradiation, histone aC8tyhtiOII focused sn r8diation damp occurring at constant mcrh-iation tease. 3csults of these studies lose but sitti chmging concentration of target and support rhe nypothesls that rdi8tlon-induced divi- m1Pcule in order to find out the rchmisr of sion delav results from a blockage of cells in the dam8p. Ze h8Ve sum dat8 that SuggLst COnCOplt8nt C, porcion of their cycle. In order to test direct and indirect action for both bue daMgC and - life the liypotkesis that irradiation of -1i.a cells Aiai II t reaks. destrovs a component or pocential for diviaion, Tfis Pear In studies of effects of r8diation on standard iicrocubulc assays have been adapted for genetic intonution transfer, we have continued the with cultured sells. Investlgaciona ertab- lnvescltationr on %-irr8di8t.d XVA polvmerrse. The use have llshed tire temporal ?actern of fncracellular micro- 9,: :alues observed for fnactfvation of enzymatic tubule prorein levels in svnchronized cultures of activit:: varted ulth different teoplate molecules rmaarlian cells. Ividencr has been obtained to isea in :he assay. In so= cues, observed in- indicate tt:ac the levels of rakrotubule protein in activation also was dependent upon substrate con- X-irradiated cells follow closelv that of other centration. Through a combination of experimental functional proteins. approocnes in vhlch sa separarelv Studied the Ltfects of S-irradlatlon on the abllltv of R!!A ;clvmcrade co bind tenplaces, to initiate iWA .hains, and to add onto chains already started. we

L,' SYrn&SIS m IWlIOSuisITInTt 01 mat DWI evaluated ua&r toot cwditiau to tbae which wadd be used for diester bod folutioo. (F. 3. Hay.., D. L. Uilll~,R. La R.tllff, C. %IO ny protectin grwpo for ph~ophate,~'~ hLiSi, C. 1. F~tL,U. B. Cod, D. E. HO8.rd. U. which 811 dasigued for r8pid isolation of the pw- Hollsteln, E. L. Xartinea, V. E. !4ltchell, and E. C. ins protected oli~anucleotide,will also be *vd- Wilmth) 6 urted. The g,~4iuthyl-~-pharyledrinowo~q Chemical Smmthesis ribonuc1eotid.r of OUs- randen the protectd oli~onuclaotid..electively Al part of our continuing program, horoollw adsorbable on A&erllte 11-45 rmsln, urd the ua- d.oxyribopucl.otidu of q specific si- am rye- reacted incod- nuclmtid. vith free phmpbate CUI thesiredl fra lonoauchotida md ud. aV8Ihbh be wuhed out, toether with -10~from the coo- 7 for radiation studies and fdrrntal biologicrl &sing agent. The 2-phenyL*ruptoethyl croup investigatiotm. llay of there polpwa ue bpt in iqarts to the protactd olfgonucleotide product stock at lou taqberacure. OUfodeo~nvclcocides of sufficient affinity for bmroylated DW-callulcm knoun bsse sequence are prcpared by stepulse chew or DEAE-Sephdex that unreacted mnonuc1.otid.s and ical condcnsacion reactions2 using functional block- by-products with free phosphate Rroups can be eluted in! groupe for duo group. and for S'-phospnue and first from the colmn. 2-Phenylrr~~ptorthyl5'- c3 -hydroxyl group.. where applicable. Protected di- thymidvlrte has been prepared adpurified for sta- ~.ucle~tiderare rynthmised and are either chedcallv bilicv evaluation bv our testing mthod. polymerized to obtain polydirrs or are used in the The extrerlp good stability of the 1.1.1- 7 synchesf* of crilgrs- or longer oligollucleotides. trichlor0-2-athy1-2-propyl (trichioro-rcrt-butyl) Chedcal polymerization 3 of the trimers, Lollwed by derivatives of nucleotides, under the coaditiouo of Mion exchap chroucography, affords poi::trimrs diester bond synthesia. is probably due in large in the size rmgc from he-r to dodecrrr in addi- part to steric hindrance. This char8cteristic of tion to the larger unresolmd polymers. the grwp is also apparent in preparation of the In :he stepuise chemical synthesis of oligo- protected nucleotides. AS illustrated in Table 1, nucleotides o€ knm base sequcnct, i€ purity of the rate of applicatloa of the trichloro-=-butyl

5ue sequeace is to be nnintained. the ideal protec- group 15 slou, and yields are poor to fair due to tive group for phosphate should rain intact during side reactions. the condensation and isolation steps md should be aemoval of the trichloro-=-butyl group from removable at the dcsired tiw without alteration of nucleotides is effected by reduction with zinc/copper ocher parts of the product structure. The widely couple in dimthvlforumide solvent at 50' C. Re- $ sred ?-cy.noethyl group does not net these removal is ementially quanticatiw from the amo- quirerota, since i; is pattiallv lost during the nucleotides in 1 hour (see fable 2). Rem0~8lIS chemical condensation step and partially removed less nearlv quantitative froa the ollgonucleoti&s during subaoqucnt hydrolysis of the )'-hydroxyl- tested but appears to reach o maximum. This appar- protecting group. Uhen two different nucleotide ently is due to fornution of a zinc salt insoluble bores are used in preparing the initial dimr, the in DMF, since the residual material is reduced when first mntioned loss o.v result in contamination of recovered and converted to a soluble trialkyl- the desired sequence ad applies to mny of the new anaonium salt. During reduction of a fully blodrd protective groups for phosphate which have appeared nucleotide, amino blocking groups such u benzovl in the recent literature. Haferrr, in no case hu and anisoyl are partlally lost. This des it n8c- the degree of retention of a suggested group, under essay to rescplate oligonucleotides concaining the rtaction conditions, Seen deraonatr8ted directly. Ye base cvtosine 5efore further condensation or chem- have evalrut.d5 a number of these grouoe which W- ical ?olvmerization reactions are carried out, since >eared to have satisfactory removal conditiom, tD- the cvcosine amino group forms phwphorrmide linkages gether with Y new group of our ovn design. Our new with nucleotide phosphate. This appannclg fs not group, l,l,l-trichloro-2-mcthyl-2-propy~, hu proven the case with guanine and adenine' (i.e., the now to be superior, in term of retention, to all those blocked dno groups are not involved in the above

fUSb442 13 TABU 1. mm01 UZEU W Rpcl.oub. LllllLol* 8-- T- (-1 I#k%Ln d*- 0,s wcb 2.5 3 28.8 0.5 Dcc 2.5 5 34.7 0.2 DCC 0.5 26 69.6 0.2 Dcc 0.4 26 61.0 s.0 mC l0.0 11 56.7 4.7 14.1 25 34.4 3.6 l8.1 26 3s .6 0.2 0.4 23 37.0 d -p&hc 4.88 #x: U.0 28 40.8

100.0 99.2 1 96.5 95 .5 2 d-PG 100.0 UO.0 2

d-pC 92.7 7.3 1 87.1 6 .l 1

51.6 44.5 93.9 59.2 24.8 96.7 67.9 22.4 100.0 Of ad m, Tdh8 1 ad2). 'ZhO fact that the ribooudrotid. occur8 intemUy YU 8tntd by nwt.rt-P.i-t -1p.S W4 -lad dvribomuclmide tripbolphcu d by rise dlatributioa before adafter &&&ne heal- pBlS (Tab 1 .nd 3). The dadribo- ad da~ribooucleotib.pdp =?a have bo- wed 88 t.gktu for WA po1prW 1. 8. C. d J. P. ViZ#dtl, Studi.8 pol~tiaW.v111. Lrp.rtuu ontbe from Eseherichi8 &. Both the deozyribmuclotid. polym8tituicm of -ti&8. 4w ad ribosucleotid. ue trmaacrfbd, 3Vh8 8 ribo- prepurioo d omparatiom of liwu thymidine pol-cidw. J. Arr. Cbr. SOC. 83. 675- nucleotide polymr vi& nucleotld. cootat aidlu 685 (1961). to the origid dprd polyrr (Tat 4). 2. S. A. nU-8, 1. U. JUOb, ad ?I.C. I[hO?a, ~lthougbno att.qt ham bean md. at thb tin Studies polyuwlmotid... mf. Roxytibe at to deternine if the polymerme from thv pol~uclrot1&8 co~uioio~repeating trlaucleo- DNA cdf tide ooqrwocu (4). Pxuparatloa of suitably will replicate the rixed ribo- addeoxyribooudeo- protected trinud.otid.8. J. kr. Chem. SOC. tide polmrs In the abamu the -89, 21562166 (1967). presence or of proper initiator, it vi11 be Interastint to c0lp.m A. Marang, T. C. Rhorum, 3. S. U. Jacob, d 11. the resulta vith previw experiuntd wod The Stdim OQ pdpWhOtib8. =XI. lhoxyribo- polpucleoti&s caotalaln~reputin8 trinuc1.o- mixed ribo- and deoxyribonucleotide polyra 8bOIdd ti& uquencaa (5). Ibe polymerization of also h8ve nom value in detenrinlnp condltiorrn for protected deoytibopolynucleor1d.r. J. her, Cha. Soc. 89, 2167-2177 (19671. chain rloagation (addition to olfgonucleotldr) .ad replicatlon of polydeoxynucleotides by DWA pol-I- 4. C. II. Tenor, 2-Cyau1ethyl ph0oph.t. .nd it. use In the 8yntbeaL of pho8ph.t. esters. J. ues fror E. & rod Micrococcus lutetu. If ch8in Arr. Soc. 83, 159-148 (1961). Chem. clongacion by the dove two enryrus depd8 entirely 5. C. R. lichmnd and C. L. Voelt, eds., Annul on the fiat feu nucleotides at the 3'41ydroxyl -port of the Biolglcrl and )(.died Research terminus, then the ribonucleotides In the piud Crow (I#-4) of tho USL 8ealth Divislm, Lm Alnoa Sdentiflc Laboratory Report U-4923- polymer nhwld only exert ma effect upoa c8tdytlc (19711, pp. 36-38. PR reaction if they are 8C or near the 3'-hydroxyl 6. T. Hat., K. Tajiu, and T. Waiyar, A simple terminus. If the riboaucleotides are at the 3'- protecting group protectton-purification hydroxyl terminw and chain elon6uion le pmvent.d, ''handle" for polynucleotide syathe8ir. I. J, Arr. Chr. Soc. z,492049930 (1971). then replication 8hould take preferemca over chain rlonytlon. This rlght h8pp.n especially If the 7. S. A. Saruy, 0. S. Bhat. J. Godchild, R. H. Wightma. adS. K. Dheer, A nn Wner8.l method ribonucleotide is not complementary to the deoxy- for the synthesis of phosphaccprotected deow- ribonucleotides [e.& , d(An,rCd.d(Tn,rCm)l. ribooligonucleotidu. ZV. J. Arr. Chem. Soc. 94, 61836189 (1972). Studies on the effect of ribonucleotide coat*nt in - A. Nar-8. 8. S. K. ftakura, and R. H. UlRhtmn. the mixed polyrrr on the above ra8ctlons, U we11 A silplificatfon In the aynthe.18 of dcoxyrlbo- oli~onuclaotides. Cmad. J. Cher. 0,769-770 w one-fold or ore replication of the Qoxyribo- (1972). nuchotide8. c8n also be determined. Additional scudfen uith d(MT),'d(ATT),, have Ft*utic Smthule been done, Acre the transcribed rlbopolyar Synthase8 of Pfnd rib- unl deoxyrlbonucleo- r(MU),,.r(AUU), has been Isolated after Pacreatlc tide polymrs have been accoq1ish.d 1 using tarrind War digestion of the deoxyribonucleotide polywr. deoyribanuc~otldyltraluferueand an oligdeoxy- Circular dichroism spectra of d(MT),-d(ATT)n and riboauclsotide laltiator. Dlpending upon the ratio r(MU)n-r(AUU)n have been determined by Dr. Daaald of deoyrlboaucleooide tripho8phate to rlbonucleo- Gray (University of Taxu at Dallas) .ad comp8red si& triphosphate, the percent of rlbonucleotfde In vith their respecclvc isorrs: dAn'dTn, d(kT),,*- the mixed polyrr can vary fra le8s than 1 percent d(ATIn, rAnvrlln, and r(AU);r(AU),. This is the to as uch as 38 perwnt (equal molar concentrations firat instance here such a coopariaon hu been f 'E;1 ay, L; 1 0 5 r; TABLE 1. !4EAREST-#GImOI MUYSZS OF d(An,rNmIa Ribonucleoside Percent of radioactivity ia triphoophate nucl conceotr8tlon

10 0.1 99.9 99.7 0.9 99.1 0.3 lo5 to 1 140 120 102 0.1 99.9 0.4 99.6 0.7 99.3 10 to 1 4 140 120 lo3 0.2 99.8 0.9 99.1 1.0 99.0 3 lo to 1 110 100 104 1.5 98.5 5.2 94.8 1.6 98.4 2 10 to 1 115 20 lo5 u.0 87.0 37.3 62.7 4.9 95.1 10 to 1 62 4

.The reaction d.ture8 coataiad the following CQ- pal ts in a totrl volu of 0.2 rl: ror, rn1.S %e reactioa dxture d88Cribd in T.bh 1 WU 3-3%dMF+D lo3 pmolms of the he- of 5'4eoxy- scaled UP 100-fold for preparation of d(h,r . thyddyllc rid, do .H, potueiu cacodylate (pH 6.81, AIter Incubation, the reactloo dxturer Yere%- 8 -12, 1 mg 2-rrcaptoeth.lwr1, 200 tmits ter- proteinired wit4 isowl alcohol ubd chloroforr md ad& d.ox]rribanucl.otldyltrurfer.re, and from 10 dialyzed ap1ll.t water. One-hdf of the i8Oht.d to Id pmoles rlbocruclealde trlphmpbte. The dx- polymer vu hydro1yr.d with 0.3 8 ICOU for 18 hr at t-8 were Incubated at 37.C for 2 hr, and neareat- 37.C and than neutrnlizmd uitb 5 E HC1. Lenshs neiwor anrlyser we= & erdnd u previourfy ac peaks of absorbance ven deterdsed for both un- described by J08s8 s&.' and Ratliff st. treated urd dkallnctreat8d glbodeoxyribocopoly- lyrs as previously described. TABLE 2. NEAREST-NEIGHBOR ANALYSES OF d(An,rHm)' TABLE 4. SUREST-NEIWBOR ANALYSES OF THE -0- Ribonucleosi& Percent of rulioactfvlty in POLYSR TRANSCUBED PRDH d(AnDrNm)a A-32p- ~d!oactivity in ribonucleoside labeled 2 - and 3'-phmph.t- (x) -RNA triphosphate 2 a 10 0.b 99.4 0.1 99.6 2.1 91.6 lo2 0.2 99.8 0.8 99.2 0.9 99.~ lo3 0.2 99.8 0.9 99.1 1.3 98.7 lo4 2.2 97.8 9.0 91.0 3.2 96.8 r(Cm,rVn) rCTP -- - 28.1 71.9 lo5 15.8 84.2 38.1 61.9 7.0 93.0 rLTP - - 26.1 73.9

%e conditions were identical to tho8e described in Tabla 1 ucept that each of the ribonucleoside tr'f- %e reaction mixtures containd per d: 00 moles phosphatw was ldelcd with A-~~P. of Tris-RCl buffer (pH 8.0). 4 -18s wlz, 1 L- mlt khtrcl?, 12 ..moles :arrcaptoech.rrolr 100 -1- possible wing spectra of double-strand RNA ribonucleosi e rriphosphotea, with each labeled in se- turn with a- 41-P, 0.5 A mlts of the mixed rlbo- quences, and the agecmt between measured and deoxvtibopolyncr, and 388 units of RNA polyrrue. approxim8ted spectra for r(MU)n*r(AUU)n is surprin- The reaction mixtura were incubated at 37.C for 6 2 hr urd were terminated by placing In boiling ingly good. water for 10 mln 8nd dialyzed against 0.01 2 Sodium The d(AAT);d(KLT), circular dichroism spectrum pyrophosphate (pH 7.0) ad then distilled water. The polwn were hvdrolyzrd with 0.3 E KOH at 37.C hu provided the first independent test of the rpec- for 18 \it and neutralized with 5 HC1, and the trd procedure of Allen for obtaining first- mnonuclegtides were separated by paper electro- s&.7 phoresis .- neighbor frequencies. In this first independrnt test of the procedure* ve found chat the spectral 2. J. Josse. A. 3. Kaiser, and A. Eornberg. analysis does Infer the existence of a avjority Of En?* at ic svncncsis of deoxyribonucleic acid. Vl11. 86 percent d(Ap.4) I(TpT), d(ApT), and d(TpA) first- Frequencies of nearest neighbor ba8e sequences neighbor a. in deoxyribonucleic acid. .I. aiol. Chm. u, 864-875 (1961). REFERENCES 3. R. i. Racliff. A. 1. Schw8rtt. 0. N. Kerr, D. L. Yilliars, D. 1;. :tt. and X. Wqes. Hetem- I. H. t. R.tliff, Synthesis of mixed polynuclee F. tides containing rib- and deomrlboaucleo- polydeoxynucleotides synthesized with remind deoxvribonucltot idvltruu€erase. Surer t tides bv teminal dcoyaucleotidyltrursfer~e. 11. Federation Proc. a,425 (1972). neighbor frequencies and extent of digestion bv tb. 0b-d ~l~uti-proti& vith ita nsocibtd (.~certaintIomcould bavo boon producod by a raw 4. of 8-h caqcD.ltiau md hctfro tha r.ly. thao that uo probdlo by pbyaicd critula. So fuuo bm .ppW nro tiid. of criteria. The 5. 1-t pnjoaid 1. to Uptkl.0 a -ut. of .1wthw Of WbCdU a&#t df8tributtm in tho 0-h; th%8 mt. to that tba p-8 that opurtr tho rP1.cul.u might dirwath ccmtiaw in tho .an of not ehuply 88 ktuoa 0 polymr of N MIL.and OIY of tho nut 6s hidor .Id 1-r ald8rO. Tho 8OCdUO. Of ~.ta100is to utth. foa of ~ienrluwoiQt distributim to that wnaratod by a rpdfic model Of tb0 prOC888 pU0r.t- it - b+~Jug.Of th. 7. polymr at randam polntl in tho CY.of Q ir- naird 8-10 - a eaeraliatioo of thm m(Lchali84bntea prows In the -8 of am18 p0rat.l by tba enzyme, cerdoal d-rib-1- Humrierl Analysi. of c.1 Pormatiorr Chmtou.PI tidyltrawforuo. Tb. cod clam8 of difficultiu Uu in defln- DU8 frar t.1 pOmatim chrarrtography arC beins rued to derlm roleculu riu dirtrlbution pat- log tho lndividwl elutioa profile8 of ucb ox- pected 8p.cim. Thro, of courmo, hmm to bo 4- LOI~Y for polydwxyribaouc&otldos. Sor of the lbntad by diroct arporlmtlr deterdnotiolr with kin& of 8qhfor which .ire dietributlow an bag.a.ocw oloeulu ersplu, but for tvo MOM valuable aro thao avly Ioohtod, thore kept loll8 it fr not femible to roly antiroly on 8xpennt.l in atoro8e, or thae 8ubjactrd to lswm rourcor of data. b the firat place, it vould mqam UI -88. ii-vor, -1 p.nuatton cbroutopaphy oomt of exporlaatal work; in the see- suffers from Incoglot. ruolutlon beoroar frar d place, if rgrlficatiai of the crrora dircwrd =ate of vulw L.n@hr. Nmmrthelo~r, If OIY dove ir to b8 dnirlud. It I8 uomtl~that the Imwo tho elutioa profile that each of tho upectod form of idividd elution profiles vllw COQ- w1ecul.r speciaa givmr vha prosat .low, la riotonrly rad ucuratoly fra OIIO rpedu to the prlndple, ho CUI find tho suporpcwltioo of tbr noxt. In the faco of oxperirrrtrl uncortrintiu, necearary to account for the oboorvrd elution pro- this latta requiromnt i. mot ndily mt by file of my 8.31pl.e of iatorut (that la, ho C.IP infar undrr8tandla~the rchd8u that dotermino tho ita cogositiod. 8h-e of tho profilo ad, thonfore. hav it. form, TO CU~thl8 throu@, tvo ClU.08 Of difficul- of noaority, varies vith m1ecul.r rite. eiu haw to be dealt vith. first, if OM trim to U. hmM& a detailed ~1.1ysI. of couplad find preci8oly utut 8- of overlappia8 contributions hydrodyndc rad dlffwlon ptocoswr by which ml- ave a particular profile, uncertalntiu and ir eculo. are trwported dam the colum, thm pncisiolu in definition of tha prof110 are SO ug- the cubing .Dd monitor, &ad into the colhcting nified in the ruult u to defeat the effort. One test tuber. The gel perticlam are rufficiontly hu errors of mauuronnt, uncertalntiea ira SMll (-100 microam) that, for the raga of poly- definition of the prof110 by a limited nder of nn of interest hero, each molecule r.9188 the finite fraetioru, and errom In definition of clu- volrr accooriblo to it rufficiontly oftar that ita tion profile. of indlvldd rpocieo prosent. A di8prr8ion dong the colum is C.wri.a, the man- typical coqomition inforred in thir way c0nai.u squm di8pormnioa being tho s- of four torr: one of luie porit1-n mnd nogrtivo nuborm of adj8cont Ln&pendmt of mlcculat dlffruim, 00. dopendent sptciu. Thorefon, w r~tinstead corrridot that

10Sbk45 17 It 8Urp-W the tbu qtUt%mCOUUhS 1-r tom Xd4tiw Cf ad co; if, 8CCO- tO Ow- tion, inrrivuiar hd accurrvd aat&mly thr#yb iu- ditrct .ctlom, tb. dtm of C obould h.rr becar meat .t hw vduu of Co.2 One vq to intor pmt the ~uult.ia that tho Unur tom in Eq. 1 la ldth dinct actlm d thu the om- tirl tam -N \mipoll? that part of the -ti** tim d by lndiact actl~o. Ihr &dated for C ro. rlwly throu$b Co vduu of 500 to 1ooo tho may V8lm of c in that rmw vn 2.26. bl p.rution chrovtoir~rW baa ob- for mriw rise dhtributi0IU Of #yIbrtb.tlC poly d.Oxyriboarcleoti& both befon d after X- Irradiuiom. Thao rob1 DUh hmtho folloUiw

S8qUO-88 poly dA, PO17 a,POI? d(A4) I d Wy d(GT). Find dysrof tho &ta udt Colpw tlm of the caputer prgrr d.acrlb8d &owe. lidnary dyai., u .xpoctd thro~&8tr.nd brulr- w, not only 8h#r that X-i?r.diatioo haaOM tho aithmtic mrn of a 8iw dbtribution &ad dau 3 thio aorvh.t In proportima to tho dou but that x-Irrdiaticm at conacme -0, quite inUtutin&. the rff~ct Irrdiatioa studiw hmm bo80 urmdod to dnQ, coatinuas. with dmat idontical dasee of blrrk.-, Poly dT, lad pOly dn tO f&Cilit.tO intOvr8t8tim Of *won up to polymer conuotrati~of 6ooo wa~-@. data that rr hmalready obt.in.a frol poly dtA4) This bp.ad.na i. quite difforrot frauht vu ad poly d(G-T) . Thu far, vo hove urriod out obrrvod in radiation-induced loma of chrolophot. Ultr8ViOht abaorptioa early808 on .U aqloa ad incurrod by d"W. Tharefora, u caacluded oxclu- 11 perration chrortwapby a1 T. atudioa 00 the aimly ha the temt of coauntratian dep.lldmcU, polpara. Cmtrolled Qadul alteration of d(C-T)20 atrand bn4Fo aolurtoa laryly ruulU from hw yielded products cont.Lptly kaom atmat. of 8 tho diroct affect. Nrther elucidatioo of chi8 type of bue dug. fornrd after irradiatiao. u-nt vi11 be amt valwblo touud incnued Uo haw ramrod tho churl;. in ahorbaa of udontrding of the effoct of ioaisiag radiation - 267 sm p.raw solute concentratiea. qma nucleic acid.. Con- &t CWtMt 25 krada of X-irtdbtioU. On0 identified rjor product of rdlatioo- rrrrr of Co vu 18 to u)oo Tho coacrntration inducod hydroxyl rdicrl attack upon the thydam of dTW that bd lost it. &-om, C*, wr fd ring in DNA mder murobic codltiom ia the 5,6- to incroue r.pldly fra t8ro urn8 tho lw8.t dihydro-S.6-dihydrory derivative .' By -0 of 5 Va1ul.r of Co .nd then to Y8Ua Mud Un8U -dum tetroxide in nmatrd phcwphate buffor u incrow.. The Unur portion oxt8nd.d bolmr~C 37' C, w ha- capvortad d(G-TIza eo product8 that 2 vduu of 400 to loo0 e. Tho rapid cham of C am idontiully hydroxylated, d(G-T*)20, .Id in €or Co mlw8 hathm 400 rCn_ i. In accord with vhich -tying parceat8pr of the thydno bum hm exp8ctrd lwering of tho rullcal ceacontrrtioa by boen altered. Go1 perration chroutography rhd 1 tocodirution at very lw roluto c~ncoatratl~na. thrt vmy little if utf 8tr.ad break+ occurrod. An oqucltion that ia tho leut-sqwro8 fit to dl the It la knwn that the -no ring ia not attack4 4Exp.drsnt81 point8 i8: by 0a.i~tatroxido.' The producta wore .nrlyxed for both ultr.viol8t ebeorptlom urd phwphonu con- C* - 0.0296 Co + 35.3 [l - exp(4.0175 Co)l- (q*1) tent fra which were cilculated c(p) nlwa; tboae 3.

UDWIoll APD (SPltRc X~ImTRAlasm

(D. A. Sdtb, P. N. 8.JnS. A. Me PkrtaS, V* E. Mitchell, 11, L. btliff, ad C. F. Stmiat.) &plicatila

fk h.rr mod dfthylur DUA polmruo to pm- pus synthotlc polynucl.otid08.~~~such pollwa hove boon wod la smml kind. of studio# including, for example, iavorti&8tiO~b#into the phyaicd .rd 3.4 tqlate propertiu of polydooxyrlbooucl.otida. The lack of nuclouo activity uaociatd dth cdf thyua DUA polmrue and tho strictly r.pdt mp- licatim property of ita catalytic utivity uk it idoal for preparation of del doub1e-atr.od.d DM8. However, the lability of thia 0- in storap 8nd tho cwridonble oxponao, ti-, and offort entailod in ita purification have caused ua to look at mA polywruor from other source. u porrible replace- mat. for calf thyua Wh polyuruo. Yo have IC cently dehighly purified prrparatioru of DNA polyuruo from nierococcw luteua. Tho ability of thia onz- to porfon accurate reprfr rapllutiw hu been dorolutratad in preliminary studio# (so0 Table I). Accurate rapair replication of pol9 dT in the proreme of a dA5 Initiator took place at

19 bedmot 1

91) 88 91 l?merirat 2

196 176 90

Erpetirnt 3

98 0 0

4. D. A. Sdth. 0. N. Hayarn, A. n. Uutinu. Re Lo &Cliff, rrd 0. L. UillfumCOOUrnhg the role of pyriddiaa clwten in trarcriptim. LIEX Abatncta of &e 160th htiord -tin8 of tho Chdcrl Soci.tYm ChiClrpe IUbda (Septder 14-18, 1970), Abbrtr8Ct No. Biol. 63. 5. C ad0.15 KCL At hwer tqaacurea ad lower KC1 concentratiau. “O*.r-repUcatioo“ vu wuallp obaemd. The ability of the e- to perforr accurate repair replication of -re cogli- cat4 raplacem ia nov unbt iaveatimtioa. wclroti& in thm RNA drab. Subwquant adeotibu hi additioa, we ue atudyin8 .OI other prop- are dded to the 3’4ydruxyl poup of AT? or CD, ertiea of s. luteu DkU pol-rue. A aitrocellc cdtbe IWA &.ia la enddby thia rch.nl.r. loae DdrMe U8.I for caplax fOrVtiab bat-= Uhan lplA haopol-ra nm u trphte for lslr the enspa .ad EUA hu been danloped. The ability of tha ea- to biad d(A-T)n*d(A-T)nm u vel1 Y ulate the rate of WA rynthesla, md thy can aene the influence of iooic atrenNh upon the amwit of to initiate Rw chdna by beins cov&atly bCot- 2 polyrwr bound, hu been ruurd. The binding porated into the grcwina BHA chdn.’ Strut =&. reectioa is vew aaaitiw to imie stn-h with recently obaemd thu c~lemnta~&oxyribmyl the mtof d(A-T)n*d(A-T)n bd&cnud by 50 perceat u the KCl coountruion in the bindinl thoudrt it urlikely that deoxpribomyl 01i-m v.Il buffer is raimd fm 0.05 to 0.3 E. V. have d80 cwaleatly Incorporated into the IINA chdn. Ye hm initiated rtudlu on the rolo of sulfhydryl aroup8 inveatl~tedthe prrarterm involved In 8tImAAtim in biadba and catalytic activttiu of E. luteum DU of UNA rynthria by deoxyriboayl olimrr md the 3 polyurue. Re8u.h to date have aham that 8- quamtlan of their covalent incorporation Into UNA. sulfhydryl re.gmt. do aoc inhibit bhdhg or activ- Deoxyribothyai&ylate oliuarrr stimulate poly P ity. Them r88vQt8 include pchlorcrrcuribematr rynchwis directad by poly A or poly dA ad cat- and t+thy~le.id&. On tho other had, sa10- dyxed by Emchorichia & RNA pol~n8e. The organic ruynta much u HyC12. which =act with awmt of rtiualatim observed is a function of sulhydyl groupsmdo inhibit C8tdytiC actirlty Of oligtmer concentration, submtretc concentrationn and the an- but not Ita ability to bind p0lydaOrY- =tal ion used. Figure 1 sh-8 stldatiolr of poly ribonucleotide#. Since rather hi& conuntratloaa +directed poly U synthesis by d(pT)* aa a functim of these inormlc reawnem .re nquind to ptdducr of UIP coacentratlm. At lau UTP ConCentraCfOm ciU effect., it yy be that the obaavd ilrhibltiOU is due to SOY w&.Ptrr other than a reaction with the stlulatory effect of d(pf)g on the ructim i8 32 putative polyurue rulhydtYl groups. hifiest. Other experiments which utiliud y- P-UTP I I 1

Fig. 1. pig. 2. Dtll-CoUulwe chrautopq~of the hydro- lysate of a d(pT) -8crlurrtd poly U symthuis reactiok A d(pT)B derw did to the owtrJised hydrolyute prior LO chmutwaphy. The rdl-ti- pulr that is .lutd after the d(pT)g la pruud U subatrate rhamd that hitiatim of poly 0 &.ins to be d(pT)g to vhich ddltiod tdti- with UCP is patly decreamed when oligodeaxythyd- actd pup r-na atucird after rllulisr hydrolyals. This pulr vu not seam in re=- dylatc is Included io the reaction Direct mixture. tiolrr uhich did not contain d(pT)a. &dance for oli~wxythymidylatrlncorporatim into poly u chdp. VU Obtaiod in the follwlag V.I. poly A, 4-ms asid IWA polyurue were incubated in 1. S. K. Uiyd and A. Stem-, Stdiu of the tw reactiom, me of uhlcb also cont.iod d(pT)8. ribooucleic acid polmerue fra Ea.cb.ficki. The ration dxturu were hydrolyzed in 0.3 at coll. Ip. Effect of OU~&Otidu 00 the -ribonuekic uld polmrue mrctiaa dth 8m- 37. C for 20 boun md mutralird, and the hydro- thetic po1fibmWl.Otid.s u t-l.tm* J* lysatu ware chrautograpbd on DEAE-cellubae. The Mol. Chr. 3,293-2598 (1965). raactim which conmind d(pT)g gave rim to a radio- 2. P. A. Straat, 0. Pmp, d P. 0. P. Ts'o, RIU active peak Vith chtoutographic properti- expected polymar... from nicmcoccua lutecr: caqrr .ti*. effect of ribom71 ad de-rSb-1 oU- ,f d(pT)gprUp (Pig. 2). Such a peak -8 not a- in 00 the h~l~rd1mct.dmUtim. the other reaction. Ye dao haw sdusi8il.r Biocba. Biophya. BAS. C-. *, 905-911 (1971). type8 of evidance that much loa-r poly dT ala0 semu to idtiate poly U synthaoir. In SOI re=- 3. D. A. Smith. A. X. Hutinea, md D. liu, Initiation of in trftro mu rpLJrfl- uia tie, a11 poly U chain# appear to be initiated with with oligodwxyrlboaucleotidu. Io: Abbrtracts deo~thymldylate011-rs. 3ligart inltiatioa of of the U4Ch Natiaml Xntiag of krricm Ch8dc.l Society, Nff Yo& Citys NwYo& RNA synthuia smto occur Indepmdontly of tha (krguat 27-Septe.bcr 1. 1972). Abbrtract No. 71. preaance of the ai- subunit of RUpolymerase. if my, DUA The role in vivo, of hiti.tion of Effects of X-Irrdiatiaa OIL IpeA Polmrue spthui8 vlrh IopA or DIU oliaora ia not k-. In the studies of P0118.d .nd c-*t.D1 it Houever, this reaction swgeats the pom~ibilltythat hu been ahom that controlled trwcriptim in dvo in vivo inftlatioa of RNA ryathmir liwt occur -- in the bacteriu Escherichia & is aemitlve to sort-. at 8ingle-#tr&nded bre& on DNA and thua ionizing rdiation: furthemon, itvu p0#8ibh to produce DW-IPU covalent hybridr. The occurrence correluc from critical target dyai. the radio- of this reaction &o requiru that atstudies of ulvitive structure rerpoaribh for utilization of in vftro initiation of IPJA synthesis be reinter- -- the RNA pncuraor uracil with a structure sirilu in -JK8Led. size to the rPlA polymrue molecule. Accordingly,

21 we mdartodr rn l.me8tiC.tIo of the radtawitivlty of thU rtrcr effect. of x-irrdi.ti00 up00 mu polmr mme fmth-0 mtr& of 5. of varyi- radio- SOMitiWkb8 hm k- bruti-d 4 of harogolyna rd a ou0r.l ddl.-.tr.ld.d PU u tql.tu.'-' ~h.rdiotupamo of -tic actitity of tb. pol~ranvu kd.p.crd.ot of the rdi-itirity of the uLl fra uhich it UY YP tracted. Dj, vduu ob- tuied according to the tqlue decul8 wd. mi. +.ri.biUg In dou-reapom. of tha mqm could mt bo attributed to .II effect (R bmof irrdhted 8nWM to the Fig. 1. traplate molocde; D3, valw for iructiv8tlao of binding to poly U or poly dl uora 4 to 8 tLrw greatu tha corrupooding Dg7 vdua for IPICtl*F ti- of enspatic activity of the e- (tip. 1 pod 2). Uhm irradiatd emYY uswad with rlbo- horopolynr tuplrtu, there vu submtrate &pen- hco of inactivation (i.e., DS7 drrm for irmcttvatioo of umyn activity of tha RUpolynr- Ye &anted u submtrate coacentration fir the reaction dxturm vu varied). At lw substruo &vel. where the IDitiatioo rtq, of RltA synthe8is 18 rat- liritin~,rdioaoaaitivlcy of the an- im h-ut. With homodamyribopolpt t.3platu, a aubatrate Indopadeuce of iMCtivatirm vu obmened. The ability of colphuntaxy ribosyl pentmra to bypus chain initiulorr rrd to stfrulate poly A rptheaim In poly U- and poly dT-dincted reactioa8 YU eumtnad. Utlli.4 thlm methud of byp4eaing the ratclfrlting step in in vitro RIU syntheeia. the in8CtintiOo of -tic activity by X-lKKdir tion YU inv8mti#atad. Tba Sub8tr8t. CmlCeTltT8tfm deprr&ncr of D37 vslun for howrlbopolyar t- vu 3 to 4 tlma vre praoocmad In poly dT4-ct.d plates is e1idolt.d uhe theme initiator mloculu routloo. caqumd to poly U4irected reaction# (aw uc included la tbo nutlocu. Furthemore, the 3). Lt I8 not Loam if damage to RNA poljrrr- 1rrdiat.d cnayn. vhen umqrd with hd.oyribo- Pig. me is II Importat effect in dvo of X-irndirtion. polyrn, becm lea. rdlwenmitive in thm pres- If 00. it MY be thar dncreued fidelity of traa- anem of oligcwr prkrn. These remults mcription cdd =count for 8- of the delay4 that the initiatiw step I8 the mst rdOS8Idtive effect8 of irradiation. in the prOWS8 Of RNA SyUthLSb. The fidelity of RNA spntheala by X-irradiated A linear inmue RNA polyneraae vu .to examined. 1. E. C. Pollard, The rffact of loairing rdiation in insertion of noncapbmntaw auclaotidu into am the rol.culu blolw of Escherichia a. -In: Current Topic8 in Ldiatioa Research s, poly A am a function of dose for borh poly U- .ad 51 (1970). X. Ebmt and A. IImard, e&., North- poly dT-directed reaction# vu obremd. mtuding Wollud Publirhl~uCoqany, Auterdr. ! 05bN i 22 I I 1 1 has not revealed aay rjor differences in cqu- baa vith base cogooitloa of the entire Du rob- de. Apol the mauy techdcd probkv in this urp.riratal approach i. the p#.lbiuty that tb. uyr my migrate to other sit80 durirrt cha div- tion prmcus. If the NU polymrue-DNA coqkr COpLa be stabilid by forutioo of a cmJ.at bd - betvea~tbe tvo, not only would aratiolr bo pn- ventad but eomre Vlmprocaduns couU be utillsed durkrg purlfiatlOa of the c-k. * hm deval0p.d 8 rthod of stebiliiiry tha polymiue-OLsA coqlu. It hu been oham pn- vioculy that dtrrviolet li*t can inducee in ddi- 1 tion to pyrimidine dimem, MJA-protein croas-lbl.. An ultraviolet-induced cmmm-link beween baud 0 2s sa yx) RNA polymrue aud a DNA right strbiliae the em DOSE ( krodrl 8t it0 idtid bind- site .nd dlW pUfifiCUim rig- 3- or mrcent fractia of QP d charuteriration of these riter. illlcotporated in a poly U- or poly dT- In control experiments, wa exulned u fuuc- dined poly A mth~i.~rtf~ by X- irradiated a. EplA polymerme u a ciao of dme the sbility of ultraviolet light to fmctioo of dam. me pareeat fraction of induce forution of a stable coclpler bmcueea iacorporated at a put1cul.r dow by either tba B/r or B,,1 Du polymerase vu Es&ar;chia BWA polmrue and poly d(A-T)'- fror tba of Q? dc0kt.d amount iocor- d(A-T). A st.blc caqlex is &find U 00. ad8 PmUsd, capued to the etOf A16 incorporated, and ultiplied by 100. malutaian it# integrity in a hie salt cwaatra- tion (2 X KCl) which totally di.Npts nodbkad 2. C. 1. Stmi.ta md D. A. Smith, Xu +It= 8tudhS polyrrw-DNA cooplexer. Eddrturcr of rucb co) 0 the 8yncheoio of ribouwleic acid rU-8 X- 2 Lrrdbted RXA polymeran. In; Abatrrtm of pleru fm detected by nitrocellulose filter maw. the Hint I(0Cltl Wuutain RegzaaL Arriurr Their forrtioa appears to be 8 biphuic rupolue, CbdcjSociety lbeting, Fort Colliru, Colordo (June *July 2, 1972). p. 17, Abrttact No. 111. conslating of UI initfal rapid forutioo fouow.d by a 10- gradual increase of stable c0ql.x 4th 3. C. F. StllPli.tee D. A. Slrith, ud F. 1. H*Jw, X-w i.rcimtim of tbe kch.iicht. DUA- dose. Irradiation of the eatfr md pdyBOP bpmdant RNA polymrue in 4uou solutioo. arately does not promote fmrrtion of stable coc 1. Stadiw 011 bindin# and subatrate concontra- tioo d.p.0d.n~ in the inactivatiaa of euv plan. upon dxing. UiC atidty. BiOchai8tv (1973). in pn#S. With poly d(A-T)*d(A-T) of .D awny length of nucltotidu, ultrsviolet-induced 4. C. P. StWte, D. A. Saith, snd F. N. Hay-, 239 we examinad x-by iructintion of tho Escherichi. DNA- stable -lex formtion at tvo enryw:polyrr ra- d.p.od.rpt BJU polyrrme in aqu.0~solution. tios (moletl~la). About 10 md 24 percent of the 11. Studiu 00 initiation end fidelity of tranocription. Biochdoty (1973) , in prers. labeled polymer ruined bound to the enxy'm in hi& salt vhen enzyu:polymet ratiam of 2.2:l.O and Ultraviolet-Induced Crws-Links between BHA Polyrr- 4.4:l.Q. respectively, were irradiated with 2 me aad DNA 150,OOO ergah . To characterize further thre eoq1elo.s. sapla the above experinrt wen "hare are cert8in specific situ on DNA mle- from csntrifuyd to cquilibriu cesiu culm vhicb bind the aApolymerue mnz]nr. Sev- In chloride. The rerults are ohom The protein- eral attmts hmboen ude in raceut year8 to in Fig. 1. stable loolate RMpolyuruo bindine sites utilizing the polyur carplex esoatially floats In theu gra- dients thur, cm be re8dily separated from property of nuchase remiaturcr of thoor reglorn d, disrociable complexes. Work is in progreor to of the DNA lolecule cowred by bound polywrue. Hwever. ban andysir of the labeled DNA fr8lpuntI characterize further theme stsble ccnp1uDu prior CmcTs OF IolIZXlKi MDII1IQI OY -IN S- AND lslh WITQTIC AtPAu'flff

(C. Rr Sb.pbord, J. If. Win, and B. J. Nolmd)

Ia0i.i- K.diUiOU .ddPi8tend at 8Uffid-t 46.. krr& dve.r -e, either diroctly or fn- dlrectly, to alterotioar in tarpt mnloculu or situ within the llViry cell. Thue alteration8 u. ewntrully expsmoud u sucb clu8icrt radiobiological phmnmna u diviwtoa doly, cdl do&, rrd &maulrborratiolu. Tha man8 by vhicb 7.0 - thole p&.aorru are effoctd U tb8 PbCUhr &d are not char, th- it is by nm quit. .pgomnt chat dtiple but intornlatod factor8 contribute to thne ph.wrna in nearly .wry we. It f. out putpoll. to (A) characttriu &* .ad in vitru olteratioolr in cdlular biochrrimtl~ bt-t &art by Ionizing radiatia; (b) detordne the -1.eular mechu~isrby which theso cbmfu ua effected; ad (c) aft-t to dotordno tho re&- of dofiaod in vitro nch.nl8.r Vith obmomd in vivo 30 40 r~d1obio1oglcalphmU8. Our tn~e8tigathMhm fmction Nvmbw nuggloced two iqottat biochemical areas in which Fig. 1. C08itm chlorldo oodlnnutiao equilibrium @&atmalysi8 of ultraviolat-idu-d iOUiZh8 rdiatloa might contribute diroctly ad rtabk comp1.r fpatlm boana l[lA aignificlarly to cell death d division delay. In polymrw ad C-poly d(A4) *d(A-T). Sqles of lrt.di8t.d polymr d ir- tho first of thac areu, the buic patuhca sup rdiuad enm:polpr coqlorrr nra mt8 that idrh8 rdi.tiOtl at SUffiCimt h*.k midwith coaiu chlorid. to A find doe sity of 1.b6 r/a3 .rd cmtrtfwd to brlags about disruption of chromatin mtructure. oquilibriu. Aftor ceotrifugbtim, rpprox- re~ulcinein ~ltcrscionnin trqlota capacity d iutely 1 ml 820 vu layered oat0 oadr grdimt. .Dd rhe grdiaat. were frac- specificity of protein-bad aenaric DNA. Chro- tiaaatd: (a) irradiated polpr; (b) ir- -tin di8ruptioa ml&c vel1 be evoked u A caw- rutderuyurpolymor (2.2:l.0); Md tc) idiatd enzyme:polymr (4.4:l.O). atlve factor in radiation-induced cell tr8arforu- Io agro-nt with the nitrocellulow fil- tion, viral inductioa, or all de8th. fa the noc- ter usq, about 10 potcent of activity WY found in the li*t peak in (b) and ad of chose &mu. the bulc postulate La thbt abaut 24 potcent in (c). rdibtim-inducod divi8lon delay stem from tho in- .billty of the cell to rndrrgo divioion aid that arultr frolr induced rltmruioor la phoo~latioo.of fractiam F2b -11 ba -1- dirl8ioa rch.lri.r. Y. b... c~oc.=od .Lid7 pummnt* vbtl. phomphorylatioa 4 do- -8.1*.8 tb. fouarlng Sndi.8 vith tboffoctm phapboryktioo of hi.- tl may roprrwrt a m- of idtixu rdiath upoa c-m of chroruin pi- procus such u tr-port or daraprouim. .Ird of tho dtotic appuatu. If .Ituuim of hi.- npru.ou tb. de- mprrriao of prior to npliutbo. in ow Effect8 Of Iddn~Wiatiolo o Chrortin Stroctore m s* li*t )&et that only "old" histonu, the ad mtrboli.. rev- si- proaUCtm of aa urllor diriaioo cy&, would CODlmt m80dUh Of pmt- bo dtud ViW 0 @*.n coll cycle. Uo hn o=l.ie rib and ability of thr. protdna to b.IDytrat& rountly that the lntmrarl popti& 13- affect tho ~UUL.ad rate of pnmic tr.nrcriptim 17 of &tan fractiai Ral la acezyhted u ruido =ti- to tho that nuelu Id .-ti- 16 uithin tho e- cell cycle in which tho hirtolv W- hmctiaa direct Pmt.b. am nguhton or u WI ~pthrisod.~Tbphi. drrpprtatiol, proridu ptoof inm-t~ mUOmOf WW mn8.h. NU- of tho priacipla that au hi8tO~mmq bs mqhtod protoiru vitb nuchar 1PrA to cabin. DIU urd foa d mdarm it ~mlikolythat .c.tylmtioa of this Chr-tiU Of cell.. AltOr8tbM th tho uriw site io mloly iovolved in tho deroproosim of DXA balance thuo cagorunu within tba cbro- of MY of vithin that coll cycl.. Thh... obsomti- .Pa0 maria or alto ratio^ propottiom of thoom CW- in with am pnriour corrolatiop of radiation-induced nmu cm ri8. chmqam .bill- cbro- @ro to in of Mlbitfat of both DUA and histono blmmthsir .od rtla to mom u t-lato in truaecriptim roac- of hi.tooo acotylatioa and rthylatiw.6 ThS, tho tlow. It tho purpose of study to (a) is this char- obaenad offects of ionizinf radlatian upolr hietam rtcrixo vivo vitro chraatin structural in d in synthais .ad structural altoratioam MY wll bo an altentioor btGI&t about by ionlahi rdiatioa; offoct rdor than a came. (b) &termlor tho molocuhr rch.ni.r by which uxn- thoso aItoratioor are effected; (c) &terriM tk4 nsult of thue cham- m normal chrovtln f-c- 1. C. 1. Shqhord, 1. J. Nolmad, J. X. Eudin, rod 1. Iyvomt, structural mdlficatiau of bimtaor ti=; and (d) eeub1i.h tho relatiwhip of those in cultured Celt. 2: Tba Ifo- effect8 to clurlcd tdiobiolopical phm-8. &.dam of C.ao C~rmssloain BierOr-- immm ReId.1, Dmdrecht, Holland (1973), in Riatonu to tho wlocular D. u8 bolioved bo prus. entltlu privrily reapocuible for coqactioa and 2. 1. Bywet, C. R. Shepherd, J. M. Hudin, and structuring of chrartin. Structural altoration8 B. J. Uoluul, Tho diatributioo .ad tuxnovmr of thw prot.lim vl. aUtJl.LiOIP, ~*~lUl~rmd of Weld rthyl mupa in histono fractioru cultutnl nUaa cells. Biochor. phmphorylatiao reactim occur at cloarly &find of Arch. Biapbym. E,558-567 (l972). tL.. within tho cell cyclo, and thue rerctiolu 3. C. R. Shogherd, 1. J. Noland, rad Hudin, appear to n&ated by wailability J. M. be of histoaos Turnover of histono acetyl group0 ia cultured within the chromatin rathor than intracolluhr nlimcellm. Arch. Biochar. Biophys. (1973), in pross. effector enzyr 10.vrla. Theso roeulte have boa 1 rrvimd recently. I. C. R. Shophod, J. K. nardin, and I). J. Ool.nd, Dophmphorylmtian of histone facti- of cul- Xf thoso rtnrctural alteratioor are po~ent, turd -fan calla. Arch. Bioch-. Biophyo. OIY might UOUT that the function thy perfom is (1973). in preas. mhtfmly COSYtmt. 8tabilitiU Of d80 m0 5. C. 1. Sh.phord, Evidonco for the biological historu acoryl, Uthyl. rad phosphoryl pmupa Y.re coupling of biosynthesis and internal acstyla- 3 tion of histone fraction FTal (IV) In culturad dstordnd. Both hlstono ~thyl'and acetyl -iM UllS. BiOchiD. B1Oph)S. Acta SOUP8 appeared to bo coarorved in r.piay dlvidlag (1973). in prua. culturod colla. Histone F2b phwphoyl mam&lian 6. C. a. Shophord, 1. A. Ualters, J. M. liardin, groups also appearod to be co~onmd,vhile histoar ad B. J. Noland, The effects of X-IrruUatioa on himtone acetylation atd methylation cul- phomph8te appeuod to during C2 rya- in F1 turn oHr in tured rrrvliur colls. Arch. Blochrr. Biophyi . chronized naW1i.n ~011s.~Thus, the bioloGd -*149 175-182 (1972). effects of hiatma acetylation and rthylatioa ad 1051345q Effect8 of Iarririlu tdl.riam m C- t. of the Hitotic AD9U 8tW

X-IRdhtiOU Of cells u intemdfue daaa htr iadDrv the pha- nonnam of dfri.1~&lay. I-td cdh typ- iC8llY C.Y. df*iSk# idl.tdy fOt 8 WaOd Of tin proportid to the Q.0 of foctdmt r.di.tiaa, rfter vhich they rmau thdr ~thputern for me or -I. mratfoss. It has ber sham that calls 1rrdirt.d at thro htr, ubi10 t.tdP ing their ability to s)pQ.ixa NA, XA. ad fuuctioorl prouhm, &cam la Cz or a C2-w" state prior to -8- divisioa. Thfr acnril.tioo hu led to the suwtion that radiation-iuduced divfsi~delay ubu frrn dyg. to cml1ul.r did- Si- rch8Uf8U Or to 8- C-t nW808r7 for initiation of d~vi~im.mi. co40-t .pp.lt. to be protein in mturo. I I I We haw detordnd tho t.qorrl pattern of e n *...k.N intracellular dcrotubulo protein lovol. io wachro- nc. 1. T.qoral profilm h 8 8pdlmZ.d CUltlKe pized culturos of usli~celt (Fig. 1). Cellu- of Iaacalt (a01 for (4DIU syn- tbris (-04-) olrd cdl cotat lar mlcrotdulo protdn l.4~dlsplay NO dbtllrct (-A-A-): (1) 1yI lue/cell (-tO-), total sol&le maxim in late S and In late C2. It nybe sem protein (-LA-). unl total inooluble pro- that thue .Ydu corrempaad closely to mxiy in tein (-); md (C) cpm h colchldm incorporated Into microtubule protoin. total sohble protein content of th8 cell.. The effect of 1600 red. of X-irradiuioa upon intracellular RIo*, total soluble protein, and ricro- tubule protein leva18 uy bo amen in Fig. 2. All level8 ro0.e to mxim benaea 15 and 30 hour8 poat irrdlatiou am cdl. ucurlatd In C2 d declimd themafter. It i. believad that ph~pho~l8tlOn uy pracede usably of microtubular protein Into racroot~ctures. Radiation hu bun sham to cause tr.ruitory effects up00 phoophorylatioa of e variety of wt.bolitu Vithin th8 UrU1i.a Cell. Teehniquu for bolation of pun microtubule protein from cultured uu1i.n call. have bem asUblishd,

and dcr0USey techniqUO8 are OW being &VdOpd LO allow 88trblishnot of phaphoryl8tiotr of tubulin u a precursor to microtubule usembly, to detmrrlne which of the subunit8 l8 ph~phoqlatmd. to &ter- mine the progrum phollphory18tion vlthln the of -11, nun-- d ultlutely to datermiua the effects of ionizin8 FIG. 2. T-rrl profilu in UI X-lrrdiated (1600- radiation upon rubulln phoaphorylation. rad) culture of manCall8 (-0) um -trated for 50 hr goat irrullatioa for (A) totrl 80lubh prot8ln: (1) wr/cell; rd(C) cpr h colchicilu iacor- poratrd into Jcrotubule protein. urmWc&Im pxsteb) of iafo-.orcl. Tbw. at P- ot fntetmata of tbe C.llul.r ldiobioloq Sectioo the Pgvttio~of infmrp.aul ru-pl- durilpl the put fur hum coatinud to tocm on i. trp. muayr #* capmbb of purieiwtiw ia pol- forr+loo. in rQota thae ula .t.~qlqirpJ cultumd dimcdl~ Th%8 Ob..rrat100 k .Ird dcrobial rfrtm m test abjoctr. tprlr with other ddau indiutiq that tm md cootral rcbaian, ull-cell interactiom, mm-8a-r d8t8 U IIuChvdns d Qmasam stmctum and function are general not u freo SPOA, prior to unocfrtioa with ribaas. futut.8 of au o*rrsll objectivm of artrbllsbiag Further chractrriratioa of dimllr* 8p.ci.n by ldantifiutlm of rino? buu hu be- *lld.fl& t88t 878t.r VhiCb e(p b. LO RNA 8-i- dotemine (1) Ir1ev-a of specific biochemical ucolp1ish.d. For awle, sever& contain dlstiactim qu&t.TMry nUdOOrid88 Uhich fOStorU to COuqC& ptomO88hU; (2) swS.PU - euily soparabt chrautogrwicdly. of pragotfia rawrdlas tho irpartace of rdhtloo rnuchar speclea contain triwthylguuuwfne, which fomd or other toxic offoctm om spdfic fmaturu of coll I8 RNAm bo dis- rtrbolinr to cell nmval; d (3) prutiul in no other spociu. Riboscrrl CIII tla@shod because 18s contab8 8i-h 7-*71- appliCUio9r Of .pwifiC f.atU-8 Of Cell Mt.bOU8B a to pr0bl.r of ttmr cell biolw rrd radiothrapy. mahe but DOO. of the other quato-- UOClao- Curmntly the wt rwful synt- nlato to I#A B~&B, vhile 2tM contains a shdo l-rtbyl- rtabolimr, bi8to~rtabolirm, call-cycle kinetiu, Bin0 .rrd no othorr. Moreover, 18s WA hu for thm cmll-surface mcoptor situ, cytopnotlc offmcts, first rims born sb#n to contain a si- bmr ad DIU syatboai.. The *nerd approach cacrtiauen vdffied bue, lithyl-3-Y-(edao-~a*q- to bo on0 of wtrbli8hin~a rystr urd thon -8sur- propyl)psoudouridine, of interrat becaure hwr wdified bues have thus far been found oxclwl*.ly lag tbe offoctn of biologically irport.at toxic in the of tRNA. Ia fact, tlPOA Of mt4 or ioniZlng radi8tlon Of V8miSlg qudlty OID anticodon region Eouad to conWn a nacho- variom utabU.hd, rrcogai~ablo,and wuurablo CtlO cells hu boon skilu fO.tU-8. side, 3-y-(cr-rrino-~-urbo~ropyl)uridina. 1 Racaat studiar r8dlation effect8 bhtono In our 1971 roport unique p.rtiCu&tO- of on 9rtdolim have revealed that phoapboryhtion ~8ocia.dPITA speclam -re characteritod, and an f2&2 is independmt of ryntho8i.8, fl phosphorylation, attqt YY DO& to ucribo hctioaal roquin- f2aZ .ad synthosis. Histone fl phosphorylation. a1- mnts for these spedu in the cyclina coll. Io DNA thou& not relotod to either fl or syntbool8, this =port we 8uurito rasdtr which deecrlbe DNA f8 npidly-ld.l~d, hi& ~hcufaw~ightlDIA from nonetholoss aa exclwivo S-phue phuroraon strongly mmcytoplur umoeiatad with protein or Mibitad by X-irradiation and, because of cycle nucirapmtoin to form two rapldly-.ediunting dependence, is probably not important in donproasion of MA trqlate activity. partidatOD (i.R. , pOly#-O ad iIIfOMI-08) .. di8tinyishablo by tholr differin8 donrities. Con- Irprwed control of experhntd COaditiOM for studying cycle-dependent bfochdcd affects ditiolu hm boon utabllshed which nstrfct -- hu been achieved by tmdem isoleucine deprivation sner or nuooryarllire lpDA a~lablofar rlbmor and hydroxyurea trostmt to yield lary cult- association to usscyr-ltke RNA of infonrwaos. suitable for biochniul uulysis fa a11 calla Under thar coaditicn$ , cycloh.xIrid.~diated poly- which am tNly in GI and rub8epurntly divide myncbroaourly some superformtion occur8 in part u a result of Biochemical criteria for assessing tho defree unodation of ribolrao with lPIA (urd prrrdly of psrturbatioa exhibited by these cultures appear to

27 h8ve merd .ppllcabiUty for rrurlng thb effect8 Of 8 VUi8w Of 8t-m QII C~tl~d8tudim Of Cell-sUrf8ce Qtop.=iU hsvm focud on attqt. to qwtltate ull-murfau bildiq of flw~eh-mj~dpl.nt l-tl- .qloJiog flw dcrofluorortrlc t.cbniquu. The surf- demlty of bindha .Ita hr boa f-d to be n-ly wtwt throudsout the cell cyele. with saeri&nce for btargndty of binafol sit- rod, ~rwrally.-tltioa for bindin6 rites be- hr.0 eoocarrrlip A md rrb..t *= a&utlaLp. AfClutianbility of rlnr-tr.luformd llnr d trypsaetreatd pueaul lln- could be mdl~ drrorutrated buc not dearly correlated vith binding. Tkm atrlking obaervatioa of DRI coostan9 dupitr mrked luldili~in chraomcr ndr in heteroploid nUaocell llnm utiliriq flar dcrofluorartrlc techniquu hu been cmfirwd indepawlently, in part by extuuive luqolo$ic (X. D. Haasr, R. A. Tobq, L. R. CurL.7. R. A. ccmpartaai of .uploid Chlwe hrrter fibroblutr Udterm, A. C. S.poruta, ad E. U. C..pbell) vi& line CHO cell8 eqdoylns mbtn Ci-0 bdn8 prouduns. Althoaah no howlogow pein of chro- Informeour in Cultur8d Chloese 8mt.r Cell8: ~0.0~8reuln in CEO alh, virtually the entirm Ctclohedd&-~i.td ?olyeor Superformtian i8 wrma can be accounted for in teru of extauive Acccrrunied by k8ociatioa of Infowroral vfth trmdocatioru md 8 perfcentric innniw; har- Ribaroru aver, mmt of 8 haploid set of chromomons hr Rapidly labeled, hi@ wlecul.r-uOi@tt UNA of tetalned nom1 coofiguration. Approxlntely rrulibn CytOplNm is uuociatui vith protein or 3 percent of the mnadssing from CRO cella hu nuclaoprotsin to folr tw types of rapidly rediunt- been ideatifid Y the arm of the hte7eplicuing ing particulate I~NC~UTU.In one imtam, the X chraaa. Thae observation8 ham important 2 W(A is wu-r BIU and the associatian is with pnetic Fqllcatiop.. rfborarr to foa pdysors. In the other iiurpLc., The evidence is nw .I.o clur thu, rlthough the -8ociation i8 with protein Of UnmflmCti08. drulcbouad DNA replicatiw rite8 do not occur 'Ihe rudtant particle (-80 percent protein) is in QIO, the 8itu for initlatioa of 8yntheSi8 referred to u an (Polymmu and rry inbod be md1r.ncu8ociated. Tho, the inforwaors arm distinguirhed by their differing spatial arranpmsnt of OlSA uithin the nucleru d&t denaitlem: 1.52 glcc and 1.4 g/cc for fiwd pu- ba ?8gUht.d by U8OCiatial Of Specific md-8 tlcles In cesiua chloride, respectiwly .] Several vith the nuclmar rmbrate. liner of evidence ru~e8tthat the MA of info- bctrih of phae mh replication following de- aoyI is .Lo rsmager. It is ridlar in rlze, reprouion of prophase by ultr&olet 1iQt in stability, hybridization propertier, and bue H.IIODhllus lnflwnru .II moat colulstsot with a C-OsitiOll to poly8ar-b-d m88sener1** .Ird i8 model in vhich both rynlthni. of a ultiple phrp- thw referred to as museuger-like RNA. There Is equivalent concuar and 8ubraqwnt reduction to about u much m888ILg8r-like RNA (mlR#A) io iafonr phrp-sired pieces occur cell mmbrane. 2 at Ea- 0.018 u there is me8s.0gar RNA in polysous. cision npdr capability of the host butsrfum dorr Circurtbntial evidence indicatu that info-- not -pur to be 888eOti.l for trigpring dorepru- uy represent the form in vhich rrsenger PNA exl8tr sion, nor dore it pre-nt d8rrprurion. Hwnr, during transport from aucl8u8 to cytoplum, during

20 IOSb451 uaociatiao vith ribaoaa to form WpOl8, lod the avrforrtim ir apparoncly du to au la- enn during trsmlation. Fiat. infomar-like C~MO~pukin8 of ribowma o polyaoua). Ua 3 particlw a-ng -cent my be found In .hat frrrthe~that polyramal 8uaay.r UIU .nd or extracted from4 nuclei. ALo. nuclur hereto- infomd.Ilu. are idontical In Wlipon RIIA, vhicb MY In put raprumt =aaewr retoatidty d tbu probably s&ua the a- e0- -- 5 precursor. ia akilarly eaaociated with protein. taat of poly A aoqlwncu. Socod, .U rLIIA of the cycoplvr ia fdto be Ln thro oxperinmu. aynahui. of ribaod particalate; no freo rlllli odats.2 Third. the lllA ia pmfarutti.l.ly inhlbltd by tnubg cd- 13 demlty of polyaw is l.u th~that of rlbooamu, turea uicb 0.05 SJIlr utwcin (nn) €OS Y) d. hdiUrim# that nucleoprotein that la predadruntly before expoah8 culturea to radioactin PU ph protein in coqaitioa wt ham uaoclated with curaon to anaura that mot of the inCOiporUiao riboaaur to fon poly-a.l Further, inforrrr bt0 CytophSdC pU%iCul8tO-u8Od8t.d i8 at0 --like partiel.. uybe diaaochted from poly- uaelyrr or uaewrlilu I1u (mllllA). Subawmot raYa wing EVfA. &‘’ Fifth, infomar-like par- to expaure to -led RNA pncuraor. furthar tlclea are relouod fropolyaara during mad n- rynthula ia rtoppd by addition of utimcin tO u8ocbted with ribomow after ritooi..lt Finally, 5 Ul/d. kcuuo nuclear-cvtopluric transpert of the kinetica of B1u precunor iacorporatloa ara not rsa-r y cmtinue for up to 15 da after. lncolulstent vith a flnr of r8aoly.r llsA through theah iablbitior, in hkk cad‘ (In gatoral. infoxmosawr to polyam. **11Bmm*.r. one cawtot transport and p-auing occur significantly lDn definftively utab1i.h such flov from kbetica rapidly in CNO), culture@are held in the hi& &vel alone, and pulae-cham exporimnu fall to demon- of actinayein for 20 dnbefon effecting aupar strate satisfactorily auch a flar, aince the incor- formation of polyeau by addition of eyclab..irid. porated lab01 in KNA of polya01~.and infornor8 (e)to 1 Thua, the firat tnatmt rith dmcaya Lo concert aubmequnt to inhibition of 8 1- levo1 of actinomycin one to eqrute the 3 further incorporation (Fig. 1). Thw, no direct etof PlsA precursor incorporatad into qt0- evidence exiata to ahow that the infoerr (or pluric paniclea of danaity 1.4 (info-) or dBLOA theroof) in the cytoplum of interphua cella 1.52 (polyaaer) vith their labeled raaagor or cut or do umociate with ribwoma to form poly- dRNA contenu. rroatunt with a high cwcentration somes. Sn thia report we ah- that polyaor ruper of after pncunor incorporation praventa my formation, &chiend under coaditiona whom info- further rpthai. of measanpr or dRNA and rlloua oa-8 are the only aource of dltMA. i8 accoquiied one (after eatabli.hiq that no free rllpu ia praa- hy a increue in polymoul-labeled JIDU and a ent) to loot for uaociation of informmaara vith decreue In informoaomd DUMA - that polyaor superforvtion ir &lend in part by uaociation of riboaawr vith informama (the vjority of

. eu

FIC. 1. Decay of IPlA in polyaaa uul inforrwoua in the prcrence of actinomycin. t05bwl :?; 29 ribourrs to form polyaw by mu- tho -t of lutorporu~-1 trufomd fra 1.b-b-iq luterial to 1.52-d.arIg ut0riJ uba rp.rforrr tion of polymau i. 0ffea.l [utmd Y tho ratio of kkl in the hro daority #pod- after 45 dn in Cx Y oppoood to holdlqin tbe hi* COD- ceutratioa of AX almn for U .bn (MI]. k ahom n& 2, Wcl0b-b tm.lrt -dU h mrkd incrrue lu poly.- emtaut md a cotruged- fag decrwo ir h.0 808 & aN1 a&- unita. u&u OPI lodu at tho .ff.ct of thla aup.r forutioa up= the r818tiv. lllDPt of Du Id.1 in KC. 4. 1.4- .lpa 1.52-d.l~Ig Swiu -ti- iU tho reglon of ull (?,I or lup (?L) polywmms, then ir indeod a cormnloa of label from inforrrwrr to polyaoaa v&n ruporforrtion OCCM (Fig. 3). "hat the 1.4- * 1.SZd.noity cammaioa effect i. not u prunounced u polymor eupetforrrtion prabdly ia became wt of the rqmrforutim im effected by a more denme packin8 of ribosrr (the effact io dum apparently to elwiw truulatlon rete relative to initiation). P.produclbllity of thi. affect aad a mors do- tailed oalpla in tom of iucluding 1.4 and 1.52 putich8 fraa all ardirut8tlaa rmys are aham In mother expotinut (flu. 4 ad 5). Aula, poll- J v' ,. IOU suporforutim (ng.4) la accogaded by a incroue in 1.52 daarlty .ad a &creme la 1.4 dm- IIycT#lLylLR 5. Iaopycnlc Myria of the effect Of pOiy- aity (Pig. 5, A * D). kcawe the effect lo rdl, nc. a- muperformtion OII MudUA cm-t ve Wt uaure ournolv8a that prerequisite cd- of hafow0rm ad poly.orr ftatho rm- @oa of tibaaa mub-ita (n), 8oB riL0- ~IOMobtata. lint, we wt hwa Q rb.nce of s01~(11, mdlpolyaomm (C), .nd lab01 lncorporatod into ribad 1DIA. Pl~ur. polymam (D). Share thi8 colrditim is It. That i8, &by *y*h* that the increme irr 1.52 density label ia sir of rRSA vould b. nomu firat in 18s RMA indip- dum entirely to .II increuod packing of rib-- QIL nom to the 405 WU submit and would produa 18be1 exlatins polyrw with caacod-t incnue in tbo in the re@oa of fraction l0; hawever, oone 1. mount of (-led) polyroul tRNA. Andyaf. of found. Second, ue mat couslder the pwalbility IWA extracted fra Aw or Cx polyrolu (lis. SA) rhwa that thim is not SO, U the hbd incm.Ur,

io in the dlu peak (5 185). A correrponding do- c-.~. la rham in the cue of dRNA extracted ftor infomama (nr.61)). Third, vo lullt verify the abeence of free riDu or rlpw at the tin ruper- formation is initiated. Thim is ah- in Fig. 7. No .RWA ir extractd Era the redimentation region (of cytoplmm) ubich would be occupied by free mu. Tbw, at lout part of the infomomon pop&- tiw CM amoociate with riboraru to form

30 be axtmly laae. probably cab- IaforrP- w nprumt either incogletely rodifled or procucHa fmr vhich Vtll be wd mutually u procam- k finlobed (the masoeapr in lafoa~roll.. I# la 8 “pip4~M”rather thm A “pd? , OT mt Of the iaferollar papoluiaa la diffenntirlly mdl- fLa to proclub utlliutioo. Such I8t-t. or dith-tial dficatiarr could lnrolva YHyIr lluI d/or thdr assodated ptotein(8). A prap.rty app8nntly p4culi.r to mRA of polp 8mla 8 coatant of poly A aufflcknt to UU.it 16 to be prtially retdnod oa Wllipolr ftrt-. I# the io- dIIA 80 modified? Th4 data mh- in ?ig. 5 Indicate that ldetd MU i. ob- tdmd fror inforrollor-dmnaity msterial Joae vhen utari8l in the 20 to 70s neim i8 OXtr&td; tkld Du obtained frm the kre p0ly.a frac- tho is prlrrily fror pdyaar-denslty matarid. Themfore. we uyuse tract^ of those frutioma ?IC. 6. w SOUX~Y of iotorr~mrlm ad po1y.d -A. By caqariaa polysarr3 -A, infomomml p1IIIu, 16 and riboovl Bw by tha procedure of be etG.# we famd that no ribomoul WA vu retdnd 0 Wlliporn but the info-orj JWA od p01JId miUU wn held up to the 8- extent (30 prcmt), iodlcatisq that the diffennce in info-oru bomd or not baund to ribom- I8 not a diffanoeo in .edification of their BbtA by preferential addition of poly A to thore usanger8 de~1gn.d for i truulation. RalmmncLs 1. A. S. Spirin, Eur. J. Blochn. 2,20 (SH).

2. C. Spdrr, N. Crmbou1.n. C. Xorel. and I(. Scbemr, Eur. J. Biochu. 2,296 (1910). 3. 0. P. S.urina, E. H. Lukanidin, J. Moh8r. aud G. P. Ceor&ev, J. Wl. Biol. 2,251 (l968). FIG. 7. U 4. K. Kobler d S. Arm&, Eur. J. Blochem. 5, 500 (1961). 5. K. Scherrer. C. Spohr, 1. Crlabwlur, C. tlorel, polysarw. Thwith tln other evidence, ueocia- J. Croeclude. .ad C. Chezzf, Syrp. Quat. tim of trw uaeqer Ipu with protein to forr Biol. x, 539 (1970). putteka of denaity 1.6 prior to uaociation of 6. R. P. Perry nd D. E. Kellry, J. Mol. Di02. zs raa.alrr with ribomma appura to be moat probable. 37 (1968). The qamtloa mu raiad CIXIUID. the relatively 7. E. C. Hemhaw. J. bl. Biol. x,001 (1968). hry roMt of IY.aetuer in thi8 form. If it np- 8. A. B-y, C. PWZr C. H.rb.i~, md B. Chm- reaa~taa atui~ticrla.qling of all r0.e-r trenne. Biochir. Biophy8. Acta tep, 228 (1969). de8tiod for truulatlou, then one would think that 9. S. OLne8. Eur. J. Biocha. JJ, a4 (1970). CoupUng between t ruucription md trardatlon would fOSb4bO 31 Qe of th. mly dimcowend b.lw to the clu8 of door burr h.*iq a qrULm-8 nit-. u. chuacterind by a bmic di.0 sociation coastmt neat neutral pU; themfore, thq ku a pomltirr Quy m&r pbt~lol08Aulcd- 12. B. Fm d S. Pm.3. m. Mol. 50, 6S5 (1970). ti-. tie wra firat d8rt.d to the preaaee of quuernary nPckotidu in MAde@ our pd- 13. R. P. Perry 4D. E. klley, J. Wl. Phymiol. h..lttf.tiw the lUCm 8piU Of ,e ,e 76 127 (1970). Of Of 1111, lo, moLCulU-ue*t , m8thyl.t.d UI (I=--) , ii. s. P-, w. p0Jvb8 -11. m.PTOC. wat. Acd. Scf. USA(I. 1878 (1970). air.. iaterudi.te borntm ad US r%bca& IPIA. Sar of th- 1wh caa+.ind wthrhtd Btulo- aidu; thetafore, to pren that thw vere not bnJ- QVO products of or tm pncurmorm, im -1-d 16. S. Y. Lee, J. !4~deckl,aad C. Bmm,Proc. MU. Acd. Sci. USA68, 1331 (1971). --rea collm chromtogra~Wof their .1L&m degradation products. Our e8rU.r 8tudi.r had 8ham mdifid brw of mu that rR#A &we different methyl di8tribution pattern fra tW by thir procedure. The patterru A UjOt dlfficule in the ia#tigatioo of nu- dmn by the vore totally ualh dircribu- cleic ad& i8 l.ah of eh.r.cteri.tic8 by vbich tiom given by eitber CRMA or rW. A larw fru- 1oleculu can be di8cri.dnat.d. nybtldiaation hu tion of the wthyl lhel warn not &.orbed by the been aployed in aoI circcrtancu to c0lp.n nu- colum rdbehaved in a mMwr errpected for the non- cleic acid sequencu, bot the technique i8 dommiding phapho~latednucle08ide at the 3'and of s poly and fr.urtrt vith the pocentlal for artifact. Cur- nucleotide ch8in. We nw koou that thi8 Undr0rb.l ther, the rrlatiauhip betwen nuclrotld. sequao material la 8 dep8datioa product derivmd from aidladti- and bybridiution c-titim hu not internally lwted qWt8rDuJ nucleotidu uhicb been defined for coqlex aequencem COLItdUh8 md- undergo ea alkali catalpxed opening of the purln8 ified Ual8081du. IlWlaiC aCi& UO k 8.p8r8t.d ring, follmmd by 1008 of the phoophate froup which according to their size .ad ahape by 8ucr0.e deruity ia re8poasible for dsorpticm to DUE-cellub8e. grdlrnt stabilized centrifugation. The technique The quatemary auclaoaide found In LW-IPU vu ha been wed ruccuafully to 8tudy ktnetiea of RWA 22 identified u N ,I ,7-triuthylgurrrori, and ue ayntbuis and to chsmcterize effects of inhlbitorr: have aham that tbt hitbarto unknown nucAeosid. havevat. rwolutioa L lidtd. The low mlecular- i8 prnsmt in throe of the irolated Lm-ar* 8pe- vci&t reeon of awme gradimtr, tradttiolully cid None of the other holm rthylatd Risk attributed to tm, in fact coadsts of a caplex cmtdna thia nucleoaide. Therefom, ve can elir alxtore of RUmleculw VNch are re8olv8bh by inate the pa8ibility that the tlDtRluI are arti- acrylnida pl electrophore.i..l Zlectropholrtic factually derived. Thur, excluding the paaibillty hili- Of pOlyWCl~ti&8f.8 ab0 &-&Ut Upon thee a clU8 of RHA uhich i8 not artract8bh by our molecular site, vhlch hu inhcrcnt difficultir u phenol procedure (erg., the chrCaO8d-~of a atandwd at.dng fra ubiquity of degruiative 0 Boaner c&. ala0 contain8 trir~ylfruooaiae, enry.u which may derive either from endogenour the three Lm-Bnk appear to be the aole Rllk 80 sucrcme or from caacrinating microorgmbr. For modified. thir reurn, we have .ought chricd charecterietiu Our studiw vith triwthyl~8ineled eo which would distinguish nucleic acid8 from one development of e simple chromatographic sy8tsl for aother. Our efforts hap. led to eluci&tim of separation of the qU8tErnary nucleoaidea of RNA. atxucture of throe preriouly mkoaun cruc&oside8. Figure 1 shwe a pattern rapreamtative of total Two of then. appear to be miqcwly loulized in ap.- RNA. The colum packfog ia phoaphoullulore which cific RNA typcm. .ad the third ia a hyperrodiflad hu been equilibrated vith buffer at pH 1.0. All nuclemide of the type klranr to be confind to the 2 of the c0-n nuclcoridas 8nd m08t of the modified mticoda~*pion O€ tRIPA.

32 FIG. 3. !mhyL.t.l quuonru) oucko8id.m prueat in 16s MU. The mqlo vu takon from I sucroaa gradient md na u ducribed. Addition of 7-ath?lflullomine, a myathot- iully prv@ nucleomide, ir ruponsible for abmrboq. nucloo8id.a uo mchu(*d at pU 7.0 ad, baawe thq h.n no affiaity for tho calm, elute in the firet 25 fraction8 which ve nfmr to Y tho void vol\u. The radioactin quetornary nucleoidem 2 folmd in Cbinuo h.rter PNA are (I) t?,N .7-trirthyl~aaorirt.,(11) 1-rthylodenodne, (121) 3- mlqtidine, and (IV) 7-athylgumoine. Their aczu~clir*~an mham in Fig. 2. -me four qut- *:=AT sifh08id~appear to be the only onom proa- cnc Ail &??A f80i8t8d frOr Chiaae hplter Cell8 by phe-1 extraction. V. ham not eacldod the pom- FIG. 4. %thyl8td quaternary auclmoiau ptumat sibility that other qWt8tmry nuclewid- , which in 28s Ipu. The r.qlr vu teka fra a sucr(re mdirnt d chr0utow.ph.d as am too labile to 8urvivo our canditim of prep- dercribd. Addition Of 8pthoticdly pro- aratian or which at. prement io RNk vhich at. not parod nucleoeldu 1-rthyld.wofm and 'I-rthylguu~abevu remponsible for ertractod by our protocol, exist in Chinese hauter abmorbacy .

00131493 04F 188 301,729 - 582 - SS30 (54.6) (0.U) (1.0) 285 472,680 - 5966 - 432 (78.9) (1.0) (0.07) 0 mm U.650 2257 - - - Lm VIK U.896 2894 - I - tm 206,027 - 39,911 6326 23,791

to correct the data presented in Tablo 1. Thlr table yu caaatructed from -1- chroutoarmphic data a& u that sham in lis. 1 and ritp. 3-6. Th. nden in palrathwer u. the calculated nur bet of methyls attributed to each of the rpeciu, Rmhm- 8S8Uily that the qauernary nucleoside pruent in FIG. 6. lbthylated qUbt8- n~l~aid88pr88-t vu i8oleted by prepua- h1fi-t ..ouot is nprereatad once. The usugtion in Is MA. 1Du ti- wcylrid. ~1 ehctrophorerir. mi8 appear8 to be corTect, rince independent utiutu fractioa Cogrisem the bulk of NApre8-t of total nubar of rtbylr per mlrcule ace 5s per in the 25 to 105 region of mucrore Ira- diaau obcaind frawholr call8 advu 1BS .ad 85 per 28s. I€ OM umethat the rib rhom to have &no acid accepturca activ- soul MAa rrpruent horomnmow ID&cUL.~. thrn ity. Mdttioa of rynthrtically prepared nucleaide 'I-rthyliaoriae io rrspouoible data in Tabla 1 indicate that each riboeoul rpe- for .b80rbmCY. ciea hu a dirtinct quatenraw nucleoside and that tri=thylgunomine in rRNA md it8 exclusive pnr- e*& hu a siaw molecule of that nudeorrid.. ence ia spwAam XV. LW mpecirr VI ad VI1 gim The umrrrption of horgeneity appoaa to be valid patternr wry similar to that rhcm €or species IV. for the ribamoul Erch.richia dl BNA of The auod clue of nucleorldem we have dir- which Felnrr et&. ham r.quen~ed.~They have #pede8 ha8 8 ria& shovn that the E. ah0 electrophoretic fraction which contain8 tRNA. Th-8 reridua of 7-wthylguuwsine per roleculo. Further- nucloorido are characterized by their conteat of dth- dl of the rr~iainsmthylatod -re, a a-..inobrryrate residua which is derivad from specie8 ua clustered in the firrt third of the athlonine probably vi8 the apacy of 2-adeno8yl- mrlecule fra the 3*-.nd, the 'I-methylgUmr@aine 10 rthionine. recornired the active 5 prnerrlly u located very arar the middle of the molecule. sthyl donor. The presence of thi8 clarr of nucleo- Filum 5 and 6 shov quoternary nucleomide dirtributioa of our iaolated LW rpeciea IV cdAD cells to incorporate rthioniae labeled in either electrophoretically purified fraction enriched for the 1. 2, or 1.2.3 and 4 cubonr.6 An illwtratiw tWA. The fiwru tiemnutrue the .b8-C# of sucroma grdient of RNA irohtrd from cells 1abel.d nc. 7. Iny&pOrUiatof (htbyl)-thidM rod 1. (2- C)rrthioaior: into totd OU of Chinue hrrter cella. toolatod a UY catri- fUpd in 8UCfO.e WOUt(UrW 8h 1. the diroctioa of 8rdiratUia1). dtb (2-lic)-th&d= 18 8ha# HI;. 7. 3. cmlote of (~-~~~)rthioainelabel from 288 OXCh&m tb pm8ibility that hbeung ?@- 6. 8Ult8 fra indi8crLdnate incorporation into purfne Or p7tiddiOr bue8. V. hmud. 8tYUttWd 5. ~ml~ntrof the nucloooidu on the bui. of (8) dmtioa of iacorpontion of iwtopic &bel 6. from pr8~r8or8,(b) &gradatIan vith .rdrydto\r hydrazine rvd corprri8on of the product8 with 8.p- 7. tb8tsc utarbl or with tho hyd?ulnoly8i. product8 of ham uridina, and (c) cogariaon of the un- knorm8 u their hydantoin derivativa8 with the 5- B-(bt-thyl)hydantoh dkylatlon product8 of 1 uridine ami of 1- cd Erthylpseudouridine. The Beawe of the involvannt of hbtonu in em- f 4s UdrDam i8 heteroyneou. V. identify one Of trol of chromaow structure .Mi pmtlc UIPrU8ioll. the colutltwntr Y 3-y-(a-rsino-a-caLbo.lpropyl)- it ir particularly important that we uadent.nd the uridine. Another of the CS cmknwtu .ppour to give ruliosemitivlty of thue protehu. 'tbr nodbio- rim to the &a*, sub8tace after hydrolysis with chemistry of histone8 la cultured Chineme hutrr 1 HCl at 100.C; themfore, it MY be m aino- cell8 hu been inw8tlgated extenriwly in thlr 8ubrtitut.d dorlvativa of that capound. A third Laborstory. 2-6 By integratlrq informatioa obtained labeled product i. rzlo prement. which we haw not from this program with knnavladp of the Chlnw 11-17 yet attapted to charactrrirr. We note that N&a- huter call .ad radiobiology Pn- 7 aishl'8 structural auivat of the "Ye' b-e. Viorvly dercribed in thlr Laboratory, an in*.8tlB8- which I8 knwn to be located in the f'-paltion to Cion Of the radiosen8itivr nature of hi.toru It- 18,19 the antlcodao rrfion of phenyl8lutiae tNAs of .bolirm was initiated. higbar orgmisu, hu 8 brivatired a-ainobutyrate Abnorwl Acc~~~htionof Hi8tone f3 fOllOUin& -.id- att8Ch.d to the 10 mition. * Sugpe8t the X-irr8distion.--Y. hmshwa previowly th8t biolog p008ibility that thia resid- is also derived from ically rieificurt domes of X-irradlatiou (800 rad.) -thionim urd that our unidentified ut8r1.1 MY, will result in UL abnormally high accdtioa of in fact. be the rl[" base or it8 derivetivr. U. 18 protein in the histone f3 fraction of chromatin. further speculate that, althou@ a11 knam hyper- Houcvmr, uhethcr thi8 abnormal esce88 of protein in modified buem djacmt to the anticodon are purine

35 fl ll*510 io,au - 6.1 f2b 24,169 27,116 + 12.2 f28l 20,775 2S.514 + 22.8 f2.l 14,982 18, b26 + 23.0 f3 12,b1 22.02s + 77.2

X-ray-induced didoion d8hy or the evenhyl death

-ry w- D.

FIG. 2. Accdtioa of pnbbskd hkt- f3 in chrovrim fOll#in# x-Irradiati cbiovr h.rtmr call8 uerm hbehd With7% -dW eci& ad tba rrsurpe&d into thmm cultures cootah- ao carbon-lb. One culttm VU irradiated with 800 rad. d h-8-d 10 hr after irradiation (+; oae W irradiated culture vuh8rrrst.d 10 hr CIYm yuyI1 after irradiation (- - -1 ; and ow *- FIG. 1. Prepatatin electropboremls of hirtonm irrrdiated culture vu hamstd in- extracted from Chino80 hamter ells u diatdl u remtir c-t-1 (--)* de8crlb.d by Curley and P.rdiniq (A) elec- Histone f3 YU laelated md rubjut.6 to ttOphOtUI8 Of 4 dXtmOf fl 4d fa; purification by preparativa electrapbot..io. (S) ehctropboruir of the f2. caqla; Rautiliutioo of lbC-dna acid. dd8the ad (C) electrophoreai. of hiotone f3 lO-hr pcwt-irrdiatlocr pedod la krdlcatod prcpared io the presence of 0.11 2- by the Increased recovery of carbon-14 in rrcaptoeth.ao1 to pra-ot dimriaatloo the 10-ht mairradiated culture ove~the of f3. zero-tir control. bductioa of X -Ih -&.-+e of tba first obwwabla etfecte of X-irrdbclm on cultured QLIDYe ktar cdt is the cusatioll of

occurripl u a molt of x-irrdiuioo is a redtat tion of hiatan fl phorpb~rylatior.~~Thir radio- mariti*. pborpborylatioo IS rpadfic far histone €1 ad d#r not occur ip other pbapho-1at.d his-

ray-induced reduction of fl phmpboqluim q be diroctly coupled with X-ray-induced divlsica

shorted the dirlsim delay pmrld to half that of mtnatd X-in.di.ted cultures (Fig. 3). Hfstone phosphoryhtiaa vu mrund by incorporating [32Pl- phosphate iuto celluhr proteins for 1 hr follarin~ irradiation md treurnt vith caffeine. We ex- tr8ct.d histoam fra these cells fractioarted ond FnctiOn FIG. 4. preparative electrophoruis of pbwpb~- rylated hbtoou extracted fraChiour hvter calls. C vere l.bdd for tw

1 hr. Cuboa-14 and phospborru-32 Y.- courted simlw.ow1y in tvo cbUmd8 Q a liquid scintillatlao spectrator. (d Electrophore~i. of the lyrltu-rfch hir- tones fl md f2b; and (B) electropbotub of the arginine-rich histones f2d, f2.2~ Md f3. The arginine-rich histom. were prepared ia the presence of 0.14 2- rrc.ptaeth.no1 to prevent diwriuth of f3.

and fourd that caffeine also prevonted X-ray-induced reduction of histotu fl phorphorylation by au .Puot similar to urlioration of division delay (Table 2). These data sugwst that radiosemirim phosphoqla- tion of histone fl uybe required for cell dlvlsiw urd that. by restoring fl phwphotylacion, on0 c.0 alleviate X-ray-inducad division delay. Caffeine is thw&t to exert its action by inhibiting che pha- phodierterue which breaks d#n cycllc An.'' thum cawing UI increase in cyclic AKP avdlebl. to hi.- cone kinme which specifically phosphoryhtrr his- Tlr (hours) tone fl.*l turn, histone stir FIG. 3. Effect of caffeine on X-rayinduced dlvi- In increued kinme lion delay. Culturos were treatod with ity vwld increue the degree of fl phmphorylstim rrdr of X-irrdiatioa and/or 2 100 e and thus reduce division delay. hrther fnvestig8- caffeine at zero ti-. (-0-1 control, no drug; (+) X-lrradiati-, no drug; tionr are being deto test thin hypothesis. (+j control, caffeine; ad (a-1 X- irradiation, caffeiue.

37 HI8 tott8 fraction fl Nme 0.226 100.00 fl X-ladirtion 0.132 58.4 fl CdhW 0.282 100.0 fl Catfob. d x-itrdiuioa 0.223 79.1 E82 Naoe 0.176 100.0 f282 X-lrrodiation 0.187 106.2 f2.2 Caffeim 0.172 100.0 f2.2 Caffelor d X-irradiation 0.172 97.7

nourly fra aa irolruclne-&fldent state into the G phue of the life welo .Id then irradi8t.d. .how 1 a sippLflcaatly roduced dlvirioa &by am e-&

thia tha cell cych aftor 8pCh~iKatiOllby mitotic relectlua. Became of tbe paribla Involve- ment of hlmtonu in X-ray-lndu-d divisioa &lay,

in tha G phue to ot.bli.h a foundatha for futuro 1 innatipatlorrs Into the rcba-r involved in reduction of X-ray-induced dlvisioa dc1.r by iroleuclno de f icleacy . #ea coll. uem syachdred In i8ohuciUO-

deficient C1-arrert, the rate of hlstoae rynthrrir .d,O;.'WPYY *. '' -asmrnw.* " mum vu reduced, u exp.ccd. but not to iero (Fig. 5A). FIG. 6. Turnover of prelabeled DM (A), hirtooo fl A rrll bur detectable lOllDt of syntbsir COD- (a), md hirtoae f2.2 (C) fou#iw ?e- tlruud for up to 50 hr In the absence of DNA rya- thesir (Pig. 6A) mad without QL iacnue Ln hirtoae acid mixture. Ti12 is the hdf-Ufa of tho loss of 14C-prelabeled hlatone from vu the tuult of hhtone turnover in the chroutin the chromatin from 16 to 56 hr. (Wg. 61 adC). then tho vuioru tuxamer rotu y.t. nmured, we famd that hi.tooe fl had tho futaat s8te of turnova? in colla urostod 5.n C1 (Tibk 3)* TMm t- rate is 011 slightly kw tha that prowlouly rrurod for fl in 0- Matirl arlsmr..'~' kcamma mA 8ynthuL is caglotaly tumd off lade? thu. toaditlw. nor- ..1 tmrwmr of hi.- fl .gp.rratly is not f -&d h abUC.tOv Vw to DNA 4, mi. aoqlvou 4 eopfirr conclrvioolr drrn ftaX-ray 8tpdiW iP vbich V. ob~~t*rrdthat fl f tmmt c~llldb at0pp.d by X-irrdIatim vithwt all StoppIBu DR* rmplicatioa. 19 EE Lou of pmhbold hist- with no luas of pm- 1.brl.d DU in C1-orrostod colla (Fig. 6) indicates i that ch. Du-hLtoM C0rpl.x is not a static 88.0- !!! ciatioa in vlv~but, rather, in oquilibrirn vith its coqonont parts. Thmrrfore, when histone syacheria 4 occutl In the absence of DIU syncheds. those nuly aynthrired histonu cm~exchonga with chromatin- bound hhtonu. This interpratation .ant. vith caoclurlaor ue hmm roached after ruuring siwf- FIG. 7. Prsporatlw eloctropho i. of vbok hL- tomu pdewwith 511ysim r~1 icmt hlmtorw tunaover In cultures vhae DNA syn- labolod for 2 hr vith [lC]lyrh.. m- thesis vu blockod by high coacentrationa of tonu wore isolated fra all. in .gewo- ti01 Forth (A), csllm tra*.-f~# C1 prrlrad thymldlno. '* ' from 1 to 3 hr foollaring sp,chroai..tim by A Cap8fhal VU ud. Of histone 8pthesiB rat0 litotic solectioa (B), md COUD -0td in $ pulsd fra 34 to 36 ht r0- in colla umtd in C1 by isoleucine bficieney rrswp.luiw in iroleucine-deficict dim ad collr rtively trmerring the C1 phue tollw- (0. ing -totic seloctioa (Fig. 7). Tho rate Of his- In GI cells travrrriaf the life cy~lo,v. could tone synth.rls in C1-arrorted cells vu mstimatd detect synthuis rate of only 3.5 to 5.0 petunt to be only 2 porcont of that of 8x1 S-phue cell. of an S-phue cell (Table 4). Therefore, w coocludo that the hi* rare of hiatone swhuldaw- ins thyddino block.de 0bu-d in uny difforent labor8torles represortts S-phue hiatone synebuh

Histom 14C-prolrbolod histone half-life' even tho- DWA synthub c.ooot procrd beemma f raetim -(hr) of feedback inhibition of droxycytidin8 phosphate synthesia. The low rate of hi.tono spthui. h 97 fl arnsted C1 celb .ad in nom1 travorslng GI colla f2b 127 further charocterirrs the isoleucino4ofici.ot €2.1 110 svnchronired cells u truly C1 in uuuro. €2.2 187 f3 145 Phosphorylation of Histone FractioM durial r! Cell Cycle.--Radiation experlwnta in thi. Labora- a tory show that hirtoa. fl pholphorylatico 1. blls ~r8l.b.l.d with %-.rinO wid# for 2 dvs radio- to prolrb.lth.ir prototlu. Tho cdlr wore thou sensitive, uhilo histone f2.2 phosphorylation is rasupaadod in i~o~~ine-doficiontrdiu vithwt not. l9 As a result * vo conclude thbt phwpboqla- 14C-dm acidr md Jlwd to ontot G~-urost. Tumotllr rat- wan ruut.d for lor8 of 1%- tion of there tvo protrinm is rclatod to tv~differ prrlabekd hirtoae fratho chromatin during the cnt f~~tio~in vivo. By relating hirtono ph08- period 16 tO 56 hr in i8O&UeiM-d.fiCiOnt rwdi- (as aham in Fiu. 6). phorylotioa to cell-cycle position, we ham dbd

39 U.941 - 46,331 - 22,302 86,532 - u.347 - 59,544 - 18,2U - 70 ,690 -

Qal 2 ,478 20.6 5.3 n3+m 3,036 13.6 3.5 f3 2,479 19.4 5 .o fl 2,707 14.9 3.8 Am8td C1 nal 1,069 9 .l 2.3 f2IZ + f2b 1,429 6.4 1.7 n 1,263 8.2 2.1 fl 1.873 10.3 2.6

I.n pho8phoryhtioo. In thir model, inter8ct* betima0 DIA ad hiatow la uult.aed by phwphowbtiorr of hiat-, ruulting ia a danpraaaion of the Du t.q&te. coacludo from our inveatipticw that hbtona fl phoaphorylatiw ia not invold in such a coarml rchanir, .I it do- not occur in 5 when W opthala is vary activa. Ua faal the fk2 phaphoqlatioo, which doer occur kr C1, should contrcirwr to be cwidarod in 8uch r0d.L concerning &tivation of MA t.glat8 activity. The S-pbe-speclfic phoaphotylaticm of histone fl haa led to 8pcculuio~that hlatone fl phoa- phoxylatioa ia involvod in DNA replication. Frm ItudiU Uia8 X-i~.dirti~,YO cOacld8 that thir 18 not the cue.1g The caffeine ulloration of X-rapinduced division &lay .ad X-rayinduced hletom fl phoaphorylation reduction su(glat8 that v. look for UI fnvolvomemt of histom fl phosphorylation in call division.

ILtnrrcts

1. V, C. Ufrey. Rmctioad atd rtabolic aspects of DMA-usoci8t.d protein8 , E: "Hiatonea 8nd Nucloohiato8m#" (0. N. P. Phlllipa, ad.),

i 41

. _- P1M-0, kr TOY& (rO71) pD. 241-294. 2. 1. IL. hrloy .Id J. M. Urdia, Tbe rt&olin of hbt- fractiom. I. S~.thui. of hi.fOM fractioru durio@the lite cyde of mll 1s. cella, Arch. Btoahr. Bbphys. 2U-292 (ma). x, 3. L. 1. -1- ud J. W. Udh, Ib. rubolism 16. of hist- f~tioru.11. Coamamtiom and tutamr of hbtotu frat- In colls, Arrch. Biseha. Biopby8. a,14 (1964). 4. L. Re cut% .Id 3. U. Hudin, Ib rt8bOl.b. of histome tnctio08. 1x1. SyathWL rd 17. t-t of bbtoru €1, Arch. Biocbr. Diophys. y4. 392-401 (UIO). 5. L. It. Curl., B. A. Ualterr, and M. D. hpr, Isolrtiaa 4 chu.cterlz~iwof himtam fl 18. in rIb0o-a. Biocba. Biophyo. Ru. Cnn. 4ps 428436 (1970).

6. L. 1. Curl-, I(. A. Waltet., ad R. A. Toboy, The rubolirm of hirtow fractiomm. IV. SyQtbril of Umtoor durlq the %*hue of 19. the maaUte cyh, Arch. Bioeha. Bio- ph~~.JAB, 633-641 (1972).

7. D. F. P.t8?80U, E. C. AadOtlOlL, rad R. A. TOW, mtatic C~US u ~OU~C.of s~achro- 20. niud cultures, &: "Method. in cell Phyo- lololy," Vol. 511 (D. I¶. Pmcott, ed.). Acdrdc Pr88#, lw YO* (l968). pp. 347-370.

8. 1. A. Tob- .od Y. D. Iroleucine-rdt.ted re@atlon of replication in variou 21. .rUl%UlCd1 lines, Caer &8. a,c6-51 (1971).

9. W. 0. Earr ad R. A. Toboy. tffects of i.0- lewfm &ficlomq a~nucleic acid and protein rtmbolirr in culturod Qilure hamter cello. Colltiurwd IWA and protein myathula in the absence of UU oyuthri., Biochdstry & 269- 277 (1972). with -1.n mu l.plicrrtiolr 10. R. A. T&oye Production ad characterization of ~~~l1.ncmlls renrmibly arruted la C1 by R.#dUiOO Of -Ul Cell prOlif8r8CaO8 1. grwth isoleucina-deflci.~t rdiu. &: in dependent to a large extent upon the c.pacity for "H.tkod. In all ~~iol~,"Pol. (D* H. Prercott, ed.), Aed.dc Pross, New York (19731, initiation of L!UA replication (that is, gmerdly In pres.. if A cell cm ropliute its WA, it can dodiridd. 11. 8. A. Ualtero ad D. I. Patemen. Bdiomennl- Very little is kuwa .bout the biochdcal event. $0 tfvlty of mluliio cell^. I.. Taw .ad GI sud at the C /S bauadazy that propan a eo11 for dcue-depadence of ruliation-hduced dlvlaion 1 d.1~~BiOphJ*. J. 8, lL75-14Bd (1968). replication of its pwtic mstefial, although such processes mt likely play .o. intows1 role In the 12. R. A. Walter# md D. P. Poteroen, Rdioomi- tivicy of .arSian cells. 11. Rdiatian rchanisr which reguhto MIA syntbuis .ad cola cf fectr rctarolecular synthesis Blophyr on , - proliferation. Part of the difficulty stm frra an J. 1, 1487-1504 (1968). 1o.bilIcy to produce sufficient quantities of pun A. Ualtma, B. Burchill, and D. 13. R. R. P. C cells synchmoumly proparing for call division. Petorsen, itodloseaoitivity of -1- cells. 1 111. Effect of ollboptid growth t..p.raturu Ue have developed a technique by which we can exn- from ruiiatioa-induced ou recmry division in. both preparative proceonr occurring during delay, Biophy~. J. 2, 1323-1328 (1969). GI and bouadsry processem and bIO~hemk41operatioxu A. Welten .ad Idiooenritir 14. R. R. A. Toby, coordinated with &enom replication and lata ity of -iur cella. IV. Effects of

92 ldfratioa of ulls in the cell cycle bud upao relar1.n WA cantent u determined with the flw dcrofluorawter.’ In ell cultuns studid to date imolvin8 a variety of ~ynchroeisin8tdIaiqul.8, the vut ujority of Celt tr8rarred the call cych in a nodfaohion; houever, in all cultures there r8Mia.d ad1SubpOpulatioru vhich, though resnh- in8 vioble for rmral days, could not carry Out normal cycle progrerrim. Figure ZA shauo the flaw liCrOflUOr~rTiCMA dlstribution pattern from 8 cultut. prepared by FIG. 2.

mitotic .rlectiaa. such cella an midally per- turbed in a biochdcrl seame. All ult pcwaweed the 4C DNA content upacted for dtotlc colla. Follawing aelectlm. thm ceU vue placed io a spinner cultun to allow continuad traverse of the cell cycle. Mt8r a11 the detacb.d cell. hde.- capd from mitab .ad eucexed Gl, colceaid vu addmi 9 hr &ter to weat nomally tra*.nbs cella in mitoria 4 to prevmnt thelr reentering C1 ad obacurlng detection of 8lmly or aoa-pro~remain~ SubpopukCiOM. krdyeb Of nlufm POpUhfiOD DNA content8 at la hr nnrled a snll 8ubpqdation with a C1 DWA cootent, charly di8tinct from the Mitotic rrlectiat 0.055 bulk of the population vhich poa8eu.d the DUA coa- Ieoleucine deficiency Oel24 tent expected of cell. arrested in Jtaris. The I8OhUCiM h f lcleacylhydrcncyutaa 0.120 non-trmrsing rubfrmtiat coqrised 5.5 percent of Iao&uciae deficiency/alng$e thymidine blockade 0.134 the population, vu hi&ly reproducible, ad Double thyddin blockade 0.170 sua?mrsrraauAm~~~ (?. N. Kwr, 8. A. CriUrn, d N. A. V~ODill.) Flal Hcmfluomrtrfc (rw) Studlu of bctia Bind- iru to ululi.ll cella

bet- 8ofh U -m A (m A) rrrd uhut lawutinin 0 sam u wful ~au in rtdiw of tho eo11 rorf.cr. kc.uo thw bind sp.citica~yto uil-nrrfaca 8-t .oi.ew.' In

OOI WU b1-8 C.WY CdlUhX 8&UtiMtioOm 2 md in othor wn it dou not. m dCroflla0r~t~(nrr) t.chaipuo YOm USO~to qUt1t.t. coll-.~rfucr bl-g of fl~iu- uin c0njyrC.d lrctilv (CS A-? adW-p) to A vid. vu103 of cultund all papulatlcw. Thue ptOp8r8timS inc1ud.d 08tabli8bd toll lim a0. ILL., md L m uoll u ouploid ull strrirv rod t-r rlnu traufoxm8ntm. te dditim, variablu 8Wh .I ~dl-~cl~p~itlom, ell1 01.0, .Id tho effocts of protrrotrnt of the cdl8 with cnryv uoro studid. Tho proparatiom warn nnrtlnoly mmltorrd by floorrscm~cedcroocopy boforo RCF dmLto uurt.ia loc8lixacion of binding to tho dl surface. Pigun 1 illutratem tho Unoticr of binding of Cm A-F to CEO all.. Ruquancy dlstrlbutiolu of cell-murf8ce bladla~,ruch a8 thcro iurutr~tod in fig. 1A, uo ruurlrrod in pig. U. It ia evf- dent that uturatfoa of cell-surfaco rite. occurred by 3 hr rxpooure .ad that dlcell-rutface flu- oreaconco. at auuratloa. em b. wed to rursrire bindi2q for any gitul all populatioa. Fiwro 2 illustrate8 the ruly.10 of binding of COQ A-F to CEQ ~011.mpnch-l~d by dtotic rdection. Won tho dU8 were 4yr.d to nullify tho &my8 in ceU 81x0, w concluded that rho surfer d.nrit9 of binding rite8 wuIrr*.bly uaifolr throu#hout the dlviaion cych. Chemlcrl hoterogoaolrp of binding situ on each individlul QIO cell vu indicrtod by apoclfic 'I I I I illhibitioa of CaA-F binding by a-wthyl unno- 0 4 e u I8 pyranoside. pigut. 3 illustrate. saturation bind- noUn, IN tuLTW ing in the proreace of 2-fold incnuoo in ddrd mc. 2. lbdd CellwWf8CO flUo-lUDCO* C-tOd -1 coll-surf~ce uu. d ratio of inhibitor. &cuo tho =ti- ritu of Con A are fluoremconco-to-area for CllO cellr rpchro- nirod Jtotlc reLoction and nlcuod n-onc~cu.~ if the collul~rbinding ritu we10 by into frooh u8rm modirn at zero tin. also hom-rnoum, ono would expect binding to decreue to me-half for each incrrmnt; thir did not In confirmtiat of othor w~rkorm,~rgglutl- occur. nability of vinu-trpuforud colX lines or 1 OSbb-19 3. L. L. 54 aad I. J. Colbtda, Protalrcubo- hydrate 2atenctloo. P. 00 tho nollba of &iiw mica 00 cos- A. the phyte -i. of rb. Jrlt b-* BiOehr. Bfe phym. kt. gs, 398-404 (UU).

(6. J. Barnhart, C. E. Hlldabrd, L. L. mor R. t. OlriW, R. A. Tab-, D. P. Petar8.o. S. It. cor, .od P. c. S.ad.rm) UU-nnbrme Ammociati- in CultdChiowe ster Cdh: Iaturo of tho Du-lllabmuo kmociatioo sad its Inwlmnt in Twrrl d Spatid Or*- lutim of DlpA in the nuclou&

'Fb. fidelity of chwau bUplic8tioa dimcelb i@iu a large de~mof orwir.r- tiw Cot npliution of mA, pa~b&~of XU hto i Chroutin, c~wationof &-tin to foir &r~- 3 moomom, and mqroyrtion of chromm- ddm atomis. It im likely th8t uo11 repdstcrd tqorrl 'i ad spatial controls at. roquirod for theae p?'o- cossei . U Torporal owairrtioa of MA rmpliutloa md *rolp.or segrrptim indlcatad by rutrictim of thue procoomw to rpocific partm of tho cell Vwth (S phuo) urd division cyclo (n). Further, tnporal cmtrol of CUA replication within S phuo hu been docuratod by ovidenco fror both cytolo#- ical uui blochdcal rtudiu.1*2 Spatial or8dra- tim of MA is iqllod in the findin8 that DNA (or chroutin) Is attachod to the nuclear u.br.ae at ultiplc sltoa. 3-16 This avidonce vu obtrined by both biochemical .nd dectron ricroocopic tech- 6-U aiqus. Rorults of sewral oC thue rtudias su~cstedthat nwly roplicatod DNA vu Comd pro- dodruatly in DMAdrane caqlears. On the buis of this rvidonce. it vu concluded that DNA teplicr- tlw oit- are attachod to the nuclear mmbrme. RtlZ-8 mre reuntly , secnral hboratoriem haw reported 1. X. Sharm md H. Lla, Loctiw: C.11 awuti- finding which aro at varlmce vith this natiq .ad sugar specific ptotoiP.* Sdenu

46 caaclwiao .l2-I* In viw of than stUdi.8, it is cocllltln8 fluid in a PacLrd liquid sdntlllation -17 thu utdrnt ritm are rpoctmrter. Re#ults of these qeduoc8 arm dimctly imol..a t mplicuioa of DRA. liarever, sham in Idle 1. It is cleu frathese ruults the pOO8tbiut7 that kritiatiocl of MA replication tbt very little noarpecific a~egarioaof free or t.L.. place .t the rrbm attachmat site is sue &natured IIA or chmvtia takm place. luted by of tbwe 8rodiu. 199UJ6 SMea Subjecting the mqluto shear by vortu air- of MA-- -10- in cultured Chiwe ho- * before or after cryst.1 forvtion rebu the stet ~ly(Qo) colla ham been nde in M effort level of nonmpocific att8chmnC cogarmd uith the to eLIILi0. tho SUM of tbe ccqluee ad to lnvn- tmsheumd mqle. For tbe coatrol srqle, vhole time thdr ftmctiomd role in the procurnu me cells or nuclei (Isol8ted either by NP40 traatmnf ti& above. of &ole cella or by hmotonic svcrlliftg foil-d by TWO iadapmuieot tachniquu haw been wed to Daurce h-iration), the DNA of which vu uni- i.olate cd cboractorire #k.rrbrure coqlexe~of fody prelabe1.d with %-TdR, were carried thd CaO cell.. Thue athodm include (a) the Wand the earkosyl lysis, ohrrr, 8nd crysta~isationpro- prdre&-loped by Earhart 1.l’ which cedures according to the preceding protocol. Shear- utilirv attrchrnt of r.bruu framnts to ing the r-lr before or after crystals are fo-d c~ul.of detorgeat, sodiu lruroyl sarcainate apparently K.IDWS KSA or chrovtin not closdy 2+ (sarkoa71). formd io the presence of Hg im, awsociued with the drme fraction. Even bftmr d (b) suc-e wentsedi~entation of cells or vortex rixins, 80 to 90 percent of the total .LI- 3 nuclei di8rupt.d by limited SatiC8tiOn. Several brae u ammayed by H-choline incorporation Into experiments were perforrd to teat for nonspecific =drrnu renins with the sarkosyl crystals. attachnnt of free DNA or cbroutin to Sark08yl To mtdy the lucrorokcular natura of the crystals. In thae studies dabeled. erpaoentially structure of the DNA drumcoqlex. both the I+ wing a0 cells wre harvested and vuhed by re- bmd technique aad aucrose grdlent s.dirnt8tion suepmiocr .nd centrIfu~tloain nt buffer at 0% of sonically disrupted cells or nuclei wen wed to [O.Ol 5 Tri8-C1 (pB 7.1 at 25*C), 0.1 KC11 d detelrin. the sensitivity of the coqlex to vat- 6 lysrd at a coocentration of 10 cellahl in TX buf- lw apnts. Msults of the +band analysis for fer with 0.1 percent satlrwyl. Either 3H-thyddin= theme treatnntm are sham in Table 2. The two (TdR) 1.bel.d DNA or %-TdR labeled chromatin in types Of 88S.y produced qUdit8tiVUly sldh t.- ‘Lx buffer YU lfnd vith the cell lysate. Sarko- sults for all ap.nts examined. In addition to cryst& verm forwd by addition of 1.0 5 MgCl, syl - TABLE 1. wnspEcmc .VIACX~ OF PRE OR to a 0.01 A11 final concentration of E. operations 3tWtVRED DNA OR QIROWIH TO !LUlXOSYL The 8ulrayl crymtrlr vere ven performd at 0%. CRYSTALS isolated In discontinuous sucrose gradiaits [6 d Total TCA-precipftable label in 20 percent suctwe (wlw) wer 4 ml 67 percent su- rarkneyl crystals (percent) cme (wh) in TKH buffer (0.01 5 Trls-C1 (pH 7.1 Xo vortex Vortex dxing u 25.C), 0.1 5 Xcl, 0.01 5 14#X2) 1 by ccntrifuy- Sawle mixing (20 rec) tun hl SpiaCO SU41 rOtOr at 15,m ?pr for *ole cells or 20 do in a Ikckua L3-50 preparative ultracentri- nuclei prelaboled fuy at 4.C. Sarkwyl crystals sedirnted to the vith 3H-fdR 80-9 5 10-20 20147 percent sucrome interface, and the grdiencs Unlabeled ceAls vere fractionated from the top. Fractioas were plw native 3H- TdR DNA 0.5 1.’ precipitated by addition of 10 percant MA (tri- chloroacetic acid).) .ad 0.01 5 sodium pyrophosphate Unlabeled cells plu &-tured %- and wre assayed by filtration throw Vhatun Gr- 1- ra DNA i.8 82 flus fiber filtera. Filter# were washed vith Lhlabeled celln 5 percent TCA d 0.01 5 pyrophoophate, dried, and plus 3H-TdR counted in toluene-POPOP-PPO liquid rclntillrtlon labeled chromatin 1.2 IOSbQTb b NOQ@ 100 100

'Cattrol 8-l~ were hrndtrd .uCCly u treated 8-h -t thU .rUpr W

b~wl.ifra ~ntlall~lraviry Celt pnl.kld for 24 hr uith 4C-TdR umre budby tmatmat of &ole call8 at 5 x u9 Celldml uitb tba m- laic dotergent M?# ia RSB (0.01 4 Trla (pa 7.4 at 25'C), 0.0U 11. Il.Cl. 0.00U -121. Ikchi vere hmted by cmttif~ia,nmmp.ndr In TI buffor at 4 final comcentratiea of 5 x lC#hl, ad qUtCk17 f-8- in DX7 IemtM. SqlW -re 8tOmd at -70.C d dtht.d to *fold b~fOm

CSqlea of rukcuyl-lysed colla or nuclel coataln- lug 0.05 snjclz voro tnatod for 1 hr at 37.C vith &o uoiu/rl of muo-frm. O#u. I. Sql.. wx8 usa).a for v&raotatt.ched Mu u d.8crlb.d in tho text.

%to rarlr#y~-1yred cell swprmioo in TK buffor vas tnated with 100 miU/d of Aloe (freed of DNase by troat~ntat 80% for lo & before UO) for 1 hr at 21% .ad mawed r &scribed in tho tom. e Cell or nuclur l~rcluea wm dl~rtedwith Pm~uo (60 uuit./ml) at 37.C for 1 ht. Tbe -le8 mi. wwed u &awlbed In the toxt.

tn8tUUt8, tho drt0rmt 8odim M-1 sulfato (SDS) eaplotoly dis8olvnd tho rdrac corplox u assayed by tho SUCIO.~ fldiart rodimaatatlar rthod. R.rdt8 of these trUtmnt8 Indiccrco cao.i&r.blc ronsltlvlty of tho UiA in the coqlax to releu0 by DIIue. Pmnua. or SD6. Tho Mu8 tnmtint shar. that no oxtended n@oru of DNA are protected fra dlgestioa by intorution with tho -&rate. lUue di-tion hrs no effect indicating th8t RNA. although fomd in ths collplu, is not requirod for ulntainlng tho structural 1. integrity of tho MIA-rdrnn. coglox. nc. To eurinc furthor tho naturo of DNA-rubrmo -rum cagleou i.olued by the aukomyl cxyatrl rthad. Hrat, cella pn-lad for approx- Lutely two tlur8tIon ttru (24 to 36 hr) vith "C- TdR y.~.aynchroniaed by dtotic aelectioo.21 At 6.5 hr after colt wra t.1.u.d from dtai., a 2- rdn %TdR polu L.kl (20 uCi/rL) VY applied. The pula. 16.1 vu atoppd by parria6 rh. aliquot over cmdfroson dim, md the cella vem hneatecl bI u de8crib.d &ova. -11 lyutv -re pmparrd mad I aubjected to vanrioum WIPU of .bur by =ex dring, rrd hy1cryatah -re forrd. Ruulta of thi. exporinrt, rhom in ?l8. 2, ahat that tha RC. 3. ratio of %/"C lncmuea ai~ificaatlyea the lysatw an aub5nt.d to increaming ahear. Ye fcnmd aidkt ruulto for e.paa.irtiJ1y *in$ cell., mumsting th8t .01 nvly labeled redoor of PA are cl-e to tbe rltr of att.chmt of PWA to mat brum md ue protected fra shear dapdation by sit.. at the -lex. If thi. ia the cam, a pula. label follolnd by a &am. perlod of grnrth In aonrdio- ti-. naulu of the we-chue experfrat auwat actin idiur 8upple~ntedwith TdR ahould rude that not dl DNA rapliution situ .I.uaod8t.d vith &rum coglena. If all replication sitem in 8 decnue fobim vortex dxing of the frac- located in tho coqlu, the munt of pb- tion of nuly -led OUin the r.br.nc caqlex labeled DRA (3H-TdR labeled DU) rarinlng with increuing t1-a in the "cheae madiua." The ammo- ciatad the aarko8yl c~atalaaftar reaulta of thir axperirat are illrutrated in with 60 da of chue wuld be conalderably lea. thm 50 perc.at of Pig. 3. Each aqtvu plrpared for the aarbayl the louat fomd iudiately after the puhe usay vi& 2hec ioteml. of vortex miring. hbd. This obaewatlon indicotu that mame nub Npli- liwro 3 ahwa that the fraction of auly labeled catad rwlna cl-e to the dram COqlax, DNA in the lipoprotein caqlax decreuee vith in- DIU the replication rite d@t cootinun to IDI. creulng "chue" ti-, while the fraction of "C- while uay fnn the sire of uaociatioa. Such a Sw- TdR prelsbaled WIA rerlm appndm8tely comtmt. tion could edit if DNA-m.braIe attachrat situ Ruulta of the two e~erirntadaacribed are chose for initfatiom of syntheab but not conaimtent vLth localiration of sow DNA mplication are DM for tloog.tion. i I I kcauu the apparent DNAeruu CI of role of -Ir coqluua in sow upect of DNA replication (pou- ribly initiation) , experiraca vere perforrd $using I culturea of CHO ce1.l. aynchronired by the dtotic i selection procedure to deterrlna if the aamt of 5 DNA In DtiAdrU8 coglaxem varied during the cell grwth urd diviaion cycle in mom vay which would provide inforution regarding fnvoluat of chc coqlu in DNA replication. CHO calla vere 2DDprelabeled for approxiuceiy two generation timi 0 S 0 I5 20 Show Tima (WC) with %-TdR. Cella vere synchronized by the Ettect of shearin8 aarkayl lyrate for ver- dtotic selecrlon procedure and re&ued from ioru tirr on the r lative dlstrlbutim of pulrcl&.l.d WA (3 ?I-DwA) .ad dfody mltosls at 0 hr by placing rhc mltotlc cella in labeled IIUA (lk-MA) betvean the n-baad surpcruion culture et 31'C. The mart of the DM bad d the frae fraction.

49 a *f f RC. 5. boat8 of di8cootinuow 8ucr0.a padieat sedimntatla .nrlymia of dtotic cell. (a) and S phase call. (b) or nuclei (c) dL- rugted by luted sonication. DNA- Tim drme coqlaxea an folad at tho intor t )uwi diu r.WI. fnr milasis I faces (l0/30 and N/55 porcont) bemeo FIG. 0. Buulta of n~urolrat.of -t of DIU- the suct#o layor.. Nucloi wore propmd drub. coq1.x at variou tkua am for by hypotonic trutrnt of 1h010 colla at mitotically aynchrmlxed all. uhich were 0.C for 15 Jn and by Douaco howg.af~- m&ud from dtalr. The qdity of cell rim in 1)( buffer fO.O1 tris (pB 7.1) , rydwoay in wing th- S phoe .ad 0.0005 H&l,]. Uuhed cells or nuchi cell dlvislan is sham in and b. All- were thon tnatod with lidtad sonication. qwu of the synckraalzed culturr y.?. Solid potuiiu cltrate vu added to a pu10cl.b.l.d vith h-TdU at 25 uCl/rl find cwcontratlon of 10 petcont. Su- (52 CItw for 10 dn at 37.C. Th -0 pies wn layorod 011 the sucroao gradionrr vu stopped by .ddition of 2 volura of in the s.r buffer rod centrifuged at 4.C Ice-cold cacodylate buffor (pU 7.1). Ihe for 4 hr at 33,000 rpr in a Splnco SUI1 colla uore hamtad by contrffuptim, rotor wing a Ikdrua L3-50 prepar8tLvo .nd the TCA-iwoluble nceri.1 vu mu- ultracmtrifupe. Ractionr were cohctod urmd u deacrlbed in the text. from thm bottom of the tube.

50 cell wplu t&.a botvmn 5 and 14 hr after rei-e from dt#i.. TUB mr%&ilitycan be attributed 1. to improduelbllig of soPiatiaa conditions from sqle to sqle. 30 such vuiability vu found in uglv thfra 0 to 4 hr or fra 14 to 17 hr 2. after t.Wo fra drcwi.. The mcmt of DNA in rapldly sdtroting -term vu low (3 to 6 per- 3. cent of total Oar) in sq1.r tako aerly or late In the cell cyclel., cmparrd vith e .uch higer lewd in 4. s..plu t&m from 5 to 13 hr (10 to 30 percent of total WA). Em-, tl.ri.biliq introduced by sod- cation vould k WII obriou phase spplu in S 5. vhor8 tk.0 lDurt of mmbrmto-usociated DNA is hrpr. Ruults of theoe rtudiu 8Ugga.t that the 6. spatial arranvnt of MPA withla the nucleus is rewhted by .uociatiw of sp8ciftc region8 of DHA 7. with the nuclear rrbram. This propod is sup- porcrd by the findial of Harriran17 that indicate 8. a pmfOlmti81 u8oeiation of cmtrorrlc hctercr chrovtin to tho nuclur r.brure. 9. FuJiwara caaeludod fra his studfu16 chat initiuioa DIU qnthuis regulated at the of is 10. nucleat m.br8m. While the reeults presented hen shwing correlation betuoa lncreue in 8880- a DNA 11. ciacod with umbrlac and met of MA Synthuir agree vith his coaclusion, other interpret8tions are 12. ccrrpatibh via these results. For a.qla, It i8 pocrslblt that Oq8ai28tiIJn of DNA within the nuclew 13. rdiated by Utachrnr of specific regions of DNA to the nuclear =.bran8 uy be required either for propar --mat of DNA to U~UIC fidelitv of 14. chrolorar duplication or for proper tidng of the seqwnca of DNA roplicatiaa. Initiation of DWA 15. cynthuir libt occur indepoadeatly of chis orga-

nizational evmt, while the 8- initiation sites 16. dght fottuitoumly close to the DNA-9.dtrme lie 17. 8ttaChUlt Situ. porolble rolu attachrat DNA to Other €or of 18. nuclear druw have bmn promrd. "J~nese 19. studies ilplicate the attachrat sites in control of emtic expression either by virtu of locallza- tion of MA vithin the nucleus or by altering the 20. activity of IWA polyrrue required €or transcrip- tion of DNA. In addition to chwe su~eotio~, other control VchMiSU such u mthybtion Of DNA 21. could functlm u signals which specify which DNA 17 should be attached to the nuclur drum. 22. Bad Analysis of tho Qiouo R-tor Cell Line CBO 1 Chfrmoe h.rter cells (line Cao) ha- bwn mod extaruively for rtabollc, radiabiolo@crl, Chrmor number and -notic rtudi~~-~vith Only a eup8rfici.l -or of cells appreciation for tho &ire. of ra.rploi9 chuacter- istic of the line. Tho mda1 ch-a ouber and I30 19 20 21 22 44 4.5 morpbdw voro briefly douibod by Um and Puck 1638 0 1 pnd clwlfied 00 tho brio of .hl8wth nuure- IA-cnd l9 20 21 22 w mnts as 12 normal ad 9 alterad ch-a~ .nd an 106 3 utiuttd 3 percent luo total &-tin than n0-1 cells. ?ticroflwroatric data Our current lack of rmdoatudin8 of auploidy, the videspreod ma of the coll lhe, and the pa- Q10 Gl peak 21.1 dour report of a relatively &rw nder of ab- G1 denormlird 0.96 5 norm81 chromoooru proqted us to \ladortake a more U-Cd c1 peak 21.9 ertoa~ivekaryol~ic .arlysto utilirin8 autordfo- c mode nOmli28d 1 .oo graphic urd Ciera banding techniques. Our observa- 1 tions demonstrate that 110 homolo- p.irs remin to be risming. and that only 8 chroweolu appear nodwhen cm- Figure 1 shws the untreated chromosorus of 6 pared vith the euploid Chine8t buter karyotype. MO and LA-CUE? arranged according to Ksu lad Tho CHO cells wd In this study wore from the knres.ll Our CHO karyotype differ8 from the one 4 liar abtrined in 1962 from Dr. T. T. Puck.' Euploid presented by Kao md Puck only by substiturfon of feule Chinaso h.rter fibroblasts were from a small metacentric chrowoome (ours.) for a sull strrin dmipated LA4& md uaro propagated fron acroeantric (theirs). We have retained thoir an 8u clipping. Autoradiop8phic dysis of DNA nplic8tion pattern# eqloyed conventional techniques ruing Kodak MI) emulsion. The C-band technique vu a dification of the nmthod ducribod by Arrighi rad Hsu8 in vhich air- dry preparationr wore treated for 3 mln vith 0.01 I.oH or saturated B8(08)2 and incllbatcd 17 hr in 6X SSC at 60.C. Preparations vere stalned 10 min with bufferod Ciera. GEmd proprratioru Y~Ntreatod vith trypsin 12X6OYK)II by a VdifiC8tiOn of Soabright'a -thod9 in which the major chute vu to troat fiamd chrarwaes vith trypsin at 0.C. Aftor dving, slide8 were stained as described for C-bd st.fning. The cold trypsin treotunt appear8 to reduco dramatically the variability charactoristic of trypsin hydrolysis, pat- ticuhrly in cells blocked with colcedd. Table 1 smriteo tho chrwor number and relative DNA cmtont of MO ad u-CHE+. Flff microfluotavtric cstlmmtes on flwroscent Feulgen- CHO-2 Untreated karyotypes of euploid Chinue stained cellsm indicate percent less CHO FIG. 1. 4 DNA in hmter fibrobluta (U-CHE$) and limo CUO than in LA-QIE$, in pod ape.mt vith the pre- cells. Tho Z-#roup classification €or alt8r.d &mors CHO boon uud vfow csthte of a 3 percent doclrmt reported in hu prevbusly6 but hu been 8ltercd on the by Kao and Puck.4 One of tho K chmm~crsappears buir of chroroclow bandins porterns. "2-groug" notuiaa but, bcaue we found evlbnce uhile neolution of bold8 in the hrger ch-8 for s.rrral e&n- too 61to be detected by ah 18 rup.rior in the mtapbue cell. length muurmnte. hmchanpd usi~nuto 'Caqarlsoa of the C-badr In CtlO md LAd the '(Z~OQ~"accordingly. reveal8 8 apparently nodChrOlOlOl8, 800. Of 1& +bandla# tehniqcu pro*.d to be met which are palred. The rdnder ha*. either ujor inforutire of the rthod. wdfor datocting or minor deletione, additions, or rearr.or#nLI. alteatioru in chrcaatia anla~mt.?igure 2 In ..ay of the 2-group &~OIO.QYS, the bad%- &am a ccqarleaa of the chrmoru of -0 and patterm permit ldentifiutioa of both the origi0 U-Cd. 'Bad patterm ara 8idl.r to thae and dretlaatla of trmmlocaced chroutln, bat 8- fnduced by urea rd IS1 or by 2-nrc.ptwchand, dteratlam which involve a 8lnae bad lack 8Uffi- ut.., .ad SDS.12 Wot.ble erteptioor aze the ter- dent Infomation and are ubiguoua. In additlm rid bab on the short CUI of chrmor 4 .nd to the Dbmd inforution, ve have used ahratio on th. 10- amof chrwar 5 which appur ua- muuraments and data from C-bd urd autoradio- rtained in our preparation. In previous prepara- graphic cdnatioa to complete the Mhy8b. u* 12 tiona. thus terrinal bed8 are heavily rtained. h8- published the detailed exulnation of the Th. dlffeawe u. pethapa daor but at. isort- karyotypu6 and pnunt here oaly mjor featuru. mt €or our 8naly.i. of the Cllo chrmorr. Two Chrowea8 2-3 and 2-7 are the product8 of 0 keryotypae of QIO 4re 8hm: a prartaphue rad a axchmap between a nodG mid a norvl 5. Zd mt&phm. -nation Of the p8ttera8 @Vrl is 8 normal 4 which hu undergone 8 pericatric ithe of the &#me of vatfuton encocmtered ad inverrim. Tha centromere 18 oriented uprid. dam indic8tee that baa& am differentiated better in u cogared with a nod4. 2-6, uhlch cant.in8 Q emre dark and light bend 8ttach.d to the long am, is of interest becu8e a sfmilar cr.luloutim CUI be reen in mother .amuploid Chinese huter 12 line rtudied by Kat0 lad Yosida.

Two generalization8 CPI be nude from the 5 band radyrls. The flrrt 18 that oaly one x chro- monome Is present in CHO cells; the other ir chat with relatively minor exceptlone (namely. a light band added to the short A- of 8, padcentric inversion of 4, ad a rull deletion In 5) one haploid ret of chracworr ha8 reruined Intact in the CIiO cell. Althwh the C-bands 8hom In Fig. 3 an lur informative than tha G-bands, they hew been useful in identifying several pairs of centrorrer. The 13 C-bmdr rerdle thoee found by Hnu urd Arrighi, but in our experience the amount of interstitial heterochromatin demonetrable vu variable and dependent on experimental conditions. By v8rying the lm6th of alkaline treatment, w8 could alter

CHo-z the nuder of burdo and conclude, therefore, that 2. TrTp.in-iaduced Clan. bmde in LA-aCg ad the intcrrtltlal are remuntr of pattern QIO chromeae. For the Q10 ChrOrOrOrl induced by the alkalincraline-CicPra techniqw of we pneent luryotypu from two ullr; the firrc &rowoar in uch group is from e Schnedl.14 In our analysis, centromeric hetrro- prometaphase cell .ad the 84cond from a ohroutin has been used for purposu of identifica- nt.phue cell. The arrow at chromoew 2-4 indicatu the point of reattachent of tion urd lnterrtitlal bands, because of variability, a pericentric innreion in a nom1 b chro- have not been relied upon. a~oy(see text). I 2 x. 4 Identiflution of late-replicating DNA (?I& 0) Indfcatr that CUO cells pooruo an alteratloa irr labellag soqueace. partlcularly in chrolrwov 1. In our had8 ad in studlea of others, ””’eupbtd Chinwe brrter cells replicate the termIn8.l 41~4s of the short am of chromos- 1 late, Hwever. in CEO thue arem are coaaiateatly free of label. In dditiaa to this differmace, 8emr.l othmr CEO chrornaro rhor late labeli~pattern. thu am iacolrlrtmt vith patterns of nomchrorr~a~ bnt 17 sirilar to patterm obserrrd by Zakharav s&. Tbse voricers sug#ostd that hte-repliC8tiag 8-u were either derived from heterochromatic portioar of the X chmacrows or that they originated by genetic iluctlvatloa of excum autosomal aegmats presat in uuqlold CRUS. Becua Z-poup chmsrr 2-6 ad 2-10 comlrteatly label later than their nod counterparts 6 8, it unlikely that Zalrh.tor’s 00-2 and is FIG. 3. Centrarrie bando In U4U? 4 a0 cell8 cxplaation applies to CRO cclle. Ue cannot rule follovisg a DYA denaturatloa-~naturatloa out positively transloution of rvll portions of procadure. The arm at the nuber 6 chro- -01 In U-Cd indicateo the telowric the 5 chracwov to 2-6 end 2-10, but the Senrr81 luterochrmatin (see text). preservation of nom1 chraoson banding aequ.aa I 2 X 4 in thue tw group wrbers sumest. that Xz trams- locatloas haw not occurred. The other hypothesis. that late-repliuting alauntr are autoaarl uce- rbl. in excess of the nodcaplernt. is coll- trery to the evidence both of the G-band analysis and the mlcroflwroutrlc e8thtu total 6 0-It of DNA contatt. Finally, if h8terochrcutiution is Involved, it NOC be facultatiPa becswe the C- brds rhar no differenma between ~rulchr- sows and their late-replicating Z-grOup counter parts. Because of the unusual nature of thur observations end their mlications with respect to ullular control of DNA agnthuir, UI escenolvm study of the replfcation sequence across the entire S period is In prOgln8S. Thue data clearly demonstrate that the rdalng X chroaoa is the early-replicating XI, in coatrmt to takharo~~s~~observation that in hypodiploid huter li~aboth X1 and 5 rWn Intact, while in both pseudo- and hyperdiploid line8 FIG. 4. ~ucor.diorapha of latrrmpliuti one or both X chroPsoua were absent. in U-QIE! md CHO chmmoorr. Thyddine (2 uCi/rl) vm added to exponea- Figure 5 is a diagrammatic reprsaentation of tlal cultures for 1.5 hr: at thi. tir th8 major G-band. arranged €or comparison of CHO and coludd (0.06 y/rLl vu d&d for 2 hr. Note the absence of label in the terdnal U-CHl!$. The arrmpunt is bued on pomitlm areu of CIlO chrarcwom 1 md the extra Ideutiflutioo of centr-res (e.8.. 2-1, 2-2. etc., lace-repllcating situ In 2-grow CEO chramosors 6 d 10. were originally cantroarem of the hGoAop now 105bqaS c 1. :*en*liritcd conditions for enxyutic dfwatfaa and bv b risi urea-marcaptoethanol treatsant ,12 La that feu bonds are broken. and it is likely th8t in the best preparations little if any material ia mmved by tne treat=nt. In each cue, the most drliute and revealing patcerru arise after che gentlest treatmznt. with tvpical fuzing of the pattemr the major consequence of prolonged treatrnt. Differentiation of chroweaul reg.nt8 into

light. intermediate, and dark strlnins area# 8- gears that metapham chromatin is oryraismd in specific way vith regard to chromoad procelnr. E, This concept is supported by studiea of isolated FIG. 5. Schutic amyof the mjor G-b.nd8 in chroautin which have demonstrated that denae chro- adCHO The first and LA-CK& cells. matin has a higher proportion of disulffde bond8 second &mora of each set are the normal diploid uid .pp.matly normal chro- than diffuse chr-tlnl' and that hirtonu are fra Chrorwaa of the 2- -am. c110. bound to template-ective and twlate-inactive ~oupare arrmyd according to centmmre of orfgla: (2-1) deletion of the terrrinal chromatin in different For thue reYO1I. portion of the upper arm; (2-2) inrema1 we conclude that G-bands are a reuult of protein deletion from short arm uith nom1 telo- rre; (2-3) nciprocd translocation with ;iydrolvsis and have little to do uith denatura- 2-7; (2-4) periuntric invaraim; (2-5) in- 11,20 version deletion; (2-6) truulocacion. tion of DNA, as proporred previously. orl&n mcertdn; (2-7) reciproul trans- Xato and Posidel' found positive cornlation8 location (2-8) translocarion, with 2-3; becueen Giemsa band8 and chrcrosac spiraliaa- ori@n uncertain; (2-9) tr.lulocation, origin uncertain; (2-10) deletion: tion. ::e have made simI1.r observations, and the (2-11) ttmslocation, origin uncertain ; studies of Stubbleficldl' of segrntod ch-ora (2-12) ublpoua. beat evfdence is ceatro- -re from X2; and (2-13) tr8mlocotion, also indicate that G-bmding patterns result from origin unctrcaln. chroaoroul condensation processes. We should note that, vhile there are similarities betwen altered in che karyotype). The first colum shws one noma1 chrOBo8ool from LA-CHE.9. The second sewnted chromoeoh~sand C-bmds (the patteram of dark and light bands are and both suggeat repmurents chose chrcwosona of CHO in which no ab- similar. that light areas are late-replicaring vhilr dark normalitlei can be detected. In colunr 3 8d 4. arens are early-replicating) there clear the chrsmoaopr with fever abnornrlitiu is displayed , are also to the left and the major tran810~atiohs~deletions, dif ferences. The sewntation patterns sumesc and reartaigewnta are indicated. that euploid Chinese h-ster 1 chromosora are noc An wtst8nding feature of the +banding p8t- haoloqous'' and that the long amof the X1 and X, chromosomes nay be Neither tern comparisons outlined here is the reproducibil- difference detected by the C-band technique. ity achieved by drmticdly reducing the hydrolytic is activity of the trppsin. The assrmption that prott- The C-band mechanism apparently le inwAvd olytic enzyms, in general, cause alterrtiona io in selective denaturation and renaturation of ,NA.13'17,13 Bianchi and Ayrcs2' contend that Ciwa staining by cleavagc of peptide bonds is sup- an increased of C-banded material (coaati- - ported by the general ob8ervatlon that banding amount .. patterns are similar follwing treatment with other cutive heterochromatin) occurs in an 8neuploid proceolvtlc en~yEier (e.g., Pronase which, on the line of African green monkey cells. Xuch of this u- one hand, should produce rather nonspecific hydrol- excess material appeared as interstitfal bands. We G ysia .ad, on the other, by trent~~ntwith urea and found no such incraaae or localization of this Ileteroctrromatin in aneuploid CHO cells but did see c- Ipcrcaptoethanol which might be expected to produce -c increaae in late-replicating sites. Xis aad more epecific effects on rulfhydrvl Linlraws). One an Subai15 concluded that chroP.tin may be rtndered notion, not yec eaphasired but supported bv both the inactlu vitboot hetorochramtic cm&aaatiaa. 3. P. A. Walter8 .ad D. F. Potarmrr, P~dlooami- titit, of mmuiir. 11. wi.tioo Takmn toyther, thue obsarvatioru 8-t that fuo ef feet. on ucroro&Cdar rynthuir , BbPb8 separate rchbni.8~uy be prueat dorilrt -laid J. 9, 1487-1104 (1968). trbwforuion. (k. nchr0L.r portlous ln&tivatu 4. 1. K.0 .od t. '1. Puck, Genetiw of somUic of tbm mwn by heto~cb~tisatiOll,the other by mu calls. X. Owntitation of .uta- by ph~~icrtad Cbdcal .*Us 2. alterfag the tiu of Irplicatlon. R~rtbot8tudi.r Cell. Pby~10l. E, 245-2S7 (1969). of .#uploid cell linr obanld elocidate tbue 5. .oa puck, -ti- of ~ovtic protamso. F. 1~.o T. T. all.: Liokp 8tudiU uith huraalnue z.Lhua S&.lr8tudiod tha Ottuctlu~Md hrter cell hybrid., Natura 3,329-332 (1970). nurlcrl miatiolr of chrommolr caplant. of 9 .Ir.rploid Chi- tmuter Ilnu md cmcludrd 6. L. L. D&waa and D. B. Pmtet.en, The chrm0- 8- of a0, .II -.uploid Chimoe hmt8r thbt "the rlablllg of thro Una doped8 OCL a cell Urn: Gbd, C-band, bud autor.dio- rather ~~TOUlldt of tba total -t of chraP- graphic &y8u , Chraomou (Berlin) (u73). in pt.88. soul materid pre8aut." A oirilar cooopt cursu fra study of the iSh coatent of 14 cell lfnu 7. T. T. Puck, P. C. Sa&-, d D. PILOI..Or Life cycb adyoio of u-uli~cell.. 11. Era 8 dlfhrutt .p.clw. total COQ- m*26 In rnA Cells fra &he Chinese hmter. Biopbp. J. 5, tent these llou cluster u points equivalent to 411-450 (1961). haploid unit. of cells. Our Du for 8. P. e. hriw and r. C. m. Localization of prCnOBt rUdtr 8hW that, Uhlh U-Iou. n- heterochroutin in huva ChrcmO8-#, CytD- poetics 2,81-86 (1971). arrlsrprntr have occurrod in the ai0 chm~y, esoa~tldlydl of the t.qlatractive maor ir 9. 3. Sebbright, A r8pid banding techniqw for huva ch-oms, Irocet & 971-972 (1971). ret8iu.d vith intoct uquencem of bmds. Also, Wh.0 the 2- chromoouo are arr.nrpd accordin8 lo. P. M. Krwr, 0. P. Pateraen, bud X. A. van Dill., DNA conmturcy in hateraploidy bud the to 8iLI, they are d8o 8rr.nrd 8ccordin~to the sta 110. theory of ttmvrs, Science 176, 714- soquonee of chroroclao in the normal karyotype 717 (1971). with respect to rh8 c.ntrai.re region (1.e.. each 11. T. C. H.u and H. T. Zenzea, U8m81im chrorp centram seem to carry e &*.a rount of chra- 8-8 in Vitro. mI. Idiogra Of th8 Chi- nese hmrer. J. Natl. Cancer Inrt. p, 857- mtin uterlal rr#ardleeo of the extent of n- 870 (1964). arrmqemt). Wne tread. auarmt that tr.luitiaa 11. 8. bto rad f. H. Yorid., B.ndln8 putem of cm- fra euploldg to aneuploidy i. rather mharply Chiao8 hrrter ChrW8am80 rended by new trolled. Both rho totd mt8nd oq.mix8tion techniqw , Chraoeou {Berlfn) s, 272-180 (1972). of cellular DIU into chnrwou -pear to be restricted. Preodly violation8 of there con- 13. T. C. UU rad F. E. Arriphi, Distribution of coustitutive heterochromatin in dim straint~are lethal. chrw-8, Chr-8- (Berlin) z, 243-253 Studlea of the kind reported here 8hmU hdp (1971). to elucidate the control achdrr involved in 14. U. SchMdl, Aa~ly~isof the humn lurpotw - evolution of meuploid lilu827 and uy ultiutrly uia8 a rruiociatioa technique, Chr-.or (Berlin) 2,468-454 (1971). a expirin the observed difference8 in ~ut4-i~be- VI hador beoreen euplold Chinese hamtcr fibroblutr 15. T. C. HDU, chrowsors in vltro. u- G XYIII. replication sequroce in the Chi- 6;: and CEO cellr. nese hawter, J. Cell Bid. p,53-62 (1964). 03 csr 16. E. Stubblefieid, Ulimchromosorr REFEEENtZS -vltro. xfx. Ch-oro of Don C, Chinue huter fibroblut strain with a part of ?u&, Cieciuta, ami A. R0biD.m. 1. T. T. S. J. aut08OW Ib tIM8lOC8ted ta the Y inroamom, hIWtiC8 8mtiC VVUP alh. Of 1x1. 3. Xatl. CmCrr 1-t. 2,799-517 !1966). Loneterr cultivation of euploid cells from huvn md mid8&jrCt8, J. Exp. Phd. 108, 17. A. F. Zakharov. E. S. Kakpakova. S. A. EspUna, 945-956 (1958). and W. E. Pogoalany, Chromosomal variability in clonal populationr of a Chinese hruter 2. D. F. Petersen. A. A. Tobey, &ad E. C. And.* cell strain, J. Natl. Cancer Inse. 2,935- son, SJochrooouly diridia8 dimCelL. 956 (1964). Federation Pmc. 28. 1771-1779 (1969).

56 u. M. C. Ord d I.. A. Stacken, !btaboUc prop- We U.O ultrariolot light irrdiuioa of lyse orriw hltaom fra .ad of rat liver th- genic cellr to daroprus tho pr0ph.w. 'ha cell almd, Bioebr. J. 98, 886-697 (1966). which harboa the prophage i. capdle of rrcisim- 19. K. nuruhir md J. Damar, Fractiotution of limr protein, Prac. htl. Acad. Sci. USA66, repair; theroforo, 8mrd obrlow qUaOtioor cea bo 2961-29h4 (l971). rkad ol thir npdr crpability relatit. to prqh.lr action pU- 20. ?I.E. 3r-to rrd 1. Y. shw, Spacific bdbg derepruoim. The of reprtt -, patt.ms of hmChroll~aao. Proc. mel. ticululy dOIL(IJU.e, on the irrrdiatmd b.ctarial Aud. Sei. USA 68* 2013-2077 (1971). chrammm could triter the prophyr darop~as~ 21. A. F* 2dh.M .rrd II. A. E@Iilu, erot. First, we c.ll uk if repdr le WU rmpliutico md dtotic opiraliratioo of for &repressioa. Seeoad, ue can con8ib.r the .laas heteroehrmucic portlono of Chiwe pos- hutor chromoar., Qnmooou (Berlin) 23, sibility that repdr does jwt the -wit.. 365-385 (1966). we wre coaeomd vith tho qt~sti~nof whather 22. H. L. Pardun adJ. c. call, chramul ropait premnts prophaw donpnssioo. Poooibh loullratioa of muse satellite DNA, scienco -*168 13%-1358 (1970). involvwnt of excisioa-npair in ultr.rlolot-li@t derepnssioa of the Ilaemouhilw pbqe Wld is 23. L. ace, S. S. Tenotbia. ud B. 1. Brinkley. n. Lnvmstipted by dyrin# ad caquias derepns- DiZfOrantaal ~llaolocentLalin# of + chrap.oru vlth antimom specific for sinfie sion in 1ysowns of a strain 4 a c-&rip 8t& DM, Exp. C.11 Rms. (l973). in prus. ative which lacks tho andanucleolytSc 8ctidty of ? 24. N. 0. Biachi adJ. Ayru, Hetorochrautin the cxciJim-rapdr sch- .- loutiaa m the chrawaaes of norrl and Lysogoa~of thase tvo rtraim were erpwad to trmuforrd cello from the Africpl green monkey (Cercopithecur eathiom). DNA varying doeos of 1iltr8viol.t li@t (254 nd and danaturatlon-triluturation rrthod, E-. Cell O... 3,253-258 (1971). usayod on a phago-#ocuitlva indicator 8tdn (nd) for infective centers (:.e., thoee cells in an ir- 25. 8. Ria .ad D. P. Kdd, Chrowoa structure, Ann. Pav. Genet. f, 263-294 (1970). r8diat.d population thot produco phae) and for the titer of infective phqr particles produfd by s.dr 26. Kraenr, Deaven, A. Crioomn. P. M. L. L. H. lnfectivo center. Froa the reoult~sham in Pig. 1 and U. A. VII Dill., MAcorutancy dupite variability In ch-ae n(Pber. s:"Ad- one CUI see that tho incroue In infoctin centem vatas in Coll ad mlecular ~iolow,'*Vol. z is similar for straily with urd without oxcision- (E. J. DuPrau, ed.), Acadedc Press, Neu York (19721, pp. 47-108. repair capability. m8 8u#gOOt8 that eXCi8iOlr repair capability nee-sary for prophqe de- 27. T. C. b,Chror#oul evohtlon in cell populc is not ti-, Int. Mv. Cytol. l2, 69-161 (1961). repression, a coacluaioo which ha. 400 bwn ro- ?orred for ph8p 1dd8EOeherichia & lpOgOM,' bDdr of Radiation Dw.N.Bd- and doer not prevont deroprossiou. In ddition, tt llcrtion of a Bate-- + -UVT strain, while quite cap8ble of excision-repaii Uo ha- studiod tho derrlwpmt of bactorio- apparently docs not repair tho.. pyrimidine dirrs vhich cause derepression. If dimri trigser phage HPlcl In tho bacteriln Hwmophilur inflwnru which prophage dareprusion tnr8 rendered iUoffOCtiW follaviag dereprusioo of the proph- by ultra- + dolot rdlation. Our msults support a -del which in the E strain, the plot of infectiva centers pk8phy DNA replication on the cell me.br.ne. versw rJdiJtiOTI dose vould be ahiftad damrud relative to the CUM, Indicative 8 dose where a trluiwly high rolecular-eight COLICJCOY? s- of reduction. Clearly, this thn -0. of phap DNA Is oynthesirod urd subsequntly de- is not hlgher doses of ultraviolet light, tho grdrd to phaspaquivalent DNA, the ujority of At frequency of derepressed lysogenr incruseo in thc vhich is rmbr.na-frec. These rerult8 led us to + uvr culture but decrouoo in che E- strain conclude that tho call mmbrure not only ia the - 2). &wing decreuing infective site of DNA replicatim during which phage DNA is (Fig. The curve centerr for the E- strain is similar to rurvi. syuthosirod in mltiple phage-equivalent concatoaers a curve ahwing that, at doses above 10 erp/m2 but is Jlo the site at which thou catc8to~mare 1 there bJCterfa 8 sufficient rider of lesio correctly sired and reduced co phagesired pieces. incur . I . x tAf -1 \ 'I \j - i

I 1 e* I I I

FIG. 2.

to reader them biowcally inutivo vi& raspact to ph.lr-prodwin8 ability. a-r, tho froqruncy of infectin ceaterm cootinuor to iact.ue with do80 3. X. Ha& rd 1. Crou, InductLon of pr0pb.e + A in 0 vtOt of 4. &Kl2 doteeti- icr in tho E cUltUm, 8-8ti- that the diffennC8 2 initiatia of DYA npliutim ot hi@ te in thoor curve8 &ovo IO erg/- Wdt8 from DNA peroture. Ibl. Con. Geaoticr 110, 299-306 (1971). ropdr, which dom indod frmction In this rtrda to renm dirrm ad podu surrir.l of ph8~- PaCkuirrS of Bacterial Chmoom: prodUCin8 .bill-. vi- A Swp0rt.d by kactivatioa of Ildiation-Dumd Our ruult. OIL proph4 derepre8oim io B~.Opbiluindieat. that eieion-aprir crp.bil- vim M ity Is not nourur) for trimring drlrplrorim or kceat erldaau su~.rutht the coqla, proventin8 derrptuoioa. Lo -ti-, it -purr fillod hod of thr brtorid virum Tb is .u.rblod .. that a coll which i8 C.p.bb Of Dw rrprir UId Which by firrt colurrueti~a protoin shell end tha door exclro pyrlmidim diwn dooo not rew~c.the kuertb8 DRA Into chi. pnfornd rtructuro.' mi8 dimer(.) vhi& rorults in derepnrrloa of the pro- 4.1 for head uturatioo stom from expcrlmntr phaB8 state, Hamvor, ercirion-ropdr i8 4Ort-t which indlcato the follariag: (1) hoedm vhich in srutainio~tho capacity of the dcnprrsred lyro- -pur to lack Cull DUA caplorot8 (by dectron gcn to eupport phav productfoa vha bacteria am microscopy .ad $UC~O.O puiient uralyrir) uo

58 65 F s the prerace .Id abomce of 9Ah. After 30 dn, the I I I control dtUN cootaid a aim&& pgokim of vipble ph4, rkih the 9M-t-d can- tained a .idle poppl.ti00 of pmearsor (dined or frwle, fillad hud.1. Rw dt U.L. tha \-’ 1 exp-ed to incmulrs door of ultmolet rdir tion and .uayed u .pgropdate tiu (domu) for aammr in 2 surrirlos infmcti*. mars. The lis. t demmutrate that inhctlva cantarm in the cmtrol “i culture am 101. semitlva to ultrh0l.t radiatioll than the OM-tnatmd cult=, 8-tiq that the DNA which rcurbtu in 9M-taatd cult- ir .om r-sible to -pair qat- thrr is the DU of phw gram mder 80-1 coodftiw. Such a findily is 101. corultent with a hd-flllins model thm 6th a fnlile, filled pmcursor del. Becclwe of the coqludg d uncertainty of radiation effects on the cell syrtem, chi. experiment wuld be more coovineins if the precise repair iQChPPi.1 thio 8Y.m We8 km. b Th. exper- RtptlMus iment vu repeated with the erci.iae-rep.irless 1. P. Lrrftia, We D. Wood, d R* T. OlrioJus atant of E8cherichi. vi& M rimif- &. -teriqw Tb hd.prpbop-i8. 1. [k icant diffe-ncu in iructivrti~c-0. CrWth the a8t- of 494foctin hub od thdr role m inte~diates.J. Hol. Diol. 2,55s- curves of phap developmt in 9M-treaud ulh 573 (1971). which wre irradiated prior to 9M removal indlcete 2. sw~nmd Piechwokt, Studiu oo that the repair rchrai.r quite rapid. n. n. n. is phai developmaat. I. k acridine s.luitL.. Experimnto ue in prognso to eotablish clock. Vir~lom26, 163-174 (1965) whether recabinatloa, OWA polymerase I repair, or 3. n. H. Piechwrki urd H. Sua=, Studiu on a phage-acdiated repair (Ten8 reactivation) lr phage bvelopunt. XI. Th. maturation of Tb phap in the pruance of puraycin, Vfrolow responmible for the bulk of rercw Era ultradokt 28, 386-395 (1966). radiation in the 9M-treated cells. The pasibil- 0. R. T. Okin&.a. Norphopnesis of the bad of ity that the dye 9-nlnoacridine protects the DNA Buteriophue Tb. Ph.D. Thuis, University of from ultraviolet irradiation is being deterrind California. Lor Aaaylu (1971). by caparing the thymine dirt conteat in control 5. E. Kcllmberger, J. Sechaud. d A. Rytu. and 9M-treated cultumo. A correlation bemeen Electroa mlcrmcog. rtudiu on phap multi- plication. IV. The eotablishmt of the ultraviolet reoiotance rrd a particular rapair DNA pool of vqetativa ph8p and matutuion svrtm ahould establioh nore distinctly the atate of phrr particles. Virolow E, 4711 (1959). of the viral chr-or prior to padragkrg into a c-act hd.

60 wtia offeeu data fro tbetvo .Lo to Mk8 .rrrcrpl.u- of Prqt * tern eff.tu fro IrC--rlr to l~ll.DL. -0 ?mj.cu ah#imatisued ipcludo (4 luy- M nrliw luiu cblr ..1 to cqra -. to -7.I .rpoclM at dffforeat h rat- md midud injury tbat w =fl.ct *e ~p.cfea1%-arm ~ffe~t#,ad (b) aUl1-L.l rt&u wadcm to 1maati-e tho kina ti^ of rdi.tia injury dptinl chasinl (build- d.aq) ad fk.4 dwrate muru.ad the effect8 of dare rate oo ra8idul injury. Bdhtioa ?mt.+tioo Cui&# (me) hmm b... ast.blJlb.d by the Intmrrutiaul Comiaaioa 00 ILdiolgicrS Protection .od afford a ready buL for edrutioo of lajury ruultily fra outrmco of tdioiaotop.. into tbe hwa body. Sow of t&e vrlua h... beon derivad aolely from obruvatia of a aaoh- ----uli~apcio beuwe no othu dUa won bvdlablo. In order to mold ire rubatiteiO8 of nonbarp data, thia Laboratory hu #ought a ratiauL bui. for utrqoktion fra mall OW to mn in the interspecific relation bdon vu- fau nlwmt rubolic parmtars and vholo-body uei&t. In a roriw deal- vith caquativa me- ab01i.m. Star. = &. coaclude, "~.~~qo&tiowto w fra.niY1 data ue ddttedly difficult ad nortima laaccurate; hobfever, the riak in not extrapohtins ate, unquutionmbly, u~hgreater.**6 Such extrapoletiolu ham be- ud. for rdio- iaotopa of zinc. iodine, man~-o, rllnr. irid- iu, niobiu, rutheni~,' and berylUum~md othar isotopu aucb u mercury-203 and aeloJniu7S ue bin8 in*.atiptod. 'Iho wtabolic patterna dater .mad with radioahnu have uaeful rm1ev.n~for qWetlOM corrernizq 8-l. i#oCop# of tb-• el.- Mnta * ThW * the MtrbOliC pathvqa f0lb.d by nonr8dioactive pollutant8 throu@ a portion of the biosphere an indicated by the#. atudiee. Detedmtioa of tho courso of uhole-body activity after a #lode dddatratioa of a radio- nuclide si*.. mtabolic data woful in eatintion of

61 2.

6. J. t. St-, 1. S. R.l.00. 1. J. Della baa, d 1. K. Brutad, C-uatl- rtabolir of radiaoudibs in m: A re'(rl.y, balth ms. 201 U3-l37 (1971). 7.

8. C. R. UJcbolri ad J. E. ruich~r,Cuiul37 body btudeum in M: Jmury 19% to Decaber 1966, B.diAtiOU h8. 2,538-5619 (1967) 9.

lo.

ll. 9. Y. Audermaa ad P. F. ktafroa, Cmcentra- tion of ce81u-U7 la hrrrn rib bmo, Sdmco -137, 668 (1962).

BIolodC81 ttfects of --Ray Doso Protractioo durlty Coutluuou Crpwure vlth Fixed and Charriq Dose Rates

(J. F. Spalding. I(. F. Archuleta, J. E. Lwdm, .Od 0. S. Joh~~n)

REYERENCES The modifying effects of dose protraction la rodrats rod larger .oL.lr ban buu reviewed axten- I. J. 8. Storor, E: Tho ProUdiaU of 4 sv riu00 Dose at. in 0di.tim rlnly iu recarit symposia rad colloqui8. To Bi01o.y (D. C. Brara. R. C. Craglo, md T. 11.

62 1I-2 f 776 8% 1185 1354 2268 2% k u-2 876 904 1272 1496 2526 3265

variau. Tdla 2 1. a threway tlbk of -.Ip murvivd ttrs. The aaalymim of varianu of M survival timu Is ahwo in Tabu 3. Tben warn 8ie nlflcutt diffenucrr in mom survival tins mtraio. mubjectod to the three do8o-r.t. h*.b &ad -&hod.. Thhr. ObWW8C%OW Coluutmdth rdiatioo-nsistaace chrracterlatla of the ~UOUM ruborrains umd .od su~estthat the bone urrw warn the critlcrl tupt organ un&r all dau- rate conditiolu. The tu0 mtbods of expomure affectad the d- dmdifferently 4t *.ch of the throe drwa lamb (1.bl.s 2 and 3). Cb.nlia$ do80 ruu V.L. .on biologically dayiw than equivalent conatant or fixed dome rates. Tha 46.4-hr doaa lovak urd in thi8 inve8tigatiaa ue .ommuhat higher th.0 those .. that My b. aCiCip8t.d during 8p.c. dSObXi0 or recmnded eaporuru in crrgenciea, U-ver. Table 2 rhara that 8a the dome rate la lararrd the difference la ...II surrivd time under the two expooure rqiru incnues. Althou@ it wupa- sible to demonstrate 8 si(aific8nt diffemnce in ran survival tL. of rice subjected to comtiilunur

00131493079 S4m of .ow lum

b13 ,ll8 b13,lU 279 ,098 279,098

l6,200 8993 8 .m,196 11.79. 21.5% 21. SO6 2.39'*'

279 ,061' 85% U.36. 207 .761 lo3*11)0 11.U' 68.l24 31.062 3. ?Sb 101,669 9,083

& Si@flcat at 0.01 -1,

bsiglliflcrrt 0.05 -1.

n.m. not r4aificat. upowre wing em eqboou+. re~Lu,pa8ible hp S. temffect differmemo from a0al.th.l domu delivered by the mo rthodr could not be a8tiuted.

6. 1.

9. 4,

64 10. J. F. Sprldi8q, N. J. Wwa, R. P. Archr lata, and 0. S. Johrron, Cwrative effects of dosa pmtractioo .L lar dome rates by frac- tiOart%Oo rob by coatinuOU8 expO8un, RdiA- tlon Ru. 2,608-611 (1972). U. C. R. ond C. L. Vdt. ads., Annul fkport of the Biolo~calrob Mid ltuurch croup (U-4) of tho USL uodth Dirislon. January throryh Docubmr 1971, Loa Az.p. Sci- urtific Laboato9 ibport U-4923-PR (April 1972). l.2. J. 1. Spddw, D. H. Papp, and R. A. Popp, A withim-atraln diffemua in radiation sen- 81tirity in tho Un me, Rdiatloa &.. +o, 37-45 (1969).

U. J. 1. Spddim, D. 1. P~pp,d R. A, POpp. A glnotic effect on radiation sensitivity cou8istent Vith a #Indo gone locus hypothesis. Miation Bar. 44, 670473 (1970).

Coqaracivo Biological Effects of Radiation Dome Protraction fn Dop aud Monkeys

(J. F. Spddin8, L. M. Hollmd, J. R. Prine, 0. S. Johmm, P. X. Ldave, and J. E. London) FIG. 1. The aiomedical Research Croup's radiacion A pljor pal In our vrrlian radiobiololy pro- exposure facility lor dose protraction studies in dogs and wmnkeys. 8r.r is to obtain d-o-rste-effects data on the monky. Thi. priute hu received little attmtion little ham. Comparative dose-rate studies on u a rmrearch animal in studying the effects of dose dogs and parkeys. two speciae vith videly differing protraction. nhi4 b parhaps pBKti8lly bccaure it acute Lajo values,' have not been reported. is 4 difficult spociw to handle urd reetrrin prop- Six beagles and six monkeys (Mac.- mularta) erly for prolonpd periods in a radiation environ- vere placed in a continuous gm-ray endtonant mnt. at a dose rate of 25 rads/dav mtil death. Blood The dog is the moat studied of out largrr samples vere obtained by venipuncture from un- rryls, adluch excellent radiation-effects data anesthetized animals before exposure and at 74av on the do( are brine acctmulatd at other labor- intervals durinE exposure. atoriu. To facilitate extrapolation of the effects !.!em ?acked-cell volumes (PO') and vhfte-blooa- of protracted vhole-body irradiation from one cell counts i'dBC) dre snom in rig. 2. These blood mamallan species to another, it is helpful to study characceristics responded rfatlarly in doe and two specia miultureowly. During the lut year. mmkeys tc vhole-boav gm-rav exposure at the H.uliur Rmlioblolow Section h88 modified Its 16 radddav, :his is in contrast to a quite dii- cobalt-60 irradiatioa facility to acehate ferent henaropoirtic response between dogs and 12 dogs and 12 mnkeyr for sirultan.oua whole-body monkeys exposed continuously at 66 rads/day. At expo8ures under nurly identical conditions. Fig- the higher dose rate, dog bone marrow appeared ure 1 sham expoourm amn~mnuused for dog- uch less resistant :o radiation injury than did 1 monkey innstigat ions currently in progress. that oi :.-.e wnicey.' ;3salute n-ers of neutro- phils, expressed a percentages of pre-exposure Hematopoietic Response of Dop and Honkoys to values for doqs md monkeys, are sham in Fig. j. Wholo-Body Pmtr8cted Exposure to Cobalt40 C.u Xlthougn :le neutropnil (the predominant grmulo- Rays at 20 rds/day.--The modifying effects of dwc cvre) :s tne principai shire cell LJ peripheral protraction by fractionation ham been report4 blood of me oog, Lt ieem nore radiosensitive for the hmver, the effects on monkeys than that of the moniev. *dhose principal white cell of d-a protraction by colltinuoru exposure are is cne 1:'snocute. ::.is difference in neutrapiiis

i 3,

lhOl.-kdr Cobdta htB.-b8idurl bjm from a&th.l t.u-ray exporuns u it ut be reflected in life short@~g huborn daolutt8td in the rodeat in n-roou. ~opotts~'~nri.vd ro- uarly. EstL.t.r of rm%dd,loaptem injw fmoxpaure to ionizing radiation in larpt nub are ndd. To eatirto rosldud hjuw spaciu such u the dgud ronlrq, it is desir3f. to wo ISL cad point other than life rborteniry. rsspoo.~is obsemd 4t hi-r *0 ratu urd has Uduction in IL.O 8UmiVd tiU durim COaC%8uoCr bua lrported wing .cute oxp~mtrer.~Ihr char- v-rsy exposure has been uaed u 4 masure of acteriatic neutrophil in this study sug- rori-1 injury in ~cs'~d in laryr mirrla gutm that la swiu-.pdfic th8t tho it ad u oh- ond 8utno.' This te&d- of ruuIiD8 smrmr roam y bo ammciatod uith acute irreparablr or bag-torm injuty in dop .Id e radiation aemitirity or & 1# v8lru in 1l)g i8 boinf investiptod. hryr mh. Twlvr be.* and 22 -s (baa wore uocd to tast for possiblo lmytoa n8idUd injury from a continwly delimrod --ray 1. conditioning exposur8 (dog8 and malreyr) .ad from largo dons rcdared by the fractionation rthod of dose pmtracrioa (hyronly). s,'x bo86l.e .od oix mnkoys wore exposed to 664 rads of 8- rays protracted o.Ir Lo d8yr of coatinww expomure. Ton dysuam 0.porr.d to 2. 2400 rads of #rurays using the frrctionatioo rthod of dcwe protractiaa. with 100-rad fracriolu boina given 8t 56-d~Intervals. Ei@ty-focr+ days after tho 660-rad expolure of doy .ad mdwys challenged by che continuous-expaour8 rrbod oad after tho 24th 100-rad fraction &ml.ai.tend to doyo challonyd by the fractloorcim mothod,

00131493082 aniulr were placed in a envlronrnt at ?Conkevs with an accuulated gM-rav dose of 30 24 r.d.1d.y continually until death. Six control 2400 rads (approxilrrtcly 4 Cirs their acute mHI) dop and deyswlth no challenp dome were also shaved no residual injury in terms of a reduction exposed continuowly at 2b rrdsiday until death. in ?ST. Challenged monkevs actually lived sli@tly Diffemncm in me- rurrivd ti-, if my, between longer in the I-rad/hr g.lu-ray field thm did un- challe-d and mdrdlenpd MIuLI Irpreunted challenged -keys; howver, the diffarance uu aoc 6 reridual injury in the rdiation-challaawd animala. stat is tically signif lcmt. Two dogs (litterutea) in the group challenged with These results are coruiatent with earlier 660 rads of sruray8 approached a "Stedy state" reports that have de.autrated that the rrco*oq condition between h.utopoietlc-s~t~cell renwal rate of heutopoietic tirrue. in terns of perlphord and radiation injury; therefore, thy were not blood valuer, remained unchanged in there ..I included in the rmsurvival tir (ST) data of monkeys troa the first 100-rad exposure them* tlw conditioned dop. 24th 100-rad exposure. Lon6-term injury sum to .%.n-survlval data are shom in Tables 1 and 2. vaw greatly uong species, and additional work *%sidual injury in do- BL days after a 660-rad remains before a rational basis for extrapolation of conditioning exposure protracted over 10 days was injury and recoverv kinetics to man can be est&- approxiutely 60 percent and YU atatirticrlly sig- liahed. nfficnt at the 0.05 level, ?(onkeys sh-d 8 percent residual injury from the s.1~conditioning REFERENCES exponure, but thi8 was not statistically significant. 1. D. Crahn and C. A. Sacher, Fractionatioa and Frotraction and the late effects of radiation 'TABLE 1. %:AN SURVIVAL TIW (mT)OF DOCS WD in gull -As, h: Proceedings of a Sympo- sium on Dowc Rate in M-1i.n Radiatioa -KEYS WITH UITXOUT A 660-RAD AND SiOlOgY (0. G. BM.R. C. Cragle, Md T. R. CONDITIONING DOSE IN A CMRIA-RAY FIELD Noonan, e& .) , UT-AEC Agricultural Remearch Laboratory and 0. S. Atomic Energy Codssion OF 1 RMIHR 3eport CONF-6W410 (July 12. 1968). (hrl ST -.9 C. D. Van Cleave, Late Somatic Effect. of Treat= nt YdL.YS & Ionizing Radiation. U. S. Atomic Energy Corission Report TID-24310 (1968). So conditioning dose 9260 +_ 260a 2428 2 334a 3. 8. >l. Fry. D. Crahn, X. L. Criem, and J. H. ::onditionlng dore Rust, Late effects of radiation, &: Proceed- (660 rad.) "070 5 149* 1461 2 2313 ings of a Colloquium held in the Center for Continuing Education. University of Chicago, :hduction In .%ST 190 96 7 >lav 15-17. 1969, Taylor and Francis, Ltd. , Publishing Company, London (1970) I pp. 101-136. ?.esidual fraction 8.4 39.8 4. J. F. Sprlding, V. C. Strani, and F. C. V. Worman, Effect of iraded acute exposurer of gam-ray6 or fission neutrons on survival in '5tandard error of the ran value. subsequent protracted gama-ray exposures, Miation Res. l3. 415-423 (1960). TABLE 2. XEAN SURV'VAL TIMES (HST) OF HDWltEYS XIM 5. Y. P. Page and E. Still. Factors dlfying AND wxma; 2400-RM CONDITIONING DOSES T. the response of large anids to Lar-inteo.ity IN A CAIO;. RAY FIELD OF 1 RM/HR radiation exposure, 3: Proceedings of the National Sylposirn on Natural and kknude Treacmant aadiatlon in Space. ha Vega. Sevda (E. A. Walun. ed.). National Aeronautics aad Space Adrlnistration Report NASA M X-2410 (Juruclry conditioning do& ::o 2260 2 260' 1972), pp. 632-662. Conditioning dose :- 30 rads) 2637 2 267 6. 3. Crahn and C. A. Sacher, The measurement of residual acute injury from exposure by 3cSduction ?SST single in -177 survival follcuing daily irrdiatim. Ann. :I.h:idwl fraction -7.8% F. P. Acad. SCI. 116, 158-168 (1966).

"Standard error of t'.ie man value.

67 Rdiobialodul tilot xmutiutiocr tm the I.r atiw ?ioa Ildiotborapy trosru

(n. R. 4u, R. 1. ~~cbul.~,L. n. iiouad, rpd J. F. Spddhg) Fig. ru. and me.ulr lam bwo wd am a rob1 for mrul tL8w ramuml. I.caro of poor depth-doae dimtributioo of tut noutzoo, wd akin ruetimm CarlR k 8erlou vhr fast neutrau arm rud In rdiorhaapy; tbanfon. tha rdi.tio0 rempoll.. of pig, rat, and l~onskin to X-r-8 md 1-5 fut IUUtM hu b..n Dtudid Ut-iUly- bopt for mom mU&t diffa~acu,thuo 8kim c11 b8 wed m a modal for 8 uU-rauaal 8fltr. Stin ro8ctioru ia pip ad hwboco mipificat 12 to 15 days aftor ruliatia oxpaum .od pulr at about 30 days. In dco rrd rata. tho mactioo bo#- 8 to 10 w8 aftor -W 4 at .bout 20 day.. A wcond wave of 8Un raactim im ob- med in hv,pip, ad rata 10 to 14 wrk. after expomure, ubeau in du no macod vau of rractiao b ob8ed. Relati*.. biological 8ff.C- tim-8 (mr) 1.l- ftm fNt UMtolu U.ll fd to bo miail8r for pig and hulrn akin; hwevor, th singlo4ose Ut vduma for tho maand rat foot am lower than thoro for pfgmkin. Thuo lw vrtvu could be due to tho pruooce of subcutmwr fat in the pig .ad h-.' Thua, the differoaco in BBg oa wefoot and htmm rltin from noutroru im uinly duo to different neutron d#r drpooitiolu in timmuem with adwithout fat. This mbdnot Ytter too ucb for nog~tivapi-. Th. maucfoot ry*tu CP be umad to obtain quantitative ruultm for n.yticn pion bwr of difforent peak vidtha wad in diffor- cllt ther8peutic mituuiolu. &mu00 Of UD.U- eaarw tramfor (LET) variuioru for dlffarnrt puir width., prmcime RBC utiutu uo n8cosm8ry in rymta much u thi. In order co mdentand md capan tho rerultm of late offectm and tumor reapomem. Kalhm ad hi. unodatu rocently wed the -e foot u mdol for cell mnvJ of nod cismue and Ym sucoy from tho a- -0. timu- platod into the thi#m of myanpnic C3H Jce, to study the potential of a radiomoasitixtng drug (Bcdl) for clinic81 radiotherapy.' IaIT im 8 marcoma that 8fO.C J~t~~OlJ~~~8t tho bua Of the 0.I Of CjK/Km wein 1962 in Kallra's khorrtorY 8t Stan- ford. Thi. lim YI. mahtallud by sorial mubcut8nooum

68 :hervat ion -Score Roaction Increuin& kactiar riubaidinp

0.5 W50; doubtful if abnorvl Hair slightly diacolored

1- (0.75) Sli+t reddaain8, definitely not nom1 Patchy .ad/or diacolored hair on foot

1.0 Definite rddening andtor alight edema Voy little half on foot and lamr leg; &/or eqor8ed blood veesela papery-thin skin

1+ (1.25) Senre r8dde.i- and e&u and/or white lo hair OD foot and 1-r le8 andlor acalinl; on bottom of foot (dry desquamation) cl& toea with no nails

1.5 tint -11 a- of roiat bred-: wully 1+ but with one small area of scab. tipa of two toea stud: (mist deaqumaticn) Nav have two toe8 stuck

1.5+ Several -11 are= of moist desqluucion Healing cmlrte except for one medium ac& or more than one ad1scab

2.0 Cadderabk mi8t de8qu.~.tion vith at Approxlutcly one-fourth of foot unhealed leaat four toes stuck together (with 8ub)

2+ Very severe 2.0 (all toes stuck with much Very severe 2.0 d..qu.ution)

2.5 Brrakdanr of hdf of the skin of foot (uluration)

2.S+ Bmrlrdwn of thrrr-fourth. of skin of root AppKOXhBtely three-fourths of foot UI- healed

3.0 Breakdam of shoot 111 skln of the foor HinFul healing of foot

3+ Very severe 3.0 Ahoat coppkte necrosis of foot

3.5 Cowplete necrosis of foot in severe ycllar Coaplete foot necroais: "clubooof mlat exudate over foot ycllw mist exudate over whole foot

plotted u a function of dame ahw the sipeoidal haparue of nrligwt Tisaw (Tumor) to X- curve airilar to plota of cure rate. md caqblica- Irradiation.--To obtain specific tumor-ciarue re- tiona vith doae In radiotherapy adagree with the spoarre to X-ray enpoaure, it la necessary to iiolate 7 rcrulta of FwLr E &.1*2*6 adBrarn tf &. --in vivo tumor tfsarw From normal ti.8~0 for exposure. Teehaiques hmbeen developed to aoln thir problem. The KXT tumor norvlly carried In C3H mice hu been lntroducd and mintairwd in a hairleaa mutant strain of RF?l llice. An - l2-m-diuter CUI)? from a donor weI8 removed, pl8c.d in nor- nul saline aolution, and minced into mull piecea 3 (- 1 E 1. Thur t-r pieera are truuphtd into the back. of hrirlesa RRI ud C3H pice by making a aull inclrion in the skin a€ter shaving the back. The back is wed in preference to the thi& to permit isolating the tumor from the re8t of the body during exposure. The twr grara to abaut 6 m in diameter on about the tenth day after trmrplmtation. When the tumrn are 6 to 8 Iin dimcter, the mice are .amthetized (0.1 cm3 Jg body weight of Nedutd), ad the tumors an expoad to FIC- 2. A*ra8e akin to 34 reactioa * X mvs by ahielding the rest of the mule. Turora each of 6 dolru of X-irradiation. ~~~~4~~ 69 1.

PIC. 3.

70 7. tor the p~ltetmof thra retentloa fua8tioor are &- in Tabla 1. Th bat fit to all data ir li-n for euh route of akhistration. The bwt fit to tbe &ta for each of tb. u mtce i. @*no in Table 2 to iwtrate Mifordb in rtaboliu of tht. 110- 8. cud. dthh woop. It is obrlorv that rrrrt uta ti. YY rb.orbed fma the rut. 1IlP.t 99 per- -t UY 1-t vith * W-tl. l~rthm 1 d8Y .nd 9. 1 p.rOrt vlth 249W-tiw. Uthe th8 f8Dt caqaao of th. retentloo hmctlolv for I? d IV data umrs dUfet.ot. in both uyu &aut 30 pmt 10. vu lat vttb a half-tin of about 90 days (.lor C-t). Tb8 rdietiarr dme fOllW* inf8-w of this nucllde by aitber route (ZP or IV) vould be the 8.1 n indicated by the ti- intatrr]. of the u. retaatia ftoction, which b proportid to radia- tioo don. Tb. effective half-ti.o @i.m=fer to tracer et- urd nd include rdlorctive d.~. 2 Hollur suguu the we of turnover tir la 12a pl8- of blolO&lcal half-tirr to .wid ..bwtr In -48. mover tin (the tin requind to tmm, Brit. J. Idiol. 4S, 237-249 -(1972). 8-e 4 pUUhtie rqU8.l CO the pool) th. reCip- ?meal of tha rum corutaat and, for the lorn$ capo- lwuuIuI lmmaxsn nont for tin in du. is dven by f - l.0/k3 - (0.693/ll8). where T is the turnover tirnkj is &tentloo of Ti-113 io )lice after Od, Intra- the rate conatoat for the loo8 caparrant (0.0077 peritoneal, ad Intrannorr Injection day-’), .ad U1 im the physical. half-life of tin- (J. 1. Looh, C. A. kd.9 J. S. UilSWI, .ad J. E. 113 and Is 8qa to 546 days. he biolo&fcrl half- hrchner) ti- CmUtOd by the Ur-1 Uthod io 379 day..

Tb5.8 fo\d.u a tMel.rnt in hmtis- sur,’ bot it ir Corvidersd to k 01y cantanant u no biolog&cal hmctlorm ue attributed to this 1. I. 1. Tipcm d H. J. Cook. Trace ekmnu in huvo timsma. Part If. Adult rubjects frum el-at, and it i. rdativrly nontoxic. Tin-113 the Unit4 Statem, haltit Phys . 9. 103445 decay Is m dectron capturr prOC888 vith an uso- (1963) . ciatd 0.39-mv ray. 11M.r physical The 2. half-life uku thi. isotope a sult.blr tracer for experiunt., Znt. J. Appl. Udiat. hOtOp8m rtabollc mtudiu. -23. 341 (1972). Appwtely 0.5 *I of ll3s~1in 0.1 nci .Ohti00 UY ddnbtOmd to f8rrk dC0. .bat 3 .OW old advd#tin@ about 26 8. Three groups of l2 da each uere inject8d by the lntravaow (J, S. Wilson md J. E. Furchnsr) (In, intrapedtonaal UP). or oral (IC) routes. Keuuronnts of lov-level activity In a nom1 Within 30 dnof Injection. the activity vu population of Haw bxlw resideatm have bwa rd. runrad in suitable datector, rrd the uuuremnu rince 19%. About 40 Irrdivldwls have bwn couated were repeated ~riodic.lly for than &yo 10- Mo on a weekly or matthly buk (about 10 and 30, re- in the Io ad XP group8 rrd for 4 days in the cue apectfwly). Ra8ults for ths 1956-1967 patiod of the IC ~oup. Th. data -re dy8.d .ad fit to have been reperted,1*2 .Id a report coveriq the a sa of tw or wza exponentlala. Avmragl d~88 period 1956-1972 is in preparation. Xonthly

D 05bSOCi i’ 71 xnt- hjoetioll

56.9 2.73 0.25 U.5 O.OS31 U.0 29.6 0.007U 97.0 44.00

&ttq.dtooul Inoj0ctioo

b3.9 1.09 0.63 25.1 0.- 11.0 30.9 0.00776 m.0 u.00

I1/2 4r .3 Ir3 52 &EL *mart) (porcont 1 (d8y#-1) (d8V) -E9

10 .O 1.10 0.63 28.6 0.0695 10 31.5 0.0077a 89 45 40.4 1.28 0.w 27.3 0.0710 9 32.2 0.W26 84 43 41.1 1.18 0.59 27.0 0.0599 12 32.0 0.00774 90 46 41. b 1.00 0.69 2s.a 0.0590 22 32.7 0.00761 91 48 35.6 1.14 0.61 32.1 0.0690 lo 32.1 0.00821 84 44 42.8 1.06 0.66 26.0 0.0590 12 31.2 0.00798 87 44 41.1 1.17 0.59 26.7 0.05S2 13 32.2 0.00769 90 47 40.6 1.56 0.44 26 -6 0.0651 11 32.7 0.00796 97 45 40.9 1.16 0.60 27.1 0.0581 12 32.1 0.00763 91 47 48.6 1.50 0.46 22.4 0.072b 10 29.1 0.00764 91 41 45.7 1.24 0.56 22.9 O.OS93 12 31.5 0.00777 89 45

a n q and ki UI tho rate 8ad intercept cautmt8 la tho rotontion function: utron t la rim la days.

bEl i. tho tir integral of tho rmtontioa function.

font pollutiar of tbo bi-phom. lrllout cui-137 and oattud potusiu l0-b in & W PkriCo pop- latiao dvria8 1972 uo roportod haro. The procduru usod in colloctlon md dysi. h8- boon roportod.' Tho rosulta (table 1) 8- prooentod u avora~.ronthly W~UOD with standard .ad mkC%VO8t-d d.vi8th. uOi@ht .ad potu- rlucontent hpdlittlo .ad sh-d no coarlrunt clunp vith tho ..urn of tho yur. Cosiua-137 activity sham a peak tvlu 0 yoar in apriag md fall (Juao .ad Decabot), ulth Cy0 htem8ain8 TABU 1. MOK vllds?02488IDI, MD CgIpCU7 DI A llIy #naO mwurxa akma (1972)' JrMIlu4E!%?SE JulrLY%?Lod E!= -Dee W vdCt (4) -x 68.2 68.0 67.8 69.6 68.6 67.9 67.8 68.1 67.9 70.0 67.2 61.5 0 ll.1 lo.8 10.8 u.7 ll.1 u.4 U.6 lO.7 1J.O 14.2 ll. 5 12.7 a/: 16.3 U.9 U.9 U.8 16.2 16.8 17.1 U.7 17.5 20.3 17.1 18.3 btemairr (3 112.3 131.4 1u.7 l29.4 128.6 125.4 l25.4 129.3 128.6 124.6 125.8 128.0 a 2Y.4 30.3 29.5 27.6 2816 31.3 28.8 27.7 20.4 31.2 22.8 28.1 a/; - 22.2 23.1 23.5 21.3 22.1 25.0 23.0 21.4 U.9 2f.0 18.1 zl.9 ?#.u~u (JL3 x 1.93 1.92 1.64 1.85 1.87 1.83 1.84 1.90 1.84 1.87 1.86 1.86 0 0.26 0.27 0.2b 0.19 0.24 0.28 0.24 0.23 0.32 0.24 0.22 0.24 01;- U.5 14.1 14.1 10.3 l2.8 15.3 13.0 12.1 17.4 l2.8 U.8 U.0 k.i\.rl37 (a)x 2.71 2.46 2.34 2.62 2.70 2.61 2.34 2.35 2.36 2.71 2.62 2.65 9 0.51 0.61 0.59 0.56 0.63 0.60 0.63 0.54 0.60 0.61 0.68 0.60 a/; M.8 24.8 25.2 21.4 23.3 23.0 26.9 23.0 25.0 22.5 25.9 22.6 cUip.U7 -x 21.1 19.0 18.8 20.6 21.5 21.0 18.6 18.6 19.3 22.5 21.0 21.1 (PWg 10 9 4.1 3.1 3.0 3.7 4.0 4.2 4.1 4.0 4.0 4.0 3.6 3.8 a/; 19.4 16.3 16.0 M.0 22.3 19.6 22.0 21.5 20.7 17.8 17.1 18.0

Cui-137 10.3 #.3 34.4 37.8 39.6 38.4 %.b 35.0 3b.9 39.5 39 .o 38.5 (PCi R3 0 7.4 7.4 6.2 6.5 7.8 6.3 7.1 7.1 7.7 7.0 7.8 9.7 a/; 18.4 20.4 18.0 17.2 19.7 16.4 20.6 20.3 22.1 17.7 20.0 25.2

1971, and 1972 vera 2.49, 2.60. and 2.U Si, re- mpecti*.ly. kcorkiog to vi^^.^,^ thie 1-1 vould produce uralvl d08er of 0.52, 0.43, and 0.42 -8 in a 70-h body, lemm than 1/30tb of tho dome delivered by 120 g potumfum to a 70+g body.

1. C. R. Richond and J. E. Furchwr, Cui-137 body burdoaa In mm: January 19s to Dmubet 1966, adfatfon I&.. x,538-509 (1967). 2. C. R. Rfclmaud .rd J. E. London, -tu-137 cooteat of Neu llrrico cwtrol eubfocut July 1966 throat& Jme l967, 4: Lao Sei- encific Idoratory Report u-3846-s (1968), pp. 75-79.

3. Section I. Hlk and Food, Mlk Surmillaucn, Jme 1971, Radlo~o~lcdhalth, Data and Raportm l2, 501-510 (1971).

4. C. R. Rfchmad. J. E. Lopdoo, .Id J. S. Uilaon, Cmi-137 cantoot of #.v mxico control mub- jecto. &: Loa Scientific Laboratory Rmport LA-3610 (19661, pp. 170-176.

5. N. S. MASO , I. 8.n. A. Fleeher, md H. PIC. 1. Uhale-body burden of ceaiur137 io a NU H.ckendorf,IYfQCs lovmlm ln huro dult popu- mcopopu&tim mqlo. Tbe clad htim ill CdifOIUia. 1960-1969. Nature 228, didmomrate the meuon: (W) vintet. zaf-284 (1970). anoooerIOIl setioa of the vat WUuth to tb. MUM Caur I~titutefor prdniul atdies for 'Ik ?hy~icrl~iolo~ Sutioa 1w f-d in rdiotberg~. Tvo of mctlm ..I cOC 4til 1972 to provld. .lurytid rd ilutnmawioa ab- th~ nltted full-tir u) the dadaotw .id r.Udiaw s\pport to tb Simlicrl bur&Geocp. Per.- portiou of this projrt. bachu attort mt de in tho B.ctiom U, a*.iltk to intaract with all scribd in Chi. raport, due to ine~LeterdtS, rU.Ucb p~op- in tbe ~roup. Spoeific efforts is A coll&oration with the Meharkr RdbbldOU of the eoctioo CM be dirld.d into the follovi~ six brod .rem: Section to atc~=~tto uadarstad pld dy8BDU "melt" curvu, M effort vhieh should bo c-htd UBq, UhiCb indudu Ubok- (1) mid& within 1973. The chsst counter for &tUtwb bog cornti-, ha8 couoting for tr-urmic el+ ansm photorm (drcribed in the pn.rloru d -to, ino~tatiom!or retastim d sxcretiw report)' hubea in rautirv use for the put yW. uperirot. (iocltdiq Met Particle Program), All Laborstory personnel uho work nctiwly Vith production of radioactivity atmd.rd sourcae. transuranic alomntm ar~nov couated at hut DIY. (2) Theoretied radiobiology, which includes per year. mole-body couatin8 efforts have u- del. d#r md respow. theoretical colputer of putded O-hAt In the put yur. All parsod doma di.trlbutioa in tis~u(..E., for hot par- who work with the Ifrum accelerator baing ce ticles and IL~OM),thoretical d-e-respome fuac- stwted at UllCr are being calmtad to obtain tiom (buod 00 .qlricrl data .ad cellular mud- oa background inforutioo that will make it pwsibl~ rod mpport to no&eul.r kdiobiolom Program. el.). to interpret my future expo~uresthat uyoccur (3) DOBtntry, which hcludu inBt11Yntatioo the accelerator is in operation. for rdiobiolgl e~p.rimnts,including daLvtry of X rays. rap. alpha puticks. sl~~troo.. ~oIyITIOt8OF DATA CoUBcTI(MI adnewcivm pions. (M. T. Butler) (6) Dmdiucsd coquter devdooprncm, uhich include .pplicrrianr for uhole-body .Id lmg count- NaI (Tl) Sp0Ctr-t.r Md Tratuuranium Ehd in& chraooou ralyeia, lmg structure ad pu- Chest Counter Interface with po?-8/I C- UtBC ticle dirtributioo studir. biophyrieal d A PM4II computer With 1ZIt COm ..rOq, qplicatioa. 32K dlsk, and orcilloacopo display hmbea inur- (5) CmBdtatios, which includss coquter faced LO the LASL totd-body speccrowtsr and 1- dtuue for use other rctiocu of the &io- by counter. The usual pularhmifit uulyeer hu bea rdical Remarch Croup, including de=loprclc, replaced with cuo .~alog-to-di~t~lconvrrtars and uint.oulce. nd inBtnICtim of the 104 in us r interface to the caputer. The interf- in- tardoal for r~moteinput to the central cogucsr clude~a clock ukd wltchr that control th. .dr facility. of operation of the symcam. Them include cod.. (6) Ehccroniu devalopent, which includes for starting and rcoppinu data collection ud for design and construction of elsctroniu specialized smlacting subgroup, cwncin~time, type of output. requind. u and counting de. There are two countin# m&s: AA illurtrated in the folloving eurplu. there me for chest counting, and OM for whole-body comt- hw been considarable progress In each area. Al- ins ruiai a 9-in. dieter by 5 in. thick Msl (TU thou& not reported belw, =.bets of the section CrJltAl. provided support in preparing the dosirtry The sad ruult is A 1054-chaanal pulse-height

00 131493.09 1

iOfIb584 built la a DSC Wire Wrap Board mi pl\yr dit.ctb into the coqutu with cwD.ctlom to the Graf-?ao.

(P. I, De- md J. 8. Jett)

Durin8 1972, the dd-watrl, phCurl*-WW 'Ihi. interf.u hu progrwnd throyh the de- 1- ,CwtU bM b# OPUaa, b 8- od b 001 h tb. CmtXUCtfm @Ye, cmtly tvo major in the wta bm ba and we e'lrpect coqletia! by euly 1973. The ise- ud.: a eet of elutraaiu is wdfor back- orated aystem will coariat of n qllfier, analog- grotad srppruaioo, and the mdtichumd puL.- to4ilit.l cmvarter (=),hteh pal, Io pra.1, height rarlyur bas bean npl-d via a ?D?4/X snd 4 l'DY-a/r coguter uith AS&-33 teletype. The coquter prosr.rud to accoquh both data colht- ADc c00rr01 aectioa viU plu8 dinctly into tba tloa and analysis. The na# metbod of b.clytd c0wut.r and control trmmfer of puhcheight suppruaioa bud 00 pukt.hrpe di.c?idrutiO fnfonvtloa from the ADC to the caquter. me centered OITE-458 pUbr.h.pe dYUr, "fnt4mapt" bua of the coqutu vill bo utilized a devi- uhich produce. .o autplt pula. whoom vith data bel- trauferred through tha .ccurl.tor. .Iplitude ia proportional to tha rin tin of tho Then will be a witch and ID pdexternal to input pulse fra the detector. The VirrQr of aa the co~puterbut conaected to it by cables. internal sin&le-ch.nMl .rulyrer (Sa) ia set to Svftchea will be available for starting utd stop- ' prdde gating pulru uhich corrempoad to intu- pins the cormting and ta set tba courting tir. actiotu in the thin YaI (Tl) portio8 of the d8t.r Other nritchu vi11 be available to oatar infoxma- tor. The SU output is wed to mtm the detactot tion ta identify the sub jsct being counted. 8lgrd iato M mdog-to-digitd conwertu (AX) Craf-Pea Interface to PgP-8lt Caputer Large overload pawcwod by cdcrays ue dr A Craf-Pen, maufactured by Scient. Accerrorles tected by an iattyrd dimcrimtnatot vbame output la wed to block the couotinl; ay8tr for 2% Corporation. hu kea interfaced to a PDP-a/f COW U8.c. puter to pmlt eoquterired on-line graphic anal- Pipre 1 rhara a block diagram of the system. The caguter caqomnt of the latmpcount%n# ysim. The unit hu a 14-b. square table vith loa8 Sy8t.r a PDP-d/I UiLh 121, VO+d. Of COn nicrophaaea dong tvo uljaceac sides. Presoina a a 32K word disk, .D oacilbmeope dl8pl.y. md a specid pat qatart the rurfaccl of the table gen- WL-built ioterfaa. The intsrfn couple8 tw erates a spark ad atarts tw scalers. Arrival of uc8 to the coquter and provide. the emutar vith tho souad wave from the spark at the microphoner stop# the scalern, the comts in uhich thco rcp- resent the X urd Y coordiorrtu of the pen. This infomation cur be remi directly into the coqutet. The integrated symcem includu ui oscilloscope for visd display of the data bein8 eotend, ao ASR-33 teletype for data printout rad coruoication with the computer, md 4 high-speed paper-cape red.?. The interface decodes coqutcr instructforu , gearrates gating aimah, inputs &ta into the CQ) puter, accepts a "d&t.-ready" signd from the Craf- Pen, notifiu the coquter that data are mady by providiag appropriate control signals, and procersw data ad control signals bemen the Crsf-Pea and caaputor to &e tha compatible. The faterfacr vu Fi8. 1. Block diagtu of electroaics. i lb 8%- cd Cob. fOt 8-i- .Id 8whs d.tb COUWSI~O, lr~p.rL.ctioa, ca~~tir SAC~~QII, oatput sdactioa, md c0l.r- a& nlectim. Tbrm u. tro car0ti.r mdu: 01 for 1- ca-ri.l Urine thm tu0 dual-eryatal uaublir, rd om for whot- dtb 8 9-in. di.rtu by 4 io. thict -1 (T1) cxymtil. A mterpmr writtr for the qrtu bu the follwha cqrbutir. In the lunycmmtin8 -, tha U- Of Cbd0.r 8\lbporrp i8 Ut, tb .ubporP fat -8 etm4 &t.rdrua fmth8 dtdr -1, .Id coutiry for a prwet ti- ir initiatd oa mupt of a sig~lfra the etch on the interface. During the comting parlod, du8 .n dhp- in a Ifvm tiu m& 80 that the -kt8 -8 8-t- U b. ob8.d. wi- t-y, #tkr 8-m CM be di8pl8yd siu~ltc rwo~rlydtber bri& or ovmrl~ppin8the actin spactnm. Upoa c'tion of the cotmt poriod, data can be priatd OL 8 .trip printer and are re8dy for and- -18. Th. analysis part of the coda proueds u feuan. After mral item of bookbeping data u8 recordd a+the teletype, the subject's chest thicknu8 nuurennto ue antomd ad amr- +,d calibration fwtors for the individual balw unmud 8x8 cdculated. The backporad 8p.C- tm ir tho 8ubtr8cted fro the subject's spectru & 8tOd U 4 net 8p.CtM. COUnt rate8 in the variou rogiom of Intarut are calculated, os are 241h hag bUrb.0 ad standard deri8tiow for * "%I, ad 23)?u. &.at8 are printed on the telt type, md net rpactra are dirplayod for photograph- ing. fn the 9 x s-h. uhoh-body cormtin8 ood..

the 8yOtU OrnU- in th. 8- U4y eXCOpt 8- Of the COIUMt8 am chanpd and the uralysis con8ist8 -r-----l of rirply calculating net eount ratos. I& me of 4 &dic.tmd -uter in advanced b8t- Or rkok-body CmtiUg 878t.m pm-d.8 rOln wrmuiuq apd allow for 0-liao deta alysI8. D.t8 em be obtalned in real time by more p8rsoanel (including tedadcirae), md radiation protection per80mnl 81. no longer dep.ldont upon om or mev- eral people do, in the put. Law the required I caputer pr-ang necessary to ob taia er tiutes of bdr cmtriU8tiW.

Pig. 2. Coquter ralyslr of a rurddy 8rariog cllo cell populatioo.

r -I 8

(P. w. noan)

A contr8l quatlon in the biol0tic.l acl.lru ia the mcb.pfn b7 vbich oulr.wotu matauin omht in their taotic mated& A t0-q- for Ob- som infomtiw ceacornfn8 the uU'~ tion of ita pnou involvu atug of the ~rpholof(- of rtaphase chrmoru of diddin8 colt. Such a technique podta comtimmua rolritoriw for pUle drlft in cultutoa cutid for ext.od.d p&& both aa a coatrol foamto end u a UUD. for doterdnfru wb0ch.r 8p.cifiC of tho mr aL. aUQT tial for aurrlval. Iqrovouat La tha techuique for elaromaoar cluslfication, doacribd horain. vea mocivatd by tho requfronnt for rtlll wro rapid, routiw k8q- otyplc ourtoation of vuioty of diploid and heteroploid llnu io culturo. Rocent rtudiu at this L.bot&tory haw rai8.d funhor queatlona con- z -: cerning the idontlty of chmorons in heteroploid lima by .Pp8rUItlY ruliw out nondiajuactioa .ad supportin8 tho id.* tbat idontical amomfa of 01A y bo incorporated in varying chrmow aderr throulh chrrt.8 in chromoaul condensation- kinatichore developrat 8y8t.u. Ooe approach to testing thls coatontion iaVOlW8 tho proclae kary- otyping of u ..rry of tbe drrooosoyo in h~toroplold

7a v2j3 Generator ELtcrmntxQ Dmxmmmm l'bere is intereat in the Biophysics Sectloo (X. Butler. J. .Id J. H. T. L. Carr, L.rkiru) for obtrining cotrelatiow of fluorescence liaht Cell Seuaretor adCommrfnt Lodw -.ion vith caU-murfece (I-. Tb.refon, sia- vu raquired uhicb vu proportional 8urf.ce Coadubing devmlopmnta in the "cell separator" ad to area. Ue decided to obtrin this by mdif?Ina the pr0g.r Vithh the 6iOphy8iu hctioa h8W MCe8Si- COUltBr VOlw 8lm& the SL3rf.u ArU Of A tatod dovelop.rrt of e- electtoaic spter. The spitere is proportid to v~'~.me 8ig-l was earlier eyatr ooluisted of uny separate mdulos, obcaimd by carrrecti~glogarfthmlc elemoats (diode- 80th. Of UhiCh Y. dml@md 8peeifidly for the tvk connoctad tramistot.) la the feedback circuits Of to VhiCh it Vu U8ig~d. 8y8ta YU OlW .od 2 Wr8CiOd upl&fie?8. m8 WtpUt Of the firBt iUOfficiat, p.rritt.d Ody 8iB(l&-ch*n#l S.p.?.t- uqlifier is proporciao81 to the loprithm of the ing in w1 direction, and had dead tire of Loo0 or This output siN m uuc. input volt4. is reduced by mm08 of a voltate divider to 213 of its full The neu aysta. designed and built by the lie VdWU rad this latter signal applied to the ia- vdical &eurch Croup, is contained in A churis is put of the secoed wlifier whose output current la 11-3/4 in. by 17 in. by 19 la. and coruistr of proportional to the anti-logarithm of the input. 3 fut singlm-chamel analyserau 3 8U1.t.. 8 A third amplifier 8e~su 8 current-to-voltaqe 20,000-volt hi&-voltage supply, poaitive ud aeg- convertor. ative charging qlifien, logics for 2 ~~.ratIn8 All the diode-camected trloristors pert chmmla with up to 20% Umec of djusteble delay, an ad lau-voltap power auppliu. All clrcuits use

80lid-8ute TTL logic8 except for the chugins .) plifier, which vu truuirtorfzed. "hi8 8y8er ham lncreued the speed of cell separating by a factor of 100 by using retrig~erlble 08e4hOt8 in the logic rodule md rhift registers to store a 8tre.r of cell pulau. Built-ie

Pig. 4. Plow chart for cell separator vith circuit. i 79 mtde~ct~ u.d driw el.etrOlric gli- €taro hma DC oftwt in tam of r0lt.p or current up00 UhbiCh th. md 8- i. Ia I- Cuu. thio 8tW-t.t. V.1- be UJ t- lbrger than the dad rd, in -dutim, my vaxy emuah to rlu 041. rubtr.crion Lpracticrf. The Wurf. ~oluti08i. Cq.eitiw C-lla& vhich block the DC effectively but introducu b.H-llru dift M t& rr)rtitioa rota incrruu. Prain cllpplol circuito ulna diodv elidlute #t of u “under- rhaot” but hm(111 offmet of several hmdrad rilli- wlta. In dditioa, the qlitud. of a closely fol- lhng prhe uill be redwd becaue recovery i. not caplet.. An active bucliaa re8torarS wing .II operational qlifiar vith fairly hi@ open loop gam. were- both probleu. The bue line con be d- jwted for aero offaet, and the ncovery tin la rmducd to a very 8ull volue becw8 the total output of the rplifier ir avdlobl. to “jark” lit- arally the bur Uru back to 2-0. Tvo rplifler8 uero rmquirod for the cell rg- aruor \Pit. Ih. siarl. to be detected cormlot of A ~~11chrrr in aperture current (Cror a Coultor counter) and a chuty in photoultiplier .Dad. current (fb~reace~codetector). ?or m~caemfulopera- tion at high Cocrnting fate8. bu8-U- re8tOrbti- ir required. One qlifier we8 301 oparatioad Pplifiers for rire t1.u of about 5 uaec. and the other we. 715 operational ~lifierafor ria. tian of 1.80 thm 0.5 we. The latter Vi11 hmdk npt 5 tition rateo up to 3 x l0 puL8.r per 8.c-d or paired pubw reparated by only 1.5 U8rb

RERIGWCE PION BEAM CXAWXERISTICS 1. C. R. Uch-d ad C. L. Vmk, .&.e W~rl (C. Rikand U. 0. Jidu) Report of the Biolo~ul.ad mdicd k.euch Croup (HA) Of th. USL Health DivL8ba, Jm- Sull milicon detector8 (lithilcdrifted and ubry throulh Docdar 1971, Lorn AlmSci- entific Laboretory report U-1923-PR (1972) , rurf8c*-b&rrier) A- being rertrd for pomibh use pp. iui-iie.

80 ktin, mimaity of Ikr Ibico, Pni-rde of .Id ooi..rsig of Caufornia DubW). Durh# the gut m, tln Bi*ysics sectiorr Color&, u Ia~totrb.w~u,ol~DYr,tlr. hu b..ll -ixod b.cwe of the forvtiom of s pumter word. ~rrsucb n.r wction, msical kcliobio~,vitllio the group. *runt uyu~llllt.mm 00 colt t=*d rLr Ihi. nY nctioa usrrd the xaapomibility for do by fluoromat Uthd. phf.kal radiobial~,caquter applicatiom, and rdm A mausor hub.r inreor- JeCtrOdu support formrly chared to the Bio- oro-color fluoruuaa poreted into our .ultipu.uter csll sortu. Ume phYSiCS &Ctim* rwl th~~Utirftiu U8 d8scrib8d of the bi-color fluorescencr doteetor hu rllcwd e1Swh.n in this report. The Biophysfcr Section ia US to rridine or8ngm-stJd hrrr;. WtU UQY sololy cm-rad with reaurcb rddeveloprrrt study that ehiblt bi-color fluor8scemca. provu in the #soen1 maof biophysics and Ua h.r irutnmntaioa wlth tbe cumnt major .ghuia on cell sort14 tachnip- to shov that thr ersk based their red fluore8cence, can be cb&f*d hlogrut md application of rapid rthod. for on lyqhocytu, DOOCytes, rdwmdoc]rtr- -11 UIalySis adsorting. mi8 vjor effort is u divided iato thrre rin areas: (1) physical inves- have also initiated vork desimed to obtdn OuChU- tiptloau of the lipt scattming ad other prop- to-cytopluric ratios bumd upom nucleu d CytW ertiu of urulim~cells that ai&t be ruurable plvmic fluorescence: ethidiu b?ad&*tdd by our in#trurntation, (2) developrat of fMCrU- yields red fluorescence, and fluoroisoth~ruL~ ataiacd protain producrs fluorescenC0. ratatiaa for cell sorting and u~alylis,and green dual-paramtar cell aalysis phot-mr VU (3) qpllutioa of these method. to bioloecal A also desi@ uui c0mstruct.d durily 1972. probla. Our success. particularly in this latter This area, hu b..n possible brcause of exc8llent device ~aaur8sfluoremcence ndsmall-ansle light cooperation with rderr of the Cellular Radio- scattering oirultaneourrly on each cell u it purr biolom Section. throu@ the luer light in e flou photct8r. Our present application tmes light scartart- to yce Ilw microphot-txy, the rthod of Ukhg rapid optical muureaats on individual biolg electronically for fluorescence analysis, prdttial M electronic "clean up" of the rignal those icd cell.. is a exciting md fqortmt emerging in technolow in cell ruearch and hu been pioneered cues where th8 riad would not b8 discsrnible fra notre present in debris-ladened biological sugles. by personnel of the Oiophymics Section at Loa theoretical rtudies ugt Am. This dsvalopwnt gmfrom our esrly More extensive on scattered cells W8?8 cductd during efforts in quantitating Coulter cwnting techniques by biological uhich resulted in iqroved Coulter volu apeerrom- 1972. High-speed CmCer8 W8m uS8d to d&te light scatter coated sphere. to sirrl.te et- mad cell sortin8 bued MI c8ll vola. Flow fra dcrofluorartry (pra) is our most iqortant con- Uslulian cells, the core representing the lluclru tributiao to dace in the area of optical masure- and the coating the cytoplasm. hsults of the calculations indicatr that the forward onts on sine cells. computet ligt scattering dominated gross sue of the During 1972. FUF wu usad routinely by USL is by personnel u well u by investigatorr from oth8r cell. Outside th8 fornard dirrctioa, the scattsr- in8 pattern io a function both nuclear ad laboratoriss who visited LASL (Salk Institute, of cytoplaauc optical properties. Experi-ntd Karoliruka Institute, Cold Spring Harbor Laboratory, scatterin8 patterns abo were obtained for suqmn- University of Houston, University of Texas at slow of various cell types. In cmes when cell

81 a UALfSW Al8D SaMIc XlBmUfW DZVIDDI-

(A, BrwmtSa@. J. R. C0dt.1, L. S. Cra, J. L. nm.J. c. mrtin, P. P. ylll~rr,J. A. st.fp

Irr), I46 A. VM DilL.. .Ird T. w-t) rnffomati.1 U&t-suturlni Sig.tur8. of Il.rc urn cell8 1 & uo reported irr tho 1971 ~n~dropott. -rli.~.cal& hm boea 8Lukted in coqutor r0d.b 8. eoUd 8pbO-8. tho COX0 npm8mdng tho nucleu urd tho coati- cytopbn. Exact oloctro- -tic chaory c.lculUinrc12 for iadiridd ticlea with cell-like optical puwtoro indicate that light scactorod in tha fonard diroctlon 18 ddnated uinly by the 8r-r ailhourtto of tho pap tido .nd contain8 inforutloo OD particlo 81- md that beyomi the forward lobo the ourteriag putem from 8 coated puticlo ia 8imificMtly differmt fra a mirplo hvaphon. In the lutor CY., the acattaring pattom I8 &@ut upon optical proportlu of both tho cor8 and coat. Durlng 1972 SOT of our thooreticrl QrdiCtiOlU ham been toatrd with awpenaioo. of rt-uliu~calk. 3 A 8-10 bur uniqw [email protected] photcmtor

YU corutructod for thtr pUrpO80 .od la ahorm 8Ch.0 mticdly io Fig. 1. Tho photometer is hacud in a 2.5-foot-dlrctor cylindor. Colrttol of 11@t from a 5-Y helium-neon lucr (Spectra Phy8k8 Ibd.1 120) iatu tho phot-ter is by 8hUtt.r. Tho light that p88.08 through 8 v8rL.ble rporturcl to rmduco oxtm- neow nonluing light ad ir nfloeted from a froot surface mlrror through a 8poci8lly doaignnd cuvetto. The ufn hoe? beu urd aoT forward auctorod

light are collocted in 4 R.yleI& horn bur duq. Light rcatrcrad by tho particloa in the cuvotto is recorded an high-spoed, red-sewitlvo film (yod4k 2479 RAR SAm. 16-m % lz-foot rolh) which is hold in 8 cr4ck on tho inaide rurface of the photortrr. ApprOXiMtdy 34 exporurom c.o bo udr from oach roll of fila. Fiducial markora on tho ftlm holder cUt sh8dwr on tho film at 30' \AZIMUTHAL UUD HORIZONTAL AoAfsIIwQsum FIG. 1. Scb- diwr of the film photmter. T& tar pmridw incidant light to tb. 8cUtersrm In the c-tte. Ib8t of the lane U&t i. deInto the hoa uh1l.e the fiL record. tho acattemd ll*t.

perdtling fib dbp.rrim to be mu- urrd QII uch oqmaure. After --, the film i. &-lopad in a It0d.L Versrrt Ibd.1 llc wing tfp. B ch..d.tq. Thi. procedure producu a typical -c rmp of three daudrr, which CM b.

extendmd by allaving 801 overlap betwoen expoaum8 and radudng intmrity in the fonrerd direction by we of neutral donsity tlltera. Ikvelopd film Is I1 nd vith a dcrodensitater. l'hrod the use of E Suiublr cdibrUian, film bluity a8 r function a of diet- m the film CUI be convmrted Into rcattering intamity u a function of auttoring a-- angle. Uslw thue technique., we haw obtrlmd scattering patternr for the angular rmp of 2.5 to 177' . It la wll kaown thet m&r poeitloru of the UIY and ddu in the sc&tteriqpattern are -0rtmt hCti0M Of p&Sticb Sid For thi8 reurn, in q suspe~uiooof partidea which af. air- e ilar in corpolitlan but not id.atical in lite, am 2. cap.riaoa theory rod experbmt auarlng out of the scattering pattern rhould FIG. A of for be the dcraphen aeattereb. A md-lol expected. l&nfon, It ia iqortmt thet the aca& of the relative iatonmity, corputd (thin) and ruured (thick), vormu meem size, alte d5strlbution. and refractive 1nd.x scetterinl angle, a, in dqrru. Theory of the particlea are well characterized. One also accotmted for the rixe dlrtributioa of the spheru uyular mrolutlon tho need8 to ucrblish that the photomter hu sufff- and of photorter. The experlmntrl curve we^ deat ruolutlon to record the eaaentld featurea corrected for photorter scatterily by proportfer tho mtte. of tho .uttering pattern of intereat. Thi8 hat trummirrioa of lheoretiul CUI*.. ue @*.n by duhed cuieideration vu accoqliahed by a calibration linos aod experlmntal by a 80lid Uw.

83 T&oirtiul rurlU am oham by tho dot- CUI.. md .gH-8t4l bv Ich. .Olia Cm. thwtho QDLIY do not much .gctl7 in .brp., their rd dJr -0 quite udl (Tabla 1). Tb agmEbe- thqal .qmirat coo- fixu tb. rcoty of nad8- dbtributiou for the mphru b& \1pa0 dcr~rcqicad Carttor .51.C*OrtdC YIIOlOQI. Wt-wb- ri.u m -tar of lO.3 ric- am coqrard ritb lO.5 rdcm u demraad by othor Vthob. nn &iffa- in alul.. iatowity kWvo tho tvo cummi (?la. 2) i. cared by ad.r of trtorr. Althm upifom in dimtor, the 8phrroo arm aet ~th,rrl optical dcramcopy .ham them to hma WlibU-LiLU 8UIfaC8. Ihot8tid 4- Cubti- parfmctly Unifo- rpk0t8. Io dditloo, tho- L an rrrcortrloty of about 5 porceat in wu-nt of fit hi- rup-0 chractor lstiu which ir not 0 UMU functiar of intoluity. Tho fact thot tho dnuml rrlniu a&?.. so vel1 demastratn tbu tho flk photortor hr ouffideot tuolutloa to -run the vin characteristics in the autteriw pattorn obt.lnod fror particlos with the dimnoiou of mlian calla. For all particle8 mtudld by thim rthod, maa size, aim dlatrlbutiaa, .nd refractin index uere ruurod. The coquter code8 udtton for individual pa-iclao ylre mdiflod to accomt for sire di8trl- butt-, Thru, at e& 0crttOd- -fie, V. Cwld obtain tho crlculatd lotamity ewtributioa from ea& particlo in the di8tributloo. Th.ao rosults wera thm vdlbbd accordin8 to tho wurod d1.r refractivm 1nd.x is not vory variablo .rmg thuo ete? distributioa ad inte(lr8tod .CI#S thia dfs- colh. Thl8 inforution vu urod to calculate tribution function to yiold tho theoretical CUWS. theoretical scattoring curves for CHO colk in tho G 8t40 of tho all cyclo. caputer program 1 The emat. the rofractivo indices of both nucha rad

CytOplrm U bein8 CONtMt for the Vd-8 8t8td rbove. The ratio of nucleu di.wter to vbole call di-ter vu also 888-d to bo con8tant and e@ to 0.73. )lun volrp. and volum distribution of 6.25 7.25 4.5 7.2 thro cell8 Y.CI dso iacorporatod into tho com- 9 .oo 10.50 9.0 1o.s peer prow-. We also calculated light ac8rterln8 11.75 13.50 11.9 13.6 from UI equlvslent hororrYw ophoro in vhich the 15.00 16.50 15.0 16.5 101- rofraetlvo index vu equJ to tho M~W- 18.00 19.50 17.9 19.3 anrapd rofractivu indox of the nuclou lad cyto- 20.15 22.25 20.8 22.1 plum.6 A coqarlson of thero tuo theorot1c.l 23.50 21.75 23.5 25.0 rrsults with erporirntal data is sham in Fig. 3. 8 ur)a *r4 OmttLIIIY FIG. 4. nmorotical plot and corrupoadiw -* --w iiat.). rosulta for the ditferontlal nc. 3. thaontical plot. d correspondiq scsttor pattmrru for U (hlneu hamtor The oquIv8lant harp. wporirntal rosult. for tho dlfferontid wary colb in H. scattot pUtora8 for GI Chime hutor 8sn.aru spherm (thin solid lim) d 8- ovary cells. Tho coated mphero (thin lrntal nsults are shova. aolid line), tho equivalent hampMour this CUO, CHO sphot. (thh duhod line) whao refractive no well-defind nuclow. In colh index ham been vol\ir-nnraed fra the in H an quit. voll modolad u homagenoow rpbuw coatad mphoro, lad tho exporimntal results (thick 80ud una) aro sham. over tho angular raga of internat. Uo sbould note that virtually a11 the rcatteriry by crLh I8 The lo( of tho 8CUtOrifu interuity is plotted as a contained In tho first 25.. function of sutterins angle. exporimntal results Thoro prelidrury studies -re conductd UU are girrn by tho huq solid cum, cortod .phon swpensionm of living cells. P.hapa tho moat thoontld t..ult. by tho lipt solld curve, .Id intermting reault io that light scattering at equlvrloat hvneoum sphere theoretical nsulta by larear aneles is Influenced by the internal st~c- the duhod cum. Expetixental results won ob- 5 ture of the cells. Ue plan to incorporrto add.- tained vi& cells in 4 concentration of 10 cellshl. mglc li&t-sutter sensor on our flov photorut. Belw 7 to 8. all thno cum8 -eo quite well. to Investigate the poaribility of wide-angle Indicating that gras si- effects primarily dd- scattering as mother par~seterfor cell Iden- nue tho scattering rasponse in this angular re#on. tification in flow-rysta U'18ly8iS. Beyond 8. tho light sutteriag from the coated rphero i. considerably leas than that fra the equir aleat hvn.oru rphen. The paition of the exporinntal uxin and minima apaes quite vsll 1. C. R. llichmnd urd C. L. Voolr, d8.n And Ibport of tho liologlul and khdiul Romurch with theoretical multr for coated spheres, a1- Group (H-4) of the LA!% Health Division, Ja- thou@ there is so- vuh-out beyond rou&ly 15', wry throu#h Docuber 1971, Lom Al- Sci- entific trboratoy report LA-4923-PR (April probably becaruo of heterogeneity not accomtd for 1972). in the del for Q10 calls. The uin feature of 2. A. b~n8tingd P. r. b(ull.n.J,4pli.d mdC8 Fig. 3 is that actual cell. behave oudr re like -11, 675 (1972). couod spborer than houmpnaow 8phOrOO. Beyond the 3. A. Brwutlng and P. F. Yullmey, R.v. Sei. f0TW.d direction, the BCatteriBS curve KOfbCtS h8tK. (1973). in pross. nuclear properties of the cell. 6. H. H. Blau. D. J. NcCleomo, and D. Uatson, Figun 4 sham exporirntal scattering €ram CUO Applied Optics 9, 2522 (1970). colls in H u coq.rmd with an equivalent homo- 5. t. S. Cram. n. J. Eulvyler, and J. D. hrrin@a, cells, thuo cells rhw grneour rphere. Unlike GI Biophysiul SOCiatY AbStT8CC8 11, 1558 (1971).

85 6.

lo.

ll.

12.

?lultip.ranter Cell Sorter

A ultipiruter cell unlysis .od sortins SyStr for we ia cell blolow research mid possible ClinifSl application8 bu been den1op.d. Cell supla 8tained vtth a fluore8cmt dye that spc cificdly 1.b.b bioch.dcrl caqolwnt8 of iUt.1Y8t at. surp.abd in phy8iological uUne .ad introduced FIG. 1. The rultiparucer cell sorter. into a flw chdr where optid and electrlcal smwors uuure ull vole (Coulter rthod). single- signals are clectro-opticdly -88Ur.d; the fluonr- or two-color fboreoceoce, md scattered light. cence sensor is a dual photomultiplier arrmy vbich Signals from the sensors are electronically pro- pedts oeasurements of rad rrd preen fluonsconce. ce8s.d In 8 mriety of ways to provide optirv cell Light acatter is measured in the fonrard direction discrimination and are displayed u frequency dls- bv blocking the laser bear vlth a optical stop and tributlaa histosram. Processed siv.lr are also focusing the forward scattered light onto a photo- collparad with preselected standards. and this trig- diode. gers sorting of the desired celh. ?OpUhtiOM of A second sheath fluld (shuth No. 2). 8- Of uul1.n tissue culturm cells, hm1eucOCYtC.. saline, flour coaxially around the cmll st-..) and other suplu h4w been andyred 8nd sorted. sliwth No. 1 flou. The total flaw jets art ioto the Figure 1 illustrates CUE-way rectlond view air from an exit nozzle. A pieweloctric tr.nrbucer of the lnrltiparuter cell sorter. Fluoresccntly mchanicrlls coupled to che flov chmber urd elec- stained cella dispersed in saline am introduced trically driven at 45 kHz produce8 mifolr liquid (lo00 cells/sec) into dual shsath flou chuber via droplets (45,tH)O/sec) by regularly disturbing the the s-le inlet tube. Flowins coaxially arourd the errglng jet. Cells are isolated effectimly into ,. inlet tube is a particle-free sbeath fluid (.heath slnple liquld droplets In this wner.' A gmup of Yo. of &ewe the flw is laner, the droplets. one of vhich will contain a cell to be c I) saline. cell stream and surroundin8 sheath do not mix but sorted, is electrically charged at the point of

g~yctoether through a Coulter volur sewlng ori- droplet formation (charging electrode) and is then fice, vhere cell voluw ia lruured electronically. deflected by a static elactrlc field into a collec- The flov next enters J fluid-filled viwlng region cion vessel. where It Intersects an argon-ion laser beu, C8US- Signals from the cell sensor# are routed to a ins light scattering and fluorescence. Both these hard vfred multipar8mater analog signal processing

a6 dt. Procanad ai- an them routd to a calls *E( upcbrmmmly. naw cell8 u. ulti- prlre-bi#lt dtsor, u&m freq-eT smxmd vith tho fluoroch- adfl.rlpr th. 2 dlstributlm hi.tmm of ~rllpalm, f1-W- fluoresceot Foul- proodun. DUA distribrti~ cow, ligt acuter, or a ccdilvrion of tho8 ab- NO put: tho flnt nprewu the calla parrrtera CII be di.playd. ?roouud si- diploid DNA (Cl -8) h&ng a ~QOffidOatOf Vd8- b triwr cell aortbg by cmhgthe -11- tiom of 4 pucmt, .ad the socod peak re?mmmau tu& of ucb proco8m.d si@ prt. to a pL'80.t cells with totraproia Du cootoat (C2 md H pb~d. atandad (1.0.) if the siw ~qlitdofdh ~hrre@m between tho put reptuonto cell. am- phuo). r& within 4 preselected ruii, II oppropriato eloc thooixily DIU (S tub of cl md c2 + trollic d.1- is UtiV8t.d VbiCh CriggaS 8 dmpbt mdal fluoruC8ace inteamiti.. of rho fuo p.Ju f. charglq pul.8, utulng the droplot Cattalliae tho 2.04, very close to the axpactd vrtu of 2.00. cell cell to bo char@ .rd mubuqwntly defloctd). A volu dlstributioo is bmd. m-d, .od t7PiJ group of droplets, usually .bout 9, is thea sorted of a cell population in e-mtid gmth- from tho dnst--. Thcre ullr failin8 to =et MA-to-cell vol- r8tiO for chi. cell poprktion, the critmria of tho presot 8trod.rd. do not triggo? sham in Fig. 2b, Is unidrl with a coofficiont Of sorting aad are dld to pum to a wutm vu8e.l. variation of If porcent. Volriu di8tributim Of c1 4 5 In a typical experlmnt 10 to 10 cella aro sorted cells (Fig. 2c) vu obtained by uralyring odJ those in a fariautu. Coulter senior ripah umociatod with the f1w-s- Ffguro 2a 8hffm the volume and fluorescent cenct signals indicating the Gl amount of DNA (so. Feulgen DWA histogr- of Chineae huter (line (30) Fig. 2.). Sirllarly , the volumm distribution of G2 uL'...I_._,_ . .,., + X cello vag obtained by analyzing only thomo Coulter senmot aimUm0CiAt.d vith thm C, + U t '*' I fluorescence peak o€ the DNA diatrlbutim. -1 voluw ratio (C, + H)/Gl la 1.7, lesa than the total volume increase over the life cycle of the cell (double) because the inotrument is ruuring avorag8 cell volur distributions of the G1 uul C2 + H phases. The red and green fluorescence of huam huco- cytes supravitally stained vith the metachroutic tluorochroae acridine oranpc3 is shwn In Flp. 3. Uhen leucocytes of diluted hole blood pro stainod according to this procedure, cytoplumlc grmulea exhibit red fluoreacence whereas the nucleus fluo- reaccs green. Erythrocytes do not talc. up the acridine orange stain. The bi-color fluoresceace sen8or waa set to muure green and red flwre8- cence. and the cell sorting logic vu adjusted to separate leucocvtu havlng a red fluonrcoace corresponding to region 1. Differential micro- scopic counts on sorted leucocvtam show chat approx- imatelv 95 percent are lymphocytes. vhon the sorting loqics 8re adjusted to reparate cella lving vlthin region6 : and 3, subsequent raicrorcqtic FTC. 2. Volmw, DNA, and DNA-to-cell volume dir- tribution histogram of Chinue h.rter counts snw that 90 percent of the cells separated ovary cells grwlng asynchronowly in sua- from region 2 are monocytes and that 95 perunt sop- p.~lacultum: (A) vol- adDUA distribution; (a) DNA-to-cell volrr di8trlbu- arated from region 3 are granulocytes. tion. and (C) vollrpc diarribution of C1 Because of the large ratlo of e1-ythroCyt88 to I 0 5 5 1 ynd C, + X cells.

I^^ hrvn and .nu(h-ter) loucacyte characterixa- tioa with the rcrhravtic fluorochra acridim oraap, lad work aa cell8 from solid t-rs d exfoliative utorial.

1. H. J. PuAvyAer, Electrmic separation of biological cella by volur, Science 150. 910- 911 (1965).

2. R. A. Toboy, H. A. Criu..II, and P. n. Kr-rl A rthod for coqarlag effects of differrnt ayachronixing protocols o dimcell-cycle traverre: The perturbation indu, J. Cell Biol. -54, 630-645 (1972).

3. 1.. H. Mos and 1.. A. Kumtsky, H8chine char- actrrlracion of huun leukocytes by acrldiar orange flwrewcence. Acts Cytol. e,289-291 (1971).

n. A. Van liilla, Y. .:. Fulvyler, and I. u. Boonv. VIII- Jlntributlon and neparation of WANWLL NUMBER nomoi liurun leukocytes. Proc. SOC. E-. FIG. 3. kd ad grnn fluormsanco distribution Hlol. l4d. 125, 367-37fl (1967). histogram of no-1 humm leucocytes supravltdly stained with acridine ormp. Durl-Parameter Cell Xicrophotoclater leucocyte# In normal hmaa blood, leucocyte voluae distributions cannot b. obtalne$ without prior I)urini 1971, a dual-panwter dcrophotorter vu developed and subjected to initial CeBting. raponin hrolysi. of eythrocyta. This difficulty The dun.1-prr.latcr tns trwnt described here repruents CUI be overcow by sortha a11 fluorescing 1 thua producing leucocytrenriched sample. a coclbinotion of the fluorescent microphotortor cells, 2 8nd liglrt-scatterin6 phocovter deweloped earlier. This sgle is tha reintroduced into the sorter. and the -1- of cells ahming only grrea fluorew- E8ch cell stained with an appropriate fluoresceat dye pulse of fluorescent light. well ceno is rasured.‘ This rthod hr che advantaw produces a u pula. scattered light it crosros a beam of yieldinB a leucocyte volume distribution without u of u 5 of blue li&c trom argon laser. These signah requiring saponin hemoly8is. an occur siultaneouly and can be rued tvo A second ultiparuter cell sorter is under u de- scriptors for each cell. After detection, each construction pmrently, with 8 coqletion date of signal amplified and fed through dual-par-ter early 1973. This unit is intended to be dedicated is a processing unft. logical conditione are to biologic81 eIIPerIIInEs, while the present sorter If certain rt. tliene two signals are then available for 8yst.l continus to be rued for SQI 1iait.d pulse-height analysis. The net result is A fre- biolo@cal experinnts coupled with inmtrtment quency hisrogru of fluorescence or light scatter development (i,e., nu cell sensing rthods 8nd of the cells of intereat. determination of optiul deteccion pararterr). .\ acheutic diagram of the dual-parameter Current exparimeats underuay on the present cell pliotortcr shown 1. The flow chuber sorter include ethidiua brafde-fluorescein iso- is in Fig. is 2 brsicallv rile s..y as chat described previously. thiOCyMaCL (FIX) staining for DNA/ce11 protein Laminar flou is established within the ChaPlbar, and studies, fluorescein-conjug8ted concurvalin X die cells are the of the main flow. bound to mmbrmr aurfacr sires, continuad studv of injected u core Just prior to entering the viewing arm, cha entire k

FIG. 2. Block diqrn of durl-parsuter mi@- PIC. 1. Sch.utlc of the dud-paruter flw micro- procumins dt. men the cordit- of fluotater. Tba mrgon-ion luer beam coincidence and proper slgnrl .qlitod. are (lwer right corner) is focused vith 8 20- ut. both mdog 8i-h are pU8d for E. imu to a 5O-micrw mpot 8t the center pdse-height malyair (PW. of the flou &.der. Fluorescent li&t is detected at 90' to the incident direction. processing unit is sham in Fig. 2. In the p-at Light scattered the fonard in direction applicaeion. uned to gate the ..cod. between the central bar stop .ad outer one sign81 is stop is collected dch a 10-cm hM and After amplification. the signal from each of the focused onto a photodiode. two detectors is Ced to separate adjustable dim-

flow Wves through a constriction orifice which crirlnrtors. If both signals are within the appro- narrow the cell stream to a colun about 20 d- priate amplitude range, they are then tested for crow in di-trr. The cells are then lined up coincidence. If both tests are positive, the am- mxh an baadm on a string aa they intersect the log signals sre pusad through a linear gate. and luer lI%t. either cr both si~dsare available for pulrc The light source for thir photo=ter is a height analysis. The logics of Pig. 2 CUL be by- Coherent Rdiation Laboratory Sadel 52cA arson passed: in which cue. the instruornt functfoM am loser operating at 1 watt at 688 nm. Laser llght a s ingle- p aruct er phot ome t er . im focused with sn l&cm convex lens co form a 50- .I particularly ueful application of the dud- micron spot at the Intersection with the cell parmter photometer is the analvmis of f1tmt.S- stream at the center of the flw chuber. After sence from weakly stained samplem. Typicdly. 5 X p~nrlngthrough the laser beam. the cells exit out IO* cellsilmin ?ass through the photo=ter, wich the top of the chuber and go to a vaste veSSe1. each cell spending bout 5 microseconds in the li*t Am each cell passes through the laser beu. it be=. T!ie total rim that cells spend in the bau 5 x seconds out of mlnuts; the produces a lO-rfcrorecond pulse of fluorescent and is then 10-l every photoneter duty cycle is percent. renin- scactered light. The -in laser beam is eliminated 0.83 The ing 99.17 percent the time the photomultiplier in a snll trap. The cone of light scattered about of lvuuring noise due to fluorescence non- this trap (approximately 0.7 to 2.0') is collected 1s of cellular material the cell Thi8 vith a 10-cm convex lens and focused on a photo- in stream. noise diode. The resulting signal io then amplified. MV be from stained cellular debris or fluorescent iye in solution Ln the cell suspending medium. rhe fluorescence signal is collected ac 90' CG ?igure 3 shcrvs the lifit-scattering frequency .. the direction of the incidact beam. Light then distribution obtained with Clrineae haester cells puses through a vellar barrier filter and is -d focused onto a 300-micron diowcer pinnolc. Ihc (line iHOj supravitally stalnza vith 13 2 acridine Xhen viewed rhe fluorescence fluorescent light eaPrging rkrough the pinhole ia orange. in nicroscope, these cells a weak green nuclear viewed with an RCA Eladel 'lii6bR :iiotomuLtiplier exhibit fluorescence ana no detectable cytoplasmic fluorer- cube selected for its crtdrcd red rrnsitivi tv. cence. The lignt-scattering signal Is seen to be This sipal is also amplified. free from any debris which is comparable In site to A box diagram of the zuai-par.mcttr signal i Next, paper ulberry poll- VU dxad vith UO- stained MO cells, .ad the lilbt-suttering di8- tributiar of tho ensd& VU wrut.d u shw in Fie. 6. Both particles 8IC the s.Y miH (.pPm- irtely 12 microns), but CHO cells shw 8 d broader site distribution (cf. Flu. 31, hac. broadening the distribution obtained from the codtination. However, if light scattering fra the colbilution is tated by the fluorescence of paper mulberry pollen, one obtri~tho scatter curve sham In Pig. 6, vhich Is id.atic.1 to tbt FIG. 6. Fluoresceaca distribution obtained for a0 of paper mulberry pollen alone. cells staimad vith 1ff8 acridine or-: This instrurnt offers the possibility of (-04) when the ilutruont is used u UI FIG; and (44-1 fluorescence gated on the iqroving the si-1-to-noise ratios on s..ph li&t-sucterin~ aim1 fra each cell. that fluoresce poorly becmuse of prep8ratioo.l difficulties and distinguishing one biological par the cells. Pigum 4 shws the fluoreaceace fre- tide from mother on the buts of differences in quency disttibution. Uhen the dusl-parmter light-scartering andlor fluorescent properties. Thr photortrr is operated u a slaw pu&meter, one obtains the upper curve which ha8 the appearance of an exponential decry typical of a noise spectrum. However, when the fluorescence signal is gated by the ll~t-seuteringsimal from the s- cells, one obtrins the larer curve. In this case, only the fluorescence of objects in the size ranp of cells is analyzed. This lower fluorescence frc- qrvncy distribution is similar to th8t obtained for greater acridine oranp concentr8tions (> 9 *ere detection is no problem. The instnunt my be used also to identify particle8 bared on the presence or rbsence of FIG. 6. Light-scattering distribution (-A-A-) obtained from a mixture of stained papor these two paruterr. In Pig. 5, the li&t- mulberry pollan and unscalned CIiO cells. SC8Cterfng and flwrescont frequency diatrlbutions Light-scatter distribution (4-0-) obtained when the scattering rignrlr from of paper dbarry pollen stained with acridine :he mixture were gated by sirulturrour ora- are shown. Both signals 8re free of noire. fluoreaccnr signala fror the pollen. t OSb5 f 90 9 --

first attribute pordu uurennu OLI suplea not pmdouly annable to FUF aalysis. The second foature greatly extend8 the RQ concept of cell identification.

1. D. N. Bok and L. S. Cram, Exp. Cell Res. (1973). in p-88.

2. P. P. Mlurey, M. A. VaDill., J. R. Coulter, and P. N. Dora, Coll slriru: A lfpt-rutterfn~ photart8r for rapid du detezmination. Rev. Sci. Imtr. 40, lO29-1032 (19699).

BI0U)cICAL APPLICATIONS OF CELL WYSIS MID SOeING

(L. S. CKU, H. A. Crissrma, J. C. Forslund, P. K. FIG. 1. DRA distributloa of W-1 t-r cellr. Horur, P. H. Kraerr. D. P. Petermen, ?1. R. Raju, animal and to CHO cells in G1. The MA-1 coll dis- A. Rorro, R. A. Tobey, and T. T. Trujillo) tribution pattern indicated the preaance of cella Fluomscent DNA Dis tributiona in .XA-1 Ttmor Cell with 3,4N. and 8N amuuts of DNA. The second Population8 population (4N) appeared to be the mmt n-rou.. The tmor cell line EA-1 vu derived in C3H 'he three peaks seen in a single s-le uy also mice by the application of the carcinogen methyl- indicate the presence of tvo cell line8 grauing cholanthmne to the skin. The resulting twr vu together: (1) normal diploid cells with th8 Us classified u a squamour cell carcinou. Because aunt of DNA, and (2) proliferating tumor cells am of our interest in developing and separating squamous indicated by the C1 (bN DNA) and C,- + N (BN DNA) cell carcinou for the cervical and uterine regions peaks. We thought it would be int8-8ti1tO to inws- of bans, we felt that this tumor would serve as a tigate whether SA-1 tumor cells could be grown good working &el. For ex.qle, use of the %A-1 in culture; this would not only provide a ready tumor would allov us to have sufficient cells which source of cells but mlght 8180 lead to raloctiva are euily dissociated eo detrrmlne che parameters line -re suitable for the model sys&em deacribd which are mast suited for cancer cell identifica- above. This has been done vith quite a degree of tion. Information gained with this modal system success. Juring the early passages (1 through 51, vould then be applied to an investigation of the the fluorescent DSA distribution rereled rhs identffication of cancer in numans. hepar-tar original rumor cells (Fig. 1); hovever, in later or cell property conaidered acceptable for identify- passages (6 through LO), we observed the dir- ing abnorP.1 cello in any cell population is the appearance of the first peak (2N) wile the cell presence and extent of polyploid cells when analyzed population was acill at a nonconfluent state >f for DNA content per cell. After vorklng out a grcuth (Fig. 2). Hatever, a confluent populacion sacis factory single-cell sumpention technique prior displaved only one peak at the 4N DNA level (Fig. 3). to fixation, fluorescent DNA distrlbutionr could be From :hem preliminaq data, ve conclude that defrom several animal tU.ors. The cells yep. animal .XA-1 tumor tissue contains both nod stained by the acriflavine Feulgen reaction method 1 diploid cells and polyploid tumor cells. with the 7 and mamured by FpiF techn1qws.- The fluorescent formar unable to survive and proliferate after a dfatribution histogram of these r.gler (Fig. 1) few passages in culture media. .'+lao, when ;ul- show thrce distinct poh in a channel ratio of cured ?fCA-l tumor cells are allowed to gcw co i first peak corresponds eo the norul confluent state. we see a tvpical arrest m 1:2:6. The G 1 diploid cells present with tumor cells. The posi- observed bv others3 in other -Ian cell lines. tion of this peak corresponds to that of diploid Because normal vaginal and cervical specimens pro- cells obtained from kidncv and spleen from the same duce t::picsl di?loid (2s DNA) histograms. it seem Twr Cell Identification .ad Sepamtioa

A 89uaDOu cell carcinoma (NU-1) growing in C311 Jce wuobtained fra Dr. R. M84r.a of th8 blrtid Cancer Institute. Tuxm fraa thue ad- mala were removed WIIUsterile prO~edun8. nincd, md placed into Ua's F-10 8upplmUd with seru rod rtiblotic8.' The ruultfng cdb am epithelial in morphology and. when implanted in CSI mice. produce twrs in approxiP.tely -third

the tiu required for the 0rigi0.1 line to produo , t-rs in theme mimds. The purpose of this Vark 1 vu to deterrine if the ultiparameter cell sorter could dirtinpiah nodtirsw from neoplutic tissue baaed on cellular DNA content meuurennts rrrd dlstln~ulshmalignant cells frar clw~rof l-ocytu. Cell identification instrwnu designed for cancer senenin8 ha*. failed beuure of their inability to distinguish rligmnt cella from clwters of lyrphocytes.* A great de81 of lymphocyte activity im found in ylly tumors; therefore, it is of par.wnmt importance to be able to dirtiuguish twr cells from white cell.. To test the capability of the instmnt to distinguish these NO cell types from each other. PL artificial mixture vu mnde from spleen cells mad =A-1 twr cell. gram in tissue culture. These cells wro subjected to trypsinitatioa, fiu- tion, and acrifl8vSne-Feulgen staining.' Figure 1. and lb shavr that the modal churnel of the C1 FIG. 3. DNA distrlbutim of E(cA-1 cultured cells spleen cells io less than the .pdd chaanel of the (coafluant). C1 tumor cells, rumestlng that it is i&ed pO8- siblc to distinguish the distribution obvious that addition of varying aaoults of el (1NI DNA of tmr t-r cells to these speciPcns will aid in deter- and spleen cells. A -cure of spleen and MA-1 mining the minimal leveir of polvploid cells cells is sham in Fig. IC. The nixed calls ven detectable vith the currently used sensing and then sorted and, am sun in Fig. 2, put 1 I8 sorting equipment. indeed reprosentative of spleen cella, peak 2 is coqrisad mostly of twr cells, and peak 3 is REFEREXES only tumor cella. To check our capability to detect t-r cell8 1. T. T. frujillo urd ?!. A. Van Dill., daptation in vivo. XA-1 twr cells prw tissue culture of the fluorescent Feulgen reaction to cells in -- in 6 $uspension for flou microfluoronrrry. hcca wen trypsinited and approxi~cely10 tuvr Cdb *J::tol. E, 26-30 (1072). inoculated rubscapularly into each of 6 C3EI nice. -. :. ?!.Holm unci L. 3. Cram. An inproved flou , After 1.5 veeks. the miuk were sacrificed and zicrolluoroaPter for rapid Jrauremenfs of the tmors remved. The twrs wers subjected to re11 fluorescence. Exp. Cell Rlrs. (L973), in 2ress. crypsinfration. fixation, and acriflavine-Feulyn - ~talning.~Resulting DNA dlstributions of tumor 3. ';. .:. 'Todaro, :. K. Lazar, and H. Green, ihe initiation of ceil division in a contact- cells are sham in Fig. ld. rhe distribution Is inhibited mliancrll line. Cell. hmp. :. coqriscd three peaks represaating tvo ?hVsfol. )a, 315-334 (1965). of

. ^PI c-e 1 1 92 fl)lsb'JL I . 0

e 4 e a

4- I i 0

0 i b. chrml llHbn (W cmkr() FIG. 1. Mal channel of sp10.n colh rad C1 twr cells: (a) lbue sploon cella which vel'. trypinired .ad fixod in forrrlin. 0 The cells wen stdad with acrifl.oint Feulgen. Colk in chutn.1 20 mprerat G1 sploen cells .ad thooo in chmml 40 % + n cells. (b) MU-1 cultund tw? cell., stand in the su fuhlon u the sploea cells. Note that the C1 dJ ch.0n.l is 45, indicating thot the Pau content of - tmor cells is m2 tius greater thao Ce 40g diploid 8phen celh. (c) Artificirl dx- Photomicrograph8 cell. described ln tun of cells from (a) mid (b). The LASL FIG. 2. of Fig. (a) Unsorted mlxture of cultumd cell sorter vu adjruted to sort cells in IC: chonnels 15 to into beaker .ad MCA-1 tmr cells and mouse sp1e.n celh. M me Sort I from distribution sham in those in channot 35 to 100 into .nother (b) beaker. (d) MA-1 twr cell. tJen from Fig. IC. (c) Sort 11 Iros dirtributlon shara in IC. a C3HJHIRej mouse. Preparatioa vu the s.r Fig. M in Fig. la. cells. From Fig. 3 it is obvious that tumor cell tnrichrnt is possible if one uses the LASL cell ovrrlapping bhodal distributions. The first peak sorter. It is also clear frol Sort I (Pig. 3) that on the loft represents noma1 diploid C.118, either diploid cells within the tumor are lvmpnocytes and fibrobluts or lyrphocyter, within the tumor. The not fibtoblutr. second or middle peak is represantativc of the C1 While these preliminary studies suggest that twr cells and the G2 + M diploid population, and mmurennt of DNA content alone is sufficiat tO the third puk is representative of C2 + ?( tumor detect twr cell8 from normal diploid cells, Mny

93 1. J. A. Stcinlrup, ?I. A. V.n Dill., md X. J. Fulvyier, A high-spwd flar system for ultiparuter cell sorting (in prsparation).

2. W. E. Tolles and H. P. msberp. Sire and shape detmai~tioain scaaalng licrwcopy, &: Scsnninp Techniques in Blolow and *dicier, Ann. 3. Y. Aud. Sci. 97, 516-536 (1962).

3. P. X. Kraaer. I.. L. Doawn, H. A. Cris-, and ?I.A. Vpa Oilla. Pardox Of DNA COll8tallCY, -In: ".Uvonces in Call 8nd nD1ecuhr Biololy," Vol. 2 (E. J. DUPrav, ed.), Acadedc Press, Sew Y&k and London (1972). pp. 47-108.

4. L. Vheelrss, University of Rochester Mdicrl School (1972). personal colrunication.

Use of Flow HicrofluoraP.trv €or Analysis and Evalu8- tion of Svnchroniring Protocols and Orup Effects on Cell-Cvcle Traverse

A nuber of cooperative studies with the Cellu- lar Radiobiology Section were unQrr8ken to determlae the effects of various protocols on DNA synthuis. Flov microfluorometry (FMF) analysis offers the unique advantage of providing DNA distrtbutioa pattern of large number8 of cells, ther8by revedfnp b. the relative rider of cells in the various phues of the cell cycle under a variety of experiwntal conditions. FPIF analysis, coupled with cell enup.ra- cion and autor8diographv, provides a powerful mothod for analyzing the effects of various synchronir- ing protocols or drugs on cell-cycle traverse. In a collaborative study with Drs. R. A. Tobey and P. X. Kraemer, :he effects of thre8 cmnly 1 used synchronizing mthods, isoleucine depriv8tioar 2 J double-thymidine blockade, and mitotic selection, were analvzcd and evaluated with respect to their effects on subsequenc DNA replication. In 8.ch caae, we found a given fraction of cells vu unrble Photonicrographs of cells d8scrlbed in ETC. 3. to coi.plete genome replication following synchro- Fig. Id: (a) Unsorted tumor cells dispersed with trypsin. (b) Sort I from nization. The tern "tr8verse perturbation indcx" distribution described in Id. Fig. WM designated for the fraction of cell8 convertrd (c) Sort I1 from distribution deecribrd in Fig. Id. to a noncycle-traversing state because of exp8r: imntal manipulation .4 Traverse perturbation in- other parameters must be investigated. Par.oater8 dices for double-t:?>midlne blockade, isoleucine such as nuclear-tecvtoplasmc ratio and tumor mes- deprivacion. and 3itotic selection were 17.0, 12.4. brane antigens currently are of interest. Perhap6 and 5.5 percent, raspectlvely. A typical DNA dis- the USB of multiparameter analysis (i.e.. DNA con- & tribution pattern revealing the aoncvcle cells cent versus nuclear-to-cvtoplsmic ratio might make follwing release €ram double-th?*rnidine blockade unequivocal tumor cell detection a redit:J. is sham in Fig. 1. A knwledgc of the travatio tUSb523 pertubacion index will perorft a direct colparison I to00 .- I I - f-t I I I A ’ 4 1000 - I I -

I I IA a I OOQ

moo 0 0 0 20i so 40s w 70.0.j DNA CONTLNT FIG. 2 DNA distribution patterns of v8riacU mtn- I It chronized cell populations: (A) Cdb were prepared bv cultivation for 30 hr in isol.uclne-dcficimt mdium, thm re- 2ooo~IO000 sumpended in fresh isoleucine-cm- log dim containing hydroxyurea to lo-’ 5; the srple for F?fF anllymis was r-d 0 IO2030 4OS0607OK) at 10 hr after rercupension of irolaucinr- DNA CONTENT deficient cell. in nom1 ..dim plUr DNA diatribution patterns shaving non- hydroxyurea. (B) Cells were pnpand by traversln6 fraction of cells after reversal cultivation for 30 hr in isolruche- of the double-thymidine blocludo synchro- deficient =dim. then resusp8ad.d in fmsh nixins technique. Broken lines represent isoleucine-containing wdim conuiniag values €or C1 and 9 + ?I DNA cclcul~tcd cytmine arrbinosid. to 5 UO/~;tho from controls. Cells were prepared via suple for Fwp anAlySi8 wam redfra the double-thyridine blockde technique. the culture at 10 hr after resurp.Miw lWF patterns in the culture at. tir of of isoleucine-deficimtt calls in 110-1 remmal of the second thyddina blockade medium plus cytosine arabinoside. The each (DNA pattern shown in A) and at 6 hr nuder of cells examined in cultura later, imdiately before the first in- was 19 ,OOO (isoleucine-def icicacy and creuc in cell number (sham in E). The hvdroxyurea) and 17 .O00 (isoleucina- nhera of cells examined in (A) ad (B) deficiencv and cytosine arabinoridd. were 51,OOO and 52,000, respectively. Broaen lines represent values €or C1 and % + ?! DYA peak values calculated from of the effects of various synchrony-induction pro- controls. tocot on cell-cycle tramtee. advurtage of providing large quantities of Cells In another study conducted vith Dr. R. A. near the il/S boundary suitable for studiu of blo- Tobey, RIF techniques wre used to evaluate a nau chcnical events associated vith coapletion of inter- protocol for preparing large quantities of synchro- phase and initiation of genov replication. nized mliancells in lace C1 of the pre-DHA In another cooperative study vith Dr. R. A. replication phue of the cell This tech- Tobev, P!F techniques were used in experlvnts nique. a modification of the nthod described by designed to determine effects of several ch-- Tobey and Lev,’ eaploys hydroxyurea (to 3) or therapeutic agents on cell-cvcle Four cytosine arabinoside (to 5 ug/ml) for 10 hr follw- agents ,iith differizg effects on cell-cycle progrcs- ing relea8c of cells from isoleucine deprivation. sion i’ere examined: hydroxyurea, cvtosine ara- Cella that are then wuhed and resuspended in fresh binoside. blenmycin, and camptothecin. Both mrdium vlthout drugs will initiate DNA synthesis Iiydronturea (lo-’ 2) and cytosine arabinoside and begin dividing within 7 hr. DNA distribution (5 Lg/nl) grosslv decreased the rote of prograssion patterns for cells synchronized bv this technique of cells into S phase. resulting in accumulation am shown in Fig. 2. This protocol offers the of cells at the Cl/S bound.?. !+either agent t 05b521r 1 ,, 95 cogletell prevented colla fra initiating Dbu syn- The eollbinuion of FW, cell an-ration, .ad auto- theair. Ble-cin (100 ug/rl) *d hitiation radiographic techaiqus pr0vid.r a n.v apprd to adcoqletion of genom replication to occur at a studying the effect8 of chevtherapwic amnL. on nearly nom1 rate. but cella accurhted in C2 and cell-cycle traverse .ad provides valuable inforu- mst cslls loet the capacity to enter dtomis. tion to the clinician rqardiag drug dorap and C.gtothmdn (1 ug/d) reduced the andl rate of tirs of applicatiw. cych progrraaion .ad allowed a f.v cella to replicate a c-lete coql.rot of DNA. Th. DNA distribution putrnu for cultures re- 1. R. A. Taboy 4K. D. by, 1soleudntrrdlat.d leu& from isolauciw-deficient Cl-amst and regulatiaa of pnome mpliution in various mancell lines. Cancer Ru. 2, 46 rmintalned in the varioum drug. am smin Fig. 3 (1971).

2. R. A. Tobw, E. C. Aad.mOa, ud D. F. Pete- sen, Properties of mitotic wlla prepamd by nrchanically shaking DOOhyer cdturu of Chinese hater cells. J. Cell. Physiol. z. 63 (1967).

3. 3. F. Petersen, E. C. Andemon, and R. A. Tobey, !Utotic cells: Source of synchrmiztd cultures, &: '?(.thods in Cell Physioloty," Vol. 3 (D. Y. Prescott, cd.), ;\cAd.de Press, Nev YZrk (1968), p. 317.

4. R. A. Tobey. H. A. Crissun, and P. X. Kraewr. A method for comparing effects of different synchronizing protocols on Ulu1i.n cell cycle traverse. J. Cell Bfol. 54, 638-645 (1972).

5. R. A. Tobey and H. A. Cris..uII, Preparation of large qwntitiee of synchronized uylian cells in late C1 In the pre-DNA mplicatlve phase of the cell cycle. Em. Cell Rem. (1973), MC. 3. DNA distribution patterns for cultures in press. reloued from isoleucine-deficimt C - arrest and uintained for 10 hr in lbo 6. R. A. Toby and H. A. CrIssnn, Us. of flw 'sg/d bleaycin (B) ; fi hydroxyurea (C) , dcroflwrolrtry in detailed analysis of or 5 Yp/d cytosine arabinoside (D). effects of chemlcal rpnts on cell-cycle pro- (A) Control. The culture vu uintained grsssion. Cancer Us. 32, 2726-2732 (1972). for 30 hr in i.ole.ucine4eficient medium, at which tin an aliquot was examined via .ipplication Biophysical Instturntation to Animal R(p analysts. (1)) B&oqfcin. The culture of vu puintained for 30 hr in isoleucine- Disease Diagnosis deficisnt acdium; then the cells were resuspended in fresh, coqlete (isoleucine- The Hog Cholera PK-11 Sys tea.--Investip.tive containing) mediua supplemnted with blao- iifort since last annual report dcicribing ycin to 100 ug/ml. and after 10 hr a year's *aJr successful "proof-of-principle" experimntr 1 sample VAS removed for MF analvsis. (C) Hydroxyurea. The culture wan nuin- tained in isoleucine-deficient medium for &.as concentrated on adaptation of this system to 30 hr; then the cells were resuspended in fresh tissue techniques. The specific rim is one fresh, conpletc lardium containing hvdroxy- cf direct detection of viral antigen fram field urea to E, and after 10 hr a sample vu rmmoved for EM€ anrlyuis. (D) Cyto- -ubmiusions. sine arabinoside. AEtcr hr in 30 iso- Yaw problems encountered in preparing leucine deficient lydium, the cells Yere were resuspended in fresh, coqlete medium heteropneous cell s.gles (pip blood and tissue containing cytosine arabinoside to 5 up/ml; biOP8v material) €or flow microfluoromtry 10 hr later M aliquot was removed for F?tF (Fnr) analysis. The bro&en lines represent c1 mwurements. The very lat fluorescence of cells ad C2 + !I DNA peak values calculated from labeled with fluorescently tagged antibodies re- the exponential culture, which uas the source of all cells used in thcue cxpcr- quires monodisperse cell suspenuionr that are free imnts. number of cells examined in The of small debris and homogeneous. Procedures were each culture vu (A) 23.000; (B) 67,000: (C) 19,OOO; and (D) 17,W. developed for isolation. fixation. and conjuguion

f 96 -.

oE spleen ad white blood cell 8rup.rriolu to aseure coq.tibllity htameu thue proud- ad Miat- Of pod 8iBW-u 8tM-b. NU- urou. mtico~l.nts,rod blood cell lymw 4au and procedurem, .Id smrd ti.;.tiao ~ti~hodrwere evaluated to prorid. proudvrv -tale vitb uint.idmS si~~e-criimp.l~ifm vich dw dobrio adstill prorid. -A1 parrubillty to fluoruuln-l.kled mtibody .ObCUb8. BdfY Coat -11 Caturn8 V8LC d80 wi&md. fithouo 8Ufe- factory protocols YLI. denloped. noahspecific bind- KC. 1. Dist?ibutioW for catrol and inf0ct.l ing of conjugate to uninfected celb decreased the Ull.. 88UdtidtY Of MI 6Y.b bel- thU Of stdd wriatim of tha puk. 1s a result of cmalbs technlqueo. 'I& prcoent level of sensitivity re- the contrlbutioa of nolupeclfic bindiq which 1s quires that 10 percent of the cella irr a rqle be proportioacll to cell MlUI. infected. Iqmnmd conjugate purtty wuld incrrue Diffenncoa in [email protected]~ pattam um=m. the e1~81-t-noiee ratio and deRp J useful tool &sewed for hog cholera Infected aad nonhfectd for studying virum replication in dimcells: PK-S cells.2 Li&t-.uttering ruure-tl trn this is being attqted. Mde with 8 nay photowter3 dercribed 818#hWm in Buic support uqrerirnts wiq the PK-15 cell this report vhfch U~Uhia-8peed film u the der- syetea to determine if other orem of RIF applic- cor. intensity of scattered radiation fra 8118- ability exist Will coatinu. For the firet ti-, a penmion8 of live infected and noninfected cell8 L qumtitative evaluation of coujug.tu CUI be ac- shwn in Fig. 2. In the u~pulrrrange of 2.5 to cwliehed uaing techaiqws developed for the proof- 4.0.. overlap of the two curves is c0~i8trafwith of-principle projsct. Quality evaluation of com- li&t-scatter theory d Coulter -1- data that arCfJlly produced conjugata is lqottant for USM indicate the two cell populaciona are very 01dl.r licenming ptocedurea and for evaluating our OWI in VOlruC. At hrpr rn8hJ (4 tO 25.). iUf8Ct.d conjugates. conjugate evaluation is bued on two par.r?ters: (1) relative cell brightness or the amount of fluoresceinatad antibody bound to a cell as corp.nd with a "st8nd.d' conjugate. urd (2) specificity am evidenced by the ratio of specifically to nonspecifically bound conjugate. Both parameters can be quantitated easily wing the M. Althou@ inotmncal development on this project hu been reduced, net techniques of rapid cell identification and inforution processing dewloped in the section ham been applied to the fluorescence ~alysisof randorly grwing PK-15 cells infected vith hog cholera vim. Recent devrlopunts pedt muuring the ratio of cell fluorescence (total amount of fluorescent antibody bound) to cell voluae. The resulting distributions (Fig. 1) for control and infected cells are both tighter (coefficient of variation dccreuw by 6'1 I approdnatelv 2.0). indicating chat larger, more s 10 is lo U SCATTERIN@ 4MLE 4 DEGREES) -Cure cells do not produce more Virus Per Cell FIG. 2. Scattered radiation intensity for infected than smaller cells. The iaprovld coefficient of and noninfected cells. colla scatter uda li@t (.Loor 2.3 tiw at a factor of 2 in Du cmtmmt but we In ch- 20.1 tha noainfoctod colla. lhir dlffmnacm in nukr) *N i.ol.td fra tho coll litn. Bm-0 lary -10 li#~t-acrttorputom la b.Ud to thwo -0 PI-15 clol~r*n irolatad from tho 8U bo dum to 801 &.ow in intoanal atnutun be- culturo, urn bmli... they either had exactly the tho tuo ull populatioor. sp. doubling tin or thmlr paeration tino umm In romrnmmo to a roqwat for help from tho not aqd but lntmrcmlluhr ut.bolic facto- uorm Emwucy Dboue Tuk Porco of thm USM, an offort boing prodwmd that diffarmutld~yreguiato c.11 f8 UIdrZVaJ to iU*..tiptO tb0 qplhXbiuq Of QUL doubling. Cella fra the two clorum we- dnd biophysical inst-atuloo for rapid dlfformtial lo diffennt ratiem .ad lllarrd to ww tbroryb diaguoof. of Ikvcutl. dimmu0 virus. h. outbrmak 6 pus-. Tbeir Poul~-EUAdlstrlbutioar w.t. Of a HlOynlc 8trh of the virum hu ruultod in thou muudto determi- if tho tu0 papulatiau the dlraam b.ioa docluod 0uiOp.l utpacy. uenn pawing u tho amrUo d Uhtaid~tho l’h dam of polyploi&y in cbickn dry0 fibr- amratioa. Bec&mo the dxhg rmtio8 rodmd 4 bluta aftor R.vcut10 infoctioa ir rmported to cwtat, we coocludo that tho NO cloau ha- tho correlate vlth the virulanu of tho virum. Thero- 4- doubling tins and raapond sinilarly to cell fON. a tapid. qU<itAtiVe muUr.wnt Of poly- density. Further experiwnt. are plmned to ploidy vith RW should proride a rapid acrmoaing chose clonu vlth regard to tbeir chrommou b.ow techaique to identify differontially strainr of p8Ctern, r08p4n8e to tg)oratuw. md rJdiStiOlr. Nawcutle diaeuo virum.

Effect. of Tomper8ture on the Utotic Cycle of REFEUEWES a0 c81h.--no purp0.e of thi8 8tudy 18 to uke 8 1. C. R, Richumd .nd C. L. Voeh, ads., kmud Roport of the Btololical and Mdiul &8OUch dotallad invemtigatiou into tho effecta of tom- Group (W-6) of tho USL bdth Divi8im. Jm- prature on diffennt phuw of tho CUO cell life ury thruqb Dodr 1971, tor Al- Sd- rntific Laboratory roport (April uaing tochniqrws for coll-cycle uulyaia. U-4923-PR cycle RQ 1972). Chinere hater cells (Q10) in swperion culturo vm grom throu% tvo exponontid growth 2. cycle. at throe trmparaturos: 34, 37, and WC. Preliminary data auggeat that during the first cycle of cell grovth aftor a 3’ teqorature change, 3. A. Brunmting ud P. F. hlhy, A 1iCt- scattering photometer wins photographic film. wt of the effect. ace an nactlon rates. During Rev. Sci. Lnstr. 63, 1510-1519 (1972). the aecond cycle, vhon steady atato situation has C. port.. A, Waterson, C. TerrY, 0. beon achiowd, the effects arm a cooaoquance of 4. P. J. Alaxurdor, rad P. hew, Cell Cuslon by N.v- chanya in concentration of chemical reactants . cutle diseue viw. J. Con. Virol. g,95- 97 (1972). This is evidonced by m apparent &ago in cell ca- poaftion which was meuured u a chmp in cell 5. P. Y. R.0 and J. &&berg, Effects of tom- perature on the mitotic uad a0 cells prwing u othor thra choir opt1.u cycle of normal .it.. synchronized uul1.a cell., &: “Cdl t.llporature vera found to incrrue in volrpr. Synchrony ,” kadomic Press, Now York (1960 , pp. 332-352. Of particular interest is the obsorvotfon that, upon a decnue in trqerature (37 to M’C), the Yowl and Twor Cell Kinetic Studle8 UsinR Flov percont of a0 cells in the C1 phue of their life Xicro f luorort ry (W) cycle tncreuea from b6 to 61 Frcent, which is just the reverse for HeLa cell. which show A decrease ?kchods for studying the kinetics of prolifora- L 5 from 66 to 36 percent. tive cells from both nom1 Md tmor tisswm & vivo arc of fundamental tntrrest in addition to InvrrtiRatioru with tho Unique Yixoploid Cell - their posalble application to the areas of chw Line, pK-lS.--lhe unique DNA diatributlon of PK-15 therapy and radiotherapy. Cell analyaia and sorting cells, a mixoploid cell line, vu reported in last instruuntotton currontly under developmnt at tho year’s annual report.’ Clonu that were diploid Los lamo or Scientific Laboratory C.D be used with ;md tetraploid in DNA content (1.e.. th.1 differ by f Q5b52-I 98 L-

relativa eu. for klaetic studir. BUtc*lly, the procedure comsists of (a) prepuing sin*-all swpeaaioaa fra the tiuw of lnterut wlng trlrp- riaizatlon procedurrs; jb) fixilpl s-e cella in rthmol adforuldahyd. dlLtura; .Id (c) striPing fimd cells with actifbdw (fluotrsmt-Feulgd for sp.cific stria(a. of ullvlu DNA. Other available tocbrripws Include the me of (a) the Coulter priaelple to d.t.tdM the dm dietributioa of cells; (b) a algnal fra suttemd U*t to pte the etctronlc ryotor Vhich wof..fl-carce, themby allminatlly nobe signah pner8ted by d.bris la cell plrp~~tiopu;(c) dt%pUmter cell separator to irolue call popululav vith different DNA distribuCloa8; ad (d) a COdiMtiOU of the &on 8yat.r to muuze dlr8ctly the DNA content per ulit cell volrru. ’2buo tedmiqcus are 1 described In detail in our andreport for 1971. Our preliml~fystudies iodiute that cell pnWt8tiOM for WY. tb8-0 8uch U Skin, LnteS- tinal epithelium, liver, splaon, md kidney that are considered ipportmt In radiotherapy can be nde and their DUA dbtributions ruund by PW Instru- mentation. Figure 1 shous DlU dlstributfons for diffrnnr normal tlsouu that arc considered to be s- of the Ilriting tlsoues la radiation therapy. The noise awed by debrls in the cell preparation and appearing in lou chdnuben vu not elec- trmically gated out in those mmuuremnts. The DNA content of a11 Chwe normal tissues la about the Sam and shars that a &re fraction of cells la the G or atate. is 0 GI Dr. 1. Y. K.llmm adusoclatcs2” haw concluded from their sndier vith IMT aarc- and Enr6 (IPIIUT~ carcinol..) that a dose of 300 rads induce8 synchrony in the t-r cell., thereby uwing cyclicdy fluctuating radiosensitivity change0 as a function of ti= after exposure. Although It will be difficult to correlate results of DNA distribution vith ruult. of radiation 8.luitivity, it will be of interest to study the dunpi in DNA diatrlbutious In tUor cell. in vivo with time after radiation exposure. IMT 18 a sarcoma that arose spataneoualy at the bue of the ear of a C3H/KM Jwae in 1962 in Dr. Kallmsn’r laboratory at Stan- ford Cnlverrity. Thii tumr line CPI be min- FIG. 1. DllA distribution of no& tluurra in da. The X a10 Is the channel number (propor- tained bv aerid rubcutmeow purap and has been tional EO DNA content in the cells), ad studied cxtmmively.4’5 A tumr of about 12 m in the Y axis is the relarive nrubar of cells per channei. --

dicrter frar 8 UrID~. ir Uspl-d In now ..limaD cl docad into m piuu .bout 3 1 m in .ol\r. 'Luor pkao um tha ti-- plmted =to the back of C% du *i. 8 amll irrcimioo in tha .uI of tha f-17 rbmn buk. Th twpan to deut 1 Q i. -tar on .bout the 12th day aftei t-lrutia. Wta~the twm wae &out 1 a io dimtar, the .lea nre .IW- thctixd (0.1 ml/g body wt Of motd),and the twrm uuo expad to X rap by &Uw the mnilrd.r 'of the we. Hane turn ..paad to XKI rd. of 2%- X-m mcflfieed u 0, 8, l2, 16, 22, 21, 2b, 4 30 br after X-ray eawoure, and cell pmparuioar v.t. rl. to mure DNA dlatributiom. A fw tiaauu vu0 &o 8rpor.d to 1500 or 3500 rads of X xmym .la vmaacrifiud at 24 hr ahar o~ura.Tv e w-tm ruggat that 3500 rdr L a curufva doe. Tvo mica wen umd for d daw lad ad fixation tin after expos-. A reterad Ught 8i& WM wd to g8te the ndae ai@ ariains from dabti. &an the cell. wra w.d in tLe RQ Lnrtrurmtatios. All -lea wore muumd to a total comt of %,- calf.. Plmre 2 rhous tha raaulta of DOA dlatribotiolu at different tima after 300 ?a& of e-ure, uhoro there an significant differoncu in DOA distribution. Micro- acopic erPilutioa ahmd that moat of the calla vere ainile and tbat.the doublet. vue lea8 than 5 percart in all arplu. Tha fiat pod io the WAdbtributim nprsrmu ooC.I.1 diploid cell. in the tw. The eecoad peak, cornrporrdiag LO tba -ut of #u nearly tviu that of noddiploid cells, is due to t-r cdl. in tho C1 phu. The third peak, corraaparding to Nice the of tmr cella in the C1 phuo, 1. dum to t-r calla in the C2 + U phue. DNA dbtributiau comrpd- lug to cella in betweon C1 .od C2 + Bl ttmor cella are due to cella irr the S ghae. Yb.n the ull. in a tmor control auplr we- sep8rbt.d according to the DNA content in the cell. the cell aep- arator, wm found that the first pJL In DNA die- trlbutioru is dru to 1eucocyt.r. It can be reen fra Fig. 2 that the &A diatributiw of tmrm 8 ad 12 hr after expoaurm to W re&. dun coqared to zero hour after expoaum, hu -?e cella In the FIG. 2. rrU diatrlbutinr of KXT sarcoma twr cella at different times after expoeure to G phue. Thi. could ba duo to C2 block cawed 2 + U XK) rod. of X-rays. by radiation. Bweuar, it can be seen that 12 hr after expomure the colt are baginning to didid. and chat at 24 hr after ereposure to 300 rad8 DNA distribution look very slmllar to that of the control. Figure 3 rhous that whan the cells ar8 cxpo8d to 1KK) rad8 therm still se- to be a smdl incraue in cell. in the Cg + fl phue at 24 hr after exposure, vhanu with 3500 rub nurly 50 percmt of the all. are still In tha t+ + f4 phue. One would orpact this trend became with increuing dooe the ritotic delay is incraud. Thase results are in 6 avo-nt with rasult8 of Kal ulna nh.b-0- sarcou cells vith wephotomter Instrumenta- tion. Variation8 in MA diatribution amng any two tuor supla8 tr8atad in the s- way were found to be remarkably sidlar. Thru, the dfffer- enc8 in DNA distribution with dome and tir after expoaure ue real. It can be condudad fromthue PreudMV 8tudiv tbt is a nq good tool

1. C. R. Riichmad lad C. L. Vdr, a&., Anad hport of the Biological od mrdiul p..aucb Croup (E-4) of tha USL Hodth Divisioa, Jan- wry through beamber 1971, Loa Sci- mmtific Moratory Beport U-4923-tR- (April 1972).

2. R. P. IlrlT-m .nd U. X. Blechm, POmt idia- tion cycUc radioauuitidty dmngu in ttmora adwrrl timo~~.p: "Procaadi~of a S-81- OU bt. in mi- Biolow (ApPril 29+y 1, 1968)," UC Divlsioa of Technical Inforution report COlW-680410, pp. 20.1-20.33. 3. S. C. Rocball, Ca11ul.r bdiosa~itivity,Call Popul.Uoa Kinetic8 and Tumor Radiuioo 1LUpoluc in Two Ut4 Ibua -ry Card-. Ph.D. mais, mimcriw ot catod. &.rltdw.

4. L. H. Van Putten and R. F. KaUnn, Oxy$auion statu8 of a truupl.ouble tumor during frac- timated radiation therapy. J. Xatl. Cancer IMC.40, 441451 (1968).

5. R. F. Kd-, G. Siliei, ad L. H. Van Puttan, Fsctors infltnncitu the quantitative artiuter of the in vivo surrival of cell. from solid tuwrs. J. Natl. Cancer Inst. 9,539-549 (1967).

6. H. B. Kd, Di8tributiau of cell volur and DNA content of lthabdaywarc- celh --in vitro and in vivo after irradiation. Eur. J. Cancer (19731, in press.

FIG. 3. DNA distribution of K?lT sarcom t-r cells after 24 hr of expomure to 300, 1500, and

101 i totally &pendoat on coo..aiar of wtpat from a Mpuuia facility into other chddfom. effort tdpublidaiq rhpotmtial of mdle oddu fra tho onriebrrrt procum 0cculmaUy can be ud dinctly; bmnr, the bulk of nqoirrrou n.eu.itatu iocorpmuiorp of .n+idld i8otagw

Perhap OIY of our difficultir h. beai 01.- into orlpnic c-&. men are rnoda for dl io tbis n@ vbicb hu sort- Oa cdiiutiacu of labaUq - a- e~r pdmut k W0-y Wled, MI 8p.cttid7 Lb.1.d in en8 or particulu rolecuru vi- tim; rrd s- dtiply l4bold with m= th.0 OD. i80top., both -1f6y .nd rpedfidy. Tor

0- appliuti-, hi, betopic enrichrot f. reqpirmd; for others, Zar arfcbot IO m- or sufficl.nt. Ib. qrvntitiw of cwollod. ao&d can vary fradlllgrrr to kl1ogt.r. iorv Wyticol nrvlas, Groq mC-4 is rto A To met there VAriOUr and mdod nqd-t. prlury collabor~cor* rpd participant in un]r of the 8ffkimC17, 8mthYu fadliw i8 U8Q.W activitir. which utillzes ad effmcti-17 cabam tho rthd Dr. Waltm V. Shreeve of the pkdicd Division, of both organic aad bio-spthui.. Each prorid.. Broolh.r*n Naticmal Laboratory, wua USL Visiting the othr with rtartlnp maerids, as vel1 u fur- SC8ff -or from JM~u~to Amt. During this nishing ultimce products. As the cap8biliv i8 tL.. dditid pluming for vuiow clinical ert.bli.hed for Synthed8 of a pmrtidar c-cPd, qpliutiolu of atable Lotopas vu carried wt, the capability for productloo of others i8 -0- d the fiat trials of carboo-13 in a glut-e tol- utidly increued. The spthetlc rout8 &OS- ermco tut vir8 perforwd. USL Visiting Staff oftan depend (la addition to other coruiderr.tlor~) Wea(Short rem) participating d aiding in on vhot experience. nterl.L.. or rthods hava V8dOW PnjOCt., elrrrdly throw Of 8 fW becor available thrwpnviocu prupbratlou, u days, include Winslar Cau(lhey (Arizona State Uniwr- well u what application mifit be lihly In future sitp), Bruce Burnhr (Utah State University), B. !I. syntheses. Tolbrt (Dnitnroity of Colorado) , C. li. Nub (Univrr- The basic inorganic starting ut8ri.k us 1 the various oxidu from the S8p.r8CiOa fmig Teuu), .nd R. T. Win (Vnlvenity of Texu). The (i.e., carbon waoxide for the carboo isotopes and section abo coatinuem to benefic from discusslaru nitric oxlde for the nitrosen 8nd oxygen isotopu). and sda8ra presented by other visitins SCi8nti8tl. Carbon dioxide ls ndily AVdl;rbh through odd.- tion of the IPonoxide. adwater and 8m PIEPABATION OF CQMWNDS LABELED WITH STMU obtained through appropriate reductiolu of nitric ISrnPES oxide. The seven isotopes available to the current program €ram the production plat are UrboO-~ (D. C. Ott, E. C. V. S. Chavrr, C. 1. Cregg, (less than 10 pp~carbon-13), carbon-13 (has thM J. L. li.Dnet., J. Y. HUtmn, V. N. Kcrr, V. H. Koll- 10 percent carbon-12), nitrogen-14 (le88 thrr 50 ppm H. A. Nevaret, T. C. Sachex, and T. 2. Whdey) nitrogen-15) , nitropn-15 (less than 5 porconc Utilization of rtdle isotopes 1s almost * nitrosen-14). oxy~n-16 (:ass L!UI 10 PPI omgln-18) The collaboration and cooperation of Or. N. A. oxygen-17 (u.10 pmrcent axy~en-16 and oxygln-181, Hatviyoff in all activltlu involving nuclear ng- and oxyen-18 (less than 5 parcent orppa-16). necic rosoomce (a well u ocher upects of che atable isotopes program) is particularly acknw- 3ost of the compounds produced by the synthuis lsdyd; J.0 Drr. 8. XcXnteer od A. Mlk B. T. facility are for in research have been usential to variow parts of the re- use Ut (includiag search progrul, in particular IUS spectrortric collaborative projects) primariiy in oiologid, fsotopic arulvses of carbon dioxide from the clin- clinic81, and chadc.1 8r;Cies irr progress fn the ical trials AS well as rupkr €tar other sources. 185b532 10: Biordtul Wearch Cm(Ed) d tin -try- infra& rpectrommpy). Cuba-U 1bUCh8t ~y Nueloar Cbmtty (=)Divioion. P+arioioa of lutic ~OSOPIILU (ax) is, of couroe, -.IT urful to -or& new in otha progr- thraybautcuSL tho mptJutie chdrt. :a dditioa, protoo ndar should be caui&red 06 *=ant ftmctioo of an -tic (or),becane of 8pieSpia up.bfiiw. coup- of ptotaam with utba-13, cm ofton bo Tbbroulb a intar- .9m-t k#.o tho wed to at* urbao-13 (for -10, tho .ortic u* s. lrtodC ww d th. #Uiw Uib, rrhrol, .d 0th- aigle ptoQcta 8- IMtitutw of Ee.lth, *.rim l.brll- mut~mmlyma~7-1 for 13c/*c ration at 0dspa- v-0 .fwhrimd ad *tpglhd to tb. chdtry ca cific &a0 ota ww0 .*le pp mp.ctrmtor). Lit. Sclacu Marot-, butch rn-0 I~ti- Veri- preparatiwr h.n ban (d0tb.m tutm, hoearch Tri-, Worth Catom, for w VtU be) rrputod uvmral tiw boumo of ua- in rUhU8 pmj.Ct0 Of the ktid fMd- otidpated mmd. or contmdemand. *thmol tuto of Ganeral lhdlcrl Scciance~. It lm fdt that md acotlc rid ham been pr-d on 8 mro or mhibr uranvnt8 could bo utually d+urtageam hacoatidxu bula, wing primarily to thoir for tho mpthwi. facility md for otbor oqad;c applicatiap. u buic rpthotic intOdiAtO8 and tiolm h mdof coqormd. labeled with stdle also bouwo of the &rge qrurrtitlu tequirod for iwtagr for varlool project. witbin the US= and yemt production. Tobacco plant. U. bola8 dn- other mnmnt apncir. duplication ChOd At VAdOW SwOf &-mot to 08SUn of offort, owrtiso, .Ird facllltiw vould be that rm~lticipatodrmquiramnt. for tho 8410 cube rvoihd, tho capabilitiu for aymthuis would hydratoa which tho7 afford CUI be mer without unduo actually be bmdaord, 8nd uny appllutlonm and dO1Ay. d.Vd-tr could proced which othonriso varld Orunlc Synthesis bo &wed or irpmmiblo. Althoryh thmra at. real rhilultieo bomoa Tblo 1 &*u A mwof lrotoplcally l~bolod mthm~with r.diwtlw I8otOm (cub--14, capom& which hove bun or are currently bola$ trltim, otc.1 ad the mt.blo laotopw, tho- at. produced, ~OYQO. for productioa, and varhu p.P P.octlOa 8mp-B- nfrrmd tO rho cm8idarablo difforena. Thlhro are primarily tilunt cmmu. h a nttot of Ida; the prepmatiocu with 8tdb the t&le for thoir prmparatioa Ue .ham fn ria. 1. isotopes am umudly eoruidorably larger. phy of NAtwA.lly, tho= 8re myaddithd #p.dfic COC the 010-t #chams developed for carbon-14 am not pounb which arm necosry latoxndiatu or which .pplIublr to arb--13 rt o.U - thw siqly VU cm be propared by tho. san rooctlom, by ob- not mcJo up matimfactorily. Oa tho othar hand. tho vim extmnmiom, or by n-raou co&iruriolu and starting mtori~lcool~l to both carbon-14 md pormutatio~of tho 18b.l; hownr, only tho80 8rO listod which have beon prepared or for there carbm-13 myathum, carbon did&, ir mor. COIL- which veaiently handlod on the largor 8c& in a cyllador fa a curront requinmt. rather than frar barium carboarto. Addltlonally, The rmportoiro of dewlopod mthodm for tha wba-'% vaoxfde la the moat radily AVrihblO efficient production of labolod cogound. incrauu dditioaai ¶tAfliag MtOdd, dhViOg UtiliUtiOa Of rOaCti0lU coatinuourly. hch nw ayathui8 ha8 th8 that are not practical for carbon-16. bonefit of siqlification of a futurm probla. for Coqorndm 1d.lod with mtable isotope axhibit Particularly valuable aro provan procodun8 diffeances from tholr Isotope imova in phymicd producing key rynthotlc interrdiator coatairria8 proportiu (0.8.. donmity, mlocular woight, and one, two, urd chroo iaotoplc atom which CUI bo wod to introduce rpacific labols into P wid. infrarod .ad nudoat ugaotic IUOIUIICO rpectra). Stocka of Thoso mmrdly woful praportlw can be utllizod VAriOCy of uarm caplicatod moleculem. in aalyticd procedun8 nocuma~for de-Aoping terrain of tho c-ly urod utorlal. are bot- synthetic rthods; th.u dlfformaum ocoriodly mintrlnod. urd soveral can be producod on a rmA- atively aula (i.o., who per vook) colplluto comm tochaiqurr which rely on cmar largo rvry when tho dmlucrd exists (vlz.. m.thAn01, acotic ~IIWvith 8tand8rdr (..I.,proof of i&ntity by

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c-: i Y a Y rl Y Y 4 0 rn ii r) r) Y Y ii Y a 1 Y Y 0 r(9 a r) 1 Y 8 li Y a rl0 t a 9 V e * Y Y e 4 3 5 Y0 rn a 0. bdY r( a dh 0 2 P,

4 a U

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0 rl 4 rlI

a 1

108 I i 83 10

a da 0 Y

m 1 0 (I 3

elU SSS

0 2 N 0 a rl 0 5 a n d

$ 14. (*a3*COI2O '" Br*aZ COOIl ** P ** x). capLcajx

1) r&*Q 31. 2) hydrolyrir

Lactobionic acid u 6-irta~ uleiu ralt f 05054 1 pu* 35- *a2 +~2-ddna

36. 'CO + 2 U2 Cu-Zn-Cr fC*W30*W A

Cu-zn-Cr *c*r30*n + 37. co2 + 3 11* A

$ 13 42. *a, - L - 6-sltomterol-3.4- C2

ll3 0 0 *Ir, 46. (** C H3)Z WR -( C M3)* " Na2*ca03, (*C*H3)2 I C X( C H3)2

3 Udlptoluidine H,CO, .*,m a llC001[300. II H 0 HOAc I

H n

0 * *I& bB. CO + 2*UR3 + S -lm. U N C NU2 + HIS acids, mthuu, cydb, rthyl iod*de, .ad certain interudiate for pyrolytic graphite-l'c urd others). paphit~-'~C. Figure 2 shars various item of nolativaly coanut1oa.l laboratory ghssvarr .pparatus devoted to relatively large-acde prOduc- and apparatu and vacuu fine techniques can bo tion of "C- and 13C-labelod carboxylic acids, uaed gaaerally for syntheses vith stable botopu. rrthanol and *thane. The unit for SynthRSb Of (kin8 to the extremsly corrmive nature of rhodium acetylene and benzene is rharn In fig. 3. carboaylr, pnparatioru of carboxylic acids via Biosynthesis arbanylation of alcohol8 are cu~iedout in Am is also encountered vith organic 8ynthesis, Hutelloy-C autochvu ~3004,1-1.. and 1-gall- C8p8Citi.S). Tb.8 VUseb am wed for 0th.r dsvelopmt of IUY procedures or clo.binatloor my typm of pressure reutionr, u vell. Stdnlur steel gu cylinders abo sem u reaction vmasels for less corrosivcl prepmatiom (e.g.. una). MethPlol is produced irr lots of about 5 des by catalytic hydrogenation of carboa dioxide in I specially cautructd stainless steel apparatus For preparation of rthan~L-~~C,a second unit wu cowtructed vhich is dedicated so&ly to carbon-12 to prevent contrinatioa of this isotope isour vith carboa-13. A 8idl.r but smaller unit vith about 1/50th the capacity, constructed of standard stainless steel fittings .ad tubing. is used for prepua- tion of sP.1l.m amornu of polpisotopically sub- stituted nth.ao~(..I. , 13~3~~~.~a especially FIG. 2. Large-scale organic synthesis apparatus. car8tnrcted apparatu l8 .l.o used for synthuis of Clockwise from lwet left: 1-1. stirred autoclave, 1-g.llon stirred autoclave, rthae which form the buis for isotopic cyaide carbon-12 mthuwl unit, carbon-13 rth- production; p.th.ar: will rl.0 be the required an01 unit. and methane unit.

114 fateWity .diu8 Vwr I-. into the 200-1. f8P mtor. This capability ir desired for ptdvctfoa of gram gumtities of dmacids throu& collborr tion vitb Dr. John Harkley of Purdw Uolmn~iIn dditiaa to application in this collaboruivm pr~joct,the ..in0 acid. are also &sired io rtdiu o hiatow biaynthesir in the Cellular .rd Ibl.C- uhr Rdioblo~Sectioaa. Other uaeful ntd0 A- coocodt.ncly produced. Nucleotidu Vi11 b. utilized for imrutiytiool vithin the ~orpd .l.o in collaboratiolu vith Dr. Uilllr Outtor of the biVA?Sie Of WifOmiA SChOOl, Prandrco. Fatty uid. are needed AS rOd.1 cow pounda of carbap-13 in tracing envicoarntd p01- lutaat. by Proferror Lou VCrpa ad Anthony brma (en Ayo grdmce student conductin8 this port- of his rerearch in the section) of OUlhoma Stue Univer8Fty. Hopefully. the steroid fractbo Vu1 serve u ureful Starting mmteriala for 8ppUC8tiOM nc. 3. Lithiu urbide ir prepamd by reacting carboo did& with mlten lithium in the in clinical stdiu .ad rasearch by Dr. Duaae Wr- reaction &der (lover left). The acet- ann of the University of Colorado Usdicrl School. ylene Uberated by treatment vith water is prrifiad .ad collected in A series of traps Th. pomribilitiae of ruing these organism for pro- (uppar portion) and can ttAMfrr?ed to be duction of glucose and galactose will be rtoraw vwsals (lower right, rear) or al.~ caavarted to benzene on the vaudfum oxide investigated. catalyot colum (lower right, front). -Yeamt.--Analysir of the data obtained durinc be mquird for productim of labeled uteri8ls the course of large-scale production of candid. through bicryathetic -tho&; often efficacious utilis (for the motme-feeding experiment conducted procedure8 perfected for radioactive isotopc label- A yur ago) Is now beinp mule. Acet1~-1,2-~%acid ing are not entirely applicable wfth the mounts served as the sole source of carbon. In Pig. 6 is needed for stable irotope synthesis. For eurple, 1 I,,,,,,,,. 1,- 1.3 A scaling up a procedure necessitating isolation bad F - "c /I purification through chromatography when dozens of gram of product are involved xsv not be practical or even feasible. One of the major problems is not so much in finding a syntem vhich producu the desired products but one vhich a180 af€ords their isolation in a rearonAble p.mer. hjor emphasis the put year has been on development of mathods and production of uriformly labeled carbon-13 suprs, in particular @ucoac~-l~~and galactose-~-~~~. for applicationa in clinical trials.

Green algae.--Algae are appealing as a source of uniformly labeled compounds of biological import- Time ~dayr) ance became of their high incorporation efficiency FIG. i. Crwth over 14 gemratlons of ywt (C. utilis) metabolizing doubly labelrd amtic- of a simple c8rbon source, carbon dioxide. Efforts acid At 92.6 At01 percent arbon-13 are being directed toward improving our previously AS the sole carbon source carparad vith yeut utaboliring now1 .bundmu 8ttained production rates (z.1 g/l./day) using (1.1 ata percent carbon-13) acetate (3 the large shaker apparatus by incorporation of high flasks each).

1U .. &a# tb0 mat of tb. fl-t Up-t rd. aa tho parth of 5. uti- o hi&ly arLehul cubao-13, fa thf. cy., a& 92.7 ata prcmt. Ihree spinwr flJu oootdnd tho oqoni8m vith amtic -10

.cid at lr~rvl d0-U c~~t~ti~ I (i.e.. 1.1 .tor parclnt)i tha other three flvlu P utilhod tha onricbod a~botrate. lb wth period -m r 0 wm oqmidt to 14 #omerrtiam, during uMch ao e effoct om lrarth tat0 of tho hi# Iaotapic ccac.1Ltra- -m t thcan be discexad. - lip- 5 oh- a tlpiul exporiulrt In vhich 8 z 'Iwt id- vu fraA 0 DoIyl cub00 &PI- dace acetate in 14-1. €.-tor ad tha tra- -O* I fedto the 200-1. fermtor d parth contipwd a¶ aa dotbly 1.b.l.d acetic acid (a0 ator percent cub-- 13 in thir eqmrlmnt). Thorn vu no appreciable

dfffoamce fa 11pori4 in the two mdia nor in the I I I 1 I 1 -0 o 4 r m*zooII mth rat... Tho.. data er. in contrast to resulu That-) aportd by other inrut~~torrr~~ww the nc. 6. Crath cur*. (doorbaa u 5% IU, A) ad - (g yurt/a 0) 1ab.l.d autic addm bu diffannt mth rate mhyield .~.t.t.. tor utili. grab8 w acotlc acid 08 sob 30 5. rurniog pracoduru; honver, It VY ahom later carbon aoaru. Ik.rrL. irdiute plmnt- y~ Of bddiq. that the culture vu, In fact. wlyc. kwmei, a typical fut wing iqurity foocnd in culture the permtuu of yurt calla budded. The initid put prOpagatiOD8 rapid rise of the yield cum vith a corres- Gtarth of g. utllis oa acetata (*ether la- docline in bdding rate vu coneistrntly obeervod. bold or not) i. biphuic, Figure 6 sham the The peak vrkm the terriaation of the period Of grwth curve .ad vwth yield curve as frmctiocu expomatla1 call g-th which, typicdly. sp.olwd of tin for a typical experinat. The nuben ac- the intenml frra 2 to 8 hr 8ft.r iaocubtim Of coqonyiag the poinu of the gmvth yield cum are the culture. Folluul~traluition frol erpon.otld . , to linear vovth, the 8rwth yidd ad perout4 of bulding cell8 droppd sharply mr a 4-hr perLad, tha fell are rlauly rntil the end of the ucpr* ivat. Clearly, optiu iocorporation of Urbaa-U fra acetate into yaut requires that gravth be uintdned sorhow fn the late erpomntlrl phue. In the course of this seriu of experlmontl. over 1.8 ke of yaut YU prodwad at an a*.r- carboa-13 earichmnt of 80 atom percent. Nearly 6 kg of acetic acid vu cozuumed for UI averap yield of 32 percent; most of tho renlniq carbon VU recovered u rerpired carbon dioxide. --Candid. utilir uu Jlo srw at 20 atm per cent carbon-13 (optidsad with rempect to arspin- spin coupling CoMideratioM) by USL Vialtins Staff Tk(mt War Dr. R. T. EJlin of the Unlnaity of Teras. FIG. 5. Crwth of yoamt In a 161. ferwator on Prom this product vu isolated 6-phoaphoglucorrrrte no& .brmd.nce acetate follouad by inoculation Into the 100-1. feruntor in a dehydrogename for kinetic urd physical rtudiu to mdiu utilizim acetat. at 80 atom percant muure the effects of varying lavela of carba~l3 cubaa-13. enrichrat. Yeut hydrolymatu wero dmo umod u ladyti4 pu1p-08. and the fi-t rtbd im .pplic- carbon soureem far grwth of a mtant of Neurmpora able for large-mcale preparations. -cruma whieh producem a high level of cytochmr c. Initi.1 mxp.rirnt# ~rLg1- to 6-LI -ti- This protein hu nw bwn purified and vill almo ti- of Giurtina did not remult la u hlgb -do be umed in Imotopr effacts ud phymical mtudiem. u expactod md. dditimally, a pater mtof endo- material vu found in the prodoct tha hriw red dge.--Lugo-sde photosynthetic 13 Altbw& the experirau tho production of @rtoeo-U- C for clinical appliu- report~d.~' sorrrd purpcwe required procadurom (rd tiom hu cafra incubation of thalli from a- of dovaloping fWttLh&B&0 lutd ..OUllt Of ut- u a-iM COmiferO Ud GiUrtiM h8rVOya. The c), minor difiuticmr of our experimental procdama rod kelp am obtained fruh lo I- to 6-kg qruntitias (particularly vitb rupoct to preincdatia aali- fmPacific Blo-lhrine Supply Coqmy, Lon hgelu, tiom) md procuramoat of uniform quality ommr .bout 26 hr or leom after they are harvomted from shouAd enh.nu production to the 8attctp.c.d l8-l the occm. Photonynthetic incubation ir carried out of 25 shook of ga.La~toeeY-~~Cat about 90 atom in the .pp.ratur mhaua in Fig. 7 over a 24-hr period, percent carbon-13. after which the non-reducing galactomide, a-pdacto- pyr.ao.pl-Z-glycem1, is extracted from tbe thalli. Crem p&nts.--Al a swrce of ulfoniy ldoled Exapt for scaling up of 4OOO- to 6000-fold. extrac- carbon-13 8lucose. sucrose, and fructomo, thke tioru brm rdc em88ntially according to publirhed green plant aymten are beiq evduaced: Nicotim8 31 procadurea . tobacm L., variety Coker 319; Swim# chard, V8rhty The crude extract im then chro~tographedon a Burpae'r Fordhook Giant; .ad Cmne indica, variety Lhmx % X8 (200- to m-sh, Ba*) colum, and Burpee's Dvarf Primrone Yellou. The wmt duirabl. the resulting pure galactomid. is hydrolyzed with mymter will be that which hu the hifieat extract- dilute trifluoroacetic acid. Galactose ir obtained Ale yield of desired carbohydrate at tho lammt by crystallizatim, and the glycerol is recovarod unpourr comt. To obtain the demired quamtitio of fraa the mther liquor. Another purification nth- product.. similar procedures dmrcribd prodoculy od fn*olves acetylation of the crude gdactomide, for carbon-13 labeling have bun suled up by 88- foll-d by imolation md deacetylation of the eral hundred fold, and nore efficient .ad desir4ble precipitated hemacetate. Folloving removal of the methods of isolation and purification have bwn acetyl groups with bame, acid hydrolyrim then developed, am illumtrated in tha following mchw.

yield. @actom and glycerol. Thia latter vthod OTIaQ LuIIl .ppUm quit. O&iCabh 011 ad8Cd8 OUd for 1. 8rolmbIlH (wt. I4 r) 1. '3%: ;l$m;lNtia

UII mmuAl. WtnetiM I(M .u.ul)

CTmmLrrmer Ips ERm 1. Rvayntlu 1. melo, -...*tam a. ?ilrrtiom ~c*~Ic.) 2. m-za 40: I 3. Blcnm --I: mm-w ITAWS-IOZM8 e SWImMIa 1. ma I. c.1- Ctwoutogner -'ntatL.. (m'I (Umm-W ma**) mRa4'% 1. Crytallraatroa I. mrrroIn&m (89 GUnS-U-'aC 1. Dowar-I: --u IIICIQR-u-"c 1. Crmt~lLlu~LII sucKm~u-~~c Clrrcplr,.."C Tobacco plants arm groun in the laboratory uring FIG. 7. Photoeynthetlc incubation apparatu. The soillear culture methodm and artificial lighting 3 Lucite chuberr ham a total volur of (Pig. 8). following publimhed procedurem. 32.33 fra -100 1. ad are munted on a c-n omcil- lating rchdrn. reed supplied by Profemmor C. D. bper, North

117 glucoaa, which vu ncryat&ird follaving e-=* ti- of the 80lutioo. Cl~~ome,susrO.e, and fnrc- tom WeN rquetd chrcutoBt.phiully in 25- qwntitiea ruin@a 5 x 100-a colm of Daru My XII (200- eo ~001.~.~a*) rrsin." me swam Vera characterfxmd by en+lrutic way, by optid rotation, by chrautogr.phy of the trirthyl- dlyl darivativem. by ion exchrrry chroutopaphy of the borate caqluu, .od by (which el80 coafid lhl rmifordty).' 'ha depc of earl&- mt of ~arbao-13 ia th. OU~U~,VU &terrimd by nss epectrortric wumntof the uC& 180- tope ratio in the CAK~OI~dioxide produced by vat co.butim. FIG. 8. Tobacco plmu .L &out 3 nth8 ~auina Eight incubation8 of tobacco leAV80 froa the in cha laboratory under soillus culture conditions. Nutrient ddition and libting firrt planting produced the carbohydrates in yields eyc- ara urtovticrlly coatroued. The sham in Table 2. me natura of 13~-cont.irriog 8xd.d leave8 .i. rued for photosynthetic pmducti- of urb01t-13 labeled SUI.?^. byproducts r.uinlng fn the leaves after extractloo hunot been deterdnd nor hu the 8m-t of ex- tractable products in the leaf vines (these were rewved from the leaves prior to extraction). With edstfng facilities, a steady-state production rate of approximately 50 8 of CWStdl.iM glucose per week cen be rintlfnod at .D estimated paaonml cost of two mm-houra p8r g. P18atinw of tob8ccO are being rrde at intervals to ensure that a sour0 of labeled glucose is readily avail8ble; the sacold crop is now mature and being uacd, lad rubreqrwnt phntinv are developtnp. Further detalls are 12 dtscribd clawhere. Experiments with Sviss chard and Cmaa haw 0 K) 20 30 40 Tkm (hour11 FIG. 9. Carbon dioxide uptake by excised tobacco leavu . Yieldb the c0uf.e of the - Carolina State University. During -Product 1 -Percent expariaents with the first planting, about 8 kg of tobacco leavrs we- taken froa 15 mature plants (pwth period greater than 75 dayr). In each exprirnt. &out 1 kg of leave. was excised and Fructore 54 l2 preincubated for 26 hr in the dark. Photoeynthetic 13 sucrose 21 5 incubation with carbon- C dioxide WAA carried out over approximately 40 hr (Fig. 9) in Lucite shaker Fm8 experlnnts which y illzed 8.2 kg of U- boxes (see' Fig. 7). .dsd leaves d 660 g of at 83 ata percent Leaves vera remved from the chambers. frozen 14 aver.^). in liquid nitroen to quench metabolism. and stored bBu.d oa %02; corrected €or s. 2 percent st ZO'C. The free sugars and starch were extracted endogenous uteri&.

eraentially u described by Put- &.13 Starch kisture content 14 percent; hydrolysis lad vu hydrolyzed with dilute trifluoroacetic acid to cryrtallization gam 130 g D-glucoea. .. .

clinicd tdb, u vdl u to hitiat. phfor uiditiaul toat., 4 ruouch phpieian 18 mot a parUaat hrOf tho 8rrff, Md rati8fUtOW dinid facility dou not &st at ML. Th& Dr. Shrnwo'r offort8, coqorati*. vantwo8 brr ba- vi& Dr. Phillip Eaton .ad Dr. Jon Sh- of th. w-aig of llrr mrdco school of mdidm bf Albyurqua, which should ontril ext8u8i* ht.r rtioa bo- dinid rtaff dorm at tbi. & h#titutiOO bd -0 L*sL 8uff. C1-e CCordiMth L bob8 atdad with D;. Sht.0~0 foUJving hi. r8t~to Brookh8m. 0th.t dhid tdd8 af. bob8 aa0Ury.d collaboration with othor iana8tiWtOr8 [viz., 3r. Po:.? Kloin of Arv National L.boratory in coaj~ctionvich Dr. Alan wftrrrr of %yo clinic (glyc~no-l~~,for &*tic- uiiti~,etc.) d vitb ~r.k'il1i.r nyb of tho lbivaaity of klifomia, SM Diogo (fiy&0-l3C, for 8td- of g1~CidOurd Addue8 iJ pd- uric bbnoditiom); Dr. Clive Sol- and Dr. E. IS. Cottoa of tho Univetriq of Coior.Oo (ugidm- Clinical Applicatioau "C/%, for studio8 in cyrtic fibtwi8); and Tho wo of non-radioactivo at~bloisotopu (In Dr, Dhd Itipnh of Wuhiagzon lhlnnity, St. 13 p.rtiCUhr carban-13) obriator vuiou8 1idtaCiOM Loui8 (8l.oinr C, for dno acid/carbohydraro mcounterod in application of r4di08CtiW Isotopu .Lt.bOUII, particularly in podiatrio) 1. in huPuu and vwld gnatly enlargo the stop. of In 1- casu, colldoratiolu involm applf- i8otopic tram tosts for pmgnmt vOIn and chil- ti- which uo liritod in clinical yolum but dren, for oloctin dla~omticprocodurm8 in hry- particularly rpecific for o~ploywntof rt~blo180- scut populatioao, and for many bulc physiological topor. Ue bdiovo that, In addition to dir8ct

and pharucol~$ical atudiu in hdthy aubjoctr. A di~~8ticAppl~CbtiOM, 4 k0d U8.p CliniUl =urroat objective of the I8otop. Appliuti4ar Soc- and pharucologful rosurch vould furrhor justify ':ion is to foatrr utd mm.p in efforts to dom- .nd rtimhto production of at&& i8otopw mad strate ouo 01 mro clinical applications with high developwnt of in8trmontatim. Tht lattor cm probabilitv of wldo8pre.d adoption urd utilization involve exploration of fcuibllity of neu or of relatinly largo amomts of c~rbon-13. Thuo unique rthodr of muuremont (8.0.. proton rdia- initial test. have bun aolected on tho bulr of ti- capture reactions or nuc1.u magnotic ro8on- bring both 8Ci*ntifiCdy mad clinically mmingful mu being actively puraued by USL Croup4 A-1 ano and technically fculble u :he preaeat rtam of QIC-4, roapoctivoly) dptod for particular biolog- devaloprnt of tho program. Slaplicity md reli- ical pmductr in blood, urino, 4nd 2erh-r tlrrwr. ability of tuting from tho standpoints of tho natura Refinownt and adaptation of eri8ting techniquu of the orgadc caqound used, clinical pmc8dure, and (..I.,pua rpoctrautry and infrarmd mu dysis) t8chnic.l wurmnt of the isotope in tho biolog- are .Ire imortant functions in support of clinical 36 ical product8 am othor important conrideratioru. a tudiu . Collrborativo nlatiormhipr are king utilizod Sar wtabolic dirouu aro cDlo0 yot diffi- to carry out the clinical procmdurer. Although cult to rocopize eithor clinically or by laboratory Dr. Wdton Shroew of the Brookhaven National Lab- to8t8. Even if recopiztd, the mtabolic catogoriu oratory vu at tAsL PI A Viriting Staff %&or from within and causer for disoue ontitior nood bmttcr Jamq to Aum8t to 08tAbli.h und conduct the fitrt eluddbtion. One of thore dirru.8 ir diabotor. RC. 10.

LZO k CY.of tbdr canna (ad fromay fatal) infant dheu. tube, sp.ctra uc6at.d at latorrrl by Bioch.d.tq d wmro the fut Fourier tramform rthod. The nrdU are mol. call rtudlw.-h intorutl~rppliutiarr of 13C onrlcbrot in biololid S78t. Vas p&d.d -ti- tha of yoat by of cmr Ipwtm (go d for pulse aemmalatioar from 56 :: 70 310 utilis) at 70 .uII p.rcmt '3~. spct- of me .Id fra 83 to 99 do. l'he fasort swrizu tho vho& callm, at rrrukbly hi* p~.dpLloO, i8 rham ruulta of tho coqlete rxpariuat. 2.8 siwd io FLg. lk. teh of tho nukrod puk cm be uai~odto a cl.H of carbon .tau io the cmlu euro. Tor -le, p..lu 19, 20, rod 21 are nthip. cubam, p.& 9 i. is the oqmctod p~itioo This si@ may ba that of inrr8ullahr alplueeme fot tb0 JphS -00 at- Of pOlwptfd. ChriM, while eo& 1 reprreena the cardual rthyl carbonr

of loopdmla fatty acid.. Further rtudiw vmre point is undar fwthrr :nrest1~8riOn. rri. om tho watar-eolubb fraction from intact yert Carbon- 13C Prroride bincinr to vorio& t..ro- colla (Fig. llc), the ruidu -alq after ex- globin, cytothrou. end rtrllopomhyrfu.4 hamsti- excrutioo with water (Pis. llbb), and nuber of studio# 01) the clt' spectra of finally with chloroforrrwthanol (not oh-). Thue "3 bd ewrirote .LI allpificaut in that rhoy mstab1i.h tho pmctimllty of e..daini tho 13C-onriched fac- observed in the smapectrd rm;iron wnetaar tha tiom of eaplu biolwcal mixtures 8-8 In aw- binding h-globin ia in hcut red C8ih. hW- p.mioa of int8ct colla or crud. ortract.. 1ysat.r , or molutiolu cf pz8vIowly cxys:rlliad k PL axternion of this work, m1.b.led yeut we= allored to wtaboliro glucose-l-l'C with 10 .lmr

. %D*--n-n-m-wB .Dub- (.rIrry*Y nc. 11. Carboa-13 nuclur upratic resonance apec- tra of 2. utilis ulls: (a) hole cella strata; the 8ir;csac suspondod in h0; (b) cellular composenu tr8ted. Siy;ulr ir. reuidng after elmawcive extraction vith -12 .ad -26 ?pm are D20; md (c) water-aolublc coqonents abundance c.--rbor\-13 nloued by oomotic ahock. other than ;-1. 105b550 El h-io ia o.ch rabbit 8-d thrt that bld Coataisd 7.5, 5.5. rrd 4.5 poruut fotd hmo- &&bin, n.p.ctim1y; hmr, thir d#. nw 8m to mtfor rhe iutomitiw of tho tu0-blrd.d rpoctm slpk. of tho blood of rabbit. 1 ad 3 hrrr baa 8mt to 01. Stepba Shabet, thimr8itT Of -76.2 cairorni. mia sch001. sr-a, -t. tb -77.4 (50 8s) h_ia c0qaat-a will be ropa8r.I. yu-d. -77.6 (55 &II) .Oa nfpID.l to Loa AMfor further 8cdy. -77.2 (K) W sin- tho- i. abmmbmt &dam for b.torog.cp.itl -76.7 (5 K8) FO both rlph d bot. duinr of rabbit heL -78.6 (5 B.) dam to palprphic .Il.lu (rub? tb- oaorlldic -71.8 (s H8) mu). rtdy of tho 13W blodiq by 8.gUat.d -7b.b (5 81) homobina vlll orphln, hopofutly, tho 0rp.P -77.5 imatal absomtioaa. -79.5 Other d rcudiw.-In cOll.boratio0 uith Dr. bithu hrrkrt, c&imof tbD.mt % ppm fra rtL..ali the aokrm ~roth. full uldtb. of the of -3- TodcolOV* P-0 Unt~~iqOf ).?me iatomity. y. haw begam a atdy of tho effocu of hw*C bCryatrlllud from poolad hum blood Sqh8 and enricbrot a0 oabryotul wwbolin fn rim. * rodiaaol*.d in D2Q. first roquinrmc of thue iovutigatioaa VU pmaoC- of .cid or bmd.lrUUr8tion Ot la 8 -8. Tho ti- of pndsoly tlrd prmgnmdw in aon 60 dce- 13 rpoctnm of the '3m bou~dto ima of cyto~hrorc In the first oxperlnut, glwoa0-U- C idday*.l or tO rfnthetlc it00 porphyrlaa im tho IQ.88 that &*.a iatr.*.aouoly ad rubrqruntly by stouch t*e Of iron Carbonyl or tho coqlox blolodcrl doculo8. to rice during tho 8th md 9th d8ys of pn~q; k ah- in ?&lo 3, lima vidtha for slngl0-ch.io in 8 socoad exp8r-t. 3ucoaa vu dvmn durlly the protoinn liko myoghbia d wtocbror c am uch loth ad 11th ~tatioarlday.. La 8 third 0rp.r- numnr tbfor intut h..Pllobin. Tba h.cdobin furit. glucoa~-U-~~Cvn glfnn during day 11 of lho vidth for rodis8ohd cryrtdllne haglobin -tation with a- of tho rrirrrl. being arcriftcod ir tho 8- n for hlrowin in hole blood or at 8 hr aftor tho fint iretop. .8daistratim packad rrd call.. vhile denuuruioa in 8 E una uul tho rouiador at 24 hr. In tho lut isotope CW8Y 8 8ub@tatlalnuroving of tho homfiobin oxporimnt, Jco ia &I. 16 of gutation uoro umd, lb8. ad C8rba-13 VU addnistond at iritemab for A mCmt p9.t3' r8.pOrC.d 8 8peCC- with tW 8.5 hr for so- of tho mklr rad for 26 hr for eqd p.lu for ''OD bdto rabbit hmglobin. tho rtufndor. Additional nmi8otoplc 0xperimuU Thoro authorm amcrib. tho tvo pod to difforoat vera porforrd to masum the offoctr on dry0 bindiry of tho 13C0 by tho h-ghbin ~lphaand naorptlon and blood glucaro lo~laof variow bot8 china. Our d8ta 8u~Oatthat tho OXpl8MtfOII routor urd tir rquencrs of ghc080 adaidstratloo. @ma by tho author8 emmot bo correct, but w ha- Aftor euh i8OCOPk oxpori.~nt, th. dry# wrlfld th8t rabbit hroglobin darj indood sh- vom rrw*.d, voll;trod, thon hom~irodurd aop- 38 tvo peaka of bo4 %. Tvo p..lu of oqd intao- aratod into a varioty of cholical fractlolu. sity r8tiO worn obaorvod in blood from tho first of l4a.torn.l blood .ad liur were coUoctad. u voll U thrro 6-mmth-old ule Dutch bhdrabbits. Tho othor rternaA tim._us in SOU experirnts. Fotd second rabbit abed UI intolulty ratio of tho po.Lu livera ven collected roparataly from rha day 16 of 4:s. whilo the third rabbic'a blood gmcvo edrvo. Labeled red blood calls voro moat to paah of intensity 1:s. The mmt likoly expla- Dr. Shohot'r laboratory in Sa8 FrMcIrco for 0- tion ir a poQulation of No h.lo@obiru in tho ination of rod toll =.branor by cmr. Carboa-13 dulr rabbit. kbuurrrot of tho pw~taof Lar.l enrichad. highly purified DNA vu isolatod Lra f 05b55 f 122 I 2.

81.0 hope to donlop motbod. for locallring the 1. mt.bolie looion caued by dryocordc writs by notint chragu in the patterm oL carbon-13 lncor- poratloa (by c-) uheu the e.bvotorin and a carboa- 13 rubetrue arm given .iultumously. Hobnover, y. oapect that the carbm-13 1d.ling ory pro- a 8. parerful tool for isolating rcrololeculoa fomed --de novo in a variety of 8ynchronized bidoglcol 9. systu, of which the nnelian embyo is the umt coqlu 8-18.

Bfonnthetlc pathvay8.--In a cdhborativa project with Dr. PtLltou Alli.00, U. S. Departrot 10. of Apicultum, a,Iwa, it YY found by applicrtica Of cp techoiqurs th8t c-2 fram acetate vu incorporated by a wnort.pirr entirely into 11. i-3 of isoleucine rather than C-2 u expected on rhe buim of kmwn rtabolfc pethwaym. Further 12. invm8tig~tloluue bdng carried out wlng both ~cerattZ-'~Cad .~ctate-l,Z-~~C. It is anticipated tbac cotmidor8ble inforution about path- my8 of Pino 8cid biaynthui8 CUI be obtained throu@ applic8tion of C8rbn-13 labeling urd car 13. in a =re straightforvard -net than is possible vith a888 rpectrcucopy or with radioactive 1rbellnE.

11.

1. R. E. Schreiber, ed., ICOWS at USL, La Ahmos Scientific Laboratory report U4739-lS 1s. (Septellber 1971).

123 --

29.

30. 11.

32.

33.

34. J. K. W. Jwmd E. A. We& The sapurtbn of syur oa i-m- ruins. Crud. J. c&.. 38, 2290 (1960). 3s. E. V. Putma d V. 2. Busid, Isoktioo d purifiutloa of rdiortiw sugars by of papor chrortoW8phy. J. Biol. Chu. 196. 749 (19S2). 36.

37.

mriour hem globiru. J. Am. Chom. Soc. s, 5093 (1972). 38. (P. n. L~uI~..s. G. Carponwr, L. n. Boltod, J. R. Prim, aad R. H. Wood) In ray radiation lethality rtdy inmldng lareor laboratov speciu such u IooLMy. ad d-, lafectioa plap ujor role in AUbut the *rrP higheot dome rate expmureo. Ovrt th Utparr DIStuE S- we hmwde m attempt to defin the brterlolop (P. X. L.lrm, H. 1. Btoolu, ad L. X. Ilollaud) ic.1 flora of rherw dwr(W.C. ul.tW before exparun to lethal irradiatioo adto doter- For rmnral yean a sur*.illaco prgrhu dne vh~t,if my, bacterial rpeciea w Le keu In affect to aid in control of Pseudaonu recovered fra the blood during the te- at80.n mruginma cd related poeudoron~dein the IQue of the experiment (see -1i.a PdiobFoLOq Sac- colog. A wetr utiliring chlorinated water tion of thir report). Tbrolt, fecal, ad blood (U ppr) plu dIspo8able bottle capo and twice cultures are obtained from each of l0 d8p jwt waybottle &m~rhu ken in use since 1969. prior to stut of rdiatim uposure. Botb aerobic C.p. of dce arm trtd at time of chmging md aawroblc culture techniqwr are mqley8d to by culturing water from the bottle. on a Sp8Ci.l Identify 8igOifiC-t spociee of bacteri8. Table 1 aeloctim rdlu (Proudcrel, BBL). In addition, shws the oprcleo identified md frequ8n:y of isol~- .tool culturea am perforwd periodically on rep- cion. re8mtative uyr. Stock md breeding rice 8.1. The presence, in uny of the monkeye, of the tooted at lust quarterly, md dce wed In radie- "trench mouth" organior without clinical swof tiar e~lporiratsarc tested before d durina the the direue io of interemt, u one of the riw of ezperimmts. often weekly. appr04ching tednal Illnus in monkyo i~ infec- Incidence of podti*. cage. was quite lar dur- tion of and h.rorrhqa frm the gur. TWO of the iq the fint 6 mth. of 1972 (5 out Of 749 Cay*)* m~i.ulshad opecieo in the threat, demon- However. in June 1972, UI outbr8.k occurred ia one stratin8 the ability of the monkey to live with the bredin6 roam rad vu traced to A dirty air condl- mutant presatcc of Shinella. Positive blood cul- tioai4 filter. Sewre culling and rewhr testlnB ture for Shiylla in one aniul .ad Staphylococcur h8v8 roduced the number of positlot calm to quite cDideraidia in three animol. vu attributed LO con- 1- lewlm, md the outbreak hu not Bprerd to tuination at the ti- the blood rqle WAS drawn.

125 6 211 HI#IMUgmmxA 9 w. c. A. J. E. L~~OQ,.od J. s. lo (L. nu, Uil.oS) 3 12 hV8l-t Of prOdWtiOQ-linr tecbPiqw8 Of 4 rdc~pbrei8j.ctta utillsing tb. r.tu pi& 1. TADIX mmpI 1u#o CULrmm AT mu11 rmquirea thu vo mtbtir hntuw OQ a *obr S t8phylocaccw epidarmid%a 1 buie. Ike of iatvfitmd p.mLobUbia &d Brteroidea 1 to death lam up to 10 to 12 peruat dud- the Clostridium perfrinpna 1 irrdiate pat-operatfve period in acu irut.acaa ad .II .dditloarJ fw &ter doarhm from inj- to Tho cormtult firrdily of ClomtrttStm pmrfrfam io .bdodnal or-. To -rear thi. 1-s. V. UW the #tool c\Jturea interutiq aot eurpda- i. but u8i kat.drw hyddrochlorib (Kmtuet, Brietol Lab- I-, a8 Clo8tridi. ue conriderad a wrrrl iabd- oatories), a nw uosthui. markmud for we in It-t the gutroiatutinal trsct of mat 2 of t-ulr. CUI. that hu thdbtinct -=tap of baiq m*rr, thi8 obBelratbm Wrthy Of UOta, U deai~dfor Intrurcuhr we. Ketmine hydro- Cloatridiu perfriagena phyr a role in thaw chloride ha @ wide urgia of 8af.V d act8 dutructioa €ound la the ladral gastric bloat ayw rapidly with an effecti*. duration of 10 to l.5 rin- 3 dror of ucaqua8. ut-. Ue have ow wdthis d- la over loa0 T&la 2 .bora the apeeiu of bacteria nearrrnd hmtera, incluUni sou that wn given a full fra blood altum8 of the am8 airrJI jrrt prior unathotic don mUy fm l2 -a&, mad h8- ob- to death. Tha largely ner).cive reaultr iadlcata 8ervad no deleteriour afteraffecta. Uitb the W that the aaLey dou not Buffer a ter- oither from of this dmg, WI amthetic looearn ham droppd mind bactereda under thue cmditionr or the tiring from -10 porernt to leas than 1 percent. A nota of 8qliag wuquetiauble. Result. .nd rthod8 on Chis application ia in propararim for publiu- of the aatiro uperiuat are to he reported else- tion. &en.

PMiloLOcI To our knovlodge, u. h8n rewined in coqu- (J. 0. Prim, S. C. Carwater, R. 11. Wood, .rd 1. n. mce with all federal 1- gonn~iaqthe care d Holland) troatint of laboratory The LASL'a Bio- Pathology atudiw bud iupport to rO.t of the rclfcd Rerureh Croup hu baen accredited by the program utiUziag animal mudeb. During the first Amriun Amsociation for Laboracoty Aaimal Care 11 moatha of c8lend.r 1972, nearly 600 coglare (MUC) rince 1966. Reprcuaucivw from Muc pathological euminatiow WON perforrd OII a de Sit. Vhit for the purpose of updatinl QUI: variety of mirl.. mt of the work lod ir use- accreditation during Novubor 1972. ctrted vftb the hot particle program which &-&

126

--.-. a*...... i2 7 D. A. Sdth, A. U. W.rtItu8, .ad D. I. w11- Urr, hitiat- of Io Vitro Du SYD~U~.with OlA@~ri~~.&: Abstract8 Of ?.Prrr of thr 164th waul m.tiag of the ~rticutcbr- icd SocietJ, Ikr Tort City, #.v York (huput 27- Septder 1, 1972)- Abmtrut No. 71. C. F. Stmfite, A. M. Hutinu, md D. A. HOLECDW IuD1~1oLocIStCTIoll Sdth, In Vitro Studla8 OD the Smth88i8 Of -0- ~ubiicatia~ nucleic Acid UBI- Idria8 1rr.dibt.d RXA P01-r- Ye. Ab8CrbCta Of the 16th bllurf 9. Bmts C. II. J. X. liUdina .nd &: weti- Of the lioph~1C.lSOdOty, Torooto, C.lud. 1. J. Nolmd, Turn0-r of L.brl.& Uathyl adAcetyl OUfbfiOs Croup. in Ubtoae Rattiom of Cultured W.n (febwry 24-27, 1972) 12. 1558 (1972), Abatract Cella. Federation Proe. J10,8% ~ba(19721, NO. SUWEl6. C. Scrnhte .Id A. Sdtb, Vitro hb8tmCt NO. 3563. F. D. In Studlv on the Syntheaia of Rlbowcleic Acid 0.i- P. Ilpoet, C. it. Shepherd, J. n. Budia, ad X-Irradiated RNA Polymerue. 3: Matractm I). J. Nohd, the Diatrlbutioll rad Turnover of of the Labelad bthyl Croup8 la Hiatom Fr8CtiOM of Cul- First Rocky Hountdn B.giOd Meeting of the Aur- IC= Chd-1 COlOr8dO State UIiiV8K8ity. tured w.yli.n Cells. Arch. Biochem. Biophys. 168. Society, 558.567 (1972). Fort Colli88, Colorado (Juac 30-July 1, 19721, U. Hollitein md P. L. Butlar, Inhibition of p. 17. Abstract No. 18. Ribowcleic Acid Synthui. by ?@In. Biochristry Hmwcriptr Submitted

L11, 1345-1350 (1972). G. H. Dbub, H. E. kkermmb, .nd r. N. 1. Rulif f Spthrir of niud Polyauch* L. , dydrw Hydrofluoric Acid u a Cyclidfig bent In ride8 Ccatdniq -0- and De~riboouclmtlduby the Pt8p8ratiOn Of SeVerbl Sub8titUt.d OXUOlU TerdnrS Dwxynuckotidyl Tramsfera88 Feder8tion . from ~-ArOyl-+Aminokrtm88. J. Otg. &a. (in hoc. 31(2), 425 Ab8 (19721, rsb.tr8Ct NO. Ll66). prema) . C. R. Shepherd, J. W. Hardin, 1. J. Nold, D. H. CK8y, R. L. Ratllff, adD. La YIUIPr, ad P. B]nrat, Turnovmr of kkthyl, Acetyl, and Phoe- The Circular Dichroir of Poly d(AIl):d(AZS) ad phoryl Croup8 Histone lactiom of Cultured in Poly r(M):r(W). A Tear of the First-Neighbor Hamalian Cells. &; Abstract8 of the First locky Hlpathe8iS. Biopol~rr(mubritted. 1YoMtain Re#on8l Meetln# of the Amri~811Chdcal R. L. UCliff, D. L. WilU.U, F. N. U-W, Sociaty, State Univcnlty, Fort cOlliMv Colorado ad D. A. Smith. Prepbr~loo.ad Propertie8 of the .. Colorado (Juue 30-July 1, 19721, p. 13. Abmtr8Ct Repeating Sequence Polymer d(A-A-T)n*d(A-T-T)n. No. 5. J. Biol. Cha. (submitted). C. R. Sh.ph8rd, R. A. Ualtet., J. n. Hardin, C. R. Shepherd, B. J. Nolaad, and J. I¶. Hardin, .ad S. J. Nolud, The Effects of X-frr8diatioa on Structural hdifiCbti0M of Xiston- in Cu1tut.d tom Acetylation and Mathylation Cultured His in Il.l.lian Cells. &: Proceediqs of the Inter- ?Irylirn Cells. Arch. Biochem. Biophys. E, 175- national %(.acing on ‘The Bioehastry of &ne la2 (lg’f’O 5 b 5 5 1 Expression in Higher Orgbnism,“ Sydney, Australfa

12 8 (Hay U-19, 1972). AuatraUm and Iw Zodand Y. 0. -or d 1(. A. Tobw, Effects of I.* BdCOqq .d 0. Wd.1 MlI8hiog Cqm,DOP hcina Daficiency 00 .Yuelmic Acid d ?rordn bt- draeht, Holland (in prou). abolin in Culturad Chinoma htorCollar Con- C. R. S&p&rd, 1. J. laird, .od J. M. Bud& thnd RNA and Protdm SymtbwL in tha Absema Of Turnmr of -tam Autyl Croups in Cultwad Y- ollli S~tbui.. Bio&datw g,269-277 (1972). lima Cella. E-. Coll p... (io proas). n. D. wr.pd E. v. cupbell, nucant C. R. Sh-rd, J. H. Budln, d 1. J. NU, 1L.unglr-Iika p111, thtabolin in Chiaua 9mur Dophcuphorylatiop of Ri.toay hactiom of Cultured 0.- ML Cult4at Elevated Tqeratorr. &l Irlfro -I&. Arch. Biochr. Biophys. (in Abstrwts of tb. 28th AanU8.l Southuest L(l4 pm.8). mati- of the Andcan Chdc8l Sociov, Baton C. P. Sbpbard, Wide- for tha Biolotfcd m,~wi.f.lu (Da-ar 6-8, 1972). p. 33, Ab- Coupliw of Bia.)ntbui. md Xatoxaal Acoglatiao stract No. 65. of Histom traction ?2d in Cultmod wU.n L. R. Gurley, R. A. Udterm, and 1. A. TOb9, Cella. Blochi.. Biophyr. Acta (in prum). The ~t&bolismof Histono Practioru. IV. Svth* J. E. Siqsm, C. 8. Daub, d F. N. Hayu, si8 of Hiotaon durlng the C:-Phue of the Th Smtb~i. of SOW 3' ,2"-Di0~th~l.n0 Brfdpd lian Life Cycle. Arch. Biocha. Biopbys. s,633- ~-Q~.torphauylaand Related Capoundo. J. Org. 6Ll (1972). ch.r. (8Ubatt.d). t. R. Gurley and R. A. Ualtom, Tho Irtaboli.m J. E. Simp.00, C. W. Dwb, and F. N. Hayu, of HistoM fractiotu. V. The -latiwhip bo-. The Syathosim of S- 2,2'-DioubrIdpod Bfphonylr Histone aud DNA Sptheais aftor X-Irrdiuim. d l,l'-BinaphthyL. J. Org. Cha. (subdtted). Arch. Biochr. BiOphy8. 153, 304-311 (1972). C. P. Strnlsto, D. A. Sdth, rod P. N. Hayom, L. K. Curley, R. A. Ualtmrs, and It. A. TObW, X-by IMCtiVation Of tho E~cherfchia&DNA- Tho Aelacionrhip of Histona Phosphoryhth to tho Dependant UBA Polymeram0 in Aqwow Solution. X. Cell Cycle. a: Abstrects of PmDors Prase& at Studiu OQ Blading ud Substrate Concentration the Tvelfth Annual Ueetlng of the A.rric.11 S0Ci.W Dapandince in the Inactivation of Enzymatic Activ- for Cell Biology, St. Louis, Uissouri (Nov..b.r 8- ity. Biochdtry (in pram). 11. 1972) r,998 (1972). Abstract NO. 198. C. P. Stmlste. 0. A. Sdth, urd F. N. Hqu, L. R. Gurloy, R. A. Ualters, md )L. A. T0b.1, X-by Inactivation of the Eschorichia 502 DNA- Tho Indepmdenco of Histone Phosphoryhtim from Dopaod.nt Ipu Polyarase in Aqurour SOlUtiOM. 11. DNA Svnthuir. &: Ab8;rrct~ of 510 28th AulUUl Studiu on Initfation and Fidelity of Trmscrip- Soufhwe8t Regiod Meeting of tho hricur Chr- tlon. Bfochditry (in presr). tcrl Society, Baton Rouge, Louioina (Dacodtar 6-8, 1972). 3. 34, Abstract No. 67. CELLULAR BADIOBIOLOGY SECTION A. E. Hampel, A. C. Saponaca, R. A. Yaltef., and H. D. Enaer, Lou Holecular-Uoight RNAa of Post- Pub fiC&tiOM Ribosomal Particles: Evidcnco that tho Partic- 8, J. Bunhart ad S. H. COX, Replication urd ulate LS RNAm Corprioe a Difforential Population Ski- Of hCtedOph40 m: b&CM8-AJ8OCi&tLd of Transfer RNA. Biochio. Blophys. Acta 269, 428- Functions in Hw hilw influenzu. &: Ab- 440 (1972). stracts of the 72d Aparvl hetiag of the American C. E. Hildobrrad and R. A. Tobey, DYA-rane Socimty for !4lcrobiology, Phila&lphfa, Penn- Associatfonr in Cultured Chime. Hwtor Colls. &: (Aprll 23-28, 1972), p. 202, Abstract sylvania Abstracts of the 16th ktnual Meting of tho Blo- Jo. V104. phy8ic.l Society, Toron:o, Ontario, Canada (Fib- L. L. Doaven .nd D. F. Petersen, Are Chroa- ww 14-21, 3972) g,24% (1972), Abstrrct No. Su- roully Aneuploid Colls Conetically Aneuploid? M-E9. In: Abstracts of Papers Presented at the Twelfth - ?. M. YraCYr, Complex Carbohydratos of m- .Annual keting of tho Anricaa Society for Cell 1i.n Cells in Culcure. &: Crovth, Sutrition ad St. LOUIB, (Nmder Biology, Ylsswri 8-11, 1972) bktabolism in Cells in Culture, Vol. -1 (C. H. a, 57. (1972). Abstract NO. 113. mthbw & v. J. CetOfdO, &.)r -C P-08, NW rorir (197~). pp. 37i-il~. P. n. Kr-r. L. L. ~.a..o, M. A. crisorra, md !f. A* PIDill.. C-tmq -pit. VUi.bi1- ity in Cbm0.0~ Uubu. g: Adt..ur in Coll oad Zbhcular Biolow, Vol. 2 (E. J. D3rW. ed.), Acddc P-0. 19.r YO* (l972), pp. 17-108. P. l4. Kraenr d 3. A. Tobay, cell-cych lMpmd.at Duqurpvtiaa of Uparm Sulfato fror tho Coll Surf-. J. call Mol. 2. 7-717 (1972). P. K. Kr-r, 11. A. Crlamm, rOa X. A. VP Dill., Flar lllcrofluarartrie (RQ) Studiu of ?kt Loctia lioding to Cultured Usmallan Celt. &: Abmtracta of Paperm Pronated at the Twelfth krnrvl hetins of tho hrlcan Sociog for Coll Biolo(J, St. Louim. Mtaoourl (loveder 8-11, 1972) 55. 141a (19721, Abmtrrt No. 281. R. A. Tobey .rrd H. A. Crluwrr. PKep.?.Ciaa of Lup-tifir of Synchroaized -1.n Cella in L8to C1 In the Pre-CUA hplicativa Phue of the Cell Cyclo. E-. Coll hm. 75, 46044 (l972). R. A. Toby. Arremt of Chinro ii~ltorCellm in G2 follwlng Treatmaat vith rhe Drug Anci-Tumor mulrcriptm Subdtced Bleomycin. J. Cell. Physiol. 79, ZR-265 (1972). 1. J. Bdut, S. H. Cam, .rd R. T. Ok-. R. A. Taboy, H. A. CrImo~a,mtd P. N. Krurr, CharactOrIzbtim of Defoctin LylOg.ny A tlethod for Coqaring Effects of Different Synchro- inf 1runw. VirOlolJ (submit td) nizing Protocolo on U--llian Cdl-Cycle Traverro: phihm B. J. Damhut .ad S. H. Cor, MA Repliatioll Tho Travarro Porturbation Index. J. Cell 8101. 56, of Induced Prophage in Ummophilulr influentu. J. 638-645 (1972). Virol. (rubritted). Re A. Tob.~, A S-1.. wid'T~chniq~O for D. P. Bi-u .ad 1. J. BaCILhart. Inhibition Dctcrrrimtioo of the Effects of Cb..othor.peutlc of Trmsforucim by Antibodies 4aimt Corpetmt Agento m wanCell-Cycle Tracnrso. Cancer Haemophllus lnflurnrao. J. Cen. l4lcrohiol. (in R... 32, 309-316 (1972). proms). R. A. Tobey, Effects of Cytosine Ar.blnomido. L. L. Dewan and D. Patermon, Tho Chramo- Daunamycln, nithruycin. Aucytidinm, Adtimycia, P. OM of CHO, ro Anouploid Chinuo H-ter Cell and C..ptothecin 011 PhneUaa Cell-Cycle Trawrre. Line: C-Bad, C-Band, urd Autoradiographic -1- Cacer UB.32, 2720-2725 (1972). ymas. Chramou (in prosd. R. A. Tobey and ti. A. Crlssun. Use of Fh L. R. Curley. R. A. Ualters, ad R. A. Tobey, Xicrofhonutry in Dotdlod Aodysis of Effects Of Phosphorylation Late Intrrphue and Chodc~lAgent. on Coll-Cycle Progression. Cancer Histone in !4itosi8. Biochr. Blophys. Res. Cc.(in prerm). Rae. 32, 2126-2732 (1972). L. R. CorlOy, R. A. Waleern* rad R. A. 1ob.l~ R. A. Tobey, H. A. Crissun, and P. N. Kraerr, The 1SItaboliam of Histon Fractions. VI. Differ- h Wthod for Caparinp Effect8 of Diffsrmnt Syachnr encu in che Phoophorylarion of HiStOM Ractioru niring Protocolm on mlirn Cell-Cyc& Tr8Wr.t. durias the Cell Cycle. Arch. Biochea. aiophyr. -In: Abmtracts of Papor. Presenud at thr Twelfth (in press). .haul kmting of the Amtican Society for Call L. R. Curley, X. D. Enper, and R. A. Ydtor8, Biology, St. ~ouio,ms.ouri (N-.ber 6-11, 1972) The %.cure of Histone fl Isolated from Polysorm. 2,99. (1972)s Abmtr8ct NO. 198. Biochomisrry (in proms). i 105b55q 130 C. 2. Ilild&d .ad It. A* TW,DlA4htol Coqluu in Cultured Chi.we Outer Ca. I. NU- of tha #Ir-%mbr8ae Auodatioo. J. Coll Biol. (aubdttod). C. E. Illldabrmid .nd 1. A. TW,DNA-- Caq1.n. in Culturd Chiare -tor Celt. 11. Inrolmat of DU-rrrm Caqha in Tqal mul Spa- Ot..PC&tia of #A. J. Call Diol. (mubdttod). P. 3. Kr-r, P. A. Tobay, md n. A. Po Dill., ?lar MLcrofluorortrlc Studlu of Put Loctia Blindin8 to VmCalla. I. Go0ar.l Faat-#. J. Coll. Phyalol. (aubdttad). P. I!. Krurr and K. A. Criaaua, Flaw nicrc- flwrartric Studiaa of PhcLectin Biccding to manCall.. 11. Interfaranco bowa.n Con- CaavUa A ami What Gorm A&utinin Situ. J. Cell. Pwaiol. (oubdtted). A. C. Sap0o.r. oad n. D. war, The fao&Clocl from Pibaouctic Acid of Subatltutad Uridlnw Containing a-ruinabutyrate lkietiw Darivad fra Xathiopiae. J. Biol. =am. (aubmittad). a. A. fobmy, Production rd Churctorir~tiocl Of WllmCalla Raveralbly Arrartad in GI by Crarth in Iaolaucine-Daflciant Hadim. 3: Uathod. in Cell Phyriolo#y, Vol. (0. n. Praacott, od.), Acdrdc Proaa, %w Yo* (in pnrs).

Pubucatio~

Y. H. L.D&U, Biolo~calImpllcatIOM of tb Trauurrriu tlwntm for Pb. Haalth Phys. 2. 963-952 (1972). C. it. Richroad md C. L. Voals, oda., Annual I)rporC of tha Bio1oglc.l d lkdicd braarch Croup (11-4) of the LASL Ikdth Division, Jlnrurg through Decoder 1971. La Sdaatific Laboratory !iaurcripta Submitted report LA-4923-Px (1972). P. w. D.-, catintloa of chut Val1 Thihwr J. F. Spddbg .ad n. R. B~OOIU, ~ooemvityamd in tung counting for Plutonium. lhalth Pbp. (iu Mortality Diatributicnr of nice with and without prer.). X-ky Expomure to 45 C.aaratiOQS of %la Progen- S. U. J0rd.a. P. N. h.09 aod J. AhlqUlrt, itors. Proc. Soc. E-. Biol. ?I.& 139, 15-18 Early Ultrutructural Effacta of Iolliti~hdia- (1972). tion. J. Lab. Invaat. (fa praaa). J. F. Spblding adM. R. Brodu, Coq-tiW Litter d Rep?oduction Characterirtiu of I(orue BIOPHYS ICS SECTION Popul&tiolu vith x-Ray Lxpoaure, Includlng 4s Con- QrbtiOM Of ?t.l. Propnitora. ProC. SOC. ?hp. BiOl. %d. iQ1, 44547 (1972). A. Ururutiag and P. F. Hullmay, Do Uta& Detail8 of Y!Um C.lb fntlunaca Thir Ulbt Scatt.dw Pattemr? 2: Abotracu of tha Uth Axmd Wntlne of the Bbphynital Sodew, Toroato, ht8rio, Cd(hbwv 24-27, 1972) 2, 141. (19721, Abetrrt lo. S.Rc16. A. Bnmmti- rrd ?. 1. MU-, Differeatid

Li&t scut.-: A ptmo.ibh mtw of ~ip Cell Id.ttifiutim. JI Colla Iotmfm Sd. 3s 492-496 (1972).

A. BMtiq.d ?a ?. A L-t- SC8tt8fiq m0tm-r ?hOtgt.phiC fib- Bn. Sa. Itutr. 43, lS14-lSl9 (1972). A, 8-t- .Oa ?. 1. ILillm, Liet SutteP in8 from Coated Spheae: A Model for Biologiul Cd&. Awl. @ti- 2,6754)D (1972). A. BNIU~~D&LiCt Sutteri~fror Cdk. PhaD. 2&.k*0.)ULPt Of P~o~u-pd L. S. Cr.r d A. B~tiRg,1hmrUCe~ ktv,Ihivmr8iq of Uw Wco, ~lbuqrwrqu, d Light Scatted* mmuraemu 011 N.v nuda. Lw A- Scimeific Laboratory report Poi Cholera- Infected PK-15 Calls. E-. Coll Roe. (in prOSd. LADC-72-974 (Jme l97,2). D. M. Uolr and L. S. Crm. An Ilprowd Pb A. BruasUin~,CaPpUter h4lylie Of Dlffonntiol Ulcrofluorowter for widI(.uur.unts of Cell Light Scattering from Coated Sphet.8. La Aluo. Fluorescence. Cell &e. (subdttd). Sclantific Laboratory report LA-5032 (Nonder Exp. 0. Kolley, T. 8. Crlmem, .ad 1972). R. 1. Baker, A. L. S. Cram end J. C. Kmulmy, Flou NIcroZluo- C. S. A. Headermoa. Illtrutructure .ad Growth of ti- ~i.bwmeachymm In Vitro. &at. Ucord -try for ?luonsceat Antlbody-&tl~n Studiu. (w) (8ubdtt.d) -In: Ahtract. of the Uth Annual -ti- of tho A. L. ~i~ch,0. y.ll.ey, it. A. crimm..o, Biophymlcll Society, Toronto , OOtario, Cde R. ad L. Puton. -0 Induced hvermion Several ('lebnury 24-27, 1972) l2, llfa (19721, Abstract L. of hatutu of Polyou Trmoforwd BHK-U Al- NO. Sa-AU. Cells: teratioam in Crwth end lbrphology. J. Call Biol. 0. N. ilok, L. S. Cram. .Id R. D. Piabert. (submitted). Iartnnotal Factors Iofl\wncin8 heolution In ?. WI11-y .rd U. 1. Uemt, A purl-Par-t*t Flw Uicrofluorortry. & Abstracto of the l6tb P. Ulcrofluorowtor for Rapid Cell Analysis. Raw. Sd. Annd betin6 of the Biophymicd Society, Toratto, txutr. (subritted). Ontario, Cad. (February 24-27, 1972) s, 141a J. A. Ste1llJL.q. U. A. Van Dill., aid M. J. (1972). Abstract No. SaPH-AU. Fuutrylor. Cell Analysis .ad Sortla: A UnWti- ;. A. St.inL.q, H. J. mbyior, .ad 2, R. parrrtor Coll Sorter. Sciacr (subdttd). Coulter, A Xeu Ilultisrolror Cell AnJysI. md SorthB J. A. Steiakug, A. XoIro, d M. A. Vrr System. &: Abetractr of the 16th &mal Wetiag Dill., Ilultiparurter Cell Sorting: S&atificUiorr Of th8 Biophyeical SOCbty, fOmLLC* hUriO* of Huva Leulrocytu by Acridlno Orang. Fluoraocelsu. Cmd. (Febnruy 24-17. 1972) 12, 8. (i972) * Ab- Acta Cytol. (in press). stract No. Fpn-A4. J. A. Steiak.lp, U. J. Fulyhr. UM l4. A. Van ISOTOPE APPLICATIQlS SECTION Dillr, A High-Speed E1ectreOpt:cai Cell U78i. Md Sortiw Symtu. 2: 6th Lbt.;-erque Src::or, Inetnuatatiw Syqool~r,Applicatioru of Optid C. T. Cregg, Sor Applications of "C Nla to Electronics in Instmntation, Inatrunt Society Biological Problem. &: Proceediags of Sednar of k.rica, AlbqWrpWr N.u Uexico (April 2625. on tho Use of Stable Isotopr in Clinical Phuucol- 1972), Abb.tr8Ct NO. 3. SudOO ow, University of Chicago, Pov8.b.r l.0-11, 1971 132 (P. 0. Rlrio md L. J. lloth, ~dr.). U. S, AtodC En.- En.- Crlrria np&t CONF-7llUS (&Wt 19721, pp. 175-161. C. I, Crra, S- hpactr of the Energy hkt- .bolim of wimnCalla. &: Grwth, Nutrition rod Ilt.bolior in Colt in Cultuto, Vd. 1 (C. Roth- b1.t rrd V. ChtOf8bn ~dr.)rACcdrdC P-8, N.V York (1972), pp. 63-136. V. E. Koll.0 md C. r. GNU, Preparation of Carbao-13 Labelod Sumrr by Photaayntberir. &: J. ColorubWyai~Ad. Sa. vLI(2-31, 20 (19721, Hmaucrlmtr Subdttod Abotrmct lo. 62. '3'. KOA-, bUMISr J. Y. bUt.00~ D. c. Ott, Ot).dC SpthWh rd aiOOynthO8i8. R. J. L. -In: Procaedinp of Semllur on tho Uro of Stmblt T. U. Uh~l~,0. C. Ott, and C. T. Crru, LuW- I8otopw in CUnlc~lF+harmacolow, UaivenlLy of sulo Photospth~ticProduction of Carboa-l3 Chicago, Ndor 10-ll. 1971 (P. D. Klein and L. J. L.be1.d Sugarr: The Tobacco Loaf System. Biocha. Rath, .do.). U. S. Attadc Coriaaion report Bi0ph.J.. ROO. C-. (~ubdtt~d). Cat-71llU (kwt 19721, pp. 15-32. Y. A. Hatvlyoff urd B. F. Burnha, C~rbco-13 Ana. Y. D. G. Ott, "IIoavy" Mco. L.b World (Jmuary Sprcttoscopy of Totr.pYrrOlO8. N. 19721, p. 72. Ac8d. Sci. (in press). D. C. Ott, V. N. UXT,.ad T. U. Uh&y, Sp- R. T. E&b, L. 0. HOiorgM, C. T. Grogan d A. %tuiyo€f, Carbon-13 Nucloar Ha.trutic Bar- thwfr vi& Stabla Irotopu. 2: Abstracts of the 3. First Rocky Hotauia C.Honal Motirq of the Arr- oouoco Spectramcopy of Liviiy Calla d Ibair Ilrt- abolirr of r Specifidly ~abo1.d 13c subrtrate. ic.a CWcdSociety. _- Colordo State Univorsitr.-. hcton (sublittod). Fort CO~UIU,Color8do (June *July 1, 19721, MS p. 35, Abotract lo. 11. CEUIloI UDXOIQbCI SIlCnoI (m64. 1972). L. R. CrU1.r. E. A. wat8ar 8d 1. A, TObWu B. J. M.tt d S. 8. C~ll.~UUCIOO Ca Irl.tA~~bipof PbmpbWl.tlo0 to tho Siring of actu&oh.l Pul Ildrmm-~ocltd Uletolu C.11 Cycle, prwatod at the lZth of F*ctiou ia 8..IQphi1- inf1ws.u. pmmnted u th k.ri- Sociee for Cell B1Olol)r St. Ld, the 72d An-1 Hwtlag of tho -duo society for Itl.eourl (Ndr8-11. 1972) Mcrablolow , Phlladdphla, ~unm~lrcllrs.(April 23- U.lter8, rpd TOW, 28, 1972). 1. 1. Catl81, P. A. IL. A. no Indepedeaco of ~ifrt~~Phmphoqbtim fra Du B- J. Buahert mad S. 11. Cor, Pr0Pb.c Exclaloo Synthasis, pruatd at tb. 28th Anmud Southvut in- UUr kfllmrlr. prU.rUd at the hSUd Mgid Wth8 of tho ChddS0dOt.I. lbow of the Satthnmt Tri-State B-ch Baton Raup, (hdor 64, of tbr hmmrluo Society for Rlicrobiol~,El PYO, Louirl.lu 1972). Illldobrd and A. Tobey, DUA-kmbroo Tuur (October U-14, 1972). c. E. P. Auociatiau in Cultured Chiocr.0 Hmtor Call8, pre- L. hroCra, A Ch8siCd 4 -OXO MPtOrch meat& at tho Anmu1 betin8 of tho Biophyslcd to Chror#ar Strrr=turd Analysis, pruented at the Society, Toromto, C& (?ebw 24-27, 1972). Departrnt of Biopnyslu, Northoan Uiuchig.n Ualver- P. U. Kr.yr, DU Colutancy in il.toroploidY, alty, Urquette, Wchlg~(Pobru~ lS, 1972). ptuentod at the Univerrlq of Colordo Hdlul L. b. Deaven, Tho Paradox of DUA cOQ8tMCy in Ceater, DeIIyIr, Colondo (-7 30, 1972). bteroploidp, pru.ntod at the Deporunt of Bio- P. K. 1r-r. Studiu of the Cell Surfau physics, Northera Wchlg8e Ualverslty, Urqwtte, Ueiliting Plw mcmfluorortly, pronentrd at Xlchigan (Febr~~q17, 1972). Dna~Evaluation Branch program revIw, WatlOMl L. L. Demea, The Paradox of #SA Cmstmcy in Cocer Inacfcute, Bethoad., I(.r~l.ad(Auwt u1, Heteroploid Call Popu&tionr, prumted at the 19 72). Deprrtrmt of Biolow, n. D. An&rsoa UapitAl and P. X. Kr-r, H. A. Crisamna, .Id H. A. Vau Tumor Kmstituco, Uainrrity of To-, Pauton, Taxu Dill., Plw ntcrofluorortric Studiu of (July 26, 1972). (m) Plant Lectin Blsdin8 to Culturod r(.ulh Cdh, L. L. Deavon, Tochnlqrus for Badlng Chromo- promentad at tho 12th Annual Meting of the -I- s-a with Clem. Stain, prasonted at tho BIomodic.1 IC-Socioty for Coll Biolow, St. Louia, Illasourl Division, Lrvrrace Llvr~roLaboratory, UOi-tritY (N-.bor 8-11. 1972). of Cdifornia, Livorroir, California (~ctobor27, D. I. Petermen, Proclinical Exp.rirntr in 1972). Pion Radiobiolo~,prcaartod at the Sumex Oacoloq L. L. Damn d D. P. Poternn, Are Chr- Course apowored by tho Edwud Wlinckrodt In8tl- somslly Aneuploid Colla Gonetidy Aawplold? tuto of R.dlol~,Uuhfngton Uafvwolty School of prommtrd at tha 12th Annual ?IrocLng of tho Arr- Pkdlclnr, Sc. Lauia, Klasarrl (August 7, 1972). ic.n Society for 1311 aiolotjy, SC. Louis. Masouri D. P. Petat.on, Cell Klnrtla In Radiobiolow (Yovesbor 8-11, 1972). (Round Table Dircwsiod and Coll-Cyclo Aaal981s X. D. Engar and E. U. C.qbei1, hmcmt md Crcurh Propartlu of Chineso Hamter Colh, !h(.caenpr-Liko RWA Ikrabolism in Chinese Hamtar proemtad at the SIlrr Oacolow Cour80 spowored Ova- Collr Cultured at EbV8t.d T.9eratue. pm- by the Edword Wlloclcrodt Inatituto of Rdlololp, srnted at rho 28cn hnwl Southweat R.&ionJ &tins Uuhinpoo Uaivoraity School of mdicine, St. LOUIS, of the ArrLc- Chrfcd SOeiet~,Baton W'Ws 105blt't" 1 ak P. Byvooc, C. P. Shophord, J. lf. fl.+din, ad B. J. Nolsad, Turn~vorof kbol~dbthyl d W-1 croup. in Hiatom rnctiom of Culturod -an Cells, prueated at the 63:d And)Irotiag Of tho mricsu Society of llologicrl Chdsts, Atlmtic City , N.u Jiraey (April lC-14, 1972). C. 8. Shopherd, P:r;cturel AltorUioor of Hi.- tonom, presonrod at ~heLoputmnt of Biodr.rirtw, Uniwralty of 3ou krico School of Hediciru, Albu- qwrqw. Nw Ikdco (Jmuu~r20. 1972). C. R. Shopherd, St?uctur.l Altoratioos in Bi.- tooam of Synchrarirod ?!.!.lim Cells, promartd at the Dopertnuts of Payckitq sod PhaNCOiO~. ~hlverrityof Colordo Yadiul Contar. Domnr, Colorado (Wch 1, 1972) , .ad at the lkputrDt of Molecular, Cellular and Cave1oprat.l Biolom, Univerrlty of Colorado, Boulder, Colorado (Urch 2, 1972). C. R. Shepherd, i. X. Wudia, 8. J. Nokod, snd P. Byvoet, Turnover of Methyl, Acetyl, and Ph=phoryl Groups In Hiatoao Proctia~sof Culturod nP.Ul1.0 Calla. presatod at the Rocky mtdu RagioOL1 %.tins of the American C!.dul Socioty, Colorado State Uaivo.%ity, Fort Col;ins, Colorado (June 30-July 1. 1972).

135 Am Bmtinjd P. P. Wllm.~rDO ttemd ktdlr of -Urn Cella fnflwoce Their Libt Secrttorina Pattenu? presented at the &mu~lIl.eting of the Bio~hysicrlSociety, Tomato, Cmd. (FebMy 24-27, 1972). L. S. Crr .Id S. C. Benoby, ?tar Kieroflu- oratry for Pluorucat Anribe-Anrt- Srudies, preunted at che Aanurl Mating of the Dhphyrlcd Society, Tomto, Clod. (February 24-21, 1972). Le S. Crn and H. A. Crbsun. DUA of Y- 1i.a Cells: Hi&-Speed Fhu Uicroepctroflwro- *try, prsaautod at Cold Spring Ilubor LaboratorY, Cold Spring tlubor, New York (July U, 1972). H. A. Crism, Cell Proparati- Conridera- ti- in the Ganeral Oprrativr Peuutw of Flw Xkrofl-try, prasatd u b Dny Lvaluti~ Branch prop- revlu, Nati-l Caur Imtltute, ihthad., WlOd (Auwt 10, 1972). H. A. crissuu, PLar l4lcmfluorortw: prin- ciples aud Applicaclonr for Call Biolow, prosrated u the Uuivoalcy of Niu brico School of ILdicine, Albuquerque, New bLdco (October 3, 1972). P. II.HO~, L. S. Cra, and R. 0. Ili.berts Inrtnrutd ?actors Iofluclncing haolution in Flw Xicrofluorautrg, pruated at tho &nun1 Il.atin&

IC I 1% *ti-. iJ in Cell Blolo~,%Ut&, UMhiEgtm (Much 27-31, kpects OC HuqChuyd Puriclra adTheir Potem- 1972). cid use la &dlotherapy, pn8entlt.d at a Eefrrmber

?(e A. VaDill., C.U kulyai8 .Oa Cd1 Sort- Course of the IL.diolo#lcd Society of Worth hwr ing, presented at the Blophyaiu Mpar-t, Univenr- IC., Chicam, Illiaoi8 (N0Vee.r 29-30, 1972). sity of cauromia, hrkelry, ufornia (thy 2, c, ILicbvn, Studylog Pion Barr for Uour 1972). Ttnrapy. prereatd at the Annual %eting of the tJw ?!.A. Van Dill., Flw Syatr Cell Arulysia, HaJ.xico Dlvlaioa of the krricm Cancer SocleCy, preraud 8t tho lo72 wnm&8 FoUodrt~OaCaO- Albuquerqrw, N.v Wxico (Sepudar 9, 1972). ferenu om %fi#aoerint fm Mdna - btovtic It. L. S~lnthOa P. N. D.m* IntOrulibnt- Cytology," spttolv Rfver, Vermnt (hgwt 7-U. for Lw-hergr Photoo IJ.uu~.ynu, preuntd at 1972). the ~aowl&etof the Health Physics Sodety, N. A. Van Dill8 and J. A. St*iok.q, Elec- tu Vegu, Nevada (June 12-16, 1972). tronic Cell Sorting od Applicatla to Cell Diol- ISOTOPE APPLICATIOtS SECHON ogy and ExfoUUiw Cytology, preaented at the C. T. CreamCarbon Pathv8ya: Clycolyair and Sy.poslu oa kmnc Developnuts in Ruearch hth- Related hMt8tiOn8, presented at the DepUtMnt ods and Irutr\mt.tlon 8pO~orodby the NUiouel of nicrobiolow, Iklveaity of N.u Mexico school of Heart and LIpg Iastltuto, latlolul Institutu of Micine. Albuquerque, Iw l4xico (October 13, 1972). Health, Bethesdr, Maryland (Novader 30, 1972). C. T. Crerp, Carbon Pathways: XA, Clywhte xAM4ALIAu IuDIoB10Locy SECTION Cycle, pre8ented at the Department of Ucrobiobw, J. F. Spaldin&, L. X. Holland, and J. R. Prlne, Univeraity of New !&xico School of .Mdicine, Albu- Caparatln Biological Effects of Protr8cted Cob8.k- querque, Neu hxlco (October 18, 1972). 60 frradiatlm In Beagle8 md I(oksy8, presurted at C. T. Grep, Electron Tramport , ptewptd U the 20th Annual !betlag of the Rdiation ReOearch the Deparcmnt of !4lcrobiolow, University of ?J8u Society, Portland. 0-g- (May 14-18, 1972). phldco School of Xedicine, Albuquerque, ?kuMxtCo (October 20. 1972). PIPlSICAL RADIOBIOLOGY SECTION V. 8. Yollva adC. T. Cregg, Prepuatloa Of P. N. De-, EItlMtfOa of Chest Us11 Thicknu8 Carbon-13 L8bel.a Sugars by Phocosynthe8i.. pre- in Lmg Counting Cor Plutoaira, prarenred at the sented at chc Annual ?Itsting of the Botraiul Scl- Annual Iketing of the Health Physics Society. Las eoces Section. So1~tttwe8temand Rocky Nountain Vegaw, Xeurd. (Jme 12-16, 1972). Division, .aericsr. .bsociation for the 1Sdv.rrc-t P. N. Dean, Visuelization of Pion Stoppfnq of Science, and the Coiorado-eodng Acdqof hgion, prementd at the Natioad hctting 00 Science, Colorado S Kate Gniversitv , Fort COlliM. Applications of Optfcal IrutruPcnta in ,Mdlcine, Colorado (April 26-29 1972) , Chicago, Illinois (November 79-30, 1972). 2. G. Ott, Strble Csotopas and the Smthetic X. R. Ralu, Physical adRdiobio1ogic.l Chenist, presented at the Departwnt of Chdstty. Aspects of n- *mau vith Reference co Radio- University of Ncu Mexico, .;lbuquerquc, Xeu Hexlco therepy, preaented at the X. D. &demon Hapital (Narch 26, 1972). md Tumor Inatitute, L'nivemity of Texu. Xoustm, 9. G. Ott. V. X. Kerr, and r. 2. Yhaley, Syn- Texu (Julv 6, 1972). cbesis vith Stable Isotopea. preaeaced at tha MCkY ?(.R. Rdu, Xegatlvc Pions in Radiotherapy: ?buntrin Regfond .Meting of the -ria Chedul Physical adRdlobl~l~gi~al Aapects, presented at Societv, Colorado State Univeraity, Fort Collins, the Sloan Ketteriog Institute, Xev York City, New Colorado (June 30-July 1, 1972). York (Julv 20, 1972). 3. G. Ott. Biwdiul Applicatiom oi Scable !L R. Uju, hgatiw Pion8 fa Rdiochermpy, ISOKO~~Sof Carbon, Oxygen, wd Xitrogen, prumtcd presented at the Third Interrutional Conf arence on at the Laboratory of Nuclur Xadicine, uniVaC8ity ?Ldical Phy8iU d &died Engineering, dt.bOrg, of California, Lor ~ngelea,California (Dec..b.r 12, Sweden (July 30-Auqurt 4, 1972). 1972). U. R. Rslu, Phy8ical snd Bdiobiol~?cel G. U. Shreeve, Lipogenesis Era Carbon-14 i t05b5b-i 138 001 31493.154 C. E. g(lrl*&, Pb.D..(-)

Syacbwi. of eoll-frea protoin -D. E. Ud,Pb,D.,V-J brmc rynntbwi. of 0l.i~~~l.Otiau Effacta of r.li.tioo aa ndicuida

L. n. ~oi~d.D.v.x.A-~ B. J. Barnhart, SC.D.,(-B L Laboratory 0i.rl mdicine Bioch.d.try of butadal gonetic tr-foru- Kauolow Oa sursery ti- U-yli.0 rdiobiolow -Photobiolog: Bacterial roapoluu to ultra- P. I. tl0r.o. ph.~..\-b violet 1-t ad #* rapair procmna m tmr all diawmis ruing LASL coll aortor tacbniquu

J. 8. Jett, Ph.D.,C-A Nuclur phy8ica .pplicationr and ilutrrr mtatlao for radloactivig courrtiu Caputer appliutiozu for dat4 &pi8 .nd systr control

P. X. Kraerr, Ph.D. m Structure and function of ..uU.n cell- surface cqlucarbohydratu

?. F. bllaaey, Ph.D.,f-p Optical coll renainp Light rcatteriq by calls ?I.tabolirm of radionuclldu Sirultuuous wuuroint of multiullulu Internal dttors parartera R.di4t101I protaction @&8 D. C. Ott, Ph.D.,l-! Organic oynthuu vith i8OtOpu Energy ruboliar of cultund celb Nucleotide chcrt8try Biochaicd appllcatiau of stable isotopu Biochdcrl appliutloma of stable irotop-

L. R. Cur&y, Ph.D.,q-b D. F. Patorson. Ph.D.,\-b Bioche.Sstry of -1i.n coll lifo cycle -Sfstme chd8try old Prtrbolirl Synchroairatioa of -1- cell cultuna F. J. Hayea, Ph.D.*{-b Radiation chaiatry and biology of nucleic add. Uch..Io.{{I c. Uch..Io.{{I m

C. R. Shopherd, Pb.D., q-

n Efforta bow lata in 1971 to ormiso ea adv%aory -ttoo for tho purpcwo of providin8 oxpatt ddco to the Director of the L&ot.toq oa tho aciontific rtit unl future direcrioo of tho more fuaantd wmts of the overill hboratory's biordi4 ronuch prosrn. Cmitioaof tho Advfsoxy Coritteo for Biordlul Rmr~chis s\rh that @vie uill bo directed twrrd the ace bric and fwukuatd upoct8 of tho overdl bIomdic8.l propn. This la *ortat becaw., afou the early l?~laadesory cdttoe roproaontm rho 000~ 1960's with oncouraprnt of tho ='a rn~i~i~t~of pool of uprrtlso .nd orporionca In tho .L.U of Biology mnd Uodicino (nw the Divlaioa of Biomdiul truuport .nd orpniration of biochomlul owenta .ed &vlwmntal b~oarch),a 8ig~ific~tfraction in livin~syatom; tumor call kirrmtiu in rdio- of the Uoratory'a biordiuf rasearch offort ham thorapy; zadiology; biochmistry; ao1ocul.r biol- boon diroctod touord a cootlauin# protar b biohm ow, tran8f.r lUU chOd8try; physlob@C8l ch- which is on fundunully ad WMrdly 0flont.d iatry; ftmdmmtal CPPCO~ reseucb, pathohm; toward AEC/DBER interoat. 011 the bui. Of tho aqu- ch.roth0rapy; bldiulixuttcP.otatioa; .nd p.nt that p-roaa ki bid- 1s ueontial to the raxym mechanism studlam. 'Phia ~oupham proprd developrat of a atrons p-ru in radiobiology. co bo extremely valuable in providing technical At tho tir of vrltinp this report, piaare beins adv-.:: ;r.d prcgras Cirec2:aE ro virtually dl Mde to hold the ..cord meting oi chis .dvisoq cnue. :L :=: sverail tiooedical ruurch prop-. cdttn. Cmpoaltion of the cdttee la riven .uorbcr smlttr thas ralatu atrmgly to bolw: Ctoup H-4 is the Cltnton P. An&noa Warn Phfaica io55510 .-

Lao A&- Zieientific Lab0r.t~ La ASMOD, Iw Lludco Dr. H. N. Kll~rman(Mr at LU-1 Loa Alnoo Scientific Laboratory Loo Alsmm, N.w lludco md Ifoi..niq of Nw Maxico Albuqtnrqu, Wow Mco

Dr. wry S. ~~.plaa(Wr at Large) Repartnut of Bdlolo~ Stmford UnicrcraiQ &die.]. Canter Stanford, California

Dr. 8. ~odneyUitben (-or at Lu-1 H. D. Andenen Hapltd .nd Tl~arLnotitutc TeaIl.dic8l Centor Hornton, fus

Dr. Louin Rooon (-or at Ute) Loo Alvw Sci.ntlflc Laborrtory Lm Alams, Nw Il.Xic0 Dt. Robert F. ICalbm Dupu+rot of Rdiolop SCanfotd UniwmltY School of ILdlJn. P8lo Alto, CdifOrni.

I 143 EE; 742 (520) IOSb513 144