structed position ofthe North Atlantic Cur-rent and the gyre boundary (Fig. 2). Thissuggests that the glacial subpolar gyre tookon polar characteristics when the subpolar-subtropical gyre boundary shifted to a morezonal orientation and the northward excur-sion of warm subtropical waters into thesubpolar region ceased. This allowed asouthern migration of water with polarcharacteristics. The polar front representsthe southern limit of this migration, whichoccurred at the subpolar-subtropical gyreboundary. This interpretation is consistentwith that of Ruddiman and McIntyre (3).We speculate on why the North Atlantic

had a more zonally oriented we=curl(r) = 0 line 18,000 years ago. In thepresent-day North Atlantic, the position ofthe line where we = curl(r) = 0 corre-sponds to the location of strong eastwardwind stresses (produced by westerlies) andoccurs where the meridional gradient ofthese stresses vanishes (19). These strongstresses are due to continental storms. Intumr, the southwest to northeast trend oftheline is due to the storm tracks, which have asimilar pattem. However, in our runs,strong eastward stresses occur only in theeastem central North Atlantic, centered onapproximately 35°W, 45°N, as if the pre-sent-day pattem had swung to the south.This suggests that the storm tracks 18,000years ago ran more zonally and farther to thesouth than they do now, which is consistentwith interpretations based on geologicaldata (20).

Central to our hypothesis is that thebuildup of a substantial ice cap is necessarybefore the paleowind fields can be expectedto shift and, in tumr, alter the orientation ofthe gyre boundary. Changes in ice volumeshould precede changes in SST. Using oxy-gen isotope analyses and assemblages ofplanktonic foraminifera from the same core,Ruddiman and McIntyre (3) have shownthat continental glaciation preceded changesin SST as far north as 60°N in the NorthAtlantic during the isotopic 5 to 4 and 5e to5d transitions approximately 120,000 yearsago. This sequence ofevents implies that theocean passively responds to continental iceconditions rather than actively driving an iceage and the consequential buildup of conti-nental ice. Our interpretation accounts onlyfor changes in the ocean over the relativelylong time scales of continental ice buildupand decay. It does not explain the muchmore rapid (approximately 1000-year)Younger Dryas event, which did not corre-spond with any substantial change in icecoverage (21).Our scenario for the role of the ocean

during glaciation can be summarized as fol-lows. Low summer insolation causes accu-

442

mulation of snow and ice through the sum-mer, which in turn increases the volume ofice and overall earth albedo. The ice volumeeventually becomes large enough to influ-ence thermal and orographic steering of thewe = curl(T) = 0 line, causing it and, byconsequence, the subtropical-subpolar gyreboundary to move southward. Finally, sucha shift shuts off the supply ofwarm and saltywaters to the high-latitude North Atlantic,resulting in a drop in SST and a cessation ofbottom water production in the high lati-tudes. Deglaciation follows a similar, butreversed, sequence (22).

REFERENCES AND NOTES

1. A. V. Bunker and L. V. Worthington, Bull. Am.Meteorol. Soc. 57, 670 (1976).

2. CLIMAP, Geol. Soc. Am. Map Ser. MC-36 (1981).3. W. F. Ruddiman and A. McIntyre,J. Geophys. Res.

82, 3877 (1977).4. B. A. Warren, J. Mar. Res. 41, 327 (1983).5. For a review, see B. H. Corliss, D. G. Martinson, T.

Keffer, Geol. Soc. Am. Bull. 97, 1106 (1985).6. W. F. Ruddiman, in North America and Adjacent

Oceans During the Last Deglaciation, W. F. Ruddimnanand H. E. Wright, Jr., Eds. (Geological Society ofArnerica, Boulder, CO, in press).

7. T. Keffer, B. H. Corliss, D. G. Martinson, Eos 67,44 (1986).

8. R. E. Newell and J. Hsiung, in Abrupt ClimaticChange, W. H. Berger and L. D. Labeyrie, Eds.(Reidel, Dordrecht, 1987), pp. 67-87.

9. W. H. Munk,J. Meteorol. 7, 79 (1950).10. H. Stommel, The Gulf Stream (Univ. of California

Press, Berkeley, 1965).11. The CLIMAP (2) geological indices have been used

to calculate present-day SSTs (12). In the region ofinterest, these geologically based SSTs are nearlyindistinguishable from the observed SSTs, exceptfor an area southeast of Newfoundland where thegeologically based estimates are warmer than ob-served by -1.20C.

12. B. Molfino et al., Quat. Res. (N. Y.) 17, 279 (1982).13. A. Leetma and A. F. Bunker,J. Mar. Res. 36, 311

(1978).14. H. U. Sverdrup, M. W. Johnson, R. H. Fleming,

The Oceans (Prentice-Hall, New York, 1942).15. I. M. Held, Icarus 50, 449 (1982).16. S. Manabe and A. J. Broccoli,J. Geophys. Res. 90,

2167 (1985).17. For examples of other atmospheric simulations for

18,000 years before present, see J. Williams et al. U.App. Meterol. 13, 305 (1974)], W. L. Gates [Science191, 1138 (1976)], and J. E. Kutzbach and P. J.Guetter U. Atmos. Sci. 43, 1726 (1986)].

18. E. J. Barron and W. M. Washington, J. Geophys.Res. 89, 1267 (1984).

19. This is because ofthe dominance ofzonal winds andthe near similarity of Ekman pumping and windstress curl. This allows Ekman pumping to beapproximated as we - curl(r) - dr(x)/ay. Hence,where the meridional gradient of zonal wind stress[ar(x)/ay] equals zero, we will tend to equal zero.

20. W. F. Ruddiman and A. McIntyre, Science 204, 173(1979).

21. W. S. Broecker, D. M. Peteet, D. Rind, Nature 315,21 (1985).

22. We thank E. Barron and L. Sloan (both at Pennsyl-vania State University) for providing wind stressvalues for 18,000 years before present. This researchwas initially supported by a grant from the WoodsHole Oceanographic Institution Center for theAnalysis of Marine Systems. Additional supportcame from NSF grants OCE 83-15369 and OCE85-15642 (T.K.), and OCE-84-19595 and OCE-86-14162 (B.H.C.). This is University of Washing-ton, School of Oceanography contribution number1776 and Lamont-Doherty Geological Observatorynumber 4299.

22 February 1988; accepted 2 June 1988

Direct Measurement of 02-Depleted MicrozonesMarine Oscillatoria: Relation to N2 Fixation

HANS W. PAERL AND BRAD M. BEBOUT

Among the nitrogen (N2)-fixing Cyanobateria, the filamentous, nonheterocystousmarine Oscillatoria spp. (Trichodesmium) appears enigmatic; it exhibits N2 fixation inthe presence ofoxygenic photosynthesis without structural protection ofthe N2-fixingapparatus (nitrogenase) from potential inhibition by molecular oxygen (02). Charac-teristically, N2 fixation is largely confined to aggregates (bundles) of filaments.Previous work has suggested that spatial partitioning of photosynthesis and of N2fixation occurs in the bundles as a means of allowing both processes to occurcontemporaneously. The probing of freshly sampled bundles with 02 microelectrodesdirectly confirmed such partitioning by showing the presence of02-depleted (reduced)microzones in photosynthetically active, N2-fixing bundles. Bundle size was directlyrelated to both the development of internal reduced microzones and cellular N2fixation rates. By enhancing microzone formation, bundles optimize N2 fixation as ameans of supporting Oscillatoria spp. blooms in surficial, nitrogen-depleted tropicaland subtropical waters.

Tt SROPICAL AND SUBTROPICAL NITRO- dish-brown aggregates or bundles. The fila-gen-depleted marine waters periodi- ments in the bundles are oriented in acally support near-surface blooms of parallel fashion (Fig. 1). Characteristically,

the filamentous oxygenic cyanobacterium N2 fixation is associated with bundles (1).Oscillatoria (Trichodesmium) spp. Blooms areconfined to calm periods when buoyant Institute ofMarine Sciences, University ofNorth Caroli-filaments accumulate at the surface as red- na, Chapel Hill, Morehead City, NC 28557.

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Previous studies concur that Oscillatoria spp.blooms are responsible for relatively highrates of planktonic N2 fixation, which con-stitute ecologically significant inputs ofcombined nitrogen (2).The mechanisms that allow Oscillatoria

spp. to fix N2 in the presence of oxygenicphotosynthesis have not been directly iden-tified. The N2-fixing enzyme complex, ni-trogenase, is readily inactivated by molecu-lar 02 in a variety of cyanobacteria andeubacteria (3). Whereas some filamentousN2-fixing cyanobacteria form biochemicallyand structurally distinct 02-devoid cellstermed heterocysts in which nitrogenase is

Fig. 1. (A) A segment ofalarge(>75 filaments) Oscillatoria spp.bundle that has a parallel ar-rangement of individual fila-ments. The tip of an 02 micro-electrode (tip diameter approxi-mately 7 ,um) is shown insertedinto the bundle. Bundles of thissize firequently revealed internal02-depleted microzones. (B) Arelatively small bundle consist-ing of approximately 30 indi-vidual filaments. Internal re-duced microzones were not evi-dent in such bundles. (C) High-magnification view of individu-al filaments arranged inbundles. Bright, gramnular intra-cellular structures are gas vacu-oles, which greatly enhancebuoyancy, thus promoting sur-face accumulations of bundlesduring calm condiions.

localized (4), Oscillatoria spp. reveal no cellu-lar differentiation. This organism is exceed-ingly difficult to culture, which has hinderedour understanding of how contemporane-ous photosynthetic 02 evolution and N2fixation can take place.

Field observations reveal that N2-fixingcapabilities of Oscillatoria spp. are closelylinked to the extent of bundle formation (1,5). Whereas large bundles composed offrom tens to several hundred filaments ex-hibit light-mediated N2 fixation, individualfilaments or small aggregates (two to tenfilaments) often fail to fix N2 (1). Accord-ingly, on calm days when filaments readily

aggregate as bundles in surface slicks, N2fixation rates per unit biomass are maximal,whereas wind-induced turbulence leads todecreases in both average bundle size andbiomass-specific N2 fixation (1, 5). Usingautoradiography, Carpenter and Priceshowed (1) that 14CO2 fixation was uneven-ly distributed along filaments; 14CO2 fixa-tion was confined to terminal portions ofthe filaments whereas intercalary regionsexhibited extremely low rates of fixation.On the basis of these observations, theysuggested that N2 fixation could accompanyphotosynthesis if the processes remainedspatially separated within filaments. Whenfilaments were aggregated as bundles, athree-dimensional 02-depleted internal por-tion, composed of parallel intercalary re-gions, might be a likely site of N2 fixation.By using 02 microelectrodes in combina-

tion with N2 fixation (acetylene reduction)measurements, we have directly confirmedthe presence of internal 02-depleted (re-duced) microzones in photosynthetically ac-tive, N2-fixing Oscillatoria spp. bundles.Both the extent ofbundle size and its config-uration are determinants in reduced micro-zone formation and resultant N2 fixationcapacity.

Unusually calm, warm, and clear weather,combined with the close proximity of sub-tropical GulfStream waters to North Caroli-na's coastline (6), led to several Oscillatoriaspp. blooms during October and November1987. These climatic conditions also favoredthe parallel development of toxic dinoflagel-late (Ptychodiscus breve) blooms (red tides)(7). Oscillatoria spp. blooms were often spa-tially separated from Ptychodiscus blooms,hence facilitating exclusive sampling andanalysis of the Oscillatoria (8, 9).We measured 02 concentrations sur-

rounding and within individual Oscillatonaspp. bundles, using microelectrodes (10).These microelectrodes had sensing tips 3 to7 ,um wide and a spatial resolution of 100pum. Bundles were placed in small plasticpetri dishes containing seawater and a glassfiber filter. The bundles were held immobilewith a few teased-up fibers on the filter.Microelectrode 02 profiles in ambient sea-water and bundles were initiated within 1 to2 min after the placement ofbundles in petridishes. Electrode tips were positioned by amicromanipulator while the specimen wasobserved under a dissecting microscope.Electrode polarization voltage was suppliedby, and current output recorded with, achemical microsensor (Diamond Electro-tech model 1201). The electrode outputwascalibrated against air-bubbled seawater(100% ofsaturation of02) and N2-bubbledseawater (0% of saturation of 02). All oxy-gen measurements were made within 5

REPORTS 44322 JULY I988

hours ofsample collection under illuminated(200 to 1140 ,iE m-2 S-') and dark condi-tions. To test for the production of abioticoxygen gradients, we heat-killed some bun-dles by placing them in a microwave ovenfor -5 s, which raised the temperature ofthe small volume of seawater in the petridish sufficiently to kill Oscillatoria spp. cellswithout destroying the structure ofthe bun-dles.

Distinct differences in the rates of chloro-phyll a-normalized nitrogenase activity(NA) were observed among bundles of vari-ous sizes, with large bundles generally ex-hibiting much greater rates than small bun-dles (Table 1). Reduction of bundle size,induced either by teasing bundles apart (byusinig a 25-gauge syringe needle) or byvigorous shaking, led to a marked reductionin NA. In all cases NA was strongly lightdependent.

Microelectrode measurements revealedregions of 02 concentration markedly lowerthan ambient both within and surroundingOscillatoria spp. bundles (Fig. 2). The lowest02 concentrations were within the centralportions ofthe bundles, whereas 02 concen-

Fig. 2. Vertical distribu- 1000tion of dissolved 02concentrations near the 800ends (O) and middle(0) segments of large , 600Oscillatoria spp. bundles, asdetermined with 02 micro- a 400electrodes. Each value indi- "cates the mean and standard a 200error of three replicate bun- Bundledles. Lower 02 concentra- 0 ____tions in the heat-killed bun-dles (A) are likely due to -200 .both a cessation of photo- 0synthesis and the reducedsolubility of 02 in the previously heated seawater.

Fig. 3. Distribution of dis-solved 02 concentrationsnear and within an Oscilla-toria spp. bundle. Points atwhich 02 microelectrodedeterminations were madeare indicated (asterisks).

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trations in terminal regions of the bundleswere only slightly lower than ambient.Highly dynamic photosynthetic processesappeared to be responsible for the distribu-tion of 02 near bundles. We recorded 02concentrations in excess of 200% of satura-tion in terminal regions immediately (within2 min) after increases in light intensity from200 to 1140 ,uE m-2 S-' of photosyntheti-cally active radiation (PAR). Completelyshading a bundle (1140 to 0 ,uE m-2 S-IPAR) reduced the 02 concentration from200% of saturation to 75% of saturationwithin 1 min. "Steady-state" 02 concentra-tions (1140 ,uE m-2 S-1) were generally 75to 100% of saturation. Under "steady-state"conditions, the bundles were able to rapidlydeplete 02 in the water immediately sur-rounding them, resulting in measurableboundary layers as far as 500 ,um from thebundle surfaces. Microelectrode profiles ofheat-killed bundles revealed neither 02-de-pleted microzones nor boundary layers (Fig.3). The 02 concentrations in the heat-killedsamples were slightly lower than in livesamples as a result of the lower solubility of02 in the previously heated seawater.

fAAA

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Bundles incubated for 48 hours in 500-mlErlenmeyer flasks (200 ,uE m-2 S-' PAR;12 hours light:12 hours dark) revealed de-creases in both NA and internal 02-depletedmicrozone formation. This response wasobserved among bundles of various sizes.These decreases were not a result of loss ofviability, because the photosynthetic 02production characteristics of bundles re-mained high. Decreases in NA may haveresulted from nitrogen regeneration by asso-ciated bacteria and protozoans, which mayhave replaced N2 fixation as the chief meansof meeting nitrogen demands among Oscil-latoria spp. bundles under confined condi-tions. In any event, a loss of NA paralleledthe disappearance of 02-depleted micro-zones among such bundles.

In concert, these results directly confirmCarpenter and Price's contention that N2fixation and oxygenic photosynthesis arecompatible in bundles of morphologicallyundifferentiated Oscillatoria spp. filaments(1). Bundle size and tightness are directlyrelated to the extent of reduced microzoneformation and NA. As such, climatic condi-tions, specifically water column turbulencecombined with adequate irradiance, whichdictate bundle size and photosyntheticallygenerated energy availability, are determi-nants that mediate marine N2 fixation inputsattributable to Oscillatoria spp. Althoughwind-induced turbulence can negate NA ofOscillatoria spp. by breaking up bundles,resultant vertical mixing of individual fila-ments and small aggregates leads to en-hanced exposure to ambient inorganic and

Table 1. Comparative acetylene reduction (AR)rates for freshly collected small versus large bun-dles of Oscillatoria spp. Each replicate represents asample containing at least 20 small or 10 largebundles. Care was taken to select bundles similarin shape and size for each treatment. The back-ground rate of ethylene production (ethylenecontamination in acetylene plus ethylene produc-tion in 0.4 ,um offiltered seawater) was 1.5 nmol/liter per hour; this rate was subtracted fromOscillatoria AR rates; chlorophyll a, Chl a.

AR AR: Chl a

Bun- Chl a (nmol/ (nanomolesdle (dg/ lter per microgramliter) per of Chl a

hour) per hour)

Small bundles (<30filaments per bundle)1 120 29.5 0.2462 125 30.5 0.2443 110 30.0 0.273

Average = 0.254,SEM = 0.016

Large bundles (>75 filaments per bundle)1 110 220.5 2.002 105 240.5 2.293 115 235.0 2.04

Average = 2.11,SEM = 0.157

SCIENCE, VOL. 24IA4

organic combined nitrogen. Among verti-cally mixed individual filaments, cellular sur-face to volume ratios are increased whiletransport to deeper, combined nitrogen-enriched waters is enhanced. In this manner,N2 fixation is optimized as a means ofobtaining combined nitrogen on calm days,whereas ambient combined nitrogen usagebecomes more feasible under turbulent con-ditions.Although we have shown the existence

and roles of 02-depleted microzones, themeans by which intercellular "division oflabor" among oxygenic photosynthesis and02-sensitive N2 fixation is induced andmaintained are currently unresolved. Trans-port of photosynthetically produced carboncompounds to 02-depleted N2-fixing cellsmust take place to provide both reductantand carbon skeletons essential as an energysource for N2 fixation and for accepting(incorporating) recently fixed NH3. Al-though transport of photosynthetically fixed14C02 to intemal 02-depleted microzonescan be shown by autoradiography, the ge-netic, physiological, and structural mecha-nisms that mediate such transport remainunknown.

NA was determined under both illuminated (200,zE m 2 S-' PAR; combined cool white and Syl-vania Grow-Lux fluorescence) and dark conditions.After incubation, all flasks were shaken to equili-brate aqueous and gas phases and a 0.3-mi head-space gas sample was withdrawn and injected into aShimadzu GC-9A flame ionization gas chromato-graph having a 2-m-long Poropak T column held at80'C. Ethylene production was calibrated againstultrahigh-purity ethylene (Matheson) standards.

9. W. D. P. Stewart, C. R. Fitzgerald, R. H. Burris,Proc. Natl. Acad. Sci. U.S.A. 58, 2071 (1967); H.

W. Paerl and P. E. Kellar,J. Phycol. 14, 254 (1978).10. N. P. Revsbech, in Polarographic Oxygen Sensors, E.

Graiger and H. Forsmer, Eds. (Springer-Verlag,Berlin, 1983), p. 265; R. G. Carlton and R. G.Wetzel, Can.J. Bot. 65, 1031 (1987).

11. Supported by NSF grants OCE 85-00740 and BSR86-14951 and by the North Carolina BiotechnologyCenter (Project 86-G-01013). We thank R. G.Carlton, L. E. Prufert, J. Garner, H. Page, and V.Page for technical assistance.

28 January 1988; accepted 20 May 1988

Translation in Mammalian Cells of a GeneLinked to the Poliovirus 5' Noncoding Region

DIDIER TRONO,* JERRY PELLETIER, NAHUM SONENBERG,DAVID BALTIMORE

The central portion (region P) of the 742-nucleotide noncoding 5' end of poliovirusallows the RNA to initiate protein synthesis in the absence of the usual 5' 7-methylguanosine capping group. Poliovirus 5' noncoding region was fused to areporter gene and transfected into cells. There was extensive augmentation of theexpression of this gene by poliovirus-mediated inhibition of cap-dependent proteinsynthesis. That the construct initiated in a cap-independent manner was verifiedthrough in vitro experiments. Smali lesions throughout region P blocked its initiationfimction, implying that a coherent functional unit, hundreds of nucleotides long, isresponsible for cap-independent initiation by poliovirus RNA.

REFERENCES AND NOTES

1. E. J. Carpenter and C. C. Price IV, Science 191,1278 (1976).

2. E. J. Carpenter and D. G. Capone, Eds., Nitrogen inthe Marine Environment (Academic Press, New York,1983), p. 521.

3. F. R. S. Postgate, The Fundamentals of NitrogenFixation (Cambridge Univ. Press, Cambridge,1982).

4. C. P. Wolk, in The Biology of Cyanobacteria, N. G.Carr and B. A. Whitton, Eds. (Blackwell, Oxford,1982), p. 359.

5. I. Bryceson and P. Fay, Mar. Biol. 61, 159 (1981).6. The location of the Gulf Stream with respect to

North Carolina's coastline is routinely determinedby satellite imagery and by field measurements con-ducted by the National Oceanographic and Atmo-spheric Administration-National Marine FisheriesService Laboratory, Beaufort, NC. During Octo-ber-November 1987, water masses representing thewestern wall of the Gulf Stream were as close as 2km to Cape Lookout and Beaufort Inlet, NC.

7. P. Tester and R. Ferguson, personal communica-tion.

8. Near-surface water samples were taken with pre-cleaned 2-liter wide-mouth polypropylene jars atseveral Atlantic coastal locations 1.6 to 4.8 kmseaward from Beaufort Inlet. Jars were transported(within 2 hours) at surface water temperature to thelaboratory. We immediately examined buoyant Os-cillatoria spp. bundles, ofvarious sizes and shapes, forN2-fixing and photosynthetic 02 production capa-bilities by using acetylene reduction assays (9) and02 microclectrode measurements (10). Bundleswere removed from jars with a large-bore polypro-pylene pipette. For acetylene reduction assays, 20-misamples, containing either large (>75 filaments) orsmall (<30 filaments) bundles, were dispensed into25-ml borosilicate Erlenmeyer flasks. Three replicateflasks were used for each size class. Flasks weresealed with rubber serum stoppers, inverted, andinjected with 2 ml of ultrahigh-purity acetylene(Matheson). Flasks were incubated on their sidesunder nonshaken conditions for 2 to 4 hours atsurface water temperatures. Chlorophyll a-specific

22 JULY I988

T HE GENOME OF POLIOVIRUS IS A

single-stranded molecule of plus-strand RNA, approximately 7500

bases long (1). Poliovirus has an unusuallylong 5' noncoding region, which consists of742 untranslated nucleotides preceding theAUG used to initiate translation (2). Ahighly significant sequence similarity ex-tends through the first 650 nucleotides ofthe three poliovirus serotypes (3), an indica-tion that the region has a crucial role in theviral life cycle. Poliovirus messenger RNA,unlike most other eukaryotic mRNAs, is notcapped at its 5' end; it terminates in pUpinstead of the usual "capping group"m7G(5')ppp(5')N... (4). It must thereforebe translated in a cap-independent manner.The virus takes advantage of its unique styleof translation initiation to inhibit cellularprotein synthesis by interfering with thecap-dependent translation of cellularmRNAs (5).Using a cDNA copy of the viral genome

(type 1, Mahoney) (6), we engineered a

D. Trono, Whitehead Institute for Biomedical Research,Cambridge, MA 02142.J. Pelletier and N. Sonenberg, Department ofBiochemis-try, McGill University, Montreal, Quebec, Canada H3G1Y6.D. Baltimore, Whitehead Institute of Biomedical Re-search, Cambridge, MA 02141, and Department ofBiology, Massachusetts Institute of Technology, Cam-bridge, MA 02139.

*To whom correspondence should be addressed.

number of mutations into the poliovirus 5'noncoding region (7). The biochemical andgenetic analysis of several phenotypicallyrecognizable viral strains thereby generatedshowed that an RNA sequence of hundredsof nucleotides (which we called region P) isinvolved in allowing viral protein synthesis(7). In vitro experiments also suggested thata large portion of the poliovirus 5' noncod-ing region is responsible for the cap-inde-pendent translation of downstream se-quences (8). So far, no cellular gene has been

SV40 p(A)

SV40 IVS

TAA

SV40Ori Polio ATG

5NC

Fig. 1. Schematic representation of the wild-typeconstruct (pWT-CAT). Nucleotides 1 to 630 ofpoliovirus type 1 (Mahoney) (1) were cloned inthe Hind III site of the pSV2CAT plasmid (9); inthe other constructs, the corresponding region ofvarious mutated clones (7) was substituted for thewild-type sequence.

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