Ribosomal ribonucleic acid sites in chicken deoxyribonucleic acid

ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS

115, 197-20.5 (1966)

Ribosomal Ribonucleic Acid Sites in Chicken Deoxyribonucleic Acid ILNIAR MERITS, WERNER SCHULZE,

AND

LACY R. OVERBY

Department of Biochemical Researcii, Abbott Loboratories, North Chicago, Illinois Received January 17, 1966 Hybridization of ribosomal RNA from chickens with DNA immobilized on membrane filters indicated that the chick ribosomal genetic area represented less than 0.02% of the genome. Competition of ribosomal RNA from differentiated tissues suggested that the same ribosomal RNA population was expressed by the cells of each tissue, including spleen cells grown in tissue culture. A saturation RNA/DNA ratio of 0.2% was found for Escherichia coU ribosomal RNA which confirmed reported values. Ribonucleic acid recovered from the hybrid was identical in base ratio to the starting RNA. Compared to the E. coli system, the hybridizing efficiency of chick ribosomal RNA was low and the base ratio of the recovered hybridized RNA indicated some susceptibility of the complex to ribonuclease attack.

Hybrid formation between DNA and labeled homologous RNA has indicated that 0.2-0.3 % of Escherichia coli and Bacillus megatel'ium DNA is complementary to the two ribosomal RNA components (1, 13, 14). About the same fraction of DNA forms specific hybrids with ribosomal RNA from Pieum sativum seedlings (3) and from D1'OSOphila melomoqaster (11, 12). However, in HeLa cells, a cell line of human origin, the fraction of DNA capable of specific hybridization with ribosomal RNA is considerably smaller. McConkey and Hopkins (8) reported values of 0.003-0.005 % for 288 RNA, and Attardi ei al. (1), 0.002-0.003 % for the 188 component. This suggested that HeLa cells reserve 40-60 times less of their total DNA for the specification ofribosomal RNA than do bacteria, higher plants, and insects. We have investigated ribosomal RNA sites in DNA of higher animals. We have also attempted to determine whether differentiated tissues express the same or different areas of their DNA as cistrons for ribosomal RNA. The system consisted of RNA from chicken spleen cells in tissue culture, RNA from chick liver or RNA from the whole chick, and DNA from chicken testicles or liver.

METHODS Escherichia coli DNA-sH. A thymineless strain was labeled with tritiated thymidine, and the DNA was isolated according to the procedure of Gillespie and Spiegelman (4). Specific activity was 400 cpm per microgram. Escherichia coli ribosomal RNA_SSP. The bacteria were grown in a glucose-salts medium and labeled with 1 mM aSP-orthophosphate (50 p.O per milliliter) for one generation and chased in 20 mM phosphate for one more generation. The RNA was isolated by the method of Hayashi and Spiegelman (5). Specific activity was 25000 cpm per microgram. Rat liver "sohtble" RNA (sRN}!). Rat liver sRNA was prepared and purified on an Ecteola column as described by Merits (9). The product was filtered through nitrocellulose :filters to remove colloidal column material. Chicken DiYA-all. A young adult male white Leghorn was given two I-mO doses of thymidine6-sH (5 0 pel' millimole) at 4-day intervals intraperitoneally and killed on the sixth day. The testicles and liver were removed, quickly frozen, and kept at -20°. The frozen organs (10 gm) were pulverized and homogenized in a Waring Blender at 4° with 100 ml of 0.15 M NaOI, 0.02 ~l EDTA, and 0.05 M Tris buffer (pH 8.0) containing 1% of sodium dodecylsulfate (SDS). The viscous solution was stirred for 5 minutes; 5.8 grn solid Na01 was added and stirring was continued for one 197

198

l\IERITS, SCHULZE, AND OVERBY

hour. The solution was shaken with 50 ml phenol for 15 minutes, the layers were separated, and the phenol layer was washed once with 25 ml of 1 M Nile!. The nucleic acids were precipitated by gently layering approximately 2 volumes ethanol on the combined water phases lind then' 'spooled" on a stirring rod, The precipitate was dissolved in 0.0015 sodium citrate (pH 7.2) + 0.015 NaCl (ho X ~sC) und incubated with heat-treated RNase (Worthington, 5X-crystallized) for4 hours at 37°. The RNase was removed by treating with pronase (Calbiochern, 50 p.g per milliliter) at 37° for 2 hours. The solution was then deproteinized by adding SDS to 0.5% and shaking 15 minutes with an equal volume of phenol. The phenol extraction was repeated twice. Finally the DNA was precipitated with 2 volumes of ethanol and ulkali-denaturated as described by Gillespie and Spiegelman (4). The specific radioactivities were 18 cpm per microgram for liver DNA and III cpm per microgram for testicular DNA. Chicken spleen RiVA-32P. Young adult male white Leghorn birds were bled to death by heart puncture, and the spleens were quickly and aseptically removed. The tissue cells were then grown as described by Patterson et al. (10), except that plastic tissue culture dishes, 100 X 20 rom (Falcon Plastics, distributed by Matheson Scientific Inc., Chicago), were used. Ten dishes were used for one spleen, and each dish contained 5 mC carrier-free crthoph.osphate-t-P in 15 ml medium. The pyrophosphate-free neutralized orthophosphate-e'P solution wall treated with 0.2 m! catalase solution (Worthington) for 10 minutes at room tempe rature prior to mixing with the medium. After 2-1-36 hours incubation ut 37° the radioactive medium was removed and the cells were grown for 36--48 hours ill fresh medium containing 5 mM unlabeled phosphate. The cells were harvested and taken up in 75-100 ml of 20 rmr Tris buffer (pH (3.7),2 mzr MgCI2, and 0.03% SDS, containing 50 mg bentonite. TIle solution was extracted 3 times at 4° with an equal volume of phenol; the phenol was removed with ethel', and the RNA was precipitated with 2 volumes of ethanol at -20°. Specific activity was 40,000 cpm per microgram. lVhole chick RN"PH and RNrP2p: Three-dayold chicks were injected once intraperitoneally with 10-15 mC carrier-free orthophosphate-UP, or 10-15 mC tritiated uri dine (22.2 mC pel' millimole) in 4: doses over a -l-day period. The 32p_ inj ected chicks were sacrificed after 2-3 days because survival was severely impaired by the radioisotope. The tritiated, uridine-treated chicks survived longer and were killed after 6-7 days labeling. The alimentary tracts were removed, cleaned, and processed with the carcass. The entire animal was homogenized in a Waring

"I

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Blender with 200 ml extraction solution (described above for spleen RNA isolation) and 200 ml phenol for 2 minutes, and then shaken for 2 hours at 4°. After separation of the water phase, the phenol layer was washed once with 100 ml extraction solution. The combined water phases were shaken twice with phenol and extracted with ethel', and the RNA was precipitated with 2 volumes of ethanol at -20 0 • Specific activit.ies were (cpm per microgram): 3000 for RNA-3H and 5000-20,000 for RNA_32P. Chick liver RNA-3H and RNA_32P. The chicks were labeled as described above; the livers were excised and the liver RNA was isolated with the same technique as for the whole chick. Specific activities were (cpm per microgram): 3000 for RNA-3H and 10,000 for RNA- 3fT.

Purification of Ribosomal RNA

(rRNA)

The RNA preparations were chromatographed on columns of methylated albumin coated on Kieselguhr (lVIAK) by the Mandell and Hershey (6) procedure; a lineal' NaCI gradient from 0.5 to 1.3 M NaCl in 0.01 MTris buffer (pH 6.7) was used. The rRNA fractions were pooled and made 1 mM with MgCb, and the RNA was precipitated with 2 volumes of ethanol at -20°. The recovered RNA was analyzed by sucrose gradient centrifugation.

H ybriclization The procedure of Gillespie and Spiegelman (4), in which DNA is immobilized on nitroeellulose membrane filters, was used. Immobilization and hybridization were carried out in 3 X SSC, the latter at 65-70° in 5 ml solution for 12-16 hours. Ribonuclease digestion (20 p.g per milliliter) was accomplished at 25° for one hour with shaking. The filters were counted in a Packard scintillation spectrometer.

Base Ratio Analyses The hybridized RNA was extracted from the membrane filters twice for 3 minutes each with hot water, and the solution was cooled quickly in ice. The yield of RNA was 85-90%. Unlabeled carrier RNA was added and the mixture was hydrolyzed in 0.3 N KOH at 37° for 18 hours, neutralized with formic acid to pH 8, and chrornatographed on a 25 X 1 em Dowex-1-formate column (8X, 200-400 mesh) using a lineal' gradient, of water to 3.5 M formic acid. The absorbanoies of the fractions were measured at 2GO mj,4 to give the base ratio of the carrier, and mdioactivities were measured by drying the liquid on planchets and counting in a gas-flow counter to give the base ratio of the labeled RNA. Duplicate runs

199

RIBOSOMAL RNA SITES iN CHIOI
Competition Ihpe1'imenls In order to compare rRNA preparations from different sources, labeled RNA was mixed with diffcrent amounts of unlabeled RNA and the mixture Wl1H hybridized with DNA. The labeled RNA wus Idwl1YH uddod in loll umount which would just aatumtc It given lt1l101111t of DNA, and this value wns obf.aincd from the satumtion curve. It was assumed thut when labeled RNA WItS diluted with nn eql1111 amount of unlabeled homologous RNA, the amount of labeled RNA in the RNA-DNA hybrid would decrease by the factor of two. Addition of twice as much unlabeled RNA would yield only one-third of the original counts in the hybrid, etc. A reduction of radioactivity in the hybrid is therefore assumed to be specific, and due to compet.ltion between the labeled and unlabeled RNA species for the same sites on the DNA. 'I'hus 011e could construct a theoretical competition eurve and compare it with actual values. RESULTS

HYBRIDIZATION P ARAME'l'ERS Conditions for hybridization were investigated to determine optimum time, temperature, and ionic strength. Periods up to 16 hours of incubation at 65° in 3 X SSC medium did not markedly affect the DNA content on the filters. However, after 90

hours of incubation considerable losses of DNA had occurred without increasing the hybridizing efficiency of the remaining DNA. The temperature for maximal hybridizing efficiency of l'RNA after 15 hours of incubation in 3 X SSC was 70°. At SO° loss of DNA from the filters was evident. The ionic strength of the incubation solution could be varied from 2 X sse to 6 X SSC without influencing annealing efficiency in 15 hours at 70°, Increase of the salt coneentration above 6 X SSC resulted in an abrupt rise in counts, but this was due to precipitation of RNA on the filters. Such apparent "hybridization" at salt concentrations above 6 X SSC occurred on plain nitro eellulose membrane filters without DNA, and the immobilized RNA was ribonuclease resistant. The optimum conditions selected for hybridization were 15 hours at 70 0 in 3 X SSC. ESCHERICHIA GOLI SYSTEM

Since the saturation plateau for E. coli rRNA had already been carefully examined by other workers it was used as a useful test for our system. Seven saturation experiments gave values of 0.22-0.27 % for the RNA/DNA ratio, figures close to the reported values. The hybridized RNA had a base composition substantially indistinguishable from that of E. coli RNA (Table I). Competition experiments clearly show that addition of homologous RNA reduced the hybridization as expected (Fig. 1), following closely the theoretical competition curve; but there was no effect of large

TABLE I NUCLEOTIDE COMPOSITION OF RIBOSOMAL RNA I-IY13RIDIZED TO HOMOLOGOUS

E. coli sys tern

Input (6)"

CMP AMP GMP UMP

24.3 25.0 31.0 19.7

DNA

Chicken system Hybridized (1)

22.2 24.8 34.8 18.3

Input (4)

28.5 19.4 35.3 16.8

" Figures in parentheses refer to the number of determinntions.

Low RNA/DNA ratio

+ RNase 18.0 17.7 50.0 14.3

(3) -RNase (2)

24.8 20.4 38.3 16.5

High fu'J:AI DNA ratio

+ RNase (3) 21.5 27.5 37.2 13.8

200

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FIG. 2. Saturation curves for hybrid formation between chicken spleen rRNA and chicken testicular DNA at low RNA/DNA input ratio. Each vial contained 112 /Lg DNA trapped on nitrocellulose membrane filter and varying amounts of 22P-labeled rRNA (specific activity 40,000 cpm/ug) in 5 rnl 3 X SSC. Incubated at 65° for 15 hours. The crude hybrid was not RNase treated.

excesses of heterologous RNA on the percentage of hybridization. CHICKEN SYSTEM

Ribosomal RNA prepared from chicken tissues yielded only one peak on MAE: columns. Sucrose density gradient (5-20 %)

centrifugation or analytical ultracentrifugation revealed that the single MAR column peak contained the 18S and 28S components of rRNA. The mixtures were used in two types of hybridization experiments: (a) low RNA/DNA ratios (less than 0.1) to detect readily hybridizable species, and (b) high

RIBOSOMAL RN A SITES IN CHICKEN DNA

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FIG. 3. Compe tition experiment in chicken system at low fu'{Aj DNA in put ratio. E a ch vial cont ain ed 112 JLg chic ken testi cular DN A trapped on nit rocell ulose membrane filter, 3 pg chicken spl een rRNA-~'P (specific activity 35,500 epm/~g) and unlab eled rRNA :from dilTerent sources in 5 ml 3 X SSC. Incub ated a t GSo f or IG hour s,

from E. coli and sn NA from rat liver did not compete. W hole chick RNA. The specific activity of A . H ybrid ization at Low RNA/DN A rRNA obt ained from whole chick was 2-5 Inpu t Ratio times lower than. R NA from spleen cells in Tissue culture spl een R N A. When about cultu re ; however, count s at saturation wer e 100 ,ug chicken testicular or liver DNA was 250-350 cpm with 100J.lg D NA. Nine exper iincubat ed w ith in vitro-labeled chicken men.ts y ielded apparen t saturation values spleen rItNA-32p t he saturation curves ranging from 0.0145 to 0.026 %. Unlabeled shown in Fig. 2 were obtained. The DNA rRNA from chicken spleen, liver, kidney, appeared to be s aturated when about and testicles inhibited comp et itively 82p_ G.on % of its weight ill RNA h ad been hy- hybrid formation ; unlabeled heterologous bridized. In three additional saturation RNA did not com pete (Fig. 4). Ch icken liver R NA behaved ve ry much experiment v alues between 0.0055 and like t he whole chick R NA an d spleen RNA. 0.012 % were obtained. In competition experi me nts it was found An apparent saturation was obtained at a that rRNA fr om ch icken spleen, liver, kid- RNA /D NA rati o of 0.01 45 %. n ey, an d testicles inhibited in a compet itive B . H ybridization at H igh RNA/DNA manner t he for mation of hybrid between in I n put Ratio vitro-lab eled chicken spleen rRNA-32P and testicular DNA (F ig. 3). Furthermore, The plateau in the foregoing experiments rUNA from nl,t liver and rabbit spleen also was taken as the point of abrupt change in compe t ed in the chick en spleen. rRNA testic- hybridizing efficiency. Careful experiments ular DNA sy stem indicati ng a close evolu- showe d t he amount of RNase-resistant comtion ary relationship . As expected , rRNA plex slowly increased with higher RNA conratios (up to 50) to study the type of interactions with large ex cesses of RNA.

202

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FIG. 5. Saturation curve for rRNA from whole chick at high RNA/DNA input ratio. Each vial contained 10.6 f,tg chicken testicular DNA trapped on nitrocellulose membrane filter and varying amounts of 3H-labeled rRNA (specific activity 2930 cpm/ug) from whole chick in 5 ml 3 X sse. Incubated at 65° for 15 hours.

centrations. Finally another "plateau" was observed with about 500 times more rRNA (Fig. 5). At this point the RNA/DNA ratio was about 0.2 %. This varied from 0.18 to 0.33 % in8 experiments with whole chick and chick liver RNA. Competition experiments, shown in Fig. 6, failed to confirm this plateau as representing a true rRNA hybrid. In this Case all rRNA preparations used reduced the radio-

activity in the hybrid, even E. coli rRNA. The competition differed considerably from the theoretical competition curve. This less efficient reaction appeared to be nonspecific and not representing true hybrid formation. HYBRIDIZATION EFFICIENCY

The efficiency of hybridization is defined here as the fraction of the totallabelecl RNA which formed a RNase-resistant complex

RIBOSOMAL RNA SITES IN

CHIC~EN

203

DNA

B ASE ANALYSES with 100 p,g DNA under standard incubation conditions. It was found that the efficiency Table I gives the base ratios of RNA-32P of hybrid formation between E. coli DNA recovered from the RNase-resistant hybrids . and RNA was directly proportional to the Ribonucleic acid from E. coli which was amount of D::\l"A presen t on filter. Therefore hybr idized on E . coli DNA had a base all efficiencies were calculated on a 100 Mg composition substantially similar to the D NA basis, The efficiency at saturation input RNA. The base compositions of the varied widely; the results are summarized hybridized chicken RNA preparations both in Table II. It can be seen that there is a at low and high RNA/DNA input levels striking difference in hybridization efficiency were different from that of the original between the E. coli system and the chicken RNA. At the high RNA/DNA input ratio system. In the chicken system the nonspe- the hybrid was rich in purine nucleotides cific complex formation at high RNA input (A and G), which suggests an attack by levels had very low efficiency. RNase on the hybridized RNA. At the low

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FlO . G. Competition experiment in chicken system at high RNA/DNA inp ut ratio . Each vial contained 11 Mg chic ken testicular DNA trapped all nit rocell ulose me mb rane filter, 130 ,ug rUN A_aH from whole chick (specific activity 293Q cpm/,ug) and unlabele d rRN A fro m different sources in 5 ml 3 X SSC . Incubated at 07° for 15 hours.

TABLE II RNA

HYBlUDIZ ATION EFFICIENCY OF RIBOSOMA L

WITH

100 /log

HOMOLOGOUS

DNA

% of RNA hyb ridi zed at saturation Source of RNA

Lab eling condi lions

Low RN A/DNA rat io

%

E. coli Chicken spleen Chicken live r Whole chick

in in in in

vitl'o vi tro vivo vivo

10-17 0 .5- 1.5

2.9 0.4 -0 .7

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Number of test.

%

7 G 1 9

0 .1 0.1-0.25

Number of tests

1

8

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FIG. 7. Sucrose gradient (linear, 5-20%) centrifugation in Spinco SW 25.1 head at 4°, 24,000 rpm for 15 hours. (... ) Unlabeled rRNA from whole chick (absorbaucy marker); ( - ) rRNA-32P from whole chick which had been incubated in 3 X SSC at 65°for 15hours.

RNA/DNA input level only the guanine content was higher than in starting material, but the base composition approached the input material when the hybrid was not treated with RNase. FATE OF RNA DUllING INCUBATION About 75% of the unbound RNA in a hybridization mixture could be recovered from the supernatant fraction by precipitation with 2 volumes of ethanol. This recovered RNA showed a sedimentation pattern unlike the original (Fig. 7). The major portion sedimented at about 10-15S. Apparently the incubation conditions had altered the sedimentation characteristics. However, there appeared to be no very small species capable of binding with nonspecific loci. DISCUSSION

Attardi et ol. (1, 2) recently published a comprehensive study about the recognition of rRNA sites on DNA of HeLa and other human cell lines. They found that DNA from HeLa cells could apparently be saturated at a level of 0.02 % with homologous 288 rRNA. However, close examination revealed the existence of partial hybrids involving RNA with base composition significantly different from that of rRNA. Attardi

and co-workers resolved the recovered RNA into fractions by sucrose gradient centrifugation and found that the heavier fractions had base ratios similar to those of the original RNA. They concluded that these fractions were derived from complexes involving rRNA sites. Thus the corrected values for 28S rRNA were calculated to be 0.0030.005 %; and for 188 rRNA, 0.002-0.003 % of total HeLa DNA. The results of our investigations are in many aspects similar to those of Attardi and co-workers. First we obtained an apparent saturation plateau for rRNA from chicken spleen and liver as well as from the whole chick at a RNA/DNA ratio of about 0.01-0.02 %. Base analyses of the recovered RNA revealed a high guanine content indicating some attack on the complex by RNase, since the crude hybrid before RNase treatment had a nucleotide composition similar to that of rRNA. However, competition experiments strongly suggested that the hybrid obtained with low RNA/ DNA input ratios contained complexes specific for rRNA sites. The similarities in saturation plateaus of the in vitro·labeled chicken spleen rRNA (where a chase with unlabeled orthophosphate provided time for the turnover of unstable RNA) and the rRNA obtained from chick liver or whole

205

RIBOSOMAL RNA SITES IN CHICKEN DNA

chick (where a chase was not possible) ensures that there was no appreciable interference by informational RNA. The "forced" saturation plateau-like results obtained at 0.2 % RNA/DNA level with very low hybridization efficiency appeared to be artifacts as demonstrated by base analyses and competition by heterologous RNA,

6. 7.

8. 9.

10.

REFERENCES

11.

1. ATTAHDI, G., HUANG, P. C., AND KABAT, 8., Proc, Nall. Acad. Sci. U.S. 00, 1490 (1965).

12.

2.

ATTARDI,

G.,

HUANG,

P.

C., AND KABAT, S.,

Proc, Nall. Acad. Sci. U.S. 54, 185 (1965). 3. CHIPCIIASE, M. 1. H., AND BIRNSTIEL, M. L., Proe, Nall. Acad. Sci. U.S. 110, 1107 (1963). 4. GILLESPIE, D., AND SPIEGELMAN, S., J. llfol. Biol. 12, 829 (1965). 5. HAYASHI, M., AND SPIEGELMAN, S., Proc. Nall. Acad. Sci. U. S. 47,1564 (1961).

J. D., AND HERSHEY, A. D., Anal. Biochem. 1, 66 (1960). MARMUR, J., J. Mol. Bio~. 3, 208 (Hl61). MCCONKEY, E. H., AND HOPKINS, J. W., Proc, Natl. Acad. Sci. U.S. 51,1197 (1964). MERITS, 1., Biochim, Biophys. Acta 108, 578 (1965). PATTERSON, R., SUSZKO, 1. M., AND PIW. ZANSKY, J. J., J. Immunol. 90, 829 (1963). RITOSSA, F. M., AND SPIEGELMAN, S., Proc, Natl. Acad. Sci. U.S. 53, 737 (1965). MANDELL,

VERMEULEN,

C.

W.,

AND

ATWOOD,

K.

C.,

Biochem. Biophys. Res. Commun, 19, 221 (1965). 13. YANKOWSKY, S. A., AND SPIEGELMAN, S., Proc. Natl. Acad. Sci. U.S. 48, 1069 (1962). 14. YANKOWSKY, S. A., AND SPIEGELMAN, S., Proe. Nall. Acad. Sci. U.S. 48, 1466 (1962). 15. YANKOWSKY, S, A., AND SPIEGELMAN, S., Proc, Natl. Acad. Sci. U.S. 49, 538 (1963).