Proceeding8 of the National Academy of Science8Vol. 65, No. 2, 349-356, February 1970

Ribosomal RNA Homologies among Distantly RelatedOrganisms*

Arnold J. Bendicht and Brian J. McCarthytDEPARTMENTS OF MICROBIOLOGY AND GENETICS, UNIVERSITY OF WASHINGTON,

SEATTLE

Communicated by Anton Lang, December 2, 1969

Abstract. The similarity in base sequences of ribosomal RNA (rRNA) inwidely divergent organisms was compared. Hybrids were prepared betweenEscherichia coli and pea rRNA on the one hand and the DNA of several orga-nisms on the other. The varying thermal stabilities of these hybrids were takenas evidence for a hierarchy of rRNA relatedness among some higher plants, andfor phylogenetic relationships among some species of bacteria, protozoa, fungi,plants, and animals. The large rRNA molecule appears to have evolved fasterthan the small rRNA molecule.

Homologies in the base sequences of ribosomal RNA (rRNA) have often beenused as a criterion for relatedness of organisms. These molecules have proved tobe highly conserved during the evolution of bacteria,1-5 insects,6 and protozoa.7There is also some evidence suggesting that this is the case for vertebrates8 andhigher plants.9 These findings have been extended by the demonstration ofpartial rRNA homology between vertebrates and invertebratesl° 11 and betweenthe toad, Xenopus, and higher plants. 10The present study demonstrates the conservative nature of plant rRNA in

evolution and compares the divergence of base sequences in rRNA moleculesamong a number of plants, animals, and microorganisms.

Materials and Methods. The following seeds were used: hexaploid wheat (Trit-icum aestivum L., cv. Seneca), barley (Hordeum vulgare L., cv. Himalaya), oats(Avena sativa L., cv. Victory), cucumber (Cucumis satiVs L., cv. National Pickling),pea (Pisum sativum L., cv. Alaska). Cedar (Cedrus deodara, Loud.) pollen and daffodil(Narcissus pseudonarcissus L.) bulbs were also used.12 DNA-filters of yeast (Saccharo-myces fragilis), razor clam (Siliqua patula, Dixon), mouse, and E. coli were donated.'3Tritium-labeled E. coli 16S and 23S rRNA's were gifts of R. L. Moore and were thesame preparations as used earlier.4DNA extraction: Seeds were germinated and grown in tap water in trays with

Pearlite. After 1-2 weeks the seedlings reached a height of 3-6 in. and the shootswere cut off 1-2 in. above the surface. Young shoots from bulbs were harvested in thesame way. The shoots were cut into approximately 1-in. segments, wrapped in aluminumfoil, and frozen at -80'C. The frozen plant material was pulverized to a powder in adry ice-chilled mill designed to make flour from grain (Rollmix blender, Universal Dis-tributors, Culver City, Calif.). Small fragments of dry ice were added to the frozen tissuein the mill just prior to pulverization. DNA was extracted from frozen powdered tissueas described earlier.'4 The tissue was ground at room temperature with a mortar andpestle with sufficient (3-7 ml/10 gm of tissue) extraction medium (0.5 M NaCl, 0.1 M

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Na-EDTA, pH 8, 1% sodium lauryl sulfate) to yield a thick paste when grinding. Pollen(0.5 gm) from Cedrus deodara was ground by hand in a chilled mortar and DNA wasextracted as described above.

Extraction of pea RNA: Pea seeds were placed in a sterile beaker and soaked for5-10 min sequentially in: 3% Roccal (a cationic disinfectant), 10% O-syl (ananionic disinfectant), and 1-1.5% sodium hypochlorite (commercial bleach). All subse-quent steps were done in a tissue culture hood. The seeds were rinsed three times withsterile water and transferred to a sterile Petri dish containing filter paper and sufficientwater to keep the seeds moist. Twenty-two germinating seeds were transferred 3 daysafter planting to a dish 6 in. high and 3 in. in diameter fitted with a lid as in a Petri plate.Then 300 ,uCi each of 3H-adenine (1 mCi/0.0199 mg) and 3H-uracil (1 mCi/0.0214 mg)(purchased from New England Nuclear), dissolved in 4 ml sterile water, were added tothe plants and additional water was added during the 5-day labeling period when needed.A 300-fold excess of unlabeled uracil and adenine was added for a 10-hr period before theplants were washed and frozen. The RNA was extracted using cold phenol by the methodof Click and Hackett"5 using their pH 5 buffer and a setting of 20 (scale of 100) on a VirTishomogenizer. The large (25S) and small (16S) rRNA's were separated by sucrosegradient centrifugation."3

After extensive investigation on the effects of bacterial contamination, procedureswere developed for axenic labeling of nucleic acids from plants.'3 There were no bacteriadetected on these plants by plate count and no radioactivity was found associated withbacterial size rRNA in sucrose gradient centrifugation.RNA/DNA hybrid formation: DNA filters (6.3 or 7.2 mm diameter containing

5-12 jg DNA each) were prepared as previously described.'6 RNA/DNA hybrids wereformed in 0.2 ml 2 X SSC (SSC = 0.15M NaCl, 0.015M Na citrate) in 40 X 10 mm vialswith 2-4 DNA filters and 0.3 ,ug E. coli or 0.5 Mg pea 3H-rRNA for 17 hr at 600 or 710C.About 0.2 ml mineral oil was added and the vials were capped. After incubation, thefilters were washed for 5 min twice in 5 ml 2 X SSC at the incubation temperature. Thefilters were then blotted, skewered on pins, dried, and counted.Thermal stability profiles: Skewered DNA-filters containing duplexed RNA were

rinsed with toluene, dried, and then immersed in 5 ml 1 X SSC at 60°C for 5 min.The pins with the filters were transferred to another 5 ml 1 X SSC and kept for 5 min at ahigher temperature. This was repeated at each of the specified temperatures. Up to10 filters were used on one pin and filters from duplicate incubations were mounted on thesame pin. Twenty ug beef serum albumin (BSA) was added to the cooled tubes whosecontents were then precipitated with 5% trichloroacetic acid, collected on Bac-T-Flexmembrane filters, type B-6, (Schleicher and Schuell), dried, and counted. The cumula-tive percentage of total radioactivity eluted at all temperatures was plotted versus tem-perature to give a thermal dissociation profile; Tm was that temperature at which 50%of the radioactivity was eluted. Radioactivity remaining on the filters after incubationat the maximum temperature (99-101°) was up to 10% of that bound for distantly re-lated organisms (e.g., E. coli and pea). This figure was about 2% for homologous rRNA-DNA reactions and about 50% for E. coli rRNA-guinea pig DNA. These counts werenot included in calculations. The differences in Tm between the homologous and heterol-ogous duplexes (ATm) is reported only for thermal dissociation profiles determinedsimultaneously.

Radioactivity measurements: All samples were counted in a Packard Tri-Carbscintillation counter in Liquifluor (purchased from Nuclear-Chicago) diluted withtoluene. Tritium counts per minute when hybridized on DNA-filters were determinedto be- 1.5-fold the counts per minute of the same amount of radioactivity when trichloro-acetic acid precipitated in the presence of 20,g serum albumim.'3

Results. The genetic relatedness of a diverse group of organisms was assessedusing hybridization of the large and small rRNA molecules with DNA as thecriterion.

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VOL. 65, 1970 GENETICS: BENDICH AND MCCARTHY 351

The thermal stability profiles of hy- 1001-brids between filter-bound DNA and E.coli 3H-rRNA are shown in Figure 1. A /summary of the binding and thermal 60 -!stability data is presented in Table 1. 40 ,//-/* YeastThe relatively strong interaction be- 1 / * ---e Peatween E. coli rRNA and pea DNA is '20-0,-- E co -perhaps surprising. Furthermore, theE. coli rRNA-pea DNA hybrid is con- 100siderably more stable than that of E.

Q

_ -coli rRNA-yeast DNA. The ATm 80 / /values listed in Table 1 are larger for Q. 60 - /the 23S RNA than for the 16S RNA. /The difference between ATm for 23S / - b. 23Sand 16S RNA is quite large for B. sub- 24, /tilis DNA and pea DNA. The interac-tion with guinea pig DNA-filters is ex- 60 70T (aC) 90 10tremely low and probably representsthe noise level in these experiments. FIG. 1.-Thermal stability profiles of RNA/DNA hybrids formed with E. coli rRNA.Analogous measurements were made 3H-labeled E. coli 16S (a) and 23S (b) rRNAusing pea 3H-rRNA. Figure 2 illus- was hybridized with DNA-filters of the in-trates the thermal dissociation profiles dicated organisms. Details are given inof both the 25S and 16 SrRNA's hy-bridized to pea DNA-filters at 600 and at 71 'C. The Tm values are independentof the temperature of incubation at least in this temperature range, and areslightly higher than those obtained with the E. coli system. The curves are alsosteeper than those in the E. coli system. The base compositions of E. colil' andpea15 rRNA's are very similar. Figure 2 also includes thermal dissociation profilesof hybrids of pea rRNA with wheat and sea urchin DNA determined in parallel.Figure 3 shows the melting curves of several other hybrids. A summary of theresults of all the rRNA comparisons made is presented in Table 2.The direct binding data may sometimes be misleading. Cucumber DNA-

filters bound more 3H-pea rRNA than did pea DNA filters. However, thestability of the cucumber hybrids was much lower than that of the homologouspea hybrids. The same situation existed for pea-yeast hybrids. It is knownthat in both E. colil8 and pea,'9 rRNA cistrons account for some 0.3 per cent of thecellular DNA. In yeast (Saccharomyces cerevisiae), rRNA cistrons constituteabout 2.4 per cent of the DNA.20 Therefore, the anomalously high binding ofpea rRNA to yeast DNA filters is readily explainable. The quality of base-pairing in the hybrids is poor but the quantity is large because of the eightfoldexcess of ribosomal DNA on the yeast filters over that on the pea filters. Asimilar situation probably exists for cucumber DNA. From the work of Mat-suda and Siegel9 it appears that the DNA of several plant species, which havedense satellite components of DNA in CsCl gradients, hybridizes more efficientlywith tobacco rRNA than does tobacco DNA itself. When cucumber DNA wasanalyzed in an analytical CsCl gradient, two dense, major satellite components

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TABLE 1. Interaction of E. coli ribosomal RNA with various DNA's.Bound Relative to

Filter- E. coli ATm 238bound (%) -Tm (0CQ- -ATm (0C)-. minusDNA 23S 168 23S 16S 23S 16S ATm 16S

E. coli 100 100 84.2 82.5B. subtilis 88 83 72.4 73.9 11.8 8.6 3.2Pea 25 19 67.3 69.2 16.9 13.3 3.6Yeast 20 13 62.0 61.1 22.2 21.4 0.8Guinea pig 1 1

Three-tenths jug 3H-labeled E. coli 23S or 16S rRNA (13,400 cpm) was incubated for 17 hr at600C in 2 X SSC with 19-23 jg filter-bound DNA. After direct binding was assayed, thermal sta,bility profiles and Tm values were determined using DNA filters from duplicate experiments. 14.5and 15.3% of the input 23S and 16S 3H-rRNA respectively was bound to E. coli DNA-filters.

were found. It is possible that one or both of these satellite bands contain rRNAcistrons: this would account for the high binding observed.DNA from bacteriophages 80 and T4, which do not contain genes for rRNA,

bound only a few per cent relative to pea DNA. This binding is taken as thenoise level in these experiments. The binding to the rodent DNA's may bemarginally greater than the noise level and the binding to razor clam DNA mayalso be significant.

loo ,~- X I

600 70 80 90 100 60 70 80 90 100

Wheateratr-TeTehrte a80-~~~~~~~80 ~ e

RNA/DN hybid 16meat6°ad7° RADAhbrdS omdwt e R

7.Pea.

the inu H1S Aor86a 7.3% peaDN-filters.Barlluesforhey

for CucumbewsbudopaDA gu eahbiswee8. nd8.°fr2itr at a.and 71° repciey2016 RAad2SrR epciey

CZ0~~~ ~~~~~~~~4

FaluG 2o therhmalogostpablt pyroiles ofFGw.-hraetaiiyprflso

RN/NAhbrd0freda 60an 10 RA/N-hbidIore wt parRA

8600 (or871 foron D. 7.0and 6% thei

filter 5sr at 60 and 710, respectively.Tr

for258rRNA at 60 and 710,brespectively

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VOL. 65, 1970 GENETICS: BENDICH AND MCCARTHY 353

TABLE 2. Interaction of pea ribosomal RNA with various DNA's.Bound Relative to

Filter- Pea ATm 258bound (%) -Tm (0C)- .ATm (0C)Y minusDNA 25S 16S 258 16S 25S 168 ATm 16S

Pea 100 100 86.4* 87.3*Cucumber 211 184 81.7 83.0 4.8 4.5 0.3Barley 46 53 80.2 81.8 6.3 5.7 0.6Oats 17 26 79.8 80.0 7.3 6.8 0.5Wheat 24 26 75.6 78.3 10.7 9.0 1.7Daffodil 12 16 76.8 80.7 8.8 7.0 1.8Cedar 20 31 76.0 78.4 10.5 9.1 1.4Yeast 105 125 75.0 78.5 12.1 8.3 3.8

(Saccharomycesfragilis)

Sea urchin 18 18 71.7 75.0 14.6 12.3 2.3Tetrahymena 51 44 72.3 73.2 14.2 14.3 -0.1Euglena 9 17 63.0 69.9 22.6 18.8 3.8B. subiilis 20 41 71.6 15.2E.coli 18 43 64.3 68.1 22.8 18.7 4.1Clam 6 11Mouse 3 6Guinea pig 3 5T4 3 4080 1 1

* Tm values for the homologous pea hybrids were 86.3, 86.5, 87.1, and 85.6 degrees for 25S rRNAand 87.3, 87.5, 86.8, and 87.7 degrees for 16S rRNA; the average value is presented in the table.

Five-tenths jg 3H-labeled pea 25S or 16S rRNA (2100 or 1900 cpm) was incubated 17 hr at 600Cin 2 X SSC with 19-23 jig filter-bound DNA. After direct binding was assayed, thermal stabilityprofiles and Tm values were determined using DNA filters from duplicate (quadruplicate for daffodiland Euglena) experiments. 8.1 and 6.6% of the input 3H-25S and 168 rRNA, respectively, was themean bound to pea DNA-filters. The ATm values in the table were derived from thermal elutionprofiles run in parallel rather than from the average pea Tm.

Using the ATm values as a criterion, it is possible to establish a hierarchy ofrelatedness among these organisms. In general, the descending order shown inTable 2 follows that expected for higher plants. The AT. values increase inprogressing down the list in Table 2. Thus the dicotyledon, cucumber, is closestto peas, with the monocotyledon and gymnosperm species following in that order.The weakness of the interaction with Euglena is perhaps surprising since bothTetrahymena and sea urbhin are closer to peas according to this criterion.In agreement with the data for E. coli rRNA, the difference between the ATm

values for 25S and 1S molecules is a positive number (Table 2). Furthermore,the magnitude of this difference increases with greater ATm values. This iscompelling evidence for .the thesis that the rate of accumulation of mutations, andtherefore the rate of evolution in the large rRNA molecule is greater than in thesmall rRNA molecule.

Discussion. The reslilts of Tables 1 and 2 may be interpreted as a measure ofthe extent of base sequpnce divergence between various rRNA cistrons of dif-ferent species. The decrease in thermal stability of nucleic acid heteroduplexesseems to be linearly related to the extent of base sequence divergence.21-23 It islikely that this relationship is only approximate and the decrease in thermalstability is a function of the nature of the actual base substitution and the distri-bution of changes along the molecule. Nevertheless, we will assume that theAT. is a quantitative approximation of the extent of sequence divergence.

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From a phylogenetic viewpoint, the findings then permit the following inter-pretation. The base sequence divergence of rRNA genes between E. coli andpea is less than between E. coli and yeast (Table 1). The rRNA divergencebetween pea and Euglena is as great as between pea and E. coli (Table 2). Thedivergence measured here depends on the degree of base substitution in the rRNAgenes of the two organisms since the time of their departure from a commonancestor. This is probably a function of the number of generation~saithattime. If fungi and higher plants have a common ancestor, many more genera-tions must have occurred since that point in the evolutionary line leading to con-temporary fungi than to present-day higher plants. We assume that the numberof generations and accumulated mutations in the line from a prokaryotic pre-cursor to the point of divergence of the pea and yeast lines is large. This wouldexplain why pea and yeast rRNA sequences are more closely related to one an-other than either is to E. coli and why E. coli rRNA is more closely related to thatof pea than that of yeast. A similar argument may be applied to the results forEuglena, although here additional factors must be invoked since pea is probablyseparated from E. coli by more generations than from Euglena. On the criterionof rRNA similarity, it is necessary to conclude that higher plants and fungi aremore closely related than are either to the Euglenophyta. These results are con-sistent with a recent, parallel study of Bicknell24 using the rRNA of the largeribosomal subunit of Saccharomyces cerevisiae in which competition hybridizationexperiments showed a closer relationship with pea DNA than with the DNA of E.coli, Euglena, or Tetrahymena.The data in Table 2, showing that the difference between ATm 25S and ATm.

16S increases with divergence between organisms, strongly support our proposalthat the large rRNA molecule evolves more rapidly than the small one. Anydegradation of the RNA's occurring during extraction and centrifugation wouldresult in contamination of the 16S fraction with 25S RNA, and not the reverse.This could only decrease the difference between ATm25S and ATm16S. Mooreand McCarthy4 reported no significant difference between ATm23S and ATm16S incomparing E. coli rRNA cistrons with those of 9 Gram negative bacteria. How-ever, their data show a large (about 3°C) difference when the 1 Gram positivebacterium, Bacillus subtilis, was compared with E. coli.

Multiple cistrons exist for both the small and large rRNA molecules. Thenumber of such cistrons has been estimated to be from three to ten in bacteria25and several hundred in Drosophila melanogaster,26' 27 HeLa cells,8' 28 or Xenopuslaevis.29 A study of the relationship among these cistrons of E. coli and rabbitwas made by Moore and McCarthy. 0 The thermal stabilities of rRNA hybridsof rabbit DNA were more dependent upon the conditions of reaction during theirformation and always lower than those of E. coli. It was concluded that rRNAcistrons in rabbit DNA exhibit far greater intercistronic base sequence divergencethan those of E. coli. In contrast, analogous experiments with pea rRNA (Fig.2) suggest a high degree of similarity of base sequence among the multiple rRNAcistrons in plants. Similar results were obtained for rRNA cistrons in Droso-phila3l and yeast.24The relative extent of binding of pea rRNA to various DNA-filters (Table 2)

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VOL. 65, 1970 GENETICS: BENDICH AND MCCARTHY 355

provides information concerning the fraction of the genome devoted to thesynthesis of rRNA. The binding with barley is about twice that with oats orwheat. Since these three species are all in the same family, Gramineae, thiskind of analysis would predict that the fraction of the genome in barley constitut-ing rRNA genes is greater than that in oats or wheat. About twice as much 3H-rRNA from Xenopus hybridizes with barley than with wheat DNA.10 Theanomalously high binding to cucumber DNA and to yeast DNA has already beendiscussed.

It should be recalled that Trewavas and Gibson32 have compared plants using32P-RNA extracted from peas and concluded that rRNA nucleotide sequencesamong plants are no more conserved than those of the total DNA. However, itis likely that their 32P-RNA preparation was dominated by labeled bacterialRNA. This could account for the unreasonably sluggish drop in their competi-tion curve.One possible source of error in our deductions must be examined. The

chloroplasts and mitochondria of eukaryotes contain DNA33 and rRNA similar insize to bacterial rRNA.34-N The possibility then arises that the observed hy-bridization of bacterial rRNA to plant DNA is an attribute of plastid DNA ratherthan of the nuclear DNA. However, as the following argument shows, only avery small fraction of the hybrid may be ascribed to plastid nucleic acids. TheDNA in chloroplasts and in mitochondria represent a few per cent of the totalcellular DNA.33 If the binding of E. coli rRNA with purified plastid DNA wereequal to the binding with the homologous E. coli DNA, which would assume nobase sequence divergence between E. coli and plastid DNA, then about 20 percent of the DNA on the pea DNA filters would have to be plastid DNA to accountfor the observed cross reaction. This is much greater than the fraction of plastidDNA in plant cells. The fraction of the total rRNA in eukaryotic cells that is ofplastid origin is also small. Plastid rRNA's have sedimentation rates distin-guishable from those of cytoplasmic rRNA's and yet are undetectable in sedimen-tation profiles of unfractionated rRNA.34 5 Since E. coli DNA bound 40 percent as much pea 3H-25S RNA as did pea DNA, there would be far too littleplastid rRNA in the pea rRNA preparation to account for this binding. Thehierarchy of direct binding and ATm in Tables 1 and 2 makes it even less likelythat plastid nucleic acids are contributing a significant effect.We conclude that the comparison of ribosomal genes may be a valuable tool for

investigating the initial divergence of the eukaryotes.* This research was supported by grant GB 6099 from the National Science Foundation.t Present address: MSU/AEC Plant Research Laboratory, Michigan State University,

East Lansing, Mich. 48823.t Present address: Departments of Biochemistry and Genetics, University of Washington,

Seattle, Wash. 98105.I Dubnau, D., I. Smith, P. Morell, and J. Marmur, these PROCEEDINGS, 54, 491 (1965).2 Doi, R. H., and R. T. Igarashi, J. Bact., 90, 384 (1965).3Takahashi, M., M. Saito, and Y. Ikeda, Biochem. Biophys. Acta, 134, 124 (1967).4Moore, R. L., and B. J. McCarthy, J. Bact., 94, 1066 (1967).6 Kohne, D. E., Biophys. J., 8, 1104 (1968).6 Laird, C. D., and B. J. McCarthy, Genetics, 60, 303 (1968).7Gibson, I., J. Protozool., 14, 687 (1967).8 Attardi, G., P. C. Huang, and S. Kabat, these PROCEEDINGS, 54, 185 (1965).

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O Matsuda, K., and A. Siegel, these PROCEEDINGS, 58, 673 (1967).10 Sinclair, J. H., and D. D. Brown, in Carnegie Inst. Wash. Yearbook 67 (1968), p. 404.11 Skinner, D. M., Biochem., 8, 1467 (1969).12 These seeds were donated: wheat, H. N. Lefever (Ohio Agricultural Research and De-

velopment Center); barley, A. Kleinhofs (Washington State University); oats and cucumber,R. E. Cleland (University of Washington). Daffodil bulbs and peas were purchased fromSears and Lilly Seed Co., respectively. Cedar pollen was collected on the University of Wash-ington campus.

13 Bendich, A. J., Ph.D. thesis, "Biochemical and Phylogenetic Studies of Plant NucleicAcids," University of Washington (1969).

14 Bendich, A. J., and E. T. Bolton, Plant Physiol., 42, 959 (1967). In place of 0.1 X SSC,0.015 M NaCl in 0.01 M Tris pH 8 was used here.

15 Click, R. E., and D. P. Hackett, Biochem. Biophys. Acta, 129, 74 (1966).16McCarthy, B. J., and B. L. McConaughy, Biochem. Genetics, 2, 37 (1968).17Midgley, J. E. M., Biochem. Biophys. Acta, 61, 513 (1962).18 Gillespie, D., and S. Spiegelman, J. Mol. Biol., 12, 829 (1965).19 Chipchase, M. I., and M. L. Birnstiel, these PROCEEDINGS, 50, 1101 (1963).20 Schweizer, E., C. MacKechnie, and H. 0. Halvorson, J. Mol. Biol., 40, 261 (1969).21 Bautz, E. K. F., and F. A. Bautz, these PROCEEDINGS, 52, 1476 (1964).22 Kotaka, T., and R. L. Baldwin, J. Mol. Biol., 9, 323 (1964).23Laird, C. D., B. L. McConaughy, and B. J. McCarthy, Nature, 224, 149 (1969).24Bicknell, J. N., Ph.D. thesis, "Nucleic Acid Homologies Among the Yeasts: A Study

Emphasizing Ribosomal Ribonucleic Acid," University of Washington (1969).26 Yanofsky, S. A., and S. Spiegelman, these PROCEEDINGS, 49, 538 (1963).21 Ritossa, F. M., and S. Spiegelman, these PROCEEDINGS, 53, 737 (1965).2 Vermuelen, C. W., and K. C. Atwood, Biochem. Biophys. Res. Commun., 19, 221 (1965).28McConkey, E. H., and J. W. Hopkins, these PROCEEDINGS, 51, 1197 (1964).29 Wallace, H., and M. L. Birnstiel, Biochem. Biophys. Acta, 114, 296 (1966).30 Moore, R. L., and B. J. McCarthy, Biochem. Genetics, 2, 75 (1968).31 Laird, C. D., and B. J. McCarthy, Genetics, in press.32Trewavas, A. J., and I. Gibson, Plant Physiol., 43, 445 (1968).3- Granick, S., and A. Gibor, Progr. Nucleic Acid Res. Mol. Biol., 6, 143 (1967).34 Stutz, E., and H. Noll, these PROCEEDINGS, 57, 774 (1967).35 Kintzel, H., and H. Noll, Nature, 215, 1340 (1967)." Loening, U. E., J. Mol. Biol., 38, 355 (1968).

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