paramecium.' (m, - pnas

VOL. 52, 1964 GENETICS: GIBSON AND SONNEBORN 869

Breast cancer shows the peak of pressure in the 20-29 to 30-39 periods with adecline in pressure to 40-49 and then with a very low ratio of increase (1.49, 1.40,1.32, and 1.29) in the four remaining age groups. The degree to which the amountof pressure toward tumorigenesis follows the amount of reproductive activity isstriking.

Conclusion.-The relationship between the rate of increase or of decrease incancer incidence at various sites, in successive age periods, may be used to give ussuggestive information concerning the nature of the host response to tumorigenicpressures of different types and derivation.

It should be emphasized that the present presentation and analysis of datais neither extensive nor exhaustive. It is intended merely to show that character-istic responses of each site exist for each sex and to call attention to differences whichneed further study and analysis.

* The present paper is based on a statistical procedure presented before the Academy someyears ago. It utilizes more recent data on incidence rather than those on mortality which werethe basis of the earlier communication.

1 Cancer in Connecticut 1935-51, Conn. State Dept. of Health (1955); Cancer in New York State1941-60, Bureau of Cancer Control, N.Y. Department of Health (1962).

2 It is recognized that other methods may be used, for example, the cohort type of tabulationand analysis. The present communication is, however, intended to raise questions rather than toanswer them and must of necessity be brief. It will therefore be confined to one method ofpresentation of -the data.

3 It should be recognized that data which include cancer of all sites are composite. Theyrepresent a mixed population of competitive trends, the composition of which may vary from yearto year. It is used here merely as an illustration of the method involved.

IS THE METAGON AN m-RNA IN PARAMECIUM AND AVIRUS IN DIDINIUM?*

BY IAN GIBSONt AND T. IM. SONNEBORNDEPARTMENT OF ZOOLOGY, INDIANA UNIVERSITY

Communicated August 10, 1964

Certain stocks of Paramecium aurelia (syngens 1, 2, 4, and 8) contain in theircytoplasm various symbiotic organisms termed lambda, kappa, mu, etc., and thepresence of these organisms results in specific "killer" phenotypes.' The mainte-nance and multiplication of some of the symbionts have been found to depend on agene or genes in the nucleus of the host paramecium.' In stock 540 (syngen 1)either of two unlinked dominant genes (M, and M2) is necessary for the mainte-nance of mu.2 These genes act via an intermediate, the metagon.3 Active metagonscan be introduced into paramecia by exposing them to cell-free extracts of metagon-bearing paramecia.4 Activity of these extracts is associated with the ribosomalfraction and with the RNA component of this fraction.4

Sonneborn5 discovered that kappa could be introduced into another ciliate,Didinium nasutum, by feeding them killer paramecia, and that kappa was thereaftermaintained and multiplied even when the didinia were fed nonkillers lacking thegene essential for kappa maintenance.

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870 GENETICS: GIBSON AND SONNEBORN PROC. N. A. S.

The present paper describes the behavior of mu in Didinium and the nature ofthe metagon in Paramecium and Didinium. The evidence indicates that themetagon replicates in Didinium and that metagon RNA hybridizes specificallywith DNA from paramecia bearing anM gene.

Materials and Methods.-(1) Cultures: The various stocks of Paramecium aurelia used have beendescribed elsewhere,3 as have the methods of culturing the cells in test tubes and larger masscultures.4 Stock 540 (genotype M1 M1 M2 M2) bears mu particles and is a killer. Branches of itexist free from mu, and these are no longer killers but sensitives, containing, metagons, however.Stocks 513 and d-200 (genotype ml ml m2 m2) are sensitive and lack both metagons and mu. Stockd-200 is largely isogenic with stock 540 except for the m genes; it was derived by back-crossingthe progeny of a hybrid (540 X 513) eight successive times to stock 540.Four stocks of Didinium nasutum were used: 1, from Twin Lakes, Indiana; 1*, derived from

1 by growth at 310C long enough to free it from kappa that had been introduced into it;5 2, fromGeneral Biological Supply House, Chicago; and 3, from Yellowood Lake, Indiana. Didiniumnasutum feeds and thrives on live paramecia. Test-tube and larger cultures of didinia were grownby adding packed paramecia in quantities and with a frequency adjusted to yield the growthrates and population densities appropriate to the various experimental needs. Routinely, 0.3 to3.0 gm/liter (wet weight) of packed didinia could usually be obtained.

(2) Preparation of ribosomal extracts from the two ciliates: The solutions, conditions of homog-enization, and the details of differential centrifugation were essentially the same as used pre-viously4 for Paramecium, with the following modifications. The microsomal pellet obtained at105,000 X g was treated with sodium deoxycholate and respun at the same force to yield a ribo-somal pellet. Ribosomal extracts from both Paramecium and Didinium were suspended in asolution consisting of 0.01 M Tris, 5 X 10-3 M MgClo, and bentonite 1 mg/ml. The suspensionwas dialyzed against this same solution overnight and was then stored at -20°C.

(3) Preparation of RNA: Ribonucleic acid was extracted from the microsomes using either oftwo techniques: sodium lauryl sulfate and phenol (technique a)4 or guanidine hydrochloride(technique b).7 These methods gave nucleic acid preparations in which neither DNA nor proteincould be detected.6 After precipitation with ethanol, the RNA was dissolved in SSC (0.015 Msodium citrate, 0.15 M sodium chloride) at a final concentration of 2 mg RNA/ml. This solutioncould be stored at +4°C and used over a period of up to 2 months.

(4) Method of detection of metagon activity: The two methods of detecting metagon activity havebeen described.4 Both involve infection of metagons into cells about to lose mu because of lossof metagons. Method a, the method of 11th-fission cells, employs cells 11 fissions past the changeat autogamy from genotype M1 ml m2 m2 to mI ml m2 mi. The number of metagons per cell of thelatter genotype decreases until by the 11th fission about 50% of the cells have none, and thereforeno mu, and the others have only one or a few metagons but a full complement of 103 or more mu.They are therefore destined to yield only one or a few descendants with a metagon, and only thesecan maintain mu. The method is therefore to infect with metagons at the 11th fission and examinefor presence of mu the eight animals produced after three more fissions. The increased numberthat possess at least one metagon and therefore mu is a measure of successful infection. Mostcritical is the number of groups with six to eight of the eight cells possessing mu, for uninfectedcontrols seldom, if ever yield, so many (see Table 1). Method b, the method of RNase treatment,destroys the metagons in M1 M1 M2 M2 mu-bearers by exposure to RNase (0.5 mg/ml) for about12 hr; mu is then lost at the next fission unless reinfection with metagons occurs earlier, in whichcase both metagons and mu persist in the clonal progeny.

(5) Nucleic acid hybridization: Paramecia were concentrated and the RNA was extracted asabove. Electrophoresis on cellulose acetate paper was then carried out under ionic conditions inwhich a metagon-containing band, a, appeared.7 This was eluted from the paper and the eluatewas concentrated to 1 ml by dialysis against polyethylene glycol. This method allowed partialpurification of metagon RNA.

Hybridization was accomplished using the DNA agar method of Bolton and McCarthy8 withcertain modifications. Details will be published in a future paper. DNA was extracted (formethod see Gibson7) from Paramecium, Didinium, Tetrahymena, Aerobacter aerogenes (the bac-terium used as food for Paramecium), and purified mu,6 incubated with agar at 1000C, treatedwith ribonuclease 10 wg/ml in 2 X SSC at 250C for 1 hr, and washed with bentonite solution

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(1 mg/ml in 2 X SSC). Following incubation of the DNA with RNA containing a known amountof metagon activity and treatment of the mixture with ribonuclease solution and bentonite asabove, it was placed in a column, and five 5-ml fractions (unhybridized RNA) were removed with2 X SSC at 60'C, then a further five with 0.01 X SSC at 750C (hybridized RNA). The fractions ineach group were pooled and concentrated to give 3 ml of RNA solution. This was then dilutedwith 2 X SSC (or in some cases-e.g., with fractions from Didinium-remained undiluted), anda 1-ml sample was used to test 20-50 cells by procedure a or b. The remainder of the solutionwas stored at -20'C and used if further tests were necessary. The percentage of cells infectedgave a relative measure of the amount of metagon activity present.

Results.-(1) Fate of mu and metagons in strains of Didinium: One didinium ofeach of the four strains was fed one killer paramecium (stock 540) and thereafteran excess of sensitive paramecia (stock 513) for 6 months, about 1,000 cell genera-tions. At the end of this period and earlier, samples of the progeny were examinedfor the presence of mu, and extracts of other samples were tested for metagon ac-tivity. Mu was abundantly present in all individuals examined in strains 1, 1*,and 3; but it was never found in individuals of strain 2. The method of examina-tion was to look at squashed cells by phase contrast microscopy. Ribosomal andRNA extracts of all four strains regularly exhibited metagon activity by both testsa and b. Sample results with strain 2 of Didinium are given in Table 1. Thus,although strain 2 of Didinium could not maintain mu, it maintained the metagon,as did the other strains.

TABLE 1INFECTION OF METAGONS INTO DIDINIUM (STRAIN 2) FROM PARAMECIUM AND INFECTION BACK

INTO PARAMECIUMNumbers of mu-bearing paramecia in groups of 8

Stocks of paramecia used as Type ot Didinium descended atter 3 fissions from single 11th-fissioninitial food of Didinium extract used animals

0 1 2 3 4 5 6 7 8Stock 540 (MK) (metagons Ribosomal 33 0 1 0 0 1 4 5 10and mu) RNA 15 0 0 0 1 0 3 1 5

Stock 540 (Sens.) (metagons, Ribosomal 46 0 0 0 0 2 3 5 26no mu) RNA 19 0 2 1 0 0 0 3 7

Stock 513 (Sens.) (neither Ribosomal 29 7 6 6 3 2 0 0 0metagons nor mu) RNA 29 3 9 11 1 0 0 0 0

None None 39 11 8 10 3 1 0 0 0

After feeding the didinia one paramecium of each of the stocks indicated, they were fed paramecia of stock 513for 6 months. Then extracts (ribosomal or RNA) of the didinia were tested by method a (11th-fission paramecia)for metagon activity. Controls (last line of table) were not exposed to extracts from didinia. The numbers in thebody of the table are the numbers of sets of eight paramecia which included 0 to 8 individuals with mu. Sens. =sensitive. MK = mate killer.

(2) Do didinia possess metagon activity and mu before feeding on killers? Fromthe start, a branch of each of the four strains of Didinium was set aside to be keptfree from paramecia bearing either metagons and mu or metagons alone. Thesebranches were therefore cultured on paramecia of stock 513. The cultures wereexamined and tested after 3 weeks, 3 months, and 6 months. Direct observationrevealed no mu. The standard tests with extracts showed no metagon activity.Thus, unless supplied with mu and metagons from paramecia, didinia do not possessthem.

(3) Does mu depend upon the metagon in Didinium? Three lines of evidencewere adduced to discover whether, in Didinium as in Paramecium, the mainte-nance of mu depends upon the presence of metagons. Exposure of didinia (stocks1 and 3), bearing metagons and mu, to RNase resulted in the loss of both metagonsand mu, the latter disappearing after the first fission. This implies the prior loss of

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metagons, but direct tests for metagons were made 10 days later; none could befound. So, as in paramecia, loss of mu is correlated with loss of metagons.The question was also tested by feeding to didinia (strains 1 and 3) paramecia

containing mu but no metagons. Such paramecia are obtained as in method b(see Methods, §4). Between RNase treatment and the first fission, when the para-mecia bear viable mu but no metagons, they were used as food for the didinia.Thereafter the didinia were fed paramecia (stock 513) lacking both mu and metagons.Mu was looked for in the didinia after 1 and 10 cell generations and after dividingfor 3 months; tests for metagons were made after 3 months. Neither metagonsnor mu could be found. Thus, again, didinia. did not maintain mu in the absenceof metagons.

Finally, the same procedure of feeding didinia RNase-treated paramecia con-taining mu but no metagons was followed, but this time the didinia used werealready carrying metagons. Such didinia are obtained by feeding them parameciaof the sensitive branch of stock 540. In this experiment, the didinia acquired muand maintained it during subsequent generations. Hence, in Didinium as inParamecium, the maintenance of mu depends upon the presence of metagons.

(4) Do didinia possess a gene comparable to the M genes of Paramecium? Threelines of evidence are pertinent. (1) When an M gene is present in paramecia,metagons are regularly formed; but they are not formed in didinia except afterintroduction from paramecia (Results, §2). (2) When an M gene is present inparamecia, loss of metagons by RNase treatment is followed by their reappearance;in didinia as in m1m1m2m2 paramecia (Gibson, unpublished), loss of metagons bythis treatment (Results, §3) is not followed by their reappearance, the loss beingpermanent. Thus, in both (1) and (2), gene M shows the capacity to initiate thegeneration of metagons, a capacity not shown by Didinium. This indicates thatdidinia do not possess a gene comparable to M, an indication supported by the thirdline of evidence, from DNA-RNA hybridization, set forth below (Results, §5, ¶3).

(5) Nature of the metagon in Didinium and Paramecium: The persistence ofmetagons in didinia for 1,000 cell generations after cutting off the supply from par-amecia implies that the number of metagons increased in didinia by a factor of21Y°° or roughly 103° without loss of activity. Since this increase occurred inthe apparent absence of the only known3 generator of metagons, the M genes, andwas dependent upon the presence of metagons for its initiation and for its continua-tion, the metagon seems to have increased by replication like an RNA virus, witha doubling time of about 4 hr (1,000 generations in 6 months). These paradoxicalconclusions about the same object, the metagon-a gene-product in one organism,a virus in another-led us to use the technique of DNA-RNA hybridization toexplore the relation of the metagon to the DNA's of the two organisms.The most striking result (Table 2) is the very high metagon activity recovered

from RNA that had been hybridized with the DNA of M1M1M2M2 paramecia (stock540). Of the RNase-treated paramecia exposed to this RNA, 90 per cent proved tohave been infected with metagons. Only 5 per cent were infected when the DNAused for hybridization came from m1m1m2m2 paramecia (stock d-200). As stocks540 and d-200 are largely isogenic, except for the M-m locus, the results are primafacie evidence for the conclusion that the DNA's of the alleles at this locus are thebasis of the differential binding of the metagon RNA. This conclusion is reinforced

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TABLE 2METAGON ACTIVITY OF RNA DISSOCIATED FROM DNA AFTER HYBRIDIZATION WITH DNA FROM

VARIOUS SOURCESMean % of metagon activity in

Source of DNA hybridized materialDidinium strain 1 (metagons, no mu) 0.67Didinium strain 2 (no metagons or mu) 1.2Mu 1.3Tetrahymena 1Paramecium (metagons, no mu), stock 540, (genotype MIMIM2M2) 90Paramecium (neither metagons nor mu), stock 513, (genotype

MImOmW272) 5Aerobacter aerogenes 1.2

For methods, see text. The percentage metagon activity is the percentage of RNase-treated paramecia (stock540) that retained mu after exposure to the various dissociated RNA's (see method b for detection of metagonactivity).

by preliminary results, to be supplemented and reported later, which indicate thatthe RNA's released from hybridization with the DNA of other isogenic stocksdiffering chiefly in the m alleles yield characteristically different percentages ofinfection, but always much less than when the DNA from MlM1M2M2 parameciais used for the hybridization. The full meaning of these characteristic differencesremains to be explored, but they at least indicate that the m genes are not totaldeletions and that the metagon hybridizes specifically with the M-m locus.

In marked contrast with the results using DNA's from paramecia are thoseobtained with DNA's from Didinium, Tetrahymena, Aerobacter, and mu (Table2). The preparations recovered after attempts to hybridize metagon RNA withthem yielded no more than 1.3 per cent "infection" under conditions in which RNAdid bind to these DNA samples. These values are not significant; they lie withinthe range (1-2%) obtained with controls, i.e., the percentage RNase-treated cellswhich yielded mu-bearing descendants in the absence of exposure to metagonpreparations. In other words, the standard RNase treatment fails to destroy allmetagons in up to 2 per cent of the treated M1M1M2M2 test cells.

These facts appear to warrant the conclusion that the metagon RNA derivedfrom Paramecium is complementary to the DNA of the geneM and to some extentof the gene m, but not to any DNA from Didinium or the other sources. This com-plementarity suggests that the metagon derived from Paramecium is the productor "messenger" of the gene M. Failure of the metagon to hybridize with DidiniumDNA indicates not only the absence of an M-like gene in Didinium, but also thatthe increase of metagons in Didinium occurs by a different mechanism, presumablyreplication. Elsewhere, evidence will be presented showing that the metagon, longafter introduction into didinia, exhibits the same specificity for hybridization withgene M, thus indicating that the presumptive replicating form of the metagon inDidinium still possesses RNA complementary to the M genes.As far as our evidence goes, therefore, the two paradoxical conclusions with which

we were confronted both appear to be correct: the metagon seems to be both anRNA product of geneM and capable of replication.Discussion.-The major feature of our results is their indication that two well-

known properties of different RNA's-origin from DNA and replication-can alsobe properties of one and the same RNA, the metagon. This is so novel and the-oretically important that alternative possibilities of explaining away one or the otherproperty need to be considered.

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Can all increase of metagons be accounted for by origin from genes withoutresort to replication? The M paramecia present no difficulty; the evidence indi-cates that their M genes do produce metagons. The m paramecia present a prob-lem. The kinetics of loss of metagons by dilution in the course of fissions afterM isreplaced by m at fertilization (Methods, §4) is consistent with the assumption of slowreplication of metagons with a doubling time of about 40 hr.'0 The kinetics datahave not been examined in relation to the assumption of slow production of meta-gons by m genes, probably because this possibility seemed excluded by the failureof extracts of m paramecia lacking mu to exhibit metagon activity.4 However, it isconceivable that very low activity would escape detection or that m genes are repres-sible, making metagons only when some are already present. Didinia present amore formidable problem. The hybridization data (Table 2) indicate that they lackDNA complementary to the metagon, i.e., any gene likeM or m, either in their owngenome or as replicates of genes derived from ingested paramecia. That didinialack functioning M genes obtained from paramecia is also shown by the failure tofind metagons in didinia after feeding them RNase-treated M paramecia beforetheir first fission (Results, §3). 11 Thus the data do not appear to allow for metagonproduction by genes in Didinium.Can then all increase of metagons be accounted for by metagon replication with-

out resort to their production by genes? Here didinia present no problem; meta-gons appear to increase in them only by replication. And ordinary m parameciaare irrelevant because they lack metagons, while those briefly possessing metagonsare indecisive: if metagons do actually increase slowly in them, the available evi-dence neither supports nor opposes interpreting the increase as due to replication.But theM paramecia present three difficult problems. (1) Metagon activity reap-pears after its loss during RNase treatment.9 Clearly not a single active metagon es-capes the treatment (which does not directly affect mu), for one active metagon suf-fices to maintain a full complement of mu,3 yet mu disappears before metagon activitycan again be detected. To explain this reappearance by replication, it would haveto be assumed that one or more metagons were only temporarily inactive and thatactivity can be regained in M paramecia, but not in didinia, for active metagons donot arise in didinia after feeding them RNase-treated M paramecia at the stagewhen the assumed temporarily inactive metagons would have to be present(Methods, §4). These are obviously ad hoc assumptions. (2) During conjugation,all gamete nuclei bearing an M gene transmit the capacity to form metagons, butnuclei bearing an m gene never do.2 To explain this by metagon replication requiresassuming that at least one metagon is carried by every gamete nucleus that possessesan M gene, while none is carried by any m-bearing nucleus. Metagon segregationwould have to parallel gene segregation at meiosis in heterozygotes. (3) The meta-gon hybridizes specifically with both M and m genes, but only M genes are clearlycorrelated with metagon-forming capacity. Hybridization with M is readily inter-pretable in terms of replication by assuming that the metagon is bound to geneM as an episome. But then, by the same reasoning, one would expect it also to bebound to gene m. Yet metagon-forming capacity is never detectably transmittedby gamete nuclei bearing any m gene, even those showing the greatest in vitrohybridization with the metagon (Results, §5), and even when the paramecia formingthe m gametc nuclei are heterozygotes possessing both M and m genes and therefore

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also metagons. This discrepancy between observations and expectation on theepisome form of the hypothesis that metagons reproduce only by replication, while itmay later be resolved, at present remains unexplained. In this connection it shouldbe emphasized that M paramecia have never lost the capacity to produce metagonsduring the 9 years they have been under observation. Unless the M gene can bedissociated from that capacity, the essential requirements for identifying themetagon as an episome is missing.The preceding attempts to explain all of the facts only by genic production

of metagons and only by metagon replication both met with difficulties. Suchdifficulties are not encountered, and no additional assumptions are needed on thehypothesis that the metagon replicates after infection into Didinium and arises asthe m-RNA ofM genes in Paramecium. There are, however, some strange featuresof the metagon, such as its peculiar high proportion of G + C (Gibson, unpublished).Nevertheless, while recognizing the need for much further evidence on both proper-ties, we tentatively adopt the m-RNA-replication hypothesis as fitting best the con-siderable evidence already available.The possibility of the conjunction of these properties in a single RNA arose with

the demonstration that viral RNA replicates and the evidence that m-RNA is theprimary product of genic action. The question has been not whether RNA can beproduced by genes or whether RNA can replicate, but why until now any givenRNA has shown one or the other feature but never both. An opportunity to attackthis problem-perhaps by relating it to a specific polymerase'3 which is blocked inParamecium but not in Didinium-appears to be provided by comparing metagonbehavior in Paramecium and Didinium. If our interpretation is correct, as nowseems likely, then the metagon would conform to Wright's'4 conception of a plasma-gene in modern terms, an m-RNA capable of controllable replication; and the releaseof control when in a foreign organism would have obvious implications for the originof RNA viruses that play so important a role in modern oncology.Summary.-The ciliate Didinium normally does not contain metagons or mu,

but can acquire them by eating paramecia which contain them. They then persistand multiply in didinia (followed for 1,000 cell generations). Although such multi-plication of mu in Paramecium depends on the continuous presence of an M gene,it occurs in Didinium even when fed paramecia lacking this gene (as well as meta-gons and mu). In Didinium, as in Paramecium, mu persists and multiplies only inthe continuous presence of the metagon. Exposure of didinia carrying metagonsto RNase is followed by permanent loss of metagons, whereas such treatment ofparamecia carrying an M gene yields only brief transient loss of metagon activity.Metagons can be extracted from didinia, as from paramecia, in the ribosomal andRNA fractions. Detector paramecia can be infected with metagons extracted fromeither organism. Regardless of the source, the metagons multiply little, if at all,when infected into m paramecia and are quickly diluted out in the course of severalfissions. Attempts were made to hybridize the metagon RNA with DNA extractsof M and m paramecia, Didinium, mu, Tetrahymena, and Aerobacter. Tests formetagon activity of the RNA released from the DNA were negative except forRNA hybridized with DNA from Paramecium. Much more activity was shown bythe RNA hybridized with DNA from M than from m paramecia. Metagon RNAthus appears to be complementary to DNA of the M locus, confirming earlier re-

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876 BIOCHEMISTRY: MACH AND TATUM PROC. N. A. S.

suits suggesting that the metagon is m-RNA of M genes. Failure of the metagonto hybridize with DNA from Didinium indicates that this organism has no genecomparable to the M genes of Paramecium. Attempts to account for all of thefacts by assuming that the metagon increases only by replication or only by produc-tion from host genes meet with difficulties. The metagon appears to replicate likean RNA virus in Didinium and to arise as the m-RNA of M genes in Paramecium.Its possible relation to Wright's concept of the plasmagene and to the origin ofRNA tumor viruses is recognized.

Note added in proof: A full and clear summary of the background work on the metagon hasjust appeared: Beale, G., in Cellular Control Mechanisms and Cancer, ed. P. Emmelot and0. Muhilbock (Amsterdam: Elsevier Publ. Co., 1964), pp. 8-18.

* Contribution no. 747 from the Department of Zoology, Indiana University. The experimentswith metagons and mu were carried out by Ian Gibson. Aided by grant to T. M. SonnebornAT(11-1)-235-10 of the Atomic Energy Commission.

t Postdoctoral fellow, Public Health Service genetics training grant. Present address:Department of Zoology, University of Washington, Seattle 5.

1 Sonneborn, T. M., Advan. Virus Res., 6, 229 (1959).2 Gibson, I., and G. H. Beale, Genet. Res., 2, 82 (1961).3 Ibid., 3, 24 (1962).4 Ibid., 5, 85 (1964).6 Sonneborm, T. M., in preparation.6 Gibson, I., in preparation.7 Gibson, I., Proc. Roy. Soc. (London), in press.8 Bolton, E., and B. McCarthy, these PROCEEDINGS, 48, 1390 (1962).9 Gibson, I., and G. H. Beale, Genet. Res., 4, 42 (1963)."Reeve, E. C. R., and G. J. S. Ross, Genet. Res., 4, 158 (1963).1" Called to our attention by Barbara McManamy.12Jacob, F., and E. L. Wollman, Sexuality and Genetics of Bacteria (New York: Academic

Press, 1961), chap. 16, pp. 319-324.13Haruna, I., K. Nozu, Y. Ohtaka, and S. Spiegelman, these PROCEEDINGS, 50, 905 (1963);

Weissman, C., L. Simon, P. Borst, and S. Ochoa, Synthesis and Structure of Macromolecules, ColdSpring Harbor Symposia on Quantitative Biology, vol. 28 (1963), p. 99; Baltimore, D., H. J.Eggers, R. M. Franklin, and I. Tamm, these PROCEEDINGS, 49, 843 (1963).

14 Wright, S., Am. Naturalist, 79, 289 (1945).

ENVIRONMENTAL CONTROL OF AMINO ACID SUBSTITUTIONSIN THE BIOSYNTHESIS OF THE ANTIBIOTIC

POLYPEPTIDE TYROCIDINE*

BY BERNARD MACH AND E. L. TATUM

LABORATORY OF BIOCHEMICAL GENETICS, THE ROCKEFELLER INSTITUTE

Read before the Academy April 27, 1964

Studies on the biosynthesis of tyrocidine, a bacterial decapeptide, have providedthe first example of the biosynthesis of a free polypeptide by mechanisms differentfrom those involved in the biosynthesis of proteins." 2 When several aspects ofprotein biosynthesis were studied in comparison with the biosynthesis of tyrocidine,it was demonstrated that (1) the enzymatic mechanisms involved in the incorpora-

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paramecium.' (m, - pnas

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