Mitochondrial DNA in Tetrahymena pyriformis

Experimental

MITOCHQNDRIAL

Cell Research 54 (1969) 143-149

DNA IN TETRAHYM’ENA C. F. BRUNKI

Biophysics Labovatovy,

PYRIFORMIS

and P. C. HANAWALT

Stanford

University,

StanJord, Cal$

94305, USA

SUMMARY Contrary to the reports in the literature, no satellite DNA bands were found in DNA isoiated from Tetvahymena pyriformis strains 6111, HSM, ST, or GL; all of the DNA from a given strain had a single buoyant density in CsCI. A technique for resolving 2 DNAs of only slightly different buoyant density using 2 different radioactive labels is described. Mitochondrial DNA was isolated from contamination-free mitochondria and shown to have a density identical to that of whole cell DNA. The molecular weight of this mitochondrial DNA as isolated was 30-40 x lo6 daltons. Attempts to demonstrate that the mitochondrial DNA existed as a twisted circle were unsuccessful. However, the relative utilization of 3H-BUdR and 14C-TdR in the synthesis of mitochondrial DNA was shown to be different from their utilization in nuclear DNA synthesis. This provides another means for distinguishing between different DNA components in the same cell when different replication systems are involved.

Within the past 10 years there have been numerous reports of the presence of DNA2 in the various organelles of eucaryotic cells. Such organelles include the fiber producers (kinetosomes or basal granules) the mitochondria and the plastids [8]. All of these have also been shown to exhibit both physical and genetic continuity [lo]. A specific gene-protein relationship in a cytoplasmic genome has been verified in the case of mitochondrial structural protein in Neurospova [25]. Independence of the mitochondrial DNA replication from the cellular mitotic cycle has also been shown by Guttes et al. [9] in Physarum polycephalum. Parsons 1151has demonstrated by autoradiography that 3H-thymidine is incorporated into DNA associated with the mitochondria of T. pyriformis. This observation was extended by 1 Present address: Department of Zoology, Los Angeles, Calif. 90024, USA. 2 Abbreviations used: DNA, deoxyribonucleic acid; TdR, thymidine; BUdR, 5-bromodeoxyuridine; EDTA, ethylenediaminetetraacetate (sodium salt); tris, tris (hydroxymethyl) aminomethane; RNA, ribonucleic acid; PPO, 2,5-diphenyloxazole; POPOP, I ,4-bis-2-(4-methylS-phenyloxazalyl)-benzene. 10 - 691816

Stone & Miller [19] using electronmicroscopicautoradiography. They showed that virtually all of the silver grains over the cytoplasm were due to label inside or near mitochondrial membranes. They further demonstrated that the labeled mitochondria were metabolically stable and that they were distributed in nearly equal numbers to the progeny during cell division Cameron [5], also using autoradiography, has indicated that mitochondrial DNA is produced during most, if not al, of the cell cycle. Mitochondrial DNA has also been isolated and characterized from T. pyn$wmis. Parsons [16] reported a buoyant density in CsCl of 1,671 g/cc for mitochondrial DNA isolated from strains HSM and 6111,while the whole cell DNA (predominantly nuclear) from these strains had a density of 1.685 g/cc. An early report of Suyama & Preer [22] showed that mitochondrial DNA from strain ST had a density of 1.682 g/cc while the whole cell DNA from this strain exhibited a density of 1.688 g/cc. When heated this mitochondrial DNA showed a hyperchromic shift characteristic of double stranded native DNA. The amount of DNA per rni~oc~~~d~~o~

144

C. F. Brunk & P. C. Hanawalt

was estimated to be 3.7 x lo-l6 g. A later report by Suyama [21] listed the densities of mitochondrial DNA from 7 strains of T. pyriformis; 3 of these strains had mitochondrial DNA that differed in density from the corresponding whole cell DNA. In this later report the buoyant density of ST mitochondrial DNA was revised to 1.686 g/cc and the whole cell DNA density was revised to 1.692 g/cc. The molecular weight of this mitochondrial DNA was estimated to be 4 x 10’ daltons. In both this report and the earlier one, a band of unknown origin was found in some of the CsCl gradients. This band may well be glycogen [3]. An early report by Sueoka [20] mentions a small satellite band in strain GL with a density less than that of the bulk of the DNA. We first attempted to isolate mitochondrial satellite DNA from T. pyriformis in order to study the relative autonomy in function and metabolism of nuclear and mitochondrial DNA. However, no satellite DNA was detected in T. pyriformis and further investigation has indicated that the density of DNA isolated from the mitochondria of T. pyriformis strains 6111, HSM, ST, and GL is identical to that of the nuclear DNA from these strains. MATERIALS AND METHODS Culturing of organisms The various strains of T. pyviformis were kindly supplied by Dr J. A. Parsons (strains 6111 and HSM), Dr Y. Suyama (strain ST) and Dr D. S. Nachtwey (strain GL). The cells were grown with vigorous aeration in the defined medium of Elliott [7], supplemented with 0.04 % proteose peptone (Difco). The DNA was radioactively labeled by the addition of 3HTdR, 14CTdR, 3HBUdR, or 32P phosphoric acid. The cells were harvested by low speed centrifugation and washed with NET buffer (0.5 M NaCl, 0.05 M EDTA, 0.05 M tris, pH 8.5).

Isolation and characteristics of DNA The cells were lysed by treatment at 60°C for + h with pronase and Sarkosyl-NL-97 (Geigy) added to a final concentration of lOOplg/ml and 0.1 % respectively. The lysate was then extracted with an equal volume of chloroform-octanol (9: 1). The buoyant density of the DNA was determined by the CsCl method of Meselson & Stahl [14]. The density of the lysate was increased to 1.71 g/cc by the addition of optical grade CsCl. Preparative gradients containing lOOpg/ml ethidium bromide (a gift of Boots Pure Drug Co. Ltd) were adjusted to a final density Exptl

Cell Res 54

of 1.206 g/cc. These solutions were placed in cellulose nitrate tubes, overlaid with mineral oil and centrifuged at 37,000 rpm for 36 h at 25°C in the 40 rotor of a Spinco model L2 ultracentrifuge. Fractions were collected through a pin hole in the bottom of the tube. The RNA content of the fractions was rendered trichloroacetic acid soluble by incubation for several hours in 1 M KOH. The DNA was precipitated with 5 ml of 5 % trichloroacetic acid, trapped on a membrane filter (Millipore, HA), and dried. The dried filters were placed in counting vials containing 5 ml toluene, 18 mg PPO and 0.45 mg POPOP and counted in a Packard Tri-Carb scintillation

spectrometer.

For analytical CsCl gradients, the DNA solution was centrifuged in the Spinco model E ultracentrifuge at 25°C and 44,770 rpm in a 12 mm 4” sector cell with a 1” negative wedge window and a Kel-F centerpiece. Ultraviolet absorption photographs were taken after 20 h of centrifugation and scanned with a Joyce-Loebl microdensitometer. The molecular weight of mitochondrial DNA was determined by zone sedimentation. 3H-labeled DNA was layered on top of a 5 to 20 % sucrose gradient (containing 1 M NaCl) and centrifuged at 25,000 rpm in a SW 39 rotor of a Spinco model L2 ultracentrifuge at 20°C for 135 min. The calculation of molecular weight was performed as described by Burgi & Hershey [4]. DNA from lambda phage (a gift from D. S. Ray) was used as a molecular weight standard.

Isolation ofrnitochondria Mitochondria were isolated from T. pyriforrnis by a modification of the procedure described by Suyama & Preer [22]. Washed cells were resuspended in RAP buffer (0.2 M raffinose, 0.25 % bovine serum albumin, 1 mM K,HPO,; pH 6.2) to a concentration of 1 g of cells (wet wt) per 4 ml of buffer. The cells were opened with a tissue homogenizer, diluted 10 fold, and centrifuged at 1000 g for 10 min. The supernatant was pipetted off, the pellet was resuspended, and this step was repeated, until the supernatant was clear. The final pellet was saved for further examination. The combined supernatants were centrifuged at 12,000 g to pellet the mitochondria [13]. The 12,000 g supernatant was decanted and the thin layer of flocculent material covering the mitochondrial pellet was removed. This 12,000 g centrifugation step was repeated until no further mitochondrial pellet was formed. The mitochondrial pellet was resuspended in RAP buffer. The flocculence and the final supernatant were saved for further assay. Thus, the cells were fractionated into mitochondria, flocculence, supernatant, and pellet. The mitochondrial content of each fraction was estimated by measuring succinic dehydrogenase activity using Nito-BT (Dajac) [15]. The isolated mitochondria were freed from nuclear DNA contamination by digestion for 30 min at 0°C with pancreatic DNase at a final concentration of 10 pg/ml 1121.As a check on the efficiency with which nuclear DNA contamination was removed, 32P-labeled DNA was mixed with the isolated mitochondria before the DNase treatment. Virtually all of this 32Plabeled DNA was removed by the DNase treatment. A typical isolation of mitochondria is shown as a flow diagram in fig. I, the figures in the brackets refer to the 32P-labeled DNA used to check the efficiency of the decontamination step. Mitochondria freed from nuclear DNA by this DNase treatment are referred to as contamination-free mitochondria. DNA was isolated from these contamination-free mitochondria and banded in a CsCl gradient.

and rebandings were performed without the detection of a satellite in any of these strains.

MASS CULTURE HARVEST and HOMOGENIZED CELLS

Increase in resolution

If the buoyant density of a satellite DNA in 7’. were very close to the density of the nuclear DNA it might be difficult to detect such a satellite band even after it was enriched for by the process described above. In order to increase the resolution, DNA from a given strain cf T. pyriformis labeled with 3H-thymidine was prepared as well as 32P-labeledDNA from the same strain. After the initial banding either the heavy or the light shoulder of the 32P-labeledband was rebanded using material from the center of the 3H-thymidine labeled band as a marker and vice versa. The use of 2 labels greatly enhances the resolution as may be seen in fig. 2. The upper graph is a tracing from an analytical CsCl gradient while the lower graph shows the fractions collected from a preparative gradient, in which

pyriformis t 12,000 g

PELLET 1.2 % + I.“.;

I

MITOCHONDRIA 2.6 % ++++

SUP. &WASH 0.6 % (98 %)

SUPERNATANT 87.6 %

FLOCCULENCE 8.6 %

CONTAMINATION-FREE MITOCHONDRIA 2.0 % (2.0 %)

Fig. 1. Flow diagram showing the steps in the isolation of

3H-labeled mitochondria. The relative amount of 3Hlabel in each fraction is expressed as the percentage of the total label incorporated into the culture. The percentage in brackets refers to the contaminating 32P-labeled DNA (see text). The succinic dehydrogenase activity of each fraction assayed with Nitro-BT is shown as f (activity) and -(no activity).

1200

RESULTS

800

/-’2oo I

-. 800

Whole cell DNA

The cells were uniformly labeled with 3zP for several generations and the DNA was isolated and banded in a CsCl gradient. The fractions corresponding to either the light or the heavy side of the resulting DNA peak were individually rebanded in a second CsCl gradient with a small amount of DNA from the center of the peak as a marker. In some cases this process was repeated several times. This technique was used in an attempt to detect a satellite band in DNA from T. pyriformis GL, 6111, HSM and ST. Numerous bandings

.. 400

400

I

0

0-a

15

20

25

30

35

0

40

Fig. 2. The upper portion of this figure is a densitometer

tracing of an analytical CsCl gradient containing DNAs from T.pyviformis strain 6111and strain GL and E. coli. The lower portion of this figure shows fractions (2 drop) from a preparative CsCl gradient containing W-labeled DNA from T. pwifoumis strain GL and 32P-labeled DNA from strain 6111.It is evident that the difference in the buoyant densities of the DNA from strain GL and 6111is more easily seen in the lower graph where each curve is shown independently. Abscissa: fraction number; ordinate: (left) SzP, cpm, (right) 3H, cpm. 0 -0 - 3, 3Hlabel; a-a-a, 32P-label.

146 C. F. Brunk & P. C. Hanawalt the 2 DNA bands had different radioisotope ferential centrifugation. The most satisfactory labels. The difference in the density of the DNA method for detecting mitochondria of T. pyrifrom these 2 strains is approx. 0.006 g/cc. It is formis proved to be staining with Nitro-BT, evident that the mean position of each peak which is specific for succinic dehydrogenase. may be more accurately determined in the Contamination of the isolated mitochondria with lower graph where each peak is shown inde- nuclear DNA was removed by treating the intact pendently than in the upper graph where a com- mitochondria with DNase. The DNase does not posite curve is traced. When this technique was penetrate the mitochondria, thus, the mitochondemployed no satellite bands were detected in any rial DNA was not degraded by this treatment. of the strains of T. pyriformis (HSM, 6111,ST, To check the efficiency of this decontamination, a large amount of 32P-labeled DNA from T. or GL). pyriformis was thoroughly mixed with the isoPossible selective loss of a satellite lated mitochondria. After the mitochondria had It is conceivable, but not likely, that a satellite been washed and resuspended, approx. 10 % of DNA was being lost selectively during the isola- the added (32P-labeled)DNA was found to be tion procedure. To check for selective loss, adsorbed to the mitochondria. The treatment cells were uniformly labeled with 14C-thymidine. with DNase removed all but a few % of the Thymidine label appears only in DNA in T. adsorbed 32P-labeled DNA, while only about pyriformis [l]. The cells were lysed and the entire 25 % of the 3H-label was removed lysate was placed on a CsCl gradient. Virtually The bottom portion of the isolation flow all of the 14C-labelwas found in the DNA band. diagram, fig. 1, shows this decontamination step. No satellite bands were detected when this The figures in brackets refer to the 32P-labeled material was rebanded with a 3H-labeled whole DNA which was added to check the efficiency cell DNA marker. Thus, it appears that satellite of decontamination. The upper graph in fig. 3 DNA was not selectively lost during the isola- shows the results of a CsCl gradient containing tion procedure. DNA from decontaminated mitochondria. The band of 3H-labeled mitochondrial DNA is Repeat of analytical procedures of narrow indicating that this DNA was not deParsons & Suyama graded by the DNase. The tiny amount of 32PThese studies were initiated using T. pyriformis labeled contaminating DNA, which remained GL, but during this investigation Parsons [16] and Suyama & Preer [22] reported the presence with the mitochondria, did not form a band in of a satellite DNA in strains 6111,HSM, and the CsCl gradient. This clearly indicates that the ST. These strains were obtained and studied as 3H-labeled DNA is associated with the mitodescribed above. For completeness an attempt chondria and is not nuclear DNA adsorbed to was made to repeat the observations of the the mitochondria during the isolation procedure. The isolated mitochondrial DNA and whole satellite DNA in these strains using the methods cell DNA were both subjected to DNase degradescribed by the authors (Parsons, personal comdation. Their sensitivity to DNase was identical. munication [22]). When these procedures were repeated using strains 6111and ST respectively, a This indicates that the protection of the mitochondrial DNA from the DNase treatment dursingle DNA band was observed. ing isolation was conferred by the mitochondria DNA isolated from mitochondria of and that this DNA is not merely insensitive to Tetrahymena pyriformis DNase. A 2 1 culture of T. pyrzfirmis was uniformly labeled with 3H-thymidine for several genera- Characteristics of mitochondrial DNA tions. The cells were opened by homogenization DNA was isolated from the mitochondria of (HSM, ST, and and the mitochondria were separated by dif- several strains of T.pyr$xmis Exptl

Cell Res 54

Mitochondrial DiVA in ~et~a~yrne~~a147 6000

6000

from the mitochondria of T. pyrifo~~~ts was determined using band velocity sedimentation. It was estimated that the molecular weight of the mitochondrial DNA was approx. JO-40 x 10” daltons. The results from a typical sedimeiltatio~ determination of the molecular weight of mitochondrial DNA are shown in the lower graph of fig. 3.

MIT0

L 3000

& NUCLEAR

5000

1000 b 0 0 800

25 MIT0

MW.

50

75

100 I

600 1 400 j

Fig. 3. Characterization of DNA isolatsd from the mitochondria of T. pyrifovmis. Graph (a) shows a 3H-labeled mitochondrial DNA peak and a background of %Plabeled contaminating DNA. Graph (b) shows 3Hlabeled mitochondrial DNA rebanded with 3ZP-labeled whole cell DNA. Graph (c) shows fractions from a band sedimentation determination of the molecular weight of mitochondrial DNA. Abscissa: fraction number; ordirzate: (a, b) (left) 32P,cpm, (right) 3H, cpm; (c) (left) 3H, cpm. 0 - 0 - 0, $H-label; a - A - n , 32P-label (except for the bottom graph where A - A - a is 3H-label).

CL). When the 3H-labeled DNA was rebanded in a CsCl gradient with DNA extracted from whole cells of the same strain, labeled with 32P, the bands from mitochondrial DNA and whole cell DNA had identical densities. The results of a typical gradient containing mitochondrial and whole cell DNA are shown in the middle graph of fig. 3. These results confirm the earlier finding that these strains do not have a mitochondrial satellite DNA band. The molecular weight of the DNA isolated

Alternate methods demonstrating the ~~~q~~~~~~s of mitochondrial DNA In order to establish that the DNA isolated from the mitochondria is unique to the mitochondria, characteristics of this DNA that might differ from nuclear DNA were examined. Often mitochondrial DNA has an unique buoyant density and may be distinguished from nuclear NA by this characteristic. The density of 16.pyr~fimnis mitochondrial DNA is identical to tha.t of the nuclear DNA. Thus, buoyant density will not establish the uniqueness of the mitochondrial DNA in this organism. The relative nucleotide ratio and the melting temperature of DNA ate directiy related to its buoyant density, thus, these characteristics of DNA would probably not establish the uniqueness of this mitochondrial DNA either. Base sequencehomology, as determined by hybridization, has been used to determine the relatedness of nuclear and mitochondrial DNA from mouse liver [6]. ever, the results from this type of study were somewhat ambivalent, indicating that the dization of mitochondrial and nuclear varies from 40-70 %. Thus, this type of measurement does not appear to be appropriate for establishing the uniqueness of mitochondrial DNA. Another characteristic of mitochondrial DNA from some organisms is the existence of this DNA in vivo as a twisted covalently bonded circle [18, 231similar to the polyoma virus DN’A described by Vinograd et al. [24]. Recently a technique has been developed for separation of twisted circular DNA in a CsCl gradient using ethidium bromide [17]. The ethidium bromide, which is a large dye molecule, intercalates into double stranded DNA and decreasesits buoyant

148

C. F. Brunk & P. C. Hanawalt

density. Radloff et al. [17] have demonstrated that less dye binds to twisted circular DNA than to linear DNA, thus, it should be possible to separate twisted circular mitochondrial DNA from linear nuclear DNA even though they would have identical buoyant densities in a CsCl gradient that did not contain ethidium bromide. For this study the cells were lysed directly in the centrifuge tubes and CsCl solution containing ethidium bromide was gently added. These experiments failed to distinguish any difference between the buoyant density of 3Hlabeled mitochondrial DNA and that of 3zPlabeled whole cell,DNA. The molecular weight of mitochondrial DNA from T.pyr$ormis is much greater than that of any twisted circular DNA for which this ethidium bromide technique has been used successfully. If the mitochondrial DNA of T. pyriformis is circular it is possible that breaks were introduced into the DNA by shear or nuclease activity.

Table 1. Differential

incorporation of BUdR and

TdR

W-TdR

3H-BUdR W-TdR

658

1182

0.56

1380 3H-BUdR Nuclear DNA w

635

2.18

3H-BUdR Mitochondrial DNA Nuclear DNA (supernatant)

3H-BUdR Mitochondrial DNA I~C-T~R

= 3.9

of different DNA synthesis systems and is perhaps a property of the DNA polymerase. Tetrahymena pyriformis were challenged with 0.10 ,uc/ml of 3H-BUdR and 0.01 PC/ml of 14CTdR to see if nuclear DNA could be distinguished from mitochondrial DNA by their differential utilization of these labels. The cells were labeled for several generations and fracMetabolic difSerence between nuclear tionated (see fig. 1). The ratio of 3H-BUdR to and mitochondrial DNA 14C-TdR found in the mitochondrial DNA and The mitochondrial and nuclear DNA can not be the nuclear DNA (supernatant) are shown in distinguished by any of the physical chemical table 1. The relative amount of BUdR incortechniques described above and in this sensethe porated into the nuclear DNA is almost 4 times mitochondrial DNA is not unique. However, greater than the relative amount incorporated the metabolism of mitochondrial DNA wasfound in mitochondrial DNA. It is evident that this to be different from that of nuclear DNA and selective utilization of BUdR makes possible a this establishes that the DNA isolated from the clear distinction between the nuclear and mitomitochondria is produced via a different meta- chondrial DNA. When cells were grown with bolic pathway than nuclear DNA. Thus, in spite large amounts of BUdR (50 ,ug/ml) the nuclear of their physical chemical similarities nuclear DNA became heavier (more BUdR) than the and mitochondrial DNA are distinct and are mitochondrial DNA. Thus, under these condiproduced by different DNA synthetic systems. tions an artificial mitochondrial DNA satellite In studies on the excision-repair system in E. was created. Such an artificial mitochondrial coli [I I] and T. pyriformis [2] it was found that DNA satellite has been banded in a CsCl grawhen this system was challenged with both dient. BUdR and TdR there was a differential utilizaDISCUSSION tion of BUdR in repair replication relative to that in normal replication. In other words, if the From our results we conclude that the buoyant excision-repair system is presented with both density of the DNA associated with the mitoBUdR and TdR, TdR will be utilized in prefe- chondria of all 4 strains of T.pyriformis (6111, rence to BUdR to an even greater extent than HSM, ST, and GL) is identical to that of the occurs during normal replication. This selection nuclear DNA from these strains, at least to the of TdR over BUdR is probably a characteristic resolution of 0.0005 g/cc. Isolation of DNA from Exptl

Cell Res 54

contamination-free mitochondria confirmed this conclusion. The mitochondrial DNA was also shown to be distinguishable from the nuclear DNA by the relative utilization of 14C-TdR and 3H-BUdR. Although this evidence appears to be quite compelling, there exist several reports in the literature to the contrary [16, 21, 221. In all of the previous reports of a mitochondrial satellite band in T. pyriformis the densities of the nuclear and the mitochondrial DNA were determined relative to a marker DNA using an analytical CsCl gradient [16, 20, 21, 221.In none of the reports was the density of the mitochondrial DNA compared directly with the density of the nuclear DNA by banding both DNAs in a single gradient. The reported difference between the density of satellite and nuclear DNA was sufficient that the 2 bands should have been easily distinguished in the same gradient. The use of 2 radioactive labels has distinct advantages over analytical gradients for this type of study. The mitochondrial DNA from T. pyviformis is not distinguishable from nuclear DNA on the basis of buoyant density, molecular weight or a twisted circular conformation. However, the selective utilization of BUdR and TdR does make possible the distinction between the mitochondrial and nuclear DNA. This method of distinguishing the DNAs produced by various different DNA synthesis systems appears to be quite general; it applies to the repair synthesis system as well as the mitochondrial synthesis system. The differential utilization of TdR and BUdR may well prove to be valuable for distinguishing the DNA produced by other DNA synthesis systems,especially the various cytoplasmic DNA synthesis systems. The absenceof a density difference associated with the mitochondrial DNA of T. pyriformis makes it much more difficult to identify the mitochondrial DNA. This makes the organism unsuitable for studies on the relative autonomy

in replication and control of the nuclear and mitochondrial components. This work is taken from a dissertation submitted by C. F. Brunk to the Biophysics Laboratory and the Committee on the Graduate Division of Stanford IJniversity in partial fulfillment of the requirements for the degree of Doctor of Philosophy. Final details of this work were completed at the Carlsberg Biological Institute, Copenhagen, Denmark.

REFERENCES 1. Albach. R A & Hull. R W. J nrotozool 10., Sunnl. __ (1963) 20. ’ ’ 2. Brunk, C F, Doctoral thesis. Biophys Lab., Stanford Univ., Palo Alto, Calif. (1967). 3. Brunk, C F & Hanawalt, P C, Exptl cell res 42 (1966) 406. 4. Burgi, E & Hershey, A A, Biophys j 3 (1963) 309. 5. Camerson. I L. Nature 209 (1966) 630. 6. duBuy, H.G & Riley, F L., Proc natl acad sci US 57 (1967) 790. 7. Elliott, A M, Brownell, L E & Gross, J A, J protozool

1 (1954) 193. 8. Granick, S & Gibor, A, Progr in nucleic acid res 6

(1967) 143. 9. Guttes, E, Hanawalt,

P C & Guttes, S, Biochim biophys acta 142 (1967) 181. 10. Jinks, J L, Extrachromosomal inheritance. PrenticeHall, Englewood Cliffs, N.J. (1964). 11. Kanner, L C & Hanawalt, P C, Fed proc 26 (1967) 872.

12. Luck, D J L & Reich, E, Proc nat! acad sci US 52 (1964) 931. 13. Mager, J & Lipmann, F, Proc natl acad sci US 44 (1958) 305. 14. Meselson. M & Stahl. F W. Proc natl acad sci US 44 (1958) 671. ’ ’ 15. Parsons, J A, J cell bio125 (1965) 641. 16. Parsons, J A & Dickson, R C, J cell biol27 (1965) 77a 17. Radloff, R, Bauer, W & Vinograd, 3, Proc natl acad sci US 57 (1967) 1514. 18. Sinclair, J H & Stevens, B J, Proc natl acad sci US 56 (1966) 508. 19. Stone, G E & Miller, 0 L, Jr, 3 exptl zoo1 159 (1965) 22 a.,. 20. Sueoka, N, Cold Spring Harbor symp quant bio126 (1961) 35. 21. Suyama, Y, Biochem 5 (1966) 2214. 22. Suyama, Y & Preer, J R, Jr, Genetics 52 (1965) 1051, 23. van Bruggen, E F J, Borst, P, Ruttenberg, C J C M, Gruber. M & Kroon. A M. Biochim biophys acta ^ 119 (1966) 437. ’ ’ 24. Vinograd, J, Lebowitz, J, Radloff, R, Watson, R & Laipis, P, Proc natl acad sci US 52 (1965) 1104. 25. Woodward, D 0 & Munkres, K D, Proc natl acad US 55 (1966) 872. Received May 13, 1968

Exptl

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