Proc. Nat. Acad. Sci. USA

Vol. 72, No. 10, pp. 3868-8872, October 1975 Biochemistry

Restriction endonuclease analysis of mitochondrial DNA from grande and genetically characterized cytoplasmic petite clones of Saccharomyces cerevisiae (agarose gel electrophoresis/EcoRI and HindIII endonucleases/antibiotic resistance markers/multiple deletions)

RICHARD MORIMOTO*, ALFRED LEWIN*, HUEY-JUANG Hsu*, MURRAY RABINOWITZ**, AND HIROSHI FUKUHARAt * Departments of Medicine, Biochemistry and Biology, and the Franklin McLean Memorial Research Institute, The University of Chicago, Chicago, Illinois 60637 USA; and t Centre de Genetique Moleculaire, Cif-sur-Yvette, France

Communicated by Hewson Swift, July 10, 1975

ABSTRACT Digestion of grande mitochondrial DNA (mtDNA) by EcoRI restriction endonuclease gives rise to nine fragments with a total molecular weight of 51.8 X 106. HindIII digestion yields six fragments with a similar total molecular weight. Specific restriction fragments can be detected despite the fact that yeast mtDNA consists of a heterogeneous distribution of randomly broken molecules. Digestion patterns of 10 genetically characterized petite clones containing various combinations of five antiobiotic resistance markers indicate that the petite mtDNA predominantly represents deletion of the grande genome. The petite mtDNAs contained up to seven EcoRI restriction fragments which comigrate with grande restriction fragments, and at least one fragment that did not correspond to any in the grande. Some strains contained multiple fragments with mobility different from that of grande; these fragments were usually present in less than molar concentrations. The genetic markers were associated with individual sets of restriction fragments. However, several internal inconsistencies prevent the construction of a definitive genetic fragment map. These anomalies, together with the digestion patterns, provide strong evidence that, in addition to single contiguous deletion, other changes such as multiple deletion and heterogeneity of mtDNA populations are present in some of the petite mtDNAs. It is now well established that mitochondrial DNA (mtDNA) of cytoplasmic petite mutants is greatly altered (1-3). Renaturation kinetic analysis (4, 5) and DNA-DNA hybridization studies (6-9) indicate that petite mtDNAs contain deletions of 20% to more than 99% of the grande (wild-type) mitochondrial genome. By electron microscopic examination, a tandem repeat arrangement of petite mtDNA has been demonstrated, and in some cases there are inverted repeats of the mitochondrial genome, i.e., palindromes (2, 3, 10). Although deletion represents the most prominent alteration, other changes may be present in some strains, including heterogeneity of mtDNA populations (10-12), amplification of some segments of the mtDNA (10-12), and base sequence change (6, 9). Based on the assumption that deletion is the predominant alteration in petite mtDNA, our laboratories have attempted to map mitochondrial tRNA and rRNA genes by using a series of petite mutants. These studies have provided a partial ordering of the tRNA cistrons (11, §). Evidence for addition-

al internal deletions in some petite mtDNAs was also obtained§. The presence of secondary or tertiary deletions, and the possibilities of other rearrangements of segments of petite mtDNA, result in some uncertainty when genetic and molecular maps are based solely on data from petite strains. Therefore, we have initiated a study in which we analyze grande and petite mtDNAs by using restriction endonucleases.

In this paper, we present data on the digestion of mtDNA. of one grande and 10 petite clones by use of EcoRI and HindIII endonucleases.1 The results confirm the molecular weight of 50 X 106 for grande mtDNA, support the hypothesis that deletion is a major change in petite mtDNA, provide further evidence that some of the petite mtDNAs contain changes other than simple contiguous deletion, and illustrate the association of genetic markers with individual groups of restriction fragments.

MATERIALS AND METHODS Strains. Three haploid grande p+ (respiratory sufficient) strains of Saccharomyces cerevtsiae containing five wellcharacterized mitochondrial antibiotic resistance markers, I and chloramphenicol,(CR), erythromycin (ER), oligomycin II (OIR and 01R), and paramomycin (pR), were constructed by M. Bolotin-Fukuhara and H. Fukuhara (manuscript in preparation). From these strains they isolated a series of stable cytoplasmic petites (p-) by ethidium bromide mutagenesis and, using procedures previously described (13), purified individual clones that retained one to three antiobioticresistant alleles. The strains were more than 90% pure with respect to genetic markers, as tested by clonal analyses after crosses to suitable grande strains (14, 15). The genetic composition of the strains examined in the present study is listed in Table 1. Strains OIP-2 and P3 contain the On locus in oligomycin-sensitive form, and strain P4 contains the OIS locus. Strains may be more homogeneous with respect to antibiotic-resistant alleles than with respect to sensitive alleles. Yeast Culture and Preparation of Mitochondrial DNA. Strains were grown to late exponential phase in a medaium containing 2% galactose, 0.1% glucose, 1% bactopeptone, and 1% yeast extract. Large-scale cultures used to prepare mtDNA involved no more than 20 cell divisions of the original genetically characterized and purified stocks. The petites were stable under these conditions.

Abbreviation: mtDNA, mitochondrial DNA. t To whom reprint requests should be addressed at The University of Chicago, Department of Medicine, Box 407, 950 East 59 Street,

Chicago, Ill. 60637.

§ H. Fukuhara, H. J. Hsu, and M. Rabinowitz, J. Mol. Biol., submit- * Nomenclature for restriction endonucleases

suggested by Smith and Nathans (22).

ted for publication. 3868

corresponds

to

that

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Morimoto et al.

Proc. Nat. Acad. Sci. USA 72 (1975)

3869

Table 1. List of strains Abbreviated name

Grande P1 P3 CEP-2 CEP-3

01P-2 OIOII-1 E01-1

Full name MH41-7B MH41-7B/P11 MH41-7B/P31 MH41-7B/AB83 MH41-7B/AC219 MH41-7B/L721 MH41-7B/C516

MH41-7B/AB25 MH32-12D

P+

PP

PP-

P-

P_ P

Grande P4

MH32-12D/H241

P+ P_

OII-2

MH32-12D/Y41

P

Grande CEP-6

TR3-15A

P+

TR3-15A/F5262

P-

C

Mitochondrial loci 0 E Oil

R 0 0 R R 0 0 0 R 0 0 R R

R 0 0 R R 0 0 R R 0 0 R R

R 0 0 0 0 R R R S S 0 R 0

Sum of mol. wt. Relative* of RI fragments

S 0 S 0 0 S S 0 R 0 R S 0

P

K2

R R R R R R 0 0 R R 0 R R

1.00 0.54

X

10-6

51.8 t t 22.5

0.67

35.2 12.1 20.8

0.061 0.11

2.3 4.2

0.57

27.0*

C, E, 0i, O , and P designate the loci carrying the drug resistance markers C321, E514 or E221, 0145 or 01, 0144, and P454, respectively. R indicates the presence of resistance allele; S, sensitive allele; and 0, deletion. Mutations 0144 and 0145 come from Dr. P. Avner, P454 from Dr. K. Wolf, and others from the C. G. M. stocks. The full genotypes of the p+ strains, from which p- clones were isolated, are the following: MH41-7B, a ade2 his1 CR321 ER514 OR145 PR454; MH32-12D, a ade2 his1 CR321 ER221 OR144 PR454; TR3-15A, a try2 his1 CR321 ER514 OR1 pR454* The second-order rate constant of reassociation of the mtDNA (k2) was determined by a procedure similar to that described by Michel et al. (4), and is expressed as the ratio of the k2 to that of grande mtDNA. t Not calculable because of multiple fragments present in nonmolar concentration. $ Assuming that the two fragments with mobilities different from grande have common sequences.

For most studies, mtDNA was prepared by CsCl centrifugation after lysis of mitochondria, washed five times, which were prepared from protoplasts as described (5, 16). Nuclear DNA contamination could not be detected by isopycnic CsCl centrifugation in the analytic ultracentrifuge after the second preparative CsCl centrifugation. Enzyme and Enzymatic Restriction of mtDNA. EcoRI and Sall endonucleases were gifts from Dr. Gary Hayward of the University of Chicago. HindIII endonuclease was prepared by the procedure of Smith and Wilcox (17). Enzyme purity was tested by comparing the digestion of lambda and T5 phage DNAs with established patterns. Samples of mtDNA were dialyzed against 0.1 X SSC (SSC is 150 mM NaCI, 15 mM Na citrate), 0.5 mM Na2-EDTA, at 40, prior to digestion. The EcoRI and Sall endonuclease reactions were carried out in 30 mM Tris-HCI (pH 7.5), 10 mM MgCI2, and 5% glycerol for 60 min at 37°. The HindIII digests were performed in 10 mM Tris-HCI (pH 7.5), 10 mM MgCl2, 10 mM KCI, 40 mM NaCI, and 10% glycerol for 2 hr at 37°. The reaction was stopped by addition of EDTA to a final concentration of 15 mM and cooling to 4°. Gel Electrophoresis. Sucrose was added to a final concentration of 20%, and electrophoresis was carried out on 0.3% or 0.5% agarose (SeaKem) tube gels (20 X 1.2 cm) at 2 V/cm for 13 hr at room temperature in 40 mM Tris, 36 mM NaH2PO4, 1 mM Na2-EDTA, pH 7.4 buffer. The molecular weights of the grande mtDNA restriction fragments were determined by electrophoresis of mixtures of grande and T5, or lambda, restriction fragments using relative DNA concentrations at which phage and mitochondrial fragments could be easily distinguished on the basis of band intensity. Calibration curves were constructed for each gel, plotting the relationship between fragment migration against logarithm of the molecular weights of the phage DNA fragments, which were based on established values (18, 1l). The molecular 1 G. S. Hayward, A. Von Gabain, and H. Bujard, manuscript in preparation.

weights of petite restriction fragments were obtained in a similar manner except that grande and petite digests were electrophoresed together, the grande restriction fragments serving as the internal molecular weight markers. RESULTS EcoRI digestion of grande mtDNA All preparations of yeast mtDNA consist of linear molecules having a heterogeneous size distribution (1, 5) complicating restriction endonuclease analysis. Despite this limitation, the specific restriction fragments can be detected if DNA preparations contain adequate numbers of randomly cleaved molecules that are larger than the largest specific restriction fragment. Restriction fragments will not be present in equimolar quantities under these conditions; the concentration of the larger restriction fragments will be lower than predicted. Cleavage of grande (p+) MH41-7B mtDNA by EcoRI restriction enzyme yields nine discrete fragments (R-A to R-I) when analyzed by agarose gel electrophoresis (Fig. 1). Another grande strain, IL8-8C§, gave a fragment pattern similar to MH41-7B. The molecular weights of the restriction fragments (Table 2) were determined by coelectrophoresis of the p+ digest with EcoRI digests of either T5 or lambda phage DNA. The sum of the molecular weights of the nine fragments is 51.8 X 106, a value that is in excellent agreeTable 2. Molecular weights of EcoRI restriction fragments

Fragment

M. Wt. x 10 -6

ment

M. Wt. x 10 -6

A B C

15.3 ± 0.87 11.9 ± 0.87 7.37 ± 0.33

D E F

5.97 ± 0.32 5.57 ± 0.29 2.32 ± 0.09

Frag-

Fragment

G H I Sum

M. Wt. X 1 0 -6 1.58 ± 1.09 ± 0.66 ± 51.76 ±

0.07 0.09 0.09 1.38

Mean 4 SD of molecular weights determined from 14 independent electrophoretic runs, with internal calibrations as described in Materials and Methods.

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Proc. Nat. Acad. Sci. USA 72 (1975)

FIG. 1. Agarose gel electrophoresis of restriction endonuclease digests of grande and petite mtDNA. All petites were run alone as well as together with grande digests to determine identity or difference in mobility of grande and petite fragments. Variable gel shrinkage prevents a direct comparison of mobilities on different gels. The molecular weight of petite fragments were calibrated by use of grande fragments as internal markers. Electrophoresis was done at 250 and bands were visualized by ultraviolet illumination of ethidium bromide stained gels. (A) Grande mtDNA: (1) EcoRI digest (0.5% gel), (2) HindIII digest (0.3% gel), (3) combined HindIII and EcoRI digest (0.5% gel). (B) P4 mtDNA (0.5% gels): (4) EcoRI complete digest, (5) EcoRI partial digest. (C) CEP-2 mtDNA (0.3% gels): (6) EcoRI digest alone and (7) with grande. (8) HindIII + EcoRI digest alone and (9) with grande. (D) P1 mtDNA (0.5% gels): (10) EcoRI digest alone and (11) with grande.

ment with the 25 ,um circles seen in mitochondrial lysates by Hollenberg et al. (19), and with the values obtained from extrapolation of the data relating the monomer circle size and kinetic complexity of a variety of petite mtDNAs by Locker et al. (5). Borst (20) and Bernardi et al. (21) have also reported a molecular weight of 50 X 106 for EcoRI and HpaII

digests, respectively, of Saccharomyces carlsbergensis. EcoRI digest of petite mtDNA

The probable circular genetic map derived from recovery of antibiotic resistance markers after mutagenesis of grande strains was -P-C-E-01-Oi-P- (Fukuhara and Bolotin-Fukuhara, in preparation). Strain P4 represents a petite mtDNA that contains a single restriction site (Fig. 1). EcoRI digestion resulted in only one major band having a molecular weight of 2.3 X 106, in agreement with the kinetic complexity (0.061) relative to p+ DNA (Table 1). The same band was present after digestion with Sall endonuclease. Several low-intensity bands of lower molecular weight were also present. Partial digests obtained by limited reaction time yielded multiple bands; four of the six partial digest bands conformed to multiples of the molecular weight of the major band in the complete digest. The single major band observed in complete digests probably

represents the unit mitochondrial genome size of this petite, whereas the presence of multiple bands in partial digests confirms previous conclusions (2, 10) that petite DNAs are arranged as tandem repeats in a circular or linear molecule. In five of nine other petites studied, a regular digestion pattern was seen with EcoRI endonuclease. These petites retained one to seven restriction bands with mobilities identical to those of grande mtDNA, and at least one additional band with a different mobility. An example of this digestion pattern is shown in Fig. 1. EcoRI digests of CEP-2 mtDNA contain one band with the mobility of grande EcoRI-D (R-D) and a second band migrating more slowly than R-D. That R-D bands in CEP-2 and grande mtDNA are identical was shown by the digestion of the DNAs with both EcoRI and HindIlI endonucleases (Fig. 1). This results in the appearance of two bands that replace R-D and correspond in mobility to bands four and nine of EcoRI + HindIII (RI + HindIII) digests of grande mtDNA. The total molecular weight of the CEP-2 fragments, 22.4 X 106, agrees well with the value derived from kinetic complexity measurements relative to grande (0.54) (Table 1). Strains OIP_2(oilS), EO-1, Oii-2, and Oi OII-1 also show similar digestion patterns with EcoRI, i.e., one or more bands have identical mobility, and one has a mobility different from that of grande (Fig. 2). Combined EcoRI and Hin-

Morimoto et al.

Biochemistry:

Proc. Nat. Acad. Sci. USA 72 (1975)

3871

30

20 A B

10 8.0 -c 6.0 D

A

I___

0*

_

B

0

C.D 2 3

co

E

0

.1.._

4.0 F

z

0

5

10

2.0 H

9 10

I

II 12

1.0

0.8 0.6 0.4

z

&w.:

7,8

x

3~

_-

m

6

F

S

_

F

13

E F

RI IH ---I

14

HM+ RI

GRANDE

tRI

RI)

P4 (0,)

H|R

RI HM, RE-R _I RI RI R I ECE-

RI

|MRIR RIR IHZ+II E-

RI HM+ RI RI I RI0RI |HZ RI| R-IR I RI |HM+ Pi P3(Oks; 0-2 o0on0 Ij

0rP-2(01,s E0I-1

-

FIG. 2. Summary of EcoRI and EcoRI + HindIII digests of petite mtDNA. Molecular weights of fragments are plotted on a logarithmic scale to simulate mobilities on gels. However, the relationship between logarithm of molecular weights and mobility is not linear above 10 X 106. Solid lines show bands comigrating with grande, and dashed lines indicate bands having a different mobility. Low-intensity bands are represented by lighter lines. tIndicates partial digest. *Indicates bands with mobility identical to that of grande, but behaving differently with respect to HindIII digestion. All molecular weights were established by comigration with DNA fragments of known molecular weights (see Materials and Methods).

dIII digestion of OI 011-1 mtDNA, however, results in two bands with characteristics different from grande. Four petite strains, P3 (Oils), P1, CEP-6, and CEP-3, showed more complex EcoRI digestion patterns (Figs. 1 and 2). The first three strains contained two to five bands identical in mobility to grande, but they also contained three to seven bands with a different mobility (Fig. 2). The latter bands differed greatly in intensity and, in many cases, were present in clearly submolar concentrations. No EcoRI fragments were detectable in CEP-3, a strain in which the loss of many tRNA cistrons located between the C and P markers strongly indicates the deletion of a second genome segment§. Localization of genetic markers Analysis of EcoRI digests of the nine petite strains did not lead to unequivocal ordering of the fragments, nor to definite localization of genetic markers on specific fragments. However, several conclusions can be derived from the results summarized in Fig. 3. The genetic markers generally segregated with individual restriction fragments or with sets of fragments. For example, only petites with CE alleles (i.e., CEP-2, CEP-6) contained the EcoRI restriction fragment R-D. Clones CEP-2 and CEP-6 each contained a high-molecular-weight "extra" fragment larger than R-A, and CEP-6 (also P1) had what was apparently an "extra" band that had a mobility identical to R-A, but differed in not containing a HindIII restriction site. Fragment R-B appears to be located in the EQ1 region of the genome since it was present in EOQ and Q1P_2(011S). Most of the remaining EcoRI fragments (i.e., R-E, R-F, R-G, R-H, and R-I) were associated with the OjO1P region of the genome, all being present in petites OIP_2(0QIS) and P3(Q11S). Therefore, it is likely that the OI and OII alleles reside in or are adjacent to one of these fragments, and that the CE region is on the

other fragments [C and E are known to be linked, as shown by genetic recombination 14]. The single marker petite OII-2 contains R-H, which is also present in all strains containing OII [i.e., OIP-2(0IIS), P3(Q11S), and OOII-1] localizing OII to R-H or to an adjacent fragment. By such analysis, one can tentatively localize several genetic markers to the vicinity of the restriction fragments. A definite map cannot be constructed from these results alone, however, because several contradictions arise if it is assumed that the petites are derived from a single contiguous deletion of grande mtDNA. One such contradiction is illustrated in Fig. 3. Strain OIOJJ-1 contains R-C, R-F, and R-H; EOI contains R-B and R-F; and strain P3(OIIS) contains R-E, R-F, R-G, R-H, and R-I. As shown in Fig. 3c, the only arrangements consistent with the results for strains OIOIJ-1 and EOI are R-C-H-F-B or R-H-C-F-B. These arrangements are not compatible with data for P3(O11s) (Fig. 3d), however, since (a) 0° 1 C H F

(b)

.HOC F

EOQ

H. F C

F'B

comptible mnapping1 (c)

CH F B

(d) P-3(0s)

H C, F B.

E.G.HI.F.

FIG. 3. Diagram illustrating fragment order inconsistency if it is assumed that all petites are derived from a single contiguous deletion of grande mtDNA. See text for explanation.

3872

Biochemistry: Morimoto et al.

fragment R-C is not present in the strain. Therefore, at least one of the three strains is anomalous and cannot be derived from a single contiguous deletion. DISCUSSION Restriction endonuclease mapping of grande mtDNA should provide a detailed analysis of the structure and formation of petite mtDNAs. The initial results of the use of these techniques, as presented in this paper, provide strong additional evidence that petite mtDNAs are derived from deletion of the circular grande mitochondrial genome. The petite restriction pattern that is frequently observed, i.e., one band differing in mobility and one or more with the same mobility as that of grande restriction fragments, is the pattern expected from a single contiguous deletion of a circular, a linear permuted, or a tandem repeated molecule. The "extra" fragment should be the site of the deletion and should contain sequences from two different grande restriction fragments. Strains CEP-2, 01P_2(011S), Oii-2, and EQ1 appear to fall into this category. Multiple deletion would result in the formation of additional bands, but only if restriction sites separate the individual deletions. "Extra" bands in less than molar-equivalent concentrations could arise from inverted repeats of the petite genome (10). Another source of submolar quantities of "extra bands" is molecular heterogeneity of the petite mtDNA. We have previously detected heterogeneity of the mtDNA population in some petite strains by using tRNA hybridization (11, 12) and renaturation kinetic analyses (12). Although the petite clones examined here were stable and reasonably pure with respect to drug resistance markers, heterogeneity of mtDNA sequences with respect to regions distant from the genetic markers may exist. The multiple "extra" bands in relatively low concentrations observed in strains P1, P3, and P4 and the two extra bands in CEP-6 probably arise from one or more of these mechanisms. The presence of secondary deletions is also indicated by contradictions in the fragment order derived from restriction analysis of different petite mtDNAs, as illustrated in Fig. 3. Further analysis of these petite mtDNAs by use of restriction enzymes that cleave at more sites should permit one to characterize and localize multiple deletion sites, and should provide additional insight into the formation of the -petite mtDNAs. We gratefully acknowledge the technical advice and the gifts of restriction endonucleases from Dr. Gary Hayward, The University of Chicago, and Dr. Daniel Nathans of Johns Hopkins University. We thank Dr. Godfrey S. Getz for discussions and critical review of the manuscript. Mary Cohen is to be thanked for her-expert technical assistance. This study was supported in part' by Grants HL04442, HL09172, and HD00174 from the National Institutes of

Proc. Nat. Acad. Sci. USA 72 (1975) Health, U.S. Public Health Service, GB41555 from the National Science Foundation, 4303 from A.T.P., a grant from'C.E.A. and the Louis Block Fund of the University of Chicago. The Franklin McLean Memorial Research Institute is operated by The University of Chicago for the U.S. Energy Research and Development Administration. 1. Borst, P. (1972) Annu. Rev. Biochem. 41, 333-376. 2. Faye, G., Fukuhara, H., Grandchamp, C.,'Lazowska, J., Michel, F., Casey, J., Getz, G. A., Locker, J., Rabinowitz, M., Bolotin-Fukuhara, M., Coen, D., Deutsch, J., Dujon, B., Netter, P. & Slonimski, P. P. (1973) Biochemie 55, 779-792. 3. Rabinowitz, M., Casey, J., Gordon, P., Locker, J., Hsu, H. & Getz, G. S. (1973) in Mitochondrial Biogenesis, eds. Kroon, A. M. & Saccone, C. (Academic Press, New York), pp. 89-105. 4. Michel, F., Lazowska, J., Faye, G., Fukuhara, H. & Slonimski, P. P. (1974) J. Mol. Biol. 85,411-431. 5. Locker, J., Rabinowitz, M. & Getz, G. S. (1974) J. Mol. Biol. 88,489-502. 6. Gordon, P. & Rabinowitz, M. (1973) Biochemistry 12, 116123. 7. Fauman, M. & Rabinowitz, M. (1974) Eur. J. Biochem. 42, 67-71. 8. Lazowska, J., Michel, F., Faye, G., Fukuhara, H. & Slonimski, P. P. (1974) J. Mol. Biol. 85,393-410. 9. Gordon, P., Casey, J. & Rabinowitz, M. (1974) Biochemistry 13, 1067-1075. 10. Locker, J., Rabinowitz, M. & Getz, G. (1974) Proc. Nat. Acad. Sci. USA 71, 1366-1370. 11. Casey, J. W., Hsu, H. J., Rabinowitz, M., Getz, G. S. & Fukuhara, H. (1974) J. Mol. Biol. 88,717-733. 12. Casey, J., Gordon, P. & Rabinowitz, M. (1974) Biochemistry 13, 1059-1063. 13. Fukuhara, H., Faye, G., Lazowska, J., Michel, F., Deutsch, J., Bolotin-Fukuhara, M. &- Slonimski, P. P. (1974) Mol. Gen. Genet. 130, 215-238. 14. Bolotin, M., Coen, D., Deutsch, J., Dujon, B., Netter, P., Petrochilo, E. &'Slonimski, P. P. (1971) Bull. Inst. Pasteur 69, 215-239. 15. Deutsch, J., Dujon, B., Netter, P., Petrochilo, E., Slonimski, P. P., Bolotin-Fukuhara, M. & Coen, D. (1974) Genetics 76, 195-219. 16. Casey, J., Cohen, M., Rabinowitz, M., Fukuhara, H. & Getz, G. S. (1972) J. Mol. Biol. 63,'431-440. 17. Smith, H. 0. & Wilcox, K. W. (1970) J. Mol. Biol. 51, 379391. 18. Thomas, M. & Davis, R. W. (1975) J. Mol. Biol. 91,315-328. 19. Hollenberg, C. P., Borst, P. & van Bruggen, E. F. J. (1970) Biochim. Biophys. Acta 209, 1-15. 20. Borst, P. (1975) in Nucleocytoplasmic Relationships during Cell Morphogenesis in Some Unicellular Organisms (Elsevier, Amsterdam), in press. 21. Bernardi, G., Brunnell, A. & Kopecka, H. (1975) in Nucleocytoplasmic Relationships during Cell Morphogenesis in Some Unicellular Organisim (Elsevier, Amsterdam), in press. 22. Smith, H. 0. & Nathans, D. (1973) J. Mol. Biol. 81,419-423.