Escherichia coli mut T1. II. Consequences of modification on the association of DNA with the cell membrane

17

Biochimica et Biophysica Acta, 5 6 3 ( 1 9 7 9 ) 1 7 - - 2 7 © Elsevier/North-Holland

Biomedical Press

BBA 99446

ESCHERICHIA COLI MUT T1 II *. CONSEQUENCES OF MODIFICATION ON THE ASSOCIATION OF DNA WITH THE CELL MEMBRANE

THERESA

CAMPANA

** and HERMAN

S. S H A P I R O * * *

Department of Biochemistry, College of Medicine and Dentistry of New Jersey, Graduate School of Biomedical Sciences, and New Jersey Medical School, Newark, NJ 07103 (U.S.A.) (Received September

27th, 1978)

Key words: DNA association; Cell membrane; (Escherichia coli rout T1)

Summary 1. Two isogenic strains of Escherichia coli, K-12 which differ by mutator gene character (rout T1) have been studied. This characteristic causes introduction of a high frequency of undirectional transversions, A-T-~ C-G, into the DNA of the strain harboring it. 2. It had been previously shown that the presence of this gene is accompanied by an alteration of a cell membrane component. Now, the nuclease susceptibility of DNA associated with membrane/DNA/DNA polymerase complexes is reported. DNA of mut T1 membranes is more sensitive towards exogenous nuclease than DNA of membrane complexes from the wild type muff strain. 3. Auto-digestion of this DNA by endogenous nuclease associated with the membrane complex is, also, more severe in preparations derived from mut T1 than from the wild-type strain, mut ÷, but to a lesser extent than observed with exogenous nucleases. 4. Nuclease susceptibility of rout ÷ membrane bound DNA is markedly influenced by the growth state of the cell. The nuclease susceptibility of membrane bound DNA from mut T1 cells, however, shows no differences between stationary and log states. 5. These differential sensitivities may be due to conformational changes in the membrane introduced as a pleiotrophic consequence of an altered membrane protein. A pertinent role of this protein in a modified replication[repair corn* Part I, Biochim. Biophys. Acta 4 4 2 (1976) 216. ** This report is abstracted from a thesis presented in partial fulfillment of the requirements for the Ph.D. in the Graduate School of Biomedical Sciences. Present address: The Memorial Sloan-Kettering Cancer Center, N e w York, NY 10021, U.S.A. ** * To w h o m reprint request should be addressed.

18 plex is an attractive suggestion, especially in the context of the mutator character of this strain.

Introduction The inner or cytoplasmic membrane of Gram negative bacteria is associated with a fraction of the cellular DNA [1--3]. Jacob et al. [4] suggested that the cell membrane plays a role in DNA replication and cell division and that there are specific attachment sites on the membrane for initiation and termination of DNA replication [4--13]. A pertinent review, with special emphasis on the Bacillus subtillis membrane complex, has been made by Sueoka and Quinn [14]. In E. coil B/r, the amount of DNA attached to the membrane fraction has been shown to vary with the pattern of DNA synthesis [10,12]. As the number of replication points increase, there is a corresponding increase in the fraction of DNA bound to the membrane. Autoradiographic analysis of mutant E. coli cells which contain a high proportion of intracytoplasmic membranes supports these interpretations [13]. Similar associations between DNA and proteins believed to be derived from membranes have been reported for SV40 [15], the col E-1 plasmid system [16], eukaryotes [17,18], as well as bacteria [19]. Mitochondrial DNA appears to be associated with the mitochondrial membrane on the basis of electron microscopic examination [20]. The results of restriction endonuclease digestion of the mtDNA-protein complexes suggested that the protein is attached at, or close to, the origin of mtDNA replication [21]. Shearman and Kalf [22] have observed a 3-fold increase of DNA associated with mitochondrial membranes derived from regenerating and foetal rat liver compared to base-level tissue. DNA-membrane complexes which can still carry out DNA replication/repair have been isolated from E. coil strains by us [23] and other investigators [24-26]. The various complexes isolated by different procedures are not comparable since artifacts of DNA associated with membrane have been reported to form during cell lysis when lysozyme is employed [ 27]. While it has been reported that most, if not all, membrane-bound DNA from E. coli is completely accessible to exogenous nucleases [25], that portion of the membrane-bound DNA corresponding to origins of replication are resistant to DNAase [6,28]. We had previously reported that a mutator strain of E. eoli K12, (mut T1); which has a molecular defect correlated to the process of DNA replication [29--33] contains an alteration in one or more membrane components [23]. We report, here, the nuclease sensitivities o f the DNA associated with the membranes relevant to the use of these probes to explore differences in the conformation of the replicon. Materlalg and Methods Bacterial strains. E. coli W3110, mut ÷, is a wild type E. coil K-12. E. coil W3110M is an isogenic strain carrying the mut T1 gene. Both were gifts from Dr. E.C. Cox of Princeton University.

19 All cells were grown in 1-1 volumes of Tripticase Soy Broth (Becton, Dickinson and Co.). Stationary phase cells were grown at 37°C for 18 h. For log-phase cells, 200 ml stationary phase cells were transferred to 1 1 fresh medium and maintained at 22°C with constant shaking for 5 h. Preparation of lysates and membrane fractions. Cells were collected by low speed centrifugation, 5000 × g for 20 min, and washed twice in a buffer containing 5 • 10 -2 M morpholinopropane sulfonate, pH 7.6; 1 • 10 -4 M ethyleneglycol-bis-(~-aminoethylether)-N,N'-tetraacetic acid; and 1 . 1 0 - 3 M 13-mercaptoethanol, hereafter designated Buffer A. The cells were resuspended in Buffer A (one-twentieth of the original volume of growth m e d i u m ) a n d the cells lysed by a single passage through a French press (Aminco, Model No. 4-3398) maintained at 4°C at 18 000 lbs/inch 2. Lysis was essentially complete on the basis of microscopic examination and the absence of sedimentable material upon low speed centrifugation. The lysates were centrifuged at 40 000 × g in a Sorvall SS-34 rotor for 30 min to prepare the membrane pellets. The membrane complexes were washed with Buffer A. Complexes washed three times are designated Membrane 3; those washed twice, Membrane 2; unwashed membrane complexes, Membrane 0. For comparison only, some lysates were prepared by incubating cell suspensions with lysozyme (0.5 pg/ml) and EDTA (0.1 M) for 30 rain. These lysates were then processed, as usual, by passage through a French press and subsequent washings. Representative analyses of cell lysates and Membrane 3 preparations are presented in Table I. Preparation of isotopically labelled membrane complexes. Membrane-DNA complexes were prepared from cells which had been grown in the presence of 3H-labeled deoxynucleosides. For this purpose, duplicate 300 ml Trypticase Soy Broth containing 0.15 mM deoxyguanosine received 15 ml overnight cell growth and were maintained at 37°C for 2.5 h with shaking. At this time 100 pCi of each 3H-labeled deoxynucleoside (spec. act. 5--12 Ci/mM were added to the medium and incubation was continued for 18 h at 37°C with

TABLE

I

NUCLEIC

ACID CONTENT

Fraction

OF LYSATES

Mode of

AND MEMBRANE Total DNA content

FRACTIONS * (mgfl)

Total RNA content

* (mg/1)

treatment

Lysate **

Membrane 3

French press or lysozyme

French press Lysozyme Lysozyme + French press ***

W3110

W3110M

W3110

W3110M

10.5 + 0.2

12.9 + 0.4

38.4 + 0.7

50.4 + 0.9

2.2 + 0.1 6.3 + 0.1

2 . 5 -+ 0 . 1 7.9 + 0.3

16.4 + 0.8 1 9 . 6 -+ 3 . 0

18.7 + 0.8 20.0 + 1.4

5.5 + 0.1

6.6 + 0.1

17.3 + 0.9

19.7 + 2.8

* N u c l e i c acid c o n t e n t w a s n o r m a l i z e d t o c o r r e s p o n d t o 1 1 o f stationary phase c e l l s . T h e m u t a t o r strain generally grows to 1.3 • cell concentration of the wild-type strain. * * T h e r e i s n o d i f f e r e n c e in nucleic acid c o n t e n t o f l y s a t e s o b t a i n e d b y either m e t h o d : data a r e c o m b i n e d average. * * * These membrane fractions lysozyme lysates.

were passed through the French press immediately

a f t e r centrifugation o f

20 shaking. Cells from one flask were collected by centrifugation and designated stationary phase cells. The duplicate culture was transferred to 5 vols. fresh, non-radioactive medium, and allowed to grow into log phase before collection by centrifugation. The conditions for cell lysis and preparation of membranes were identical to those described previously [23]. Membrane 3 preparations were employed for the studies on nuclease sensitivity of membrane-bound DNA. Nuclease digestion. Membrane 3 fractions from 3H-labeled cells were resuspended in Buffer A. Each 10 ml sample contained 50--100 mg of membrane protein. A pretreatment of these samples with pancreatic RNAase was done (50 ~g RNAase/ml + 20 mM EDTA final concn.). After 18 h incubation at 37°C, the membranes were collected by centrifugation at 40 000 × g for 30 min and washed three times with 20 ml Buffer A. The washed membranes were then resuspended in 2 ml Buffer A and appropriate additions were made. Controls consisted of membrane samples in Buffer A containing 20 mM EDTA and others containing 20 mM MgC12. The third set of samples contained 20 mM MgC12 and 100 ~g/ml final concentration of pancreatic DNAase I; while the fourth set of samples contained 20 mM MgCl2 and 4U/ml final concentration of Neurospora crassa endonuclease. Incubation of these mixtures was done at room temperature (25°C) for 5 h, while 0.2-ml aliquots were removed at intervals during this period and treated by addition of 1 vol. ice-cold 10% trichloroacetic acid. The precipitates were collected by low speed centrifugation in the cold, and washed twice with cold 5% trichloroacetic acid. The total wash volume was 2.5 ml. 1 ml of this pooled wash fluid was counted in Hydromix fluor in a Beckman scintillation counter. Estimation o f DNAase activity dissociable from the membrane complexes. Membrane 3 fractions derived from 1-1 cultures of both strains (logarithmic as well as stationary growth situations) were resuspended in 5 ml buffer (20 mM Tris-HC1 (pH 7.5), 5 mM dithiothreitol, 20 mM MgC12). After 18 h at 4°C, the samples were centrifuged for 30 min at 15 000 rev./min. The protein concentrations in the supernatant fluids from the stationary and log phase complexes were 400 pg/ml and 150 pg/ml, respectively. 0.5 ml supernatant showed no detectable nuclease activity, measured as acid-soluble ultraviolet-absorbing material, towards 500 pg T7 DNA after 3 h incubation at 37°C. Other methods. DNA contents were determined by the Burton modification of the diphenylamine reaction [34]. RNA assays were based on the orcinol procedure [35]. Protein contents were determined by the method of Lowry et al. [36]. Source o f materials. Morpholinorpropane sulfonate, ethyleneglycol-bis-(~aminoethylether).N,N'-tetraacetic acid and EDTA were obtained from Sigma Chemical Company. RNAase (EC 3.1.4.22} and DNAase (EC 3.1.4.5) were obtained from Worthington Biochemicals. N. crassa endonuclease was purchased from Boehringer Mannheim Biochemicals. Tritium labelled deoxyribonucleosides were obtained from Schwartz/Mann Biochemicals. Guanosine was purchased from Sigma Chemical Company. T7 phage DNA was prepared from 20-1 of E. coli B lysate following the procedure of Shapiro and Bassett [37]. Hydromix, LSC mixture, was purchased from Yorktown Research.

21

Results and Discussion

Retention o f nucleic acids by membrane fractions. The DNA content of lysates and washed membranes is shown in Table I. Membranes prepared from cells lysed in the presence of lysozyme retained three times more (P < 0.001) cellular DNA than those prepared from cells lysed by passage through a pressure cell. This DNA was strongly attached to the membranes, since further washing removed less than 10% of the bound DNA. When complexes isolated from cells lysed in the presence of lysozyme were passed through a French press, only 15% of the bound DNA could be further removed. These observations complement the report that lysozyme promotes a non-specific binding of DNA to membranes [27]. Our results, reported here, are based on studies of DNA/membrane complexes derived from cells disrupted by passage through a French press. The extent of retention of RNA by the membrane complexes is also reported in Table I. The amount of RNA retained shows no correlation with the method used for cell lysis (P > 0.01). In all cases, about 40% of the total cellular RNA was found associated with the membranes. This KNA was routinely removed prior to use of the membranes, and has not been further characterized. Abe et al. [28] have found that pretreatment of their E. coli membrane fraction with RNAase does not influence subsequent release of DNA by nucleases. It is possible that this fraction of KNA represents mRNA and ribosomes associated to DNA; transcription and translation occurring simultaneously with replication in this prokaryote [38]. The mutator strain grows to a slightly greater density than does the parental strain as shown by (a) cell count at stationary phase, (b) total cellular protein, and (c) total cellular nucleic acids [23]. Table I shows that both DNA and RNA content between the two strains correspondingly differ by about 30%; data which correlate with t h e observation of Gibson et al. [33] that the mutator strain grows more efficiently in a chemostat than does the wild type strain. This could be a consequence of and increased activity level of an obligatory polymerase (replicase) in the mutator strain membrane. The consensus, however, is that the membrane-bound enzyme is DNA polymerase II, which has not been shown to participate directly in replication [24,25]. In addition, no evidence for any structural alteration in this enzyme associated with this mutator gene has been found [23]. An alteration in the conformation of a component of the total membrane-bound replication complex may, however, account for the differences in growth characteristics of the mutator strain. Comparison of the susceptibility to nuclease would allow detection of some subtle modifications in the conformation of the membrane bound DNA among these strains. Since it had been reported that membrane-bound DNA contains some single-strand regions [5] our protocol included use of a nuclease specific for such regions; N. crassa nuclease. General observations on the effects o f nuclease upon membrane bound DNA. All membrane-bound RNA may be removed by 18 h incubation with RNAase (5% enzyme concentration, w/w, with respect to substrate). Limit digestion with deoxynucleases, however, removed a b o u t one-third; (mut *,

22

stationary); to one-half; (mut T1, stationary); of the membrane-bound DNA (Table II) from fractions prepared in the absence of lysozyme. When fractions derived from lysozyme treated cells were similarly treated, significantly more DNA could be released, suggesting, again, a non-specific association of a proportion of this DNA with the membrane. An observation that 90% of membrane-bound DNA can be released on exposure to endonucleases may be a reflection of the mode of preparation of these DNA/membrane complexes as well as differences between our analytical methods [28]. No nuclease activity could be detected in supernatant fluids of washed Membrane 3 fractions, indicating that the endogenous nuclease is strongly associated to the membrane complex. In the presence of Mg~÷ all membrane fractions showed endogenous nuclease activity towards the bound DNA (Table II). When the membrane fractions were incubated in the presence of Mg2÷, the endogenous membrane-DNA underwent digestion (Fig. 1) at a rate which appeared to be biphasic (semi-log plot) with time. The initial reaction may represent limited digestion; perhaps the formation of large, acid-insoluble, fragments; followed by extensive digestion to acid-soluble fragments by the endogenous nucleases. This suggestion is supported by other observations [28]. Exogenous nucleases also released acid-soluble fragments from the membrane-DNA complexes. The reaction is closer to first-order kinetics than the release of digestion products by endogenous nucleases. Both DNAase I and N. crassa nuclease each solubilize between 30--60% of the membrane-bound DNA (Fig. 2, 3 and Table II), in consort with the endogenous nucleases. Specific observations on the accessibility o f membrane-bound D N A to nucleases: Strain W3110, m u t ÷. Stationary phase cultures contain a low proporT A B L E II R E L E A S E OF D N A ON E X P O S U R E OF MEMBRANE-3 F R A C T I O N S TO N U C L E A S E S S a m p l e s w e r e p r e p a r e d f r o m F r e n c h p r e s s lysates and incubated for 5 h w i t h the additions listed. Strain

Additions

Percent m e m b r a n e 3H-labeled DNA r e n d e r e d trlchloroacetic acid s o l u b l e *

Stationary phase

p**

Log p h a s e membrane

membranes W 3 1 1 0 , rout*

W3110M, m u t T1

EDTA Mg 2+ Mg 2+ + D N A a s e Mg 2+ + N . ¢ r a u a nueleese EDTA Mg 2+ Mg 2+ + D N A a s e Mg 2+ + N . c r a u a

8.0 31.1 33.7 33.8

± ± + ±

0.7 2.0 1.2 1.2

7.6 36.9 41.8 42,8

-+ 0.4 ± 1.0 -+ 0 . 5 ± 1.0

N.S. <0.05 <0.001 <0.001

9.6 45.1 53.6 55.0

+ ± ± ±

0.8 1.5 3.0 3.6

11.0 41.2 53.5 54.0

-* 0 . 5 ± 0.8 -+ 1.4 ± 1.7

N.S. N.S. N.S. N.S.

nuclease * I n b o t h m a m b r a n e p t ~ p m t t o n s the c o n t e n t o f original celhtlar D N A ~ a p p r o x . 20% ( T a b l e I). *e p was deterlnhled b y a 2*taned t - t e s t e d . P-values s h o w n refer to c o m p a r i s o n o f stationary and log phase stationary and log phase samples. N.S;'= n o t significant. See t e x t for P v a l u e s w i t h i n g r o u p s and between strains.

23

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TIME, mln of incubotion Fig. 1. A m e a s u r e o f m e m b r a n e - b o u n d D N A accessible t o e n d o g e n o u s nuclease. P e r c e n t o f 3 H - l a b e l e d D N A c o n v e r t e d t o acid soluble m a t e r i a l w h e n m e m b r a n e - 3 f r a c t i o n s were e x p o s e d o n l y t o B u f f e r A + 20 m M MgCI 2. A, E. coli W 3 1 1 0 M , log p h a s e m e m b r a n e f r a c t i o n ; o, E. coli W 3 1 1 0 M , s t a t i o n a r y p h a s e m e m b r a n e f r a c t i o n ; o, E. coil W 3 1 1 0 , log phase m e m b r a n e f r a c t i o n ; e, E. coZi W 3 1 1 0 , s t a t i o n a r y phase m e m brane fraction.

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TIME, rain of incubation Fig. 2. A m e a s u r e o f m e m b r a n e - b o u n d D N A accessible t o D N A a s e I. P e r c e n t o f 3H-labelled D N A c o n v e r t e d t o acid soluble m a t e r i a l w h e n m e m b r a n e - 3 f r a c t i o n s were e x p o s e d t o B u f f e r A + 2 0 m M MgCI 2 + p a n c r e a t i c D N A a s e I. % E. coil W 8 1 1 0 M , log p h a s e m e m b r a n e f r a c t i o n ; o, E. coli W 3 1 1 0 M , s t a t i o n a r y p h a s e m e m b r a n e f r a c t i o n ; o, E. coil W 3 1 1 0 , log phase m e m b r a n e f r a c t i o n ; e, E. co|i W 3 1 1 0 , s t a t i o n a r y phase membrane fraction.

24

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22

50

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T I M E , rain of inl:ullNdton Fig, 3. A measure of membrane-bound DNA acceulble to N. c r a ~ a endonuclease. Percent of 3H-labeled DNA converted to acid soluble mateldal when membrane-3 f~sctionJ were e x p o N d to Buffer A ÷ 20 mM MgCl 2 + N. c r a ~ a endonuclease. A, E. coZi W3110M, log phase membrane f~lmt/on; o, E. coU WS110M, statiozutry phase membrane fraction: o, E. ¢oli W3110, log phase rnembnme fraet/on; a, E. col• W8110, stationary phase membrane fraction.

tion of cells which are actively dividing. Membrane fractions from such cells might contain DNA which differs in physical organization from that of complexes derived from an actively dividing culture (log phase). The susceptibility of membrane-bound DNA from stationary and log phase cultures toward exogenous nucleases was, indeed, significantly different (Table II). This difference is, moreover, maintained when comparison is made between the action of the endogenous nuclease on the membrane-bound DNA from stationary and log phase cells of strain rout* (Table II). The proportion of the cellular DNA bound to the membrane fractions is the same for cells at either growth state; (20% of total cell DNA), and the base composition of this membrane bound DNA is the same; A + T/G + C = 1.00 (unpublished results). The difference in susceptibility to nuclease, therefore, does not appear to be a function of the chemical compositions of the bound DNA, but may be a consequence of the conformation of the DNA associated to the membrane structure. During the log phase of growth, the DNA may be associated in such a way to be accessible to the action of replication enzymes, including any nucleases which might participate in mechanisms which can be postulated for replication. In cells not actively dividing, one or more membrane components may change conformation to screen the DNA from these enzymes. A mechanism of this type can account for both the increased growth rate and the differential susceptibility of the DNA to nuelesse ~ . The action of endolFmeus nuelesse would be dependent on the physical proximity of the bound DNA to the enzyme. Strain W3100M, rout T1. Unlike the rout* membranes, the membrane-DNA

25 complexes from stationary and log phase cells of the mutator strain show no significant differential susceptibility toward nuclease (Table II). We assign this to a change in conformation of some component(s} of the membrane induced by the mutator gene. The organization of the membrane in log phase mut T1 cells may then be maintained during transition to a stationary growth stituation. This could explain all of our observations. Our previous studies on membrane-bound polymerase activity from stationary phase cells of mut ÷ and mut T1 support this hypothesis [23]. The mutator strain membrane-bound DNA is used more effectively than by its membrane polymerase than mut ÷ membrane-bound DNA, implying a differential accessibility in the two strains. Comparison between W3110, m u t ÷, and W3110M, rout T1 membranes exposed to nucleases. The time course of exogenous nuclease digestion on mutator and wild-type membranes is shown in Figs. 2 and 3. Our observations may be summarized as follows: All membrane fractions subjected to pancreatic DNAase I show digestion patterns which essentially follow first-order kinetics (Fig. 2). Wild type membranes from log and stationary phase cells show a significant different in susceptibility of membrane-bound DNA when exposed to nucleases (Table II, Figs. 2 and 3). Mutator membranes from log and stationary phase cells show similar susceptibility of membrane-bound DNA when exposed to nucleases (Table II, Figs. 2 and 3). Action of Neurospora endonuclease. The extent of digestion of membranebound DNA by N. crassa endonucleases is the same as by pancreatic DNAase I. The initial rates of formation of acid soluble fragments from the DNA are, however, lower during N. crassa endonuclease digestions, suggesting a transition from longer to shorter products of digestion (Fig. 3). A reasonable interpretation of these results relies on one or more of the following situations. (a) Initially, the action of N. crassa nuclease is impeded by the conformation of the membrane-DNA complex. (b) The initial lag could be due to a limited hydrolysis of double-stranded DNA by the N. crassa nuclease. In later stages, as single-stranded fragments become available, digestion occurs at a faster rate. (c) Large, acid insoluble, single-stranded fragments may be released by endogenous nuclease activity during the early stages. These subsequently serve as substrates for the N. crassa nuclease. We favor the last option. To the limited extent that comparisons between different membrane systems may be made, the observations of Abe et al. [28] on the release of DNA fragments having a molecular weight of approx, l 0 s by single-strand specific endonucleases is compatible with our results. Conclusions

Limit nuclease digestion of the DNA-membrane complexes inavariably leaves a resistant core of DNA, amounting to about 10% of the total cellular DNA. This DNA does not appear to differ from the bulk of the cellular DNA in composition. Thus, we conclude that the resistance is conferred by some

26 peculiarity in the organization and association of this DNA with a particular membrane component(s). Comparison of the mutator strain with its wild parent support this contention. There are greater differences between stationary and log phase membranes from mut* cells than between membrane complexes from log phase mut ÷ and stationary phase mut T1 complexes (Table II). This implies that the conformation of the mutator strain DNA-membrane complexes during stationary phase, shares some characteristics with the log phase complexes isolated from the wild-type. On the other hand, membranes from the mutator strain show no significant difference in nuclease susceptibility during the stationary and log phases of growth (Table II), implying that the DNA-membrane complex retains a particular conformation during the growth cycle. We have previously reported that a new, basic protein band can be identified on isoelectric focusing of mutator strain membrane protein extracts [23]. This alteration could be responsible for the altered nuclease susceptibility of the two strains. This protein could alter the conformation of other membrane components closely associated with membrane-bound DNA, or might itself be the binding protein responsible for such a conformational change. Isolation and further characterization of this protein may allow resolution of this problem.

Acknowledgements These studies were supported in part by an allocation from the American Cancer Society institutional research grant 1N-92C of the New Jersey Medical School and, in part, by an allocation from the Office of the Graduate School of Biomedical Sciences.

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