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Jun 14, 1977 - POLSINELLI, M. (I976). A recombination test to classify mutants of Bacillus subtilis of identical phenotype. Genetical Research 27, 47-58.
J. gen. Virol. (I978), 39, 81-9o

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Printed in Great Britain

PBSX Induction in a Temperature-sensitive Mutant of Bacillus subtilis By U M B E R T O C A N O S I , F R A N C O A. F E R R A R I , E U G E N I O U. F E R R A R I , G I O R G I O M A Z Z A AND A N T O N I O G. S I C C A R D I * Istituto di Genetica, Universitgl di Pavia and Laboratorio di Genetica Biochimica ed Evoluzionistica del CNR - Via Sant'Epifanio, t 4 - 27IOO Pavia, Italy (Accepted 26 October I977) SUMMARY

A temperature-sensitive mutant of Bacillus subtilis is described which produces PBSX phage at a non-permissive temperature (47 °C) • The mutant is, in the properties tested, phenotypically identical to the mutant tsi23 reported by Siegel & Marmur (I969). Its mutation (tsi85) maps in the same chromosomal region (dal-purB) in which tsi23 is located; the two mutations are shown to be distinct but probably affect the same function. Double mutants carrying the mutations tsi and xin (which blocks the induction of PBSX by mitomycin C) do not produce PBSX at a non-permissive temperature but retain their thermosensitivity. Tsi mutants display a reduced rate of RNA synthesis at the non-permissive temperature. Such a phenotype is lost in tsi-xin double mutants, revealing that it is associated with PBSX induction. The nature of the primary lesion that leads to PBSX induction in tsi mutants remains to be ascertained.

INTRODUCTION

All strains of Bacillus subtilis are able to produce phage-like particles, either spontaneously at a low rate, or at a high rate through the action of inducing agents such as mitomycin C (Seaman et aL I964) or hydrogen peroxide (Stickler et aL 1965). The main phage produced, PBSX, is a fully defective virus since it contains B. subtilis DNA (Okamoto et al. 1968 a, b) and is devoid of infectivity, although it is bactericidal for suitable sensitive host bacteria such as the B. subtilis strains W23 and $3I (Okamoto et al. I968 a). The DNA extracted from PBSX particles obtained by mitomycin C induction has peculiar properties (Haas & Yoshikawa, I969b; Garro et al. I976); the markers purA (proximal to the replicative origin) and metC (proximal to the phage-specific markers xin, xtl, xhd) (Thurm & Garro, I975) are more frequent, as measured by transformation, than all other host markers and display a high degree of differential renaturability after heat denaturation (Haas & Yoshikawa, I969b; this paper Table 4). The hypothesis has been put forward that the PBSX genome is integrated in the B. subtilis chromosome in a 'scattered' fashion resulting in the inability of the phage DNA to be * Present address: Cattedra di Microbiologia II, Facolth di Medicina, Universit~t degli Studi di Pavia, Italy. 6-2

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Table i. List of strains of Bacillus subtilis used Strain

Genotype*

SB 19 W23 Mu8u5u6 Ts85

Prototroph Prototroph purB6 leu8 metB5 leu8 metB5 ts8 S

MB5oo 6o935 BC37

metB5 leu8 tsi23 dal metC trpCz purA16 argA3 phe12 lys2~ metB asal o pyrA metC argC metC xin thyA thyB arg purB6 leu8 metBs hisA1 lys21 thr(saeA) purB6 leu8 hisA1 lys21 purB6 leu8 metB 5 tsi85 purB6 leu8 lys21 tsi8S tsi8 S xin tsi23 xin thyA thyB tsi8S dal purB6 lys21 hisA1 trpC2 metB5

GB64 GB63 PB566/2 PBI665 PBI753 PBI754 PB1755 PBI756 PBI757 PBI758 PB336o

Origin J. Lederberg E. W. Nester N. Sueoka From Mu8u5u6 as described by by Siccardi et al. (1976) J. Marmur E Freese A. Adams A. Garro A. Garro

From PB1665 + DNA SBI9 From PBI665 + DNA Ts85 From PBI753 + D N A 1754 From GB63 + DNA PBI755 From GB63 + D N A MB5oo From PB566/2 + DNA PBI755

* Symbols: pur, leu, met, his, lys, thr, dal, trp, arg, phe and pyr indicate, respectively, requirement for adenine, leucine, methionine, histidine, lysine, threonine, D-alanine, tryptophan, arginine, phenylalanine and uracil; saeA, inability to utilize sucrose as carbon source; asalo, resistance to arseniate; tsi, thermosensitive induction of PBSX; xin, inability to induce PBSX by mitomycin C.

excised and packaged as a single infective unit. The tsi23 mutation, described by Siegel & Marmur (I969) as a probable PBSX mutation Ieading to heat-induction of PBSX phage, supports such an hypothesis since it has been mapped in the purB region, far from the PBSX specific markers (xin, xtl, xhd) which are located close to metC. Two lines of evidence indicate that tsi is not a phage marker: (I) tsi23 strains lysogenic for SPO2 and ~IO5 display heat-induction of these phages as well as of PBSX; (2) another ts (temperature-sensitive) mutant of B. subtilis, xhi-z479 (Buxton, I976), has been described in which PBSX only, and not ~5io5, is induced at the non-permissive temperature in ~blO5 lysogens. The latter mutation is located in the argC-metC region with the other PBSX markers (Buxton, 1976). This suggests the alternative hypothesis that tsi23 is a host marker whose defect non-specifically leads to phage induction. In other bacterial systems (Kirby et al. 1967; Schuster et al. 1973), temperature-sensitive bacterial mutations have been associated with prophage induction. This work describes a second tsi mutation distinct from tsi23 but possibly affecting the same function and supports the hypothesis that tsi is a host marker with an indirect effect on phage induction. METHODS

Bacterial and phage strains. The origin and description of the strains used in this work are given in Table I. The phage PBS-I was used for transduction experiments (Takahashi, 1963). Culture media. Spizizen minimal medium MT (Spizizen, 1958) was used to prepare competent cells. Medium Y (Yamagishi & Takahashi, I968), Penassay broth (PY) (anti-

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biotic medium no. 3, Difco) and Tryptose Blood Agar Base (Difco) were used in transduction experiments. The minimal medium of Davis & Mingioli (I95o) supplemented by the appropriate requirements was used for selection of transformants and transductants. Bouillon Nutritif Complet (Bio Kar) solidified with nutrient agar was used to determine viable counts and temperature-sensitive phenotype (ts). Reagents. Mitomycin C (MitC) was purchased from Sigma Chemical Co. 5-3H-Uridine (sp. act. 28 Ci/mmol) and methyl-ZH-thymidine (sp. act. 18 Ci/mmol) were purchased from the Radiochemical Centre, Amersham, England. P B S X induction. An exponential culture in PY broth (34 °C) at E56o = o.I to o.2 was treated with 4 #g/ml MitC for I5 rain. The cells were collected by centrifugation and resuspended in the same volume of PY broth. PBSX induction in ts mutants was also obtained by shifting an exponential culture (34 °C) at 4o to 5o Klett units or E56o = o.I to o.2 to 47 °C. In both cases the turbidity usually reached a peak at about 9o rain after treatment with MitC, or after the shift to the non-permissive temperature, and then declined falling to a minimum at about 3 to 4 h. The lysed cultures were then centrifuged at 8ooo g for IO rain to remove cell debris and unlysed cells and the supernatants were stored at 4 °C with a few drops of chloroform. Evaluation of P B S X activity. PBSX has been recognized by its killing activity on B. subtilis W23. A o'5 ml sample of a lysate was mixed with o'5 ml of a culture of B. subtilis W23 taken at the end of the exponential phase and diluted to Io 5 cells]ml. After IO min at room temperature the reduction in cell titre was determined. Under these conditions the bactericidal activity of PBSX-containing lysates, obtained either by MitC or thermal induction, reduced the viability of W23 to about 1% of the control. Concentration of phage lysates. The lysates obtained by MitC or temperature induction were centrifuged for 2 h at 5oooo g in a Spinco rotor 3o at I5 °C. The pellets were dissolved in a few ml of o'I5 M-NaCI plus o'oi5 M-sodium citrate (SSC). After centrifuging for I o rain at I o ooo g the lysates were treated with 5o/zg/ml of deoxyribonuclease, 25/zg/ml of pancreatic ribonuclease and 2 units/ml of T1 ribonuclease. After 3° min of incubation at 37 °C the lysates were filtered on Millipore filters (o'45/zm). Preparation of DNA. Bacterial D N A was prepared according to the method described by Marmur (I96I). D N A from PBSX was extracted from concentrated phage as described by Haas & Yoshikawa (i969a). Denaturation and renaturation of DNA. Heat denaturation of phage D N A in SSC was accomplished by heating the D N A (IO #g/ml) at ioo °C for i2 min in a screw-cap tube. The sample was then quickly cooled in ice with mild shaking. Renaturation was accomplished by incubating the denatured D N A at 65 °C for I6 h. Preparation of antisera. Anti-PBSX antiserum was prepared by immunizing a rabbit with a suspension of concentrated temperature-induced PBSX and complete Freund's adjuvant (Difco). The rabbit received three weekly subcutaneous injections. One week after the final inoculation, an antiserum active on PBSX was obtained. The antiserum activity was measured as follows: to a sample of PBSX lysate an equal volume of a I[2oo dilution of antiserum was added. The mixture was incubated for I5 min at 37 °C and the residual activity of PBSX was assayed. In these conditions the antiserum totally suppresses the bactericidal activity of PBSX as compared to the control. Transformation and transduction. Competent cells were prepared as described by Stewart (I969). The transformation procedure was that described by Mazza et al. 0975). For marker congression experiments the D N A concentration was 2 to 3 #g/ml. PBS-I transduction was performed according to the method of Hoch et aL (I967).

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Fig. I. Effect of a 34 to 47 °C shift on the growth of PBI754 ( O - - O ) 34 °C; ( C ) - - O ) 47 °C.

(tsi85)

in PY broth.

Genetic mapping. The linkage of the tsi85 mutation with other markers of known location on the genetic map was measured by PBS-I transduction; the ts phenotype was tested on nutrient agar plates incubated at 47 °C. Recombinants for auxotrophic markers were selected on minimal medium supplemented with 0"5 % glucose and the appropriate auxotrophic requirements (25 #g/ml). After 2 days of incubation at 37 °C, recombinants were picked, re-isolated and tested for their phenotype. Distances between markers are expressed as percentage of recombination according to the convention: % recombination = (I - c o t r a n s f e r ) × Ioo. Macromolecular syntheses. An overnight culture in PY was diluted Ioo-fold in the same medium and grown at 34 °C. At about E560 -- oq to 0.2, one-half of the culture was shifted to 47 °C. The rate of D N A synthesis was estimated by adding 0.2 ml of culture to a test tube containing I5 #1 of 5o #Ci/ml ~H-thymidine and incubating for 2 min at the same temperature. The reaction was stopped by adding 2 ml of cold io % trichloroacetic acid (TCA) and the tubes were then stored in an ice bath. After 3o rain, samples were filtered on G F / C Whatman glass fibre paper and washed with 30 ml of cold 5 % TCA and 2 ml of 95 ~ ethanol. Radioactivity was determined in a liquid scintillation counter. The rate of R N A synthesis was estimated as described for DNA, except that 3H-uridine was used instead of thymidine. RESULTS

Characterization of the ts85 mutant We have isolated, by nitrosoguanidine co-mutagenesis (Siccardi et al. I976), several temperature-sensitive mutants linked to a given biochemical marker. In this paper we describe the biochemical and genetic characterization of a temperature-sensitive mutant (ts85) linked to purB. Fig. I shows the growth of the mutant at 34 °C and after the shift to 47 °C. After about 90 rain of growth at 47 °C, the turbidity of the culture decreases and cell lysis is observed. PBSX-like bactericidal activity against B. subtilis W23 can be demonstrated in the supernatant of the lysed culture (see Methods). The PBSX-like activity observed in the lysed

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T a b l e 2. Three-factor crosses by PBS-I mediated transduction to map the tsi85 d a l and p u r B markers* Selection Dal +

Pur +

Pur + Dal +

Non-selected markers

No. of transductants

Dal + Pur + tsDal + Pur + ts + Dal + Pur- tsDal + Pur- ts + Pur + Dal + tsPur + Dal + ts + Pur + Dal- tsPut + I3al- ts+ Pur + Dal + tsPur+ Da 1+ ts +

4 I 9 35 I I 33 I9 42 9

* Donor strain: PBI758 thyA thyB tsi85; recipient strain: PB 3360 dal purB6 lyszl hisA1 trpC2 metB5. T a b l e 3. Analys& of transformation* crosses by the recombination index method Transformantst ~ Tsi + PurB +

Donor DNA

~ Tsi+/Pur+ ratio

SBI9 (prototroph)

7"6 × Io 4

3"3 × Io 4

2'3 (A) [

MB5oo tsi23 metBJ leu8

8.6 × IO3

3"8 × IO4

o.226 (B)J

!

RI:~ o.o98

* Transformation was performed at o'I/zg/ml; cell titre was 2"I7 × IOs cells/ml. Recipient strain: PBI755 purB6 leu8 lys21 tsi85. t Tsi+ transformants have been corrected for spontaneous reversion frequency, i.e. I'5 × IO-5. :~ Recombination index (RI) = (B)/(A). culture is several o r d e r s o f m a g n i t u d e higher t h a n t h a t o b s e r v e d in the s u p e r n a t a n t o f s t a t i o n a r y phase cultures, where P B S X particles are n o r m a l l y present in low c o n c e n t r a t i o n ( S u b b a i a h et al. 1965). T h e m o s t obvious e x p l a n a t i o n o f these d a t a is t h a t the t e m p e r a t u r e shift induces the defective phage. A similar p h e n o m e n o n is o b s e r v e d in wt strains after t r e a t m e n t with m i t o m y c i n C ( S e a m a n et al. I964; H a a s & Y o s h i k a w a , I 9 6 9 a ) a n d in a n o t h e r t e m p e r a t u r e sensitive m u t a n t (tsi23) described b y Siegel & M a r m u r (I969). T o test the identity o f the b a c t e r i c i d a l activity released at the n o n - p e r m i s s i v e t e m p e r a t u r e b y the ts85 m u t a n t with PBSX, we p r e p a r e d a r a b b i t a n t i s e r u m against the bactericidal activity o b t a i n e d after t e m p e r a t u r e i n d u c t i o n a n d tested its inactivating p o w e r on lysates o b t a i n e d b y M i t C i n d u c t i o n a n d b y t e m p e r a t u r e induction. B o t h activities were suppressed at high serum dilutions, i n d i c a t i n g serological cross-reactivity.

Genetic studies with ts85 mutant W e have c o n f i r m e d the l o c a t i o n o f the ts85 m u t a t i o n in the dal-purB region using PBS-I t r a n s d u c t i o n . T h e results o b t a i n e d b y three-factor crosses, involving dal, purB a n d ts85, are r e p o r t e d in T a b l e 2. The o r d e r o f m a r k e r s d e d u c e d f r o m these results is dal ts85 purB a n d the distances o f ts85 f r o m dal a n d purB are 74 a n d 38, respectively. Siegel & M a r m u r 0 9 6 9 ) m a p p e d the tsi23 m u t a t i o n , which is also responsible for temperature-sensitive i n d u c t i o n o f PBSX, in the same c h r o m o s o m a l region with similar

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U. CANOS[ AND OTHERS Table 4. Marker ratios obtained with P B S X and bacterial DNA native and renatured* Ratio~ r

DNA source

DNA type

PurA+/MetB +

MetC+/TrpC +

PBt753 (parental)

Native Renatured Native Renatured Native Renatured Native Renatured

2'2 4"6 2.8I 6"94 2 7"5

I'34 3'57 o'98 z.68 I "9 2.3

PB1755 (tsi85)

tsi-PBSXt MitC-PBSXt

22" 7

I I" 8

12-7

111'6

* For denaturation and renaturation technique see Methods. tsi-PBSX: temperature-induced from strain PBI755; MitC-PBSX; mitomycin C-induced from strain PBI753. :~ To determine purA/metB and metC/trpC marker ratios BC37 and 60935 were respectively used as recipient strains. Transformation was performed at I #g/ml DNA. distances. In order to establish whether the two mutations are in the same or in different genes, we tested them by the recombination index method described by Lacks & Hotchkiss (I96o); competent cells of strain PBI755 (ts85) were used in transformation crosses using as D N A donors the strains SBI9 (control) and MB5oo (tsi23). Selection was carried out for Pur + (outside reference marker) and Ts + transformants. The results of these experiments are reported in Table 3. Since the recombination index obtained in repeated experiments is less than I (but more than o), the two mutations are found to be distinct but closely linked. Considering the identical phenotype and the closeness on the genetic map, the possibility that these two mutations belong to the same cistron can not be excluded. We thus consider ts85 as a tsi mutation (tsi85).

Marker frequency analysis of the DNA of P B S X obtained by various methods We have demonstrated that PBSX induced by mitomycin C (MitC-PBSX) is inactivated by the antiserum prepared against the phage obtained by temperature induction of tsi85 (tsi-PBSX). However, differences exist in the marker ratios of the D N A s of the phages obtained by the two methods. In MitC-PBSX D N A there is an enhancement of the frequency ofpurAz6 and metC markers compared to other chromosomal markers (Haas & Yoshikawa, I969b; T h u r m & Garro, I975). An increase in the purAI6 marker is not observed in tsiPBSX (Siegel & Marmur, i969). Haas & Yoshikawa (I969 b) have demonstrated that the purAz6 marker in MitC-PBSX D N A is more renaturable after temperature denaturation compared to other markers. We have studied marker ratios and renaturability of tsi-PBSX D N A . The results reported in Table 4 indicate that the purA/metB and metC/trpC marker ratios in tsi-PBSX D N A are similar to those observed in bacterial D N A and that the purA and metC markers do not renature any better than other bacterial markers after heat denaturation.

Macromolecular syntheses in tsi85 strain In the tsi23 strain, Siegel & M a r m u r (I969) have observed, using cumulative incorporation methods, a slight decrease after shift to the non-permissive temperature in R N A and protein synthesis, while D N A synthesis is not affected. We have studied R N A and D N A

tsi temperature-sensitive induction

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Fig. 2. Growth and macromolecular synthesis of PB566/2 (wild type) (upper panel) and PBI758

(tsi85) (lower panel) at 34 °C ( Q - - 0 ) and 47 °C ( O - - O ) . (a) E~0; (b) 5-SH-uridine incorporation; (c) 3H-thymidine incorporation.

syntheses in PBt758 (tsi85) and its parental PB566/2 by pulse-label incorporation (Fig. 2). We have confirmed that, on shifting to high temperature, the rate of RNA synthesis does not increase as in the control culture while the rate of DNA synthesis does not appreciably differ from that of the control up to the time when the tsi85 culture starts lysing. The lack of increase in the rate of RNA synthesis is likely to be due to PBSX induction since an analogous and even greater reduction in the rate increase is observed during MitC induction of wt strain GB64 (see Fig. 3) and upon temperature induction of xhi mutant (R. S. Buxton, personal communication). This hypothesis is substantiated by the results obtained with GB63 xin described by Thurm & Garro 0975) which does not produce any phage-specific proteins after MitC treatment; in this strain the rate of RNA synthesis is much less affected by the MitC treatment (Fig. 3). We have been able to obtain strains carrying tsiz3 or tsi85 in addition to the xin mutation (see Table x); these temperature-sensitive strains are unable to produce PBSX either with temperature or MitC treatment. We have checked the rate of RNA synthesis in these strains (PBr756 and PBI757) and the results reported in Fig. 4 indicate that the rate of RNA synthesis is not affected at the non-permissive temperature, i.e. that the primary therrnosensitive lesion is not responsible for the reduction in RNA synthesis. DISCUSSION

The hypothesis formulated by Siegel & Marmur 0969) that PBSX genes might be integrated in a scattered manner in the B. subtilis genome, was mainly based on two arguments, the enrichment in marker frequency of two different chromosomal regions, purA

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tsi temperature-sensitive induction

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and metC, and the location of the tsi23 mutation which was linked to purB, and not to metC, where the PBSX xtl gene was mapped (Thurm & Garrow, x975). The following recent findings make such a hypothesis less likely: (~) all phage-specific markers including xhi (thermal induction of PBSX) are clustered in a region proximal to metC (Buxton, I976); (2) mitomycin C induces re-initiations of replication with purA marker enrichment even in xin mutants; however the xin + function is necessary for metC marker enrichment (Thurm & Garro, I975; Garro et al. I976); (3) thermal induction of tsi23 is not specific for PBSX, affecting other prophages as well (Siegel & Marmur, I969; Thurm & Garro, I975). The present work describes tsi85, a mutation genetically distinct from tsi23, which causes a very similar phenotype. We have investigated a common feature of PBSX induction (both in temperature-sensitive mutants and in MitC-treated wild type strain) which is the reduction in the rate of R N A synthesis; such a reduction disappears in tsi-xin double mutants indicating that it is due to phage induction and not to the tsi mutation. The xin mutation had been shown previously to block induction of PBSX by mitomycin C (Thurm & Garro, I975). The fact that xin also blocks thermal induction of PBSX in the tsi85 (and tsi23) strains but does not alter the temperature-sensitive phenotype clearly shows that cell death at the restrictive temperature is not the result of PBSX replication but rather a direct effect of tsi mutations. Several other thermosensitive mutants, dnaH, dnaI, dnaG (Karamata & Gross, I97o) and tseAx, tsc49 (Galizzi et al. I976) liberate, at a non-permissive temperature, substantial PBSX activity even in the absence of rapid lysis. It is therefore advisable to utilize a xin background for the phenotypic analysis of ts mutants to avoid PBSX dependent contributions to the phenotype. Thanks are due to Professor S. Jayakar for reading the manuscript. We gratefully acknowledge the technical assistance of Sandro Costa and Giuseppe Alloni.

REFERENCES BUXTON,R. S. (I976). Prophage mutation causing heat inducibility of defective Bacillus subtilis bacteriophage PBSX. Journal of Virology zo, 22-28. DAVIS, B.D. ~ MIN~IOLI, r.S. (I950). Mutants of Escherichia coli requiring methionine or vitamin BI2. Journal of Bacteriology 60, 17-28. GALIZZI, A., SICCARDI,A. G., MAZZA,G., CANOSI,13".& POLSINELLI,M. (I976). A recombination test to classify mutants of Bacillus subtilis of identical phenotype. Genetical Research 27, 47-58. OARRO, A. J., HAMmerER,F. & RECHT, B. (I976). Biochemical and genetic analysis of the defective Bacillus subtilis bacteriophage PBSX. In Microbiology, pp. 34o-349. Washington, D.C. : American Society for Microbiology. r~AAS, M. ~ YosrnKAWA,H. (I969a). Defective bacteriophage PBSX in Bacillus subtilis. I. Induction purification and physical properties of bacteriophage and its DNA. Journal of Virology 3, 233-247•

HAAS,M. & YOSmKAWA,rI. (I969b). Defectivebacteriophage PBSX in B. subtilis. III. Properties of adenine-i6 marker in purified bacteriophage DNA. Journal of Virology 4, 844-850. trOtH, J. A., BARAr,M. & ANAGNOSTOPOULOS,C. (I967). Transformation and transduction in recombinationdefective mutants of Bacillus subtilis. Journal of Bacteriology 93, I925-~937. KARAMATA, D. & GROSS, J.D. (I970). Isolation and genetic analysis of temperature-sensitive mutants of B. subtilis defective in D N A synthesis. Molecular and General Genetics xo8, 277-287. KIRBY, E. P., JACOB,F. & GOLDTHWAIT, D. A. (I967). Prophage induction and filament formation in a mutant strain of Escherichia coli. Proceedings of the National Academy of Sciences of the United States of America 58, I9o3-I9IO. LACKS, S. & HOTCHKISS, R. (I960). A study of the genetic material determining an enzyme activity in Pneumococcus. Biochimica et Biophysica Acta 39, 5o8-5x7.

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(Received 14 June 1977)