MGi3

Molec. gen. Genet. 175, 351 357 (1979)

© by Springer-Verlag 1979

The Genome of B. subtilis Phage SPP1 Physical Arrangement of Phage Genes B. Behrens, G. Ltider, M. B e h n c k e * , a n d T.A. T r a u t n e r Max-Planck-Institut ffir molekulare Genetik, Abt. Trautner, IhnenstraSe 63/73, D-I000 Berlin 33 A.T. G a n e s a n Department of Genetics, Stanford University Medical School, Palo Aito, California, USA

Summary. 41 genes o f SPP1 have been d e l i n e a t e d by using c o m p l e m e n t a t i o n a n a l y s e s o f 75 c o n d i t i o nally lethal (ts a n d sus) m u t a t i o n s . T h e p h y s i c a l locations o f these genes on the SPP1 c h r o m o s o m e have been d e t e r m i n e d by t r a n s f e c t i o n / m a r k e r rescue exper i m e n t s in w h i c h r e s t r i c t i o n e n d o n u c l e a s e g e n e r a t e d f r a g e m e n t s o f S P P I D N A were used as d o n o r D N A . The p h y s i c a l o r d e r o f these f r a g m e n t s has been prev i o u s l y e s t a b l i s h e d ( R a t c l i f f et al., 1979).

infected with a c o n d i t i o n a l l y lethal phage, e x p o s e d to i s o l a t e d SPP1 W T r e s t r i c t i o n e n d o n u c l e a s e generat e d fragments, a n d then p l a t e d u n d e r c o n d i t i o n s n o n permissive for the g r o w t h o f the m u t a n t phage, U s i n g this t e c h n i q u e we have d e t e r m i n e d the p h y s i c a l a r r a n g e m e n t o f the a v a i l a b l e SPP1 genes. The s a m e technique has been used by C r e g g a n d S t e w a r t (1978) to localize genes on the c h r o m o s o m e o f B. subtilis p h a g e SPO1.

Materials and Methods 1 Introduction W e have p r e v i o u s l y d e s c r i b e d e x p e r i m e n t s w h i c h led to the p h y s i c a l c h a r a c t e r i z a t i o n o f the g e n o m e o f SPP1 ( M o r e l l i et al., 1978; M o r e l l i et al., 1979; R a t c l i f f e t al., 1979). T h e D N A o f SPP1 has a m o l e c u l a r weight o f 28.6 M d a l , the D N A is p a r t i a l l y circularly p e r m u t e d a n d t e r m i n a l l y r e d u n d a n t . T h e s a m e conclusions were r e a c h e d b y M c I n t o s h et al. (1978). M a n y SPP1 m u t a n t s , i n c l u d i n g p l a q u e t y p e m u t a n t s a n d c o n d i t i o n a l l y lethal s u p p r e s s o r a n d t e m p e r a t u r e sensitive m u t a n t s , have been isolated. These m u t a t i o n s define a p p r o x i m a t e l y 50 cistrons o f SPP1, a n d prelim i n a r y d e t e r m i n a t i o n s o f gene a r r a n g e m e n t s b y p h a g e crosses have been r e p o r t e d ( S p a t z a n d T r a u t ner, 1970; Burger a n d T r a u t n e r , 1978). S o m e SPP1 genes have been c h a r a c t e r i z e d in t e r m s o f the p r o t e i n s o r f u n c t i o n s w h i c h t h e y affect (Esche et al., 1975; Burger et al., 1978; M e r t e n s et al., 1979). P a r t o f o u r p h y s i c a l studies o f the SPP1 g e n o m e e s t a b l i s h e d the order of restriction fragments generated by a number o f r e s t r i c t i o n e n d o n u c l e a s e s ( R a t c l i f f et al., 1979). G e netic m a r k e r s can be rescued f r o m such f r a g m e n t s in a s i t u a t i o n where c o m p e t e n t B. subtilis cells were * Part of this work is from the doctoral thesis submitted by M Behncke to the Freie Universitfit Berlin (1973). For offprints contact: T A. Trautner

Plates. Media and Buffers used in these experiments were described in previous publications (Biswal et al, 1967 : Rottl/inder and Trautner, 1970~ Esche et al, 1975, Ratcllff et al., 1979). Bacteria. The stratus of B subtds used in this study are described

in TabIe 1 Bacteriophage. SPP1 WT was from our collection. Phage mutants isolated in our laboratories are mentioned under "Results". Several hydroxylamine induced ts-mutants of SPP1 were kindly made available to us by Professor M. Polsinelli of the University of Florence. These mutants are designated by the prefix "I". Mutagenesls and Isolatton of Mutants. SPP1 WT stocks were either

mutagenized by treatment with hydroxylamine (Spatz and Trautner, 1970) or (for ts-mutants) by growth of SPP1 WT in a mutator strain ofB subtlhs (Montenegro et al., 1976). ts-mutants designated by the prefix A were made using N-methyl-nitrosoguanidine treatment of SPP1 WT infected cells according to Cerd~-Olmeda et al. (1968). Temperature sensitive (ts) mutants were identified by plating the mutagenized phage stocks at 30° and then replica plating the plaques obtained on plates at 46°. A similar technique was used to identify suppressor sensitive (sus) mutants : the mutagenized stock was first plated with the permissive indicator from which plaques were replica plated onto plates seeded with the nonpermlssive strain. Only mutations which reverted to WT at a frequency of less then 1 in 10 4 w e r e used in this study Growth of Mutant Phage Stocks Is-Mutants. A fresh single plaque was suspended m 1 ml of TY

broth and the contents spread on 6 LTT plates with MCB plating Abbreriattons used: pfu=plaque forming umts; sus=suppressor sensitive: ts= temperature sensitive; WT = wild type

0026-8925/79/0175/0351/$01.40

B. Behrens et al " Physical Arrangement of SPP1 Genes

352 Table 1. Bacterial strains used (all strains are derivatives of B subnks 168)

Strain

Genotype

Purpose in this investigation

Reference

MCB

trp C2

Growth of sus + phage stocks, plating bacteria

Spatz and Trautner (1971)

MCB su3 + leusus 3+

Plating bacteria for sus phages

Behncke (1973)

HAI01B

[his met leu] su + try C2

Growth o f s u s phage stocks

Okubo and Yanagida (1968)

222

arg trp C2

Transfectlon experiments

Trautner et al. (1974)

trpC2 thr5

Production of mutants

D. Dubnau quoted in Montenegro et al (1976)

endouuclease digestions of individual fragments or with marker rescue. The purified DNA fragments were stored at 4 ° and remained biologically active for many months after isolation.

with the addition of 50 gg tryptophane/ml. The cells were collected by centrifugatlon, resuspended in the same volume of NB medium, and incubated at 37°. A single isolated sus mutant plaque from an overnight plate was inoculated into 100 ml of such bacteria. Lysls of the culture was observed after approximately 7 h at 37 °. These crude lysates contained 109-101° pfu/ml, reversion frequencies were between 10 -6 and 10 5.

Marker Rescue Experiments. In these experiments, 222 cells grown to competence according to Rottl~inder and Trantner (1970), were incubated for 10 min at 30 ° in the presence of 0.1 M MgC12. These cells were distributed into 0.5 ml aliquots (Eppendorf cenmfnge tubes) to which were added small volumes of concentrated phage to give an input multiphclty between 1 and 5 pfu per cell. Adsorption was allowed for 10 rain at 37°. Equal volumes of these helper infected cells (HIC) (between 10 and 30 gl) and extracted DNA were mixed and incubated for 30 rain at 37 ° The DNA uptake reaction was stopped by placing the reaction tubes at 0 ° and by adding 200 gl of an overnight culture of indicator MCB cells. Aliquots of 20 ~tl from this tube and/or from appropriate dilutions were plated on LTT plates When helper phages of the sus type were used the plates were incubated at 37 °. Incubation at 46 ° was reqmred if ts helper phages were employed. Control experiments, run with each experiment were i) determination of the revertant background in HICs in the absence of added DNA and i0 determination of the helping capacity of a particular helper. The latter parameter was assayed by adding undegraded SPPI WT DNA at a concentration of 5 x 10 .3 gg/ml to the HICs. No plaques were detectable at this DNA concentration in 20 ~tl of the transfectlon mixture with uninjected competent cells, whereas large numbers of infective centers were produced with HICs. A marker was assigned to a particular endonuclease generated fragment if the marker rescue in a marker/ fragment combination exceeded the background by at least a factor of twenty. This posttive assignment of a gene to a particular fragment was supported by the absence of marker rescue in combinations with other biologically active fragments of the particular digest under investigation. Recombination studies with phage mutants which had preceeded these analyses allowed us to make rough predictions of the location of a given mutation. Hence in many cases not all possible fragment/mutant combinations were analyzed.

Purtfication ofLysates. SPP1 lysates were concentrated by centrifu-

Restriction Endonucleases used and their suppliers were those de-

BD337

mutl

bacteria. After overnight incubation at 30°, leading to confluent lysis, the top layer of each plate was collected with 1 ml of TBT. The scrapings of the plates were pooled, centrifuged at low speed to remove debris and unlysed cells, and then millipore filtered (~Z~0.45 g). Stocks gave titers of ~ 2 × 10 l°. sus-Mutants. HA101B cells were grown overnight m MIII medium

gatlon and subsequently purified by sedimentation through a preformed CsCI step gradient (p= 1.7; 1.5; 1.3 g/ml). Centrifugation was in a Splnco SW41 rotor for 150 mln at 28 000 rpm. Phages accumulating in a sharp band within the central layer were collected, extensively dialyzed against TBT and stored at 4 °.

scribed by Ratcliff et al. (1979). An additional restriction enzyme, XbaI, was purchased from Bmlabs (Bethesda, Md.) and used as

suggested by the supplier.

Results and Discussion

Complementatlon Tests. Equal volumes of conditional lethal mu- '

tants were combined at a concentration of 107 pfu/ml each in wells of microtiter plates. The mixtures were spotted on TY plates seeded with MCB. Plates were incubated overnight at 46 °. Clearing of the spotted regmn in ts × ts, ts × sus, and sus x sus mutant combinations indicated complementation. As a control, all mutants m each test were also spotted alone. Restrtctlon Enzyme Digestzons and Isolation o f D N A Fragments.

Resmction endonuclease digests of SPPI DNA were performed according to the conditions given by the commercial suppliers. Endonuclease generated fragments were separated by vertical slab gel electrophoresIs m the presence of 0.5 gg ethldium bromide/ml (Ratchff et al., 1979). At least three aliquots of a digest were subjected to electrophoresis in adjacent positions. The outside tracks were photographed to identify band posxtions, whilst DNA was isolated from the central track(s) which had been shlelded during the long wave UV irradiation reqmred for photography. Agarose pieces, containing the desired DNA fragments, were cut out from the central track(s), washed extenswely with 5 mM TRIS/HC1 (ph 7.5) and DNA was extracted from them by the freeze/squeeze method of Thuring et al. (1975). EtBr, still present in isolated DNA fragments, did not interfere with either subsequent restriction

1. C o m p l e m e n t a t i o n

Analysis

41 g e n e s o f S P P 1 w e r e c h a r a c t e r i z e d b y c o m p l e m e n t a t i o n t e s t s b e t w e e n t h e 75 c o n d i t i o n a l l y l e t h a l m u t a n t s isolated. The results of these tests are summarized i n T a b l e 2. T h e g e n e s t h u s c h a r a c t e r i z e d a r e n u m b e r e d 1 t h r o u g h 41, f r o m t h e r i g h t e n d o f t h e m a p t o t h e left, i.e. s t a r t i n g w i t h t h e g e n e c l o s e s t t o t h e p a c site (see Fig. 1). It is o b v i o u s f r o m T a b l e 2 t h a t t h e r e is a w i d e fluctuation in the numbers of mutations which characterize an SPP1 gene. A combination of the effects o f g e n e size, m u t a g e n s p e c i f i c i t y , h o t s p o t s o f m u t a tion and our selection of mutation with a low revers i o n f r e q u e n c y , is p r o b a b l y r e s p o n s i b l e f o r t h i s f l u c t u ation. The number of genes mentioned here therefore represents a minimum estimate of the total number of SPP1 genes. A more reliable approximation of

353

B. Behrens et al.. Physical Arrangement of SPP1 Genes J

Table 2. Gene assignments Gene No.

ts mutants

1

2 3 4 5 6

37 38 39 40 41

HindIII

KpnI

2, 87, 106, 114, 119 62 19

1

1

1 1

1 1 1 1

1

2+4 2÷4 2+4 2+4 2+4

2b 2b

3 3

4 2+4 1

2b

1 3 3 3

M10 86 100, 115, 118, 126, 129 70 7 61, 64, 92, 98, 102, 112, 122, 124, 131 32

7 8 9

10 11 12 13 14 i5 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36

sus mutants

bM, 2M, 170M M20. G I 9

A12 M5

5b

4b

KpnI SalI

SalI

Sinai

SmaI EcoRI

EcoRI

1

2+4

2

I

1+2 1

1

117, 128 31

4b

82 666 46

5a

2b 2b 2b 2b 2b

3

1 1

1 1 1

1

1

3

1 1 12 9 9

4

1 1

G6 45 I10 222 76 79

2b 1 1 2a 2a 2a

1 1 1 1

2 2

3 1 1 1

1 1 1 1

3 3 3

3 3

1

2

2

1

2

1 1

53 A40 I13, 128 A74, A80, A81 A14 I2I, A38, A66

1 12, 109

99 5l 22

BglI

9 3 3

1 4b

I5, dM M28 I15, IM, 5M A58 3M G7 I17 I20F B3

EcoRI XbaI

1

1

1

I1, 6M

1

1

3 3 3 3 3 6 6 6 6 6 6 6 11 7 2 2 2 2 2 5 4 4 4 l i 1

1 1 1 1

1 1 1 1 1

The 75 m u t a n t s analyzed were assigned to genes 1 through 41 by complementatlon analyses (Columns 1-3). C o l u m n s 4-12 give the fragment n u m b e r s of the restriction endonuclease digests indicated to which a gene was assigned oil the basis of marker rescue experiments.

the number of SPP1 genes is given by the identification of SPP1 encoded polypeptides. 44 such polypeptides, representing 84% of the coding capacity of SPP1, have been identified (Mertens, Amann and Reeve, 1979). Some of these peptides, and also of those identified by Esche et al. (1975) in in vivo analyses could be assigned to genes described here. We do not know whether the sus mutants isolated here are of the " a m b e r " type. We can, however, distinguish two types of sus-mutants. The majority

of mutants selected on strain HA101B will also plate on the strain characterized by the suppressor su 3+, originally described by Georgopoulos (1969), but this is not the case for mutants sus222, sus666, susl9, sus70. 2. Ordering of SPP1 Genes

We have determined the position of SPP1 genes on restriction endonuclease generated fragments of SPP 1

=~ ~.~ ~ ..~

~~

g ~c~ ~.~

_N

I

o

ce~-

,o

.

~-oa

_--W->

X

-m

ua

ua

tat;

¢.

*~g~.

l

o

t3 0

t,a

~#q'~ r,a

g,..

~

~o5

r,a

~'~-

t,a -ca

=

-¢.a

l::r' D

~.

O

~"


5000

>5000

92

350

~ 2000

14

69

332

2

9

21

4

6

2

~3000 ~3000 ~1100 ~2200 ~3000 ~3000 >5000 >5000 >5000 >5000 >5000 >5000 347 266 319 96 180 135 13 19 77 i1 30 2 7 85 808 46 37 5

936 960 18 12 1

107 154

736 832

21

10

34

10

278 ~1500 ~3000 ~3000 241 ~2000 ~1500 ~1500

396

520

646

2990

1920

4300

79

37

1

Rescue with

Undegraded DNA Fragment 1 Fragment 2a and 2b Fragment 3 and 4 Fragment 5 EcoRI

Helper background

509 >5000 ~1100 220 >5000 >5000 6 12 67 8 0

38

26

79

37

1912 ~3000 82 ~900

~3000 ~1500

2

1

9

Rescue with :

Undegraded DNA Fragment 1 Fragment 4 Fragment 9 Fragment 12 HindIII Helper background

47 ~ 1500

83 ~ 2000 10

84

19

5

13

10

1

5

20

2

2

~3000 ~3000 6 26 4 20 8 19 600 284 383 19 41

1760

40

3 1

0 3

Rescue with.

Undegraded DNA Fragment 1 Fragment 2 Fragment 3 Fragment 4 Fragment 5

960 >5000 13 >5000 12 23 8 191 40

750 ~3000 >5000 1136 149 > 5000 11 15 20 29 8 24 6 73

750 ~4000 1160

Plaque numbers (from 20 gl of the incubation mixture) counted in marker rescue experiments and in controls which led to the gene assignments of Table 2 and Fig 1. In the case of degradation with Sinai we have also entered marker rescue values (plus controls) obtained with an unfractionated digest of SPP1 DNA. a S i n a i X b a I d o u b l e digest. F r o m Fig. 1 a n d T a b l e 2 it is c l e a r t h a t g e n e s l, 2, 3, 4, 5, 6, (7), 8 a r e carried on the minor Sinai fragment 4 and on Sinai f r a g m e n t 2. C o m p l e t e l y a n a l o g o u s r e s u l t s w e r e r e p o r t e d by C r e g g a n d S t e w a r t (1978) f o r S P O 1 D N A , a phage with unique ends and terminal redundancy. T h e use o f t h e m i n o r b a n d s in m a r k e r r e s c u e also a l l o w s m a r k e r l o c a t i o n s to be a s s i g n e d w i t h i n E c o R I f r a g m e n t 1. G e n e s 1, 2, 3 w h i c h c a n be r e s c u e d f r o m both EcoRI fragment 1 and the minor fragment of t h e S i n a i digest m u s t be l o c a t e d b e t w e e n the left e n d

of EcoRI fragment 1 and the DNA sequence determ i n i n g t h e p a c site. T h e p a c site is 0.4 M d a l (size o f E c o R I f r a g m e n t 13 t) d i s t a n t f r o m t h e E c o R I c l e a v age site s e p a r a t i n g E c o R I f r a g m e n t s 1 a n d 12. I n a d d i t i o n t o e s t a b l i s h i n g a p h y s i c a l m a p o f SPP1 g e n e s we h a v e m a d e t h e f o l l o w i n g o b s e r v a t i o n s : (1) Neither the molecular ends of restriction fragments ( s t a g g e r e d , flush, o r m i x e d ( f r o m d o u b l e digests)) u s e d in a m a r k e r r e s c u e e x p e r i m e n t , n o r t h e l o c a t i o n o f a m a r k e r w i t h i n s u c h a f r a g m e n t w i t h r e s p e c t to t h e m o l e c u l a r e n d s effects t h e e f f i c i e n c y o f m a r k e r rescue.

B. Behrens et ai. : Physical Arrangement of SPP1 Genes

(2) The same is true of the presence of partial nonhomology caused by the use of deletion mutants between phage genome and restriction fragment: e.g. the efficiency of marker rescue from SPP1 WT EcoRI fragment 1 and from the corresponding fragment isolated from deletion mutants of SPP1 are identical. This finding allowed the use of restriction endonuclease digests of deletion carrying SPP1 D N A in those cases (Sinai, BgII) were the resolution of fragments in electrophoresis could be enhanced by the use of deleted D N A instead of WT DNA. (3) With a few markers we were unable to detect marker rescue after digestion of SPP1 D N A with a restriction enzyme although such markers were readily rescuable before enzymatic digestion or following digestion with other restriction enzymes. For example, susl09 from gene 36 is readily detectable on fragments from EcoRI, KpnI or HindIII digests, but could not be located on any SmaI fragment. Presumably the Sinai cut which separates SmaI fragments 2 and 7/8 leads to destruction of the gene. The physical ordering of what must be the majority of SPP1 genes presented here raises the question of whether the established marker distribution reflects a meaningful arrangement in terms of regulation of gene expression. In this connection it is of interest to refer to the analysis of Burger and Trautner (1978) who demonstrated that seven genes involved in SPP1 D N A synthesis are located in a contiguous region at the junction of EcoRI fragments 1 and 4. In contrast to these genes, which are only described in terms of the function they effect, a few genes have been identified which code for structural proteins of the phage (Esche et al., 1975 : Mertens et al., 1979). These genes are also located in distinct regions of the genome. Biochemical studies are in progress to identify the proteins encoded by individual restriction fragments (Amann and Reeve, in preparation), to localize promoter sites in SPP1 D N A and to follow the kinetics and specificity of SPP1 m - R N A (Montenegro, Morelli and Trautner, in preparation). The results of these experiments should provide a coherent picture of the regulatory processes operative in SPP1 development and their relationship to structural arrangement. Acknowledgement. Discussions with G. Morelli, J. Reeve, E. Amann and M. Montenegro are gratefully acknowledged. We thank Professor M Polsmelli (University of Florence) for his gift of temperature sensitive SPP1 mutants. The part of this work which was performed in Stanford was aided by grants from the National Institutes of General Medical Sciences, GM-14108 to A.T.G. We wish to thank Ms. C.C. Yuh, J. Lub, and Dr. J Gillen for their help in the isolatmn of mutants and in certain marker rescue experiments.

Untersuchungen von Heteroduphces aus komplement~iren DNA-Str/mgen mit Wlldtyp- und konditional-lethalem Mutantentyp. Dissertation Frele Unlversitfit Berlin (1973) Biswal, N., Kleinschmidt, A.K., Spatz. H.Ch., Trautner, T.A.: Physical properties of the DNA of bacteriophage SP50. Mol. Gen. Genet 100, 39 55 (1967) Burger, K.J., Trautner, T.A. : Specific labelling of replicating SPP1 DNA. Analysis of viral DNA synthesls and identification of phage dna-genes. Mol. Gen. Genet. 166, 277-285 (1978) Cerdfi-Olmeda, E., Hanawalt, P.C., Guerola, N : Mutagenesis of the replication point by nltrosoguanidine: map and pattern of replication of the Escherichm coli chromosome. J Mol. Biol. 33, 705-719 (1968) Cregg, J M., Stewart. C.R : Terminal redundancy of "'high frequency of recombination" markers of Bacillus subtdis phage SP01. Virology 86, 530-541 (1978) Esche, H.. Schweiger. M , Trautner, T.A.: Gene expression of bacteriophage SPPI. I. Phage directed protein synthesis. Mol. Gen. Genet. 142, 45-55 (1975) Georgopoulos, C.P. : Suppressor system in B. subtihs 168. J. Bacteriol. 97, i397 1402 (1969) McIntosh, P., Dunker, R., Mulder, C., Brown, N.C.: DNA of Bacilhts subtdts bacteriophage SPP1. Physical mapping and Iocahzatlon of the origin of replication J. Virol. 28, 865-876 (1978) Mertens, G., Amann, E., Reeve, J.N.. Bacteriophage SPP1 polypeptIdes synthesized in infected mlmcells and m vm'o. Molec. Gen Genet. Mol Gen. Genet 172, 271 279 (1979) Montenegro, M.A., Esche, H., Trautner, T.A. : Induction of mutations in B. subtdis phage SPP1 by growth on host cells carrying a mutator DNA polymerase III. Mol Gen. Genet. 149, 131 134 (1976) Morelll, G , Fisseau, C., Behrelas, B., Trautner, T.A., Luh, J., Ratcliff, S.W., Allison, D.P, Ganesan, A.T.' The genome of B. subtilis phage SPP1 ' The topology of DNA molecules. Moi. Gen. Genet. 168, 153-164 (1979) Morelli, G., Montenegro, M.A., Hillenbrandt. G , Scherzinger, E., Trautner, T.A.: The genome of B. subtilis phage SPPI: Assignment of 5'-3'-polarity to the complementary strands of SPP1 DNA. Mol. Gen Genet. 164, 93 97 (1978) Okubo, S., Yanaglda, T . Isolation of a suppressor mutant in B. subtilis. J. Bacterlol. 95, 1187 1188 (1968) Ratchff, S.W., Luh, J., Ganesan, A.T., Behrens, B , Thompson, R., Montenegro, M.A., Morelh. G , Trautner, T.A ' The genome of Bacillus subtilis phage SPP1 : The arrangement of restriction endonuclease generated fragments. Mol. Gen. Genet. 168, 165-172 (1979) Rottlgnder, E., Trautner, T.A.: Genetic and transfectlon studies with B subtdis phage SP50. I Phage mutants with restricted growth on B. subtlhs strata 168. Mol. Gen Genet. 108, 4 7 4 0 (1970) Spatz, H.Ch., Trautner, T.A.: One way to do experiments on gene conversion? Mol. Gen. Genet. 109, 84-106 (1970). Spatz, H.Ch, Trautner, T.A. : The role of recombinauon in transfection of B. subtilis. Mol. Gen. Genet. 113, 174-190 (1971) Thurlng, R.W.G., Sanders, J.P.M., Borst, P.. A freeze squeeze method for recovering long DNA from agarose gels. Anal. Biochem. 66, 213-220 (1975) Trautner, T.A., Pawlek, B., Bron, S, Anagnostopoulos, C.: Resmction and Modification in B subtilis. Biological Aspects. Mol Gen. Genet 131, 181-191 (1975)

Communicated by E. Bautz

References Behncke, M.

357

Genkonverslon bei dem B. subtzhs Phagen SPP1

Received May 28, 1979