J. Xol.

Biol.

(1977) 113, 181-191

Electron Microscope Visualization of the Products of Bacillus subtilis Transformation SANDRO

L. FORNILI~ Department

Massachusetts

Cambridge, (Received 27 July

AND

MAURICE

X. Pox

of Biology

Institute of Technology Nass. 02139, U.X.A.

1976; and in revised form 31 January

1977)

The reduced alkalinity required to denature DNA in which bromouracil has replaced thymine has permitted the visualization of the molecular products of transformation of Bacillus subtilis with bromouracil-substituted DNA. The appearance of these molecules suggests that several distinct segment’s of a single bound DNA molecule can be integrated into the genome of a recipient bacterium.

1. Introduction For a number of bacterial systems, Bacillus subtilis (Bodmer & Ganesan, 1964; Bresler et aE., 1964; Ayad & Barker, 1969; Dubnau & Davidoff-Abelson, 1971), Xtreptococcus pneumoniae (Fox & Allen, 1964), and Hemophilus injluenzae (Notani & Goodgal, 1966), the products of genetic transformation ha#vebeen shown to be heteroduplex regions in the DNA of the recipient bacterium in which segments of singlestrand donor DNA are paired with their complementary recipient strands. The product of pneumococcal transformation has been shown to contain continuous single-strand DNA segments of an average molecular size of 2 x lo6 with some segments exeeeding 5 x 10” daltons in length (Gurney & Fox, 1968). Estimates of the molecular size of the integra,ted single-strand DNA segment in B. subtilis ranges from 4.9 x lo5 (Bodmer, 1966) to 2.8 x lo6 (Dubnau & Cirigliano, 1972; Dubnau et al., 1973). The question of the number of such single-strand insert,ions that may result from the interaction between a competent cell and a large molecule of donor DKA remains unanswered by these investigations. Dubnau et al. (1973) suggested that in B. subtilis donor DNA is integrated in clusters of two to five segments, while Darlington & Bodmer (1968) concluded that the single segment model was consistent with the results of their investigation involving four linked markers in B. subtilis transformation. The fact that the alkalinity required to denature DNA is reduced as a result. of replacement of thymine by bromouracil (Inman & Baldwin, 1962 ; Baldwin & Xhooter, 1963; Inman & SchnGs, 1970) permitted a new approach to the characterization of the product’s of transformation. DNA isolated from bacteria transformed with BrUra$substituted donor DNA has been examined by electron microscopy, following treatment that results in denaturation of only BrUra-containing molecules (Inman $ 7 Present address: Instituto di Fisica dell’Universit8 and G-ruppi Naeionali Struttura della. %terial Consi$io Nazionale delle Ricerche, Via Archirafi, 36, 90123 Palermo, Italy. $ Abbreviations used: BrUra, bromouraeil; hyDNA, hybrid DNA; BB-DNA, “fully” substituted DXA; LT-DNA, BT-DNAs, see Materials and Methods, section (e) (iii). N-DNA, unsubstituted DNA. 181

S. L.

182

S&n&,

FORNILI

AND

1970). This procedure permitted

segments that have been integrated genome.

M. S. FOX

us to trace out the BrUra-labeled

into the thymine-containing

donor DNA of the recipient

The observations described in this paper (1) confirm the conclusion that donor DNA is inserted as single-stranded segments into the recipient chromosome; (2) enable us to estimate

the average

size of the segment

of donor

DNA

that

i.s integrated;

(3)

reveal the insertion of more than one donor segment within individual recipient DNA molecules, presumably as the result of coupled events in DNA uptake. The multiple inserted segments can be accounted for as the product of uptake events that initiate at several of the sites at which a single large donor DNA molecule is bound to cell receptors.

2. Materials and Methods (a) Bwcterial strains SB25 (trpC2 hisB2) was used as a recipient for transformation. Donor DNA was isolated from B. subtilis 168 (h-p+) and a mutant strain, 5-bromouracil-tolerant B. subtilis 168 (trp tky But), isolated by Bishop & Sueoka (1972), was used to prepare BrUra-substituted DNA. B. subtilis

(b) Media

and competent

cells

Penassay broth (Difco Antibiotic medium no. 3) was used for growing overnight cultures. The minimal salts medium LS and growth media 1 and 2 (referred to hereinafter as TM1 and TM2), described by Cahn & Fox (1968), were used to prepare competent cultures. Transformants were scored for the trp + or his + marker on LS medium supplemented with 20 pg histidine or tryptophan/ml, 0.5% (w/v) gl ucose and 1.2% (w/v) Bacto agar (Difco). LB agar (1% Bacto Tryptone (Difco), 0.5% Bacto yeast extract (Difco), 1% NaCl and 1.2% Bacto agar, pH 7.0) was used to determine the titer of viable bacteria. Bacteria from overnight cultures , grown at 30°C under good aeration for about 20 h wera collected by centrifugation, resuspended at about 2 x lo7 bacteria/ml in TM1 and shaken at 37°C until the culture growth, measured with a Klett calorimeter, reached the end of exponential phase. This culture was diluted 5 times into TM2 and incubated with moderate shaking at 30% for about 135 min, this being the approximate time of maximal competence under our conditions. (c) Transformation

procedure

After adding MgSO, (0.02 M) to a sample of competent culture it was mixed with about 10 pg donor DNA/ml and incubated at 30°C for 30 min with moderate shaking. Transformation was terminated by treating the culture with 25 pg deoxyribonuclease l/ml (Worthington Biochemical Corp., Freehold, NJ.) for 5 min at 30°C. (d) Ikxtionation

of competent cultures

The method for separating competent from non-competent cells on Renografin (E. R. Squibb and Sons, New York, N.Y.) gradients is similar to that described by Haseltine & Fox (1971). For each tuba in a Spinco SW27 rotor, 30 ml of competent culture were gently layered on a g-ml Renografin solution whose refractive index was 125 = 1.370. The tubes were centrifuged at 25,000 revs/min for 15 min at 2O”C, the bacteria separated into a fraction lying on top of the Renografin solution and into a heavier fraction pelleting to the bottom of the tube. The top fractions were removed with Pasteur pipets and thoroughly washed by centrifugation at 8000 revsjmin in a Sorvall 5534 rotor for 5 min at 5°C and resuspended first in LS medium, then in O-02 M-MgCl, and then in LS medium. Cells were finally resuspended in TM2 and tested for competence. Compared to the unfractionated culture, this procedure resulted in a 12-fold increase in the frequency of transformation for the trp+ marker.

PRODUCTS

OF B. SUBTILIS (e) DlVA

TRANSFORNATIOK

183

preparatiolzs

13. subtilis 168 (trp+) was grown to about 3 x 10s cells/m! at 37°C in LX medium supplemented with 0.027/, vitamin-free Casamino acids (Difco), 20 pg t,ryptophan/ml and 0.59; glucose. Bacteria were harvested, washed 3 times with cold Tris/EDTA buffer jO.01 M-Tris (Sigma, St. Louis, MO), 0.01 M-Na,EDTA (Sigma), pH 8.0), resuspended into the same buffer with the addition of sucrose to 0.3 M and lysozyme (egg white, 3 x crystallized; Sigma) to 1 mg/ml, incubated at 37°C for 10 min and lysed by adding Sarkosyl (Geigy Industrial Chemicals, Ardley, N.Y.) to 0.3%. The 1ysat.e was then treated with 200 pg ribonuclease/ml (bovine pancreatic, RASE, Worthington), previously heated for 20 min at 65”C!, and 20 units T1 ribonuolease/ml (Calbiochem, Los Angeles, Cdif.). After 30 min incubation at 3i”C, Pronase (B grade, Calbiochem) was added to a concentrat,ion of 1 mg/ml end incubation continued for 16 h at 42°C. The DNA in the lysate was then purified by isopyenic separation in CsCl gradients. The fractions containing DNA m-ere pooled and extensively dialyzed against SE solution (0.02 nf-NaC1, 0.005 IX-EDTA, pH 74) (SchnBs & Inman, 1970). (i)

(ii) Br Ura-substituted

DNA

An overnight culture of B. subtilis 168 BrUra-tolerant,, grown in Penassay brot,h supplemented with 25 pg thymine/ml, was diluted into G medium (LS medium, 0.020/, charcoal-filtered vitamin-free Casamino acids, 20 ,ug tryptophan/ml, 20 pg uracil/ml and 0.5% glucose), supplemented with 50 pg thymine/ml, and grown at 38°C t,o 150 Klett units (green filter). These exponential cells were centrifuged, resuspended in 2 vol. G medium supplemented with 20 pg B-bromouracil/ml and 1 &‘i 5-[2-1”C]bromouracil (1.2 Ci/mol ; Calbiochem) and incubated further. When this culture had doubled, half of the tot-al culture volume was taken for isolation of hybrid DNA in the manner outlined in (i) for DNA preparation. The same procedure was applied to isolate DNA from the remaining culture when the culture had grown to stationary phsse, reflecting about a 5.5.fold increase in cell density. BrUra-substituted DNA preparations were fra.ctionated iit C&O, gradients and the fractions conta,ining hybrid or “fully” substit,uted DNA were pooied and exhaustively dialyzed against SE solution. Operations involving bacterial growth in the presence of BrUra and handling of BrUraSo enhance resolution (Flamm et al., 1966). The gradients were collected, alterna,ting 4-drop fractions, with P-drop fractions collected under I ml mineral oil for refractive index measurements. The radioactivity analysis of these gradients enabled us to locate the bands of hybrid and fully BrUra-substituted DNAs, on the basis of the refraetive index

S.L.FORNILP

184 measurements et al., 1963).

which

coincided

with

AND 3l.S.FOX

the values previously

reported

(Opara-Kubinska

(g) Determination of radioactivity Radioactive samples were deposited on 21 mm filter discs (Schleicher and Schuell Co., Keene, N.H.), dried, washed twice with cold 5% trichloroacetic acid, and once with 95% ethanol. The dried filter discs were placed in glass vials (Wheaton, Milville, N.J.) containing 5 ml of scintillation fluid, prepared by adding 160 ml Liquifluor (New England Nuclear, Boston, Mass.) to 1 gallon toluene. Radioactivity of these samples was measured with a Nuclear Chicago mark I scintillation counter. (h) Partial denaturation of DNA In order to denature hybrid DNA without denaturing unsubstituted DNA, the procedure of Schnijs & Inman (1970) was followed. The pH of a high pH buffer with a reduced content of formaldehyde (0.068 M-Na,COe, 18% HCHO (Matheson, Coleman and Bell, Norwood, Ohio) and 0.011 M-EDTA) was carefully adjusted, using 10 M-NaOH: 3 ~1 were added to 7 ~1 of DNA solution in SE and left at 2O’C for 10 min. The mixture was then chilled in an ice-bath and prepared for electron microscopy. (i) Electron microscopy Samples were prepared following the procedure described by Schnijs & Inman (1970), modified in that the formamide concentration was reduced to 40%. To 10 ~1 of DNA high pH buffer mixture, 7 ~1 of formamide (Matheson, Coleman and Bell) and 1 ~1 of a 0.1% solution of cytochrome c (Type II, Sigma) were added. The spreading of ~-PI samples of this mixture on water drops was carried on as outlined by Inman & Schnos (1970). Samples were then picked up on collodion-coated grids, stained with 10e5 M-Uranyl acetate in 95% ethanol, rinsed in isopentane and rotary shadowed at an angle of 6” with platinum/ palladium alloy (80 : 20), in order to provide good contrast and discrimination between single and double strands (Davis et al., 1971). An RCA 3G electron microscope was used at 50 kV. Micrographs were taken at a magnification of 7000x, calibrated on the basis of photographs of standard grating replicas and of +X174 DNA. ( j) Measurement and computation of electron microscope data Micrographs of partially denatured DNA molecules were printed at an additional IO-fold enlargement (final linear magnification, 70,000 x ) and measured with a curvimeter (Minerva, Villaret, Switzerland). A Hewlett-Packard 2100A computer was used to carry out all computations. Figures were drawn by a Hewlett-Packard 7210A digital plotter on line with the computer.

3. Results The work of Inman & Baldwin (1962), Baldwin $ Shooter (1963) and Inman & SchnGs (1970) demonstrated that BrUra substitution reduces the pH required to denature DNA. In the presence of formaldehyde the configuration of partially denaturated DNA is stabilized as a consequence of its reaction with the single-strand structure (von Hippel & Wong, 1971). Exposure to the appropriate pH in the presence of formaldehyde permits visualization by electron microscopy of “open” DNA regions (“loops”, “ bubbles”) (Beer & Thomas, 1961; Inman, 1966). In order to determine the maximum pH value such that LT-DNA is not significantly affected while BT-DNA displays segments with loops, LT-DNA and BT-DNA were exposed to buffers at various adjusted pH values for ten minutes at 20°C in the presence of formaldehyde. Samples of these DNAs were then spread and examined by electron microscopy. Parallel experiments were carried out with unsubstituted, hybrid, and fully substituted DNAs. The qualitative results reported in Table 1 show that the pH threshold at which open regions become detectable is lowest for BB-DNA, higher

PRODUCTS

OF

B. SUBTILIS

TRANSFORMATION

TABLE

185

1

is’cazning by electron microscopy of allculi denatwation of native, Br Ura-substituted and transformant DNA in the present of soi0 form&delLyde 9.8 DXA s LT hY BT BB

-

10.8

10.9

-

-

-

+

+ -t +

+ + +

0 Jr 0

A + 0

10.1

+

PH

10.3

9.9

Unsubstituted (N), hybrid (hy), fully BrUra substituted (BB) DNAs and DNAs isolated from top-fraction bacteria transformed with N-DNA or BB-DNA, indicated by LT and BT, respectively, were held for 10 min at 20°C at different pH values in the presence of 5% formaldehyde, and then prepared for electron microscopy. The symbols (-) and (+) indicate the absence or presence of donaturation loops, respectively. The symbol (0) indicates that more than 90% of the DNA molecules were in single-stranded form.

for hyDNA and BT-DNA and highest for X-DNA and LT-DNA. -4t pH 99 some denaturation loops are apparent in BB-DNA preparations but in none of the others. At pH 10.1 to 10.3 denaturation loops are seen in hyDNA, BT-DNA and BB-DNA preparations but not in N-DNA or LT-DNA preparations. At pH lOG3, BB-DNA molecules are completely denatured, as are most of the hyDNA molecules. BT-DXA is predominantly native but as many as 10% of the molecules contain denatured loops. Neither N-DNA nor LT-DNA molecules contain denatured loops, though rare molecules show t.erminal single-stranded segments. At pH 10.9 hyDNA and BB-DNA are completely denatured. BT-DNA exhibits loop structure similar to that seen at pH 10.8, and N-DNA and LT-DNA molecules remain native, though they occasionally contain a few small bubbles as well as showing terminal single-st’randed regions. On the basis of t,hese findings, we conclude that: (1) BT-DNA seems to have a BrUra composition similar to hyDNA in the tracts in which partial denaturation occurs. sequences of (2) At a pH of 10% we are able to visualize the bromouracil-containing the DNA isolated from bacteria transformed with BrUra-substituted DNA. At this pH value, in fact, neither N-DNA nor LT-DNA molecules exhibit loops or sequences of loops (Fig. 1) and only rarely show terminal single-stranded segments. On the other hand, BT-DNA molecules exhibit internal clusters of open regions often spaced by long stretches of double-stranded DNA (Fig. 1). Such structures have not. been seen in either N-DNA or LT-DNA preparations at any of the pH values examined. Following exposure of hyDNA to pH 10.8, in most of the fields examined, the great majority of the molecules (more than 90%) were completely denatured. Occasionai fields contain molecules retaining some duplex structure. The schematic tracing of several such molecules exhibiting extensive loop sequences are shown in Figure 2. The rare molecules harboring long continuous sequences retaining duplex structure that have been seen could be unsubstituted DNA molecules contaminating the hyDNA preparation or could represent hyDNA segments with exceptionally high (C + C) content. In either case, since they represent a rare species we will ignore them in the discussion that follows. For each BT-DNA preparation the number of fields scanned by electron microscopy included about 20,000 pm of DKA or the equivalent

PRODUCTS

OF B.SUBTILIS

isi

TRANSFORMATION hy-DNA

BT3-DNA

YTZ-3NA

,-I. I) __a

.-,_-*--.Y *a-

-. c.--, 0

I

IO

20

I L.--_L-_Lip

30

40

50

60

70

.-,.

I

I

0

IO

20

-

* I

I

I

I

I

30

40

50

60

70

Length (pm)

1. Schematic representation of partially denatured BT-DNA and hy-DNA molecules he!d as pH 10.8 for 10 min at 20°C in the presence of 5% formaldehyde. For each molecule the lower level lines represent double-stranded segments, while the upper level lines indicate open regions. The label * means that the indicated end of the molecule was outside the micrograph field. Length scales (in pm) are drawn at the bottom. FIG.

of about 20 bacterial chromosomes. Micrographs of molecules containing loop sequences were taken and analyzed as describedin Materials and Methods. Figure 2 shows these _molecules, displaying their partial denaturation pattern. For BT-3 DNA about one molecule in ten contained denatured sequences. For comparison, severa,] molecules of hyDNA that retain some double-stranded structure are displayed in Figure 2. In agreement with earlier reports, no double-strand donor DNA (BB) is found in IF-sates of the transformed bacteria (Fox & Allen, 1964; Piechowska & Fox, 1971). We propose that the molecules exhibiting internal clusters of open regions often spaced by long stretches of duplex DNA are the products of the insertion of BrUra,substituted transforming DNA into the continuity of the bacterial chromosome. In support of this proposal it is worth noting that in roughly the same amount of DNA examined by electron microscopy, the number of molecules found to contain loop sequences in BT-DNAs corresponded to the transformation frequency of the cultures from which the DKA was isolated. The pH threshold for the appearance of denaturation loops seems to be the same for BT-DNA and hyDNA (Table l), but the distribution of loop sequences is strikingly

186

S. L. FORNILI

AND

M. S. FOX

different. For hyDNA, treatment at pH 10.8 resu.lts in denaturation of most of the molecules and most of those that retain some duplex structure include only short duplex regions connecting denaturation loops. On the other hand, the BT-DNA molecules are predominantly duplex, and those harboring denaturation loops are composed predominantly of duplex DNA connecting limited regions that are denatured or clusters of denatured regions. The appearance and abundance of the denaturation loops in BT-DNA is consistent with that to be expected a,mong normal DNA molecules, portions of which have participated in a genetic exchange involving single-strand displacement with BrUra-substituted transforming DNA. Features which these BT-DNAs share are that loop sequences are often connected to long double-stranded DNA segments and that many of the molecules harbor more than one well-separated loop sequence. These characteristics are not evident among partially denatured hyDNA molecules (Fig. 2). We will therefore assume that the BT-DNA loop sequences without long duplex interloop segments can provide us with an estimate of the length of the continuous donor DNA segments that are inserted. In order to make this estimate, however, we have to establish a criterion that permits us to discriminate between double-stranded interloop segments that connect two distinct loop sequences and those that are part of the same loop sequence. This can be done on the basis of the distribution described in Figure 3 of interbubble distances found in partially denatured hyDNA molecules. If we select a probability value less than 1% as a reasonable limit, we see from Figure 3 that we have to consider every duplex segment exceeding about 2.5 pm as one which probably separates two distinct loop sequences. With this criterion of continuity for a loop sequence, the estimated average length of the inserted DNA segments for each of the BT-DNA preparations is estimated (Table 2). Despite the fact that the three DNA preparations represent widely different X (Frn)

Fro. 3. P represents the frequency of duplex DNA segments, connecting consecutive loops on partially denatured hyDNA molecules, having B length larger than X (in pm).

PRODUCTS

OF B. SUBTILIS

TRANSFORMATIOX

TABLE 2

-~lveragelelzgth of connected loop sequencesand estimated molecular integrated single-&and segments of donor BNA

BT-1DNA BT-2DNA BT-3DNA

Average length (pm)

Molecular weight (x10-61

3.5 3.0 4.0

3.4 2.9 3.9

weight of

yields of transformants as well as different frequencies of loop-containing molecules, the estimated lengths of the inserted DNA segments are very nearly the same and correspond to a, sequence of about 10,000 bases. The size distribution is broad and a substa.ntial fraction of the loop clusters include sequences whose lengt,h corresponds to an inserted segment of 30,000 bases or more. Although most of the denatured regions conta,in clusters of small loops, a substantial number of loops are large enough to correspond to a completely denatured sequence of 15,000 bases. A striking feature of the observations is the presence in all of the BT-DNA preparations of substantia’l numbers of molecules harboring two or more well-separat’ed loops or loop clusters (Fig. 2). The abundance of such molecules may be greater among RT-2 and BT-3 molecules than among BT-1 molecules.

4. Discussion Bnalysis of the clusters of denaturation loops, present in otherwise native molecules isolated from bacteria transformed with BrUra-substituted DNB, provides an estimate of about 10,000 bases for the average length of the segment of DNA inserted as the result of transformation. This estimate is in rea’sonable agreement with estimates made on the basis of physical analysis of DNA isolated from bacteria transformed with 2H-labeled DNA in both pneumococcus (Gurney & Fox, 1968) and B. subtilis (Dubnau & Cirigliano, 1972). We believe that the presence and abundance of molecules harboring widely separated denaturation loops or clusters of denaturation loops reveal some interesting fea,tures of the transformation process. They do not appear to be the products of independent interactions between a single bacterium and several DNA molecules. If each independent event were to involve insertion of a DNA length of 3.5 pm into a bacterial chromosome whose length was lo3 pm (Wake, 1975), and the efficiency of transforma,tion, per genome equivalent of DNA taken up, were close to unity (Cahn & Fox, 1968; Xomma & Polsinelli, 1970), then a 5% frequency of transformation (BT-3) would involve insertion of about 15 such segments per chromosome with an average distance between them of about 70 pm. For the BT-2 case the expected separation between loop clusters would be expected to be five times larger. These values are large compared to the total lengths of the DNA molecules examined (Fig. 2) and render the possibility of independent events unlikely. In both BT-2 and BT-3 preparations, half or more of the molecules that are longer than 10 pm display evidence of two or more

190

S. L. FORNILI

AND

31. 8. FOX

distinct loop clusters. The abundance of molecules harboring more than one loop cluster excludes the possibility that our size distribution selects for only the shorter intervals between presumed independent events. We conclude that the multiple loop clusters are the products of coupled integration events. If single molecules of the high molecular weight transforming DNA were to bind at several sites on a competent bacterium and single-strand uptake were often to be initiated at more than one of these sites, the integration of these single strands would result in the kinds of molecules displayed in Figure 2. On the basis of the observed reduction in molecular weight of transforming DNA that results from binding to pneumococcus, Lacks & Greenberg (1976) have made a similar proposal. These considerations suggest a modification of the interpretation of genetic linkage in transformation. In addition to linkage that is manifested by markers that are sufficiently close to each other that they are integrated as part of a single segment, more remote markers may exhibit linkage because they were both present in a larger bound DNA molecule but were taken up and integrated as separate segments. Such a mechanism would account for two paradoxical reports in the literature. Transforming DNA heavily labeled with 32P loses biological activity as the result of 32P disintegration and single markers are inactivated at a rate consistent with a target size of about 1500 bases (Fox, 1963). Despite the fact that 32P disintegrations result in the interruption of the continuity of the single strand t.hat had harbored the 32P atom (Rosenthal Bt Fox, 1970), linkage between distant markers does not seem to be interrupted by such events (Fox, 1963). If these markers were sufficiently distant to represent separate integrated segments of a single bound DNA molecule, single-strand interruptions in the polynucleotide chain connecting the markers would have little effect on the degree of linkage that they exhibited. Roger (1972), in her report on the transforming activity of artificially constructed heteroduplex molecules, observed linkage between markers present on opposite strands. If, as has also been suggested by Lacks $ Greenberg (1976), the segment entry of single strands of different portions of the bound molecule could involve either strand, transformants could arise in which one strand were displaced in one region of the recipient genome and in a neighboring region the complementary strand were displaced. Some base pair mismatch correction in this product could result in the appearance of linked transformants (White & Fox, 1974; Tiraby & Fox, 1973).

5. Conclusions Making use of the difference in the pH required to denature BrUra-substituted DNA, the molecular products of bacterial transformation have been visualized by electron microscopy. The appearance of these products supports the earlier evidence for single-strand insertion of donor DNA into the continuity of the recipient chromosome. On the basis of the dimensions of the BrUra-containing regions, the average size of the inserted segments is estimated to be about 10,000 bases, with about 10% exceeding 30,000 bases in length, in agreement with earlier estimates (Gurney & Fox, 1968 ; Dubnau & Cirigliano, 1972). Molecules harboring more than one well-separated loop or loop cluster are common. Their appearance and abundance support the proposal that large donor DNA molecules are bound at several sites and that each of the sites may initiate

resulting in coupled integration

events.

an uptake

event

PRODUCTS

OF B. SUBTILIS

TRANSFORXATXON

191

This work was supported by grants BMS72-02020 from the Nationa. Science Foundation and A105388 from the National Institute of Allergy and Infectious Diseases Do one author CM. s. F.). A CNR-NATO fellowship and a subsequent grant from the Consiglio Nazionale delle Ricer&e have supported part of the stay of one of us (S. L. F.) at the Massachusetts Institute of Technology. We gratefully acknowledge help from S. E. Luria, L. Cordone, 21. White, E, Lenk and M. Ahlquist. REFERENCES Ayad, S. R. & Barker, G. R. (1969). Biochem. J. 113: 167-174. Baldwin, R. L. & Shooter, E. M. (1963). J. Mol. Biol. 7, 511-526. Beer, M. & Thomas, C. A., Jr (1961). J. Mol. Biol. 3, 699-700. Bishop, R. J. R: Sueoka, N. (1972). J. Bacterial. 112, 870-876. Bodmer, W. F. (1966). J. Gen. Physiol. 49, 233-258. Bodmer, W. F. & Ganesan, A. T. (1964). Genetics, 50, 717-738. Bresler, S. E., Kreneva, R. A., Kushev, V. V. & Mosevirskii, M. I. (1964). Z. Vererbamgsl. 95, 288-297. Calm, F. H. & Fox, M. S. (1968). J. Bacterial. 95, 867-875. Darlington, A. J. & Bodmer, W. F. (1968). Genetics, 60, 681-684. Davis, R. W., Simon, M. & Davidson, N. (1971). In Methods in Enzymology (Grossman, L. & Moldave, K., eds), vol. 21, part D, pp. 413-428, Academic Press, New York. Dubnan, D. & Cirigliano, C. (1972). J. Bacterial. 111, 488-494. Dubnau, D. & Davidoff-Abelson, R. (1971). J. Mol. BioZ. 56, 209-221. Dubnau, D., Davidoff-Abelson, R. & Cirigliano, C. (1973). In Abstracts of th,e Symposium on, Genetic Recombination, held at the Roche Institute of Molecular Biology, Nutley, N.J., May 2-4, 1973, Roche Inst. of Molecular Biology, Nutley, NJ. Flamm, W. G., Bond, H. E. & Burr, H. E. (1966). Biochim. Biophys. Acta, 129, 310-319. Fox, M. S. (1963). J. Mol. BioZ. 6, 85-94. Fox, M. 5. & Allen, M. K. (1964). Proc. Nat. Acad. Xci., U.X.A. 52, 413-419. Gurnev, T., Jr & Fox, M. S. (1968). J. Mol. Biol. 32, 83-100. Haseltine, F. P. & Fox, M. S. (1971). J. Bacterial. 107, 8899899. Inman, R. B. (1966). J. Mol. BioZ. 18, 464-474. Inman, R. B. & Baldwin, R. L. (1962). J. Mol. BioZ. 5, 172184. Inman, R. B. & Schnos, M. (1979). J. Mol. BioZ. 49, 93-98. Lacks, S. & Greenberg, B. (1976). J. Mol. BioZ. 101, 255-275. Notani, N. & Goodgal, S. H. (1966). J. Gen. Physiol. 49, 197-209. Opara-Kubinska, Z., Kurylo-Borowska, Z. & Szybalski, W. (1963). Biochim. Biophys. Acta, 72, 298-309. Piechowska, M. & Fox, M. S. (1971). J. Bacterial. 108, 680-689. Roger, M. (1972). Proc. Nat. Acad. Sci., U.S.A. 69, 466-470. Rosenthal, P. N. & Fox, 111. S. (1970). J. MoZ. BioZ. 54, 441-463. S&n&, M. & Inman, R. B. (1970). J. Mol. BioZ. 51, 61-73. Somma, S. & Polsinelli, I\I. (1970). J. Bacterial. 101, 851-855. Tiraby, J-G. & Fox, M. S. (1973). Proc. Nat. Acad. Sci., U.S.A. 70, 3541-3545. von Hippel, P. H. & Wong, K.-Y. (1971). J. Mol. BioZ. 61, 587-613. Wake, R. 6. (1975). J. Mol. BioZ. 77, 569-575. M'llite, R. L. & Fox, M. S. (1974). Proc. Nat. Acad. Sci., U.S.A. 71, 1544-1548.