Molecular fate of DNA in genetic transformation of Pneumococcus

J. ll'J ol. Biol. (1962) 5, 119-131

Molecular Fate of DNA in Genetic Transformation of Pneumococcus S. LACK S Biology Department, Brookhaven National Laboratory, Upton , L.I., N .Y., U.S.A . (Received 12 February 1962, and in revised f orm 23 A pril 1962) E xt racts · of pneumococcal cells wh ich had incorp orated [32P]DNA were fractionat ed in order to determ ine t he fate of t he in corpora t ed radioac tivity . Immed iately aft er en t ry, about half of the DNA was found t o b e conver ted to a sing le -st r an de d form; the other half was degraded to d ialysable oligonucleotides and inorganic phosphate. Very lit tl e native [32P]DNA was present. On incubation of the cells for various lengths of time a ft er introducti on of the DNA, radioactivity was rapidly incor p orated into t he n ative DNA fr action. The sour ces of this radioactivi ty were (1) t he fragments in itially presen t which app arently were in corporated by m eans of normal syntheti c processes, and (2) t he single-stran ded DNA some of which may have b een t rans fer red in t act. This la t ter t ransfer a ppears to b e t he rout e by wh ich gen etic informat ion is t rans ferred. The in cr eas e of radi oa ctivity in nativ e DNA and the depl etion of the single-stranded DNA corresp ond ed in t ime with recovery of a genetic fa ct or in troduced by t he donor DNA. The extent to wh ich t h is genetic fac t or was recovered , det ermined bo th by d irect m easurem en t of it s t ransforming activ ity an d b y m easur em ent of t he fre que n cy of transformed cells, corresponded to a physical integr ation of about one-qu art er of the donor DNA taken up by the cells.

1. Introduction Recent st udies of t he transform ati on pr ()cess in Pneumococcus have shown that imm ediately after ent ry into t he cell ·the t ransfor ming ability of the introduced DNA disappears, only t o be gradually recovered within several minutes (F ox, 1960). Newly introduced DNA also appears to be un able to exert its phenotypic fun ction in enzyme form ation pri or to perm an ent integration in th e host cell (Lacks & Hotchkiss, 1960b). The pr esent st udy was undertak en in order t o learn about the molecul ar events responsible for these observat ions . The results indicate that, very soon after or during its introduction into the cell, DNA is conv erted partly into low-molecular-weight fragments and partly into single-st randed polynucleotides and that incorp oration of a portion of the latter into native DNA is the physical basis und erlying th e geneti c tran sform ati on .

2. Methods 32P·labeled DNA was pre pare d fr om cells of a strepto mycin- resistant strain grown in a medi u m previously d escr ibed (Lac ks & Hotchkiss, 1960a) m od ified in t he following manner. Phosphate was rem ove d from t he casein h ydrolysate prior to its addition t o t he medium by treatment with excess ca lci um chl oride and removal of the precipitate ; 0'1 M-tris was substitut ed for the phosphate used t o buffer the m edium; and carrier -free [82P]phosphoric acid was added. The procedure for ex t ract ing and pu r ify in g the DNA was essentially that of McCarty & Aver y (1946). 119

120

S. LACKS

A recipient culture was prepared by growing a streptomycin-sensitive strain in 2 1. of medium to an optical density of 0·1 at 650 mfL. The cells were concentrated twenty-fivefold, incubated at 30°C, and treated for 5 min with [32P]DNA at 5 fLg/mi. The reaction was terminated by treatment with DNase at 1 fLg/mi. for 1 min. Immediately thereafter, aliquots were diluted either into chilled medium or into flasks at 37°C which were chilled after 3, 10 and 40 min incubation. Smaller aliquots were also diluted appropriately to allow growth for 100, 140, 180,220, 260 and 300 min at 37°C before chilling. These were used to measure the frequency of transformant colony-forming units at different times by means of serial dilution in medium with and without streptomycin according to the method of Hotchkiss (1954). Recovery of the transforming ability of the newly introduced streptomycin-resistance factor was measured in samples removed from the aliquots incubated for 0, 3, 10 and 40 min. These samples were centrifuged, and the cells were suspended in 0·2 ml. of a solution containing 0·15 M-NaCl, 0·1 M-sodium citrate, and 0·1 % sodium deoxycholate. After incubation at 37°C for 15 min the lysates were diluted ten-fold with 0·15 M-NaCI and frozen to kill any residual cells. Transforming activity in the lysate!'! with respect to the newly introduced st.reptomycin-resistance marker and to an intrinsic pyrimidineindependence marker was tested with a streptomycin-sensitive, pyrimidine-requiring strain as recipient. Details of the transformation and scoring procedures have been previously given (Lacks & Hotchkiss, 1960a). Extracts of the aliquots incubated for 0, 3, 10 and 40 min were prepared, after washing the cells 5 times with medium and once with a solution of 0·15 M-NaCI and 0·1 M-sodium citrate, by resuspending the cells in 10 ml. of the latter and adding sodium deoxycholate to 0·1 %. Lysis occurred within 5 min at room temperature. The lysates were shaken with chloroform, centrifuged, decanted, and shaken with chloroform again to remove protein. Subsequently the extracts were shaken 3 times with ether, following which air was passed through the solutions to remove residual ether. This served partially to remove the deoxycholate. The extraction procedure resulted in less than a 5% loss of either the radioactivity or DNA content of the original lysates. Columns composed of methylated albumin-coated kieselguhr were used to fractionate the extracts. Material for the columns was prepared according to the procedure of Mandell & Hershey (1960). Washed columns composed of 12 g of kieselguhr suspended in 50 m1. of buffered 0·1 M-saline and treated with 3 ml. of I % methylated albumin gave satisfactory fractionation of samples containing 0·2 mg of DNA. Buffered saline solutions were prepared by dissolving the appropriate amount of NaCI in 0·05 M-sodium phosphate at pH 6·7. Following application of the extract to the column 40 ml. of buffered 0·3 M-saline were passed through. This was followed by 300 ml. of a gradient of buffered saline established by passing 300 ml. of 1·5 M-saline into a closed mixing chamber containing 300 m1. of 0·3 M-saline from which fluid passed to the column. Lastly, a gradient of increasing pH was set up with 200 m1. of a solution containing 0·9 M-saline and 1·5 M-ammonium hydroxide in the reservoir and 200 m1. of buffered 0·9 M-saline in the mixing chamber. Flow rates through the column were maintained between 0·5 and 1·0 ml.jmin, Four-mi. fractions were collected. The optical density of the fractions was measured at 260 and 280 mfL in a Beckman spectrophotometer. Radioactivity was determined by plating 0·2 ml. samples and counting with a Nuclear-Chicago thin-window gas-flow counter. DNA was determined by the Dische (1955) method using as standards purified solutions of pneumococcal DNA dissolved in salt solutions of concentration equivalent to that of the sample tested. The DNA content of these standard solutions was determined from their optical density by equating an optical density at 260 mfL of 20 with a concentration of I mg/rnl. The pH of fractions was measured with a Beckman pH-meter. Determination of inorganic [32P]phosphate was made by selective precipitation of the magnesium ammonium phosphate salt. The sample was treated with 1/3 vol. of a solution of 5% MgCI 2,6H 20 and 10% NH 4Cl, ammonium hydroxide was added in excess, the mixture was chilled, and the precipitate was separated by centrifugation. After redissolving the precipitate in 0·05 M-acetic acid the precipitation was repeated. Again the precipitate was dissolved and an aliquot was plated for determination of its radioactivity.

FATE OF D NA I N G E N E T I C TRA NS F O RMA T ION

121

In order to determine monoest erifi ed phosphate t he sam p le was first depleted of inorganic phosphate in t he manner described above. The supe rnat a nt fluid was t he n brought t o pH 8·2 b y addition of acetic ac id . Aliquot s were t re ated for 30 , 60 a nd 90 m in at 37°C wi th 0·1 p.g/m!. of alkaline phosphatase (Worthington Bi och em ical Co. ch ro mato graphically purified preparation). The r eaction was t erminated by ch illing. Carrier phosphate was added , and the inorganic [32P]pho sphat e r eleased on in cubation wi th the enzyme was precipitated and measured as described above. A density gradient analysis of the t ype described by Meselson, Stahl & Vinograd (1957) was carried out on ex t rac ts of cells which had taken up [32P]DN A for 5 min a t 30°C. Solid CsCI and wat er were added to a samp le of a de pro tein ized ex t ract t o brin g t he concentration of CsCI to 7·7 molal and the volume t o 5 m!' The m ix ture was placed in a plastic tube which w as t he n cen t ri fuged for 46 hr at 37,000 eev J tn u: in t he SW 39 r ot or in the Spinco model L cen t rifuge. Fraction s were collecte d by puncturin g t he b ott om of the t ub e an d tak ing two drops (t o give a vo lume of about 0·1 6 m !.) p er fr act ion. Each fraction was diluted with 2 m!' of wat er , and t he optica l d en sit y a t 260 mp. and t he radioactivity were m easured .

3. Results (a) Uptake of [32 p ]DN A

Other workers have shown that in genetic transform ation [32P]DNA is irreversibly fixed by the cells (Lerm an & Tolm ach , 1957 ; Fox, 1957). Table 1 gives t he amount of radioactivity found in t he extracts of cells su bject ed t o a 5-minute treatment with [32P]DNA t erminated by the addition of deoxyribonuclease and subsequently incubated for various lengths of time. (In ord er t o facilitate compa rison referen ce will henceforth be mad e to the aliquot placed on t he column which in th e zero-tim e sample contained 0·20 mg DNA.) It is evident that there is no substantial net gain or release of radioactivity to the medium on incub ation in the medium containing deoxyribonuclease-degraded fragments. TABLE

Uptak e and retention of Time of incubat ion (min)

o 3 10 40

1 32 P-labeled

DNA

Radioactivit y of extrac t (ctsjmin)

D NA cont en t of ex t rac t (mg)

10, 200 10,300 11, 000 11,200

0·20 0·20 0· 18 0·40

Aliquots of a culture treated for 5 min at 30 °C with 0·06 m g of [32P]DNA (spec ific radioactivity 1·48 x 10' cts /m in /m g , ag e of preparation = 4 days), an d t he n with l/Lg/ml. DNase for 1 min, were inc ub ated at 37 °C as indicated and were processed as describ ed under Methods. =

(b) Relationship between extent of transformation and up take of radioactivity

One way of determining the extent of transformation is to measure the frequ ency of transformed colony -forming units in the population. Since t he colony-forming unit of the pneumococcal strains used, even in well shake n cultures, consists of a chain of two to eight cells each of which may contain two nu clei which in turn may be heterozygous, a true index of transformation is obtained only after sufficient growth of the population so that the colony-forming units attain genetic homogeneity. A risk inherent in this method is that the transformed genot ype may not multiply as rapidly

122

S. LACKS

as the untransformed one either temporarily or permanently; this would give a spuriously low value. The results obtained, presented in Table 2, demonstrate, however, that the eventual rate of growth of streptomycin-resistant cells is equal to TABLE

2

Frequency of transformed colony-forming units Time incubated Total at 37°C c.f.u.t (min) ( x 10-11 ) 100 140 180 220 260 300

t Total

1·3 2·3 5·2 10·0 23·0 45·0

Streptomycin-resistant c.f.u, (x 10- 8 )

Frequency of streptomycin -resistants

1·8 2·5 3·6 6·7 14·0 29·0

0·14 0·11 0·069 0·067 0·062 0·065

(%)

e.f.u, = colony-forming units in equivalent of aliquot used in column fractionation.

that of the original type (both types grow with a generation time of 40 minutes), and a constant ratio of transformed colony-forming units to the total is attained. There is thus no permanent difference in the rate of multiplication, and the ratio obtained can be used as a measure of the extent of transformation. Another way of determining the extent of transformation depends on measurement of the transforming activity of the newly acquired marker in extracts of samples of the treated population. In testing the extracts, an indicator strain which was both streptomycin-sensitive and pyrimidine-requiring was used. Thus it was possible to measure transforming activity both with respect to a marker originally present in the population (pyr+) and one which was introduced in the transformation (str f ) . Sufficient quantities of extract were used so that saturating concentrations of DNA were present in the test cultures; under these circumstances the amount of transformation observed should be proportional to the proportion of marker molecules among the total of homologous DNA molecules (Hotchkiss, 1957). This proportion can then be determined by comparison with the amount of transformation observed when an extract of a population wholly resistant to streptomycin is tested under identical conditions. The results are given in Table 3. As expected, the ability to transform the pyr+ marker is uniform in the extracts tested. Activity of the newly introduced marker, sir", which is initially almost entirely masked, recovers rapidly to attain ultimately a ratio of 0'092% with respect to the activity of DNA from a homogeneously strf-marked strain. Once the extent of genetic transformation is determined, by making the assumptions (a) that the DNA taken up is a random sample of all the molecular species and (b) that incorporation of the marker considered is representative of all genetic determinants introduced, it is possible to calculate the corresponding amount of physical incorporation of [32P]DNA into host DNA which could account for the extent of transformation observed. This is done by multiplying the extent of transformation by the quantity of DNA in the aliquot and by the specific radioactivity of the introduced DNA. Using the ultimate frequency of streptomycin-resistant colony-forming units as the gauge of extent of transformation, the corresponding 32p

:FATE OF DNA IN GENETIC TRANSFORMATION

123

incorporation would be 0·00065 x 0·20 x 1·48 x 10' = 1920 counts/min. This value is only 18% of the total radioactivity taken up (mean value from Table 1 = 10,600 counts/min). Similarly, calculation based on the transforming activity of extracts as TABLE

3

Transforming activity of extracts at different times Number of bransformante/rnl. test culture

Minutes at 37°C

Extract tested

pyr+ str B cells treated with pyr+ str r DNA

Proportion % of of str r maximal marker recovery

Pyrimidine independence (x 10-5 )

Streptomycin resistance (x 10- 2)

9·5 9·0 9·5 9·5

0·4 4·0 8·0 8·0

0·005 0·046 0·092 0·092

10·0

8700

100

0 3 10 40

pyr+ str r cells

(%) 5 50 100 100

The designated extracts were incubated with cultures of pyr- str B cells for 30 min at 30°C. Subsequently the reaction was terminated by DNase and the cultures were incubated for 100 min at 37°C, after which they were scored for pyrimidine-independent and streptomycin-resistant transformants.

a gauge of extent of transformation gives: 0·00092 x 0·20 x 1·48 x 10' = 2700 counts/ min or 25% of the radioactivity taken up. So it appears that only one-fifth to onequarter of the DNA taken up during transformation exerts its genetic activity. Only this fraction of the total taken up need have retained genetic integrity. (c) Column fractionation of newly introduced [32 P]DNA

The behavior of bacterial extracts on methylated albumin-coated kieselguhr columns has been accurately described by Mandell & Hershey (1960). The peaks of ultraviolet absorbing material seen in Fig. 1 correspond, in order of elution, to (1) low-molecular-weight substances such as nucleotides, (2) low-molecular-weight "soluble" RNA (two low peaks are usually seen), (3) native DNA, and (4) a broad peak of high-molecular-weight "ribosomal" RNA. In Figs. l(b) and (d) the peak immediately following the initial one appears to be soluble RNA which is poorly TABLE

4

Radioactivity in different fractions as a function of time of incubation Time of incubation at 37°C after introduction of [32P]DNA Fraction

I II III

IV

o min eta/min 3900 240 0 3720

% 50 3 0 47

3 min eta/min] 4000 2420 540 1560

% 47 29 6 18

1'0 min cts/minj 3010 5140 910 270

% 32 55 10 3

40 min eta/mint 1790 5650 1370 300

t Corrected for 32p decay to correspond to time of counting of O-min samples.

% 20 62 15 3

S. LACKS

124

retained by the column when the extract is insufficiently depleted of deoxycholate. In more recent experiments in which deoxycholate was completely removed by lowering the pH of the extract to 6·2 before ether extraction fractionation gave sharper peaks and better separations. The ribosomal RNA, in particular, eluted as (0)

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two well-defined peaks. Recoveries of radioactivity and DNA in the fractions ranged between 80 and 90%. Immediately after treatment of the cells with [32P]DNA almost none of the radioactivity introduced is present as native DNA. About half of the DNA appears in a rapidly eluted fraction which is not at all retained by the column (Fig. l(a) and Table 4): the other half is very strongly retained and is only released by raising the pH of the eluent. These fractions will be referred to as Fractions I and IV respectively.

FATE OF DNA IN GENETIC TRANSFORMATION

125

On incubation of the treated cells for 3 minutes at 37°C the radioactivity is redistributed (Fig. l(b)). A sizeable fraction appears under the native DNA peak; this will be called Fraction II. A smaller amount of radioactivity appears to be associated with the broad RNA peak; this will be referred to as Fraction III. By 3 minutes Fraction IV has lost more than half of its radioactivity.

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FIG. 1. Column fractionation of extracts of cells incubated for (a) 0, (b) 3, (c) 10 and (d) 40 min after introduction of [32P]DNA. Aliquots of a culture treated with [32P]DNA for 5 min at 30°C were incubated at 37°C for the time indicated. Deproteinized extracts containing the nucleic acids were prepared and applied to the column as described under Methods. The column was eluted with a gradient of increasing salt concentration until fraction 90, after which a gradient of increasing pH was applied. 0-0-0 radioactivity, optical density, x - x - x DNA content.

e-e-e

9

S. LACKS

126

By 10 minutes (Fig. l(c)) Fraction I, which appeared undiminished after 3 minutes, is noticeably reduced, while Fraction II now incorporates the majority of the radioactivity. Fraction III has augmented more slightly and Fraction IV has virtually disappeared. At the end of 40 minutes (Fig. l(d)) the DNA and RNA fractions are somewhat further increased at the expense, apparently, of Fraction 1. (d) Nature of Fraction I

The radioactivity in Fraction I must be associated with low-molecular-weight substances since, as well as not being bound by the column, it is found to be entirely dialysable. About one-quarter to one-third of the radioactivity appears to be in the form of inorganic phosphate; the remainder appears to be in the form of oligonucleotides. Results of an analysis of Fraction I of the O-minute aliquot are given in Table 5. Here 23% of the radioactivity appeared as inorganic phosphate. Similar TABLE

5

Inorganic [32p]phosphate content of Fraction I Time of incubation (min)

0 3 10

eta/min cts/min in total sample as phosphate

390 390 270

88 U8

62

% free phosphate 23 30 23

Samples of Fraction I of aliquots incubated for different periods after introduction of [33P]DNA were assayed for inorganic [32P]phosphate as described under Methods.

proportions of free phosphate were found in Fraction I of the 3- and lO-minute samples. In other experiments this proportion has been found consistently to be between 20 and 40%. The reliability of this method is substantiated by similar results obtained by selective precipitation of the phosphate as ammonium phosphomolybdate or as calcium phosphate. The remainder of Fraction I appears to consist of small fragments of DNA. The number-average chain length of these fragments was roughly determined by measuring the inorganic [32P]phosphate released after treatment with phosphatase and comparing this amount with the total bound phosphate. Figure 2 gives the results of this procedure applied to a sample of Fraction I of the O-minute aliquot. There appears to be a rapid release of phosphate followed by a slow increase with time. The latter may be due to slight contamination of the phosphatase preparation by a diesterase capable of splitting deoxy-oligonucleotides. The contribution of this slow increase may be eliminated by extrapolation to O-time which gives a minimal value of 27% for the proportion of phosphatase-sensitive 32P. From this value a number-average chain length of four nucleotides is obtained. (e) Nature of Fraction IV

Fraction IV is so well retained by the column that it is not released even by elution with 1·5 M-saline. It can be released by raising the pH to 10. On neutralization of such an eluate and re-chromatography the fraction is again tightly bound. Since it appears that retention by the column is greater for flexible polymers like RNA rather

FATE OF DNA IN GENETIC TRANSFORMATION

127

than rigid structures like DNA, and that among flexible polymers, those with longer chains are more strongly retained (ribosomal RNA as opposed to soluble RNA), it is reasonable to expect that Fraction IV corresponds to high-molecular-weight, 100,------,-----,-------.,----,

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Fraction number FIG. 3. Column behavior of Fraction IV compared to heat-denatured DNA. An extract prepared from cells treated for 10 min at 30°C with [32P]DNA was mixed with 10 ml. of pneumococcal DNA at 70 p.gjml. in 0·015 M-NaCl which had been heated for 10 min at 100°C. This mixture was applied to the column and eluted successively with salt and pH gradients. The figure depicts elution by the pH gradient of the material which was retained by the column after salt elution. Symbols are as in Fig. 1. The contribution of the extract itself to the optical density of this fraction is negligible (less than 10%) compared to that of the heated DNA which was added.

S. LACKS

128

single-stranded DNA. Heat-denatured DNA, which appears to be single-stranded (Doty, Marmur, Eigner & Schildkraut, 1960), shows the same behavior on the column as does Fraction IV. This correspondence is depicted in Fig. 3. Density gradient analysis of newly introduced [32P]DNA supports the contention that Fraction IV consists of single-stranded material. The findings of Meselson & Stahl (1958) indicate that single-stranded DNA can be separated from its corresponding double-stranded form by virtue of its higher density. Figure 4 shows

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Fraction number FIG. 4. Density gradient analysis of newly introduced [32P]DNA. A deproteinized extract of cells treated for 5 min with [32P]DNA at 30°C was centrifuged in the presence of 7·7 molal CsCI for 46 hr at 37,000 rev.zmin. The centrifuge tube was punctured and fractions containing 2 drops (about 0·016 mI.) were collected and diluted with 2 ml, of water. Radioactivity (0) and optical density (.) of the fractions were measured.

that about half of the radioactivity incorporated as [32P]DNA appears as a peak of greater density than the peak of ultraviolet absorption corresponding to the native DNA of the recipient cells. The separation between the two peaks is about what would be expected for heated and unheated DNA which differ in density by 0·015 glml. (Guild, 1961). Most ofthe rest of the radioactivity appears to be uniformly distributed in the tube; such behavior would be expected of the small fragments corresponding to Fraction 1. Only a small proportion of radioactivity appears to be associated with the native DNA band. The rather high base-line of optical density can be ascribed partly to low-molecular-weight substances and partly to trailing of the high peak due to RNA at the bottom of the tube. (f) Conversion of newly introduced DNA with time

Table 4 summarizes the amount of radioactivity in each fraction after different times of incubation following introduction of [32P]DNA. Initially, the radioactivity is divided between low-molecular-weight fragments and single-stranded DNA. Inasmuch as large segments of genetic loci which must involve over a hundred nucleotides have been shown to be transformed with appreciable frequency (Lacks & Hotchkiss, 1960a), it is unlikely that the dialysable fragments of Fraction I can contribute genetic factors to the host. It is more likely that the single-stranded DNA of Fraction IV is the source of genetic information. It would appear from the calculation

129

FATE OF D NA I N GENETI C TRA N SFO R MATION

of the radioactivity corresponding to the physical integration of the genetic factor that only half of the radioactivity of this fraction is transferred intact to the native DNA fraction; the rest may only appear t here afte r fragmentation and pa ssage through Fraction 1. Th ere is no proof here that any of the introduced DNA is incorporated intact; it may all be in corporated as fra gments in the normal synt hesis of DNA after having fulfilled its information transfer by means of a copy mechanism. However, the contrary appears more lik ely . It should be noticed that the removal of radioactivity fr om Fraction IV and its appearance in native DNA correspond closely t o the recovery of the stre pt omycin resistance transforming factor (compare Tables 3 and 4). Initi ally , only 5 % of the eventual act ivity of the latter is present compa red to 5% of the radioactivity present in F raction II at 10 minutes. Afte r 3 minutes, when just 50 % of the st reptomyc inresistance marker ha s been recovered, Fraction II stands at 50% of its 1O-minut e mark, and Fraction IV is ju st about 60 % depleted. Nevertheless, it is evid ent that the fragments of Fraction I are also extensively used for DNA synthesis since more radioactivity appears in the DNA than can be accounted for by Fraction IV. Incorporation into the RNA of Fraction III is presumably mediated by the inorganic phosphate component of Fraction 1. Figure 5 is a flow diagr am which represents the mole cular conversions of the introduced DNA and it s ph osphoru s atoms as envisaged by the abo ve considerations. Fraction

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F IG. 5. Flow diagram showing con ve rs ions undergone b y ssp in trod uce d as [32P]DNA. H eavy arrows d epict presumed t rans fer of genetic informat ion as well as ssp with correspon din g quantities calculated from the extent of recovery of the introduced marker. Li ght arrows depict transfer s of radioactivity calculated to give the ex pe rimen t ally observed perc entages. Dotted arrows indicate retention of radioactivity by t he fraction.

4. Discussion The disruption of DNA following ent ry into the recipient cell offers an explana t ion for it s inab ility t o exert its phenotypic function wit hout further int egration (Lacks & Hotchki ss, 1960b). Immediately after ent ry t he information is present in singlestranded DNA which may be un abl e to transfer inform ation in prot ein synthesis. Th is is suggested by t he findin g of W ood & Berg (1962) that single-st ra nded DNA is unabl e to make " messe nger" R NA which is active in stimulat ing amino acid incorporati on into prot ein in an in vitro system . Furthermore, t he existence of single-stranded DNA in the recipient cell is transitory , for within 10 minutes it has

130

S. LACKS

either been integrated more or less intact or else destroyed by fragmentation. The latter would entail destruction of the information. Hence, that part of the introduced genetic material which is not permanently integrated rapidly loses its functional potential. Similarly, the inability of newly introduced DNA to function effectively as a transforming agent (Fox, 1960) appears to be due to the presence of the genetic factor in the form of single-stranded DNA. Recent studies have shown that heat-denatured DNA, which is apparently single-stranded, has a very low efficiency in transformation (Rownd, Lanyi & Doty, 1961; Guild, 1961). The investigations of Roger & Hotchkiss (1961) as well as the findings of Lerman & Tolmach (1959) suggest that heat-denatured DNA is a poor transforming agent because the single-stranded form is not readily taken up by the cells. This consideration, and also the fact from the present study that the entering DNA is divided just about in half between fragments and single strands, leads one to think that this fragmentation process may be an essential step in the entry of the DNA into the cell. A rather speculative model for such a process might be the following: the end of a DNA molecule outside the cell penetrates the cell membrane, at the interior of which is fixed a molecule of deoxyribonuclease. If this enzyme then alternately attaches to and splits linkages along one strand (it may not act on the other strand because it is of opposite polarity), the other strand will be dragged into the cell. Such a process would not require an external source of energy. This model, in any event, suggests a role for the deoxyribonuclease known to be present in Pneumococcus (Avery, MacLeod & McCarty, 1944). Such a model would not hold for Hemophilus injluenzae transformation since it has been found that two genetic markers present on different strands of a single, artificially annealed molecule may both be integrated in the cell (Herriott, 1961). However, there does not appear to be any masking of a newly introduced marker in Hemophilus (Voll & Goodgal, 1961), so the process of entry may well differ from that in Pneumococcus. The finding that single-stranded DNA is an intermediate in genetic transformation explains why artificially produced single-stranded DNA is able to transform once it is incorporated into the cell (Rownd et al., 1961; Guild, 1961; Roger & Hotchkiss, 1961). It is not known whether the single strands are partially fragmented before integration into host DNA. If such intermediate-length chains are short-lived there may be no observable accumulation at any time. Furthermore, they would be expected to elute in the region of the ribosomal RNA fraction where their presence would be obscured by the incorporation of 32p into the RNA. Although single-stranded DNA is found apparently free in the deproteinized extracts, it is possible that within the cell it is associated with other structures. Ephrussi-Taylor (1960) has proposed that newly introduced DNA is bound to protein. Another possibility is that the single strand is bound to a native DNA molecule to form a three-chain structure of the type proposed by Zubay (1962) for messenger RNA formation. According to such a model switches of phosphate bonds could transfer segments of the third strand to host DNA. This would account for the genetic recombination within molecules which is found to occur in transformation (see review by Ravin, 1961). Whatever the precise mechanism of the genetic recombination in Pneumococcus may be, it would appear to involve breakage and insertion of the single-stranded DNA which carries the introduced genetic information. Meselson & Weigle (1961) and

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Kellenberger, Zichichi & Weigle (1961) have found that genetic recombination in bacteriophage involves the physical transfer of molecular segments, although the evidence of Meselson & Weigle indicates that such transfer occurs between two double-stranded structures. , Another implication of the present work with respect to genetic recombination stems from the finding that about one-fifth of the introduced DNA is genetically integrated. (It should be mentioned that Lerman & Tolmach (1957) and Fox (1957) by a somewhat different method have estimated this value to be as high as one-half and two-thirds, respectively.) This would set a lower limit of 20% to the extent of linkage observable in Pneumococcus; for any markers in a single molecule, no matter how far apart, would have this probability of simultaneous integration. Findings to the contrary might indicate either that a high efficiency of integration did not prevail under the conditions of the test, or that the transforming DNA "molecules" are not uniform, but rather fragments of indefinite length and marker composition torn off randomly from the host genome. This research was carried out at the Brookhaven National Laboratory under the auspices of the United States Atomic Energy Commission. REFERENCES Avery, O. T., MacLeod, C. M. & McCarty, M. (1944). J. Exp. Med. 79, 137. Dische, Z. (1955). In The Nucleic Acids, ed. by E. Chargaff & J. N. Davidson, vol. 1, p. 285. New York: Academic Press. Doty, P., Marmur, J., Eigner, J. & Schildkraut, C. (1960). Proc, Nat. Acad. Sci., Wash. 46,461. Ephrussi-Taylor, H. (1960). C.R. Soc. Biol., Paris, 154, 1951. Fox, M. S. (1957). Biochim. biophys. Acta, 26, 83. Fox, M. S. (1960). Nature, 187, 1004. Guild, W. R. (1961). Proc. Nat. Acad. Sci., Wash. 47, 1560. Herriott, R. M. (1961). Proc. Nat. Acad. Sei., Wash. 47, 146. Hotchkiss, R. D. (1954). Proc, Nat. Acad. Sci., Wash. 40, 49. Hotchkiss, R. D. (1957). In The Chemical Basis of Heredity, ed. byW. D. McElroy & B. Glass, p. 321. Baltimore: Johns Hopkins Press. Kellenberger, G., Zichichi, M. L. & Weigle, J. J. (1961). Proc. Nat. Acad. Sci., Wash. 47, 869. Lacks, S. & Hotchkiss, R. D. (1960a). Biochim. biophys. Acta, 39, 508. Lacks, S. & Hotchkiss, R. D. (1960b). Biochim. biophys. Acta, 45, 155. Lerman, L. S. & Tolmach, L. J. (1957). Biochim. biophys. Acta, 26, 68. Lerman, L. S. & Tolmach, L. J. (1959). Biochim. biophys. Acta, 33, 371. Mandell, J. D. & Hershey, A. D. (1960). Anal. Biochem, 1, 66. McCarty, M. & Avery, O. T. (1946). J. Exp. Med. 83, 97. Meselson, M. & Stahl, F. W. (1958). Proc, Nat. Acad. Sci., Wash. 44, 671. Meselson, M., Stahl, F. W. & Vinograd, J. (1957). Proc, Nat. Acad. Sci., Wash. 43, 581. Meselson, M. & Weigle, J. J. (1961). Proc, Nat. Acad. Sci., Wash. 47, 857. Ravin, A. W. (1961). Advanc. Genetics, 10, 62. Roger, M. & Hotchkiss, R. D. (1961). Proc. Nat. Acad. Sci., Wash. 47, 653. Rownd, R., Lanyi, J. & Doty, P. (1961). Biochim. biophys. Acta, 53, 225. Voll, M. J. & Goodgal, S. H. (1961). Proc. Nat. Acad. Sci., Wash. 47, 505. Wood, W. B. & Berg, P. (1962). Proc. Nat. Acad. Sci., Wash. 48, 94. Zubay, G. (1962). Proc. Nat. Acad. Sci., Wash. 48, 456.