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Transformation of phage T4 by small denatured DNA fragments
VIROLOGY
41, 175-199 (1070)
Short Transformation
of
Phage
Communications T4 by Small
The T4 phage transformation system (1, 2) is well suit.ed for investigating the mechanism of genet.ic recombination and for assaying the genetic content of chromosomal fragments since the T4 chromosome is well marked and easily isolated intact, @), the strands are separable (4), and small fragments of denatured DNA from either strand can donate genetic markers to the recipient phage (5,6). On the ot,her hand this transformation system has t.he disadvantage of a low efficiency of transformation (7) when compared with most bacterial systems. In this report, we will define the relation between lengt,h of single-stranded donor DNA and efficiency of transformation and determine the smallest polynucleotide fragment with which genetic activity can still be determined. In the T4 transformation system, penicillinittduced spheroplasts of Escherichia co/i B are infected with urea-t,reated ~11 recipient phage in the presence of denatured T4r+ donor DNA. Recipient phage growing in the spheroplasts rescue markers from denat.lcred donor DNA. For a detailed descript.ion of the methods employed in this system see Veldhuisen and Goldberg (2). To investigate the relation between DNA fragment length and transformat,ion, we prepared solutions of I>NA fragments homogeneous in size. Typical preparations are described in Table 1. Degradat,ion by physical methods was more reproducible than by nuclease digestion (however, even for duplicate nuclease digestions t,he peak fractions generally varied less than twofold in molecular weight). Since several fractions from the same gradient were often used in the transformat.ion assays, it was import,ant to demonstrate the resolution of the gradient. To test the homogeneity of t.he fractions, we resedimented a fraction from one alkaline gradient on a second alkaline gradient. Data from the two gradients are plotted in Fig. 1. Less t,han 1% of the DNA in the original peak fraction is greater than twice the molecular weight of t.he peak of the rerun fract,ion. This agrees with Studier’s finding (8) that there is lit.tle aggregation of denatured DNA in alkali. The relation between transformation frequency and I)NA concentration for long and short fragments of denatured 11X.4 is shown in Fig. 2A. Short fragments transform less efficiently per rg
Denatured
DNA Fragments
DNA than long fragments. The relation between transformat,ion eficiency (initial slope of the transformation curve for short DNA/initial slope of the transformation curve for long DNA) and DNA fragment weight is plotted in Fig. 2B. Transformation efficiency is independent of fragment size down to approximately 5 X lo5 daltons. It then declines to 507, at about 3 X lo5 daltons and to about 37, at 1 X lo5 daltons. The shape of the curve shown in Fig. 2B can be expected to depend on the homogeneity of the DNA samples. Contamination with larger fragments would lead to an overestimation of the transformation efficiency of the fractions. However, as was shown in Fig. 1, a homogeneous fraction contained less t.han 1% DNA with twice the molecular weight of the mode and would therefore not significantly affect the average transformation efficiencies calculated for pieces of various sizes. The mutation in the recipient phage T4T1~1173 , which was used in these experiments, is presumably a small deletion. However, since large deletions behave abnormally in transformation (E. Goldberg, unpublished experiments), we also used the revertible point mutant T4T)rII;7 as the recipient phage and obtained similar results. Therefore, the relation between size and transformation efficiency is the same when t,he recipient phage marker is a point mutant, as when it is a short. deletion. The reason for the decline in transformation efficiency as a function of molecular weight is not known. It is known that nucleases are associated with the surface of E. coli and are released upon preparat,ion of spheroplasts (9). We therefore investigated degradat.ion of donor I)NA in the incubation mixture with the spheroplast,s. Less than lOTo of sonicated DNA (mol. wt. = 100,000) is degraded to acid-soluble length after 1 hour of incubation at 37”. Long DNA is degraded to a lesser extent since there are fewer ends per unit weight susceptible to exomuzleases with increasing molecular weight of t.he DNA. Endonuclease activity was checked by alkaline sucrose densitygradient centjrifugation of long DNA and of sonicated I)NA before and after a 60.minute incubation in the spheroplast medium. There was no decrease in size of t,he sonicated DNA. But long DNA, which sediments heterogeneously, was 175
176
SHORT TABLE FRAGMENTATION
Treatment Long DNA Virtis homogenizer 11,000 RPM 36,ooO RPM Pancreatic DNase 0.02 units/ml 0.03 units/ml Micrococcal DNase 4 X 1O-4 pg/ml French pressure cell 15,000 lbs/in2 25,000 lbs/in2 Sonication
COMMUNICATIONS
I OF
DNA Molecular weight of peak fraction ca 10' 7.2 X lo5 6.2 X lo5 7.4 x 3.5 x
105
105
7.2 X lo6 3.1 x 1.45 x 1.0 x
105 105 106
Note: Preparation and characterizat,ion of typical T4 DNA fragments of different. sizes. The DNA was fragmented and then fractionated. Native DNA was fragmented in the following ways: (1) Pancreatic DNase digestion: 1 ml of DNA solution (0.5 mg/ml in 0.01 M sodium phosphate buffer, pH 7.0, 0.002 M MgCln) was mixed for 15 minutes at 4” on a rot,ator (Scientific Industries, Model 150) with 0.01-0.04 units DNase (Worthington, DSV). The mixture was incubated 3.5 minutes at 37” and boiled for 10 minutes. (2) MicrococcaZ Dh’ase digestion: 0.1 ml of DNA solution (0.5 mg/ml in 0.02 M Tris buffer, pH 8.0, 0.001 M CaC12) was mixed as above with 1 to 4 X lOA4 pg DNase (Worthington, NFCP). The mixture was incubated 6 minutes at 29”, and the reaction stopped by adding 0.1 ml EDTA (0.1 M) and boiling for 10 minutes. For fragments with both 3’- and 5’-hydroxyl termini, samples were treated with micrococcal DNase as in (2), but no EDTA was added and the mixture was boiled for 10 minutes and then 1 unit of alkaline phosphatase (Worthington BAPC) was added. The mixture was then incubated for 30 minutes at 37”. (3) Sonication: 2 ml of DNA solution (0.5 mg/ml in 0.01 M sodium phosphate buffer, pH 7.0) was sonicated at maximum power for 3 X 20 seconds at 0” (MSE, Model 60 with 3/8 in. probe). (4) Shear in French pressure cell: 2 ml of DNA solution, as above, was passed through a French pressure cell three times at 15,000 or 25,000 lbs/in2. (5) Shear in homogenizer: 1 ml of DNA solution, as above, was sheared with a rotating blade in a homogenizer (Virtis 45 with microblade and cup No. 16-111) for 45 minutes at several speeds. The fragmented DNA was sedimented through alkaline sucrose density gradients (11.5 ml, &2070 sucrose gradients containing 0.35 M NaOH,
1 8
DISTANCE FROM TOP (CM) FIG. 1. Homogeneity of fractions from alkaline sucrose gradients. 32P-DNA was degraded with 0.03 units pancreatic DNase and fractionated as described in Table 1. The peak fraction was rerun on a similar gradient. The peak of marker 3HDNA (1) was 9.2 X lo6 mol wt (obtained by shearing 3H-labeled T4 DNA in a virtis homogenizer), and the peak of both gradients corresponded to 1.9 X lo* mol wt. Hydrolyzed DNA---; peak fraction rerun- - -.
0.65 M NaCl) at 40,000 rpm (Spinco SW 41 rotor) were for 17 hours at lo”, and 0.9-ml fractions collected from the bottoms of the tubes. Some sonicat,ed tritiated T4 DNA (S = 5.36; mol. wt. = 1.05 X 105 as determined by boundary sedimentation in alkali on the analytical centrifuge) was included as a marker in each sample. Radioactivity and optical density (at 260 mp) of aliquots were measured. Fractions containing sufficient DNA were diluted and dialyzed against 0.01 M sodium phosphate, pH 7.0, before being used in transformation. These DNA fractions were called short DNA as opposed to the DNA not purposely fragmented which is called long DNA. Long DNA is native DNAwhich has been boiled for 3 minutes and rapidly chilled on ice in order to denature it. Analysis on alkaline sucrose gradients have demonstrated that long DNA is heterogeneous in length and has an average molecular weight, of about 107. Relative molecular weights of all DNA fractions were determined by the relation (1),/D?) = (MI/M$‘.4D (8). In some cases the molecular weights of fractions were also determined by boundary sedimentation in alkali (0.9 A4 NaCI, 0.1 M NaOH) in an analytical ultracentrifuge. The values obtained by the two methods agreed in all cases.
SHORT
177
CObZMUn’ICBTIONS
I.0
g5
0.8
G iii h 0.6 8 i= Q 0.4 z i? i!2 0.2 L I0.5
I .o [DNA]
1.5
2.0
2.5
(pglml)
2
I
I
1
4
6
8
MOLECULAR
WEIGHT
’
IO
II--”
1
100
X lO-5
FIG. 2. Relation between transformation and size of DNA. A. Variation of transformation frequency with DNA concentration and fragment size. Different concentrations of denatured r+DNA of varying sizes were incubated with spheroplasts for 20 minutes at 37”. Then rIIT3 recipient phage was added, and on the mixture was incubated and then titrated for total phage on E. co& B and for T+ transformants E. coli K(x). The ratio r+/rII is called the transformation frequency. DNA was fragmented in a French pressure cell and fractionated in an alkaline sucrose gradient. Long DNA-O; 2.8 X 105 mol wt,-X ; 1.7 X lo6 mol wt-0. B. Relation between molecular weight of DNA fragments and transformation efficiency. For each point, the transformation efficiency was calculated (see text) from a plot of transformation frequency vs. DNA concentration for long DNA and for fragments of relevant size. Recipient phage, ~73, and r+ donor DNA were used. Long DNA-m; Virtis fragments-0 ; pancreatic DNase fragments-e; French pressure cell fragments-A; micrococcal DNase fragments-O ; micrococcal DNase + phosphatase fragments-X ; sonicated fragments-A. reduced to about half its molecular weight. This finding is compatible with previous experiments (5) which showed that linkage of genetic markers is dependent on lengths of donor DNA molecules much larger than those discussed here. Thus, neither exonucleases nor endonucleases in the incubation mixture are sufficient to explain the decrease in transformation efficiency of small denatured fragments of DN,4. The linkage experiments (5) also rule out the role of ettdonucleases within the cell. 1~1keeping with this, unpublished experiments by one of us (E. U.) with an endoE. coli B, ER22 nuclease I negative bacterium, (IO), showed that neither transformation frequency nor linkage of the r7g-ulcp region using long donor DNA is altered in this host,. Base pairing does not seem to be a limit.ing factor since the minimum length necessary for pairing of homologous DNA strands is well below 300 nucleotide residues and has been estimated at 10-20 nucleotide pairs (11). Unpublished inhibition experiments with 1‘73and 480 DNA show that both will inhibit transformation by T+ DNA in an apparent,ly competit.ive manner. 680 DNA inhibits about half as well as ~73 , indicating that at least
part of the competition is not at, the level of base pairing. There are still two obvious possibilities of processes which might reduce transformation efficiency of short fragments more than long ones: (1) intracellular exonucleases which reduce fragments to asize below that required for integration, I.nd (2) size dependence of DNA uptake. (1) As fragments approach the minimum size for integration, less exonucleolytic degradation per fragment would be required to inactivate them. Fragments with different termini are differentially sensitive to exonuclease (12). Therefore we attempted to see if transformation efficiency depended upon phosphorylation of either the 5’or 3’.hydroxyl termini. Figure 2B shows the transformat,ion efficiency of DNA fragments prepared from digests of pancreatic DNase (5’phosphate terminus) or micrococcal DNase (3’phosphate terminus) or microccal DNase plus alkaline phosphatase (3’- and 5’-hydroxyl termini) as well as DNA fragmented in the French pressure cell. The transformation efficiencies are independent. of the termini. These experiments are inconclusive in ruling ollt intracellular exonucle-
17s
SHORT
COMMUNICATIONS
olytic degradation as a factor limiting transformation efficiency of small fragments, however, since the termini of the donor l>NA fragments might be modified either before or after penetrating the spheroplasts. (2) (:llrney (IS) and Cato and Guild (14) have in Pneumococcus, for double-stranded shown DNA, that, large DNA fragments are taken up bet,ter than smaller ones at excess DNA conceni.ration. We have not yet succeeded in measuring directly DN.4 uptake in infected or uninfected spheroplasts and are therefore unable to evaluate the role of DNA uptake in limiting the transformation efficiency of small donor fragments. Guild it al. (15) have discussed a quantit,ative model, relating size of donor DNA to transformation efficiency, which is based on exonuclease action and switching frequencies. Our data fit their model well. We suggest that the reduction of efficiency of t,ransformation with smaller fragments may be related to the intracelllllar kinetics of pairing (16) as well as to exonuclease action and to the probability of switching. We are now trying to determine directly the role of intracellular exonllclease and DNA uptake in controlling the efficiency of donor DNA of various sizes, using both E. coli and ;lerobacfer aerogenes (17) as hosts. SCKNOWLEDGMENTS We are grateful to Dr. J. Eigner for the strain E. coli B EK22 and to Dr. M. Malamy for the phage 480. This work was supported by grants from the National Science Foundation (GB-5923) and the National Institutes of Health (GM-13511). One of [IS (E. G.) is a Career Development Awardee (GM-7567) of the National Institutes of Health. REFERENCES 1. Vas de POL, J. H., VELDHUISEX, G., and COIIEN, J. A., Biochem. Biophys. Arts 48, 417-418 (1961). VELDHUISEN, Cr., POELM.IN, n2. C., and COHEN, J. A., Biochim. Biophys. tlcta 161, 9C109 (1908). 2. VELDHUISEN, CT., and GOLDBERG, E. B., Nucleic Acids, Part B. In “Methods in Enzymology,” (I,. Grossman and K. Mol-
dave, eds.), pp. 858-863. Academic Press, New York, 1968. 3. HERSHEY, A. I)., and BUMSI, E., J. Xol. Biol. 2, 143-152 (1960). 4, GuH.\, A., and SZYB.LLSKI, W., I’irology 34, 608-616 (1968). 5. GOLDRERG, IS. B., Proc. Nat. Acad. Sri. I’.S. 56, 1457-1463 (1966). 6. Jz\~.~~I~~l\~, lt., and GOLDBERG, E. B., Proc. ~Yat. Ad. Sci. U.S., 64, 198-204 (1969). Y. GOLDP.\RB, I>. M., AVDEEV~, A. V., BLINOVA, S. V., SERGEEV~, A. N., and LEVIN.Z, G. A4., Genetika 7, 148-153 (1966). 8. STUDIER, F. W., J. Mol. Biol. 11, 373-390 (1965). 9. HEPPEL, L. A., Science 156, 1451-1455 (19G7). 10. EIGNEH, J., and BLOCK, S., J. Virology 2, 320-326 (1968). il. THOMAS, C. A., JR., In “Progress in Nucleic Acid Research and Molecular Biology” Vol. 5. (J. N. Davidson and W. E. Cohn, eds.), p. 323. Academic Press, New York, 1966. 12. LEHM.IN, I. R., In “Progress in Nucleic Acid Research” Vol. 2. (J. N. Davidson and W. 15. Cohn, eds.), pp. 83-134. Scademic Press, New York, 1963. 13. GURNEY, T., JR., Thesis, Yale University, New Haven (1965). 14. CATO, A. E., JR., Thesis, Duke University, Durham (1966); GUILD, W. R., and CATO, A., Jn., J. Mol. Biol. 3, 157-178 (1968). 16. GUILD, W. R., CATO, A., JR., and LXKS, S., Cold Spring Harbor Symp. of Quanf. Biol. 33, 643 (1968). 16. WETMUR, J. G., and I>.~VIDSON, N., J. Mol. Bid. 31,3*9-370 (1968). lY. W.\IS, A., and GOLDBERG, E. B., Virology 39, 153-161 (1969). HANS ZWEERINK’ ED~.\RD B. GOLDUERG Departmenl of Molecular Biology and Microbiology l’ujfs University School of Medicine Boston, Massachusetts 02111 Accepted January 30, 19YO 1 Present address : Department of Microbiology and Immunology, Duke University Medical Center, Tjurham, N.C. 27706.
41, 175-199 (1070)
Short Transformation
of
Phage
Communications T4 by Small
The T4 phage transformation system (1, 2) is well suit.ed for investigating the mechanism of genet.ic recombination and for assaying the genetic content of chromosomal fragments since the T4 chromosome is well marked and easily isolated intact, @), the strands are separable (4), and small fragments of denatured DNA from either strand can donate genetic markers to the recipient phage (5,6). On the ot,her hand this transformation system has t.he disadvantage of a low efficiency of transformation (7) when compared with most bacterial systems. In this report, we will define the relation between lengt,h of single-stranded donor DNA and efficiency of transformation and determine the smallest polynucleotide fragment with which genetic activity can still be determined. In the T4 transformation system, penicillinittduced spheroplasts of Escherichia co/i B are infected with urea-t,reated ~11 recipient phage in the presence of denatured T4r+ donor DNA. Recipient phage growing in the spheroplasts rescue markers from denat.lcred donor DNA. For a detailed descript.ion of the methods employed in this system see Veldhuisen and Goldberg (2). To investigate the relation between DNA fragment length and transformat,ion, we prepared solutions of I>NA fragments homogeneous in size. Typical preparations are described in Table 1. Degradat,ion by physical methods was more reproducible than by nuclease digestion (however, even for duplicate nuclease digestions t,he peak fractions generally varied less than twofold in molecular weight). Since several fractions from the same gradient were often used in the transformat.ion assays, it was import,ant to demonstrate the resolution of the gradient. To test the homogeneity of t.he fractions, we resedimented a fraction from one alkaline gradient on a second alkaline gradient. Data from the two gradients are plotted in Fig. 1. Less t,han 1% of the DNA in the original peak fraction is greater than twice the molecular weight of t.he peak of the rerun fract,ion. This agrees with Studier’s finding (8) that there is lit.tle aggregation of denatured DNA in alkali. The relation between transformation frequency and I)NA concentration for long and short fragments of denatured 11X.4 is shown in Fig. 2A. Short fragments transform less efficiently per rg
Denatured
DNA Fragments
DNA than long fragments. The relation between transformat,ion eficiency (initial slope of the transformation curve for short DNA/initial slope of the transformation curve for long DNA) and DNA fragment weight is plotted in Fig. 2B. Transformation efficiency is independent of fragment size down to approximately 5 X lo5 daltons. It then declines to 507, at about 3 X lo5 daltons and to about 37, at 1 X lo5 daltons. The shape of the curve shown in Fig. 2B can be expected to depend on the homogeneity of the DNA samples. Contamination with larger fragments would lead to an overestimation of the transformation efficiency of the fractions. However, as was shown in Fig. 1, a homogeneous fraction contained less t.han 1% DNA with twice the molecular weight of the mode and would therefore not significantly affect the average transformation efficiencies calculated for pieces of various sizes. The mutation in the recipient phage T4T1~1173 , which was used in these experiments, is presumably a small deletion. However, since large deletions behave abnormally in transformation (E. Goldberg, unpublished experiments), we also used the revertible point mutant T4T)rII;7 as the recipient phage and obtained similar results. Therefore, the relation between size and transformation efficiency is the same when t,he recipient phage marker is a point mutant, as when it is a short. deletion. The reason for the decline in transformation efficiency as a function of molecular weight is not known. It is known that nucleases are associated with the surface of E. coli and are released upon preparat,ion of spheroplasts (9). We therefore investigated degradat.ion of donor I)NA in the incubation mixture with the spheroplast,s. Less than lOTo of sonicated DNA (mol. wt. = 100,000) is degraded to acid-soluble length after 1 hour of incubation at 37”. Long DNA is degraded to a lesser extent since there are fewer ends per unit weight susceptible to exomuzleases with increasing molecular weight of t.he DNA. Endonuclease activity was checked by alkaline sucrose densitygradient centjrifugation of long DNA and of sonicated I)NA before and after a 60.minute incubation in the spheroplast medium. There was no decrease in size of t,he sonicated DNA. But long DNA, which sediments heterogeneously, was 175
176
SHORT TABLE FRAGMENTATION
Treatment Long DNA Virtis homogenizer 11,000 RPM 36,ooO RPM Pancreatic DNase 0.02 units/ml 0.03 units/ml Micrococcal DNase 4 X 1O-4 pg/ml French pressure cell 15,000 lbs/in2 25,000 lbs/in2 Sonication
COMMUNICATIONS
I OF
DNA Molecular weight of peak fraction ca 10' 7.2 X lo5 6.2 X lo5 7.4 x 3.5 x
105
105
7.2 X lo6 3.1 x 1.45 x 1.0 x
105 105 106
Note: Preparation and characterizat,ion of typical T4 DNA fragments of different. sizes. The DNA was fragmented and then fractionated. Native DNA was fragmented in the following ways: (1) Pancreatic DNase digestion: 1 ml of DNA solution (0.5 mg/ml in 0.01 M sodium phosphate buffer, pH 7.0, 0.002 M MgCln) was mixed for 15 minutes at 4” on a rot,ator (Scientific Industries, Model 150) with 0.01-0.04 units DNase (Worthington, DSV). The mixture was incubated 3.5 minutes at 37” and boiled for 10 minutes. (2) MicrococcaZ Dh’ase digestion: 0.1 ml of DNA solution (0.5 mg/ml in 0.02 M Tris buffer, pH 8.0, 0.001 M CaC12) was mixed as above with 1 to 4 X lOA4 pg DNase (Worthington, NFCP). The mixture was incubated 6 minutes at 29”, and the reaction stopped by adding 0.1 ml EDTA (0.1 M) and boiling for 10 minutes. For fragments with both 3’- and 5’-hydroxyl termini, samples were treated with micrococcal DNase as in (2), but no EDTA was added and the mixture was boiled for 10 minutes and then 1 unit of alkaline phosphatase (Worthington BAPC) was added. The mixture was then incubated for 30 minutes at 37”. (3) Sonication: 2 ml of DNA solution (0.5 mg/ml in 0.01 M sodium phosphate buffer, pH 7.0) was sonicated at maximum power for 3 X 20 seconds at 0” (MSE, Model 60 with 3/8 in. probe). (4) Shear in French pressure cell: 2 ml of DNA solution, as above, was passed through a French pressure cell three times at 15,000 or 25,000 lbs/in2. (5) Shear in homogenizer: 1 ml of DNA solution, as above, was sheared with a rotating blade in a homogenizer (Virtis 45 with microblade and cup No. 16-111) for 45 minutes at several speeds. The fragmented DNA was sedimented through alkaline sucrose density gradients (11.5 ml, &2070 sucrose gradients containing 0.35 M NaOH,
1 8
DISTANCE FROM TOP (CM) FIG. 1. Homogeneity of fractions from alkaline sucrose gradients. 32P-DNA was degraded with 0.03 units pancreatic DNase and fractionated as described in Table 1. The peak fraction was rerun on a similar gradient. The peak of marker 3HDNA (1) was 9.2 X lo6 mol wt (obtained by shearing 3H-labeled T4 DNA in a virtis homogenizer), and the peak of both gradients corresponded to 1.9 X lo* mol wt. Hydrolyzed DNA---; peak fraction rerun- - -.
0.65 M NaCl) at 40,000 rpm (Spinco SW 41 rotor) were for 17 hours at lo”, and 0.9-ml fractions collected from the bottoms of the tubes. Some sonicat,ed tritiated T4 DNA (S = 5.36; mol. wt. = 1.05 X 105 as determined by boundary sedimentation in alkali on the analytical centrifuge) was included as a marker in each sample. Radioactivity and optical density (at 260 mp) of aliquots were measured. Fractions containing sufficient DNA were diluted and dialyzed against 0.01 M sodium phosphate, pH 7.0, before being used in transformation. These DNA fractions were called short DNA as opposed to the DNA not purposely fragmented which is called long DNA. Long DNA is native DNAwhich has been boiled for 3 minutes and rapidly chilled on ice in order to denature it. Analysis on alkaline sucrose gradients have demonstrated that long DNA is heterogeneous in length and has an average molecular weight, of about 107. Relative molecular weights of all DNA fractions were determined by the relation (1),/D?) = (MI/M$‘.4D (8). In some cases the molecular weights of fractions were also determined by boundary sedimentation in alkali (0.9 A4 NaCI, 0.1 M NaOH) in an analytical ultracentrifuge. The values obtained by the two methods agreed in all cases.
SHORT
177
CObZMUn’ICBTIONS
I.0
g5
0.8
G iii h 0.6 8 i= Q 0.4 z i? i!2 0.2 L I0.5
I .o [DNA]
1.5
2.0
2.5
(pglml)
2
I
I
1
4
6
8
MOLECULAR
WEIGHT
’
IO
II--”
1
100
X lO-5
FIG. 2. Relation between transformation and size of DNA. A. Variation of transformation frequency with DNA concentration and fragment size. Different concentrations of denatured r+DNA of varying sizes were incubated with spheroplasts for 20 minutes at 37”. Then rIIT3 recipient phage was added, and on the mixture was incubated and then titrated for total phage on E. co& B and for T+ transformants E. coli K(x). The ratio r+/rII is called the transformation frequency. DNA was fragmented in a French pressure cell and fractionated in an alkaline sucrose gradient. Long DNA-O; 2.8 X 105 mol wt,-X ; 1.7 X lo6 mol wt-0. B. Relation between molecular weight of DNA fragments and transformation efficiency. For each point, the transformation efficiency was calculated (see text) from a plot of transformation frequency vs. DNA concentration for long DNA and for fragments of relevant size. Recipient phage, ~73, and r+ donor DNA were used. Long DNA-m; Virtis fragments-0 ; pancreatic DNase fragments-e; French pressure cell fragments-A; micrococcal DNase fragments-O ; micrococcal DNase + phosphatase fragments-X ; sonicated fragments-A. reduced to about half its molecular weight. This finding is compatible with previous experiments (5) which showed that linkage of genetic markers is dependent on lengths of donor DNA molecules much larger than those discussed here. Thus, neither exonucleases nor endonucleases in the incubation mixture are sufficient to explain the decrease in transformation efficiency of small denatured fragments of DN,4. The linkage experiments (5) also rule out the role of ettdonucleases within the cell. 1~1keeping with this, unpublished experiments by one of us (E. U.) with an endoE. coli B, ER22 nuclease I negative bacterium, (IO), showed that neither transformation frequency nor linkage of the r7g-ulcp region using long donor DNA is altered in this host,. Base pairing does not seem to be a limit.ing factor since the minimum length necessary for pairing of homologous DNA strands is well below 300 nucleotide residues and has been estimated at 10-20 nucleotide pairs (11). Unpublished inhibition experiments with 1‘73and 480 DNA show that both will inhibit transformation by T+ DNA in an apparent,ly competit.ive manner. 680 DNA inhibits about half as well as ~73 , indicating that at least
part of the competition is not at, the level of base pairing. There are still two obvious possibilities of processes which might reduce transformation efficiency of short fragments more than long ones: (1) intracellular exonucleases which reduce fragments to asize below that required for integration, I.nd (2) size dependence of DNA uptake. (1) As fragments approach the minimum size for integration, less exonucleolytic degradation per fragment would be required to inactivate them. Fragments with different termini are differentially sensitive to exonuclease (12). Therefore we attempted to see if transformation efficiency depended upon phosphorylation of either the 5’or 3’.hydroxyl termini. Figure 2B shows the transformat,ion efficiency of DNA fragments prepared from digests of pancreatic DNase (5’phosphate terminus) or micrococcal DNase (3’phosphate terminus) or microccal DNase plus alkaline phosphatase (3’- and 5’-hydroxyl termini) as well as DNA fragmented in the French pressure cell. The transformation efficiencies are independent. of the termini. These experiments are inconclusive in ruling ollt intracellular exonucle-
17s
SHORT
COMMUNICATIONS
olytic degradation as a factor limiting transformation efficiency of small fragments, however, since the termini of the donor l>NA fragments might be modified either before or after penetrating the spheroplasts. (2) (:llrney (IS) and Cato and Guild (14) have in Pneumococcus, for double-stranded shown DNA, that, large DNA fragments are taken up bet,ter than smaller ones at excess DNA conceni.ration. We have not yet succeeded in measuring directly DN.4 uptake in infected or uninfected spheroplasts and are therefore unable to evaluate the role of DNA uptake in limiting the transformation efficiency of small donor fragments. Guild it al. (15) have discussed a quantit,ative model, relating size of donor DNA to transformation efficiency, which is based on exonuclease action and switching frequencies. Our data fit their model well. We suggest that the reduction of efficiency of t,ransformation with smaller fragments may be related to the intracelllllar kinetics of pairing (16) as well as to exonuclease action and to the probability of switching. We are now trying to determine directly the role of intracellular exonllclease and DNA uptake in controlling the efficiency of donor DNA of various sizes, using both E. coli and ;lerobacfer aerogenes (17) as hosts. SCKNOWLEDGMENTS We are grateful to Dr. J. Eigner for the strain E. coli B EK22 and to Dr. M. Malamy for the phage 480. This work was supported by grants from the National Science Foundation (GB-5923) and the National Institutes of Health (GM-13511). One of [IS (E. G.) is a Career Development Awardee (GM-7567) of the National Institutes of Health. REFERENCES 1. Vas de POL, J. H., VELDHUISEX, G., and COIIEN, J. A., Biochem. Biophys. Arts 48, 417-418 (1961). VELDHUISEN, Cr., POELM.IN, n2. C., and COHEN, J. A., Biochim. Biophys. tlcta 161, 9C109 (1908). 2. VELDHUISEN, CT., and GOLDBERG, E. B., Nucleic Acids, Part B. In “Methods in Enzymology,” (I,. Grossman and K. Mol-
dave, eds.), pp. 858-863. Academic Press, New York, 1968. 3. HERSHEY, A. I)., and BUMSI, E., J. Xol. Biol. 2, 143-152 (1960). 4, GuH.\, A., and SZYB.LLSKI, W., I’irology 34, 608-616 (1968). 5. GOLDRERG, IS. B., Proc. Nat. Acad. Sri. I’.S. 56, 1457-1463 (1966). 6. Jz\~.~~I~~l\~, lt., and GOLDBERG, E. B., Proc. ~Yat. Ad. Sci. U.S., 64, 198-204 (1969). Y. GOLDP.\RB, I>. M., AVDEEV~, A. V., BLINOVA, S. V., SERGEEV~, A. N., and LEVIN.Z, G. A4., Genetika 7, 148-153 (1966). 8. STUDIER, F. W., J. Mol. Biol. 11, 373-390 (1965). 9. HEPPEL, L. A., Science 156, 1451-1455 (19G7). 10. EIGNEH, J., and BLOCK, S., J. Virology 2, 320-326 (1968). il. THOMAS, C. A., JR., In “Progress in Nucleic Acid Research and Molecular Biology” Vol. 5. (J. N. Davidson and W. E. Cohn, eds.), p. 323. Academic Press, New York, 1966. 12. LEHM.IN, I. R., In “Progress in Nucleic Acid Research” Vol. 2. (J. N. Davidson and W. 15. Cohn, eds.), pp. 83-134. Scademic Press, New York, 1963. 13. GURNEY, T., JR., Thesis, Yale University, New Haven (1965). 14. CATO, A. E., JR., Thesis, Duke University, Durham (1966); GUILD, W. R., and CATO, A., Jn., J. Mol. Biol. 3, 157-178 (1968). 16. GUILD, W. R., CATO, A., JR., and LXKS, S., Cold Spring Harbor Symp. of Quanf. Biol. 33, 643 (1968). 16. WETMUR, J. G., and I>.~VIDSON, N., J. Mol. Bid. 31,3*9-370 (1968). lY. W.\IS, A., and GOLDBERG, E. B., Virology 39, 153-161 (1969). HANS ZWEERINK’ ED~.\RD B. GOLDUERG Departmenl of Molecular Biology and Microbiology l’ujfs University School of Medicine Boston, Massachusetts 02111 Accepted January 30, 19YO 1 Present address : Department of Microbiology and Immunology, Duke University Medical Center, Tjurham, N.C. 27706.