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Single-burst analysis for semiconserved phage
DISCUSSION
AND PRELIMINSRY
L. M., Phytopathology 45, 2@216 (1955). 6. BLACK, L. M., BRARKE, M. K., and VATTER, A. E., (Abstract) Phytopathology 42, 3 (1952). 7. BLACK, L. M., BRAKKE, M. K., and VATTER, A. E., Virology 20,120-130 (1963). 8. BRAKKE, M. K., Advan. Virus Res. 7, 193-224 (1960). 9. FUKUSHI, T., SHIKATA, E., and KIMURA, I., Virology 18, 192-205 (1962). 10. MILLONIG, G., J. Biophys. Biochem. Cytol. 11, 736-739 ( 1961). 11. MORGAN, C., GODMAN, G. C., BREITENFELD, P. M., and ROSE, H. M., J. Exptl. Med. 112,373-382 (1960). M. M. MARTIN Department of Plant Pathology and Microbiology 6. BLACK,
Faculty of Agriculture University of Natal Pietermaritzburg, South Accepted December
$1, 1963
Single-Burst
for
Analysis
Africa
Semiconserved
Phage
Studies of Arber and Dussoix (1, 2) on host-induced modifications of bacteriophage h have provided a method for detecting single semiconserved phages. The method consists of modifying phage h by growth on Escherichia coli K12(Pl) and infecting K12 with the modified phage. The work of Arber and Dussoix has demonstrated that among the progeny phage produced by this latter infection only the conserved or semiconserved phage produce plaques when plated on the bacterial strain C6OO(Pl). This method suggests the possibility of determining directly, through a single-burst analysis, whether a bacterium infected with a single phage is capable of producing two semiconserved functional offspring. The “wild type” h of Kaiser (3) was modified by growth on E. coli strain C6OO(Pl) (donated by W. Arber), as described by Arber and Dussoix (1, 2). Bacteria 3110 (a X-sensitive derivative of E. coli K12 obtained from J. Lederberg), were grown to saturation in tryptone broth, centrifuged, and resuspended in 0.01 M MgS04. After 30 minutes’ aeration at 37”, the culture was inoculated with the modified A at a multiplicity of infection of 0.01. After 10 minutes’ adsorption at 37”, the bacteria were treated
REPORTS
649
with anti-h serum and washed repeatedly through a 0.45~ Millipore filter. Assays for unadsorbed phage revealed lessthan 10V3% of the input phage. The bacteria were diluted into “A” medium (4) plus yeast extract, 1 mg/ml, and distributed into small test tubes (by syringe) or into capillary tubes (by direct capillary action) at an average concentration of 3 bacteria per tube. The tubes were incubated at 37” for 2 hours, then spot tested on K12 to identify those that had yielded bursts. Approximately 10% of the contents of each tube were used in the spot tests. The remaining contents of the tubes containing bursts were then plated on C6OO(Pl). In order to determine the total burst size, a number of tubes were plated directly on K12. The burst size found ranged from 30 to 65, with a mean of about 50. Extreme precaution was taken against the possibilities of contamination and reversion of the C6OO(Pl). The maximum efficiency of plating tolerated for unmodified A was 3 x 10-5. Out of a total of 17,610 tubes, 510 yielded bursts. Of these, 85 revealed 1 phage capable of plating on C600 (Pl), 10 revealed 2, and the rest none. At a leakage rate of 3 x 1O-5 and a burst size of 65 (the highest observed), the total expected number of plaques due to leakage would be about 1. The work of Arber and Dussoix (1, 9) suggests that the remaining plaques can be associated with semiconserved or conserved parental phage. With a multiplicity of infection as low as 0.01, the proportion of conserved phage should be negligible (5). The following question arises: In how many of the tubes containing 2 presumably semiconserved phages are the phages of different parental origin, due either to a bacterium receiving more than one input phage, or a tube receiving more than one infected bacterium? Assuming that the distributions of parental phages among bacteria and of bacteria among tubes are Poissonian, the answer is calculated to be 0.27. [See reference (6) for details of the calculation as well as for a discussion of the effects of deviations from the Poisson distribution.] This is to be compared with the observed value of 10. If the assumptions made in the calculation are valid, and if no important
DISCUSSION
650
AND PRELIMINARY
sources of error have been ignored, the hypothesis that one parental phage can give rise to only one semiconserved daughter can be excluded with confidence well over 99%. This result is of particular interest when interpreted in. the light of the experiments previously reported by Fox and Meselson (4). The results of photoinactivation studies of 5-bromodeoxyuridine-labeled semiconserved phage suggested that there was some vital function of h DNA which could be performed by only one strand, either unique or randomly chosen. The results reported here suggest that the vital function in question is not replication. A likely candidate might be the synthesis of some early enzyme. BCKNOWLEDGMENT This work was supported by a grant from the National Science Foundat.ion. REFERENCES W., and DCSSOIX, D., J. Mol. Biol. 5, 1% 36 (1962). 2. DUSSOIX, D., and ARBER, W., J. Mol. Viol. 5, 3749 (1962). 3. KAISER, D., ViroIogy 3,42-61 c.1957). 4. Fox, E., and MESELSON, M., J. Mol. Biol. 7, 5831. ARBER,
589 ( 1963 )
M., and WEIGLE, J., Proc. N&Z. Acad. Sci. U.S. 47,857-868 (1961). 6. Fox, E. Ph.D. Dissertation, “Photoinactivation and the Expression of Genetic Information in Bacteriophage x,” Harvard University, 1963. EVELYN Fox KELLER Courant Institute of Mathematical Sciences 5. MESELSON,
New York New York, Accepted
Recombination
University New York
January 18, 1964
between
Coliphages
X and
980
We have observed genetic recombination between coliphage h and the serologically unrelated coliphage $30 (1) . Preliminary characterization of recombinants for the immunity and host-range markers indicates that the locus governing the prophage attachment site is linked genetically to the host-range locus, and that immunity determines neither the location of prophage at-
REPORTS
tachment nor the specificity of specialized transduction. Crosses.After either mixed infection and lytic growth, or induction of doubly lysogenie strains with ultraviolet light, recombinants with the immunity of one of the parents and the host range of the other, i.e., Ahso and @Ohi , are isolated by plating on the appropriate bacterial indicator strains. The frequency of this recombinant-pair is of the order of 0.1 to 1.0% output. Density gradient centrifugation. The densities of h and 480 are, respectively, 1.508 (S?) and 1.495. After passage of the cross output through sensitive bacteria to eliminate phenotypic mixing of the host range character, phages of each of the immunity-host range recombinant types are found in cesium chloride density gradients at two different densities; hhsoat 1.490 and 1.495, and @Ohx at 1.505 and 1.510 (all densities + 0.001). The purified rocombinants retain their characteristic densities upon subsequent growth; however, those of the cp80hx type give rise in addition to phages of the same type but lighter in density. Prophage attachment. Prophages h and 480 are located on the genetic map of Escherichia coli near galactose (3) and tryptophan (I), respectively. Phage Pl cotransduces x with galactose at a frequency of S%, and 480 with tryptophan at a frequency of 42%. The recombinant prophage Ahso, density 1.495, is localized in the tryptophan region by time of entry (about 25 minutes after the leucine marker), in HfrHayes-Fbacterial crosses.Furthermore, cotransduction by phage PI of the h immunity of this prophage occurs with tryptophan (42%), but not with galactose (less than 0.5%). This prophage occupies the $30 site on the E. coli chromosome, and from specialized transduction data (below), we infer that the same is true for prophage hhso! density 1.490. Hence attachment specificity, even for the portion of hhsooriginating from the h parent, is linked genetically to the host range rather than to the immunity character. The same has been found by *Jacob (4) for recombination between h and coliphage 21, and independently in this laboratory by Brenner (personal communication) ; how-
AND PRELIMINSRY
L. M., Phytopathology 45, 2@216 (1955). 6. BLACK, L. M., BRARKE, M. K., and VATTER, A. E., (Abstract) Phytopathology 42, 3 (1952). 7. BLACK, L. M., BRAKKE, M. K., and VATTER, A. E., Virology 20,120-130 (1963). 8. BRAKKE, M. K., Advan. Virus Res. 7, 193-224 (1960). 9. FUKUSHI, T., SHIKATA, E., and KIMURA, I., Virology 18, 192-205 (1962). 10. MILLONIG, G., J. Biophys. Biochem. Cytol. 11, 736-739 ( 1961). 11. MORGAN, C., GODMAN, G. C., BREITENFELD, P. M., and ROSE, H. M., J. Exptl. Med. 112,373-382 (1960). M. M. MARTIN Department of Plant Pathology and Microbiology 6. BLACK,
Faculty of Agriculture University of Natal Pietermaritzburg, South Accepted December
$1, 1963
Single-Burst
for
Analysis
Africa
Semiconserved
Phage
Studies of Arber and Dussoix (1, 2) on host-induced modifications of bacteriophage h have provided a method for detecting single semiconserved phages. The method consists of modifying phage h by growth on Escherichia coli K12(Pl) and infecting K12 with the modified phage. The work of Arber and Dussoix has demonstrated that among the progeny phage produced by this latter infection only the conserved or semiconserved phage produce plaques when plated on the bacterial strain C6OO(Pl). This method suggests the possibility of determining directly, through a single-burst analysis, whether a bacterium infected with a single phage is capable of producing two semiconserved functional offspring. The “wild type” h of Kaiser (3) was modified by growth on E. coli strain C6OO(Pl) (donated by W. Arber), as described by Arber and Dussoix (1, 2). Bacteria 3110 (a X-sensitive derivative of E. coli K12 obtained from J. Lederberg), were grown to saturation in tryptone broth, centrifuged, and resuspended in 0.01 M MgS04. After 30 minutes’ aeration at 37”, the culture was inoculated with the modified A at a multiplicity of infection of 0.01. After 10 minutes’ adsorption at 37”, the bacteria were treated
REPORTS
649
with anti-h serum and washed repeatedly through a 0.45~ Millipore filter. Assays for unadsorbed phage revealed lessthan 10V3% of the input phage. The bacteria were diluted into “A” medium (4) plus yeast extract, 1 mg/ml, and distributed into small test tubes (by syringe) or into capillary tubes (by direct capillary action) at an average concentration of 3 bacteria per tube. The tubes were incubated at 37” for 2 hours, then spot tested on K12 to identify those that had yielded bursts. Approximately 10% of the contents of each tube were used in the spot tests. The remaining contents of the tubes containing bursts were then plated on C6OO(Pl). In order to determine the total burst size, a number of tubes were plated directly on K12. The burst size found ranged from 30 to 65, with a mean of about 50. Extreme precaution was taken against the possibilities of contamination and reversion of the C6OO(Pl). The maximum efficiency of plating tolerated for unmodified A was 3 x 10-5. Out of a total of 17,610 tubes, 510 yielded bursts. Of these, 85 revealed 1 phage capable of plating on C600 (Pl), 10 revealed 2, and the rest none. At a leakage rate of 3 x 1O-5 and a burst size of 65 (the highest observed), the total expected number of plaques due to leakage would be about 1. The work of Arber and Dussoix (1, 9) suggests that the remaining plaques can be associated with semiconserved or conserved parental phage. With a multiplicity of infection as low as 0.01, the proportion of conserved phage should be negligible (5). The following question arises: In how many of the tubes containing 2 presumably semiconserved phages are the phages of different parental origin, due either to a bacterium receiving more than one input phage, or a tube receiving more than one infected bacterium? Assuming that the distributions of parental phages among bacteria and of bacteria among tubes are Poissonian, the answer is calculated to be 0.27. [See reference (6) for details of the calculation as well as for a discussion of the effects of deviations from the Poisson distribution.] This is to be compared with the observed value of 10. If the assumptions made in the calculation are valid, and if no important
DISCUSSION
650
AND PRELIMINARY
sources of error have been ignored, the hypothesis that one parental phage can give rise to only one semiconserved daughter can be excluded with confidence well over 99%. This result is of particular interest when interpreted in. the light of the experiments previously reported by Fox and Meselson (4). The results of photoinactivation studies of 5-bromodeoxyuridine-labeled semiconserved phage suggested that there was some vital function of h DNA which could be performed by only one strand, either unique or randomly chosen. The results reported here suggest that the vital function in question is not replication. A likely candidate might be the synthesis of some early enzyme. BCKNOWLEDGMENT This work was supported by a grant from the National Science Foundat.ion. REFERENCES W., and DCSSOIX, D., J. Mol. Biol. 5, 1% 36 (1962). 2. DUSSOIX, D., and ARBER, W., J. Mol. Viol. 5, 3749 (1962). 3. KAISER, D., ViroIogy 3,42-61 c.1957). 4. Fox, E., and MESELSON, M., J. Mol. Biol. 7, 5831. ARBER,
589 ( 1963 )
M., and WEIGLE, J., Proc. N&Z. Acad. Sci. U.S. 47,857-868 (1961). 6. Fox, E. Ph.D. Dissertation, “Photoinactivation and the Expression of Genetic Information in Bacteriophage x,” Harvard University, 1963. EVELYN Fox KELLER Courant Institute of Mathematical Sciences 5. MESELSON,
New York New York, Accepted
Recombination
University New York
January 18, 1964
between
Coliphages
X and
980
We have observed genetic recombination between coliphage h and the serologically unrelated coliphage $30 (1) . Preliminary characterization of recombinants for the immunity and host-range markers indicates that the locus governing the prophage attachment site is linked genetically to the host-range locus, and that immunity determines neither the location of prophage at-
REPORTS
tachment nor the specificity of specialized transduction. Crosses.After either mixed infection and lytic growth, or induction of doubly lysogenie strains with ultraviolet light, recombinants with the immunity of one of the parents and the host range of the other, i.e., Ahso and @Ohi , are isolated by plating on the appropriate bacterial indicator strains. The frequency of this recombinant-pair is of the order of 0.1 to 1.0% output. Density gradient centrifugation. The densities of h and 480 are, respectively, 1.508 (S?) and 1.495. After passage of the cross output through sensitive bacteria to eliminate phenotypic mixing of the host range character, phages of each of the immunity-host range recombinant types are found in cesium chloride density gradients at two different densities; hhsoat 1.490 and 1.495, and @Ohx at 1.505 and 1.510 (all densities + 0.001). The purified rocombinants retain their characteristic densities upon subsequent growth; however, those of the cp80hx type give rise in addition to phages of the same type but lighter in density. Prophage attachment. Prophages h and 480 are located on the genetic map of Escherichia coli near galactose (3) and tryptophan (I), respectively. Phage Pl cotransduces x with galactose at a frequency of S%, and 480 with tryptophan at a frequency of 42%. The recombinant prophage Ahso, density 1.495, is localized in the tryptophan region by time of entry (about 25 minutes after the leucine marker), in HfrHayes-Fbacterial crosses.Furthermore, cotransduction by phage PI of the h immunity of this prophage occurs with tryptophan (42%), but not with galactose (less than 0.5%). This prophage occupies the $30 site on the E. coli chromosome, and from specialized transduction data (below), we infer that the same is true for prophage hhso! density 1.490. Hence attachment specificity, even for the portion of hhsooriginating from the h parent, is linked genetically to the host range rather than to the immunity character. The same has been found by *Jacob (4) for recombination between h and coliphage 21, and independently in this laboratory by Brenner (personal communication) ; how-