- Email: [email protected]
Physical analysis of the genomes of hybrid phages between phage P1 and plasmid p15B
J. Mol. Biol. (1983) 165, 191-195
Physical Analysis of the Genomes of Hybrid Phages Between Phage P1 and Plasmid p15B The genomes of three plaque-forming recombinant phages between phage P1 and plasmid pl5B were characterized by restriction cleavage analysis and electron microscopic heteroduplex studies. The structure of all three Pl-15 hybrid genomes differs from that of Pl DNA in the res mad region coding for restriction and modification systems EcoPl5 and EcoPl, respectively. Pl-15 hybrid 2 shows an additional major difference to Pl around the site of the residential IS/element of P1 and it does not carry an IS/ in its genome.
In cells lysogenic for the temperate bacteriophage P1 the prophage replicates as an autonomous plasmid of 90 k b t (Ikeda & Tomizawa, 1968). As special structural features it contains one copy of the insertion element I S / (Iida el al., 1978) and the so-called C-loop, a 3 kb long invertible segment flanked by 0"6 kb inverted repeats (Lee et al., 1974; Yun & Vapnek, 1977). The P1 genome also carries the genetic information for the EcoP1 restriction and modification system (Lederberg, 1957; Arber & Dussoix, 1962). Plasmid pl5B found in Escherichia coli 15T- has extensive sequence homology to P1 DNA (Ikeda et al., 1970). Together with phages P1 and P7 it belongs to incompatibility group Y (Hedges et al., 1975). This plasmid is considered to be a Pl-related defective prophage. However, it codes for its own restriction and modification system, EcoP15 (Arber & Wauters-Willems, 1970), and cells carrying pl5B are not P1 immune. During growth of P1 in cells containing plasmid pl5B, recombination between the two genomes occurs. Arber & Wauters-Willems (1970) have isolated plaque-forming P l - 1 5 hybrid phages, which specify P1 virions and P1 immunity, but EcoP15 restriction and modification functions. We report here on physical differences in the genomes of three such P l - 1 5 hybrid phages. Their restriction cleavage maps were constructed (Fig. 1) by the methodology used to establish the maps of phage P1 and P7 DNA (Bgchi & Arber, 1977 ; Iida & Arber, 1979). The restriction cleavage patterns of DNA from P l - 1 5 hybrids l, 2 and 3 are quite similar to that of DNA from P1. However, there is a uniform, marked difference in the BgIII sites within the res mod region coding for EcoP15 and EcoP1, respectively. These functions are determined by the respective B a m H I - 4 DNA fragments (Mural etal., 1979; Iida etal., 1983, accompanying paper), which are indistinguishable in size for the four genomes compared. Redigestion of B a m H I - 4 of all three P l - 1 5 hybrids with BglII revealed four fragments, of which two were identified as end fragments not seen in BglII single digestion, while a 2"4 kb and a 0"2 kb fragment were also obtained in BglII digestion of the whole P l - 1 5 DNA. The relative order of these internal fragments t Abbreviation used: kb. l0 a base-pairs. 0022-2836/83/090191-05 $03.00/0
19l
© 1983 Academic Press Inc. (London}Ltd.
192
J . M E Y E R . S. I I D A A N D W. A R B E R poc c l Iox P r e s m o d ./'SI
C-loop
poc c l lox P
P1 5kb
l, P1-15
I II
l
Ill
i
II
I
ag,
Born HI
hybrid 1
hybrid 2 hybrid 3
A~
A
vv
0
N~
Fit:. I. EcoRI, B.q/II, BnmHI and PstI restriction cleavage ]naps of the genomes of PI and P I - P 5 hybrid phages. The Pl maps are redrawn fl'om the published ]naps (B~chi & Arber, 1977: Iida & Arber. 1979). The long arrow above the line indicates the PstI site within the I S / o f PI. the short bat~ above the line indicate EcoRI sites, those below the line B,q/II sites, and the long bars below the line BamHI sites. The numbers 3. 4 and 9 indicate B¢ImHI-3, BamH]-4 and BglII-9 fragments, respectively. The markers above the PI restriction map are referred to in the text, except for pac (start of DNA packaging; BiCchi & Arber. 1977), c/(repressor; Scott, 1970) and IoxP (locus of crossing over; Sternberg et al., 1980). In the Pl-15 maps only alterations relative to the PI maps are shown: open triangles denote sites missing, filled triangles indicate new sites, those above the line in the EcoRI maps, those below the line in the BgllI maps. The open rhombus in the P l - ] 5 hybrid 2 map gives the missing PstI site. The EcoRI maps of the Pl-15 hybrids include only changes in the large fl'agments. Note that the EcoRI site missing in P l - ] 5 hybrid 2 is also absent in phage P7, and the new EcoRI site on Pl-15 hybrid 2 is close to or identical to that described for P7 (Iida & Arber, 1979). The kb scale refers to the Pl genome, size difference of Pl-15 hybrids is discussed in the text and the Pl-15 hybrid 2 map is not entirely drawn to scale.
as shown in Figure 1 was inferred from the analysis of a P l - 1 5 hybrid 2 derivative carrying a tetracycline resistance transposon TnlO (Kleckner et al., 1975) insertion in this region (data not shown). The DNA sequence relationship between P1 and the PI-15 hybrids was also studied by an electron microscope heteroduplex analysis. DNA of a P l - 1 5 hybrid 2 derivative containing TnlO within fragment BglII-9 was hybridized to Pl DNA as described (Meyer & Iida, 1979). The snap-back structures of TnlO and, if present, of the C-loop allowed propel" orientation of heteroduplex molecules and provided intramolecular length standards. There are two regions in the genomes with marked differences: one is a 1"2 kb segment of non-homology, called Modloop (Fig. 2(a) and (b)), in which the altered BglII sites of the res mod region locate. This alteration is common to all three P l - 1 5 hybrid genomes. Detailed mapping of this segment with respect to the modification and restriction functions is presented by Iida el al. (1983) (accompanying paper). The other gross sequence difference from P1 is revealed by P I - I 5 hybrid 2 only, and locates in the region of P1 carrying the IS/element. Heteroduplex molecules between P l - 1 5 hybrid 2 and P1 DNA show in this region two deletion/insertion loops of 0"74kb (loop Bi) and 1-77 kb (loop B2) separated by a homologous segment of 0"80 kb (Fig. 2(a), (b) and (c)). The same loops are also seen in heteroduplex mo|ecules between phage P1 DNA and plasmid pl5B (M. Streiff & J. Meyer, unpublished results). The B1 loop and the homologous segment between loops B1 and B2 were also observed in heteroduplex molecules between the
LETTERS TO THE EDITOR ~
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Fro. 2. (a) Electron micrograph of a heteroduplex molecule between a Pl-15 hybrid 2 derivative carrying TnlO within fragment BqlII-9 and Pl DNA illustrating the genome differences (loops Mod, Bl and B2) discussed in the text. The bar represents 1 kb of DNA. (b) Diagrammatic representation of the heteroduplex molecule shown in (a) with measurements in kb based on 15 molecules. (c) and (d) Sequence relationship between P1, Pl-15 hybrid 2 and P7 in the segments corresponding to the IS/ region of P1. The heteroduplex molecule shown was made between (c) P1 and Pl-15 hybrid 2 and (d) Pl-15 hybrid 2 and P7. See the text for interpretation.
genomes of P l - 1 5 hybrid 2 and a P1 d e r i v a t i v e with a : D N A s e g m e n t inverted a d j a c e n t to the right of I S / (Iida & Meyer, 1979). Therefore, the B2 loop m u s t represent P1 D N A and contains the I S / e l e m e n t plus material i m m e d i a t e l y to its right. This conclusion agrees well with the distance of 7"8 kb measured between this loop and the C-loop, which is a b o u t 1-2 kb shorter t h a n the distance between I S / a n d the C-loop in P1 D N A (Iida et al., 1978). F u r t h e r m o r e , f r a g m e n t B a m H I 3 of P1-15 hybrid 2 lacks the P s t I site (Fig. 1), and neither this f r a g m e n t nor a n y other region of the genome of this phage hybridizes to a 32P-labelled A:: I S / probe, while the corresponding f r a g m e n t B a m H I - 3 of P1 a n d of P l - 1 5 hybrids 1 and 3 do (data not shown). The B2 loop can thus clearly be assigned to the P1 genome and includes I S / . Because the B a m H I - 3 f r a g m e n t of P l - 1 5 hybrid 2 is only a b o u t 1 kb shorter t h a n t h a t of P1 DNA, the two loops B1 and B2 seen in the heteroduplex c a n n o t represent material carried on the same D N A strand. The B1 loop m u s t therefore be due to an insertion carried in P I - I 5 h y b r i d 2 DNA. W h e t h e r it represents an unidentified IS element remains to be determined. Absence of an I S / e l e m e n t and a shorter segment non-homologous to 1)1 D N A in this p a r t of the genome are also features of the P l - r e l a t e d phage P7 (Iida & Arber, 1979). Nevertheless, P7 and 1)1-15 hybrid 2 are not identical in this region, H e t e r o d u p l e x molecules between P l - 1 5 h y b r i d 2 : : T n l O and P7 D N A clearly d o c u m e n t non-homologous segments of 1-8 kb on one side and 2.2 kb on the other (Fig. 2(d)). The 2"2 kb segment m o s t likely represents the P7 p a r t described as the
194
J. MEYER, S. IIDA AND W. ARBER
2"4 kb loop B by Yun & Vapnek (1977). The 1"8 kb part probably represents P115 hybrid 2 DNA and corresponds to the 0-74kb insertion plus the 0.80kb segment homologous to P1. In addition, all the non-homologous regions between P1 and P7 DNA, namely the segments A, D, E, F, G defined by Yun & Vapnek (1977) and the two additional segments described by Chow et al. (1978), together with the pl5B substitution in the res mod region (Iida el al. (1983), accompanying paper), are also observed in heteroduplexes between Pl-15 hybrid 2 and P7 DNA (data not shown). Thus, both P7 and P1-15 hybrid 2 lack the IS/ element, but have different substitutions in this part of the genome, and neither of those affect phage propagation and lysogenization. However, the presence of an I S / e l e m e n t on a phage genome may facilitate the formation of cointegrate structures with other plasmids or the bacterial chromosome which eventually might lead to an increase in transduction frequency (Iida, 1980; Iida & Arber, 1980; Iida etal., 1980,1981). The expert technical assistance of Margaretha St~lhammar-Carlemalm and Solveig Schrickel is gratefully acknowledged. We thank Tom Bickle for discussions and reading the manuscript. This work was supported by grant no. 3.479.79 from the Swiss National Science Foundation. Department of Microbiology Biozentrum of the University of Basel Klingelbergstrasse 70 CH-4056 Basel, Switzerland
Jt~RG MEYER SHIGERU IIDA W E R N E R ARBER
Received 21 September 1982 REFERENCES Arber, W. & Dussoix, D. (1962). J. Mol. Biol. 5, 18-36. Arber, W. & Wauters-Willems, D. (1970). Mol. Gen. Genet. 108, 203-217. B~ehi, B. & Arber, W. (1977). Mol. Gen. Genet. 153, 311-324. Chow, L.-T., Broker, T. R., Kahmann, R. & Kamp, D. (1978). In Microbiology 1978 (Schlessinger, D., ed.), pp. 55-56, American Society for Microbiology, Washington, D.C. Hedges, R. W., Jacob, A. E., Barth, P. T. & Giinter, N. J. (1975). Mol. Gen. Genet. 141, 263-267. Iida, S. (1980). Plasmid, 3, 278-290. Iida, S. & Arber, W. (1979). Mol. Gen. Genet. 173, 249-261. Iida, S. & Arber, W. (1980). Mol. Gen. Genet. 177, 261-270. Iida, S. & Meyer, J. (1979). Experientia, 35, 968. Iida, S., Meyer, J. & Arber, W. (1978). Plasmid, 1,357-365. Iida, S., Meyer, J. & Arber, W. (1980). Cold Spring Harbor Syrup. Quant. Biol. 45, 27-43. Iida, S., Meyer, J. & Arber, W. (1981). Mol. Gen. Genet. 184, 1-10. Iida, S., Meyer, J., B~ichi, B., St~lhammar-Carlema]m, M., Schrickel, S., Bickle, T.A. & Arber, W. (1983). J. Mol. Biol. 165, 1-18. Ikeda, H. & Tomizawa, J. (1968). Cold Spring Harbor Syrup. Quant. Biol. 33, 791-798. Ikeda, H., Inuzuka, M. & Tomizawa, J. (1970). J. Mol. Biol. 50, 457-470. Kleckner, N., Chan, R. K., Tye, B. K. & Botstein, D. (1975). J. Mol. Biol. 97, 561-575. Lederberg, S. (1957). P~rology, 3, 496-513. Lee, H. J., Ohtsubo, E., Deonier, R. C. & Davidson, N. (1974). J. Mol. Biol. 89, 585-597.
LETTERS TO THE EDITOR
195
Meyer, J. & Iida, S. (1979). Mol. Gen. Genet. 176, 209-219. Mural, R. J., Chesney, R. H., Vapnek, D., Kraft, M. M. & Scott, J. R. (1979). I4rology, 93, 387-397. Scott, J. R. (1970). Virology, 41, 66-71. Sternberg, N., Hamilton, D., Austin, S., Yarmolinsky, M. & Hoess, R. (1980). Cold Spring Harbor Symp. Quant. Biol. 45, 297-309. Yun, T. & Vapnek, D. (1977). Firology, 77, 376-385.
Edited by S. Brenner
Physical Analysis of the Genomes of Hybrid Phages Between Phage P1 and Plasmid p15B The genomes of three plaque-forming recombinant phages between phage P1 and plasmid pl5B were characterized by restriction cleavage analysis and electron microscopic heteroduplex studies. The structure of all three Pl-15 hybrid genomes differs from that of Pl DNA in the res mad region coding for restriction and modification systems EcoPl5 and EcoPl, respectively. Pl-15 hybrid 2 shows an additional major difference to Pl around the site of the residential IS/element of P1 and it does not carry an IS/ in its genome.
In cells lysogenic for the temperate bacteriophage P1 the prophage replicates as an autonomous plasmid of 90 k b t (Ikeda & Tomizawa, 1968). As special structural features it contains one copy of the insertion element I S / (Iida el al., 1978) and the so-called C-loop, a 3 kb long invertible segment flanked by 0"6 kb inverted repeats (Lee et al., 1974; Yun & Vapnek, 1977). The P1 genome also carries the genetic information for the EcoP1 restriction and modification system (Lederberg, 1957; Arber & Dussoix, 1962). Plasmid pl5B found in Escherichia coli 15T- has extensive sequence homology to P1 DNA (Ikeda et al., 1970). Together with phages P1 and P7 it belongs to incompatibility group Y (Hedges et al., 1975). This plasmid is considered to be a Pl-related defective prophage. However, it codes for its own restriction and modification system, EcoP15 (Arber & Wauters-Willems, 1970), and cells carrying pl5B are not P1 immune. During growth of P1 in cells containing plasmid pl5B, recombination between the two genomes occurs. Arber & Wauters-Willems (1970) have isolated plaque-forming P l - 1 5 hybrid phages, which specify P1 virions and P1 immunity, but EcoP15 restriction and modification functions. We report here on physical differences in the genomes of three such P l - 1 5 hybrid phages. Their restriction cleavage maps were constructed (Fig. 1) by the methodology used to establish the maps of phage P1 and P7 DNA (Bgchi & Arber, 1977 ; Iida & Arber, 1979). The restriction cleavage patterns of DNA from P l - 1 5 hybrids l, 2 and 3 are quite similar to that of DNA from P1. However, there is a uniform, marked difference in the BgIII sites within the res mod region coding for EcoP15 and EcoP1, respectively. These functions are determined by the respective B a m H I - 4 DNA fragments (Mural etal., 1979; Iida etal., 1983, accompanying paper), which are indistinguishable in size for the four genomes compared. Redigestion of B a m H I - 4 of all three P l - 1 5 hybrids with BglII revealed four fragments, of which two were identified as end fragments not seen in BglII single digestion, while a 2"4 kb and a 0"2 kb fragment were also obtained in BglII digestion of the whole P l - 1 5 DNA. The relative order of these internal fragments t Abbreviation used: kb. l0 a base-pairs. 0022-2836/83/090191-05 $03.00/0
19l
© 1983 Academic Press Inc. (London}Ltd.
192
J . M E Y E R . S. I I D A A N D W. A R B E R poc c l Iox P r e s m o d ./'SI
C-loop
poc c l lox P
P1 5kb
l, P1-15
I II
l
Ill
i
II
I
ag,
Born HI
hybrid 1
hybrid 2 hybrid 3
A~
A
vv
0
N~
Fit:. I. EcoRI, B.q/II, BnmHI and PstI restriction cleavage ]naps of the genomes of PI and P I - P 5 hybrid phages. The Pl maps are redrawn fl'om the published ]naps (B~chi & Arber, 1977: Iida & Arber. 1979). The long arrow above the line indicates the PstI site within the I S / o f PI. the short bat~ above the line indicate EcoRI sites, those below the line B,q/II sites, and the long bars below the line BamHI sites. The numbers 3. 4 and 9 indicate B¢ImHI-3, BamH]-4 and BglII-9 fragments, respectively. The markers above the PI restriction map are referred to in the text, except for pac (start of DNA packaging; BiCchi & Arber. 1977), c/(repressor; Scott, 1970) and IoxP (locus of crossing over; Sternberg et al., 1980). In the Pl-15 maps only alterations relative to the PI maps are shown: open triangles denote sites missing, filled triangles indicate new sites, those above the line in the EcoRI maps, those below the line in the BgllI maps. The open rhombus in the P l - ] 5 hybrid 2 map gives the missing PstI site. The EcoRI maps of the Pl-15 hybrids include only changes in the large fl'agments. Note that the EcoRI site missing in P l - ] 5 hybrid 2 is also absent in phage P7, and the new EcoRI site on Pl-15 hybrid 2 is close to or identical to that described for P7 (Iida & Arber, 1979). The kb scale refers to the Pl genome, size difference of Pl-15 hybrids is discussed in the text and the Pl-15 hybrid 2 map is not entirely drawn to scale.
as shown in Figure 1 was inferred from the analysis of a P l - 1 5 hybrid 2 derivative carrying a tetracycline resistance transposon TnlO (Kleckner et al., 1975) insertion in this region (data not shown). The DNA sequence relationship between P1 and the PI-15 hybrids was also studied by an electron microscope heteroduplex analysis. DNA of a P l - 1 5 hybrid 2 derivative containing TnlO within fragment BglII-9 was hybridized to Pl DNA as described (Meyer & Iida, 1979). The snap-back structures of TnlO and, if present, of the C-loop allowed propel" orientation of heteroduplex molecules and provided intramolecular length standards. There are two regions in the genomes with marked differences: one is a 1"2 kb segment of non-homology, called Modloop (Fig. 2(a) and (b)), in which the altered BglII sites of the res mod region locate. This alteration is common to all three P l - 1 5 hybrid genomes. Detailed mapping of this segment with respect to the modification and restriction functions is presented by Iida el al. (1983) (accompanying paper). The other gross sequence difference from P1 is revealed by P I - I 5 hybrid 2 only, and locates in the region of P1 carrying the IS/element. Heteroduplex molecules between P l - 1 5 hybrid 2 and P1 DNA show in this region two deletion/insertion loops of 0"74kb (loop Bi) and 1-77 kb (loop B2) separated by a homologous segment of 0"80 kb (Fig. 2(a), (b) and (c)). The same loops are also seen in heteroduplex mo|ecules between phage P1 DNA and plasmid pl5B (M. Streiff & J. Meyer, unpublished results). The B1 loop and the homologous segment between loops B1 and B2 were also observed in heteroduplex molecules between the
LETTERS TO THE EDITOR ~
~,.~ •
~ ~.
193
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loop B1
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C- loop
Fro. 2. (a) Electron micrograph of a heteroduplex molecule between a Pl-15 hybrid 2 derivative carrying TnlO within fragment BqlII-9 and Pl DNA illustrating the genome differences (loops Mod, Bl and B2) discussed in the text. The bar represents 1 kb of DNA. (b) Diagrammatic representation of the heteroduplex molecule shown in (a) with measurements in kb based on 15 molecules. (c) and (d) Sequence relationship between P1, Pl-15 hybrid 2 and P7 in the segments corresponding to the IS/ region of P1. The heteroduplex molecule shown was made between (c) P1 and Pl-15 hybrid 2 and (d) Pl-15 hybrid 2 and P7. See the text for interpretation.
genomes of P l - 1 5 hybrid 2 and a P1 d e r i v a t i v e with a : D N A s e g m e n t inverted a d j a c e n t to the right of I S / (Iida & Meyer, 1979). Therefore, the B2 loop m u s t represent P1 D N A and contains the I S / e l e m e n t plus material i m m e d i a t e l y to its right. This conclusion agrees well with the distance of 7"8 kb measured between this loop and the C-loop, which is a b o u t 1-2 kb shorter t h a n the distance between I S / a n d the C-loop in P1 D N A (Iida et al., 1978). F u r t h e r m o r e , f r a g m e n t B a m H I 3 of P1-15 hybrid 2 lacks the P s t I site (Fig. 1), and neither this f r a g m e n t nor a n y other region of the genome of this phage hybridizes to a 32P-labelled A:: I S / probe, while the corresponding f r a g m e n t B a m H I - 3 of P1 a n d of P l - 1 5 hybrids 1 and 3 do (data not shown). The B2 loop can thus clearly be assigned to the P1 genome and includes I S / . Because the B a m H I - 3 f r a g m e n t of P l - 1 5 hybrid 2 is only a b o u t 1 kb shorter t h a n t h a t of P1 DNA, the two loops B1 and B2 seen in the heteroduplex c a n n o t represent material carried on the same D N A strand. The B1 loop m u s t therefore be due to an insertion carried in P I - I 5 h y b r i d 2 DNA. W h e t h e r it represents an unidentified IS element remains to be determined. Absence of an I S / e l e m e n t and a shorter segment non-homologous to 1)1 D N A in this p a r t of the genome are also features of the P l - r e l a t e d phage P7 (Iida & Arber, 1979). Nevertheless, P7 and 1)1-15 hybrid 2 are not identical in this region, H e t e r o d u p l e x molecules between P l - 1 5 h y b r i d 2 : : T n l O and P7 D N A clearly d o c u m e n t non-homologous segments of 1-8 kb on one side and 2.2 kb on the other (Fig. 2(d)). The 2"2 kb segment m o s t likely represents the P7 p a r t described as the
194
J. MEYER, S. IIDA AND W. ARBER
2"4 kb loop B by Yun & Vapnek (1977). The 1"8 kb part probably represents P115 hybrid 2 DNA and corresponds to the 0-74kb insertion plus the 0.80kb segment homologous to P1. In addition, all the non-homologous regions between P1 and P7 DNA, namely the segments A, D, E, F, G defined by Yun & Vapnek (1977) and the two additional segments described by Chow et al. (1978), together with the pl5B substitution in the res mod region (Iida el al. (1983), accompanying paper), are also observed in heteroduplexes between Pl-15 hybrid 2 and P7 DNA (data not shown). Thus, both P7 and P1-15 hybrid 2 lack the IS/ element, but have different substitutions in this part of the genome, and neither of those affect phage propagation and lysogenization. However, the presence of an I S / e l e m e n t on a phage genome may facilitate the formation of cointegrate structures with other plasmids or the bacterial chromosome which eventually might lead to an increase in transduction frequency (Iida, 1980; Iida & Arber, 1980; Iida etal., 1980,1981). The expert technical assistance of Margaretha St~lhammar-Carlemalm and Solveig Schrickel is gratefully acknowledged. We thank Tom Bickle for discussions and reading the manuscript. This work was supported by grant no. 3.479.79 from the Swiss National Science Foundation. Department of Microbiology Biozentrum of the University of Basel Klingelbergstrasse 70 CH-4056 Basel, Switzerland
Jt~RG MEYER SHIGERU IIDA W E R N E R ARBER
Received 21 September 1982 REFERENCES Arber, W. & Dussoix, D. (1962). J. Mol. Biol. 5, 18-36. Arber, W. & Wauters-Willems, D. (1970). Mol. Gen. Genet. 108, 203-217. B~ehi, B. & Arber, W. (1977). Mol. Gen. Genet. 153, 311-324. Chow, L.-T., Broker, T. R., Kahmann, R. & Kamp, D. (1978). In Microbiology 1978 (Schlessinger, D., ed.), pp. 55-56, American Society for Microbiology, Washington, D.C. Hedges, R. W., Jacob, A. E., Barth, P. T. & Giinter, N. J. (1975). Mol. Gen. Genet. 141, 263-267. Iida, S. (1980). Plasmid, 3, 278-290. Iida, S. & Arber, W. (1979). Mol. Gen. Genet. 173, 249-261. Iida, S. & Arber, W. (1980). Mol. Gen. Genet. 177, 261-270. Iida, S. & Meyer, J. (1979). Experientia, 35, 968. Iida, S., Meyer, J. & Arber, W. (1978). Plasmid, 1,357-365. Iida, S., Meyer, J. & Arber, W. (1980). Cold Spring Harbor Syrup. Quant. Biol. 45, 27-43. Iida, S., Meyer, J. & Arber, W. (1981). Mol. Gen. Genet. 184, 1-10. Iida, S., Meyer, J., B~ichi, B., St~lhammar-Carlema]m, M., Schrickel, S., Bickle, T.A. & Arber, W. (1983). J. Mol. Biol. 165, 1-18. Ikeda, H. & Tomizawa, J. (1968). Cold Spring Harbor Syrup. Quant. Biol. 33, 791-798. Ikeda, H., Inuzuka, M. & Tomizawa, J. (1970). J. Mol. Biol. 50, 457-470. Kleckner, N., Chan, R. K., Tye, B. K. & Botstein, D. (1975). J. Mol. Biol. 97, 561-575. Lederberg, S. (1957). P~rology, 3, 496-513. Lee, H. J., Ohtsubo, E., Deonier, R. C. & Davidson, N. (1974). J. Mol. Biol. 89, 585-597.
LETTERS TO THE EDITOR
195
Meyer, J. & Iida, S. (1979). Mol. Gen. Genet. 176, 209-219. Mural, R. J., Chesney, R. H., Vapnek, D., Kraft, M. M. & Scott, J. R. (1979). I4rology, 93, 387-397. Scott, J. R. (1970). Virology, 41, 66-71. Sternberg, N., Hamilton, D., Austin, S., Yarmolinsky, M. & Hoess, R. (1980). Cold Spring Harbor Symp. Quant. Biol. 45, 297-309. Yun, T. & Vapnek, D. (1977). Firology, 77, 376-385.
Edited by S. Brenner