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Integration sites of foreign genes in the chromosome of coliphage P1: A finer resolution
VIROLOGY
73, 299-302 (1976)
Integration
Sites of Foreign Genes in the Chromosome of Coliphage Pl: A Finer Resolution G. SCHULZ’
Department
of Microbiology,
Loyola
AND
M. STODOLSKY*
University Stritch School of Medicine, Maywood, Illinois 60153 Accepted April
2160 South First Avenue,
23,1976
Several foreign genetic elements have been united with the chromosome of coliphage Pl in the cat-gene 2 chromosomal region. Chromosomes of the resultant specialized transducing derivatives Pldlac, Pldcatsulstr, Pleat, and PlargF have been compared with that of coliphage Pl by cleavage analysis, using the Eco . Rl restriction endonuclease. There is an Eco.Rl susceptible site separating the loci of integration ofargF and cat from those of lac and catsulstr. It is confirmed that the Pldlac chromosome is much too large to be encapsidated genetically intact in Pl virions.
This note establishes two points concerning Pl chromosomal transactions. The Pl prophage is a plasmid and the bacteriophage DNAs are (at least partially) permuted (1). The genetic map produced through bacteriophage crosses is linear (2) and may have several recombinational hotspots (3). Of primary interest herein is one involved in aberrant recombination. Several specialized transducing derivatives of Pl have been characterized. In each case, at least one of the sites of union of Pl and foreign gene sequences of these hybrids is in the cat3-gene 2 region. The mapping data have most recently been summarized by Walker and Walker (3) and an additional case has been presented by Scott (4). We are employing chromosome cleavage analysis as one tool for the elucidation of Pl chromosomal transactions. Through a comparison of several hybrids, we have resolved some genetic end-
points in the cat-gene 2 region. Attendant plasmid molecular weight estimates confirm a deduction concerning the size of a Pldlczc chromosome (5). Virions were produced in 500-ml cultures of thermally induced lysogens and collected by low speed centrifugation in the presence of PEG-6000 (6). We consider this procedure preferable to collection by high speed centrifugation, because there is less (if any) preferential loss of Pl small capsid morphology variants. The small capsid vu-ions comprise about 20% of the total virion population (7). They are well separated from the main virion band (respective densities of 1.42 and 1.47) in the isopycnic CsCl gradients used in the final step of purification. Some qualitative information concerning defects of the hybrids was obtained by visual comparisons of virion band thickness after isopycnic fractionation. The yield of virions from the nondefective Pleat lysogen (8) is indistinguishable from that of the Pl lysogenic control. The PlargF (9) shown herein is also without manifest defects in virion production. Iida et al. (10) are analyzing a family of Pl derivatives with drug-resistance genes from R plasmid, and have provided us with the lysogen 3507 (Pldcatsulstr). In both our labora-
’ In partial fulfillment of the M.S. degree. e Author to whom requests for reprints should be addressed. 3Abbreviations: cat, sul, and str for genes of R plasmid origin controlling resistance to chloramphenicol, sulfonamides, and streptomycin, respectively; argF and lox for Escherichia coli K12 genes controlling ornithine transcarbamylase synthesis and lactose catabolism; md for 10” daltons. 299 Copyright All rights
0 1976 by Academic Press, Inc. of reproduction in any form reserved.
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tories, we have observed an excessive production of small virions (about equal thickness of small and normal virion bands) by the lysogen, even though the total virion yield is similar to that of the Pl lysogenic control. Lastly, the PLO100 (Pld2e.c) lysogen (5) virion yield is only about 20% that of the Pl control; the fruction of small virions is about twice that of the Pl control. Thus, the Pldcatsulstr and Pldlac both have mutations affecting the virion capsid size distribution. In addition, the Pldlac is severely affected in net virion production, as previously reported (5). DNA was purified from the virions by phenol extraction. Chromosome cleavage was accomplished by treatment with the Eco . Rl nuclease, which was kindly provided by Dr. G. Haywood. Digests were fractionated through agarose slab gels in an EC490 electrophoresis apparatus. Gels were stained with ethidium bromide and illuminated with a long wave-length uv light to excite fluorescence (11). Photographs were made with Polaroid Type 57 film. To resolve all relevant features, it has been necessary to use a few different fractionation conditions. High molecular weight features are best revealed in the upper photographs. To clearly show lower molecular weight features of the Pldlac digest, photographs of gels with differing amounts of DNA have been joined together. The molecular weight “labels” of the fragments are taken from the first description of Eco *Rl digests of phage Pl and X DNAs (12) and our extrapolations from them. The absolute error for fragments of greater than 8.0 and less than 3 .O md is probably of the order of 20%, because of poor resolution and/or lack of several standards in these md ranges. When fragments of a substrate chromosome are present in equimolar quantities, there is a uniform decrease in the DNA content of (resolved) bands with the molecular weight of the fragments. While this trend is evident in the digests of Pl and its hybrids, there are exceptions which could complicate interpretation of the results. Of primary concern was the following possibility: the absences of some Pl fragments from digests of the hybrid DNAs might be due solely to chromosome encapsidation
processes, rather than to features of the plasmid chromosomes. Through a comparison of plasmid and virion DNAs of Pl and Pldlac, we have distinguished true plasmid features from encapsidation artifacts. These results will be presented in detail in another report. We do, however, mention a few particular points which are relevant to this study. The partially resolved 15-, 12-, and lomd Pldlac triplet contains less DNA than expected for its aggregate molecular weight; the 4.8-md band is also DNA poor (Fig. 1). The following bands are either unresolved doublets, or contain DNA fragments from components of a chromosomal
-
9.6
-4.3 -4.2 43.0 -2.9 -1.6
-0.78 -0.68 '0.65 FIG. 1. Eco’Rl digests of PI and derivative hybrid DNAs. For Pl, the fragment molecular weights in 10” dalton units are 9.6, 6.5, 6.2, 4.3, 4.2, 3.8; 3.5, 2.1, 2.0, 1.8, 1.6, 1.0, 0.87, 0.78, 0.68, 0.66, 0.56, and 0.38. Molecular weight of the novel Pldloc fragments are 15, 12, 10, 8.3, 4.8, 3.3, 3.0, 2.9, 1.5, and 1.4 md. The lower Pldlac photograph is a composite derived from photographs of gels through which 2, 10, and 30 pg of DNA were fractionated. The following electrophoretic conditions were employed: top right, 30 V for 36 hr through a 0.5% gel; top left, 40 V for 24 hr through a 0.6% gel; bottom, 40 V for 18 hr through a 0.7% gel.
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duplication: the 2.9-md PldZac band, the 3.0-md Pldlac band, and the 1.6-md Pl band. In contrast, the 1.6-md Pldlac band is a singlet. Fragments with one end produced during virion packaging must be present in much smaller quantities than the fragments produced solely by Eco . Rl digestion; all plasmid digest bands thus far detected are present in the virion DNA digests and vice versa. Thus, for the Pldlac digest shown herein, absences of Pl fragments do reflect a deletion or alteration of corresponding segments of the Pldlac plasmid chromosome. We assume that the Pl fragment absences from digests of the other (less defective) hybrids are subject to the same interpretation. The Pldlac digest lacks the 0.7%md fragment, one member of the 1.6-md band (as mentioned above), and the 9.6-md fragment. To unambiguously establish that there is no Pldlac fragment at the 9.6-md position, Pldlac and Pldlac + Pl digests are compared. The appearance of the 9.6md fragment in the combined digest shows that the Pldlac fragment with the 8.3-md designation is indeed novel. Iida et al. (10) have found that the digests of Pldcutsulstr lack the 9.6- and 0.78md bands, as also shown in Fig. 1. We expect that the 1.6-md bands of Plcutsulstr, PlurgF, and Plcut are doublets. PlurgF lacks only the 4.3-md Pl band (as do nine other PlurgF thus far characterized). Pleat also lacks solely the 4.3-md band. Heretofore, there has been no fine structure information on the cut-gene 2 region. Gene 2 function is altered in Pldlucs (3,5), but there has been no simple assay for the presence or absence of the cut insertion locus in the Pldlacs. Our results show that there is an Eco *Rl cut site(s) separating loci of cut and urgF elements from those of Zuc and catsulstr elements. Cat and urgF are encoded in recombinant derivatives of the 4.3-md Pl segment to the left of this site, while Zuc and cutsulstr are to its right. Cutsulstr presumably has termini in 9.6- and 0.78-md segments and Zuc in 9.6- and 1.6-md segments. The first fractionation of Eco *Rl digests of bacteriophage Pl DNA revealed the presence of 15 fragments with a molecular
301
weight greater than 0.6 md (12). As mentioned above, the 1.6-md band is now recognized as an unresolved doublet. The 0.67-md “band” has been resolved as a doublet (Fig. 1). In addition we have detected eight lower molecular weight fragments, employing fractionation through gradient pore acrylamide gels. This latter result is not documented here, because the digests of the hybrids do have the lower molecular weight Pl fragments, and because of the lack of low md-fragment references for molecular weight extrapolations. The fragments detected on the gel shown above have an additive molecular weight of about 53 md. The additional molecular weight of the lower molecular weight fragments is probably not more than two md. From purely genetic data, Rae and Stodolsky (8) deduced that the Pldluc plasmid chromosome is larger than the chromosome of Pl virions. The additive molecular weights of all the Pldluc Eco *Rl fragments is about 110 md. However, we suspect that this estimate is excessive. There is a 47-md (~:&-Zuc-p~a~ E. coli chromosome segment, where a& and &x:, are inverted repeats of one another (13). If this linkage group is encoded in the bacterial segment of the Pldluc, the Pldluc plasmid population could become genetically heterogeneous as a consequence of recombinational inversion. With respect to the Eco. Rl digests, each of the ~$3 sequences would be represented on two distinct fragments, which would only together be present in the same molar quantity as the other (singlet) fragments. Thus, the presence of an invertible sequence may be responsible for the anomalous DNA content of the l&,12-, lo-, and 4.8-md bands mentioned above. A minimal Pldluc molecular weight estimate is obtained by assuming that the 15 and 4.8-md segments are present in one form of the P1dZa.cplasmid, and the 12- and lo-md segments are present in the inverted recombinant. With these assumptions, molecular weights of Pldluc, the Pl segment, and the bacterial segment are, respectively, 84 md, 40 to 55 md, and 35 to 50 md. It is apparent that the data confirm deductions of Rae and Stodolsky (5) that the Pldluc plasmid chromosome is too large to be transduced genetically intact,
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and that even the bacterial segment of the Pldlac must be frequently split during scission of the Pl~&c chromosome into 60md virion chromosomes. The increased genetic resolution of the cat-gene 2 region will be of immediate utility for choosing chromosomal pairs to be used in DNA heteroduplex construction. For instance, a heteroduplex of Pleat and Pldlac should have the structure: cat insertion loop-duplex DNA region-lac insertion loop/corresponding gene 2 deletion loop; measurements by electron microscopy should reveal the size of the two foreign genetic elements, the distance between the sites of integration, and the size of the Pl gene deletion. Heteroduplexes of other hybrid pairs with foreign genetic elements to the right and left of the Eco . Rl cut site will provide corresponding information. For continuing studies of Pl aberrant recombination, the following possibilities must now be considered for the cat-gene 2 hot spot: either there is an extended chromosomal region, as opposed to a single locus, in which Pl and foreign genetic elements are united, or there are at least two distinct hot spots for the aberrant recombinational processes.
ACKNOWLEDGMENTS This research was supported by USPHS General Research Support Grant No. RR05368 and USPHS Grant No. 1 ROl GM21435-01. M. Stodolsky is the recipient of Research Career Development Award No. NIGMS OADPA l-K04-GM-00113-01. Restriction endonuclease was generously supplied by Dr. G. Haywood. REFERENCES 1. IKEDA, H., and TOMIZAWA, J., Cold Spring Harbor Symp. Quant. Biol. 33, 791-798 (1968). 2. SCOTT, J. R. Virology 36, 564-574 (1968). 3 WALKER, D. H., JR., and WALKER, J. T., J. Viral. 16, 525-534 (1975). 4. SCOTT, J. R., Virology 65, 173-178 (1975). 5. RAE, M. E., and STODOLSKY,M., Virology 58,3254 (1974). 6. YAMAMOTO, K. R., and ALBERTS, B. M., Virology 40, 734-744 (1970). 7. WALKER, D. H., JR., and ANDERSON, T. F., J. Virol. 5, 765-782 (1970). 8. KONDO, E., and MITSUHASHI, S., J. Bacterial. 88, 1266-1276 (1964). 9. STODOLSKY, M., Amer. Sot. Microbial. (Ab&acts) V151 (1973). 10. IIDA, S., BACHI, B., and ARBER, W., In progress. 11. SHARP, P. A., SUGDEN, B., and SAMBROOK, J., Biochemistry 12, 3055-3063 (1973). 12. HELLING, R. B., GOODMAN, H. M., and BOYER, A. W., J. Viral. 14, 1235-1243 (1975). 13. Hu, S., OHTSUBO, E., and DAVIDSON, N., J. Bacteriol. 122, 749-763 (1975).
73, 299-302 (1976)
Integration
Sites of Foreign Genes in the Chromosome of Coliphage Pl: A Finer Resolution G. SCHULZ’
Department
of Microbiology,
Loyola
AND
M. STODOLSKY*
University Stritch School of Medicine, Maywood, Illinois 60153 Accepted April
2160 South First Avenue,
23,1976
Several foreign genetic elements have been united with the chromosome of coliphage Pl in the cat-gene 2 chromosomal region. Chromosomes of the resultant specialized transducing derivatives Pldlac, Pldcatsulstr, Pleat, and PlargF have been compared with that of coliphage Pl by cleavage analysis, using the Eco . Rl restriction endonuclease. There is an Eco.Rl susceptible site separating the loci of integration ofargF and cat from those of lac and catsulstr. It is confirmed that the Pldlac chromosome is much too large to be encapsidated genetically intact in Pl virions.
This note establishes two points concerning Pl chromosomal transactions. The Pl prophage is a plasmid and the bacteriophage DNAs are (at least partially) permuted (1). The genetic map produced through bacteriophage crosses is linear (2) and may have several recombinational hotspots (3). Of primary interest herein is one involved in aberrant recombination. Several specialized transducing derivatives of Pl have been characterized. In each case, at least one of the sites of union of Pl and foreign gene sequences of these hybrids is in the cat3-gene 2 region. The mapping data have most recently been summarized by Walker and Walker (3) and an additional case has been presented by Scott (4). We are employing chromosome cleavage analysis as one tool for the elucidation of Pl chromosomal transactions. Through a comparison of several hybrids, we have resolved some genetic end-
points in the cat-gene 2 region. Attendant plasmid molecular weight estimates confirm a deduction concerning the size of a Pldlczc chromosome (5). Virions were produced in 500-ml cultures of thermally induced lysogens and collected by low speed centrifugation in the presence of PEG-6000 (6). We consider this procedure preferable to collection by high speed centrifugation, because there is less (if any) preferential loss of Pl small capsid morphology variants. The small capsid vu-ions comprise about 20% of the total virion population (7). They are well separated from the main virion band (respective densities of 1.42 and 1.47) in the isopycnic CsCl gradients used in the final step of purification. Some qualitative information concerning defects of the hybrids was obtained by visual comparisons of virion band thickness after isopycnic fractionation. The yield of virions from the nondefective Pleat lysogen (8) is indistinguishable from that of the Pl lysogenic control. The PlargF (9) shown herein is also without manifest defects in virion production. Iida et al. (10) are analyzing a family of Pl derivatives with drug-resistance genes from R plasmid, and have provided us with the lysogen 3507 (Pldcatsulstr). In both our labora-
’ In partial fulfillment of the M.S. degree. e Author to whom requests for reprints should be addressed. 3Abbreviations: cat, sul, and str for genes of R plasmid origin controlling resistance to chloramphenicol, sulfonamides, and streptomycin, respectively; argF and lox for Escherichia coli K12 genes controlling ornithine transcarbamylase synthesis and lactose catabolism; md for 10” daltons. 299 Copyright All rights
0 1976 by Academic Press, Inc. of reproduction in any form reserved.
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COMMUNICATIONS
tories, we have observed an excessive production of small virions (about equal thickness of small and normal virion bands) by the lysogen, even though the total virion yield is similar to that of the Pl lysogenic control. Lastly, the PLO100 (Pld2e.c) lysogen (5) virion yield is only about 20% that of the Pl control; the fruction of small virions is about twice that of the Pl control. Thus, the Pldcatsulstr and Pldlac both have mutations affecting the virion capsid size distribution. In addition, the Pldlac is severely affected in net virion production, as previously reported (5). DNA was purified from the virions by phenol extraction. Chromosome cleavage was accomplished by treatment with the Eco . Rl nuclease, which was kindly provided by Dr. G. Haywood. Digests were fractionated through agarose slab gels in an EC490 electrophoresis apparatus. Gels were stained with ethidium bromide and illuminated with a long wave-length uv light to excite fluorescence (11). Photographs were made with Polaroid Type 57 film. To resolve all relevant features, it has been necessary to use a few different fractionation conditions. High molecular weight features are best revealed in the upper photographs. To clearly show lower molecular weight features of the Pldlac digest, photographs of gels with differing amounts of DNA have been joined together. The molecular weight “labels” of the fragments are taken from the first description of Eco *Rl digests of phage Pl and X DNAs (12) and our extrapolations from them. The absolute error for fragments of greater than 8.0 and less than 3 .O md is probably of the order of 20%, because of poor resolution and/or lack of several standards in these md ranges. When fragments of a substrate chromosome are present in equimolar quantities, there is a uniform decrease in the DNA content of (resolved) bands with the molecular weight of the fragments. While this trend is evident in the digests of Pl and its hybrids, there are exceptions which could complicate interpretation of the results. Of primary concern was the following possibility: the absences of some Pl fragments from digests of the hybrid DNAs might be due solely to chromosome encapsidation
processes, rather than to features of the plasmid chromosomes. Through a comparison of plasmid and virion DNAs of Pl and Pldlac, we have distinguished true plasmid features from encapsidation artifacts. These results will be presented in detail in another report. We do, however, mention a few particular points which are relevant to this study. The partially resolved 15-, 12-, and lomd Pldlac triplet contains less DNA than expected for its aggregate molecular weight; the 4.8-md band is also DNA poor (Fig. 1). The following bands are either unresolved doublets, or contain DNA fragments from components of a chromosomal
-
9.6
-4.3 -4.2 43.0 -2.9 -1.6
-0.78 -0.68 '0.65 FIG. 1. Eco’Rl digests of PI and derivative hybrid DNAs. For Pl, the fragment molecular weights in 10” dalton units are 9.6, 6.5, 6.2, 4.3, 4.2, 3.8; 3.5, 2.1, 2.0, 1.8, 1.6, 1.0, 0.87, 0.78, 0.68, 0.66, 0.56, and 0.38. Molecular weight of the novel Pldloc fragments are 15, 12, 10, 8.3, 4.8, 3.3, 3.0, 2.9, 1.5, and 1.4 md. The lower Pldlac photograph is a composite derived from photographs of gels through which 2, 10, and 30 pg of DNA were fractionated. The following electrophoretic conditions were employed: top right, 30 V for 36 hr through a 0.5% gel; top left, 40 V for 24 hr through a 0.6% gel; bottom, 40 V for 18 hr through a 0.7% gel.
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duplication: the 2.9-md PldZac band, the 3.0-md Pldlac band, and the 1.6-md Pl band. In contrast, the 1.6-md Pldlac band is a singlet. Fragments with one end produced during virion packaging must be present in much smaller quantities than the fragments produced solely by Eco . Rl digestion; all plasmid digest bands thus far detected are present in the virion DNA digests and vice versa. Thus, for the Pldlac digest shown herein, absences of Pl fragments do reflect a deletion or alteration of corresponding segments of the Pldlac plasmid chromosome. We assume that the Pl fragment absences from digests of the other (less defective) hybrids are subject to the same interpretation. The Pldlac digest lacks the 0.7%md fragment, one member of the 1.6-md band (as mentioned above), and the 9.6-md fragment. To unambiguously establish that there is no Pldlac fragment at the 9.6-md position, Pldlac and Pldlac + Pl digests are compared. The appearance of the 9.6md fragment in the combined digest shows that the Pldlac fragment with the 8.3-md designation is indeed novel. Iida et al. (10) have found that the digests of Pldcutsulstr lack the 9.6- and 0.78md bands, as also shown in Fig. 1. We expect that the 1.6-md bands of Plcutsulstr, PlurgF, and Plcut are doublets. PlurgF lacks only the 4.3-md Pl band (as do nine other PlurgF thus far characterized). Pleat also lacks solely the 4.3-md band. Heretofore, there has been no fine structure information on the cut-gene 2 region. Gene 2 function is altered in Pldlucs (3,5), but there has been no simple assay for the presence or absence of the cut insertion locus in the Pldlacs. Our results show that there is an Eco *Rl cut site(s) separating loci of cut and urgF elements from those of Zuc and catsulstr elements. Cat and urgF are encoded in recombinant derivatives of the 4.3-md Pl segment to the left of this site, while Zuc and cutsulstr are to its right. Cutsulstr presumably has termini in 9.6- and 0.78-md segments and Zuc in 9.6- and 1.6-md segments. The first fractionation of Eco *Rl digests of bacteriophage Pl DNA revealed the presence of 15 fragments with a molecular
301
weight greater than 0.6 md (12). As mentioned above, the 1.6-md band is now recognized as an unresolved doublet. The 0.67-md “band” has been resolved as a doublet (Fig. 1). In addition we have detected eight lower molecular weight fragments, employing fractionation through gradient pore acrylamide gels. This latter result is not documented here, because the digests of the hybrids do have the lower molecular weight Pl fragments, and because of the lack of low md-fragment references for molecular weight extrapolations. The fragments detected on the gel shown above have an additive molecular weight of about 53 md. The additional molecular weight of the lower molecular weight fragments is probably not more than two md. From purely genetic data, Rae and Stodolsky (8) deduced that the Pldluc plasmid chromosome is larger than the chromosome of Pl virions. The additive molecular weights of all the Pldluc Eco *Rl fragments is about 110 md. However, we suspect that this estimate is excessive. There is a 47-md (~:&-Zuc-p~a~ E. coli chromosome segment, where a& and &x:, are inverted repeats of one another (13). If this linkage group is encoded in the bacterial segment of the Pldluc, the Pldluc plasmid population could become genetically heterogeneous as a consequence of recombinational inversion. With respect to the Eco. Rl digests, each of the ~$3 sequences would be represented on two distinct fragments, which would only together be present in the same molar quantity as the other (singlet) fragments. Thus, the presence of an invertible sequence may be responsible for the anomalous DNA content of the l&,12-, lo-, and 4.8-md bands mentioned above. A minimal Pldluc molecular weight estimate is obtained by assuming that the 15 and 4.8-md segments are present in one form of the P1dZa.cplasmid, and the 12- and lo-md segments are present in the inverted recombinant. With these assumptions, molecular weights of Pldluc, the Pl segment, and the bacterial segment are, respectively, 84 md, 40 to 55 md, and 35 to 50 md. It is apparent that the data confirm deductions of Rae and Stodolsky (5) that the Pldluc plasmid chromosome is too large to be transduced genetically intact,
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and that even the bacterial segment of the Pldlac must be frequently split during scission of the Pl~&c chromosome into 60md virion chromosomes. The increased genetic resolution of the cat-gene 2 region will be of immediate utility for choosing chromosomal pairs to be used in DNA heteroduplex construction. For instance, a heteroduplex of Pleat and Pldlac should have the structure: cat insertion loop-duplex DNA region-lac insertion loop/corresponding gene 2 deletion loop; measurements by electron microscopy should reveal the size of the two foreign genetic elements, the distance between the sites of integration, and the size of the Pl gene deletion. Heteroduplexes of other hybrid pairs with foreign genetic elements to the right and left of the Eco . Rl cut site will provide corresponding information. For continuing studies of Pl aberrant recombination, the following possibilities must now be considered for the cat-gene 2 hot spot: either there is an extended chromosomal region, as opposed to a single locus, in which Pl and foreign genetic elements are united, or there are at least two distinct hot spots for the aberrant recombinational processes.
ACKNOWLEDGMENTS This research was supported by USPHS General Research Support Grant No. RR05368 and USPHS Grant No. 1 ROl GM21435-01. M. Stodolsky is the recipient of Research Career Development Award No. NIGMS OADPA l-K04-GM-00113-01. Restriction endonuclease was generously supplied by Dr. G. Haywood. REFERENCES 1. IKEDA, H., and TOMIZAWA, J., Cold Spring Harbor Symp. Quant. Biol. 33, 791-798 (1968). 2. SCOTT, J. R. Virology 36, 564-574 (1968). 3 WALKER, D. H., JR., and WALKER, J. T., J. Viral. 16, 525-534 (1975). 4. SCOTT, J. R., Virology 65, 173-178 (1975). 5. RAE, M. E., and STODOLSKY,M., Virology 58,3254 (1974). 6. YAMAMOTO, K. R., and ALBERTS, B. M., Virology 40, 734-744 (1970). 7. WALKER, D. H., JR., and ANDERSON, T. F., J. Virol. 5, 765-782 (1970). 8. KONDO, E., and MITSUHASHI, S., J. Bacterial. 88, 1266-1276 (1964). 9. STODOLSKY, M., Amer. Sot. Microbial. (Ab&acts) V151 (1973). 10. IIDA, S., BACHI, B., and ARBER, W., In progress. 11. SHARP, P. A., SUGDEN, B., and SAMBROOK, J., Biochemistry 12, 3055-3063 (1973). 12. HELLING, R. B., GOODMAN, H. M., and BOYER, A. W., J. Viral. 14, 1235-1243 (1975). 13. Hu, S., OHTSUBO, E., and DAVIDSON, N., J. Bacteriol. 122, 749-763 (1975).