JOURNAL OF VIROLOGY, Sept. 1979, p. 645-656 0022-538X/79/09-0645/12$02.00/0
Vol. 31, No. 3
New Classes of Viable Deletion Mutants in the Early Region of Polyoma Virus BEVERLY E. GRIFFIN* AND CHRISTINE MADDOCK Imperial Cancer Research Fund, London WC2, England
Received for publication 20 March 1979
Viable mutants of polyoma virus have been isolated which have deletions in defined parts of the early region of the genome. One class of mutants has deletions (less than 1% of viral genome length) located between 71.5 and 73.5 on the physical map of polyoma virus DNA, near the origin of replication. These mutants appear to grow and to transform cells in a manner indistinguishable from wildtype virus. A second type of mutant with deletions (about 2% of viral genome length) located between about 88 and 94.5 units on the physical map of polyoma virus DNA have altered transformation properties. One of the latter (which maps between 88 and 91.5 units) also has altered growth characteristics, whereas another (which maps between 91.5 and 94.5 units) resembles wild-type virus in its growth properties. The regions with deleted sequences have been defined by cleaving mutant DNAs with restriction endonucleases and analyzing pyrimidine tracts.
Polyoma virus transforms cells in tissue culture and produces tumors in susceptible animals. In spite of intensive study, relatively little is known at the molecular level about the process by which this occurs and what roles the viral genes play in transformation. One of the classical methods for answering questions about biological functions involves the isolation and characterization of mutants with lesions affecting these functions. For polyoma virus, there are very few mutants available for studying transformation (or the in vivo production of tumors). Those that exist fall into two classes, the temperature-sensitive mutants which have lesions in the early region of polyoma virus (20) and appear to be defective for the initiation of, but possibly not for the maintenance of, transformation, and the host-range transformation-defective (hr-t) mutants (1) which are also early mutants and do not transform cells or produce tumors. (For a review, see reference 4.) These two classes of mutants complement each other for transformation (2, 6). In the present study, biological and chemical properties of several new mutants in the early region of polyoma virus are described. They are clearly different from the previously known types of mutants. Some of these mutants have short deletions near the viral origin of replication. Such deletions seem to have little effect either on lytic growth or transformation. The deletions in these mutants probably lie in a noncoding part of the genome (30). However, other mutant isolates (designated dl-8 and dl-23)
have deletions within the coding part of the early region which affect transformation and in one case (dl-8) also lytic growth. MATERIALS AND METHODS Virus strains. Wild-type strains of polyoma virus used were the large-plaque strains designated A2 and A3 isolated from the same (Pasadena strain) viral stock (5). One strain may be presumed to be a variant of the other which arose spontaneously. The DNA sequence of the A2 strain has recently been determined (30; manuscript in preparation), and A3, a closely related derivative strain, has been found by sequence analysis to contain a small deletion in the early region of the DNA. Viral DNA. Subconfluent layers (about 5 x 106 cells per 90-mm dish) of 3T6 mouse cells were infected with polyoma virus at about 5 to 10 PFU/cell. Infected cultures were incubated at 37°C in Dulbecco-modified Eagle (E4) medium containing 5% fetal calf serum for 60 to 72 h. Viral DNA was extracted by the Hirt procedure (11) and purified by equilibrium centrifugation with cesium chloride-ethidium bromide density gradients, followed by purification on sucrose density gradients (9). Only form I polyoma virus DNA was used in subsequent experiments. For analysis of mutants, 3P-labeled DNA was prepared from each viral mutant as in the procedure described by Griffin et al. (10) and was analyzed with restriction endonucleases HpaII, HaeIII, Hinf, MboI, MboII, and AvaI. Construction of mutants. The two protocols used for mutant construction are schematically illustrated in Fig. 1. In method 1, polyoma virus DNA (A3 strain) was treated with restriction endonucleases HaeII or BglI (usually at 1 pl of enzyme per ug of DNA in 10 mM
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GRIFFIN AND MADDOCK Si nuclease (Tj Linear DNA
infect cells e* 4 etc.
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FIG. 1. Schematic diagram illustrating the methods by which the deletion mutants described in this paper were made. In method 1, Si nuclease should remove nucleotides from linear DNA at the original restriction endonuclease site (27) and lead to slightly truncated linear DNAs. By this method deletions are expected around the restriction endonuclease cleavage site. In method 2, a mixture of different DNAs (either A2 and A3 DNAs, or di-51 and A3 DNAs) were linearized with the restriction endonuclease BamI, which cleaves polyoma virus DNA at 58.5 map units (7) and gives single-stranded ends. After denaturation, homoduplex and heteroduplex (designated A2, A3) species should be obtained, the latter containing a single-stranded region, indicated by V, and sequence extensions at the ends of the duplex DNA. These species could be recircularized by T4-ligase, and only the circles with single-stranded (mismatched) regions (A2, A3) would be susceptible to cleavage with the single-strand-specific SI nuclease. The latter would yield linear duplex molecules with blunt ends, which had been cleaved at the site of the mismatch, that is, at a position equivalent to V in the heteroduplex DNA. The linear molecules were used to transfect cells in an attempt to produce deletion mutants.
Tris-hydrochloride [pH 7.5]-10 mM MgCl2 for 2 h at
370C) to produce linear DNAs. When only form III
DNA could be seen to be present, an additional amount of restriction endonuclease was added, and incubation was continued for a further 4 h at 370C. Form III DNA was separated by electrophoresis on agarose gels as below. Isolated form III DNA was incubated with Si nuclease, isolated by alcohol precipitation, and used in subsequent studies (see below). In method 2, an adaptation of the method of Shenk (26), an equimolar mixture of viral DNAs from two different strains was cleaved to full-length linear molecules with restriction endonuclease BamI. The DNAs in solution (concentration about 1 Itg of DNA per 50 jil of solution) were heated at 700C for 5 min to allow denaturation to occur and then were permitted to cool slowly to room temperature to renature. Linear double-stranded DNA was recovered by alcohol precipitation from a solution made 1 M with respect to NaCl. Ligation of linear mixtures was carried out essentially as described elsewhere (26) in the presence of T4 ligase. Ligation was followed by observing the change of linear (form III) DNA to circular (form II) DNA by electrophoresis on analytical cylindrical agarose gels (9). When no more form III DNA could be seen to be present, an additional quantity of T4 ligase was added, and the solution was left for a further 24 h at 40C to ensure that ligation was as nearly complete as possible. DNA was reisolated by alcohol precipitation. Form II DNA was then separated from any remaining form III DNA by electrophoresis on preparative 1% agarose slab gels. Forn II DNA was excised, isolated by electrophoresis as described elsewhere (9), and subsequently alcohol precipitated. It was then treated with the single-strand-specific nuclease, S1, as previously described (28, 34) to cleave the DNA at any single-
stranded or non-base-paired sites. Form III DNA, which resulted from cleavage with S1 nuclease, was separated on preparative 1% agarose slab gels from form II DNA and then isolated. This material should have contained mainly linear DNA of heteroduplex origin (Fig. 1). This was confirmed with analysis by gel electrophoresis on analytical cylindrical agarose gels. Dishes (50 mm) of slightly subconfluent, secondary whole mouse embryo cells (WME) were transfected with about 1 jug of the linear DNA (made by either method 1 or 2 above) per dish by the dextran procedure as described elsewhere (19). Cells were covered with soft agar and left at 320C for periods up to 14 to 18 days. Plates were stained with neutral red, and individual plaques were picked. Virus stocks were made from single plaques by infecting dishes (2 by 50 mm) of WME cells with plaque-isolated material. When maximum cytopathic effects were observed (6 to 8 days), cells were freeze-thawed three times and harvested. These stocks were used to make polyoma virus DNA as described above. The DNAs made from each stock were analyzed by agarose gel electrophoresis after cleavage with either endonuclease HpaII or HaeIII (see Fig. 2B). Patterns were compared with those obtained from wild-type DNA (7, 10). When patterns were obtained that appeared to differ from wild-type DNA, larger quantities of viral stocks were made with WME cells. Stocks were titrated by the haemagglutination and plaque assay methods. Transformation with virions. Transformation of a permanent line from Fisher rat embryos (21) with virus (wild type or viable deletion mutant) was assayed by using either the soft agar method (18) or the dense focus assay. The latter was carried out essentially as described elsewhere (24). Depurination analysis. Polyoma virus mutant
DELETION MUTANTS IN POLYOMA VIRUS
VOL. 31, 1979
DNAs were depurinated and labeled with [32P]phosphate with [y-'P]ATP and T4 polynucleotide kinase (33). The products were separated in two dimensions by the method of Ling (17) used for polyoma virus DNA by Griffin (7). Restriction endonuclease maps. Physical maps of polyoma virus DNA cleaved with either restriction endonuclease MboI, MboII, or Hinf were determined by methods described elsewhere (7, 10) (see Fig. 2A). In vivo-labeled polyoma virus [32P]DNA (wild type, A2 strain) and mutants made by procedures described above were cleaved with individual restriction endonucleases, as well as with mixtures (MboI, MboII, or Hinf plus either HpaII or HaeIII) which allowed cleavage assignments to be correlated with existing physical maps. Enzymes. Restriction endonucleases HpaII, HaeIII, BamI, PvuII, Hinf, MboI, and MboII were isolated by standard procedures. HaeII and AvaI were purchased from New England Biolabs Inc. BglI was a generous gift of J. R. Arrand. DNA ligase (from T4infected Escherichia coli) was purchased from Miles Laboratories, Inc. T4 polynucleotide-kinase was purchased from Boehringer. S1 endonuclease was purchased from Sigma. [32P]phosphate and [y-32P]ATP (high specific activity) were purchased from the Radiochemical Centre, Amersham, England.
RESULTS Several novel types of deletion mutants of polyoma virus have been isolated by the two procedures outlined in Fig. 1. The rationale is this. Shenk et al. (28) showed that when Si endonuclease was used to cleave duplex DNA at the site of a deletion loop, a further 30 or so base pairs were also cleaved from the ends of the duplex DNA. Moreover, additional base pairs were lost from the ends of DNA during cellmediated closure of blunt-ended linear molecules to generate circular DNA (27). In the protocol adopted here in which cells were first transfected with linear DNA (method 1, Fig. 1), mutants were therefore expected which had deletions at the ends of the linear duplex DNA used in the transfections. That is, when cells were transfected with linear DNA made by cleavage with a particular restriction endonuclease followed by Si endonuclease treatment, mutants with deletions around that restriction site should be obtained. For these studies, the A3 (largeplaque) strain of polyoma virus was used. This strain has a "deletion" of 11 base pairs relative to another large-plaque viral strain designated A2 (5). Sequence studies (30) show this deletion to be in a noncoding region of the polyoma virus genome, at around 71.5 map units (Fig. 2A). To expand deletions generated by the method above, a modified procedure based on the protocol of Shenk (26) was adopted (see method 2, Fig. 1). Heteroduplex polyoma DNA molecules were made either between a deletion mutant (dl-
647
51) generated by method 1 (above) and DNA from the A2 strain of polyoma virus or between the DNA from A2 and A3 strains; in the former, two mismatched regions were therefore expected to exist in the resulting duplex DNA, and in the latter, one was expected. That is, between dl-51 and A2 there should be mismatched regions in the heteroduplex DNA at the site of difference of the two strains and at the original restriction endonuclease site, both of which should be susceptible to cleavage by S1 nuclease. Heteroduplex DNA from the A2 and A3 strains should contain S1 nuclease susceptibility only at the site of strain difference. Cells were transfected with linearized DNA in an attempt to obtain mutants with enlarged deletions. Mutants were selected for viability. In practice, a number of viable polyoma virus mutants were obtained by the procedures outlined in Fig. 1. As expected, most of these had deletions in the early region of the genome near the viral origin of replication (mutants dl-51, -6, -28, and others; Fig. 3). In addition, two mutants, dl-8 and dl-23, were isolated which had deletions located between about 88 and 94.5 units on the physical map of polyoma virus (see Fig. 3). For simplicity, the properties of the various classes of mutants will be considered separately. Mutants from early region DNA near the viral origin of replication (dl-51, -1, -6, -21, -28). Mutants with sequence deletions near the viral origin of replication were isolated after transfection of mouse cells with linear DNAs made by cleaving polyoma virus DNA (A3 strain) first with either the restriction endonuclease HaeII or BglI and then with S1 nuclease (method 1 of Materials and Methods, see Fig. 1). HaeII was reported previously to cleave the DNA at about 72.4 map units (7) and Bgll to cleave at about 72.5 map units (J. R. Arrand, personal communication). Subsequent sequence data have shown, however, that polyoma virus DNA (A2 or A3 strains) contains two HaeII sites which are 11 base pairs apart (30). Linear DNA made by HaeII cleavage is therefore not full length. The origin of replication lies somewhere near the HpaII-3-5 junction at 70.7 map units (10). Mutants made by transfection with DNA treated as described above were found to contain deletions of about 0.4% of the viral genome, as analyzed by comparing restriction endonuclease digestion patterns (HpaII and HaeIII) of wildtype and mutant DNAs (7). Some of the mutants made from HaeII-cleaved polyoma DNA retained the BglI site; others did not. One of the mutants, dl-51, used in subsequent experiments (see below), retained the BglI site. All mutants, whether made from HaeII- or Bgll-cleaved polyoma DNA, had lost both HaeII sites (and the
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corresponding HhaI sites which are part of HaeII sites). The mutant viruses did not require helper for growth. The mutant, designated dl-51, with a deletion of about 20 base pairs in the restriction fragment HpaII-5 (Fig. 3) was used in the following experiments. To extend the size of the deletion, a mixture of DNAs from dl-51 and from wild-type (A2 strain) virus was treated with BamI, which cleaves polyoma virus DNA in the late region at about 58.5 map units (7). The DNA mixture was denatured and allowed to renature to form a heteroduplex species which was then ligated to produce circular DNA. This heteroduplex circular DNA was treated with Si nuclease to produce linear DNA which was used to transfect mouse cells as described in Materials and Methods and illustrated (method 2, Fig. 1). DNA was then made from viral stocks grown from individual plaques which exhibited characteristic large, small, or minute plaque morphology. (Control dishes transfected with A2 DNA cleaved with BamI produced mainly large plaques.) DNA, made from viral stocks from about 30 plaques and analyzed after cleavage with restriction endonucleases, could be roughly divided into four types, illustrated by the mutants dl-1, -6, -21, and -28. (i) Large-plaque mutant dl-1 contained a deletion in HpaII-5 and was indistinguishable from dl-51. (ii) Minute-plaque mutant dl-6 contained a deletion in HpaII-5 of about 0.8% of the genome. An analysis of depurination products of dl-6 DNA showed it to contain the large depurination product characteristic of HpaII-5 (7). This has been shown previously to lie within restriction endonuclease fragment HaeIII-14' (7). (iii) Large-plaque mutant dl-21 had a deletion in HpaII-5. Analysis of DNA with restriction enzymes suggested the presence of a mixture of products. One had a restriction pattern similar to that found for the common polyoma virus-defective species D-50 (8), and one gave a pattern which appeared to be related to that of dl-28 (see below and Fig. 2B). (iv) Large-plaque mutant dl-28 was indistinguishable from dl-6 by restriction endonuclease and depurination analysis. It had a deletion in restriction endonuclease fragment HaeIII-14' (Fig. 2B). All the mutant DNAs lacked the HaeII (and corresponding HhaI) sites, and all except dl-1 lacked the BglI restriction endonuclease site. Further analysis showed that all of the mutants lacked a wild-type MboII fragment (MboII-14; Fig. 2A) of about 2.0% genome size but retained the MboII fragments on either side of MboII-14 (data not shown). All the deletions could therefore be located somewhere between 71.4 and 73.4 units on the physical map of polyoma virus DNA (Fig. 3). Since all mutant DNAs contained an
J. VIROL.
FIG. 2. (A) Physical maps of polyoma virus DNA. The HpaII physical map and the position of the EcoRI and BamI endonuclease cleavage sites are taken from Griffin et al. (9, 10). See text for discussion of BglI and HaeII sites. The origin of replication (OR) is taken from Griffin et al. (10). Three other maps, those of Hinf, MboI, and MboII, were determined relative to the HpaII and the HaeIII physical map (7) by methods elsewhere described (9). The very small MboI fragment (MboI-8) in HpaII-7 (7 base pairs) was first discovered by J. R. Arrand during sequence analysis of this region of the DNA. (B) Autoradiogram of 32P-labeled DNAs from wild-type (A2) and mutant (dl-8, -23, -21, and -28) polyoma virus, cleaved with the restriction endonuclease HaeIII and fragments separated on a two-phase (4%/ 8%) polyacrylamide-bisacrylamide slab gel (20 by 40 cm) by electrophoresis (7). The physical map of polyoma virus DNA cleaved with HaeIII has been described earlier (7). In mutants dl-8, -23, and -21, HaeHI-1 is missing, and a new band is seen as indicated by arrows (B, left). In dl-28, HaeIII-14' is missing and a new band is seen, as indicated by arrow (B, bottom left). Sites that give fragment HaeIII-1 are found at 86.2 and 3.6 units on the physical map of polyoma virus; sites that give fragment HaeIII-14' are found at 70.8 and 72.5 units (7). Deletions were further localized by cleavage with numerous other restriction endonucleases (data not shown), and the results are discussed in the text and summarized on Fig. 3.
18-residue-long depurination product which lies in HpaII-5 between 71.4 and 71.8 map units (7, 30), the deletions could be further defined as lying between 71.8 and 73.4 map units. If plaque morphology is meaningful, mutants of similar size need not have the same sequences deleted. For example, dl-6 (small plaque) and dl-28 (large plaque) have deletions of similar size. Other
VOL. 31, 1979
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DELETION MUTANTS IN POLYOMA VIRUS
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Vol. 31, No. 3
New Classes of Viable Deletion Mutants in the Early Region of Polyoma Virus BEVERLY E. GRIFFIN* AND CHRISTINE MADDOCK Imperial Cancer Research Fund, London WC2, England
Received for publication 20 March 1979
Viable mutants of polyoma virus have been isolated which have deletions in defined parts of the early region of the genome. One class of mutants has deletions (less than 1% of viral genome length) located between 71.5 and 73.5 on the physical map of polyoma virus DNA, near the origin of replication. These mutants appear to grow and to transform cells in a manner indistinguishable from wildtype virus. A second type of mutant with deletions (about 2% of viral genome length) located between about 88 and 94.5 units on the physical map of polyoma virus DNA have altered transformation properties. One of the latter (which maps between 88 and 91.5 units) also has altered growth characteristics, whereas another (which maps between 91.5 and 94.5 units) resembles wild-type virus in its growth properties. The regions with deleted sequences have been defined by cleaving mutant DNAs with restriction endonucleases and analyzing pyrimidine tracts.
Polyoma virus transforms cells in tissue culture and produces tumors in susceptible animals. In spite of intensive study, relatively little is known at the molecular level about the process by which this occurs and what roles the viral genes play in transformation. One of the classical methods for answering questions about biological functions involves the isolation and characterization of mutants with lesions affecting these functions. For polyoma virus, there are very few mutants available for studying transformation (or the in vivo production of tumors). Those that exist fall into two classes, the temperature-sensitive mutants which have lesions in the early region of polyoma virus (20) and appear to be defective for the initiation of, but possibly not for the maintenance of, transformation, and the host-range transformation-defective (hr-t) mutants (1) which are also early mutants and do not transform cells or produce tumors. (For a review, see reference 4.) These two classes of mutants complement each other for transformation (2, 6). In the present study, biological and chemical properties of several new mutants in the early region of polyoma virus are described. They are clearly different from the previously known types of mutants. Some of these mutants have short deletions near the viral origin of replication. Such deletions seem to have little effect either on lytic growth or transformation. The deletions in these mutants probably lie in a noncoding part of the genome (30). However, other mutant isolates (designated dl-8 and dl-23)
have deletions within the coding part of the early region which affect transformation and in one case (dl-8) also lytic growth. MATERIALS AND METHODS Virus strains. Wild-type strains of polyoma virus used were the large-plaque strains designated A2 and A3 isolated from the same (Pasadena strain) viral stock (5). One strain may be presumed to be a variant of the other which arose spontaneously. The DNA sequence of the A2 strain has recently been determined (30; manuscript in preparation), and A3, a closely related derivative strain, has been found by sequence analysis to contain a small deletion in the early region of the DNA. Viral DNA. Subconfluent layers (about 5 x 106 cells per 90-mm dish) of 3T6 mouse cells were infected with polyoma virus at about 5 to 10 PFU/cell. Infected cultures were incubated at 37°C in Dulbecco-modified Eagle (E4) medium containing 5% fetal calf serum for 60 to 72 h. Viral DNA was extracted by the Hirt procedure (11) and purified by equilibrium centrifugation with cesium chloride-ethidium bromide density gradients, followed by purification on sucrose density gradients (9). Only form I polyoma virus DNA was used in subsequent experiments. For analysis of mutants, 3P-labeled DNA was prepared from each viral mutant as in the procedure described by Griffin et al. (10) and was analyzed with restriction endonucleases HpaII, HaeIII, Hinf, MboI, MboII, and AvaI. Construction of mutants. The two protocols used for mutant construction are schematically illustrated in Fig. 1. In method 1, polyoma virus DNA (A3 strain) was treated with restriction endonucleases HaeII or BglI (usually at 1 pl of enzyme per ug of DNA in 10 mM
645
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GRIFFIN AND MADDOCK Si nuclease (Tj Linear DNA
infect cells e* 4 etc.
-*** truncated linear DNA
Bam, Baml Polyoma virus DNA + (A2, A3 strains) endonuclease -
MUTANTS
~~~~~~~~~A2
-
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A3 renature
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A2,A3
Iv
ligase 4,Si nuclease
Separate, infect mouse cells
MUTANTS
+4 --------with A2,A3 linear DNA etc.
A2,A3
+
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A3,
A2
FIG. 1. Schematic diagram illustrating the methods by which the deletion mutants described in this paper were made. In method 1, Si nuclease should remove nucleotides from linear DNA at the original restriction endonuclease site (27) and lead to slightly truncated linear DNAs. By this method deletions are expected around the restriction endonuclease cleavage site. In method 2, a mixture of different DNAs (either A2 and A3 DNAs, or di-51 and A3 DNAs) were linearized with the restriction endonuclease BamI, which cleaves polyoma virus DNA at 58.5 map units (7) and gives single-stranded ends. After denaturation, homoduplex and heteroduplex (designated A2, A3) species should be obtained, the latter containing a single-stranded region, indicated by V, and sequence extensions at the ends of the duplex DNA. These species could be recircularized by T4-ligase, and only the circles with single-stranded (mismatched) regions (A2, A3) would be susceptible to cleavage with the single-strand-specific SI nuclease. The latter would yield linear duplex molecules with blunt ends, which had been cleaved at the site of the mismatch, that is, at a position equivalent to V in the heteroduplex DNA. The linear molecules were used to transfect cells in an attempt to produce deletion mutants.
Tris-hydrochloride [pH 7.5]-10 mM MgCl2 for 2 h at
370C) to produce linear DNAs. When only form III
DNA could be seen to be present, an additional amount of restriction endonuclease was added, and incubation was continued for a further 4 h at 370C. Form III DNA was separated by electrophoresis on agarose gels as below. Isolated form III DNA was incubated with Si nuclease, isolated by alcohol precipitation, and used in subsequent studies (see below). In method 2, an adaptation of the method of Shenk (26), an equimolar mixture of viral DNAs from two different strains was cleaved to full-length linear molecules with restriction endonuclease BamI. The DNAs in solution (concentration about 1 Itg of DNA per 50 jil of solution) were heated at 700C for 5 min to allow denaturation to occur and then were permitted to cool slowly to room temperature to renature. Linear double-stranded DNA was recovered by alcohol precipitation from a solution made 1 M with respect to NaCl. Ligation of linear mixtures was carried out essentially as described elsewhere (26) in the presence of T4 ligase. Ligation was followed by observing the change of linear (form III) DNA to circular (form II) DNA by electrophoresis on analytical cylindrical agarose gels (9). When no more form III DNA could be seen to be present, an additional quantity of T4 ligase was added, and the solution was left for a further 24 h at 40C to ensure that ligation was as nearly complete as possible. DNA was reisolated by alcohol precipitation. Form II DNA was then separated from any remaining form III DNA by electrophoresis on preparative 1% agarose slab gels. Forn II DNA was excised, isolated by electrophoresis as described elsewhere (9), and subsequently alcohol precipitated. It was then treated with the single-strand-specific nuclease, S1, as previously described (28, 34) to cleave the DNA at any single-
stranded or non-base-paired sites. Form III DNA, which resulted from cleavage with S1 nuclease, was separated on preparative 1% agarose slab gels from form II DNA and then isolated. This material should have contained mainly linear DNA of heteroduplex origin (Fig. 1). This was confirmed with analysis by gel electrophoresis on analytical cylindrical agarose gels. Dishes (50 mm) of slightly subconfluent, secondary whole mouse embryo cells (WME) were transfected with about 1 jug of the linear DNA (made by either method 1 or 2 above) per dish by the dextran procedure as described elsewhere (19). Cells were covered with soft agar and left at 320C for periods up to 14 to 18 days. Plates were stained with neutral red, and individual plaques were picked. Virus stocks were made from single plaques by infecting dishes (2 by 50 mm) of WME cells with plaque-isolated material. When maximum cytopathic effects were observed (6 to 8 days), cells were freeze-thawed three times and harvested. These stocks were used to make polyoma virus DNA as described above. The DNAs made from each stock were analyzed by agarose gel electrophoresis after cleavage with either endonuclease HpaII or HaeIII (see Fig. 2B). Patterns were compared with those obtained from wild-type DNA (7, 10). When patterns were obtained that appeared to differ from wild-type DNA, larger quantities of viral stocks were made with WME cells. Stocks were titrated by the haemagglutination and plaque assay methods. Transformation with virions. Transformation of a permanent line from Fisher rat embryos (21) with virus (wild type or viable deletion mutant) was assayed by using either the soft agar method (18) or the dense focus assay. The latter was carried out essentially as described elsewhere (24). Depurination analysis. Polyoma virus mutant
DELETION MUTANTS IN POLYOMA VIRUS
VOL. 31, 1979
DNAs were depurinated and labeled with [32P]phosphate with [y-'P]ATP and T4 polynucleotide kinase (33). The products were separated in two dimensions by the method of Ling (17) used for polyoma virus DNA by Griffin (7). Restriction endonuclease maps. Physical maps of polyoma virus DNA cleaved with either restriction endonuclease MboI, MboII, or Hinf were determined by methods described elsewhere (7, 10) (see Fig. 2A). In vivo-labeled polyoma virus [32P]DNA (wild type, A2 strain) and mutants made by procedures described above were cleaved with individual restriction endonucleases, as well as with mixtures (MboI, MboII, or Hinf plus either HpaII or HaeIII) which allowed cleavage assignments to be correlated with existing physical maps. Enzymes. Restriction endonucleases HpaII, HaeIII, BamI, PvuII, Hinf, MboI, and MboII were isolated by standard procedures. HaeII and AvaI were purchased from New England Biolabs Inc. BglI was a generous gift of J. R. Arrand. DNA ligase (from T4infected Escherichia coli) was purchased from Miles Laboratories, Inc. T4 polynucleotide-kinase was purchased from Boehringer. S1 endonuclease was purchased from Sigma. [32P]phosphate and [y-32P]ATP (high specific activity) were purchased from the Radiochemical Centre, Amersham, England.
RESULTS Several novel types of deletion mutants of polyoma virus have been isolated by the two procedures outlined in Fig. 1. The rationale is this. Shenk et al. (28) showed that when Si endonuclease was used to cleave duplex DNA at the site of a deletion loop, a further 30 or so base pairs were also cleaved from the ends of the duplex DNA. Moreover, additional base pairs were lost from the ends of DNA during cellmediated closure of blunt-ended linear molecules to generate circular DNA (27). In the protocol adopted here in which cells were first transfected with linear DNA (method 1, Fig. 1), mutants were therefore expected which had deletions at the ends of the linear duplex DNA used in the transfections. That is, when cells were transfected with linear DNA made by cleavage with a particular restriction endonuclease followed by Si endonuclease treatment, mutants with deletions around that restriction site should be obtained. For these studies, the A3 (largeplaque) strain of polyoma virus was used. This strain has a "deletion" of 11 base pairs relative to another large-plaque viral strain designated A2 (5). Sequence studies (30) show this deletion to be in a noncoding region of the polyoma virus genome, at around 71.5 map units (Fig. 2A). To expand deletions generated by the method above, a modified procedure based on the protocol of Shenk (26) was adopted (see method 2, Fig. 1). Heteroduplex polyoma DNA molecules were made either between a deletion mutant (dl-
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51) generated by method 1 (above) and DNA from the A2 strain of polyoma virus or between the DNA from A2 and A3 strains; in the former, two mismatched regions were therefore expected to exist in the resulting duplex DNA, and in the latter, one was expected. That is, between dl-51 and A2 there should be mismatched regions in the heteroduplex DNA at the site of difference of the two strains and at the original restriction endonuclease site, both of which should be susceptible to cleavage by S1 nuclease. Heteroduplex DNA from the A2 and A3 strains should contain S1 nuclease susceptibility only at the site of strain difference. Cells were transfected with linearized DNA in an attempt to obtain mutants with enlarged deletions. Mutants were selected for viability. In practice, a number of viable polyoma virus mutants were obtained by the procedures outlined in Fig. 1. As expected, most of these had deletions in the early region of the genome near the viral origin of replication (mutants dl-51, -6, -28, and others; Fig. 3). In addition, two mutants, dl-8 and dl-23, were isolated which had deletions located between about 88 and 94.5 units on the physical map of polyoma virus (see Fig. 3). For simplicity, the properties of the various classes of mutants will be considered separately. Mutants from early region DNA near the viral origin of replication (dl-51, -1, -6, -21, -28). Mutants with sequence deletions near the viral origin of replication were isolated after transfection of mouse cells with linear DNAs made by cleaving polyoma virus DNA (A3 strain) first with either the restriction endonuclease HaeII or BglI and then with S1 nuclease (method 1 of Materials and Methods, see Fig. 1). HaeII was reported previously to cleave the DNA at about 72.4 map units (7) and Bgll to cleave at about 72.5 map units (J. R. Arrand, personal communication). Subsequent sequence data have shown, however, that polyoma virus DNA (A2 or A3 strains) contains two HaeII sites which are 11 base pairs apart (30). Linear DNA made by HaeII cleavage is therefore not full length. The origin of replication lies somewhere near the HpaII-3-5 junction at 70.7 map units (10). Mutants made by transfection with DNA treated as described above were found to contain deletions of about 0.4% of the viral genome, as analyzed by comparing restriction endonuclease digestion patterns (HpaII and HaeIII) of wildtype and mutant DNAs (7). Some of the mutants made from HaeII-cleaved polyoma DNA retained the BglI site; others did not. One of the mutants, dl-51, used in subsequent experiments (see below), retained the BglI site. All mutants, whether made from HaeII- or Bgll-cleaved polyoma DNA, had lost both HaeII sites (and the
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corresponding HhaI sites which are part of HaeII sites). The mutant viruses did not require helper for growth. The mutant, designated dl-51, with a deletion of about 20 base pairs in the restriction fragment HpaII-5 (Fig. 3) was used in the following experiments. To extend the size of the deletion, a mixture of DNAs from dl-51 and from wild-type (A2 strain) virus was treated with BamI, which cleaves polyoma virus DNA in the late region at about 58.5 map units (7). The DNA mixture was denatured and allowed to renature to form a heteroduplex species which was then ligated to produce circular DNA. This heteroduplex circular DNA was treated with Si nuclease to produce linear DNA which was used to transfect mouse cells as described in Materials and Methods and illustrated (method 2, Fig. 1). DNA was then made from viral stocks grown from individual plaques which exhibited characteristic large, small, or minute plaque morphology. (Control dishes transfected with A2 DNA cleaved with BamI produced mainly large plaques.) DNA, made from viral stocks from about 30 plaques and analyzed after cleavage with restriction endonucleases, could be roughly divided into four types, illustrated by the mutants dl-1, -6, -21, and -28. (i) Large-plaque mutant dl-1 contained a deletion in HpaII-5 and was indistinguishable from dl-51. (ii) Minute-plaque mutant dl-6 contained a deletion in HpaII-5 of about 0.8% of the genome. An analysis of depurination products of dl-6 DNA showed it to contain the large depurination product characteristic of HpaII-5 (7). This has been shown previously to lie within restriction endonuclease fragment HaeIII-14' (7). (iii) Large-plaque mutant dl-21 had a deletion in HpaII-5. Analysis of DNA with restriction enzymes suggested the presence of a mixture of products. One had a restriction pattern similar to that found for the common polyoma virus-defective species D-50 (8), and one gave a pattern which appeared to be related to that of dl-28 (see below and Fig. 2B). (iv) Large-plaque mutant dl-28 was indistinguishable from dl-6 by restriction endonuclease and depurination analysis. It had a deletion in restriction endonuclease fragment HaeIII-14' (Fig. 2B). All the mutant DNAs lacked the HaeII (and corresponding HhaI) sites, and all except dl-1 lacked the BglI restriction endonuclease site. Further analysis showed that all of the mutants lacked a wild-type MboII fragment (MboII-14; Fig. 2A) of about 2.0% genome size but retained the MboII fragments on either side of MboII-14 (data not shown). All the deletions could therefore be located somewhere between 71.4 and 73.4 units on the physical map of polyoma virus DNA (Fig. 3). Since all mutant DNAs contained an
J. VIROL.
FIG. 2. (A) Physical maps of polyoma virus DNA. The HpaII physical map and the position of the EcoRI and BamI endonuclease cleavage sites are taken from Griffin et al. (9, 10). See text for discussion of BglI and HaeII sites. The origin of replication (OR) is taken from Griffin et al. (10). Three other maps, those of Hinf, MboI, and MboII, were determined relative to the HpaII and the HaeIII physical map (7) by methods elsewhere described (9). The very small MboI fragment (MboI-8) in HpaII-7 (7 base pairs) was first discovered by J. R. Arrand during sequence analysis of this region of the DNA. (B) Autoradiogram of 32P-labeled DNAs from wild-type (A2) and mutant (dl-8, -23, -21, and -28) polyoma virus, cleaved with the restriction endonuclease HaeIII and fragments separated on a two-phase (4%/ 8%) polyacrylamide-bisacrylamide slab gel (20 by 40 cm) by electrophoresis (7). The physical map of polyoma virus DNA cleaved with HaeIII has been described earlier (7). In mutants dl-8, -23, and -21, HaeHI-1 is missing, and a new band is seen as indicated by arrows (B, left). In dl-28, HaeIII-14' is missing and a new band is seen, as indicated by arrow (B, bottom left). Sites that give fragment HaeIII-1 are found at 86.2 and 3.6 units on the physical map of polyoma virus; sites that give fragment HaeIII-14' are found at 70.8 and 72.5 units (7). Deletions were further localized by cleavage with numerous other restriction endonucleases (data not shown), and the results are discussed in the text and summarized on Fig. 3.
18-residue-long depurination product which lies in HpaII-5 between 71.4 and 71.8 map units (7, 30), the deletions could be further defined as lying between 71.8 and 73.4 map units. If plaque morphology is meaningful, mutants of similar size need not have the same sequences deleted. For example, dl-6 (small plaque) and dl-28 (large plaque) have deletions of similar size. Other
VOL. 31, 1979
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