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Characterization of DNA from polyhedral inclusion bodies of the nucleopolyhedrosis single-rod virus pathogenic for Orgyia pseudotsugata
VIROLOGY
83, 474-418 (1977)
Characterization of DNA from Polyhedral Inclusion Bodies of the Nucleopolyhedrosis Single-Rod Virus Pathogenic for Orgyia pseudotsuga ta G. F. ROHRMANN Department
of Argicultural
Chemistry,
G. S. BEAUDREAU
AND
Oregan State University,
Corvallis,
Oregon 97331
Accepted August 16,1977 A nucleotide sequence complexity of 88.5 x 10s was determined for the DNA of the nucleopolyhedrosis single-rod (unicapsid) virus of Orgyiu pseudotsugata using optical renaturation. In addition, the genome size was determined to be 85 x lo6 by comparison of EcoRl restriction endonuclease fragments with markers of known size using agarose gel electrophoresis. A G+C concentration of 44% for the viral DNA was estimated from its melting properties and buoyant density in CsCl. Evidence from buoyant density in C&l indicates that DNA which is occluded in the polyhedral matrix but not associated with virions is of viral origin.
Two nucleopolyhedrosis viruses (Baculovirus subgroup A) have been isolated from the Douglas fir tussock moth (Orgyiu pseudotsugutu). They are designated the nucleopolyhedrosis bundle (multicapsid) virus (NPBV) and the nucleopolyhedrosis single-rod (unicapsid) virus (NPSV) (I). The NPBV has been shown to have a genome size and complexity of approximately 86 x lo6 and have a G+C concentration of 54% (2). Although the NPBV and NPSV have polyhedrin molecules of close physical and antigenic structure (31, they show 1% or less genetic relatedness (Rohrmann, in preparation). This report describes studies on the NPSV DNA. The genome size was determined by comparison of restriction enzyme fragments of the genome with markers of known size, the genetic complexity was determined by examining its reassociation kinetics, and the G+C concentration was estimated by examining the DNA’s buoyant density in CsCl and by measuring its
was confirmed with dark-field microscopy of polyhedra using alkaline hydrocolloid (51, as well as by extraction of viral DNA and the determination of its buoyant density in CsCl.
T
/5h, l.OOO-65
70
75 TEMPERATURE
I
I
80
85
I
90
(“Cl
FIG. 1. Melting
profiles of NPSV DNA (01 and E. coli DNA (a) in 0.1 x SSC. The T, of NPSV DNA was estimated to be 72.1 based on the T, of E. coli of 74.5. The midpoint of the absorbance temperature profile was determined using a Gilford recording DU spectrophotometer at 260 nm (13). DNA concentration was approximately 15 *g/ml.
“I;1 addition, DNA found in the polyhedra but which did not sediment with virus was characterized to determine its origin. Viral polyhedra were produced and purified by the methods of Martignoni (4). The purity of the polyhedra preparations 474 Copyright 0 1977 by Academic Pres, Inc. All rights of reproduction in any form reserved.
ISSN 0042-6622
475
SHORT COMMUNICATIONS
Virus isolation, DNA preparation, and 32P-labeling of viral DNA are described elsewhere (2; Rohrmann, in preparation). To obtain [3H]DNA from Orgyia pseudotsugutu, 120 mg larvae (strain GL) (6) were each injected with 200 &i of L3Hlthymidine. After 24 hr at 25”, the larvae were frozen at -20”. Nuclei were isolated (7) and lysed with 2% SDS, 0.15 M NaCl, 0.100 M EDTA, pH 8.0 buffer and then digested for 2 hr at 37” with 200 pglml of proteinase K. The DNA was purified by phenol extraction followed by ethanol precipitation. The precipitate was resuspended in 0.02 M Tris, 0.005 M EDTA, pH 7.2, buffer containing RNase (50 PgI ml) and incubated for 45 min; then pro-
teinase K (200 pg/ml) was added and further incubated for 2 hr. The digest was phenol-extracted to separate the DNA. This DNA had a specific activity of 4500
cpmh5
To correct for the influence of the base composition of the viral DNA on the renaturation kinetics, the G+C content of the NPSV DNA was estimated from its melting temperature and buoyant density in CsCl. A T, of 72.1 -t 0.6” in 0.1x SSC based on three melting profiles indicated a G+C concentration of 44% (Fig. 1) (8). NPSV DNA had a buoyant density in CsCl of 1.704 -+ 0.001 g/ml (Fig. 2) based on five replicate gradients. This buoyant density also corresponds to a G+C concen-
f. 1.800
3000 r
1.700
3 ;
v, y ,”
4000
2000
In=
2000
IO00
5
15
IO
25
20
FRACTION
1E a 2
1.600
I5 o
35
30
NO.
FIG. 2. Buoyant density of NPSV DNA: 5000 cpm of 32P-labeled NPSV DNA and 16,000 cpm of 3Hlabeled E. coli marker DNA (1.710 g/ml) in 2.45 ml of 0.02 M Tris were mixed with 7.8 ml of saturated C&l and centrifuged in a 50-Ti angle rotor at 145,000g for 40 hr at 20”. Fractions of 0.3 ml were collected by dripping the tube from the bottom. NPSV DNA density is 1.704 g/ml. 01 0%
,
I,,,, I,,,,
1
1
,I”“, 11
O”‘,
I
. . . . .
20 -
l *.
.
.
.
..
.-g 406 '3 on :: B 8 60-
.
. ‘0 '0 '.‘. l. %
:
..
.
. .@
60-
1001 I11111' 100 ’ 1 IllI’ 0.003 0.003 0.01 0.01
.
1
I
I
I.11fiOl 0.1 0.1
1.0
COT (mole~rec/liter)
FIG. 3. Reassociation kinetics of NPSV DNA in SSC at T, -25”. DNA concentration was 15.4 wg/ml.
476
SHORT
COMMUNICATIONS
tration of 44% (9). Renaturation kinetics of the viral DNA were examined by optical methods using the procedures of Seidler and Mandel (10) with Escherichia coli DNA as a standard. A nucleotide sequence complexity of 88.5 f 3.3 x lo6 was calculated from a C$,,, of 0.23 mole *set/liter (Fig. 3) after correcting for G+C content (Table 1). The size of the NPSV genome was estimated by comparing an EcoRl digest of the NPSV DNA with DNA markers of known size (Fig. 4) using agarose gel electrophoresis (11). EcoRl produced 23 fragments and a genome size of 85.4 ? 2.6 x lo6 based on three separate digests was estimated by this procedure (Fig. 5). The agreement between the sequence complexity and the molecular size indicates that the viral genome contains predominantly non-repeated DNA. Three other restriction endonucleases were used to cleave the NPSV DNA (Rohrmann, in preparation); however, EcoRl produced the most satisfactory number and distribution of fragments for molecular weight determination. To determine if host DNA fragments are occluded in polyhedra, polyhedra purified from infected insects labeled with 32P were suspended in 0.01 M Tris, 5 mM MgCl*, pH 7.0, buffer and DNase-treated (50 pg/ml for 1 hr). The polyhedra were then sedimented at 12,000 g for 15 min, resuspended in distilled water, made 10 mM in EDTA, and dissolved with 0.1 vol of 1 M Na&O,, 0.5 M NaCl and the resulting suspension was sedimented at TABLE
1
DETERMINATION OF SEQUENCE COMPLEXITY OF NPSV DNA USING OPTICAL RENATURATION Source of DNA
C,t,,,”
Molecular weight x 106
Molecular weight x lo8 (corrected for %GC concentration)*
E. coli NPSV
5.00 ? 0 0.23 2 0.01
2200 101.2
2200 88.5
n All reactions done in SSC at T, -25”. DNA concentrations were 50 pg/ml for E. coli and 15 pg/ ml for NPSV. C$,,l for NPSV DNA was averaged from four renaturation experiments. b Based on the equation of Seidler and Mandel (10).
FIG. 4. NPSV genome size. NPSV DNA was digested with EcoRl restriction endonucleases (middle slot). EcoRl digests of h phage DNA (14) (right slot) and Haelll digests of PM 2 phage DNA (15) (left slot) were run as molecular weight markers. Digestion, electrophoresis, and photographic procedures are described elsewhere (11). Gels were electrophoresed at 50 V for 18 hr.
477
SHORT COMMUNICATIONS
75,000 g for 45 min. The supernatant was adjusted to pH 8.0 with HCl, made 1% in SDS, proteinase K-treated (200 pglml for 1 hr at 37”), and then phenol-extracted and ethanol-precipitated. The DNA was then treated with RNase (50 pg/ml) for 45 min, proteinase K-treated (200 pglml) for 1 hr, and then phenol-extracted. The extraviral DNA was shown to be doublestranded by resistance ot S1 nuclease and comprised from 8 to 30% of the total DNA recovered from different polyhedra preparations. The extraviral DNA’s buoyant density of 1.704 g/ml (Fig. 6) is identical to NPSV viral DNA and indicates it is not of host origin. In addition, when the neutralized iupernatant described above, which had not been treated to remove proteins, was put directly onto a CsCl
Relative
Distance
gradient containing 3H-labeled tussock moth and E. coli marker DNAs and centrifuged as described in Fig. 2, a buoyant density profile similar to Fig. 6 was produced. Therefore, it appears that a substantial quantity of unencapsulated viral DNA fragments is trapped in the matrix during polyhedra formation. The genome of the NPSV is similar in size and complexity to that previously determined for the NPBV pathogenic for the same insect (2). The polyhedrin molecules of both viruses are closely related in size and antigenic structure (3). However, their DNAs are readily distinguishable due to the 10% difference in their G+C concentration. Other studies, which included DNA-DNA hybridization and comparative analysis of restriction endonucle-
Migrated
------)
FIG. 5. Scan of EcoRl cleavage fragments of NPSV DNA. Numbers indicate molecular weight x 106. The molecular weights of DNA fragments were estimated by scanning the photographs of agarose gel electrophoresis profiles with an ORTEC Model 4310 densitometer and comparing the NPSV scans to those of marker DNA fragments (Fig. 4). The asterisks indicate two fragments with the same molecular weight. I.800 ; . z 1.700 > !z z
8000
1 1.600: ;
6000 :
5
IO
15 FRACTION
20 NO.
25
30
FIG. 6. Buoyant density of extraviral DNA. 32P-labeled extraviral DNA, 3H-labeled tussock moth DNA (1.695 g/ml) and SH-labeled E. coli marker DNA (1.710 g/ml) in 0.02 M Tris, pH 7.0, were mixed with CsCl and centrifuged as described in Fig. 2. The extraviral DNA density is 1.704 g/ml.
478
SHORT COMMUNICATIONS
ase fragments, further confirms that the evolution of these viruses is widely divergent (Rohrmann, in preparation). In nature, the NPSV and NPBV are often found mixed in the same insect population (12). The presence of two viruses in one population of susceptible insects may be explained by their different genetic character.- One of the most evident differences is the natholoav of the infections produced. The NPSF requires a longer period to kill insects than the NPBV (6). Insect larvae grow rapidly and therefore a slower infection would have eventual access to a greater number of insect cells. In nature, the NPSV may be capable of producing a significantly larger quantity of polyhedra per insect because of its slower pathology and therefore would have an advantage over the NPBV at low insect population densities. If insects escape initial infection by the NPSV and a more dense population develops, the NPSV would then be at an advantage because it would rapidly produce polyhedra which would readily spread to other insects because of the high insect population density. ACKNOWLEDGMENTS We thank Dr. R. H. McParland of the Department of Biochemistry, Oregon State University for assistance with the restriction endonuclease analysis. We also thank Dr. M. E. Martignoni, Forestry Sciences Laboratory, USDA, Corvallis, Oregan, for
supplying pure virus isolates, and larvae of the !aboratory strain GL of Orgyia pseudotsugoto, as well as for his advice and review of this manuscript. This project was supported by. Grant No. PL 89-106 from the Forest Service, United States Department of Agriculture. This is Oregon Agricultural Experiment Station Technical Paper No. 4582. REFERENCES 1. HUGHES, K. M., and ADDISON,R. B., J. Invertebr. Pathol. 16, 196-204 (1970). 2. ROHRMANN, G. F., CARNEGIE, J. W., MARTIGNONI, M. E., and BEAUDREAU, G. S., Virology 80, 421-425 (1977). 3. ROHRMANN, G. F., Biochemistry 16, 1631-1634 (1977). 4. MARTIGNONI, M. E., J. Viral. 1, 646-647 (1967). 5. MARTIGNONI, M. E., J. Znvertebr. Puthol. 19, 281-283 (1972). 6. MARTIGNONI, M. E. and IWAI, P. J., J. Invertebr. Puthol. 30, 255-262 (1977). 7. LAIRD, C. D., and MCCARTHY, B. J., Genetics 60, 303-322 (1968). 8. DE LEY, J., J. Bacterial. 101, 738-754 (1970). 9. MANDEL, M., IGAMBI, L., BERGENDAHL, J., DODSON, M. L., JR., and SCHELTGEN, E., J. Bacteriol. 101, 333-338 (1970). 10. SEIDLER, R. J., and MANDEL, M., J. Bacterial. 106, 608-614 (1971). 11. MCPARLAND, R. G., BROWN, L. R., and PEARSON, G. D., J. Viral. 19,1006-1011 (1976). 12. HUGHES, K. M., Canad. Ent. 108,479-484 (1976). 13. MARMUR, J., and DOTY, P., J. Mo2. Biol. 5, 109118 (1962). 14. THOMAS, M., and DAVIS, R. W., J. Mol. Biol. 91, 315-328 (1975). 15. SHAW, B. R., HERMAN, T. M., KOVACIC, R. T., BEAUDREAU, G. S., and VAN HOLDE, K. E., Proc. Nut. Acad. Sci. USA 73, 505-509 (1976).
83, 474-418 (1977)
Characterization of DNA from Polyhedral Inclusion Bodies of the Nucleopolyhedrosis Single-Rod Virus Pathogenic for Orgyia pseudotsuga ta G. F. ROHRMANN Department
of Argicultural
Chemistry,
G. S. BEAUDREAU
AND
Oregan State University,
Corvallis,
Oregon 97331
Accepted August 16,1977 A nucleotide sequence complexity of 88.5 x 10s was determined for the DNA of the nucleopolyhedrosis single-rod (unicapsid) virus of Orgyiu pseudotsugata using optical renaturation. In addition, the genome size was determined to be 85 x lo6 by comparison of EcoRl restriction endonuclease fragments with markers of known size using agarose gel electrophoresis. A G+C concentration of 44% for the viral DNA was estimated from its melting properties and buoyant density in CsCl. Evidence from buoyant density in C&l indicates that DNA which is occluded in the polyhedral matrix but not associated with virions is of viral origin.
Two nucleopolyhedrosis viruses (Baculovirus subgroup A) have been isolated from the Douglas fir tussock moth (Orgyiu pseudotsugutu). They are designated the nucleopolyhedrosis bundle (multicapsid) virus (NPBV) and the nucleopolyhedrosis single-rod (unicapsid) virus (NPSV) (I). The NPBV has been shown to have a genome size and complexity of approximately 86 x lo6 and have a G+C concentration of 54% (2). Although the NPBV and NPSV have polyhedrin molecules of close physical and antigenic structure (31, they show 1% or less genetic relatedness (Rohrmann, in preparation). This report describes studies on the NPSV DNA. The genome size was determined by comparison of restriction enzyme fragments of the genome with markers of known size, the genetic complexity was determined by examining its reassociation kinetics, and the G+C concentration was estimated by examining the DNA’s buoyant density in CsCl and by measuring its
was confirmed with dark-field microscopy of polyhedra using alkaline hydrocolloid (51, as well as by extraction of viral DNA and the determination of its buoyant density in CsCl.
T
/5h, l.OOO-65
70
75 TEMPERATURE
I
I
80
85
I
90
(“Cl
FIG. 1. Melting
profiles of NPSV DNA (01 and E. coli DNA (a) in 0.1 x SSC. The T, of NPSV DNA was estimated to be 72.1 based on the T, of E. coli of 74.5. The midpoint of the absorbance temperature profile was determined using a Gilford recording DU spectrophotometer at 260 nm (13). DNA concentration was approximately 15 *g/ml.
“I;1 addition, DNA found in the polyhedra but which did not sediment with virus was characterized to determine its origin. Viral polyhedra were produced and purified by the methods of Martignoni (4). The purity of the polyhedra preparations 474 Copyright 0 1977 by Academic Pres, Inc. All rights of reproduction in any form reserved.
ISSN 0042-6622
475
SHORT COMMUNICATIONS
Virus isolation, DNA preparation, and 32P-labeling of viral DNA are described elsewhere (2; Rohrmann, in preparation). To obtain [3H]DNA from Orgyia pseudotsugutu, 120 mg larvae (strain GL) (6) were each injected with 200 &i of L3Hlthymidine. After 24 hr at 25”, the larvae were frozen at -20”. Nuclei were isolated (7) and lysed with 2% SDS, 0.15 M NaCl, 0.100 M EDTA, pH 8.0 buffer and then digested for 2 hr at 37” with 200 pglml of proteinase K. The DNA was purified by phenol extraction followed by ethanol precipitation. The precipitate was resuspended in 0.02 M Tris, 0.005 M EDTA, pH 7.2, buffer containing RNase (50 PgI ml) and incubated for 45 min; then pro-
teinase K (200 pg/ml) was added and further incubated for 2 hr. The digest was phenol-extracted to separate the DNA. This DNA had a specific activity of 4500
cpmh5
To correct for the influence of the base composition of the viral DNA on the renaturation kinetics, the G+C content of the NPSV DNA was estimated from its melting temperature and buoyant density in CsCl. A T, of 72.1 -t 0.6” in 0.1x SSC based on three melting profiles indicated a G+C concentration of 44% (Fig. 1) (8). NPSV DNA had a buoyant density in CsCl of 1.704 -+ 0.001 g/ml (Fig. 2) based on five replicate gradients. This buoyant density also corresponds to a G+C concen-
f. 1.800
3000 r
1.700
3 ;
v, y ,”
4000
2000
In=
2000
IO00
5
15
IO
25
20
FRACTION
1E a 2
1.600
I5 o
35
30
NO.
FIG. 2. Buoyant density of NPSV DNA: 5000 cpm of 32P-labeled NPSV DNA and 16,000 cpm of 3Hlabeled E. coli marker DNA (1.710 g/ml) in 2.45 ml of 0.02 M Tris were mixed with 7.8 ml of saturated C&l and centrifuged in a 50-Ti angle rotor at 145,000g for 40 hr at 20”. Fractions of 0.3 ml were collected by dripping the tube from the bottom. NPSV DNA density is 1.704 g/ml. 01 0%
,
I,,,, I,,,,
1
1
,I”“, 11
O”‘,
I
. . . . .
20 -
l *.
.
.
.
..
.-g 406 '3 on :: B 8 60-
.
. ‘0 '0 '.‘. l. %
:
..
.
. .@
60-
1001 I11111' 100 ’ 1 IllI’ 0.003 0.003 0.01 0.01
.
1
I
I
I.11fiOl 0.1 0.1
1.0
COT (mole~rec/liter)
FIG. 3. Reassociation kinetics of NPSV DNA in SSC at T, -25”. DNA concentration was 15.4 wg/ml.
476
SHORT
COMMUNICATIONS
tration of 44% (9). Renaturation kinetics of the viral DNA were examined by optical methods using the procedures of Seidler and Mandel (10) with Escherichia coli DNA as a standard. A nucleotide sequence complexity of 88.5 f 3.3 x lo6 was calculated from a C$,,, of 0.23 mole *set/liter (Fig. 3) after correcting for G+C content (Table 1). The size of the NPSV genome was estimated by comparing an EcoRl digest of the NPSV DNA with DNA markers of known size (Fig. 4) using agarose gel electrophoresis (11). EcoRl produced 23 fragments and a genome size of 85.4 ? 2.6 x lo6 based on three separate digests was estimated by this procedure (Fig. 5). The agreement between the sequence complexity and the molecular size indicates that the viral genome contains predominantly non-repeated DNA. Three other restriction endonucleases were used to cleave the NPSV DNA (Rohrmann, in preparation); however, EcoRl produced the most satisfactory number and distribution of fragments for molecular weight determination. To determine if host DNA fragments are occluded in polyhedra, polyhedra purified from infected insects labeled with 32P were suspended in 0.01 M Tris, 5 mM MgCl*, pH 7.0, buffer and DNase-treated (50 pg/ml for 1 hr). The polyhedra were then sedimented at 12,000 g for 15 min, resuspended in distilled water, made 10 mM in EDTA, and dissolved with 0.1 vol of 1 M Na&O,, 0.5 M NaCl and the resulting suspension was sedimented at TABLE
1
DETERMINATION OF SEQUENCE COMPLEXITY OF NPSV DNA USING OPTICAL RENATURATION Source of DNA
C,t,,,”
Molecular weight x 106
Molecular weight x lo8 (corrected for %GC concentration)*
E. coli NPSV
5.00 ? 0 0.23 2 0.01
2200 101.2
2200 88.5
n All reactions done in SSC at T, -25”. DNA concentrations were 50 pg/ml for E. coli and 15 pg/ ml for NPSV. C$,,l for NPSV DNA was averaged from four renaturation experiments. b Based on the equation of Seidler and Mandel (10).
FIG. 4. NPSV genome size. NPSV DNA was digested with EcoRl restriction endonucleases (middle slot). EcoRl digests of h phage DNA (14) (right slot) and Haelll digests of PM 2 phage DNA (15) (left slot) were run as molecular weight markers. Digestion, electrophoresis, and photographic procedures are described elsewhere (11). Gels were electrophoresed at 50 V for 18 hr.
477
SHORT COMMUNICATIONS
75,000 g for 45 min. The supernatant was adjusted to pH 8.0 with HCl, made 1% in SDS, proteinase K-treated (200 pglml for 1 hr at 37”), and then phenol-extracted and ethanol-precipitated. The DNA was then treated with RNase (50 pg/ml) for 45 min, proteinase K-treated (200 pglml) for 1 hr, and then phenol-extracted. The extraviral DNA was shown to be doublestranded by resistance ot S1 nuclease and comprised from 8 to 30% of the total DNA recovered from different polyhedra preparations. The extraviral DNA’s buoyant density of 1.704 g/ml (Fig. 6) is identical to NPSV viral DNA and indicates it is not of host origin. In addition, when the neutralized iupernatant described above, which had not been treated to remove proteins, was put directly onto a CsCl
Relative
Distance
gradient containing 3H-labeled tussock moth and E. coli marker DNAs and centrifuged as described in Fig. 2, a buoyant density profile similar to Fig. 6 was produced. Therefore, it appears that a substantial quantity of unencapsulated viral DNA fragments is trapped in the matrix during polyhedra formation. The genome of the NPSV is similar in size and complexity to that previously determined for the NPBV pathogenic for the same insect (2). The polyhedrin molecules of both viruses are closely related in size and antigenic structure (3). However, their DNAs are readily distinguishable due to the 10% difference in their G+C concentration. Other studies, which included DNA-DNA hybridization and comparative analysis of restriction endonucle-
Migrated
------)
FIG. 5. Scan of EcoRl cleavage fragments of NPSV DNA. Numbers indicate molecular weight x 106. The molecular weights of DNA fragments were estimated by scanning the photographs of agarose gel electrophoresis profiles with an ORTEC Model 4310 densitometer and comparing the NPSV scans to those of marker DNA fragments (Fig. 4). The asterisks indicate two fragments with the same molecular weight. I.800 ; . z 1.700 > !z z
8000
1 1.600: ;
6000 :
5
IO
15 FRACTION
20 NO.
25
30
FIG. 6. Buoyant density of extraviral DNA. 32P-labeled extraviral DNA, 3H-labeled tussock moth DNA (1.695 g/ml) and SH-labeled E. coli marker DNA (1.710 g/ml) in 0.02 M Tris, pH 7.0, were mixed with CsCl and centrifuged as described in Fig. 2. The extraviral DNA density is 1.704 g/ml.
478
SHORT COMMUNICATIONS
ase fragments, further confirms that the evolution of these viruses is widely divergent (Rohrmann, in preparation). In nature, the NPSV and NPBV are often found mixed in the same insect population (12). The presence of two viruses in one population of susceptible insects may be explained by their different genetic character.- One of the most evident differences is the natholoav of the infections produced. The NPSF requires a longer period to kill insects than the NPBV (6). Insect larvae grow rapidly and therefore a slower infection would have eventual access to a greater number of insect cells. In nature, the NPSV may be capable of producing a significantly larger quantity of polyhedra per insect because of its slower pathology and therefore would have an advantage over the NPBV at low insect population densities. If insects escape initial infection by the NPSV and a more dense population develops, the NPSV would then be at an advantage because it would rapidly produce polyhedra which would readily spread to other insects because of the high insect population density. ACKNOWLEDGMENTS We thank Dr. R. H. McParland of the Department of Biochemistry, Oregon State University for assistance with the restriction endonuclease analysis. We also thank Dr. M. E. Martignoni, Forestry Sciences Laboratory, USDA, Corvallis, Oregan, for
supplying pure virus isolates, and larvae of the !aboratory strain GL of Orgyia pseudotsugoto, as well as for his advice and review of this manuscript. This project was supported by. Grant No. PL 89-106 from the Forest Service, United States Department of Agriculture. This is Oregon Agricultural Experiment Station Technical Paper No. 4582. REFERENCES 1. HUGHES, K. M., and ADDISON,R. B., J. Invertebr. Pathol. 16, 196-204 (1970). 2. ROHRMANN, G. F., CARNEGIE, J. W., MARTIGNONI, M. E., and BEAUDREAU, G. S., Virology 80, 421-425 (1977). 3. ROHRMANN, G. F., Biochemistry 16, 1631-1634 (1977). 4. MARTIGNONI, M. E., J. Viral. 1, 646-647 (1967). 5. MARTIGNONI, M. E., J. Znvertebr. Puthol. 19, 281-283 (1972). 6. MARTIGNONI, M. E. and IWAI, P. J., J. Invertebr. Puthol. 30, 255-262 (1977). 7. LAIRD, C. D., and MCCARTHY, B. J., Genetics 60, 303-322 (1968). 8. DE LEY, J., J. Bacterial. 101, 738-754 (1970). 9. MANDEL, M., IGAMBI, L., BERGENDAHL, J., DODSON, M. L., JR., and SCHELTGEN, E., J. Bacteriol. 101, 333-338 (1970). 10. SEIDLER, R. J., and MANDEL, M., J. Bacterial. 106, 608-614 (1971). 11. MCPARLAND, R. G., BROWN, L. R., and PEARSON, G. D., J. Viral. 19,1006-1011 (1976). 12. HUGHES, K. M., Canad. Ent. 108,479-484 (1976). 13. MARMUR, J., and DOTY, P., J. Mo2. Biol. 5, 109118 (1962). 14. THOMAS, M., and DAVIS, R. W., J. Mol. Biol. 91, 315-328 (1975). 15. SHAW, B. R., HERMAN, T. M., KOVACIC, R. T., BEAUDREAU, G. S., and VAN HOLDE, K. E., Proc. Nut. Acad. Sci. USA 73, 505-509 (1976).