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Effect of nuclear polyhedrosis virus infection on polyribosome content of gypsy moth larvae (Porthetria dispar)
Copyright 0 1972 by Academic Press, Inc. All rights of reproduction in my form rescrwd
Experimental Cell Research 74 (1972) 519-524
EFFECT
OF NUCLEAR
POLYRIBOSOME
POLYHEDROSIS CONTENT
VIRUS
OF GYPSY
INFECTION
MOTH
ON
LARVAE
(PORTHETRIA DISPAR) ALVA A. APP and R. R. GRANADOS Boyce Thompson Institute for Plant Research, Inc., Yonkers, N.Y. 10101, USA
SUMMARY Polyribosomes have been purified from whole gypsy moth (Porthetriu &spar) larvae and from excised fat body tissue. These polyribosomes have been characterized by their sedimentation on sucrose density gradients, their sensitivity to ribonuclease, electron microscopy, and their ability to support in vitro amino acid incorporation. Approx. 85 % of the ribosomes purified from gypsy moth tissue were in the polyribosome fraction. Only 40 % of these polyribosomes were extracted from the tissue in the absence of 0.5 % sodium deoxycholate. There was a steady decrease in the polyribosome content of gypsy moth larvae after infection with nuclear polyhedrosis virus.
Protein synthesis in vivo is believed to be accomplished by clusters of ribosomes attached to a strand of messengerRNA. These aggregateshave been detected in both eucaryotic and procaryotic cells and are termed polyribosomes. In insects, polyribosomes have been studied in the genus Drosophila
METHODS AND MATERIALS Insect larvae rearing GYPSYmoth larvae were obtained from eggs collected from-the field during the fall of 1970 e&ntially as described by Doane I1 11.Eggs were sterilized with0.2% sodium hypochlorite and-the larvae were reared aseptically on sterile artificial diet.
[I, 21, Musca [3], Trichoplusiu [4], Acheta [5],
and Bombyx [6]. As part of a study of the synthesis of virus and viral inclusion body proteins in insect tissue, a method has been developed for the isolation of polyribosomes from gypsy moth larvae (Porthetria dispar). The method may be employed for isolation of polyribosomes from either intact larvae or from excised fat body tissue. Some of the characteristics of the polyribosomes have been investigated and the effect of nuclear polyhedrosis viral (NPV) infection on the polyribosome content of gypsy moth larvae has been examined. 34 - 721810
Infection of larvae with nuclear polyhedrosis virus The nuclear polyhedrosis virus was obtained from diseased gypsy moth larvae collected in the field (Mahopac, New York). The virus-containing polyhedral inclusion bodies (PIB) were extracted from dead larvae by grinding them in distilled water and allowing the tissue homogenate to putrefy. The PIB were clarified by differential centrifugation and stored at - 10°C as stock suspensions containing 2.6 x lo7 PIB ml. Second to third instar larvae (av wt 28 mg) were infected per OSby allowing them to feed on a small piece of artificial diet contaminated with 20 ~1 of the stock PIB suspension (5.2 x lo5 PIB/larvae). After 48 h the residual inoculum was removed and replaced with fresh sterile diet. The polyribosomal content of infected larvae was determined every 24 h during a 4-day test period. Exptl Cell Res 74 (1972)
520 A. A. App h R. R. Granados Polyribosome isolation Either intact larvae (generally 0.4-l .Og) or the excised fat body tissue was frozen in liquid nitrogen and crushed-by mortar and pestle. The disrupted tissue was gently resuspended in 5.0 ml of the following medium at 2 C: 0.45 M sucrose; 0.02 M Tris, pH 8.7; 10 mM M&l,: 4 mM dithiothreitol; 16 mM KCI; 100 @g/ml c$l
In vitro amino acid incorporation The in vitro amino acid incorporation assays were performed as previously described [lo]. The reaction mixtures contained the following components in a total volume of 0.5 ml: 50 mM Tris, pH 7.8; 5 mM Mg acetate; 60 mM KCI; 0.1 mM GTP; 2.3 mM phosphoenolpyruvate; 2 pug phosphoenolpyruvate kinase; 4 mM dithiothreitol; 10 000 cpm of V-aminoacyl-tRNA (290 cpm/wg .- ..~- yeast tRNA); 25 ,ul of larva supernatant fraction; and 2.7 Az6, &ts df polyribosomes. The ‘*C-amino acid mixture was purchased from New England Nuclear Corp., Boston: Mass.
Electron microscopy The polyribosome preparations were kept on ice prior to examination by electron microscopy. Small droplets of the unfixed ribosomes were placed on carbon coated Formvar-covered conper grids for 2 min before the excess fluid was bldtied off with a filter paper strip [12]. For negative staining, the grids were covered with a drop of 2 % aqueous uranyl acetate, pH 4.0. After 2 min, the excess stain was removed and- the grid allowed to dry. All ribosome samples were examined within 1 h after preparation in a Zeiss EM9IIS electron microscope. Exptl Cell Res 74 (1972)
RESULTS Polyribosomes were prepared from intact larvae and subjected to sucrose densitygradient centrifugation (fig. 1). Addition of RNase to the preparation before centrifugation resulted
in a shift of much of the ultra-
violet absorption material from the heavier region to the 80s region of the gradient. Intact larvae that were frozen in liquid nitrogen and stored at -20°C also yielded polyribosomal preparations whose absorption profiles were similar to those depicted in fig. 1. Similar absorption patterns were obtained with polyribosome preparations from excised fat body tissue (fig. 2). An indication of the reproducibility of the method can be obtained by examining the absorption patterns of duplicate preparations in fig. 2. The polyribosome preparations exhibited breakdown in vitro into smaller size aggregates and monomeric ribosomes upon storage for 20 h at 0°C (fig. 3). The total ribosomal population from excised fat tissue was prepared as described in Methods and examined by density-gradient
0.4
I I
80 s J :i!! ;\
t
Figs. 1-3. Abscissa: effluent (ml); ordinate: Azao. Fin. 1. Density gradient centrifugation of a volvribo-so&e preparation from whole larvae. 100 pl bf preparation containing 6.3 Azaounits of material was layered. on a 15-30% sucrose gradient containing 20 mM Tris, pH 7.6; 15 mM KCI; 5 mM dithiothreitol; and 1 mM Mg acetate. The gradients were spun at 40 000 rpm in a SW 41 rotor for l& h at 2°C. The absorbance tracing was obtained with a 10 mm flow cell on an Isco density gradient fractionator. For convenience, the approximate position of the 80s ribosome is noted. A portion of the preparation was treated with 0.3 pg of ribonuclease for 5 min at 30°C before layering.
NPV Injection and gypsy moth polyribosomes
521
Table 1. In vitro amino acid incorporation b? polyribosomes from Porthetria dispar
Conditions
0
5
IO
Fig. 2. Density gradient centrifugation
of duplicate polyribosome preparations from excised fat body tissue. The procedure was identical with that described in fig. 1 except that 150,uI of each preparation containing 6.2 A,,, units was layered on separate gradients.
centrifugation. Approx. 85 % of the ribosomes were found in aggregates larger than 80 S. Similar values were also obtained for the total ribosomal population from intact larvae. It was also observed that approx. 60% of the polyribosomal population remained in the pellet after centrifuging at 12 000 g of a crude homogenate prepared from intact larvae in the absence of 0.5 % sodium deoxycholate. This suggests that a good portion of the polyribosomes is attached to membranes. The poiyribosomal preparations are capable of supporting in vitro amino acid incorporation into material insoluble in trichloroacetic acid (table 1). This process requires GTP, is sensitive to ribonuclease, and is enhanced by the addition of either larvae or rice supernatant fraction. Electron microscopy of the total ribosome 04
0.22
:
._-* 0’
0
I 5
IO
Fig. 3. Effect of storage at 0°C on a polyribosome preparation from excised fat bodies. The procedure was identical with that described in fig. 1 except that a portion of the preparation was stored at 0°C for 20 h before being subjected to density gradient centrifugation (---). 100 ,A of preparation containing 4.2 AzSOunits were layered on the gradients.
Complete - GTP -Larval supernatant + RNase - Polyribosomes -Larval supernatant + Rice supernatant
W amino acid incorporation (cm) 988, 1 071
191 415 55
61 I 553
fraction (fig. 4) indicated both monosomes and polysomes were present. Some ribosomes were clearly differentiated into the large and small subunit. The large subunit usually appeared as a sphere flattened on the surface adjacent to the small subunit (fig. 4a). In contrast, the small subunit was frequently seen as a flattened cap. A furrow or line of high density across the body of the ribosomes probably corresponds to the region of apposition of the two ribosomal subunits (fig. 4a, b). Small round structures approx. 5 nm in diameter were observed on many ribosomes (fig. 4b). The dimensions of individual ribosome particles were approx. 33 x 28 nm. A number of experiments were performed to examine the effect of nuclear polyhedrosis virus infection on the polyribosome content of third instar gypsy moth larvae. Inclusion body formation was evident 3 days after infection (per OS).By the 4th day, the infected larvae were very inactive and hardly able to move. Most were dead by the 5th day. Little growth or weight increase was observed with infected larvae. On the other hand, control larvae continued normal growth. Thus, the longer the infection period, the greater the difference between infected and healthy larvae in size and physiological development. There was a steady decrease in polyribosome content during the first 4 days as a result of Exptl Cell Res 74 (1972)
Fig. 4. (a), Electron micrograph of gypsy moth larvae polyribcsomes. A few single 80 S monosomes (M)and many polyribosomes (P) are shown. The large (L) and small (S) subunits can be clearly differentiated in some ribosome particles. x 180 000; (b), Electron micrograph of a gypsy moth larvae ribosome population consisting primarily of monosomes. The cleavage furrow (arrows) between the small and large subunit(s) is noticeable in many ribosomes. x 180 000. Inset: Several small round structures (awows) can be seen on some ribosomes. x 269 500. Expil Cell Res 74 (1972)
NPV Infection and gypsy moth polyribosomes
_. b
523
1 80 s infected
(48
h)
(96
h)
” 04-
c
__ d 80 S
80 Infected
O0
(72
5
h1
S Infected
IO
0
5
IO
Fig. 5. Abscissa: effluent (ml;) ordinate A,,,. Effect of nuclear polyhedrosis virus infection on the polyribosome content of whole larvae, (a) 24 h, (b) 48 h, (c) 72 h, and (d) 96 h after infection. Polyribosomes were prepared from healthy and infected larvae, resuspended in 300 ,A of resuspension buffer and examined by density gradient centrifugation with the procedure described in fig. 1. 100 ~1 of preparation containing the following amounts of ALaounits were layered: (a) contol 3.4, infected 2.9; (b) 1.9; (c) 1.7; (d) 1.0.
nuclear polyhedrosis virus infection (fig. 5). The polyribosome content of control larvae (fig. 5a) remained essentially the same throughout the test period. No increase in polyribosome content in infected larvae was noted during the period that inclusion body formation was in progress. One might have expected an increase in polyribosome content at this time in order to account for the massive amount of inclusion body protein that is formed. Under these experimental conditions, therefore, it was not possible to distinguish a change in polyribosome content which could be related to PIB synthesis from the general decline of cellular functions which accompany a terminal infection.
DISCUSSION A method has been described for the preparation of polyribosomes from gypsy moth larvae. The identification of the polyribosomes is based on the following criteria: rate
of sedimentation on sucrose density gradients electron microscopy, sensitivity to RNase, and their ability to support in vitro amino acid incorporation. This method is not applicable to all Lepidoptera insect tissue since it failed to yield polyribosomes from the fat body of Estigmene acrea larvae. There appears to be a steady decrease in the polyribosome content after infection of gypsy moth larvae with nuclear polyhedrosis virus. One might have expected to see a decline in the content of host polyribosomes in the early stages of infection and a subsequent increase in the level of polyribosomes as viral proteins and inclusion body proteins are synthesized [13-1.51. This assumes that there is a degree of synchrony of infection in the larva tissue and that the frequency of sampling was sufficient to detect subtle shifts in polyribosome content. It should be noted that a recent report has also indicated that the polyribosome content of Trichoplusia is reduced late in the course of a granulosis virus infection [4]. Expfl Cell Res 74 (1972)
524 A. A. App & R. R. Granados We are indebted to Dr Charles C. Doane of the Connecticut Agricultural Experiment Station, New Haven, Conn., for supplying the gypsy moth eggs used in part of this work and for his advice on insect rearing procedures. The capable technical assistance of Mrs Barbara Di Gregorio, Mrs Elena Manasse, and Mrs Marybeth Naughton is acknowledged. This research was supported in part by Grant No. ROI-AI 08836 from NIH.
REFERENCES 1. Stafford, D W, Whitney, J B, III & Lucchesi, J C, Exptl cell res 64 (1971) 29. 2. Boshes, R A, J cell bio146 (1970) 477. 3. Tsiapalis, C M, Hayashi, Y & Chefurka, W, Nature 214 (1967) 358. 4. Webb, S R & Young, S Y, J invertebrate path01 18 (1971) 148.
Exptl Cell ResI74 (1972)
5. Kaulenas, M S, J insect physiol 16 (1970) 813. 6. Prudhomme, J-C, Guelin, M, Grasset, L & Daillie J, Exptl cell res 63 (1970) 373. 7. Wettstein, F 0, Staehelin, T, Nell, H, Nature 197 (1963) 430. 8. Ellem, K A 0 & Colter, J S, J cellular camp physiol 58 (1963) 267. 9. Laga, E M, Baliga, B S & Munro, H N, Biochim biophys acta 213 (1970) 391. 10. App, A A, Bulis, M d & McCarthy, W J, Plant physiol 47 (1971) 81. 11. Ddane, C C, J invertebrate path01 14 (1969) 199. 12. Bruskov. V I & Kiselev. N A. J mol bio137 (19681 . , 367. 13. Sydiskis, R J & Roizman, B, Science 153 (1966) 76. 14. Watanabe, H & Kobayashi, M, J invertebrate path01 14 (1969) 102. 15. Watanabe, H, J invertebrate path01 9 (1967) 428. Received February 15, 1972
Experimental Cell Research 74 (1972) 519-524
EFFECT
OF NUCLEAR
POLYRIBOSOME
POLYHEDROSIS CONTENT
VIRUS
OF GYPSY
INFECTION
MOTH
ON
LARVAE
(PORTHETRIA DISPAR) ALVA A. APP and R. R. GRANADOS Boyce Thompson Institute for Plant Research, Inc., Yonkers, N.Y. 10101, USA
SUMMARY Polyribosomes have been purified from whole gypsy moth (Porthetriu &spar) larvae and from excised fat body tissue. These polyribosomes have been characterized by their sedimentation on sucrose density gradients, their sensitivity to ribonuclease, electron microscopy, and their ability to support in vitro amino acid incorporation. Approx. 85 % of the ribosomes purified from gypsy moth tissue were in the polyribosome fraction. Only 40 % of these polyribosomes were extracted from the tissue in the absence of 0.5 % sodium deoxycholate. There was a steady decrease in the polyribosome content of gypsy moth larvae after infection with nuclear polyhedrosis virus.
Protein synthesis in vivo is believed to be accomplished by clusters of ribosomes attached to a strand of messengerRNA. These aggregateshave been detected in both eucaryotic and procaryotic cells and are termed polyribosomes. In insects, polyribosomes have been studied in the genus Drosophila
METHODS AND MATERIALS Insect larvae rearing GYPSYmoth larvae were obtained from eggs collected from-the field during the fall of 1970 e&ntially as described by Doane I1 11.Eggs were sterilized with0.2% sodium hypochlorite and-the larvae were reared aseptically on sterile artificial diet.
[I, 21, Musca [3], Trichoplusiu [4], Acheta [5],
and Bombyx [6]. As part of a study of the synthesis of virus and viral inclusion body proteins in insect tissue, a method has been developed for the isolation of polyribosomes from gypsy moth larvae (Porthetria dispar). The method may be employed for isolation of polyribosomes from either intact larvae or from excised fat body tissue. Some of the characteristics of the polyribosomes have been investigated and the effect of nuclear polyhedrosis viral (NPV) infection on the polyribosome content of gypsy moth larvae has been examined. 34 - 721810
Infection of larvae with nuclear polyhedrosis virus The nuclear polyhedrosis virus was obtained from diseased gypsy moth larvae collected in the field (Mahopac, New York). The virus-containing polyhedral inclusion bodies (PIB) were extracted from dead larvae by grinding them in distilled water and allowing the tissue homogenate to putrefy. The PIB were clarified by differential centrifugation and stored at - 10°C as stock suspensions containing 2.6 x lo7 PIB ml. Second to third instar larvae (av wt 28 mg) were infected per OSby allowing them to feed on a small piece of artificial diet contaminated with 20 ~1 of the stock PIB suspension (5.2 x lo5 PIB/larvae). After 48 h the residual inoculum was removed and replaced with fresh sterile diet. The polyribosomal content of infected larvae was determined every 24 h during a 4-day test period. Exptl Cell Res 74 (1972)
520 A. A. App h R. R. Granados Polyribosome isolation Either intact larvae (generally 0.4-l .Og) or the excised fat body tissue was frozen in liquid nitrogen and crushed-by mortar and pestle. The disrupted tissue was gently resuspended in 5.0 ml of the following medium at 2 C: 0.45 M sucrose; 0.02 M Tris, pH 8.7; 10 mM M&l,: 4 mM dithiothreitol; 16 mM KCI; 100 @g/ml c$l
In vitro amino acid incorporation The in vitro amino acid incorporation assays were performed as previously described [lo]. The reaction mixtures contained the following components in a total volume of 0.5 ml: 50 mM Tris, pH 7.8; 5 mM Mg acetate; 60 mM KCI; 0.1 mM GTP; 2.3 mM phosphoenolpyruvate; 2 pug phosphoenolpyruvate kinase; 4 mM dithiothreitol; 10 000 cpm of V-aminoacyl-tRNA (290 cpm/wg .- ..~- yeast tRNA); 25 ,ul of larva supernatant fraction; and 2.7 Az6, &ts df polyribosomes. The ‘*C-amino acid mixture was purchased from New England Nuclear Corp., Boston: Mass.
Electron microscopy The polyribosome preparations were kept on ice prior to examination by electron microscopy. Small droplets of the unfixed ribosomes were placed on carbon coated Formvar-covered conper grids for 2 min before the excess fluid was bldtied off with a filter paper strip [12]. For negative staining, the grids were covered with a drop of 2 % aqueous uranyl acetate, pH 4.0. After 2 min, the excess stain was removed and- the grid allowed to dry. All ribosome samples were examined within 1 h after preparation in a Zeiss EM9IIS electron microscope. Exptl Cell Res 74 (1972)
RESULTS Polyribosomes were prepared from intact larvae and subjected to sucrose densitygradient centrifugation (fig. 1). Addition of RNase to the preparation before centrifugation resulted
in a shift of much of the ultra-
violet absorption material from the heavier region to the 80s region of the gradient. Intact larvae that were frozen in liquid nitrogen and stored at -20°C also yielded polyribosomal preparations whose absorption profiles were similar to those depicted in fig. 1. Similar absorption patterns were obtained with polyribosome preparations from excised fat body tissue (fig. 2). An indication of the reproducibility of the method can be obtained by examining the absorption patterns of duplicate preparations in fig. 2. The polyribosome preparations exhibited breakdown in vitro into smaller size aggregates and monomeric ribosomes upon storage for 20 h at 0°C (fig. 3). The total ribosomal population from excised fat tissue was prepared as described in Methods and examined by density-gradient
0.4
I I
80 s J :i!! ;\
t
Figs. 1-3. Abscissa: effluent (ml); ordinate: Azao. Fin. 1. Density gradient centrifugation of a volvribo-so&e preparation from whole larvae. 100 pl bf preparation containing 6.3 Azaounits of material was layered. on a 15-30% sucrose gradient containing 20 mM Tris, pH 7.6; 15 mM KCI; 5 mM dithiothreitol; and 1 mM Mg acetate. The gradients were spun at 40 000 rpm in a SW 41 rotor for l& h at 2°C. The absorbance tracing was obtained with a 10 mm flow cell on an Isco density gradient fractionator. For convenience, the approximate position of the 80s ribosome is noted. A portion of the preparation was treated with 0.3 pg of ribonuclease for 5 min at 30°C before layering.
NPV Injection and gypsy moth polyribosomes
521
Table 1. In vitro amino acid incorporation b? polyribosomes from Porthetria dispar
Conditions
0
5
IO
Fig. 2. Density gradient centrifugation
of duplicate polyribosome preparations from excised fat body tissue. The procedure was identical with that described in fig. 1 except that 150,uI of each preparation containing 6.2 A,,, units was layered on separate gradients.
centrifugation. Approx. 85 % of the ribosomes were found in aggregates larger than 80 S. Similar values were also obtained for the total ribosomal population from intact larvae. It was also observed that approx. 60% of the polyribosomal population remained in the pellet after centrifuging at 12 000 g of a crude homogenate prepared from intact larvae in the absence of 0.5 % sodium deoxycholate. This suggests that a good portion of the polyribosomes is attached to membranes. The poiyribosomal preparations are capable of supporting in vitro amino acid incorporation into material insoluble in trichloroacetic acid (table 1). This process requires GTP, is sensitive to ribonuclease, and is enhanced by the addition of either larvae or rice supernatant fraction. Electron microscopy of the total ribosome 04
0.22
:
._-* 0’
0
I 5
IO
Fig. 3. Effect of storage at 0°C on a polyribosome preparation from excised fat bodies. The procedure was identical with that described in fig. 1 except that a portion of the preparation was stored at 0°C for 20 h before being subjected to density gradient centrifugation (---). 100 ,A of preparation containing 4.2 AzSOunits were layered on the gradients.
Complete - GTP -Larval supernatant + RNase - Polyribosomes -Larval supernatant + Rice supernatant
W amino acid incorporation (cm) 988, 1 071
191 415 55
61 I 553
fraction (fig. 4) indicated both monosomes and polysomes were present. Some ribosomes were clearly differentiated into the large and small subunit. The large subunit usually appeared as a sphere flattened on the surface adjacent to the small subunit (fig. 4a). In contrast, the small subunit was frequently seen as a flattened cap. A furrow or line of high density across the body of the ribosomes probably corresponds to the region of apposition of the two ribosomal subunits (fig. 4a, b). Small round structures approx. 5 nm in diameter were observed on many ribosomes (fig. 4b). The dimensions of individual ribosome particles were approx. 33 x 28 nm. A number of experiments were performed to examine the effect of nuclear polyhedrosis virus infection on the polyribosome content of third instar gypsy moth larvae. Inclusion body formation was evident 3 days after infection (per OS).By the 4th day, the infected larvae were very inactive and hardly able to move. Most were dead by the 5th day. Little growth or weight increase was observed with infected larvae. On the other hand, control larvae continued normal growth. Thus, the longer the infection period, the greater the difference between infected and healthy larvae in size and physiological development. There was a steady decrease in polyribosome content during the first 4 days as a result of Exptl Cell Res 74 (1972)
Fig. 4. (a), Electron micrograph of gypsy moth larvae polyribcsomes. A few single 80 S monosomes (M)and many polyribosomes (P) are shown. The large (L) and small (S) subunits can be clearly differentiated in some ribosome particles. x 180 000; (b), Electron micrograph of a gypsy moth larvae ribosome population consisting primarily of monosomes. The cleavage furrow (arrows) between the small and large subunit(s) is noticeable in many ribosomes. x 180 000. Inset: Several small round structures (awows) can be seen on some ribosomes. x 269 500. Expil Cell Res 74 (1972)
NPV Infection and gypsy moth polyribosomes
_. b
523
1 80 s infected
(48
h)
(96
h)
” 04-
c
__ d 80 S
80 Infected
O0
(72
5
h1
S Infected
IO
0
5
IO
Fig. 5. Abscissa: effluent (ml;) ordinate A,,,. Effect of nuclear polyhedrosis virus infection on the polyribosome content of whole larvae, (a) 24 h, (b) 48 h, (c) 72 h, and (d) 96 h after infection. Polyribosomes were prepared from healthy and infected larvae, resuspended in 300 ,A of resuspension buffer and examined by density gradient centrifugation with the procedure described in fig. 1. 100 ~1 of preparation containing the following amounts of ALaounits were layered: (a) contol 3.4, infected 2.9; (b) 1.9; (c) 1.7; (d) 1.0.
nuclear polyhedrosis virus infection (fig. 5). The polyribosome content of control larvae (fig. 5a) remained essentially the same throughout the test period. No increase in polyribosome content in infected larvae was noted during the period that inclusion body formation was in progress. One might have expected an increase in polyribosome content at this time in order to account for the massive amount of inclusion body protein that is formed. Under these experimental conditions, therefore, it was not possible to distinguish a change in polyribosome content which could be related to PIB synthesis from the general decline of cellular functions which accompany a terminal infection.
DISCUSSION A method has been described for the preparation of polyribosomes from gypsy moth larvae. The identification of the polyribosomes is based on the following criteria: rate
of sedimentation on sucrose density gradients electron microscopy, sensitivity to RNase, and their ability to support in vitro amino acid incorporation. This method is not applicable to all Lepidoptera insect tissue since it failed to yield polyribosomes from the fat body of Estigmene acrea larvae. There appears to be a steady decrease in the polyribosome content after infection of gypsy moth larvae with nuclear polyhedrosis virus. One might have expected to see a decline in the content of host polyribosomes in the early stages of infection and a subsequent increase in the level of polyribosomes as viral proteins and inclusion body proteins are synthesized [13-1.51. This assumes that there is a degree of synchrony of infection in the larva tissue and that the frequency of sampling was sufficient to detect subtle shifts in polyribosome content. It should be noted that a recent report has also indicated that the polyribosome content of Trichoplusia is reduced late in the course of a granulosis virus infection [4]. Expfl Cell Res 74 (1972)
524 A. A. App & R. R. Granados We are indebted to Dr Charles C. Doane of the Connecticut Agricultural Experiment Station, New Haven, Conn., for supplying the gypsy moth eggs used in part of this work and for his advice on insect rearing procedures. The capable technical assistance of Mrs Barbara Di Gregorio, Mrs Elena Manasse, and Mrs Marybeth Naughton is acknowledged. This research was supported in part by Grant No. ROI-AI 08836 from NIH.
REFERENCES 1. Stafford, D W, Whitney, J B, III & Lucchesi, J C, Exptl cell res 64 (1971) 29. 2. Boshes, R A, J cell bio146 (1970) 477. 3. Tsiapalis, C M, Hayashi, Y & Chefurka, W, Nature 214 (1967) 358. 4. Webb, S R & Young, S Y, J invertebrate path01 18 (1971) 148.
Exptl Cell ResI74 (1972)
5. Kaulenas, M S, J insect physiol 16 (1970) 813. 6. Prudhomme, J-C, Guelin, M, Grasset, L & Daillie J, Exptl cell res 63 (1970) 373. 7. Wettstein, F 0, Staehelin, T, Nell, H, Nature 197 (1963) 430. 8. Ellem, K A 0 & Colter, J S, J cellular camp physiol 58 (1963) 267. 9. Laga, E M, Baliga, B S & Munro, H N, Biochim biophys acta 213 (1970) 391. 10. App, A A, Bulis, M d & McCarthy, W J, Plant physiol 47 (1971) 81. 11. Ddane, C C, J invertebrate path01 14 (1969) 199. 12. Bruskov. V I & Kiselev. N A. J mol bio137 (19681 . , 367. 13. Sydiskis, R J & Roizman, B, Science 153 (1966) 76. 14. Watanabe, H & Kobayashi, M, J invertebrate path01 14 (1969) 102. 15. Watanabe, H, J invertebrate path01 9 (1967) 428. Received February 15, 1972