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Ribonucleic acid synthesis in nuclei isolated from Musca domestica
Insect Biochem., 1971, x, 363-372. [Scientechnica (Publishers) Ltd.]
363
RIBONUCLEIC ACID SYNTHESIS IN NUCLEI ISOLATED FROM MUSCA DOMESTICA* MOISES AGOSIN Department of Zoology, University of Georgia, Athens, Georgia 30601, U.S.A.
(Received 15 .~an., I971) ABSTRACT Nuclei isolated from adult houseflies by centrifugation in 2"2 M sucrose3 mM CaCI~ and filtration through io-gm, pore nylon bolting cloth have an RNA/DNA ratio of 3"o8. This ratio is 4"05 in nuclei isolated from larval tissues. N/~clei catalyse the incorporation of labelled nudeoside triphosphates into acid-insoluble RNA. The RNA polymer,meof nuclei depends on the presence of four nucleoside triphosphates and either Mn a+ or Mg m+. RNA synthesis is inhibited by DNase, RNase, and actinomycin D. DDT added in vitro has no effect on the polymerase activity. The reaction product from nuclei isolated from adult insects and in the presence of Mg 2+ is high in adenine and uridine, with a U/G ratio of 1.77. In the presence of Mn 2+ the U/G ratio decreases to t'33. The product from larval nuclei in the presence of Mg 2+ is high in guanine, whereas in the presence of Mn 2+ it is high in uridine. Nuclear preparations from insects pretreated with DDT have a higher polymerase activity than control preparations. The increase in activity is only evident during the first hour of treatment. CYTOPLASMIC and nuclear RNA in insects is substantially increased as the result of treatment with chlorinated insecticides (Balazs and Agosin, i968a; Litvak and Agosin, 1968; Litvak, Tarago-Litvak, Poblete, and Agosin, 1968 ). RNA obtained from 2,2-bis(p-chlorophenyl)-i,i,x-trichloroethane (DDT)-treated insects has a higher template activity when compared to non-stimulated RNA (Balazs and Agosin, 1968b ). This observation suggests that D D T induces the synthesis of mRNA, which is supported by the appearance in DDT-treated insects of a rapidly labelled RNA with a sedimentation coefficient of I6S (Balazs and Agosin, i968a ). Stimulation of RNA synthesis in Calliphora has also been demonstrated after ecdysone injection, and the participation of m R N A seems to be fairly well substantiated (Karlson and Sekeris, 1963). RNA's have been characterized in houseflies by sucrose density gradient analysis and by methylated albumin-kieselguhr column chromatography, and some observations on their synthesis have been reported (Balazs and Agosin, i968a, b). Litvak and Agosin (1968) have shown that D D T is preferentially bound to nuclear proteins, probably histones. It is then possible to speculate that increased m R N A production may be due to a derepression phenomenon involving the weakening of ionic bonds between D N A and histones. It is assumed that a single enzyme, DNA-dependent RNA polymerase (E.C. 2.7.7.6), is responsible for the synthesis of all classes of RNA (Igarashi and Yura, 1969). It is then pertinent to note that RNA polymerase is apparently involved in the * This work was supported in part by U.S. Public Health Service Grant No. AI-o99o2-oi.
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Insect Biochem.
effect of steroid hormones on R N A synthesis (Stackhouse, Chetsanga, and Tan, 1968 ). I n order to study R N A synthesis more directly than is possible with intact insects, a nuclear system capable of incorporating ribonucleoside triphosphates into R N A has been prepared. T h e present report concerns some properties of the system, the characterization of the R N A synthesized in vitro in terms of base composition and behaviour in sucrose density gradients, and the effect which D D T has on the nuclear system. MATERIALS AND METHODS REAGENTS ATP, CTP, GTP, and UTP were purchased from Mann Research Laboratories, New York. [SH]ATP (i8'o c. per mmole), [3H]CTP (13'5 c. per mmole), [3H]GTP (1"o9 c. per mmole), and [sH]UTP (I7"8 c. per mmole) were obtained from Schwarz BioResearch, Orangeburg, New York. DNA (Type I) and RNA were purchased from Sigma Chemical Co., St. Louis, Missouri. DNase-I, DPFF, and RNase were obtained 3 × crystallized from Mann Research Laboratories, New York. Nylon bolting cloth (Nytex), 1o-I~m. pore size, was purchased from Tobler, Ernst, and Traber Inc., New York. Sucrose was 'density gradient' grade from Mann Research Laboratories. Actinomycin D was obtained from Calbiochem, Los Angeles, California. INSECTS
Adults 5 days after emergence and 4-day-old larvae of the Fc and DDT-45 housefly strains were used throughout. The latter strain, highly resistant to DDT, was obtained through the courtesy of Dr. Albert S. Perry (CDC, Savannah, Georgia). The characteristics of the Fc strain have been reported elsewhere (Litvak and Agosin, I968). PREPARATIONOF NUCLEI Adult insects were rapidly chilled and then homogenized in a Sorvall homogenizer (30 seconds at 45 V. ; 30 seconds at maximum speed) in lO volumes of 0"25 M sucrose containing 3 m m CaClv Larvae were collected, carefully rinsed with the same medium, and then homogenized as above. All these and the following operations were carried out at 2-4 ° C. The homogenate was filtered through 4 layers of cheese-cloth, and then centrifuged at 800 g for lO minutes. The supernatant fluid was discarded and the inner walls of the tubes were carefully cleaned. The pellet was homogenized in an all-glass Potter-Elvehjem homogenizer with the same amount of the same medium, and the centrifugation repeated until the supernatant fluid was clear. The supernatant fluid was again discarded and the pellet was suspended in a small amount of 2"2 M sucrose-3 mM CaClg, and homogenized with I stroke in a hand-driven all-glass Potter-Elvehjem homogenizer. Following this, 2"2 M sucrose-3 m m CaCI2 was added to the nuclei homogenate to a volume of about 60 ml. per 1o g. original fresh weight, and centrifuged at 25,500 r.p.m, for 60 minutes in an L2-65B Spinco centrifuge rotor No. 42. The supernatant fluid was discarded and the inner walls of the tubes were carefully cleaned. The pellet was then homogenized in about 20 ml. per g. original weight of 0"25 M sucrose--3 mM CaC12, and the homogenate filtered by gravity through io-gm, pore size Nytex. The slightly yellowish filtrate was centrifuged at 8oo g for io minutes, the supernatant discarded, and the pellet homogenized with about 20 ml. of i'3 M sucrose-3 mM CaClv The homogenate was centrifuged at 5ooo g for 1o minutes. The resulting white nuclear pellet was finally suspended in o'25 M sucrose to give a final protein concentration of 2o rag. per ml. Microscopical examination showed that the nuclei were reasonably clean. The yield of nuclei in adult insects was o'o33 mg. DNA per g. fresh weight, while in larvae the yield was about o.o6 mg. DNA per g. fresh weight. ANALYTICALPROCEDURES DNA was determined according to Burton (I968) and RNA by the orcinol procedure (Mejbaum, I939). Protein was estimated by the Folin method (Lowry, Rosebrough, Farr, and Randall, x951).
i97i, t
RNA SYNTHESIS IN HOUSEFLY NUCLEI
365
In vitro SYNTHESISOVRNA Enzyme activity was measured by following the incorporation of [aH]ATP into trichloro-acetic acid-insoluble material. Standard conditions correspond to 'Tris' buffer, pH 7"8, Ioo graoles; MgCl 2 or MnCls, o'25 gmole; B-mercaptoethanol, 5 gmoles; CTP, GTP, UTP, 0"5 ltmole each; [sHO]ATP, 0. 5 gmole (specific activity, 4.0 gc. per gmole); nuclei, o'x ml. containing o'5-I"o rag. protein. The final volume was 0"5 ml. The reaction was started by the addition of nuclei and, unless otherwise stated, incubation was for 5 minutes at 38° C. The reaction was stopped by the addition of I'o ml. io per cent trichloro-acetic acid containing o.i M sodium pyrophosphate. After the addition of trichloro-acetic acid, the reaction mixtures were left standing at 2-4 ° C. for at least 30 minutes and then filtered through millipore filters. The filters were washed with three 15-ml. portions of 5 per cent trichloro-acetic acid/o- t M sodium pyrophosphate, and dried overnight at room temperature. The filters were then placed into scintillation vials containing 5 ml. of scintillation mixture [o'4 per cent 2,5-diphenyloxazole (PPO), o'ox per cent 1,4-bis-[2-(5-phenyloxazolyl)]-benzene (POPOP)], and counted in a Nuclear Chicago Mark I scintillation counter. SUCROSEDENSITYGRADIENTS Linear sucrose density gradients from Io to 40 per cent sucrose were prepared as described by Litvak and Agosin (I968). RNA obtained from nuclei or synthesized in vitro was dissolved in I'o ml. o'oI M acetate buffer, pH 5.x, carefully layered on top of a 25-ml. gradient, and centrifuged at 23,500 r.p.m, in the rotor of a SW-25.I Spinco centrifuge. The gradients were fractionated as previously described (Balazs and Agosin, x968a). RNA ISOLATION Nuclear or in vitro synthesized RNA was isolated by phenol-dodecyl sulphate as described by Balazs and Agosin (x968a, b). BASE COMPOSITION OF RNA SYNTHESIZED in vitro
The base composition of the polymerase product was estimated by following the incorporation of each nucleotide labelled with tritium in the presence of the other three corresponding unlabelled ones. RESULTS CHEMICAL COMPOSITION OFNUCLEI As has been shown for whole Drosophila (Hastings and Kirby, i966 ) and for crude nuclei preparations from housefly (Litvak and Agosin, i968), the amount of R N A in purified nuclei is much higher than D N A (Table I). This is exactly opposite to what is found in mammalian tissues(Hogeboom, Schneider, and Striebich, i952 ) and in Triatorna infestans (Litvak and others, I968 ). This striking difference is even more evident in larval tissues. On the other hand, the ratio of D N A to protein in larvae is essentially the same as in rat liver, but the amount of protein in relation to D N A in adult insects is much higher. Similar high ratios of protein to D N A have been reported also for t u m o u r nuclei (Muramatsu, Smctena, and Bush, 1963)and for salivary gland nuclei from Drosophila hyda" (Boyd, Berendes, and Boyd, i968 ). It is possible that the differences between adult and larval between tissues may correspond to transition characteristics between these two developmental stages. AssAY OF DNA-DEPENDF_~NTR N A POLYMERASEACTIVITY U n d e r the conditions described in Materials and Methods the R N A polymerase activity of nuclei isolated from adult insects was higher in the presence of M n 2+ than M g 2+, but the reverse was observed in larval nuclei (Table H). Congote, Sekeris, and
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Insect Biochem.
AGO$IN
Karlson (i969) have reported that the RNA polymerase of nuclei isolated from fat body of Calliphora is activated by Mg ~+ rather than M n 2+, which indicates that species differences may be involved in this phenomenon or that different polymerases are present. Preliminary experiments showed that optimal activity was obtained with o.2-o. 5 ~tmole Table/.--CHEMICAL COMPOSITION OFNUCLEI ISOLATED FROMADULTANDLARVALHOUSEFLY MASS RATIO COMPONENT
Adults DNA RNA
Protein
Larvae
I "O
3"o8 14'5 [
Rat Liver
I "O
I'O
4"65 3"92
0'27 4"05
Values for rat liver nuclei have been included for comparison purposes. Results obtained with the DDT-strain. See under Materials and Methods for experimental details.
Table//.--AssAy FOR R N A POLYMERASE IN ISOLATED NUCLEI FROM ADULT AND LARVAL HOUSEFLY
SYSTEM
Complete Minus nucleotides Minus CTP Minus GTP Minus UTP Plus (NH4)2SO~, 1o #moles Minus Mg or Mn 2+ Plus DNase, Ioo pg. Plus actinomycin D, ioo pg. Plus RNase, lOO pg. DDT, 2"0 × 1o- s M DDT, 5"8 × Io- 8 M Larvae, complete FI strain, Adults
INCORPORATIONOF [3H]ATP INTO TCA-INSOLUBLE MATERIAL(m/tmoles [3H]ATP per mg. protein) M g 2+
Mn2+
o.9 I o'I4 o'o9 0.28
1"78 o'18 o'I8
O'O I'IO
o'38 0'26 O"12
o'92 o'9o 1"78
O'O O'O
2"56 0"0 0"32 0'02 0"2 1"8I i "80 0"72 1 "73
See Materials and Methods for details of the complete system. of MgCI~ per reaction mixture, but concentrations above 0,25 ~tmole of MnCI2 were inhibitory. A similar observation has been made for rat liver nuclei (Novello and Stirpe, i969). T h e activity was dependent on the presence o f t h e four nudeotides, was inhibited by either DNase, RNase, or actinomycin D, and was stimulated by (NH4)2SO4. As shown for rat liver (Novello and Stirpe, 1969), (NH4)2SO4 exerted a stronger stimulation
i97t,
i
367
RNA SYNTHESIS IN HOUSEFLY NUCLEI
on the Mn-activated polymerase. Fig. I shows the kinetics of the reaction, with maximum incorporation of [SH]ATP within 5 minutes. The activity is quite comparable to mammalian and bacterial tissues (Krakow and Ochoa, t963 ; Nakamoto, Fox, and Weiss, i964). When the activity is expressed in terms of DNA, larval tissues seem to have less enzyme than the adult nuclei (Table H). As shown in Fig. z, the activity was proportional, 1.6'
•
~ 1.2.
i
~. o.o<
~
0.4Mg ~+ I
"w Ma~+
I 0'
' 20
' 1o 30 T/me (minutes)
;0
' 60
FIG. x.--Kinetics of RNA synthesis by isolated nuclei of adult insects (DDT-45 strain). Conditions as the complete system of Table II. MgC1z and MnCla as indicated, o'z5 I~mole each.
1.0 r-
0,5
1.0
I
Nuclei concentration (rng. p~otein)
FIO. z.--Effect of nuclei concentration on the rate of RNA synthesis. Complete system as in Table II, but with MnCI~ (o'z5 limole) added instead of MgCI~. DDT-45 strain, nuclei from adult insects. within certain limits, to the amount of nuclei added, and hence to DNA. Optimal temperature for the Mn~+-activated enzyme was about 380 C. (F/g. 3). However, this parameter was not further explored because it has been shown to depend on the concentrations of cations and nucleotides (Novello and Stirpe, i969). Contrary to what has been reported for rat liver nuclei (NoveUo and Stirpe, i969) , the Mn~+-activated enzyme had
368
Insect Biochem.
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the same nucleotide concentration requirement as the MgZ+-activated one (Fig. 4). Higher nudeotide concentrations appeared to be inhibitory for the MnZ+-enzyme, but not for the Mg~+-enzyme. Data not shown here indicate that the optimal pH in both cases was broadly 7.8.
o
~1.0.
' 20
,'0
30 Ternt~erature {'C3
FIG. 3.uEffect of temperature on the rate of RNA synthesis by isolated nuclei. Conditions as in Fig. z.
2.0
E
r.~ 1.0 O
-6
0.5
1.0
It'5
MgT M
2~'0
Nuclei concentration
F I c . 4.--Effect of nucleotide concentration on the rate of R N A synthesis by isolated nuclei. Conditions as in Fig. z. x ' o = o ' 5 lmaole of each nucleotide; z ' o = x ' o lamole of each nucleotide.
COMPOSITION OF THE R N A SYNTHESIZED in vitro
•When the base composition of the polymerase product was determined by the procedure outlined under Materials and Methods (Table III), it was found that only the RNA synthesized by adult nuclei in the presence of Mg displayed base pairing in accord with the G-=C and A = U rule, which is similar to what has been found for
~97 I, x
m~A SYNTHESIS IN HOUSEFLY NUCLEI
369
housefly ribosomal RNA (Balazs and Agosin, i968b ). The U/G ratio for the Mg-RNA was 1.77, which is close to the U/G ratio of housefly ribosomal RNA (Balazs and Agosin, i968b ). However, in the case of the Mn-RNA, the U/G ratio was lower, 1.33. This resembles the rapidly labelled RNA reported by Wyatt and Linzen (i 965) in Hylophora which is induced by ecdysone and has a U/G ratio of I"3. As to the larval product, neither the Mg- nor the Mn-RNA had any resemblance to housefly ribosomal RNA or DNA. Whether the RNA synthesized by adult nuclei in the presence of Mn may have messenger characteristics, as in Hylaphora (Wyatt and Linzen, i965) , remains to be established. Table III.--NucLEOTIDS COMPOSITION OF THE R N A SYNTHESIZEDin vitro CONDITIONS
ATP
CTP
GTP
UTP
A+G C+U
Adults
Mg2+-activated Mn~+-activated
28"03 30"55
x8.z 28"43
x9"9 x7"65
33"3 23"8
0'9I I'I2
i .60 I'I8
Larvae
M g 2+-activated M n a+-activated
25"5 25"o
x9"7 I5"o
4z'5 x8.o
x3"3 4z'o
I'39
0"70
o'6z 2"0 3
3z'4 I7"8 z9"6
I8"7 32"2 I9"Z
x6'z 33"o x9"7
32"4 I7"O I9"7
I "00 I "0I 0"96
SOURCE
Housefly z8s r R N A Rat liver z8s r R N A Housefly D N A
A+U C+G
z.85 0"53 z'54
T h e composition of housefly and rat liver ribosomal R N A as well as housefly D N A are included for comparison purposes. See under Materials and Methods for experimental details. Nuclei from adults and larvae of the D D T - 4 5 strain; the values correspond to the average of 5 closely agreeing determinations. SUCROSE DENSITY GRADIENT ANALYSIS
Total nuclear RNA obtained by phenol extraction (Balazs and Agosin, i968b ) at 2-4 ° C. showed the presence of the z8s and x8s ribosomal RNA's, and the 4s peak corresponding to transfer RNA. A small component of 35-4os was also detected. This profile resembles the one reported for nuclear RNA from Tenebriopupae (Ilan, i969). The RNA synthesized in ~itro showed no definite peak, but rather the radioactivity was dispersed between 8s and 35s. The same profile was obtained in the case of Mg- or Mn-RNA formed by adult nuclei, although the Mn-RNA tended to be of lower molecular weight. It should be pointed out that the xo--4o per cent sucrose gradient did not separate satisfactorily the various nuclear RNA components. A 5-zo per cent sucrose gradient was even less adequate. Preliminary experiments suggest that the temperature of extraction may not be ideal in this case (Ilan, I969). EFVECT OF D D T
ON THE POLYMERASE SYSTEM
Evidence suggests that steroid hormones may act at the nuclear level (Dukes and Sekeris, i965). Thus, cortisol increases RNA synthesis when added in vitro to a rat liver chromatin preparation (Stackhouse and others, 1968). In insects Berendes and Boyd (i969) could not show any effect of ecdysterone added in vitro to a nuclear preparation isolated from salivary glands of Drosop/u'/a hydei. However, Congote and others (i969)
37 °
Insect Biochem.
AGOSIN
have shown some evidence indicating that ecdysone stimulates Mg-activated RNA polymerase from Calliphora nuclei when added in vitro. As shown in Table II, D D T added in vitro had no effect on the polymerase activity. Concentrations of D D T higher than those shown in Table H were clearly inhibitory. In view of these results, attempts to enhance the housefly RNA polymerase in vivo were undertaken. Adult insects of the 250 -
o-:
~s
200-
7E
i. IO0 .
.
.
.
.
I
I
!
30
60
90
.
.
!
I
I
120
150
180
Time of intoxication (minutes)
FIG. 5.--Effect of pretreatment with DDT on the rate of RNA synthesis by isolated nuclei from adult housefly. Insects of the Fc strain were topically treated with z.o lal. of acetone containing i.o lag. DDT. After the indicated intervals nuclei were prepared and the polymerase assayed as indicated in Table I1, in the presence of MnCI 2.
Fc strain were topically treated with I.O lag. D D T in acetone as described previously (Gil, Fine, Dinamarca, Balazs, Busvine, and Agosin, i968), and nuclei were obtained at timed intervals. As shown in Fig. 5, RNA synthesis was increased in the polymerase system by pretreatment with D D T . T h e changes occurred markedly after 3 ° minutes, and then the activity started to decline. After 9 ° minutes of intoxication no effect of D D T on the polymerase was evident.
DISCUSSION Nuclei isolatedfrom housefly are clearlydifferentfrom mammalian nuclei in their D N A content. The amount of D N A is lower in housefly,and whereas the R N A / D N A ratio in rat livernuclei iso.27, in adult fliesit ismuch higher. However, the synthesisof RNA, as catalyscd by DNA-dcpcndcnt R N A polymcrasc, is quite similar to that in mammalian tissues. The synthesis requiresD N A as primer (as shown by the effectof DNasc), the product is sensitiveto RNasc, and the reactionis inhibitedby actinomycin D. However, the product of the reaction seems to be quite heterogeneous in housefy, whereas in liveran R N A of 18s is essentiallysynthesized (Widncll and Tata, 1956). As indicated before, the polymerasc product in the presence of M n s+ was mainly of low molecular weight. This may be duc to the degradationphenomenon observed with total R N A isolatedin the absence of M g s+ (Balazsand Agosin, x968b ). The composition of the product deservessome comment. Total R N A from Drosophila and housefly (Hastings and Kirby, 1966; Balazs and Agosin, 1968b) has high contents
I97I, I
RNA SYNTHESISIN HOUSEFLYNUCLEI
371
of AMP and UMP, whereas rat liver RNA is high in G M P and CMP. RNA from silkmoth tissues is high in G M P (Wyatt and Linzen, 1965). In the in vitro system the RNA produced in the presence of Mg s+ had a base composition similar to housefly 28s RNA, high in AMP and UMP. However, the MnS+-activated system yielded a product that did not resemble either housefly ribosomal RNA or DNA. On the other hand, larval nuclei incubated with Mg ~+ gave a product with a composition similar to housefly transfer RNA, but when Mn ~+ was present the composition of the product was again different. Intact nuclei should provide an environment similar to the whole cell, which should allow the possibility of studying the effects of compounds such as D D T or hormones on the complex formed by DNA, RNA, histones, and non-histone proteins. Although no effect of D D T was found in vitro, the increase in polymerase activity of nuclei isolated from DDT-treated insects indicates that the effect of this compound could be at the nuclear level. It is interesting to note that in vivo D D T increases the incorporation of labelled uracil into RNA (Balazs and Agosin, x968a). This increase parallels the kinetics of D D T enhancement of the polymerase. Furthermore, the above changes precede the synthesis of induced proteins by D D T (Litvak and Agosin, i968 ). The above results clearly implicate DNA-dependent RNA polymerase as the enzyme responsible for the increased RNA synthesis in response to D D T treatment. Topically applied D D T is rapidly localized at the nuclear level (Litvak and Agosin, i968), and it is then plausible that the compound produces the above changes, stimulating transcription. However, whether D D T or other compounds act at the chromatin level by derepression of D N A genetic loci or by direct action on the polymerase remains to be established. REFERENCES BALAZS,I., and AGOSIN,M. (x968a), ' The effect of 2,2-bis-(p-chlorophenyl)-i,t,x-trichloroethane on RNA metabolism in Musca domestica (L.)', Biochim. biophys. Acta, x579 x-7. BALAZS, I., and AGOSlN, M. (I968b), 'Isolation and characterization of ribonucleic acid from Musca domestica (L.)', Comp. Biochem. Physiol., 27, 227-237. BER~NDm,H. D., and BOYD,J. R. (x969), 'Structural and functional properties of polytene nuclei isolated from salivary glands of Drosophila hydei',J. Cell Biol., 4x~ 59x-599. BoYD,J. R., BIZR~Dm,H. D., and BOYD,J. H. (I968), ' Mass preparation of nuclei from the larval salivary gland of Drosophila hydei', J. Cell Biol., 38j 369-376. BURTON,K. (I968), 'Determination of DNA concentration with diphenylamine', in Methods in Enzymology (ed. GROSSMAN,L., and MOLDAVE,K.), vol. XII (B), pp. 163-169. New York: Academic Press. CONOOTE,L. F., SEKEmS,C. E., and KAm~SON,P. (x969), 'On the mechanism of hormone action. XIII. Stimulatory effects of ecdysone, juvenile hormone, and ions on RNA synthesized in fat body cell nuclei from Ualliphora erythrocephala', Expl. Cell Res., 56, 338-346. DUKES, P. P., and SEKmUS,C. E. (I965), 'Stimulierung des Einbans von (14C)-Uracil in Ribonucleis~iure von Ratten Leberkernen durch Cortisol in vitro', Z. physiol. Chem., 34x, x49-I 5 x. Gv, L., FI~, B. C., DIN^M^RCA,M. L., B~AZS, I., Busvl~m, J. R., and AoOSIN, M. (x968), 'Biochemical studies on insecticide resistance in Musca domestica', Entomologia exp. appl., xx, 15-29.
HASTiNesS,J. R., and KmB~', K. S. (i 966),' The nucleic acids of Drosophila melanogaster', Biochem. 3., IOO~532-539. Hoo~ooM, G. E., SCHNEIDEa,W. C., and STEmBICH,I. (X952), ' Cytochemical studies. V. On the isolation and biochemical properties of liver cell nuclei',.J ¢. biol. Chem., I96, i I x-I2O. IGARASHI,K., and YURA, T. (x969), 'The role of RNA polymerase in genetic transcription', Biochem. biophys. Res. Commun., 34, 65--69.
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ILAN, J. (i969) , 'Studies on template-active ribonucleic acid from nuclei of insect pupae', Biochemistry, 8, 4825-483 x. KARIaON, P., and SEKERIS,C. E. (1963), ' Eedysone and insect steroidal hormones, and its mode of action', Prog. Horm. Res., 22, 473-502. KRAKOW, J. S., and OCHOA, S. (1963), 'Ribonucleic acid nucleotidyl transferase of Azotobacter vinelandi', Biochem. Z., 338, 796-806. LITVAK, S., and Ac.osm, M. (1968), 'Protein synthesis in polysomes from houseflies and the effect of 2,2-bis-(p-ehlorophenyl)- I, I, l-trichloroethane ', Biochemistry, 7, 1560- x567. LITVAK, S., TAaAco-LITVAK, L., POBL~a'E,P., and ACOSlN, M. (I968), 'Evidence for the D D T induced synthesis of messenger ribonucleic acid in Triatoma infestans', Comp. Biochem. Physiol., 26, 45-56. Low~Y, O. H., ROSEBaOUOH,N. J., FAme, H. C., and RANDALL,R. J. (I95I), 'Protein measurement with the Folin reagent',.7, biol. Chem., I93, 265-275. MEJBAOM, W. (Z939), ' l~ber die Bestimmung kleiner Pentosemengen insbesondere in Derivaten der Adenyls~aure', Hoppe-Seyler's Z. physiol. Chem., 258, i x7-120. MUaAMATSU, M., SME'rANA, K. S., and BUSCH, H. (1963), 'Quantitative aspects of isolation of nuclei of the Walker carcinosarcoma in liver of the rat', Cancer Res., 23, 51o-518. N^KAMOTO, T., FOX, C. F., and WEIss, S. B. (I964), 'Enzymatic synthesis of ribonucleie acid. I. Properties of ribonucleic acid polymerase from extracts of Micrococcus lysodeihticus', ft. biol. Chem., 239, 167-I74. NOWLLO, F., and STIRPE, F. (I969), 'Experimental conditions affecting ribonucleic acid polymerase in isolated rat liver nuclei. Effect of nucleoside phosphate concentration, temperature, ammonium sulphate and heparin', Biochem. ft., xI2, 721-727. STACKHOUSE,H. L., CH~rSANOA,C. J., and TAN, C. H. (1968), ' T h e effect of cortisol on genetic transcription in rat liver chromatin', Biochim. biophys, Acta, 155, 159-168. WIDN'XLL,C. C., and TATA, J. R. (1966), ' Studies on the stimulation by ammonium sulphate of the DNA-dependent RNA polymerase of isolated rat liver nuclei', Biochim. biophys. Acta, I23, 478-492. WYATT, G. R., and LINZEN, B. (1965), ' T h e metabolism of ribonuclei acid in Cecropia silkrnoth pupae in diapause, during development and after injury', Biochim. biophys, Acta, xo3, 388-400.
Key Word Index: Nucleic acid synthesis, RNA polymerase, isolated nuclei, D D T , Musca domestica, mRNA.
363
RIBONUCLEIC ACID SYNTHESIS IN NUCLEI ISOLATED FROM MUSCA DOMESTICA* MOISES AGOSIN Department of Zoology, University of Georgia, Athens, Georgia 30601, U.S.A.
(Received 15 .~an., I971) ABSTRACT Nuclei isolated from adult houseflies by centrifugation in 2"2 M sucrose3 mM CaCI~ and filtration through io-gm, pore nylon bolting cloth have an RNA/DNA ratio of 3"o8. This ratio is 4"05 in nuclei isolated from larval tissues. N/~clei catalyse the incorporation of labelled nudeoside triphosphates into acid-insoluble RNA. The RNA polymer,meof nuclei depends on the presence of four nucleoside triphosphates and either Mn a+ or Mg m+. RNA synthesis is inhibited by DNase, RNase, and actinomycin D. DDT added in vitro has no effect on the polymerase activity. The reaction product from nuclei isolated from adult insects and in the presence of Mg 2+ is high in adenine and uridine, with a U/G ratio of 1.77. In the presence of Mn 2+ the U/G ratio decreases to t'33. The product from larval nuclei in the presence of Mg 2+ is high in guanine, whereas in the presence of Mn 2+ it is high in uridine. Nuclear preparations from insects pretreated with DDT have a higher polymerase activity than control preparations. The increase in activity is only evident during the first hour of treatment. CYTOPLASMIC and nuclear RNA in insects is substantially increased as the result of treatment with chlorinated insecticides (Balazs and Agosin, i968a; Litvak and Agosin, 1968; Litvak, Tarago-Litvak, Poblete, and Agosin, 1968 ). RNA obtained from 2,2-bis(p-chlorophenyl)-i,i,x-trichloroethane (DDT)-treated insects has a higher template activity when compared to non-stimulated RNA (Balazs and Agosin, 1968b ). This observation suggests that D D T induces the synthesis of mRNA, which is supported by the appearance in DDT-treated insects of a rapidly labelled RNA with a sedimentation coefficient of I6S (Balazs and Agosin, i968a ). Stimulation of RNA synthesis in Calliphora has also been demonstrated after ecdysone injection, and the participation of m R N A seems to be fairly well substantiated (Karlson and Sekeris, 1963). RNA's have been characterized in houseflies by sucrose density gradient analysis and by methylated albumin-kieselguhr column chromatography, and some observations on their synthesis have been reported (Balazs and Agosin, i968a, b). Litvak and Agosin (1968) have shown that D D T is preferentially bound to nuclear proteins, probably histones. It is then possible to speculate that increased m R N A production may be due to a derepression phenomenon involving the weakening of ionic bonds between D N A and histones. It is assumed that a single enzyme, DNA-dependent RNA polymerase (E.C. 2.7.7.6), is responsible for the synthesis of all classes of RNA (Igarashi and Yura, 1969). It is then pertinent to note that RNA polymerase is apparently involved in the * This work was supported in part by U.S. Public Health Service Grant No. AI-o99o2-oi.
364
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Insect Biochem.
effect of steroid hormones on R N A synthesis (Stackhouse, Chetsanga, and Tan, 1968 ). I n order to study R N A synthesis more directly than is possible with intact insects, a nuclear system capable of incorporating ribonucleoside triphosphates into R N A has been prepared. T h e present report concerns some properties of the system, the characterization of the R N A synthesized in vitro in terms of base composition and behaviour in sucrose density gradients, and the effect which D D T has on the nuclear system. MATERIALS AND METHODS REAGENTS ATP, CTP, GTP, and UTP were purchased from Mann Research Laboratories, New York. [SH]ATP (i8'o c. per mmole), [3H]CTP (13'5 c. per mmole), [3H]GTP (1"o9 c. per mmole), and [sH]UTP (I7"8 c. per mmole) were obtained from Schwarz BioResearch, Orangeburg, New York. DNA (Type I) and RNA were purchased from Sigma Chemical Co., St. Louis, Missouri. DNase-I, DPFF, and RNase were obtained 3 × crystallized from Mann Research Laboratories, New York. Nylon bolting cloth (Nytex), 1o-I~m. pore size, was purchased from Tobler, Ernst, and Traber Inc., New York. Sucrose was 'density gradient' grade from Mann Research Laboratories. Actinomycin D was obtained from Calbiochem, Los Angeles, California. INSECTS
Adults 5 days after emergence and 4-day-old larvae of the Fc and DDT-45 housefly strains were used throughout. The latter strain, highly resistant to DDT, was obtained through the courtesy of Dr. Albert S. Perry (CDC, Savannah, Georgia). The characteristics of the Fc strain have been reported elsewhere (Litvak and Agosin, I968). PREPARATIONOF NUCLEI Adult insects were rapidly chilled and then homogenized in a Sorvall homogenizer (30 seconds at 45 V. ; 30 seconds at maximum speed) in lO volumes of 0"25 M sucrose containing 3 m m CaClv Larvae were collected, carefully rinsed with the same medium, and then homogenized as above. All these and the following operations were carried out at 2-4 ° C. The homogenate was filtered through 4 layers of cheese-cloth, and then centrifuged at 800 g for lO minutes. The supernatant fluid was discarded and the inner walls of the tubes were carefully cleaned. The pellet was homogenized in an all-glass Potter-Elvehjem homogenizer with the same amount of the same medium, and the centrifugation repeated until the supernatant fluid was clear. The supernatant fluid was again discarded and the pellet was suspended in a small amount of 2"2 M sucrose-3 mM CaClg, and homogenized with I stroke in a hand-driven all-glass Potter-Elvehjem homogenizer. Following this, 2"2 M sucrose-3 m m CaCI2 was added to the nuclei homogenate to a volume of about 60 ml. per 1o g. original fresh weight, and centrifuged at 25,500 r.p.m, for 60 minutes in an L2-65B Spinco centrifuge rotor No. 42. The supernatant fluid was discarded and the inner walls of the tubes were carefully cleaned. The pellet was then homogenized in about 20 ml. per g. original weight of 0"25 M sucrose--3 mM CaC12, and the homogenate filtered by gravity through io-gm, pore size Nytex. The slightly yellowish filtrate was centrifuged at 8oo g for io minutes, the supernatant discarded, and the pellet homogenized with about 20 ml. of i'3 M sucrose-3 mM CaClv The homogenate was centrifuged at 5ooo g for 1o minutes. The resulting white nuclear pellet was finally suspended in o'25 M sucrose to give a final protein concentration of 2o rag. per ml. Microscopical examination showed that the nuclei were reasonably clean. The yield of nuclei in adult insects was o'o33 mg. DNA per g. fresh weight, while in larvae the yield was about o.o6 mg. DNA per g. fresh weight. ANALYTICALPROCEDURES DNA was determined according to Burton (I968) and RNA by the orcinol procedure (Mejbaum, I939). Protein was estimated by the Folin method (Lowry, Rosebrough, Farr, and Randall, x951).
i97i, t
RNA SYNTHESIS IN HOUSEFLY NUCLEI
365
In vitro SYNTHESISOVRNA Enzyme activity was measured by following the incorporation of [aH]ATP into trichloro-acetic acid-insoluble material. Standard conditions correspond to 'Tris' buffer, pH 7"8, Ioo graoles; MgCl 2 or MnCls, o'25 gmole; B-mercaptoethanol, 5 gmoles; CTP, GTP, UTP, 0"5 ltmole each; [sHO]ATP, 0. 5 gmole (specific activity, 4.0 gc. per gmole); nuclei, o'x ml. containing o'5-I"o rag. protein. The final volume was 0"5 ml. The reaction was started by the addition of nuclei and, unless otherwise stated, incubation was for 5 minutes at 38° C. The reaction was stopped by the addition of I'o ml. io per cent trichloro-acetic acid containing o.i M sodium pyrophosphate. After the addition of trichloro-acetic acid, the reaction mixtures were left standing at 2-4 ° C. for at least 30 minutes and then filtered through millipore filters. The filters were washed with three 15-ml. portions of 5 per cent trichloro-acetic acid/o- t M sodium pyrophosphate, and dried overnight at room temperature. The filters were then placed into scintillation vials containing 5 ml. of scintillation mixture [o'4 per cent 2,5-diphenyloxazole (PPO), o'ox per cent 1,4-bis-[2-(5-phenyloxazolyl)]-benzene (POPOP)], and counted in a Nuclear Chicago Mark I scintillation counter. SUCROSEDENSITYGRADIENTS Linear sucrose density gradients from Io to 40 per cent sucrose were prepared as described by Litvak and Agosin (I968). RNA obtained from nuclei or synthesized in vitro was dissolved in I'o ml. o'oI M acetate buffer, pH 5.x, carefully layered on top of a 25-ml. gradient, and centrifuged at 23,500 r.p.m, in the rotor of a SW-25.I Spinco centrifuge. The gradients were fractionated as previously described (Balazs and Agosin, x968a). RNA ISOLATION Nuclear or in vitro synthesized RNA was isolated by phenol-dodecyl sulphate as described by Balazs and Agosin (x968a, b). BASE COMPOSITION OF RNA SYNTHESIZED in vitro
The base composition of the polymerase product was estimated by following the incorporation of each nucleotide labelled with tritium in the presence of the other three corresponding unlabelled ones. RESULTS CHEMICAL COMPOSITION OFNUCLEI As has been shown for whole Drosophila (Hastings and Kirby, i966 ) and for crude nuclei preparations from housefly (Litvak and Agosin, i968), the amount of R N A in purified nuclei is much higher than D N A (Table I). This is exactly opposite to what is found in mammalian tissues(Hogeboom, Schneider, and Striebich, i952 ) and in Triatorna infestans (Litvak and others, I968 ). This striking difference is even more evident in larval tissues. On the other hand, the ratio of D N A to protein in larvae is essentially the same as in rat liver, but the amount of protein in relation to D N A in adult insects is much higher. Similar high ratios of protein to D N A have been reported also for t u m o u r nuclei (Muramatsu, Smctena, and Bush, 1963)and for salivary gland nuclei from Drosophila hyda" (Boyd, Berendes, and Boyd, i968 ). It is possible that the differences between adult and larval between tissues may correspond to transition characteristics between these two developmental stages. AssAY OF DNA-DEPENDF_~NTR N A POLYMERASEACTIVITY U n d e r the conditions described in Materials and Methods the R N A polymerase activity of nuclei isolated from adult insects was higher in the presence of M n 2+ than M g 2+, but the reverse was observed in larval nuclei (Table H). Congote, Sekeris, and
366
Insect Biochem.
AGO$IN
Karlson (i969) have reported that the RNA polymerase of nuclei isolated from fat body of Calliphora is activated by Mg ~+ rather than M n 2+, which indicates that species differences may be involved in this phenomenon or that different polymerases are present. Preliminary experiments showed that optimal activity was obtained with o.2-o. 5 ~tmole Table/.--CHEMICAL COMPOSITION OFNUCLEI ISOLATED FROMADULTANDLARVALHOUSEFLY MASS RATIO COMPONENT
Adults DNA RNA
Protein
Larvae
I "O
3"o8 14'5 [
Rat Liver
I "O
I'O
4"65 3"92
0'27 4"05
Values for rat liver nuclei have been included for comparison purposes. Results obtained with the DDT-strain. See under Materials and Methods for experimental details.
Table//.--AssAy FOR R N A POLYMERASE IN ISOLATED NUCLEI FROM ADULT AND LARVAL HOUSEFLY
SYSTEM
Complete Minus nucleotides Minus CTP Minus GTP Minus UTP Plus (NH4)2SO~, 1o #moles Minus Mg or Mn 2+ Plus DNase, Ioo pg. Plus actinomycin D, ioo pg. Plus RNase, lOO pg. DDT, 2"0 × 1o- s M DDT, 5"8 × Io- 8 M Larvae, complete FI strain, Adults
INCORPORATIONOF [3H]ATP INTO TCA-INSOLUBLE MATERIAL(m/tmoles [3H]ATP per mg. protein) M g 2+
Mn2+
o.9 I o'I4 o'o9 0.28
1"78 o'18 o'I8
O'O I'IO
o'38 0'26 O"12
o'92 o'9o 1"78
O'O O'O
2"56 0"0 0"32 0'02 0"2 1"8I i "80 0"72 1 "73
See Materials and Methods for details of the complete system. of MgCI~ per reaction mixture, but concentrations above 0,25 ~tmole of MnCI2 were inhibitory. A similar observation has been made for rat liver nuclei (Novello and Stirpe, i969). T h e activity was dependent on the presence o f t h e four nudeotides, was inhibited by either DNase, RNase, or actinomycin D, and was stimulated by (NH4)2SO4. As shown for rat liver (Novello and Stirpe, 1969), (NH4)2SO4 exerted a stronger stimulation
i97t,
i
367
RNA SYNTHESIS IN HOUSEFLY NUCLEI
on the Mn-activated polymerase. Fig. I shows the kinetics of the reaction, with maximum incorporation of [SH]ATP within 5 minutes. The activity is quite comparable to mammalian and bacterial tissues (Krakow and Ochoa, t963 ; Nakamoto, Fox, and Weiss, i964). When the activity is expressed in terms of DNA, larval tissues seem to have less enzyme than the adult nuclei (Table H). As shown in Fig. z, the activity was proportional, 1.6'
•
~ 1.2.
i
~. o.o<
~
0.4Mg ~+ I
"w Ma~+
I 0'
' 20
' 1o 30 T/me (minutes)
;0
' 60
FIG. x.--Kinetics of RNA synthesis by isolated nuclei of adult insects (DDT-45 strain). Conditions as the complete system of Table II. MgC1z and MnCla as indicated, o'z5 I~mole each.
1.0 r-
0,5
1.0
I
Nuclei concentration (rng. p~otein)
FIO. z.--Effect of nuclei concentration on the rate of RNA synthesis. Complete system as in Table II, but with MnCI~ (o'z5 limole) added instead of MgCI~. DDT-45 strain, nuclei from adult insects. within certain limits, to the amount of nuclei added, and hence to DNA. Optimal temperature for the Mn~+-activated enzyme was about 380 C. (F/g. 3). However, this parameter was not further explored because it has been shown to depend on the concentrations of cations and nucleotides (Novello and Stirpe, i969). Contrary to what has been reported for rat liver nuclei (NoveUo and Stirpe, i969) , the Mn~+-activated enzyme had
368
Insect Biochem.
AGOSIN
the same nucleotide concentration requirement as the MgZ+-activated one (Fig. 4). Higher nudeotide concentrations appeared to be inhibitory for the MnZ+-enzyme, but not for the Mg~+-enzyme. Data not shown here indicate that the optimal pH in both cases was broadly 7.8.
o
~1.0.
' 20
,'0
30 Ternt~erature {'C3
FIG. 3.uEffect of temperature on the rate of RNA synthesis by isolated nuclei. Conditions as in Fig. z.
2.0
E
r.~ 1.0 O
-6
0.5
1.0
It'5
MgT M
2~'0
Nuclei concentration
F I c . 4.--Effect of nucleotide concentration on the rate of R N A synthesis by isolated nuclei. Conditions as in Fig. z. x ' o = o ' 5 lmaole of each nucleotide; z ' o = x ' o lamole of each nucleotide.
COMPOSITION OF THE R N A SYNTHESIZED in vitro
•When the base composition of the polymerase product was determined by the procedure outlined under Materials and Methods (Table III), it was found that only the RNA synthesized by adult nuclei in the presence of Mg displayed base pairing in accord with the G-=C and A = U rule, which is similar to what has been found for
~97 I, x
m~A SYNTHESIS IN HOUSEFLY NUCLEI
369
housefly ribosomal RNA (Balazs and Agosin, i968b ). The U/G ratio for the Mg-RNA was 1.77, which is close to the U/G ratio of housefly ribosomal RNA (Balazs and Agosin, i968b ). However, in the case of the Mn-RNA, the U/G ratio was lower, 1.33. This resembles the rapidly labelled RNA reported by Wyatt and Linzen (i 965) in Hylophora which is induced by ecdysone and has a U/G ratio of I"3. As to the larval product, neither the Mg- nor the Mn-RNA had any resemblance to housefly ribosomal RNA or DNA. Whether the RNA synthesized by adult nuclei in the presence of Mn may have messenger characteristics, as in Hylaphora (Wyatt and Linzen, i965) , remains to be established. Table III.--NucLEOTIDS COMPOSITION OF THE R N A SYNTHESIZEDin vitro CONDITIONS
ATP
CTP
GTP
UTP
A+G C+U
Adults
Mg2+-activated Mn~+-activated
28"03 30"55
x8.z 28"43
x9"9 x7"65
33"3 23"8
0'9I I'I2
i .60 I'I8
Larvae
M g 2+-activated M n a+-activated
25"5 25"o
x9"7 I5"o
4z'5 x8.o
x3"3 4z'o
I'39
0"70
o'6z 2"0 3
3z'4 I7"8 z9"6
I8"7 32"2 I9"Z
x6'z 33"o x9"7
32"4 I7"O I9"7
I "00 I "0I 0"96
SOURCE
Housefly z8s r R N A Rat liver z8s r R N A Housefly D N A
A+U C+G
z.85 0"53 z'54
T h e composition of housefly and rat liver ribosomal R N A as well as housefly D N A are included for comparison purposes. See under Materials and Methods for experimental details. Nuclei from adults and larvae of the D D T - 4 5 strain; the values correspond to the average of 5 closely agreeing determinations. SUCROSE DENSITY GRADIENT ANALYSIS
Total nuclear RNA obtained by phenol extraction (Balazs and Agosin, i968b ) at 2-4 ° C. showed the presence of the z8s and x8s ribosomal RNA's, and the 4s peak corresponding to transfer RNA. A small component of 35-4os was also detected. This profile resembles the one reported for nuclear RNA from Tenebriopupae (Ilan, i969). The RNA synthesized in ~itro showed no definite peak, but rather the radioactivity was dispersed between 8s and 35s. The same profile was obtained in the case of Mg- or Mn-RNA formed by adult nuclei, although the Mn-RNA tended to be of lower molecular weight. It should be pointed out that the xo--4o per cent sucrose gradient did not separate satisfactorily the various nuclear RNA components. A 5-zo per cent sucrose gradient was even less adequate. Preliminary experiments suggest that the temperature of extraction may not be ideal in this case (Ilan, I969). EFVECT OF D D T
ON THE POLYMERASE SYSTEM
Evidence suggests that steroid hormones may act at the nuclear level (Dukes and Sekeris, i965). Thus, cortisol increases RNA synthesis when added in vitro to a rat liver chromatin preparation (Stackhouse and others, 1968). In insects Berendes and Boyd (i969) could not show any effect of ecdysterone added in vitro to a nuclear preparation isolated from salivary glands of Drosop/u'/a hydei. However, Congote and others (i969)
37 °
Insect Biochem.
AGOSIN
have shown some evidence indicating that ecdysone stimulates Mg-activated RNA polymerase from Calliphora nuclei when added in vitro. As shown in Table II, D D T added in vitro had no effect on the polymerase activity. Concentrations of D D T higher than those shown in Table H were clearly inhibitory. In view of these results, attempts to enhance the housefly RNA polymerase in vivo were undertaken. Adult insects of the 250 -
o-:
~s
200-
7E
i. IO0 .
.
.
.
.
I
I
!
30
60
90
.
.
!
I
I
120
150
180
Time of intoxication (minutes)
FIG. 5.--Effect of pretreatment with DDT on the rate of RNA synthesis by isolated nuclei from adult housefly. Insects of the Fc strain were topically treated with z.o lal. of acetone containing i.o lag. DDT. After the indicated intervals nuclei were prepared and the polymerase assayed as indicated in Table I1, in the presence of MnCI 2.
Fc strain were topically treated with I.O lag. D D T in acetone as described previously (Gil, Fine, Dinamarca, Balazs, Busvine, and Agosin, i968), and nuclei were obtained at timed intervals. As shown in Fig. 5, RNA synthesis was increased in the polymerase system by pretreatment with D D T . T h e changes occurred markedly after 3 ° minutes, and then the activity started to decline. After 9 ° minutes of intoxication no effect of D D T on the polymerase was evident.
DISCUSSION Nuclei isolatedfrom housefly are clearlydifferentfrom mammalian nuclei in their D N A content. The amount of D N A is lower in housefly,and whereas the R N A / D N A ratio in rat livernuclei iso.27, in adult fliesit ismuch higher. However, the synthesisof RNA, as catalyscd by DNA-dcpcndcnt R N A polymcrasc, is quite similar to that in mammalian tissues. The synthesis requiresD N A as primer (as shown by the effectof DNasc), the product is sensitiveto RNasc, and the reactionis inhibitedby actinomycin D. However, the product of the reaction seems to be quite heterogeneous in housefy, whereas in liveran R N A of 18s is essentiallysynthesized (Widncll and Tata, 1956). As indicated before, the polymerasc product in the presence of M n s+ was mainly of low molecular weight. This may be duc to the degradationphenomenon observed with total R N A isolatedin the absence of M g s+ (Balazsand Agosin, x968b ). The composition of the product deservessome comment. Total R N A from Drosophila and housefly (Hastings and Kirby, 1966; Balazs and Agosin, 1968b) has high contents
I97I, I
RNA SYNTHESISIN HOUSEFLYNUCLEI
371
of AMP and UMP, whereas rat liver RNA is high in G M P and CMP. RNA from silkmoth tissues is high in G M P (Wyatt and Linzen, 1965). In the in vitro system the RNA produced in the presence of Mg s+ had a base composition similar to housefly 28s RNA, high in AMP and UMP. However, the MnS+-activated system yielded a product that did not resemble either housefly ribosomal RNA or DNA. On the other hand, larval nuclei incubated with Mg ~+ gave a product with a composition similar to housefly transfer RNA, but when Mn ~+ was present the composition of the product was again different. Intact nuclei should provide an environment similar to the whole cell, which should allow the possibility of studying the effects of compounds such as D D T or hormones on the complex formed by DNA, RNA, histones, and non-histone proteins. Although no effect of D D T was found in vitro, the increase in polymerase activity of nuclei isolated from DDT-treated insects indicates that the effect of this compound could be at the nuclear level. It is interesting to note that in vivo D D T increases the incorporation of labelled uracil into RNA (Balazs and Agosin, x968a). This increase parallels the kinetics of D D T enhancement of the polymerase. Furthermore, the above changes precede the synthesis of induced proteins by D D T (Litvak and Agosin, i968 ). The above results clearly implicate DNA-dependent RNA polymerase as the enzyme responsible for the increased RNA synthesis in response to D D T treatment. Topically applied D D T is rapidly localized at the nuclear level (Litvak and Agosin, i968), and it is then plausible that the compound produces the above changes, stimulating transcription. However, whether D D T or other compounds act at the chromatin level by derepression of D N A genetic loci or by direct action on the polymerase remains to be established. REFERENCES BALAZS,I., and AGOSIN,M. (x968a), ' The effect of 2,2-bis-(p-chlorophenyl)-i,t,x-trichloroethane on RNA metabolism in Musca domestica (L.)', Biochim. biophys. Acta, x579 x-7. BALAZS, I., and AGOSlN, M. (I968b), 'Isolation and characterization of ribonucleic acid from Musca domestica (L.)', Comp. Biochem. Physiol., 27, 227-237. BER~NDm,H. D., and BOYD,J. R. (x969), 'Structural and functional properties of polytene nuclei isolated from salivary glands of Drosophila hydei',J. Cell Biol., 4x~ 59x-599. BoYD,J. R., BIZR~Dm,H. D., and BOYD,J. H. (I968), ' Mass preparation of nuclei from the larval salivary gland of Drosophila hydei', J. Cell Biol., 38j 369-376. BURTON,K. (I968), 'Determination of DNA concentration with diphenylamine', in Methods in Enzymology (ed. GROSSMAN,L., and MOLDAVE,K.), vol. XII (B), pp. 163-169. New York: Academic Press. CONOOTE,L. F., SEKEmS,C. E., and KAm~SON,P. (x969), 'On the mechanism of hormone action. XIII. Stimulatory effects of ecdysone, juvenile hormone, and ions on RNA synthesized in fat body cell nuclei from Ualliphora erythrocephala', Expl. Cell Res., 56, 338-346. DUKES, P. P., and SEKmUS,C. E. (I965), 'Stimulierung des Einbans von (14C)-Uracil in Ribonucleis~iure von Ratten Leberkernen durch Cortisol in vitro', Z. physiol. Chem., 34x, x49-I 5 x. Gv, L., FI~, B. C., DIN^M^RCA,M. L., B~AZS, I., Busvl~m, J. R., and AoOSIN, M. (x968), 'Biochemical studies on insecticide resistance in Musca domestica', Entomologia exp. appl., xx, 15-29.
HASTiNesS,J. R., and KmB~', K. S. (i 966),' The nucleic acids of Drosophila melanogaster', Biochem. 3., IOO~532-539. Hoo~ooM, G. E., SCHNEIDEa,W. C., and STEmBICH,I. (X952), ' Cytochemical studies. V. On the isolation and biochemical properties of liver cell nuclei',.J ¢. biol. Chem., I96, i I x-I2O. IGARASHI,K., and YURA, T. (x969), 'The role of RNA polymerase in genetic transcription', Biochem. biophys. Res. Commun., 34, 65--69.
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ILAN, J. (i969) , 'Studies on template-active ribonucleic acid from nuclei of insect pupae', Biochemistry, 8, 4825-483 x. KARIaON, P., and SEKERIS,C. E. (1963), ' Eedysone and insect steroidal hormones, and its mode of action', Prog. Horm. Res., 22, 473-502. KRAKOW, J. S., and OCHOA, S. (1963), 'Ribonucleic acid nucleotidyl transferase of Azotobacter vinelandi', Biochem. Z., 338, 796-806. LITVAK, S., and Ac.osm, M. (1968), 'Protein synthesis in polysomes from houseflies and the effect of 2,2-bis-(p-ehlorophenyl)- I, I, l-trichloroethane ', Biochemistry, 7, 1560- x567. LITVAK, S., TAaAco-LITVAK, L., POBL~a'E,P., and ACOSlN, M. (I968), 'Evidence for the D D T induced synthesis of messenger ribonucleic acid in Triatoma infestans', Comp. Biochem. Physiol., 26, 45-56. Low~Y, O. H., ROSEBaOUOH,N. J., FAme, H. C., and RANDALL,R. J. (I95I), 'Protein measurement with the Folin reagent',.7, biol. Chem., I93, 265-275. MEJBAOM, W. (Z939), ' l~ber die Bestimmung kleiner Pentosemengen insbesondere in Derivaten der Adenyls~aure', Hoppe-Seyler's Z. physiol. Chem., 258, i x7-120. MUaAMATSU, M., SME'rANA, K. S., and BUSCH, H. (1963), 'Quantitative aspects of isolation of nuclei of the Walker carcinosarcoma in liver of the rat', Cancer Res., 23, 51o-518. N^KAMOTO, T., FOX, C. F., and WEIss, S. B. (I964), 'Enzymatic synthesis of ribonucleie acid. I. Properties of ribonucleic acid polymerase from extracts of Micrococcus lysodeihticus', ft. biol. Chem., 239, 167-I74. NOWLLO, F., and STIRPE, F. (I969), 'Experimental conditions affecting ribonucleic acid polymerase in isolated rat liver nuclei. Effect of nucleoside phosphate concentration, temperature, ammonium sulphate and heparin', Biochem. ft., xI2, 721-727. STACKHOUSE,H. L., CH~rSANOA,C. J., and TAN, C. H. (1968), ' T h e effect of cortisol on genetic transcription in rat liver chromatin', Biochim. biophys, Acta, 155, 159-168. WIDN'XLL,C. C., and TATA, J. R. (1966), ' Studies on the stimulation by ammonium sulphate of the DNA-dependent RNA polymerase of isolated rat liver nuclei', Biochim. biophys. Acta, I23, 478-492. WYATT, G. R., and LINZEN, B. (1965), ' T h e metabolism of ribonuclei acid in Cecropia silkrnoth pupae in diapause, during development and after injury', Biochim. biophys, Acta, xo3, 388-400.
Key Word Index: Nucleic acid synthesis, RNA polymerase, isolated nuclei, D D T , Musca domestica, mRNA.