Isolation of complementary DNA unique to the genome of avian myeloblastosis virus (AMV)

VIROLOGY

103, 112-122 (1980)

Isolation

of Complementary DNA Unique to the Genome Avian Myeloblastosis Virus (AMV)

J. H. CHEN,“,’ M. GIOVANNELLA

MOSCOVICI,t

AND

of

CARLO MOSCOVIC1-t

*Laboratory of Molecular Biology, Life Sciences Biomedical Research Institute, St. Petersburg, Florida 33710, and +Tumor Virology Laboratory, Research Service, Veterans Administration Medical Center, and Department of Pathology, College of Medicine, University of Florida, Gainesville, Florida 82602 Accepted January 18, 1980 Avian myeloblastosis virus (AMV) can transform avian cells of hemopoietic origin, such as bone marrow, embryonic yolk sac, and circulating macrophages. The AMV is defective in its replication and can only replicate in the presence of helper viruses. This defectiveness in replication is probably due to a deletion or substitution of nucleotide sequences in its genome. The AMV genome contains no sequences homologous to the src gene, which is responsible for the transforming function of the sarcoma viruses. We attempted to identify the AMV sequences that may contain sequences which are responsible for its transforming function. We isolated a complementary DNA (cDNAAMV) that hybridized preferentially to the RNA of the transforming AMV but not to the RNA of the helper virus. Using this as a probe, we determined the size of the AMV genome to be 33-34 S with a cDNA,,v molecular weight of 2.6 x 10G. A similar molecular weight estimation of the AMV genome size was obtained by methylmercury-agarose gel electrophoresis of AMV RNA. In AMVproducer myeloblasts we can detect about 6000-7000 copies per cell of AMV-specific RNA, whereas fewer than 2 copies per cell of AMV RNA are found in helper virus-infected cells. In AMV nonproducer myeloblasts, about 2000 copies of AMV-specific RNA are detected. Furthermore, we find that RNA of AMV NP myeloblasts can only hybridize to 55% of cDNA complementary to helper virus genome. In uninfected hemopoietic cells, e.g., bone marrow cells, about 20 copies per cell of AMV-specific RNA are present, whereas in uninfected chick embryo fibroblasts less than 1 copy per cell is found. INTRODUCTION

Avian RNA tumor viruses have been classified into three main groups (Hanafusa, 1977): (i) the sarcoma viruses which can transform chick embryo fibroblast (CEF)’ cultures and induce solid tumor in vim; (ii) Iymphoid leukosis viruses which do not transform cells in culture but induce lymphoma ’ To whom reprint requests should be sent. 1 Abbreviations used: AMV-B, AMV (MAV-2) subgroup B; SR-B, Schmidt-Ruppin-subgroup B sarcoma virus; NP, nonproducers obtained after infection of either YS macrophages or bone marrow cells with a low m.o.i. of AMV-B; CEF, chick embryo cell resistant to subgroup E virus (C/E); QEF, quail embryo cell resistant to subgroup B and C viruses (Q/BC); gs-, chf?, chicken cells negative for groupspecific antigen and for helper activity; ds DNA, double-stranded DNA; ss DNA, single-stranded DNA; YS, yolk sac-derived macrophages. 0042.6822/80/070112-11$02.00/O Copyright All rights

% 1980 by Academle Press, Inc. of reproduction in any form reserved.

in vivo after a long incubation period (generally 4-8 months postinfection); and (iii) the acute leukemia viruses which can transform cells in culture and induce leukemia in viva within l-4 weeks postinfection. Sarcoma viruses have been widely used to study the mechanism of cell transformation by RNA tumor viruses. In sarcoma viruses a specific viral gene called the “WC” gene has been identified as being responsible for their transformation function (Stehelin et al., 1976). The avian acute leukemia viruses comprise avian myeloblastosis virus (AMV) (Beard, 1963;Moscovici, 1975),avian erythroblastosis virus (AEV) (Engelbreth-Holm and RotheMeyer, 1935),myelocytomatosis virus (MC29) (Ivanov et al., 1964), and Mill Hill 2 virus (MHZ) (Begg, 1927; Moscovici et al., 1978). These viruses share the following common properties: (a) the ability to induce leukemia in vivo after a short incubation period; (b) 112

NUCLEOTIDE

SEQUENCES

IN AMV

113

either LSI flocks or line 6 chickens maintained at the Veterans Administration Medical Center in Gainesville, Florida. Viruses. The following viruses were used in these experiments: AMV plasma of subgroups A and B were kindly provided by Dr. J. W. Beard. AMV (td B77) was prepared by infecting nonproducer (NP) myeloblasts with td B77; virus was then harvested from mass culture of AMV (td B77) myeloblasts. MAV-1, MAV-2 (Moscovici, 1975), RAV-2 (Hanafusa et al., 1964), td B77 (Toyoshima et al., 1970), and td SR-D/21 (Vigne et ul., 1979) were harvested from roller bottle cultures of infected CEF. RAV-60 (Hanafusa et al., 1970)was harvested from roller bottle cultures of infected QEF. MC29 and SR-B RSV were harvested from fully transformed CEF cultures infected with these viruses. Reverse transcriptase assay. We used the synthetic template primer, poly(rC) . oligo(dG),-,, and followed published methods for the procedure (Weissbach et al., 1972;Kawai and Hanafusa, 1973). Briefly, 5-10 ml of medium from infected cultures or control cultures was centrifuged at 10,000 g for 10 min to remove cell debris. The supernatant fluid was then centrifuged at 36,000 rpm for 90 min in a SW 41 rotor. The viral pellet was then resuspended in polymerase reaction mixture. The polymerase reaction was carried out in 100~~1quantities containing 0.05 M Tris-HCl, pH 8.3; 6 mM magnesium acetate; 0.06 M NaCl; 0.1%’ NP40; 15 m&f dithiothreitol; 10 Fg poly(rC) . oligo(dG) (7:3); and 10 ~1 [:‘H]dGTP (specific activity, 25 Ci/mmol). The reaction mixtures were incubated at 37” for 60 min. They were then chilled in an ice water bath, and 0.1 ml of MATERIALS AND METHODS 0.1 M sodium pyrophosphate and 25 pg Cells. For the growth of transforming bovine serum albumin were added. The resarcoma virus and nontransforming leukosis action mixtures were precipitated by adding 0.2 ml of 25% trichloroacetic acid and kept viruses, C/E chick embryo fibroblasts (CEF) and quail embryo fibroblasts (QEF) were in ice for 20 min. The acid-precipitable radioprepared from gs, chf- embryos obtained activity was collected in Whatman GFiC from SPF flocks maintained at Life Sciences, glass-fiber filters. Toluene-based scintillaInc. (LSD. For the growth and transformation fluid was added to the filters and counted tion assay for AMV, the yolk sac (YS) cells in a Beckman liquid scintillation counter. and bone marrow cultures were prepared as Preparation qfviral RNAs. Viruses were describea previously (Dodge and Moscovici, pelleted from culture media by centrifuga1973; Moscovici and Moscovici, 1973; Mos- tion at 21,000 rpm for 2 hr in a Beckman covici et al., 1975). Embryos for YS and type 21 rotor. Viral RNAs were then exbone marrow cultures were obtained from tracted as described by Hayward (1977).

the ability to transform hemopoietic cells; (c) the defectiveness in viral replication. MC29, AEV, and MH2 can transform both hemopoietic and fibroblastic cells in culture and induce solid tumors in viva (Beard, 1963; Ishizaki and Shimizu, 1970; Langlois and Beard, 1967; Graf, 1973; Graf et al., 1976; Hu et al., 1978; Moscovici et al., 1978), whereas AMV transforms exclusively cells of hemopoietic origin, such as chick embryonic yolk sac (YS) and bone marrow cells (Moscovici and Moscovici, 1973; Moscovici et al., 1975). MC29, AEV, and MH2 have been shown to contain 28 S RNA as their genomes (Duesberg et al., 1977; Kamahora et al., 1979; Hu et al., 1979). Furthermore, no genomic RNA of MC29 and AEV was found to be homologous to the arc gene of sarcoma viruses (Duesberg et al., 1977; Stehelin and Graf, 1978). It became obvious that the viral sequence responsible for the transformation functions of the acute leukemia viruses is different from src. Indeed, a viral sequence specific for the transforming component of MC29 has been identified (Sheiness et al., 1978). In this study, we have attempted to isolate a complementary DNA (cDNA) unique to the AMV genome that may contain the sequence responsible for its transforming function. This cDNA hybridizes preferentially to RNA of transforming AMV over that of its helper virus. It hybridizes to cellular RNA of AMV-transformed myeloblasts but not to those of helper virus-infected cells. Using this AMV cDNA as a probe, we are able to estimate the size of the AMV genome to be 33-34 S RNA.

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Briefly, viral pellets were dissolved in RNA extraction buffer containing 0.01 M TrisHCl, pH 7.4,O.Ol M EDTA, 0.2% SDS, and 200 pg/ml of proteinase K and incubated at 37” for 30 min. Viral RNAs were then extracted twice with equal volumes of phenolchloroform-isoamyl alcohol mixture (1:1:0.5 (v/v) containing 0.05% hydroxyquinoline) and once with chloroform. RNAs were then precipitated with ice-cold ethanol for at least 6 hr. Viral RNAs were recovered by centrifugation at 10,000 rpm for 45-60 min in a Beckman 521 centrifuge. For the preparation of 60-70 S viral RNA, viral RNA pellets were dissolved in RNA buffer containing 0.02 M NaCl; 0.01 M Tris-HCI, pH 7.4; 1 mM EDTA; and 0.2% SDS and layered onto 15-30% sucrose gradient and centrifuged at 40,000 rpm in an SW 41 rotor for 210 min. The 60-70 S RNA peak fractions were pooled and precipitated with ethanol. For the preparation of 35 S viral RNA, the viral 60-70 S viral RNAs were dissolved in RNA buffer and heated at 90” for 1 min, then chilled rapidly in ice and layered onto a 15-30% sucrose gradient and centrifuged at 40,000 rpm for 7% hr in an SW 41 rotor. The 35 S RNA peak fractions were pooled and precipitated with ethanol. For the preparation of poly(A)-containing 70 S AMV (td B77) RNA, 70 S viral RNA was loaded onto an oligo(dT)-cellulose column that had been equilibrated with 0.01 M Tris-HCl, pH 7.4, and 0.5 M NaCl. After extensive washing, poly(A)-containing RNA was eluted with 0.01 M Tris-HCl and precipitated with ethanol. The poly(A)-containing viral RNA was then centrifuged through 15-30% sucrose gradient as described above, and 60-70 S RNA fractions were pooled and precipitated with ethanol. Preparation of cellular RNA. Cellular RNAs were extracted by the procedures described by Hayward (1977) and Wang et al. (1977). Preparation of “2P-labeled AMV(td B77) RNA. AMV (td B77)-transformed myelo-

blasts at log growth phase were incubated in phosphate-free MEM media for 8-12 hr at l-2 x lo7 cells/ml. The medium was then changed to MEM that contained 2 mCi/ml of 32P. Twelve hours after being labeled with 32P,culture media were harvested and

AND MOSCOVICI

myeloblasts resuspended in complete media and harvested at 3-hr intervals for 6- 12 hr. All “‘P-containing media from AMV (td B77) cultures were pooled and processed for viral RNA extraction as described in the foregoing section. The specific activity of viral RNA obtained was 8- 10 x lo6 cpm/pg RNA. Ribosomal RNA marker. The “2P-labeled infected culture, after collecting the labeled virus, was used for cellular RNA extraction as described (Hayward, 1977). The 32Plabeled cellular RNA was chromatographed on an oligo(dT)-cellulose column. The flowthrough fractions from the column were collected and precipitated with ethanol. The majority of RNA in these fractions was 28 and 18 S RNA. Calf thymus DNA primer. The primer was prepared as described by Taylor et al. (1976) with a modification by Dr. Sheiness (personal communication). Briefly, about 1 g of calf thymus DNA was dissolved in a buffer of 0.01 M Tris-HCl (pH 7.4) and 0.01 M MgCl,. DNase I was added to 25-30 pg/ml and the whole mixture was incubated at 37 for 2 hr. The DNA solution was then adjusted to contain 1% SDS, 15 mM EDTA, and 20 pgiml proteinase K and incubated at 37” for 45 min. The DNA was then extracted twice with a phenol-chloroform mixture and precipitated with ethanol. The precipitated DNA primer mixture was then dissolved in 5 mM Tris-HCl (pH 7.4) and 1 mM EDTA. It was heated at 100” for 10 min, and chilled in ice; then NaCl was added to 0.1 M. The DNA primer mixture was loaded on a DEAE-cellulose column equilibrated with 5 n&’ Tris-HCl, pH 7.4, 1 mM EDTA, and 0.1 M NaCl. The column was washed extensively with buffer and the DNA primer eluted with 5 mM Tris-HCl (pH 7.4) and 0.3 M NaCl and precipitated with ethanol. The stock solution of primer was adjusted to a concentration of 50 mgiml and stored at -20”. Preparation qf cDNAs. cDNA,, RT7and cDNA,,,-, were synthesized in endogenous reaction as described (Hayward and Hanafusa, 1973). cDNA,~,.,.:cDNA,~,,was prepared according to the procedure described by Hayward (1977), using depurinated high molecular weight cDNA prepared from SR-B RSV and then selected with RAV-2 RNA.

NUCLEOTIDE

104

lo”

100

115

SEQUENCES IN AMV

IO’

Crttmol-set/l) FIG. 1. Hybridization of cDNA,,,. with SR-B RSV, RAV-2, and AMV (td B7’7) RNA. About 800 cpm of cDNA was mixed with viral RNA in a solution containing 30% formamide, 3 x SSC, 0.296, and 5 mM EDTA in a volume of 10 pl and incubated at 50” for various times. Degree of hybridization was determined by the resistance of hybrids to S, nuclease treatment. Viral RNA concentrations: SR-B RSV (O), 15 pgiml; RAV-2 (A), 20 pgiml; AMV (td B77) (01, 20 @g/ml.

cDNA,~~:cDNA,,, was prepared following the procedure similar to the one described for the synthesis of cDNA,,,, (Sheiness et al., 1978). About l-2 pg of 60-70 S poly(A)containing AMV (td B77) was heated at 90 for 1 min and then chilled in ice. The RNA was added to 250 ~1 of reaction mixture consisting of 50 mJ4 Tris- HCI, pH 8.1; 4 mM dithiothreitol; 8 mM MgCl,; 48 m&ZKCl; 35 pg actinomycin D; 0.4 mM each of dGTP, dCTP, and dATP; 800 pg primer; 100 units of purified AMV reverse transcriptase (kindly provided by Dr. Beard); and 1 mCi of [H]dTTP. After the mixture was incubated at 37” for 45-60 min, the reaction was stopped by adding SDS to the final concentration of 1% and 100 kg proteinase K and was incubated for another 15 min at 37”. The cDNA was then extracted once with a phenolchloroform mixture and once with chloroform and then precipitated with ethanol. cDNA had a specific activity of 2.0-2.5 x lo7 cpml pg. cDNA was passed through a Sephadex G-50 column to remove unincorporated nucleotides. About 80-90% of this cDNA was separated as single-stranded (ss) cDNA when chromatographed on a hydroxyapatite column. Gel electrophoresis of RNA. Methylmercuric hydroxide-agarose gel electro-

phoresis was adapted with slight modification from the method of Bailey and Davidson (1976). Briefly, 60-70 s viral RNA samples were dissolved in 0.5X E buffer (E buffer: 50 mM boric acid, 5 mM sodium borate, 10 mM sodium sulfate, and 1 mM EDTA, pH 8.19) containing 5 mM methylmercuric hydroxide and 10% glycerol. Samples were applied onto horizontal 1% agarose slab gel containing 5 mM methylmercuric hydroxide and electrophoresis was carried out at 50 mA/gel for 5 hr. After electrophoresis gels were dried and processed for autoradiography . Nucleic acid hybridization. Hybridization was carried out under moderately stringent conditions (Hayward and Hanafusa, 1975). Each hybridization reaction mixture contained 30% formamide; 0.45 M NaCl; 0.045 M sodium citrate; pH 7.2; 5 m&ZEDT&, 0.2% SDS; 700-1000 cpm of cDNA; and appropriate amounts of RNA as indicated in the figure legends. The hybrid mixture consisting of 5-10 ~1 in sealed 20-p; capillaries was incubated at 50” for various times. The degree of hybridization was determined by the resistance of the hybrid to SI nuclease treatment as described (Hayward and Hanafusa, 1973). All data were corrected for background levels of 1.8-4.2% for cDNAs. RESULTS

Lack of Homology Sequences

in

AMV RNA with WC

qf Sarco,ma Viruses

To determine whether AMV-B and AMV (td B77) contained src-related sequence, AMV-B and AMV (td B77) viral RNAs were

hybridized to DNA,,,.. Neither of these RNAs contained sequences hybridizable to cDNA,,, prepared from SR-B, RSV (Fig. 1). In AMV-B the titer of the helper virus was 2 to 3 logs higher than that of the transforming virus whereas in AMV (td B77) the titer of helper virus, td B77, was only threeto fivefold greater than that of the transforming virus (see below). It can be argued that, in carrying out hybridization kinetics experiments between cDNA,,,. and AMV RNA, especially in AMV-B, the helper virus would mask the possible presence of a srcrelated sequencein the RNA of transforming AMV. This argument would not hold in the

116

CHEN, MOSCOVICI, AND MOSCOVICl

case of AMV (td B77). In carrying out hybridization kinetics to lOI mol-see/liter, AMV (td B77) RNA still did not hybridize to cDNA,,,.. Therefore, we concluded that AMV (td B77) RNA did not contain a sequence related to that of the arc of sarcoma viruses.

Choice of AMV (td 3 77) ,for Isoltrtion qf AMV-Spec@ic Sequences Since AMV is defective in replication, most of the AMV preparations contain more helper virus than transforming virus. In order to make the isolation of AMV-specific cDNA easier, it was essential to use viral preparations with lower concentrations of helper virus. As shown in Table 1, AMV-B preparations usually contain a considerable excess of helper viruses while the AMV (td B77) showed a lower ratio of helper virus versus the transforming virus. Therefore, AMV (td B77) was a better choice than AMV-B for the isolation of AMV-specific DNA.

FIG. 2. Scheme for cDNA,>,, isolation.

sentially the same as the one used for the cDN&.w Synthesis isolation of MC29-specific cDNA (Sheiness The method for the isolation of eDNAAX et al., 1978). cDNA was first synthesized is outlined in Fig. 2. This method was es- in vitro using poly(A)-containing 70 S AMV (td B77) RNA as the template in the presence of calf thymus DNA primer. ss cDNA TABLE 1 was separated from double-stranded (ds) COMPARISONOFREVERSETRANSCRIPTASEACTIV- cDNA by hydroxyapatite chromatography. ITIESANDTITERSOFTRANSFORMINGVIRUSINAMV The ss cDNA was then hybridized to AMV (MAV-2) AND AMV (fd B77) (td B77) RNA to select homologous DNA sequences. The hybrid thus formed was diAMV (MAV-2) AMV (td B77) gested with S, nuclease and hydrolyzed with alkali to remove viral RNA. This AMV (td Reverse Reverse tranTranstranTransB77) cDNA was then subjected to three Virus scripWipformaformacycles of selection by the use of td B77 RNA dilution tion tasen tion” tase to remove DNA sequencescommon to helper virus. After a third cycle of selection of + + + f 10-1 cDNA, about 3-5% of starting cDNA that + + + + IOwas homologous to AMV (td B77) RNA was + + + + 10-3 + + + + 10-q recovered as ss cDNA. The ss cDNA showed + f 10-s preferential hybridization with AMV (td + 10-e B77) RNA to td B7’7 RNA as shown below. .IO-’ + This cDNA was designated as cDNA,,,. 10-n Media

-

-

-

-

0 Reverse transcriptase assay was carried out as described under Materials and Methods. b Transformation assay was carried out in secondary YS cultures as described (Moscovici et al., 1975).

Specificity

of

cDNA,,~,~

When cDNA,,v was hybridized to AMV (td B77) RNA and td B77 RNA (as shown in Fig. 3), cDNA,,, showed preferential

NUCLEOTIDE

SEQUENCES

Crt ( mol-sedl) FIG. 3. Hybridization of eDNAAMY and cDNA,,, 877 with AMV (td B77) and td B7’7 RNA. Hybridization condition was the same as in Fig. 1. cDNA,,, u-as used at 900 cpmireaction and cDNA,,, Bi7 at 1000 cpm/ reaction. Concentration of viral RNA: AMV (td B77), 10 pgiml; td B77, 14 pgiml. (O--O) cDNA,,, vs AMV (td B77) RNA; (a ~ A) eDNAAM\. vs td B77 RNA: (0 ~ 0) cDNA,,, 877vs td B7 RNA; (X - - - X) cDN&, sii vs AMV (td B77) RNA.

hybridization to AMV (td B7’7) RNA. About 80% of cDNA was hybridized to AMV (td B77) RNA with Crtli2 of 5.5 to 6 x lo-” molsee/liter, whereas less than 10% of this cDNA

could hybridize to td B77 RNA. These small amounts of cDNAAMv that were hybridized to td B77 RNA may represent cDNA sequences homologous to td B77 RNA which were not removed by the selection procedure described above. When cDNA,~ 877was used to hybridize with AMV (td B77) and with td B77 RNAs, it hybridized to the full extent as expected with CrtliZ of 1.8-2.0 x lo-’ mol-see/liter. These results also indicate that AMV (td B77) preparation used in these experiments contained threeto fivefold more helper than transforming particles. Because the cDNA,,, showed preferential hybridization to RNA from transforming AMV (td B77) than that from nontransforming td B77, we considered that cDNA,,v had a high specificity. Content of AMV Sequences in Other Asian RNA Tumor Viruses

cDNA,,v was used for hybridization with RNAs extracted from avian RNA tumor viruses of subgroup range A to E. As shown in Table 2, cDNA,,v can hybridize only to RNAs from AMV (td B77) and AMV plasma. Less than 10% of cDNA,,, was hybridizable to different nontransforming leukosis

TABLE HYBRIDIZATION

RNA AMV-c AMV-plasma” MC29-B’ MAV-1 MAV-2 td B77 td SR-D/21 RAV-60 SR-B RSV td B77 + MAV-2 td B77 + MAV-1 td B77 + RAV-60

117

IN AMV

2

OF VIRAL

cDNA,,,

cDN&,,

76 73 6 4 3 4 5 5 4 3 4 4

92 80 67 85 65 94 75 85 80

RNA TO cDNAs” wi

cDNA MAV-2 70 90 75 75 97 73 80 80 96 -

cDN&r, 3 4 2 5 3 2 2 1 90 2 1 2

u For cDNA,,,: hybridization of viral RNA to cDNA was carried out to C,t = 1.0 mol-set/liter. For other cDNAs the values listed are values of hybridization kinetics reaching plateau levels, at C,t = 5 x 10’ moliseciliter. * AMV-plasma RNA contained both subgroups A and B and was hybridized to cDNA,,, to reach C,t = 10 mol seciliter. c MC29-B was recloned and had a helper/MC29 ratio of 5/l using biological assay. MC29-B RNA was hybridized to cDNAAMv and cDNA,<,,. to C,1 = 5.0 mol-see/liter to cDNA ,,, a,, and cDN.4,,,. 2 to C,t = 0.7 mol-seciliter.

118

CHEN.

MOSCOVICI.

viruses or transforming viruses. Table 2 shows also little sequencehomology between the RNA of MC29-B and cDNA,,,. Since the helper virus of AMV (td B77), i.e., td B77, and AMV (td B77) were not known to be congenic, we combined RNAs from different subgroups of helper viruses and then hybridized them to cDNA,,v. The data in Table 2 show that the mixture of RNAs of different subgroups of helper viruses had very little sequence homology to cDNA,,,,. Therefore, we considered it unlikely that the sequences in cDNA,,v were due to the accumulation of sequences of different subgroups of avian RNA tumor viruses.

AND MOSCOVICI

c

Size o-f the AMV Genome

Among avian acute leukemia viruses the size of the viral genomes of MC29, MH2, and AEV had been determined to be a 2830 S RNA with a MW of about 2.0 x lo6 (Duesberg et al., 1977; Sheiness et al., 1978; Kamahora et al., 1979; Hu et al., 1979). Using viral RNA extracted from AMV preparations with the lower helper/AMV ratio and analyzing this RNA on methylmercury gel electrophoresis, the size of the AMV genome may be resolved from that of the helper virus and compared with those of other acute leukemia viruses. As shown in Fig. 4, RNA of several AMV (td B77) clones, under denaturing conditions, was resolved into two components: one migrated as 35 S helper virus RNA whereas the secondcomponent moved slightly faster. Since the RNA of AMV (td B77) clones examined possessed this second component, it seemed that this second RNA component of AMV (td B77) represented the AMV genome. In the same experiment MC29 RNA was also resolved into two components: one migrated as 35 S RNA and another migrated slightly slower than 28 S ribosomal RNA. Using 35 S helper virus RNA, ribosomal 28 and 18 S RNAs as markers, the second component of AMV (td B77) RNA was estimated to have 7.7 to 7.9 kilobases (kb), or with a MW of 2.7 x 106. The second component of MC29 RNA had 5.7 to 5.9 kb or with a MW of 2.0 x 106. The size of AMV genome was also estimated by rate zonal centrifugation of viral RNA. The viral RNA in the fractions collected after centrifugation was hybridized

FIG. 4. Methylmercury agarose gel electrophoresis of viral RNA. “2P-Labeled 60-70 S viral RNAs of td B77, AMV (td B77) clones and MC29-B, and chicken ribosomal RNA were electrophoresed in 1% agarose slab gel containing 5 mM methylmercuric hydroxide at 50 mA per gal for 5 hr. After electrophoresis, gel was dried and processed for autoradiography. Exposure time: 14 hr. Lanes 1 and 9, ribosomal RNA; lanes 2 and 8, td B77 RNA; lane 3, MC29-B; lane 4, MC29AV-B RNA; lane 5, AMV (td B77) clone FR449 RNA; lane 6, AMV (td B77) clone 248-9 RNA; and lane 7, AMV (td B77) clone 248-2 RNA.

with cDNA,,v and cDNA,, B77.As shown in Fig. 5, cDNA,~ 877was hybridized to fractions peaking at 35 S whereas eDNAAM” hybridized to fractions with a peak of 33-34 S. This sedimentation value corresponded to a MW of 2.6 x lo6 as calculated from Spirin’s formula: MW = 1550 x S2.1(Spirin, 1963). Therefore we considered the size of the AMV genome to be slightly smaller than the genomes of leukosis viruses, yet larger than those of other avian acute leukemia viruses, i.e., AEV, MC29, and MH2. The gene complexity of cDNA,,, sequences was not determined, due to the small quantity of cDNA,,, available and the presence of the excess of td B77 in AMV (td B77). Content of AMV RNA in AMV-Transformed Myeloblasts

To determine the quantities of AMVspecific RNA in AMV-transformed cells, total cell RNAs were extracted from trans-

NlJCLEOTIDE

SEQUENCES

IO

119

IN AMV

20

Fraction Number FIG. 5. Size analysis of AMV RNA by sucrose density gradient centrifugation. :“P-Labeled 70 S AMV (td B77) RNA (1 x 1w cpm) was mixed with 10 pg of 70 S AMV (td B77) RNA and 40 pg of chick ribosomal RNA, heated at 90” for 1 min, chilled, and layered onto 15-30s sucrose gradient and centrifuged at 40,000 rpm for 7% hr in an SW 41 rotor. Fractions were collected and precipitated with ethanol. The RNA in each fraction was then resuspended in 25 ~1 of 0.2 x ETS buffer (ETS: 0.01 M Tris-HCI, pH 7.4, 0.01 M EDTA, and 0.2% SDS). Five microliters of each RNA fraction was mixed with 5 ~1 of appropriate concentration of hybridization buffer containing 500 cpm of cDNAs and sealed in 20-~1 capillaries. The hybrids were incubated at 50” for 2-5 hr. Degree of hybridization was determined by S, digestion of hybrids. (0) ‘Y’ AMV (td B77) RNA; (a) cDNA,,, ,ii7; ((2) cDNA ,h,\.

formed cells, helper virus-infected CEF, uninfected CEF, and bone marrow cells. The bone marrow cells and uninfected CEF were used as uninfected cell control. These cellular RNAs were then hybridized to cDNA,,,~ and cDNA,,. B77in kinetics experiments. As shown in Fig. 6, RNA extracted from AMV-transformed cells contained about 6000 to 7000 copies of AMV-specific RNA per cell [calculated from the formula: number of RNA copies = (C,t,,:! of viral RNA/C,t,,, of cell RNA) x (g of RNA per cell/g of viral RNA per subunit), based on the following figures: g of RNA/CEF = 1.0 x lo-“; g of RNAmyeloblast = 3 x lo-“, g of AMV viral RNA = 3.3 x 10m’x],whereas td B77infected cells had less than 1 copy per cell of AMV RNA. In contrast, both cell RNAs from producer myeloblasts and helper virusinfected CEF contained 8000 to 10,000 copies of td B77 RNA. In AMV-transformed NP cells about 2000 to 3000 copies of AMV RNA per cell were found. In NP cells, cellular RNA could only hybridize to about 55% of cDNA,,! Bi7. In addition, we also found that uninfected bone marrow cells also contained about 20 copies of AMV RNA,

whereas uninfected CEF had fewer than two copies per cell. This indicated the presence of an endogenous AMV sequence in uninfected cells. DlSClJSSION

The avian acute leukemia viruses, MC29, MH2, and AEV, can transform both avian fibroblasts and hemopoietic cells in culture (Beard, 1963; Langlois and Beard, 1967; Ishizaki and Shimizu, 1970; Graf, 1973; Graf et al., 1976; Hyet al., 1978; Moscoviciet al., 1978), whereas AMV transforms exclusively avian hemopoietic cells (Moscovici and Moscovici, 1973; Moscovici et al., 1975). Several reports had shown that viral genomes of MC29, MH2, and AEV did not possess sequences in their genomes homologous to the SK gene, which had been shown to be responsible for the transformation of fibroblasts by avian sarcoma viruses (Sheiness et al ., 1978; Stehelin and Graf, 1978). It has been suggested that a specific nucleotide sequence is responsible for the transforming activity of MC29 (Duesberg et al., 1977; Sheiness et al., 1978; Duesberg and Vogt, 1979). Our data indicate similar results

120

CHEN,

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which show that the genome of AMV does not have sequences homologous to src sequences of avian sarcoma viruses. These data were obtained by the hybridization kinetics between RNA of AMV (td B77) and cDNA,$,,.. Since the AMV genome lacked the arc sequence, it was presumed to have different sequences responsible for its transforming function. Our objective was to isolate the sequences that might be responsible for AMV transformation. We were able to isolate an AMV-specific DNA (cDNA,,,), although the yield of AMV-specific cDNA recovered was very low. This cDNA preferentially hybridized to the RNA of AMV (td B77) and least to the RNA of td B77 or to the RNA of MAV-1 and MAV-2. This cDNA,,, also did not hybridize with RNA of RAV-60 or RNA from mixtures of different subgroups of leukosis viruses. These results rule out the possibility that cDNA,,v sequences were derived from endogenous RNA tumor virus sequences or were due to the contribution of small amounts of sequences from different leukosis viruses. We could not determine the complexity of cDNA,,, isolated and the excess of helper virus in AMV (td B77) preparation. Neither do we know if these sequences are contiguous in the genome of AMV. The data reported here indicate that cDNAmv represents the sequences complementary to the region of AMV genome that is not homologous to that of the helper viruses. The sequences coded for the AMV transforming function may be present in this unique sequence. This argument is provided by the evidence of preferential hybridization of AMV-specific cDNA to AMV (td B77) RNA, rather than to td B77 RNA. Furthermore, in hybridization kenetics experiments, cDNA,,~~ hybridized to cellular RNA extracted from AMV-transformd myeloblasts about 200 times faster than to cellular RNA of the uninfected bone marrow. The hybridization rate between cDNA,,, and the cellular RNA of myeloblasts is 1000 times faster than the one between cDNA,,, and RNAs of helper virus infected CEF or uninfected CEF. Whether this unique AMV sequence is the one responsible for the transforming function of AMV remains to be proven directly from genetic studies.

AND MOSCOVICI

Crt (mol-set/l)

FIG. 6. Hybridization of cDNA.,,, and cDNA,,! Ui7 with total cell RNA. Hybridization condition was the same as in Fig. 1. cDNA,,, was used at 800 cpm/ reaction and cDNA,,, ,%,,at 1000 cpmireaction. Concentration of RNA: AMV producer myeloblasts, 3 mgiml: AMV NP myeloblasts 5 mgiml; td B7’7-infected cells, 2 mgiml; uninfected CEF and bone marrow cells, 10 mg/ml each. (O---O) cDNA,,, u,7 vs td BWinfected cells RNA; (O---O) cDNAtdHii vs AMV (td B77) myeloblast RNA; (U---U) cDNA,, H7i vs NP myeloblast RNA; (a-0) cDNA,,,,, vs AMV (td B77) myeloblast RNA; (O-00) cDNA,,, vs td 877-k fected cell RNA; (Cl __ q ) cDNAAuv vs NP myeloblast RNA; (A-A) cDNA.,,,,~ vs uninfected bone marrow- cell RNA; (X-X) cDNA,\,,, vs uninfected CEF RNA.

Attempts to isolate mutants of AMV are in progress. Among the avian acute leukemia viruses, the genome sizes of MC29, MH2, and AEV have been shown to be 28-30 S RNA (Duesberg et al., 1977; Duesberg and Vogt, 1979; Kamahora et al., 1979; Hu et al., 1979) while the genome size of AMV has not been determined. Using gel electrophoresis, under denaturing condition, AMV (td B77) RNA was resolved into two components. The first component was attributed to the genome of the helper virus having a MW of 3.0 x lo6 (about 8.5 kb). The second component had an estimated MW of 2.7 x 10” (about 7.7-7.9 kb). Using cDNA,,,~ to hybridize with RNA in fractions from the rate zonal centrifugation of AMV (td B77) RNA, the size of AMV RNA was estimated as 33-34 S. The latter method yielded an estimated molecular weight of the second component of AMV (td B77) RNA comparable to that obtained from gel electrophoresis. Based on the close agreement of molecular weight estimations of the two methods, we considered the size of AMV

NUCLEOTIDE

SEQUENCES

genome to be 33-34 S with a MW of2.6-2.7 x 106. The difference in size between the helper virus and AMV RNA is only 600-800 bases, yet AMV is defective in replication. This may indicate that in the AMV genome, there are extensive substitutions in nucleotides which can account for the defect in replication of AMV. The NP cell RNA could only hybridize to 55% of cDNA,~ R77.A similar result has also been obtained by Roussel et al. (19’79). This may represent the sequences of AMV RNA in NP cell homolgous to those of helper viruses. The incomplete hybridization between NP cell RNA and cDNA,,, H77may explain the defectiveness of AMV replication. In NP cells the quantity of AMVspecific RNA per cell is about 50% of that found in producer myeloblasts. Whether the defectiveness in replication has some effect on the production of AMV-specific RNA in NP cells remains to be determined. Our experiments have also indicated the presence of an endogenous AMV sequence in uninfected chicken cells. The presence of endogenous AMV sequences has also been reported recently (Roussel et al., 1979). The cDNA,,, hybridized appreciably to the RNA extracted from uninfected hemopoietic cells and less to RNA extracted from uninfected CEF (Fig. 6). This unexpected finding suggests that a differential expression of endogenousAMV sequencesexists in chicken cells and might be related to some specific transforming events occurring only within the myeloid lineage. This problem is currently under investigation. With the availability of cDNA,,, it is now possible to investigate the expression of the AMV sequence during the viral leukemogenesis induced in chicken by AMV. ACKNOWLEDGMENTS This work was supported by funds from the American Cancer Society, Florida Division, Project Nos. ACS-78-079 (to C.M.), and F79 LS-1 (to J.H.C.), from the National Cancer Institute CA-10697 (to C.M.), and from the Medical Research Service of the Veterans Administration. J.H.C. thanks Drs. J. W. Beard and M. Nonoyama for their critical comments on this manuscript. We wish to thank Gordon Thompson, Ana Ramirez, and Lydia Camacho for technical assistance and Alice Cullu for editorial assistance.

121

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