Biochemical properties and replication of murine intracisternal A particles during early embryogenesis

Europ. J. Cancer Vol. 11, pp. 131-138. Pergamon Press 1975. Printed in Great Britain

Biochemical Properties and Replication of Murine Intracisternal A Particles During Early Embryogenesis* STRINGNER S. YANG,'~ PATRICIA G. CALARCO + and NELSON A. WIVEL~" "~Laboratory of Cell Biology, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20014, U.S.A. +Department of Anatomy, University of California, School of Medicine, San Francisco, California 94143, U.S.A. Abstract Intracisternal A particles exhibit features which are characteristic of oncogenic RNA viruses and features which are reminiscent of fetal antigen expression. Evidence has been developed by ultrastructural study which points toward a vertical transmission of these particles, a situation analogous to known RNA tumor viruses. Intracisternal A particles are readily found in certain preimplantation mouse embryos; they are found occasionally in a variety of neoplasms. Biochemical properties of highly purified intracisternal A particles isolated from murine neuroblastoma cells or plasma cell tumor include high molecular weight 60-70S RNA and an RNA-dependent D NA polymerase. The enzymatic characterization of the A particle D N A polymerase is reported. The enzyme is found analogous to other C-type viral reverse transcriptase by virtue of its p@rence for Mn 2 + activation and certain D NA-RNA hybrid templates, oligo dT12"polyrA (dT12"rA") and oligo da12"polyrC (dG12"rC"), for the stimulation of its activity. The endogenous reaction of the A particle RNA-dependent D N A polymerase produces a D N A transcript of its intrinsic RNA with base complementarity as demonstrated by the results of hybridization studies. The control of the expression of this gene entity and its relevance to malignant transformation"constitute a problem worthy of further investigation.

INTRODUCTION

transformation of the cell. Intracisternal A particles (IAP), known primarily for their association with tumors in mice [3-6], gerbils [7], rats [8], and guinea pigs [9], are most probably vertically transmitted from maternal oocyte to fertilized embryo [10, 11]. Their appearance in large numbers during murine preimplantation development has been reported recently [11-13]. In the cell, IAP consist of two concentric shells surrounding a relatively electron lucent core; they measure between 70 and 100 nm in outer diameter when appearing singly (Fig. 1). They are confined exclusively within the cisternae of the endoplasmic reticulum and are formed by budding at the reticulum membrane [14]. The purposes of this communication are two-fold, 1, to detail the mode of appearance and subsequent fate

NEOPLASTIC transformation has been viewed as a departure from the normal process of differentiation. Frequently the reappearance of fetal characteristics accompanies the onset of the neoplastic state; for example, proteins like serum alpha-fetoprotein and carcinoembryonic antigen can serve as diagnostic markers for hepatocellular carcinoma [1] and carcinoma of the digestive tract [2] respectively. Such phenomena have been attributed to the reactivation of the fetal genes during neoplastic Accepted 19 November 1974. *This work was presented earlier at the Third Conference on Embryonic and Fetal Antigens in Cancer, Nov. 4-7, 1973, Oakridge, Tenn., U.S.A. 131

132

Stringner S. Yang, Patricia G. Calarco and Nelson A. Wivel

of IAP in preimplantation mouse embryos, and 2, to enumerate nucleic acid and DNA polymerase characteristics which indicate that IAP are an expression of an RNA tumor virus genome.

MATERIAL AND METHODS

Electron microscopy Oocytes were obtained from CF-1 female mice. Preimplantation stages of development were collected from Swiss Webster, ICR, BALB/c, CF-1, New Zealand Black and A K R strain females. Preparative techniques were as outlined previously [11].

Sourcesfor IAP A mouse plasma cell tumor (MOPC-104E) was maintained by s.c. transplantation in BALB/c mice in this laboratory. Neuroblastoma N-18, a continuous tumor cell line described previously [15], was cultured in Dulbecco's modified Eagle medium supplemented with 10 % heat inactivated fetal calf serum, penicillin and streptomycin as described earlier. All tissue culture materials were obtained from GIBCO, Grand Island, N.Y., U.S.A.

Isolation and purification of IAP Isolation and purification of IAP have been well documented [14-15]. Briefly, a pellet of mitochondria and microsomes was prepared by centrifugation of cytoplasmic extracts obtained from tumor homogenates at 13,000 xg for 20 min. Intracisternal A particles were then sheared from the microsomal vesicles in the presence of Triton X-100. The freed A particles were then purified by sucessive discontinuous and continuous linear sucrose gradient centrifugations for three or more times. The methods employed allow for the preparation of a highly homogenous concentrate of IAP. Appropriate control experiments established that these purified isolates were free of any C-type particle contamination [15]; therefore the demonstrated biochemical properties can be considered as intrinsic to IAP alone. It should also be noted that no comparable preparation could be isolated from normal tissues uninfected with intracisternal A particles.

DNA polymerase assays Details of DNA polymerase assays have been described earlier [16-18]. Some minor varia-

tions were allowed to optimize the assay conditions. Briefly, the assay mixture contained Tris-HC1, pH 8.1, 50 m M ; Mg acetate, 10 m M or MnC12, 0.8raM; dithiothreitol (DTT), 8 m M ; KC1, 75 m M ; and specified amount of purified IAP protein treated with 0.018% Triton X102 for 10 min at 0% For assaying the endogenous reaction, cold dCTP, dGTP, and dATP at a final concentration of 8 x 10-5 M were added along with 5 ~Ci of 3H-TTP (New England Nuclear) in a 50pl reaction was carried out at 37 ° . In assays with synthetic templates, the appropriate substrates as specified in the Table were used; the reaction was carried out at 30 ° . The reaction of DNA polymerase was terminated with additions of 2 ml of ice cold 0.08 u sodium pyrophosphate in 12.5% trichloroacetic acid (TCA) and 20 ttg of yeast nucleic acid as carrier. The radioactive precipitates were collected on Millipore filters (Millipore Filter Corp., Mass.), washed extensively with 5% TCA, then 80% ethanol, dried and counted by a liquid scintillation method. All chemicals were obtained from Calbiochem, Calif. and Fischer Scientific Co., New Jersey. Triton X-100 or X-102 detergents were obtained from Sigma, Mo. All isotopes were purchased from New England Nuclear, Mass. Bovine pancreatic RNase, at 0"25 mg/ml and boiled prior to use, was obtained from Worthington, Germany.

Virus purification Avian myeloblastosis virus (AMV) and Rauscher leukemia virus (RLV) at 1011 particles/ml were generously supplied by Drs. J. W. Beard and D. P. Bolognesi and Electronucleonics, Md. respectively via the Special Cancer Virus Program of the National Cancer Institute. Further purification of these viruses was described [19]. Materials banded at the 1.16 g/ml area after isopycnic sucrose density gradient centrifugation were used for RNA extraction.

RNA extraction and purification High molecular weight, 60-70S, RNA was extracted from A M V and R L V and purified by velocity gradient centrifugation prior to being used as standards and templates in DNA polymerase assays. Details of the extraction and purification procedures were described elsewhere [15]. Purified IAP from N-18 neuroblastoma cells, labelled with all four 3H-ribonucleosides at 5 #Ci/ml of media for 48 hr,

Fig. 1.

Intracisternal A particles in a CF-1 mouse oocyte, x 106,000.

Fig. 2.

Intracisternal A particles in a Balb/c 4 cell stage. Note associated crystalloid materials (C). x 45,500.

Fig. 3. Blastocyst of Swiss-Webster mouse embryo. Arrow denotes small particle which appears to be intracisternal, C, crystalloid materials ; P, plaques along endoplasmic reticulum, x 39,000. (to face p. 132)

Fig. 4. Secondpolar bodyfrom an IGR blastoeyst. Note the intracisternal A particles, fibrous lined ER cisternae and the lack of ribosomes. E. embryo proper. × 19,700. Fig. 5. Intraeisternal A particles and budding intracisterhal A particles in cell from mouse embryo placed in culture as a blastocyst and grown for 4 days. × 28,000.

Biochemical Protierti~s and Replica'f%~ oJ'~MUrink,Intr~temal A Particles were subjected to RNA extraction and purification by the same method [15]. The purified 3H-RNA from IAP was then analyzed by velocity glycerol gradient along with standard 18S rRNA and 70S RNA in a SW 40 rotor at 40,000 rev/min for 90 min. Temp/ates All natural templates were purchased from Miles Laboratory, Ill. Activated salmon sperm DNA was prepared as described by Schlabaeh et al. [20]. Copo]lymers and homopolymers of various synthetic oligo-nucleotides with specified chain lengths were obtained from either Miles LaboratoEr or Collaborative Research Corp., Mass.

eurificaaon and analysis of the DNA products The radioactive DNA products from an endogenous RNA-dependent DNA polymerase reaction of IAP were purified by sequential extractions with phenol-SDS and ether, and then precipitated in ethanol-NaC1 a t - 2 0 °. The radioactive DNA precipitates were redissolved in 0.3M Na-acetate and further purified by gel filtration in a sulphopropyl Sephadex C-50 column in the same buffer. The radioactive fractions at the void volume were pooled and reprecipitated in ethanolNaG1 prior to cesiium sulfate isopycnic centrifugation analysis at 40,000 rev]min for 60 hr at 4 ° in a SW 50 rotor. At the end of the equilibrium run, the sample was fractionated. Density was determined by direct weighing and the samples were precipitated in 12.5 % TCA and processed for radioactive determination as described. Purified DNA product was also hybridized back to IAP 60-70S RNA by the 50% formamide method at 37 ° for 18 hr. At the end of the hybridizatior~L the reaction mixture was adjusted for S-1 exonuclease digestion according to the method described earlier [21].

..7-1 Exonuclease The preparation of S-1 exonuclease from

Aspergillus oryzae crude a-amylase (Sigma, Mo.) has been described earlier [21].

Protein determination Protein was determined by the Lowry method [22]. Samples were first precipitated and washed with 10% perchloric acid prior to alkali digestion.

133

RESULTS

Replication of intracisternal A particles during early embryogenesis IAP are seen in small numbers in dictyate oocytes immediately after their release from ovarian follicles. They are always enclosed within agranular portions of the endoplasmic reticulum (Fig. 1). IAP are only rarely observed in oocytes after break-down of the germinal vesicle has begun. None were seen in oocytes which reached metaphase II either in vitro or in vivo, nor were any such particles observed in fertilized eggs. At approximately the 4 cell stage, large numbers of complete IAP reappear within the endoplasmic reticulum (Fig. 2). Quantitation of IAP at different stages of development was not feasible, but the number of particles seems to peak at the 4-8 cell stage. By the 16 cell stage (morula) and the 32 + cell stage (blastocyst), A particles are only rarely encountered within the now granular encoplasmic reticulum, and no cytoplasmic densities or budding particles are seen (Table 1). In tracing the morphologic sequence of events preceding the development of IAP, the following observations were made. Crystalloid aggregates (C) composed of units about 40 nm wide, 300 nm long, with a cross striation every 12 nm [23], and usually contiguous with the endoplasmic reticulum, appear first in the fertilized egg (Fig. 3). These structures seem to increase in number and size until the morula stage. During the blastocyst stage crystalloid units often form densely staining plaques (P) along cisternae of encoplasmic reticulum (Fig. 3). The very small particles seen occasionally within the endoplasmic reticulum at this stage may be cross sections of these plaques (Fig. 3). Stages in IAP formation have also been observed within the first and second polar bodies. Polar bodies possess most of the organelles of the embryo but are notably deficient in ribosomes. Crystalloid formation often proceeds in the second polar body until one or a few very large crystalloids are formed; these fill much of the polar body and are easily visible in sections cut for the light microscope. Interestingly, at the blastocyst stage when IAP are rare within the embryo proper, IAP as well as cisternae of endoplasmic reticulum lined with fibrous material can still be observed in the surviving second polar body (Fig. 4). When mouse embryos are cultured beyond blastulation they hatch from their zonas, attach to the substrate, and cell proliferation

134

Stringner S. }rang, Patricia G. Calarco and Nelson A. Wivel

begins, i.e., the outgrowth stage begins. These proliferating cells show an occasional budding or complete IAP (Fig. 5) but crystalloid material is notably absent. The time of appearance and approximate frequency of occurrence of crystaUoids and IAP from fertilized egg to the outgrowth stage is summarized in Table 1. Table 1. Occurrence of intracisternal A particles and associated structures in preimplantation mouse embryos

J

Fertilized egg* 2 cell (early) 2 cell (late) 4 cell

+ + + + +

8 cell

+ + +

Morula Early blastocyst Expanded blastocyst Outgrowth

+ + + + + 0

+ + + + + + +

0 0 + + + + +

+

+ + + +

0 0 0 +

+ + + +

*Examined at the stage just prior to the first cleavage, approximately 24 hr after fertilization.

Table 2.

1.64

1.42

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_~_2'000

i

t ,.

_N 400

i

27 io

T

20 3o 40

Ib 2'0 -'30"- - ~

o

±

FRACTIONNUMBER

Fig. 6. Profiles of high molecular weight RNA (60-70S) associated with intracisternal A particles purified from N-18 neuroblastoma cells. 6 A. Velocity glycerol (10-35%) gradient analysis of a typical preparation of 3H-RNA of intradsternal A particles, 0 - - 0 - - 0 . Standard 3H-188 ribosomal RNA was from Miles Lab., - - O - - C - - , and standard 708 RNA from RL V (arrow) was run in an accompanyinggradient simultaneously. 6B. Cesium sulfate isopycnic centrlfugation analysis of the 60-70S 3H-RNA of intracisternal A particles from the bracketedfractions in 8A, 0 - ' - 0 - - 0 . Standards are aH-18S rRNA and 3H-DNA purified from 3T3 cells, © - - © - - © , ran simultaneously in another tube. Density was designated by A . . A . . ~.

(Counts/rain per 30 rain/50 gl) 864 1478 25 18 560 42 228 23 1150

3H-substrate used

3H-TTP

Stimulation by synthetic RNA, D N A and R N A - D N A d T 12- XS'poly rA (50/tg/ml) dT 12-1S.poly dA (50/lg/ml) poly dT-poly rA (50/tg/ml) poly rA.poly rU (50/~g/ml) Endogenous--none dG x2- XS'poly rC (50/~g/ml)

20,000~

i 0,ooo 5o

TCA-insoluble tritium incorporation

Complete with Mg 2+, 10 ham Complete with M n 2+, 0.8 m~t Minus Mg 2+ or M n 2+ Minus D T T Minus Na + or K + Minus dA, dG, dC, T P Minus Triton X-102 treatment Preincubation with RNase at 37°]2 hr Preincubation at 37°/2 hr

40.oo0

3O,OOo~

Characteristics of an RNA-dependent D N A polymerase activity associated with intracisternal A particles

Conditions

B

"...

~.,,ooo oo~s

Crystal- Budding A Complete A loids particles particles

Stage

I--

A

8825 940 5075 812 410 2050

SH-TTP

3H-dGTP

All assay conditions were described in the text. Complete condition referred to inclusions of all critical requirements such as K + or Na +, M n 2+ or Mg 2+, DTT, all four deoxyribonucleoside triphosphates, and 0-018% Triton X-102 treatment, at 37 °. Each assay contained 12.5/~g of IAP protein from plasma cell tumor (MOPC-104E). Preincubation with RNase (25/tg/ml) was carried out in 0.15 M NaC1 and the subsequent assays were in the presence of M n 2+. Assays with synthetic templates were carried out with 100/tCi/ml of the specified substrate and in the presence of M n 2+, at 30 °. SH-TTP was 13,000 counts/rain per pmole and 3H-dGTP was 2560 counts/rain per pmole.

Biochemical Properties and Replication of Murine Intradstamal A Particles High molecular weight RNA as intrinsic nucleic acid of IAP IAP are observed occasionally in normal differentiated murine tissue. Upon neoplastic transformation :however, these viral particles can be recovered in sufficient amounts from such tissues as the plasma cell tumor or neuroblastoma so thai: biochemical analyses may be carried out. It has been established earlier that IAP purified from N-18 neuroblastoma cells showed the ability to incorporated tritiated ribonucleosides !into intrinsic RNA [15]. This intrinsic RNA ]~as a sedimentation value of 60-70S (Fig. 6A) since it co-sedimented with RLV 70S RNA, and it exhibits a density of 1.64 g/cm 3 in cesium sulfate isopycnic centrifugation analysis (Fig. 6B), typical of an RNA.

D NA polymerase activities associated with IAP Purified IAP exhibited an endogenous RNAdependent DNA polymerase that can be stimulated to much greater activity by the addition of exogenous templates. Table 2 summarizes the basic requirements for maximal endogenous activity of the A particle polymerase; they included the presence of DTT, Mn 2+ or Mg 2+, Na + or K +, and all four deoxyribonucleo,dde triphosphates. Analogous to other C-type 'viral reverse transcriptase, the RNA-dependent DNA polymerase of IAP was sensitive to digestion with a low concentration of RNase even at a high concentration of NaC1, and it preferred Mn 2+ over Mg 2+ for its divalent cation requirement [24-26]. It is interesting to note that preincubation of the IAP in the same condition as that of RNase digestion resulted in a minor loss of RNAdependent DNA polymerase activity suggesting that the enzyme is rather unstable at 37 °. The stimulatory effects of a variety of natural and synthetic templates on the activity of the RNA-dependent DNA polymerase of IAP have also been investigated. Among the natural RNA templates tested at 50/~g/ml, which included AMV 70S RNA, RLV 70S RNA. MS-2* RNA, VSV~ R NA, Influenza viral RNA and QB~ RNA, only AMV showed some stimulation. Others either did not stimulate or showed slight inhibitory effect. Likewise, the natural DNA templates, T2 phage DNA and activated salmon sperm DNA at 50 /lg/ml, showed little or no stimulatory effect on the A particle RNA-dependent DNA polymerase. *MS-2 phage; tV.SV is vesicular stomatitis virus; **OB phage.

135

Among the synthetic templates studied (Table 2), dTX2"rA" exhibited the greatest stimulatory effect on A particle DNA polymerase activity, but not dT~ 2.dAn. When the activities of the polymerase stimulated by these two templates were compared, a characteristic ratio of dT12"rA"/dTa2"dA" greater than 8.5 was observed. Poly dT-poly rA, the universal template that stimulates both viral and cellular DNA polymerases [20, 27] also stimulated the IAP DNA polymerase. But the DNA copolymer, poly d(A-T), which was used efficiently by cellular DNA polymerase [20] did not stimulate A particle DNA polymerase. The diagnostic synthetic hybrid template oligo dG 12"poly rC [28] also stimulated A particle DNA polymerase.

Kinetics and product analyses of the endogenous reaction of the RNA-dependent DNA polymerase of IAP Kinetics of the endogenous RNA-dependent DNA polymerase of lAP differed in the presence of actinomycin D. The endogenous reaction of a representative preparation of IAP was generally linear up to 50-60 rain under the optimal assaying conditions described above (Fig. 7A). In the presence of 5 pg/ml of actinomycin D, the initial rate of the endogenous reaction became slightly slower although its linearity extended up to 120min before it slowed down (Fig. 7B). The DNA products labelled in tritium synthesized at different points during the course

B

12000\ I0000 z o

8000

o

6000

4000

~

2000

Z i

~

0

'

2'o

'

4o

' MIN

6'0 AT

20 37 °

40

60

Fig. 7. Kinetics of the endogenous reaction of the RNAdependent D NA polymerase of intracisternal A particles from MOPC-IO4E plasma cell tumor, 0 - " - 0 - - 0 (A), as compared with the same in the presence of 5 pg/ml*of actinomycin,.1), A - - A "~'.-'A (B).

136

Stringner 8. Yang, Patrida G. Calarco and Nelson A. Wivel

of the endogenous reaction were examined by cesium sulfate isopycnic gradient centrifugation analysis. It was found that the DNA products obtained at short-term reaction (5 min) were primarily linked to the endogenous RNA of the A particles (Fig. 8) and hence sedimented at the areas of both the RNA (1.65 g/cm 3) and the RNA-DNA hybrid (1.52-1.58 g/cm3). At the 15 min point, the distribution of the newly synthesized DNA was at all three regions, the RNA area, the RNA-DNA hybrid area, and the DNA area (1.42-1.44g/cm3), although the majority of the DNA was now at the hybrid

approximately 3500 counts/min of input 3HDNA product (equivalent to about 6.4 x 10-* pg) and 2"0 gg of A particle RNA. There was no hybridization of the 3H-DNA product of IAP endogenous DNA polymerase reaction with other heterologous RNA such as Q B RNA. Details of this study will be reported elsewhere. These results indicated that the newly synthesized DNA in an endogenous RNA-dependent DNA polymerase reaction of IAP represented a faithful transcript of the intrinsic RNAs of the A particles as reflected by their base complementarity in the hybridization results.

.45' RNA

15' 5'RNA

3ooj... I

DNA

RNA

DNA

1

1

DNA

"8000

- * "A • • -A

"6000

~"1.

1400

200Q_ ._c E %.

"4000

.~

: 2000

._ E

I00"

Q_

0

I0

15 20 25

.30

J

I

"L . . . . . . . . . . .

; ,o ,; 2'0 ~ 3'o,;

15 20

L

g 0

25 50

Fraclion number

Fig. 8. Cesium sulfate isopycnic gradient ¢entrifugation analysis of the D NA produas of the endogenous reaction of the RNA-dependent DNA polymerase of intracisternal A particles. Approximately 2"5 mg of highly purified intracisternal A particle protein was assayed as described in the text at 37° with I00 gCi of SH-dA, dC, dG, T, TP at equal molar ratio and adjusted to the same specific activity, 5 mCi[O.05 rag, in a final volume of 1.0ml. Samples of 200 ld were withdrawn at 5 min and at 15 min, and the remaining 600 ld at 45 min. The reaction was terminated by adjustment to 0.3 M Na-acetate and phenol-SDS extraction, followed by extensive purification of the 3H.DNA products as described in the text. 3H-DNA distribution was designated by Q - - - O w O and density was designated by A-

- -A-

and DNA areas (Fig. 8). At the end of a reaction of 45 min, the newly synthesized DNA sedimented totally at the DNA area (Fig. 8). The sedimentation pattern of the newly synthesized DNA, dependent on the time of the reaction in this case, also resembled those for other oncomaviruses [29-32]. When the long-term DNA product was purified exhaustively and hybridized back to the 35S and 60-70S RNAs of IAP in 50% formamide at 37 ° for 18 hr and the percentages of hybridization was analyzed by the Aspergillus S-1 exonuclease digestion method [21], a 70-90% efficiency was observed, using

- -A.

DISCUSSION The presence of intracisternal A particles in oocytes, their disappearance during maturation, and their reappearance at the 2--4 cell stage suggests a vertical mode of transmission. Furthermore, the observation of A particle formation in the first polar body at the late 2 cell stage suggests that information for gene expression was present in the maternal ovum. The time and place of appearance of crystalloid material accumulated during preimplantation period suggests a possible relationship with intracisternal A particle formation; how-

B~;ochemical Properties and Replication of Murine Intradsternal A Particles

ever, the proof of this relationship remains to be developed. Complete intracisternal A particles, when isolated in quantity from tumors, possess biochemical properties analogous to R N A tumor viruses, such as 60-70S R N A and an RNA-dependent DNA polymerase that synthesizes a DNA transcript of the intrinsic R N A with base complementarity. Therefore, in inl:racisternal A particles, there is an experimental system which provides some fundamental aspects of viral gene expression during embryoger:esis on both the ultrastrucrural and biochemical levels, and which is quite possibly related to the problem of neoplastic transformation. Whether intracisternal A particles, similar to that of fetal antigen,

represent a particular gene entity accidentally co-expressed with the other molecular anomalies in tumor development, or whether they might themselves be carcinogenic remains to be resolved. The relationship between the expression of this gene entity and malignant transformation constitutes a problem worthy of further investigation. Biochemical properties of intracisternal A particles, when better understood, might lead to development of diagnostic tools such as labelled DNA probe for early detection of viral synthesis and a more accurate tracing of events during tumor growth in an animal model system.

Acknowledgement--We thank Mr. R. Steinberg for his excellent assistance in photography.

~ C E S 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16.

137

K . R . MclI~rrIRE, C. L. VOOEL, G. L. PRINCLER and I. R. PAI'EL, Serum Alpha-fetoprotein as a biochemical marker for hepatoceUular carcinoma. Cancer Res. 32, 1941 (1972). E . D . HOLYOKE,G. RAYNOSO and T. CHU, Carcinoembryordc antigen in patients with carcinoma of the digestive tract. In Embryonic and Fetal Antigens in Cancer, p. 215-220. Oakridge National Laboratory (1972). A . J . DALTON and M. D. FELIX, The electron microscopy of normal and m;dignant cells. N.Y. Acnd. Sci. 63, 1117 (1956). A.J. DALTON,M. POTTERand lZ. M. MERWm, Some ultrastructural characteristics of a series of primary and transplanted plasma-ceU tumors of the mouse, d. nat. Cancer Inst. 26, 1221 (1961). E. DEHARVEN and C. FRIEND, Electron microscopy study of a cell-free induced leukemia of the mouse, jr. biophys, biochem. Cytol 4, 151 (1958). N.A., WIVEL and G. H. SMITH, Distribution of intracisternal A particles in a variety of normal and neoplastic mouse tissues. Int. J. Cancer 7, 167, (1971). J . J . TUMILOWlCZ and J. J. CHOLON, Intracisternal type A particles and properties of a continouus cell line originating from a gerbil fibroma. Proc. Soc. exp. Biol. 136, 1107 (1971). A . J . NOVIKOFF and L. BmMPICA, Cytochemical and electron microscopic examination of Morris 5123 and Reuber H-35 Hepatomas after several years of transplantation. Gann 1, 65 (1966). E.W. NADEL, W. BANFIELD,S. BURSTEINand A. J. TousiMm, Virus particles associated with strain 2 guinea pig leukemia (L2c/N-B). d. nat. Cancer Inst. 38~, 979 (1967). W. BxczvsKo, D. SOLTER, C. GRAHAM and H. KOPROWSKI, Synthesis of endogenous type-A virus particles in pathenogenetically stimulated mouse eggs. J. nat. Cancer Inst. 52, 483 (1974). P.G. CALARCO, and D. SZOLLOSI,Intracisternal A particles in ova and preimplantation stages of the mouse. Nature New Biol. 243, 91 (1973). W. BiczvsKo, M. PIENKOWSKI,D. SOLTERand H. KOPROWSKI,Virus particles in early mouse embryos, or. nat. Cancer Inst. 51, 1041 (1973). D . G . CHASE and L. PIK6, Expression of A- and C-type particles in early mouse embryos. J. nat. Cancer Inst. 51, 1971 (1973). E.L. KuFF, N. A. WIVEL and K. K. LEUDERS,The extraction ofintracisternal A particles from a mouse plasma-cell tumor. Cancer Res. 28, 2137 (1968). S.S. YANO and N. A. WIVEL, Analysis of high molecular weight ribonucleic acid associated with intracisterual A particles. J. Virol. 11~ 287 (1973). D. BALTIMORE,Viral RNA-dependent DNA polymerase. Nature (Lond.) 226, 1209 (1970).

138

Stringer S. Yang, Patrida G. Calarco and Nelson A. Wivel 17.

18.

19. 20. 21. 22. 23. 24. 25.

26. 27.

28. 29.

30.

31. 32.

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