Chicken leukosis virus genome sequences in DNA from normal chick cells and virus-induced bursal lymphomas

Cell, Vol. 4, 311-319,

April

1975,

Copyright01975

by MIT

Chicken Leukosis Virus Genome Sequences in DNA from Normal Chick Cells and Virus-Induced Bursal ,ymphomas Paul E. Neiman Department of Medicine University of Washington School of Medicine Seattle, Washington 98195 H. Graham Purchase and Willlam Okazaki U.S. Department of Agriculture Agricultural Research Service Regional Research Laboratories East Lansing, Michigan 48823

Genome sequences of two recent field isolates of avian leukosis viruses in the DNA of normal and neoplastic chicken cells were studied by DNARNA hybridization under conditions of DNA excess. Comparisons were made between 60-70s RNA from these viruses and that of a chicken endogenous type C virus (RAV-0), and of a series of “laboratory” leukosls and sarcoma viruses, by competitive hybridization analysis. A minimum of 16% of the genome sequences of both ALV isolates detected in DNA from iymphomas they Induced were not detected in normal chicken DNA. The vast majority of the fraction of RNA sequences from ALV which do form hybrids with normal chick DNA appear to be reacting with the endogenous provirus of RAV-0. The genomic representation of a variety of avian leukosis and sarcoma viruses in normal chicken cells could not be distinguished by these methods (except that 13% of the RAV-0 genome was not shared with any of the other viruses). In contrast, the portion of the ALV genome exogenous to the normal chicken genome showed signiflcant divergence from that of two sarcoma viruses (Pr RSV-C and B-77). The Increased hybridization of ALV RNA with iymphoma DNA was used to detect the appearance of ALV specific sequences in the bursa of Fabriclus following infection. Increased hybrldization was correlated wlth both the time after infection and the extent of replacement of the bursa by lymphoma. About one half of the increase in hybridization preceded histologic evidence of transformation. Introduction Specific proviral DNA sequences derived from RNA tumor viruses (Temin, 1964) of the chicken leukosis and sarcoma complex have been detected in host cells by a number of nucleic acid association techniques, including direct hybridization of viral RNA to cellular DNA under conditions of initial DNA excess (Neiman, 1972; Varmus et al., 1973; Shoyab, Baluda, and Evans, 1974a). Previous studies from our laboratory have focused on cellular DNA sequences related to helper independent strains of

Rous sarcoma virus (RSV) and to genetically transmitted subgroup E endogenous leukosis viruses represented by Rous-associated virus type 0 (RAV-0) (Vogt and Friis, 1971; Weiss et al., 1971). We concluded that all or nearly all of the sequences of the 70s RNA genome of RAV-0 were present in the DNA of all normal chicken cells with a very low (l-4) average frequency per haploid genome (Neiman, 1973). An apparently smaller fraction of the genome of Prague strain of RSV subgroup C (Pr RSV-C) was also detectable in the DNA or normal cells by this hybridization technique (Neiman, 1972). Study of the ability of viral RNA to compete for proviral sequences indicated that the vast majority of the RSV-related DNA sequences in normal ceils were accounted for by homology with the endogenous provirus of RAV-0 (Wright and Neiman, 1974). When chicken cells are infected and transformed by RSV the frequency of RSV-related sequences is increased and a larger fraction of the sarcoma virus genome is detectable in the genome of tumor cells (Neiman, 1972; Neiman et al., 1974a; Varmus, Heasley, and Bishop, 1974). Hybridization competition studies involving a transformation-defective deletion mutant of Pr RSV-C (td Pr RSV-C) provided evidence in support of the hypothesis that this sarcoma virus adds transformation specific sequences to the DNA of normal cells (Neiman et al., 1974a). Qualitatively similar observations have been reported for cellular DNA sequences related to avian myeloblastosis virus studied by a similar technique (Shoyab, Evans, and Baluda, 1974b). While these observations suggest that oncogenesis by these avian viruses is mediated by the introduction of viral genes exogenous to the normal chick genome, it should be noted that all the viruses studied were isolated many years ago from malignancies which arise only rarely in chickens, and might now be considered to be “laboratory” viruses. The common type C virus-associated neoplasm arising in chicken flocks is an undifferentiated lymphocytic lymphoma which arises in lymphocytes of bursal origin (Cooper et al., 1968). The etiologic avian leukosis viruses (ALV) are maintained by congenital infection under natural conditions, and can be readily isolated from embryos (Rubin, Cornelius, and Fanshier, 1961). In congenitally infected chicks, or following inoculation of newly hatched susceptible “leukosis free” birds, such viruses produce viremia which is suppressed with the development of specific antibody (Rubin et al., 1962; Rubin, 1962). In chickens which subsequently die of lymphoid leukosis, the first signs of neoplasm are foci of large anaplastic lymphoblasts in individual follicles in the bursa of Fabricius as early as 8 weeks of age (Cooper et al., 1968). The tumor grows locally until the onset of sexual maturi-

Cell 312

ty at 4-6 months when the lymphoma metastases widely. Removal of the bursa at any time after infection, but before metastases occur, prevents systemic lymphoma (Peterson et al., 1964; Peterson et al., 1966) but not the systemic viremia (Purchase and Okazaki, unpublished observations). In this article we report hybridization and hybridization competition studies involving the 60-70s RNA genome of new ALV isolates and DNA from normal chicken cells and from virus-induced lymphomas. We observed that, like RSV, ALV inserts exogenous viral gene sequences into the DNA of bursal lymphoid cells which undergo malignant transformation. Results Induction of Neoplasms by ALV Two recent independent field isolates of ALV were selected for this study (ALV 5938 and 5951). In a controlled test of pathogenecity, all 26 white leghorn isolator-reared chickens receiving ALV 5938 developed lympoid leukosis, and one bird also developed erythroblastosis. Of 22 birds receiving ALV 5951, 59% developed lymphoid leukosis, 27% erythroblastosis, 4% hemangiomas, and 4% osteopetrosis. A simultaneous cohort of birds receiving a standard subgroup A leukosis virus, Rousassociated virus type 1 (RAV-1) showed 83% lymphoid leukosis and 17% erythroblastosis, while uninoculated control birds showed no evidence of disease even at necropsy following 239 days of observation. It was significant to note that, despite susceptibility to exogenous infection with subgroup E viruses, this line of chickens did not develop neoplasms of any description following inoculation with RAV-0 (Purchase and Vogt, unpublished observations). Kinetics of Hybridization of W-labeled Viral 6070s Genome RNA wlth DNA ALV genome sequences in the DNA of virus-induced lymphomas were detected by hybridization with ALV 60-70s I*sl-labeled RNA under conditions of modest DNA excess as outlined in Experimental Procedures. Tumors used for DNA extraction were composed of greater than 95% malignant lymphoblasts. Figure 1A shows the kinetics of these reactions plotted as a function of Cot, where Co is the concentration of bulk DNA nucleotides and t is the time in seconds (Britten and Kohne, 1968) without correction for sodium concentration. As shown, about 70% of the radioactive leukosis virus RNA in both instances enters ribonuclease-resistant hybrids at Cot levels above 4 x 104. Except for a slightly faster rate of reaction of the first 15% or so of the sequences, the data points conform reasonably closely to the theoretical hybridization ki-

0

1 I02 C,t

Figure 1. Kinetics of Hybridization with DNA from Normal Chick Lymphomas

I

IO3 I04 (mole -set/liter)

I 10s

of 1WLabeled Viral 60-70s RNA Embryos and from Virus-Induced

Formation of ribonuclease resistant hybrids formamide at 49°C was measured as described ences cited.

in 5 x SSC, 50% in the text and refer-

Pane/A describes hybridization reactions between u+labeled RNA from ALV 5936 (0) and DNA from bursal lymphomas which it induced, as well as reactions between RNA from RAV-0 (A) or ALV 5951 (m) and DNA from lymphomas induced by that field isolate. The computer-assisted calculations of a theoretical kinetic curve for viral RNA reacting with sequences present with an average frequency of one complete copy per haploid genome (-) and a derivative two copy curve (---) are shown. Panel 6 describes hybridization reactions between RNA from ALV 5936 and ALV 5951 (same symbols) and DNA from normal chicken embryos. Reaction between RAV-0 RNA and the same uncharacterized normal chicken embryos (A) as well as with DNA from chf and gs antigen negative line 15 embryos (V) are also depicted and can be compared with the theoretical 1 copy kinetic curve (as for panel A).

Chicken 313

Leukosis

Virus

Genes

in Bursal

Cells

netic curve for RNA hybridizing with proviral DNA sequences present with an average frequency of two complete copies per haploid genome. RNA from RAV-0 formed hybrids with tumor DNA to a similar extent. Labeled RNA from the endogenous virus formed hybrids with DNA from normal chick embryos as extensively as it had with tumor DNA, but at a slightly slower rate (Figure 1 B), reaching 70% at Cot values of about 7-8 x 104. This observation was not significantly different in the reaction of DNA from otherwise uncharacterized embryos from leukosis-free flocks or from line 15, viral group specific (gs) antigen and chicken helper factor (chf) free embryos. In contrast the same figure shows that RNA from both ALV isolates formed hybrids much less extensively with normal chicken DNA than had been noted in reactions with lymphoma DNA, not quite reaching 50% at the highest Cot value studied. These observations are consistent with the proposition that the ALVs contribute genome sequences to the lymphoma cells which are not detectable, by this technique, in normal chicken DNA. Further evidence in support of this proposition was sought by competitive hybridization experiments.

Competitive RNA-DNA Hybridization Studies With Proviral Sequences Endogenous to Normal Chlcken DNA Figure 2 depicts the competitive effects of unlabeled 60-70s RNA from various chicken leukosis and sarcoma viruses in hybridization reactions between ‘+labeled ALV RNA from both field isolates and normal chicken DNA. The extent of hybridization on the ordinate was measured at Cot values of 1.5 x 104 in the presence of increasing added RNA up to an estimated 200 fold excess (for a proviral sequence frequency of 1 per haploid genome). Competition by homologous RNA from either ALV isolate appears to follow closely the theoretical curve for identical RNA (see Experimental Procedures). RNA from other exogenous viruses such as B-77, a subgroup C sarcoma virus, and RAV-7, a laboratory subgroup C leukosis virus, also produced complete competition, suggesting the fraction of the ALV genome represented in normal chicken DNA is shared by a wide variety of exogenous leukosis and sarcoma viruses. As is shown in the same figure, RNA from RAV-0 also competed extensively for ALV related sequences in normal DNA, but reached a plateau about 2.5% above that expected for complete competition. This difference is at the limit of significance. However, a similar result was observed in measurements of the competitive effect of RNA from RAV-0 in hybridization reactions between labeled RSV RNA and normal

1,

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Figure 2. Competition Studies in the Reaction between ALV RNA and Normal Chicken Embryo DNA

r WLabeled

Reaction mixtures with radioactive RNA from ALV 5936 were incubated to a Cot value of about 1.5 x 104 in the presence of increasing quantities of unlabeled 60-70s RNA from the same virus (o), RAV-7 (0, B-77 (O), and RAV-0 (A). The competitive effect of unlabeled RAV-0 RNA was also tested in reactions with ‘2Wabeled RNA from ALV 5951 (A). The data are compared with a theoretical competition curve (---), the derivation of which is described in the text. The lower abscissa is plotted relative to the theoretical competition curve appropriately for 1 proviral copy per haploid genome.

chicken DNA (Wright and Neiman, 1974). In any case we can conclude that, within the limits of the method, all but a very small fraction of the ALV related sequences in the genome of normal chicken cells are accounted for by the endogenous provirus of RAV-0. Figure 3 depicts the competitive effects of RNA from the same viruses on reactions between 125llabeled RNA from RAV-0 and normal DNA. Since we presumed that normal chicken DNA contains a complete endogenous provirus for RAV-0, the 60% hybridization reaction was normalized on the ordinate. Homologous RNA from RAV-0 was observed to compete completely along the theoretical curve while RNA from ALV 5951, as well as from the other exogenous viruses, competed to a plateau value of about 13% less than that expected if they contained all of the RAV-0 sequences detected in normal DNA.

Hybrldization Competitlon Studles Wlth ALV-Related Provlral Sequences in Lymphoma DNA Competition by unlabeled RNA from several sources with I*sl-labeled ALV RNA for proviral DNA

Cdl 314

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Figure 3. Competition in the Hybridization Labeled RAV-0 RNA and Normal Chick

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Reaction between Embryo DNA

The extent of competition at Cot = 1.5 x 104 of RNA from (A), ALV 5951 (m), RAV-7 (0, and B-77 (0) are depicted.

0.008

‘2%

RAV-0

sequences in lymphoma DNA is described in Figures 4 and 5. A normalized ordinate was also used to display this data. Unlabeled RNA from both ALV isolates competed with radioactive RNA from either virus close to the theoretical curve for identical sequences. Thus the genomes of ALV 5938 and ALV 5951 could not be distinguished by this technique. By contrast, RNA from RAV-0 competed incompletely with a plateau value, suggesting that about 18% of the genome sequences of the oncogenic leukosis viruses detected by hybridization are not present in the genome of the endogenous virus. The relationships of genomic RNA from three subgroup C laboratory viruses to that of the field ALVs is detailed in Figure 5. The leukosis virus RAV-7 eventually competed completely with ALV for complementary sites in lymphoma DNA, but required a higher RNA-DNA ratio to achieve this level of competition than was expected. The reason for this anomalous competition curve is not readily apparent, but the possibility of an excess of defective (deleted) RNA molecules in the RAV-7 stock has not been excluded. RNA from both sarcoma viruses B-77 and Pr RSV-C failed to compete completely, plateauing about 1.2% above the value for identical RNA. These observations are in striking contrast to the competition studies of ALV sequences in normal chick embryo DNA, and indicate that the heterogeneity detected in this experiment is related to oncornavirus genes which are exogenous to the genome of normal chicken cells.

DNA

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: RNA

0.18 I RNA

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Figure 4. Competition Studies of Homologous RNA and Endogenous Virus RNA in the Hybridization Reaction between ALV-Radio.active RNA and DNA from ALV-Induced Lymphomas The extent of hybridization of radioactive RNA from ALV 5938 with DNA from a lymphoma induced by this agent was measured as in Figures 2 and 3 in the presence of increasing quantities of unlabeled RNA from the same virus (0) ALV 5951 (m), and RAV-0 (A). A similar study was performed for competition with radioactive RNA from ALV 5951 by cold RNA from ALV 5938 (0). RAV-0 (A), and rat liver polysomal RNA(V) as a nonspecific control. The lower abscissa has been adjusted relative to the theoretical competition curve appropriately for 2 proviral copies per haploid genome.

The Appearance of Avian Leukosis Virus Specific Sequences in Bursal Lymphocyte DNA of Infected Chickens The detection of ALV-specific DNA sequences in lymphoma cells provided an opportunity to begin to study the sequence of events in the chicken bursa following infection with lymphomagenic C type viruses. The appearance of infectious leukosis virus and of ALV-related DNA sequences was studied in bursas removed at various times from a cohort of chickens following inoculation with a “stansubgroup A leukosis virus, RAV-1. dard” Radioactive RNA from ALV 5951 was used as a probe for leukosis virus-related sequences rather than RNA from RAV-1 because of technical difficulties in obtaining large quantities of RAV-1 RNA, and because, on the basis of the foregoing experiments, we expected sufficient homology between leukosis viruses to allow detection of the specific RAV-1 sequences in bursal DNA. Table 1 lists the data which describe the appearance and quantity of cells acting as infectious centers and of detectable tumor in the bursas of infected chickens during the 26 weeks observation period. The proportion bursal

Chicken 315

Table

Leukosis

1. Viral

Virus

Infection

Genes

in Bursal

Cells

and Transformation

Infectious Units Inoculated

of Bursal

Cells

in Newly

Hatched

Line

15 X Line 7 Chicks

Injected

with

Estimate of Fraction of Eursa Replaced by Tumor

Proportion of Infected Cells’

Time (Weeks)

Wing Band

0

10

J5819

0

Number

RAV-1

0

0

10

J5824

0

103

10

J5793

2.8

x

103

10

J5765

2.2

x 10-S

0

103

10

J5777

7.5

x 10-S

0

103

18

J5774

1.3 x 100

0

103

18

J5752

2.6

x

1%

103

18

J5780

1.7

x 10-S

10”

18

J5782

1.6 x

10-Z

10’

26

J5767

4.8

x

10-I

70%

10’

26

J5779

1.5 x

IO-’

100%

*Proportion

of washed

bursal

cells

producing

virus

detected

by endpoint

h

4I

80-

.s 3 70:S $ 60-s

4I I

I

I

00004

0008

I:40

Viral

DNA

0.16 pg

Figure 5. Competition Viruses in the Reaction and Lymphoma DNA

I:20

0.32

Competing

I:60

I:80

I:100

I:IE

: RN A

0.48

0.64

0.8 ‘??

RNA

by RNA from Other between I+Labeled

Sarcoma RNA from

Leukosis ALV 5938

Conditions were identical to those shown in Figure 6. Extent of hybridization was measured in the presence of unlabeled competitor from ALV 5938 (O), RAV-7 (0, B-77 (0), and Pr RSV-C (Q).

cells producing infectious particles in our assay system varied widely over a 5 log range, but did tend to increase, in general, with time and the appearance of gross lymphomatous transformation. We note that the extent of transformation of the whole organ was estimated on the basis of gross tumor mass and obvious geographic replacement of the normal bursal architecture on histologic examination. Small foci of neoplastic cells may be

dilution

0 10-I

0

10-i

in the phenotypic

25% 25%

mixing

assay.

found as early as 8-10 weeks (Cooper et al., 1968), but these were difficult to quantitate and appeared to occupy a negligible fraction of the gland. The appearance of proviral sequences of the infecting leukosis virus was recognized by the increasing extent of hybridization of ALV ‘25l-labeled RNA with bursal DNA depicted in Figure 6. DNA from uninfected bursas was indistinguishable from normal chick embryo DNA in this reaction (data not shown). Bursal DNA extracted 10 weeks after infection from specimens showing little if any evidence of transformation yielded a slight increment in the extent of hybridization over contol, more clearly noticeable with a specimen yielding a high virus titer (J5793, Table 1) than with a sample of DNA extracted from a pool of two bursas with low virus titer (J5765, J5777). At 18 weeks bursas showed a spectrum of transformation from virtually none to 25% replacement with grossly obvious tumor. This was associated with a further increment in the extent of hybridization which varied over a narrow range. Finally at 26 weeks two bursas, about 70 and 100% transformed, respectively, were studied which yielded DNA giving further increases in the extent of hybridization observed up to a level of reaction with DNA from the bursa completely transformed by RAV-1, which was indistinguishable from that achieved with DNA from ALV induced lymphomas. Aside from the slight effect seen with DNA from bursas taken at 10 weeks, the widely ranging levels of virus producing cells detected by endpoint dilution in the phenotypic mixing assay and listed in Table 1 seemed to correlate poorly with the extent of hybridization. Theexcellent correlation between increasing hybridization with time after infection is shown in Figure 7. The same figure also depicts the correlation of the same parameter with the extent of transforma-

Cell

316

. . A

l.O20 Percent

Figure fection

0 A --I burso replaced

by tumor

:

IO 20 30 Weeks after Infection

7. Relationship of Increased Hybridization to Time and Replacement by Malignant Lymphoma

after

In-

The scale on the ordinate is the ratio of the extent of hybrid formation with DNA from infected bursas over that obtained with normal DNA. The data points represent the average of the four determinations above Cot values of 7 x 103, and the symbols are identical with those in Figure 6.

/ /

I

lo2 C,t Figure 6. Kinetics 5951 with Bursal

I

lo4 IO3 ( mole - set /liter)

of Hybridization DNA Following

of r*rl-Labeled RNA from Infection with RAV-1

I

lo5 ALV

Formation of ribonuclease-resistant hybrids was measured in reactions with DNA from bursas 10 weeks after infection with no tumor and high virus titer (J5793, A), 10 weeks after infection with no tumor but high virus titer (J5765 and J5793, 0), 18 weeks after infection with no tumor but low virus titer (J5774 q ), 18 weeks after infection with 1% replacement by clinically apparent lymphoma (J5752, n ), 18 weeks after infection with 25% replacement by lymphoma (J5780 and J5782, A), and 26 weeks after infection with 70% (J5767, v), and 100% (J5779, 0) replacement by tumor. The wing band “J” numbers correspond to those in Table 1. The upper curve (---) is identical with that for two complete genome copies in Figure 1, and the lower curve (***a) represents the hybridization of ALV RNA with DNA from normal chick embryos (Figure 1) and from uninfected bursas (not shown).

tion. We were interested to note that about one half of the increase in hybridization which was observed occurred before obvious tumor nodules were detected in the bursa. Discussion Interpretation of the foregoing hybridization studies must be qualified by the inherent limitations of the DNA-RNA hybridization technique which have been discussed previously (Bishop, 1972; Straus and Bonner, 1972; Hutton and Wetmur, 1973; Neiman, Wright, and Purchase, 1974b). The most important qualification follows from the observation that the most extensive hybridization achieved by viral RNA and DNA from host cells involved about 70% of the radioactivity in the reaction mixture. There is evidence to support the assumption that the nucleic acid sequences involved in these exten-

sive, but partial, hybridization reactions between ALV RNA and infected cell DNA, or between RAV-0 RNA and normal chick DNA, are representative of the entire genome. First, transfection experiments demonstrate that complete proviruses for both exogenous and endogenous chicken oncornaviruses are present in cell DNA extracted from appropriate sources (Hill and Hillova, 1972; Cooper and Temin, 1974a,b). Second, the hybridization reactions reported here conform to the predictions of theoretical hybridization kinetic curves based on best available estimates of hybridization rate constants adjusted for the effects of thermal scission and modest levels of DNA excess (Neiman et al., 197413). This later observation is, however, only suggestive since there is no independent means of confirming the estimates for rate constants in this viral system. Definite assignment of the fraction of viral genome involved in these studies therefore must be limited to whatever portion between 70 and 100% is actually represented in these reactions. The central conclusion from these studies is that the more extensive hybridization of ALV 60-70s RNA with DNA from virus induced lymphomas, in comparison with that observed with DNA from normal chicken tissue, is caused not only by some slight increase in the frequency of proviral sequences but, principally, by the insertion of new viral genes. This conclusion receives its strongest support from the competitive hybridization studies. These experiments demonstrate, first, that the vast majority of the genetic representation of ALV endogenous to the normal chicken cell is accounted for by the provirus of RAV-0, and, second, that RAV-0 RNA as a measure of the endogenous portion of the ALV genome lacks at least 18% of the proviral sequences for ALV detected in virus-induced lym-

Chicken 317

Leukosis

Virus

Genes

in Bursal

Cells

phomas. These findings suggest that, analogous to the transforming functions of RSV, this exogenous portion of the ALV genome mediates lymphoid leukosis in chickens. Additional circumstantial support for this hypothesis is derived from the observation that RAV-Q present either as an endogenous provirus, or when inoculated into this line of C/O chickens, appeared to lack the capacity to produce lymphoma (or any other tumor) (Purchase and unpublished observation). vogt, However, transformation-defective deletion mutants analogous to those used to study the nature of the Rous sarcoma virus transformation functions are not available in the leukosis system. The assignment of transforming functions to any segment of the ALV genome remains speculative. The studies described here shed some further light upon the genetic relationships between some representatives of the chicken leukosis-sarcoma complex. In the competitive hybridization analysis of viral genes present in normal chick DNA, we were unable to discriminate between the endogenous sequences of several leukosis and sarcoma viruses despite their widely variant backgrounds. This is perhaps not surprising, since normal chick cells can express both viral envelope (chf) and structural protein (gs antigen) functions which are widely shared. It should be noted, however, that even the hybrids of high thermal stability formed in this viral system can harbor up to 1 or 2% mismatched base pairs (Laird, McConaughy, and McCarthy, 1969) providing a basis for structural and functional differences which may occur in these functions among the several viruses. Further, the data give the barest suggestion of the presence of cellular sequences related to a very small fraction (2-4%) of the oncogenic ALV isolates which are not shared with RAV-0, and a clear demonstration of about a 13% portion of the endogenous provirus of RAV-0 which is not represented in the genomic RNA of any of the leukosis and sarcoma viruses which we have studied. The relative roles in these differences of such factors as simple sequence divergence as opposed to differences in gene function (for example, subgroup envelope determinants, regulatory functions for endogenous viruses, or oncogenicity) between endogenous and exogenous viruses remain to be determined. In contrast to the homology of their endogenous genes, hybridization competition studies indicate a striking divergence between the portion of leukosis and sarcoma virus genomes exogenous to normal chick DNA. Previous studies of this type have failed to discriminate between two independently isolated sarcoma viruses of the same subgroup, B-77 and Pr RSV-C (Wright and Neiman, 1974), and in this instance we could not distinguish the genomes of

two field isolates of ALV. However, there are clearly distinct unshared sequences in the exogenous portions of both classes of viruses. It is reasonable to speculate, again, that such differences underlie differences in oncogenic spectrum. Finally, the quantitative differences in hybridization of ALV genomic RNA with DNA from normal and lymphomatous tissue provided an opportunity to explore the sequence of events in the bursa of Fabricius in the long interval between the onset of infection and neoplastic transformation. The mechanisms interposed between the insertion of the infecting viral genome and the formation of clinically apparent lymphomas are likely to be many and complex, raising issues of molecular, immunologic, and humoral controls. The specific purpose of this experiment was to distinguish between two possibilities: -either the genome of the infecting ALV is found primarily in transformed lymphoid target cells, -or the leukemogenic viral genes are widespread in the normal cell population of the bursa for long periods of time before malignant transformation occurs. The observations reported here indicate that the iatter alternative is correct. Nearly one half of the detectable increase in hybridization of ALV RNA with bursal DNA which ultimately occurred was apparent by 8-10 weeks after infection at a time when there was extensive viral implication, but well before any extensive neoplastic transformation. The very wide range of virus-producing cells in the bursa at all times after infection suggests that ALV genome-carrying cells may or may not produce infectious virus. Assuming a reasonably even distribution of viral genome amongst the infected cells, these observations suggest a slow spread of virus infection, and/ or clonal growth of apparently normal virus-infected cells, within the organ for a considerable period of time, eventually affecting a large fraction of the cell population. Gross clinical transformation appeared then to occur as a secondary event in some (probably small) subpopulation of infected cells which grow slowly, but relentlessly, to replace the organ and metastasize. Experimental

Procedures

Viruses and Viral Propagation ALV 5938 and 5951 are recent field isolates from normal embryos from field flocks with a low spontaneous incidence of lymphoid leukosis. These viruses were isolated and propagated on chick embryo fibroblasts following a single endpoint dilution according to previously described methods (Rubin, 1960) but retained both A and B subgroup envelope determinants when tested by interference with RSV transformation (Vogt, 1970). RAV-0 was spontaneously released from line 7 chick embryo cells of the C/A phenotype originally supplied by Dr. Peter Vogt. Stocks of RAV-7, Pr RSV-C, and S-77 were also originally obtained from Dr. Vogt. All virus-

Cell 318

releasing cultures were grown on 75 cm3 tissue F-10 (GIBCO) supplemented growth medium.

culture

dishes

in

Viral Tumors and Normal Control Tissues Bursal lymphomas were obtained by injecting 104 and 105 tissue culture-infecting doses of ALV 5951 and ALV 5938, respectively, intraabdominally into newly hatched chicks of a cross between line 15 and 7 (Crittenden, 1968). These ch’icks are of the C/O phenotype, are positive for chf, and are susceptible to exogenous infection with subgroup E viruses (for example, RAV-0). Viremia with RAV-0 is not present. All chicks were vaccinated against Marek’s disease (Okazaki, Purchase, and Burmester, 1970) and reared in isolation for 10 weeks. Procedures for serial study of the introduction of ALV genomes into bursal DNA involved inoculation of RAV-1, a standard subgroup A leukosis virus, into the same line of chickens. Cohorts of chickens were sacrificed at the time intervals indicated and bursas removed. The extent of transformation was estimated both by gross observation and by examination of histologic sections of the whole organ. Small aliquots of bursal tissue were removed, and a washed cell suspension prepared. The proportion of virus-producing cells was estimated by endpoint dilution in the phenotypic mixing assay in which duplicate 2 fold dilutions of bursal cells were co-cultivated with RSV (RAV-0) infected C/O cells for 7 days. Phenotypically mixed sarcoma virus is subsequently assayed by focus formation on C/E cells. This technique has recently been described in detail (Okazaki, Purchase, and Burmester, submitted for publication). The remainder of the bursa was frozen at -20°C for subsequent DNA extraction. Normal 10 day chick embryos from leukosis-free flocks (Heisdorf and Nelson Farms, Redmond, Washington) line 15 embryos that are both gs antigen and chf negative (contributed by Dr. Lyman Crittenden), and bursas from uninfected control chicks at 10 weeks of age from experimental flocks, were used as normal control tissues for extraction of DNA. Extraction and Preparation of Cellular DNA and Viral RNA Whole cell DNA extraction from appropriate materials followed the technique of Marmur (1961). High molecular weight DNA was fragmented to an average single strand of molecular weight of 200,000 by limited depurination followed by alkaline hydrolysis (McConaughy and McCarthy, 1967; Neiman, 1973) and dissolved in 0.01 x standard saline citrate (SSC, 1.5 mM NaCI, 0.15 mM sodium citrate) until use. Unlabeled viral RNA from all viruses used was extracted by the sodium dodecyl sulfate-phenol method (Robinson, Pitkanen, and Rubin, 1965) from viral pellets recovered from l-3 I of tissue culture fluid, as recently described by Wright and Neiman (1974). Viral 60-70s RNA genome was separated from lower molecular weight species on 5 ml 1530% glycerol gradients, located by absorption at 260 nm and precipitated by 2 vol of cold ethanol as described in the same report. Viral RNA was stored in distilled water at -176°C until use. Polysomal RNA from rat liver, used as a nonspecific control, was a gift from Dr. Stephen Wright. Labeling of Viral RNA With 1251 The technique used to label viral 60-70s RNA with 1251to specific activities between l-4 x 107 cpm/pg was modified from that originally described by Commerford (1971) and by Tereba and McCarthy (1974). The specific procedures used and properties of r251labeled viral RNA in hybridization reactions has been reported (Neiman et al., 1974a). The iodinated RNA was found to have an apparent mean molecular weight of 60,000 by electrophoresis on mixed agarose acrylamide gels (Neiman and Henry, 1971) and was rapidly frozen in liquid nitrogen and stored at -176°C in distilled water until use. RNA:DNA Hybridlxation Kinetic analysis of hybridization reactions between viral RNA cellular DNA were performed in free solution under conditions

and of

modest DNA excess (Gelderman, Rake, and Britten, 1971; Melli et al., 1971; Grouse, Chilton, and McCarthy, 1972; Straus and Bonner, 1972). We have recently presented a detailed description of the hybridization conditions, their effect upon the size of the reacting polynucleotides, and the computer assisted generation of theoretical hybridization kinetic curves for the reaction described in this study (Neiman et al., 1974a). Briefly reiterated, reaction mixtures consisted of 1000-2000 cpm of labeled viral RNA (about 10-d pg) and 0.5 mg of single-stranded cellular DNA fragments (about 3 x 10-S pg of proviral DNA, assuming a minimum viral genome size of 3 x 106 daltons present with a frequency of 1 copy per haploid cell genome) in 0.05 ml of 5 x SSC, 50% formamide. These mixtures were incubated for various periods of time at the Top,, 49°C (Neiman et al., 1974a), for the hybridization reaction, and hybridization of labeled RNA was assayed by acquisition of resistance to ribonuclease. In order to achieve (uncorrected) Cot values of 6 x 104, incubations were carried forward for periods of up to 28 days. Degradation of nucleic acids under these conditions, estimated by electrophoretic migration of single-stranded nucleic acids on mixed agarose acrylamide gels, appeared to occur principally in the viral RNA that decreased in apparent mean size by a factor of 0.5 every 7 days. The simultaneous reassociation of nonrepetitive bulk cellular DNA fragments was independently measured by optical methods and found to have an apparent rate constant, K,, of about4.35 x IO-a(Britten and Kohne, 1968) under our hybridization conditions. An initial rate constant for viral RNA:DNA hybridization, KP, was assumed to be 0.75 K,, as suggested by the detailed studies of Hutton and Wetmur (1973). This computation was made following correction of K, for the approximately 4 fold larger size of the DNA fragments (Wetmur, 1971) yielding a value of Kz of about 1.83 x 1 O-4. Theoretical hybridization kinetic curves were generated by the use of a simple computer program which solves the general expressions for simultaneous RNA-DNA hybridization and DNA-DNA reassociation for small increments of time(Straus and Bonner, 1972) and included an adjusting equation which assumed that KZ decreases as a function of the square root of the mean size of the slowly degrading viral RNA (Hutton and Wetmur, 1973) over the period of the hybridization reaction. Competltlve Hybrldlzatlon Studles The technique for determining the sequence relationships of viral RNAs by hybridization competition analysis, including the derivation of the theoretical competition curves, has recently been described (Wright and Neiman, 1974; Neiman et al., 1974b). The method involves the addition of unlabeled viral RNA to reaction mixtures sufficient to vary the reaction conditions from the initial modest DNA excess to a modest RNA excess, thereby reducing the fraction of ‘25l-labeled RNA entering hybrids in proportion to homology with the unlabeled competitor. Theoretical curves for competition by homologous RNA were obtained by observing the decrement in extent of hybridization predicted by the computer program by increasing the RNA:DNA ratio in the reaction mixture. A convenient Cot level of about 1.5 x 104 was chosen for these studies (about 7 days of incubation), at which point DNA:RNA ratio of viral nucleic acids of 1:l was predicted to result in a 50% reduction in radioactivity entering the hybrids under conditions of these experiments. Both the computer-generated theoretical curves and the experiment data closely conform to: X y= x+1 where y equals the fraction of radioactive RNA entering hybrids and x equals the viral DNA:RNA ratio in the reaction mixture. Thermal Stablllty of the Hybrids The thermal stability of viral RNA-cellular DNA hybrids formed under the conditions of these experiments as measured by thermal chromatography on hydroxylapatite columns has been previously

Chicken 319

Leukosis

Virus

Genes

in Bursal

Cells

described (Neiman, 1972, 1973; Neiman et al., 1974a). Hybrids formed between r25l-labeled RNA from both ALV isolates and DNA from virus-induced lymphomas demonstrated an apparent T, of 82” in 0.12 M sodium phosphate by these methods.

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