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STUDIES OF GENETIC TRANSMISSION OF MURINE LEUKEMIA VIRUS BY AKR MICE

AKR mice, which regularly contain infectious murine leukemia virus, were mated with four Fv-1n strains of mice which show little or no expression of virus. F1, F2, and first and second backcross generation hybrids were tested for virus in tail tissue at 2 and 6 wk of age. The segregation data indicate that the AKR mouse contains two unlinked, autosomal, chromosomal loci, either of which suffices to induce detectable levels of infectious virus in Fv-1n progeny by 6 wk of age. One of the loci (tentatively referred to as V1) is on linkage group I, 25–30 map units from the locus for albino; the gene order tentatively appears to be N1-c-Hbb.

(Received for publication 27 June 1972) Over the past 20 yr, evidence has progressively mounted that murine leukemia virus (MLV) ~ or its genetic determinants are ubiquitous in both highleukemic and low-leukemic strains of mice, and that viral genetic determinants are heritable components present in all of the cells (1)(2)(3)(4)(5)(6)(7)(8)(9). However, little is known of the state of the viral genetic determinants in the cells, i.e. if they are present as one or more D N A and/or R N A copies, and if they can be carried as elements of the host chromosome. In high-leukemic strains, such as AKR, infectious virus and viral antigens are present throughout life, while lowleukemic strains generally show little or no antigen or infectious virus until late in life. In some low-leukemic strains, virus-related antigens can be detected in embryos or in certain postnatal tissues in the absence of demonstrable infectious virus (10)(11)(12)(13). The differences between inbred strains of mice in frequency and age when viral expression becomes detectable are sufficiently marked to allow genetic analysis of the transmission of viral antigen or infectivity.
The previously reported genetic studies have been based on viral antigen expression; in these, the segregation patterns differed with the antigen under study. The Gix antigen, a possibly virus-coded cell surface al|oantigen which in normal animals is expressed primarily on thymocytes, requires two unlinked genes for its expression (12). One o[ these genes is semidominant, and in crosses between 129 and C57BL/6 mice was found to be located in linkage group IX. Its expression requires the presence of a dominant gene, whose linkage is not yet established.
In recent studies of the expression of the group-specific (gs) antigen of MLV in the spleen of offspring obtained from crosses between AKR and C57L mice, Taylor et al. (14) observed two patterns. In F1, F2, and first-generation backcross mice, the occurrence of antigen indicated that the AKR mouse was contributing two unlinked dominant genes for its expression, either of which alone caused the appearance of gs 1 Abbreviations used in this paper: Bcl, first backcross; gs, group specific; GSA, Gross soluble antigen; IdU, 5-iododeoxyuridine; METC, mouse embryo tissue culture; MLV, murine leukemia virus; NIH, National Institutes of Health; p.f.u., plaque-forming units.
antigen. However, the frequency distribution of spleen gs antigen in multiple inbred lines derived from the F2 generation indicated that there was only one gene for gs antigen expression, and that another AKR gene must also be present in order for infectious virus to be formed.
Genetic studies have also been reported (15) on the occurrence of the Gross soluble antigen (GSA), a virus-specific antigen detectable in the serum of high-leukemic mice. Although GSA was transmitted by AKR to F1, F~, and backcross mice, the penetrance was not complete and showed a marked maternal influence. Consequently, no attempt was made to interpret the results in terms of gene segregation ratios.
The naturally occurring MLV of AKR mice is readily detected as infectious virus by tissue culture techniques (16). Since virus is uniformly present, is in high titer, and appears much earlier in life in AKR than in low-leukemic mouse strains, it was feasible to carry out genetic studies of transmission of MLV by AKR mice using appearance of virus infectivity in young animals as an end point. Particularly useful in this regard was the finding that the bone of the tMl was high in virus, providing a readily accessible tissue which could be sampled repeatedly.
We have studied progeny of crosses of a number of mouse strains with AKR by quantitating the amount of virus in the tail at 2 wk and again at 6-12 wk of age. The low-virus mouse strains used in these crosses were chosen to allow evaluation of the influence of several genes known to be of importance in MLV infection or in experimental or spontaneous leukemogenesis, i.e. 1t-2, Fv-1, and Fv-2, and to follow a viral genetic marker by using mouse strains known to carry virus of a host range type different from that carried by AKR. Table I shows the strains which were used. This report describes the results of virus testing of F1, F2, and first and second backcross generations of crosses of AKR with other mouse strains having the Fv-1 '~ allele (17,18), in which the AKR strain of MLV (N-tropic) (19) should have maximal opportunity for replication. Crosses with Fv-1 b mice are described in the accompanying report.

Materials and Methods
Mice.--The parental strains, their sources, and relevant genetic markers were shown in Table I. The National Institutes of Health (NIH) Swiss mouse is not an inbred strain, but was used because it was the only strain which combined albino, Fv-1 n, and apparent absence of endogenous infectious MLV. The virologic characteristics of the low-virus parental Fv-1 '~ strains are shown in Table II; the comparable studies of AKR were described previously 06).
Hybrid mice were bred in our laboratory; the majority were first litters, though some second litters are included. The designations of the hybrid strain combinations are given with the maternal strain first. Mice were tested for virus in the tail at 2 wk and again at 6 wk of age. Specimens were obtained by clipping about 1 cm off the tail; the specimen was briefly immersed in diethyl ether to destroy any surface virus. Scissors were also decontaminated between animals by dipping in ether.
Virus Testing.--Tail specimens were held on ice until the ether had evaporated, and 2% suspensions were prepared by grinding with a mortar and pestle and suspending in Eagle's basal medium with 20cfv veal infusion broth. After clarification by low speed centrifugation  (7). Embryos were immersed briefly in diethyl ether to remove possible contaminating surface virus, and were then minced and the cells dispersed with 0.25% trypsin. Petri dishes (50 ram; Falcon Plastics, Oxnard, Calif.) were seeded with 2 X 106 cells; plating efficiency was 10%. On the following day, two or more cultures were treated with 5 meg/ml IdU, which was held in the medium for 42 hr. 6 or 7 days after beginning Id'U treatment, treated and control cultures were exposed to UV irradiation (1800 ergs/mm 2) and overlaid with 4 X 106 NIH METC. 4 or 5 days later the cultures were developed for plaques by the UV-XC procedure (20). * Positive mice are those with virus at either time point. Thus, a mouse who was negative at 2 wk and positive at 6 wk is included in the calculation of the median titer at 2 wk. Titers are expressed as logi0 p.f.u./0.4 ml of 2% extract.

R E S U L T S
Virus, in Fi, F2, and First Backcross Generations.--Fi hybrid mice of crosses between A K R and C57BR or C57L closely resembled A K R both in the regular early appearance of virus and the titers attained (Table I I I and Fig. 1).
(NIH X AK)F1 mice showed a tendency for appearance of virus to be somewhat delayed and for the titer at 6 wk to be slightly lower than in the other crosses. There also was a slight delay in appearance of virus in some ( D 2 ) < A K ) F i  mice, but the titers at 6 wk were essentially the same as those in the (BR X AK)F1 and (L X AK)F1 mice. Stated in genetic terms, the tests of F1 mice showed that the inheritance of the AKR virus phenotype was dominant, highly penetrant, and contributed equally well by both sexes.
Tests of the first backcross (Bcl) and F2 generations showed clear-cut segregation, which was not dependent on the maternal direction of the cross. The segregation ratios (approximately 3:1 in the Bcl generation and 15:1 in the F2 generation) are those expected for two independently segregating loci, either of which gives a positive phenotype. The results are incompatible with models in which presence of virus is determined by a single gene or requires the joint presence of two genes from AKR. For convenience, we will refer to the two loci indicated by the segregation data as "AKR virus-inducing loci" and designate them as "VI" and "V2"; this is not meant to imply their mechanism of action or to be a formal proposal for nomenclature. "Vt" will be used to refer to the locus on linkage group I, as described below.
The quantitative data ( Fig. 1) give further support to a multiple independent gene model, in that 6-wk virus titers in the low and intermediate categories, and delayed appearance of virus, which were infrequent in the Fa mice, were common in the segregating generations. This would be expected if presence of both V loci tended to lead to earlier and higher titer of virus than either one alone.
Comparison of virus titers in the different backcrosses again suggested that there was more often a delay in development of titer in the hybrids with DBA as compared with those with C57BR and C57L, as was seen in the F1 generation. The C57L backcross mice were unique in that, although titers were as high as in the C57BR hybrids at 2 wk, in many mice the titer remained the same or had declined somewhat in the 6-wk test. Since this pattern was not seen in the Fx generation, it suggests that C57L mice may have one or more recessive genes, not shared by C57BR or DBA, which partially inhibit production or spread of virus in mature mice.

Second Backcross Generation of C57BR Hybrids.--To obtain further in-
formation on the number of AKR genes leading to appearance of infectious virus, and to obtain each of these genes in isolation from the others, secondbackcross generation mice were studied. 29 unselected BR X (BR X AK) mice were mated with C57BR; many of the males were also given N I H partners, and in a few cases C57L and DBA also. Females having a litter by a C57BR male were generally remated with an N I H male. The offspring of these crosses were tested for virus in the tail at 2 and 6 wk.
19 families with more than 10 offspring were obtained. Table IV shows the results of the 6-wk virus testing and their interpretation in terms of the number of AKR virus-inducing genes carried by the Bcl parent. Three families showed 3:1 ratios of virus to no virus; 12 families had ratios compatible with 1 : 1 ; and three families were virus negative. One family, No. 3-3, gave a pattern compatible with either a 3:1 or 1:1 segregation ratio. Interpreted in terms of the    number of AKR virus-inducing loci carried by the Bcl parent, these results indicate that three or four mice carried two loci, 12 or 13 carried one, and 3 carried none. This is in full agreement with the two-locus model inferred from the data in the preceding section, which predicts that the 19 families would show two, one, and zero locus inheritance in a ratio of 4.8:9.5:4.8.
Localization  determines whether the hemoglobin migrates as a single or diffuse, multiple band pattern on electrophoresis (21). The results, shown in Table V, indicate that Bcl mice which had received the c-Itbb region from AKR were consistently more likely to be virus positive than those without the AKR linkage group I markers. It should be kept in mind that linkage relationships are less easily discerned where two independently segregating genes give the same phenotype than in the usual one-gene system. In this type of two-gene system, the closest possible linkage of one of the two genes with a given marker would give only a 2:1 ratio between the proportions positive in mice with and without the marker.
The association with linkage group I was fully confirmed by the second backcross families (Table IV). Four of the families showing one-gene segregation ratios (Nos. 1-4, 2-4, 4-5, and 1-2), as well as the family with the intermediate segregation ratio (No. 3-3) showed clear-cut linkage to group I. Since BR X (BR X AK) mouse No. 2-4 is a recombinant between c and Hbb, having the AKR c locus, it appears that the gene order is Vl-c-Hbb; however, it is possible that he is a double recombinant. The father of family 3-3 is a recombinant between c and VI. Calculation of map distance from the data on both backcross generations gives an estimate that the gene is about 30 units from the c-ttbb region.
In contrast, four other one-locus families (Nos. 3-5, 4-1, 4-2, and 4-3) showed no association of virus with linkage group I, and presumably carry only the second locus (V2). The remaining one-locus families either did not have the AKR linkage group I markers, or had too few progeny to evaluate for linkage.
There was no evidence of linkage with the other coat color markers (brown, dilute, and leaden). There was no close linkage with H-2, though distant linkage camnot yet be ruled out.

Generation Parents
No. of embryos yield-Strain  (7). In contrast, cultures from low virus strains of mice rarely release detectable infectious virus after IdU treatment; this has been shown with BALB/c (9), C57BR, DBA, NIH, C57BL, and B10.D2 (J. W. Hartley and W. P. Rowe, unpublished data). Cultures from F1 hybrids between AKR and BALB/c react like AKR cells (22). Studies were made of the proportion of backcross embryos in which infectious virus could be induced by IdU; the embryos were from the Be1 generation and from the Bc3 generation of three of the one-locus families (Nos. 1-4, 1-2, and 4-1 in Table IV) which are considered to carry V1 or V2. Table VI shows the results.
In full agreement with the in vivo results, Bcl embryos gave results compatible with a 3:1 segregation ratio, while the one-locus families gave 1:1 ratios. Although the numbers tested are small, these findings provide an important confirmation of the tail test results, and indicate that the spontaneous development of virus in vivo and the inducibility of infectious virus by IdU in vitro are functions of the same genetic loci. DISCUSSION The data presented here are completely compatible with a model in which the high-virus characteristics of the AKR mouse are the result of its carrying two unlinked chromosomal loci, both of which have the capacity to induce virus synthesis early in life. One of the loci is located 25-30 map units from the albino locus on linkage group I, while the linkage of the other is not known.
Our results are in full agreement with the Bcl and F2 segregation data which Taylor et al. (14) observed in their studies of gs antigen in the spleen of hybrids between AKR and C57L; however, our data are incompatible with their findings in the F2-derived inbred lines. This discrepancy may indicate that different genetic loci are being followed in the two studies, or may be a reflection of the different tissues studied and the difference in techniques used.
While the data fit a two-locus model very well, and are incompatible with a single-locus model, it cannot be rigorously excluded that more than two virusinducing loci are segregating. If there were one locus with high penetrance and several other loci with low penetrance, the over-all segregation ratios might mimic a two-locus pattern. Further studies of the Bc2 families will be needed to clarify this point. Isolation of the virus-inducing loci in the backcross families will also be important for determining the linkage of the second locus, for testing for possible genetic differences between the MLV strains induced by the different loci, for testing for allelism with comparable loci in other high-virus mice, and for clarifying the basis of the quantitative differences observed in mice in the F1, Bcl, and F2 generations (Fig. 1).
The type of genetic system under study here should be thought of in a different frame of reference than most classical genetic systems. It is likely that we are not dealing with the usual type of cross involving the presence or absence of genetic material capable of making the functional gene product under study, since two of the four low-virus parental strains studied here show a low incidence of endogenous MLV infection. Rather, the high-and low-virus parental strains differ in the probability that the genetic material will be expressed. An appropriate analogy might be the high efficiency yielder and low efficiency yielder adenovirus type 2-SV40 hybrid virus particles (23). Both of these viruses contain the complete SV40 genome integrated into a defective Ad.2 genome, but they differ by a factor of 104 to 10 ~ in the probability that an infected cell will produce SV40 virus.
A further difference from classical genetic systems is that the end result of the expression of the genetic element being studied is production of infectious virus, which can convert other cells to a positive phenotype independently of their own genetic makeup and regulation. This carries the implication that a positive phenotype, instead of resulting from the normal functioning of the locus in a large number of cells, could be the result of the misfunctioning (mutation, misreading, excision, etc.) of the locus in a very small number of cells, or even in a single cell. This concept also applies to genetic studies of 1ViLV antigen expression if the crosses involve a high-virus parent.
In the crosses studied here there are no known virus genetic markers which would differentiate whether the MLV found in the various hybrids is derived from the AKR or the low-virus parent. Consequently, the studies reported here do not bear on the question of whether the two chromosomal loci detected are the viral genomes themselves, or expression genes which increase the probability of activation of virus determinants elsewhere in the cell. Attempts to utilize a virus host range marker for this purpose will be described in the companion paper (22).
It has long been known that spontaneous leukemogenesis in the mouse is influenced by a large number of genetic factors (24,25). In two respects, the studies reported here are of importance toward unraveling these genetic complexities. First, identification of two loci for expression of infectious virus affords a degree of control over this fundamental, early step in the disease process. By introducing a known set of virus-inducing genes into various genetic backgrounds, the genes affecting the later stages of the pathogenetic process may be amenable to identification and analysis. Second, the ability to monitor mice sequentially for degree of virus activity provides a means of evaluating the role of the early virologic events in determining the risk of developing leukemia. Questions such as whether leukemia incidence correlates with early appearance of virus, maximal titer attained, or the linkage group from which the virus derived can now be studied.
It was striking that for the most part the virologic patterns in the various hybrids were very similar, both qualitatively and quantitatively. This implies that there are no major undefined genetic influences, from either dominant or recessive genes, inhibiting virus activation or spread in the four Fv-l" strains studied here. Minor effects were suggested by the slight delay in virus development in DBA and NIH hybrids, and in the failure to attain maximal virus titers seen in the L X (L ;< AK) backcross mice. The latter could represent an effect of the H-2 b allele, which has been shown to confer partial resistance to viral leukemogenesis (26)(27)(28). However, there was clearly no significant effect of H-2 type on the virologic pattern of the F1 hybrids.
The lack of correlation of virologic status with the dilute locus, which is closely linked with Fv-2 (29), indicates that Fv-2 is not one of the V loci. Fv-2 type also appeared to be without effect on the expression of endogenous virus as judged by the similarity of the virologic findings in Bcl mice of the C57BR and C57L crosses (half Fv-2r/Fv-2 r and half Fv-2S/Fv-2 r) to those in the DBA crosses (all Fv-2S/Fv-2S). In contrast, Fv-1 has a marked influence, as will be shown in the companion paper (22).

SUMMARY
A K R mice, which regularly contain infectious murine leukemia virus, were mated with four Fv-1 ~ strains of mice which show little or no expression of virus. F1, Fe, and first and second backcross generation hybrids were tested for virus in tail tissue at 2 and 6 wk of age. The segregation data indicate that the A K R mouse contains two unlinked, autosomal, chromosomal loci, either of which suffices to induce detectable levels of infectious virus in Fv-1 n progeny by 6 wk of age. One of the loci (tentatively referred to as V1) is on linkage group I, 25-30 map units from the locus for albino; the gene order tentatively appears to be Vl-c-Hbb.