THE NUMBER OF GENES CONTROLLING THE RESPONSE OF CHICK EMBRYO CHORIOALLANTOIC MEMBRANES TO TUMOR INDUCTION BY ROUS SARCOMA VIRUS1

Transcription

1 THE NUMBER OF GENES CONTROLLING THE RESPONSE OF CHICK EMBRYO CHORIOALLANTOIC MEMBRANES TO TUMOR INDUCTION BY ROUS SARCOMA VIRUS1 RAYMOND K. BOWER'. N. ROY GYLES AND CONNELL J. BROWN Departments of Microbiology. School of Medicine, Little Rock, and Animal Industry and Veterinary Science, Universiry of Arkansas. Fayetteuille Received December 28, 1964 WO previous workers have estimated the number of pairs of genes controlling Tthe response of the chicken to Rous sarcoma virus. PRINCE (1958) proposed (a) that a single pair of autosomal alleles controlled susceptibility and resistance of chorioallantoic membranes to Rous sarcoma virus and (b) that susceptibility (allele S) was dominant to resistance (s). This proposal was based on the proportion of nonreacting chorioallantoic membranes in the Pure Hope White Leghorn strain and the Fayoumi strain, and in their reciprocal crosses. Assuming one pair of autosomal alleles, in which susceptibility was dominant to resistance, the product of the gene frequencies of the recessive gene (s) in the two parental strains would give the proportion of the nonreacting chorioallantoic membranes in the reciprocal crosses. This calculated proportion of nonreacting membranes was found to approximate closely the observed proportion. WATERS and BURMESTER (1961) reported that a single pair of alleles was responsible for susceptibility and resistance of the day-old chick to intracerebral inoculations of Rous sarcoma virus. Progeny from five unrelated inbred lines, and their reciprocal crosses, were challenged with this virus. Susceptibility appeared dominant to resistance, because 100% mortality occurred among the reciprocal inbred-cross progeny from certain crosses between resistant and susceptible lines of chickens. A supposedly heterozygous sire (8s) mated to resistant dams (ss) gave full-sib families, each with a ratio of resistant to susceptible progeny closely approximating 1 : 1. The same sire mated to supposedly heterozygous dams (8s) gave 3:l ratios of susceptible to resistant progeny. DHALIWAL (1963) in a study of the mode of inheritance of resistance of the chorioallantoic membrane to inoculation with Rous sarcoma virus stated that resistance was dominant to susceptibility. However, it could not be determined from his results whether resistance was due to a single gene or number of genes. CRITTENDEN, AKAZAKI and REAMER (1963) found that the genetic response of chorioallantoic membranes (CAMS) of chick embryos to Rous sarcoma virus (RSV) was similar to that of tissue cultures of embryos to this virus. In both This inwstlgation was supported in whole by Public Health Senrice Researrh Grant CA-049W-04, from the National Cancer Institute. Present Address J. K. and Suai~ I.. W+DI.EY Research Institute and Graduate Research Institute of Raylor University. Dallas, Texas. Genetics 51 : i May 1965

2 740 R. K. BOWER, et al. cases susceptibility was completely dominant to resistance. Apparently the amount of resistance exhibited by the various lines and crosses was not imparted by maternal transmission of the leukosis resistance-inducing factor (RIF), because this resistance was overcome by mating resistant hens to susceptible males. The success of tissue cultures in studying genetic variability of susceptibility to RSV has opened another means of investigation. In light of the studies made by these investigators, the present work was planned to give a direct estimate of the number of pairs of genes controlling the response of the chorioallantoic membrane to tumor induction by Rous sarcoma virus. This estimate was based on the average difference between counts of tumors on chorioallantoic membranes of chick embryos from susceptible and resistant parental lines of chickens and the difference in the variance of the counts between the F, and F, generations. MATERIALS AND METHODS Strains of chick embryos: The two parental strains of chick embryos were from a purebred White Leghorn (W.L.) and a purebred Giant Jungle Fowl (G.J.F.) strain. The W.L. strain has been maintained as a closed flock at the Arkansas Agricultural Experiment Station for over 20 years. It is considered highly inbred, but the coefficient of inbreeding is unknown. The G. J. F. strain was introduced from Southeast Asia about 15 years ago and has been maintained as a closed flock. It is probably highly inbred. This strain was obtained from DR. J. N. THOMPSON of the Thompsm Poultry Breeding Farm, Pottsville, Arkansas. Purebred W.L. fertile eggs were obtained from 47 hens artificially inseminated with pooled semen from eight males. Pooled semen from five G.J.F. males was used to inseminate artificially 20 G.J.F. hens for the production of purebred fertile eggs. The F, generation of crossbred eggs was produced by pooling semen from five G.J.F. males and artificially inseminating about 50 W.L. females. The F, gmeration of crossbred eggs came from F, generation adult birds produced the previous year by artificially inseminating the W.L. females with poded G.J.F. semen. The fertile eggs obtained for the F, generation came from a pen mating of eight F, males with 60 F, females. In each of s:x experiments, eggs representing the four breeding groups (W.L., G.J.F., F, generation cross and F, generation cross) were incubated at C in the University of Arkansas farm hatchery. The only exception was that in the last two experiments, no purebred G.J.F. eggs were available because the hens had ceased production. The eggs were candled on the ninth day of incubation and shipped by air to the University of Arkansas Medical Center. Strain of uirus: Rous sarcoma virus (RSV), identified as CT-905, was received from DR. W. RAY BRYAN at the National Cancer Institute. On chick embryos of the Arkansas W.L. strain, this stock of virus titered 1.63 x 107 tumor-forming units (tfu) per milliliter. Dilution of uirus: A single dilution of RSV was used. 1:2500 was chosen, because it yielded a good tumor counting range-from 0 to e00 tumors present on any single CAM. Based on the stock titer, this dilution contained approximately 326 tfu per 0.05 ml. Dilutions of virus were made in EAGLE S standard medium (1955) which contained a 2% normal calf serum. The dilution tubes were kept chilled by immersion in an ice-water bath during inoculation; 0.05 ml was used as a standard volume of inoculum. Inoculation of eggs: Embryonated eggs were prepared for inoculation on the 11th day of incubation by techniques utilized customarily for introduction of virus onto the CAM. Detailed procedures for the inoculation of eggs, harvesting and examination of CAM s have been described (BOWER 1962). Counting of tumors: Difficulty was experienced in making precise counts of tumors on CAMS where the tumors were very close to one another or confluent. DHALIWAL (1963) reported the

3 GENES AND MEMBRANE RESPONSE TO VIRUS 741 same difficulty and considered it impossible to enumerate accurately numbers of tumors exceeding 350 per CAM. PRINCE (1958) had counts of tumors greater than 1000 per CAM in a comparative evaluation of different strains of chick embryos. In the present study, special notations were made of CAMS on which precise tumor counts were not possible and only estimated counts were obtained. The only strain of chick embryo in which confluency of tumors was encountered on a number of CAMs. necessitating estimated counts, was the W.L., which is more susceptible than the G.J.F. For each experiment, a t-test was conducted according to SNEDECOR (1956) to determine whether there was any significant difference between the average number of tumors on CAMS of White Leghorns from the precise counts as compared with all counts (precise and estimated). In each experiment there was no significant difference between precise counts and all counts. The values for t were.25,.45, 64,.14,.05 and.05 for experiments 1. 2, 3, 4, 5 and 6 respectively. All these t values were substantially lower than the value required for significance at the (P <.05) level. In view of this similarity between precise counts and all counts, all counts were used in statistical analyses and in estimates of gene number. During the winter and spring of 1963, six experiments were conducted with W.L., G.J.F., F, and F, generations of chick embryos. The counts of tumors on the CAMs did not conform with a normal distribution. On this account, three transformations of the data were tried according to BARTLETT (1947) to determine which one would stabilize the variance most effectively. Logarithmic transformations to log,, (z + l), the square root of (z + 0.5) and the hyperbolic inverse sine transformations of the actual numbers were made. The logarithmic and hyperbolic inverse sine transformations were slightly more effective than the square root transformation. The transformation to log,, (z + 1) was chosen because of its suitability for these data and because it had been used previously in a similar investigation (DHALIWAL 1963). An analysis of variance according to SNEDECOR (1956) was conducted to determine whether there were significant differences between experiments for mean tumor counts. When the mean square between experiments was compared with the mean square within experiments, there was a highly significant difference between experiments for the W.L., F, and I?, crossbred embryos, but no significant difference between experiments for the G.J.F. On account of these differences between experiments, the number of pairs of genes controlling resistance and susceptibility was estimated for each experiment separately, recognizing that the small numbers within an experiment would be subject to greater chance variation. Also, since there was no difference between experiments for the G.J.F., the average value of Experiments 1 through 4 was used in Experiments 5 and 6 (where there was no representation of the G.J.F. because they were out of egg production). The reason for the differences between experiments in the average tumor response of the W.L., F, and F, embryos is not known. Incubated eggs were transported by airplane from Fayetteville to Little Rock on the ninth day of incubation. The treatment of these eggs in transit varied considerably between experiments for such factors as length of time out of incubators and outside weather conditions, particularly temperature. RESULTS A sample of the data (Experiment 4) is given in Table 1 to illustrate the actual numbers of tumors on each chorioallantoic membrane among the four breeding groups. The pattern of variation within and between the groups exhibited by this sample was similar to that shown by the entire data. The nonreactor CAMs (those with zero counts of tumors) for all experiments amounted to 0.46% for White Leghorns, 02.50% for Giant Jungle Fowls, 2.67% for F, crosses, and 19.88% for F, generation crosses. Histograms (Figure 1) show the distribution of tumor counts for the two parental strains of chick embryos and the F, and F, generations of crossbred chick embryos. They demonstrate the wide differences in response

4 742 R. K. BOWER, et al. TABLE 1 A sample of the actual numbers of tumors (Experiment 4) on indiuidual chorioallantoic membranes of purebred White Leghorn, Giant Jungle Fowl, and their F, and F, generation crossbred embryos White Leghorn Giant Jungle Fowl F, Crossbreds F2 Crossbreds Precise counts: 44,49,87,106 0,0,0,0,0, 26,33,41,61, 0,0,3,3,19,20,22, 115,131,136, O,~,O,O,O, 95,116,153, %,51,69,76,92, 137,152~ 57, 0,0,3,4,4, 170,185,185, 101,111,114,122, 168,170,180, 5,6,6,9,10, 198,208,213, 144;156,187,202, 203,208,216, 1435,74 219,232,255, 202,257,257,303, 217,223,223, , ,233,243, 260,282,303, 310,320,321, 347,434,445 Estimates: * 190,200,200, 200,200,200, 200,200,200, 200,200,214, * These numbers represent only estimates of tumor counts So far as possible, individual tumors were counted on all membranes showing confluency of tumors. between the resistant G.J.F. and the susceptible W.L. strains, with an intermediate F, performance slightly inclined towards susceptibility and a large variation in the F, distribution. For each experiment, the number of CAMs examined, average tumor count (i), standard error (SE) and variance (s2) are reported in Table 2 for each of the parental strains, the F, and the F,. The CAMs of the F, embryos had an average nontransformed tumor count of 96.90, which was slightly higher than the midpoint of 92.97, between for the W.L. and 3.75 for the G.J.F. lines. This F, generation was from a mating of pooled semen from the G.J.F. males with W.L. females. The reciprocal cross was not made because insufficient numbers of G.J.F. females were in egg production. The intermediate response of the F, crossbreds agrees with work previously reported on reciprocal crosses by BOWER (1964). CASTLE ( 1921 ) showed that the minimum number (n) of pairs of genes controlling a trait may be estimated according to the formula: A2 n= 8(& - S'F, ) in which (A) is the difference between the extreme parental strains. The F, variance is a combination of the genetic variance and the nongenetic variance, and the latter may be taken as measured by the variation of the F,. The main usefulness of this formula has been to estimate the minimum number of gene pairs

5 GENES AND MEMBRANE RESPONSE TO VIRUS 743 WHITE LEGHORN - GIANT JUNGLE FOWL -U- F, GENERATION CROSSBRED Fz GENERATION CROSSBRED 30. '02 I S IO II I2 I I6 17 I B FIGURE 1.-Distribution of tumor counts per chorioallantoic membrane among parental strains, F,, and F2. Ordinate: Percentage of embryos. Abscissa: Log,, (I + 1) of actual tumor count per CAM. responsible for determining the expression of a character. Also, the following assumptions are necessary for the application of this formula: (a) The genes concerned with the trait under observation act additively, and there is no dominance or epistasis; (b) the parental lines are homozygous; (c) one line has all the contributing alleles and the other all the noncontributing alleles; (d) all gene pairs are independent; (e) environmental differences combine additively with the genetic ones and furnish the same amount of environmental variance to each of the crosses or generations being compared; (f) numbers are sufficiently large that sampling errors are small. The degree to which these experiments may meet these assumptions is important to the interpretation of the results. BOWER, GYLES and BROWN (1964) showed that there was no difference between the average tumor responses of reciprocal crosses between resistant and susceptible parental lines, and that the F, crosses gave an intermediate susceptibility suggesting additive gene action. It is clear that the W. L. and G.J.F. lines may not be homozygous, but certainly it is

6 744 R. K. BOWER, et al. TABLE 2 A summary of the average (Z), standard error (SE) and variance (9) of counts of tumors on the CAMS of W.L., G.J.F. and their F, and F, generation crossbred embryos* White Leghorn Experiment Number - No. embryos I SE , , , ,1849 Giant Jungle Fowl Number embrvos i SE 9 18,254.lo ,092, , I F, Crossbreds F, Crossbreds Experiment Number Number No. embryos 5 SE s embryos E SE SI , , , I21, ,145.5# , T ,110, I * All statistical calculations are on log,, (x+l) of the actual counts of tumors. reasonable to assume a high degree of homozygosity for loci controlling resistance or susceptibility to Rous sarcoma virus. Should susceptibility be conditioned by a single pair of genes, it is likely that most parents used in this study were homozygous. It is not known whether the gene pairs are independent if several genes are involved, but should there be only one pair of alleles as suggested by these data then this is of no concern. The parental lines, F, and F, embryos were all handled and treated alike and subject to the same environment within each experiment. Six separate experiments offer replications that add confidence to the overall evaluation of results. Within each experiment, the numbers of embryos tested are in accord with the usual numbers reported for this type of genetic study with RSV, but the numbers were small enough to permit sampling errors. The circumstances of these experiments do not fulfill completely the requirements stated in all the assumptions required by CASTLE S formula, but they seem to meet them sufficiently well to permit estimates of the numbers of pairs of genes to be made subject to interpretation in view of the limitations. The application of this formula to these data yielded estimates of.92, 2.31, 1.04,.92,.95 and.58 pairs of genes for Experiments 1, 2, 3, 4, 5 and 6 respectively. DISCUSSION This investigation suggests that the minimum number of pairs of genes controlling susceptibility is one, because five of the six experiments yielded an esti-

7 GENES AND MEMBRANE RESPONSE TO VIRUS 745 mate of 1.04 or less. This agrees with the work of PRINCE (1958) who indicated a single pair of genes based on a gene frequency analysis, and with the work of WATERS and BURMESTER (1961) who indicated monofactorial inheritance based on analysis of genetic ratios among progeny of selected matings. Thus, three different procedures have indicated that susceptibility is conditioned by a single pair of genes. The distributions of the tumor counts per CAM of the parental strains, F, and F, do not fall into discrete classes as might be expected for classical monofactorial inheritance (see Figure 1 ). The variability in tumor counts among embryos within an experiment and within the parental strain, F, or F,, as shown in Table 1, indicate considerable variability in the expression of these alleles. SANG (1963) has defined penetrance and expressivity of genes as properties of populations in which individuals may or may not show the character. The exact reason for the variability in the present study, and the extent to which it is conditioned by differences in penetrance, expressivity and minor within-experiment environment, is not clear. BOWER (1962), in a previous study with pedigreed Jungle Fowl-Cornish chick embryo CAMs, obtained a heritability estimate of.28 from a paternal half-sib analysis. PRINCE (1958) pointed out that partial dominance of the allele for susceptibility could account for some of the variation among CAMS with intermediate tumor counts. RUBIN (1962) has demonstrated that leukosis viruses may inhibit RSV expression, and has developed the procedure known as the RIF test (Rous inhibiting factor) for determining the presence of leukosis virus in individual birds. This test was not applied to any of the birds in these experiments. It seems unlikely that the extreme resistance shown by the purebred G.J.F. embryos is due to the presence of leukosis in these birds because when G.J.F. males were mated to W.L. susceptible females, the F, embryo CAMs gave an intermediate tumor count response. RUBIN, CORNELIUS and FANSHIER (1961) state that there is no evidence to support transmission of RIF from males to their progeny. The authors are indebted to DR. GEORGE R. DUBES for helpful criticism of this manuscript. Grateful appreciation is expressed to DR. E. L. STEPHENSON for cooperation in the use of the White Leghorn strain of chickens. SUMMARY Chorioallantoic membranes from a total of 219 White Leghorn, 72 Giant Jungle Fowl, 112 F, generation crossbred and 176 F, generation crossbred chick embryos were examined for the numbers of tumors induced by Rous sarcoma virus. The difference between the average tumor counts of the two parental strains and the difference between the variance of the F, and F, generations were used to estimate the minimum number of pairs of genes controlling susceptibility and resistance of the membranes to the virus; the estimated number was one. LITERATURE CITED BARTLETT: M. S., 1947 The use of transformations. Biometrics.3: BOWER, R. K., 1962 A quantitation of the influence of the chick embryo genotype on tumor production by Rous Sarcoma virus on the chorioallantoic membrane. Virology 18:

8 746 R. K. BOWER, et al. BOWER, R. K., N. R. GYLES, and C. J. BROWN, 1964 Tumor induction by Rous sarcoma virus on the chorioallantoic membranes of reciprocal crosses between resistant and susceptible strains of chickens. Virology 24: CASTLE, W. E., 1921 An improved method of estimating the number of genetic factors concerned in cases of blending inheritance. Science 54: 223. CRITTENDEN, L. B., W. AKAZAKI, and R. REAMER, 1963 Genetic resistance to Rous sarcoma virus in embryo cell cultures and embryos. Virology 20: 541. DHALIWAL, S. S., 1963 Resistance of the chorioallantoic membrane of chick embryos to ROUS sarcoma and MH, reticuloendothelioma viruses. J. Natl. Cancer Inst. 30: PRINCE, A. M., 1958 Quantitative studies on Rous sarcoma virus. 11. Mechanism of resistance of chick embryos to chorioallantoic inoculation of Rous sarcoma virus. J. Natl. Cancer Inst. 20: RUBIN, H., 1962 Response of cell and organism to infection with avian tumor viruses. Bacteriol. Rev. 26: RUBIN, H., A. CORNELIUS, and L. FANSHIER, 1961 The pattern of congenital transmission of an avian leukosis virus. Proc. Natl. Acad. Sci. U.S. 47: SANG, J. H., 1963 SNEDECOR, G. W., 1956 Penetrance, expressivity and thresholds. J. Heredity 54: Statistical Methods. 5th ed. Iowa State College Press, Ames. WATERS, N. F., and B. R. BURMESTER, 1961 Mode of inheritance of resistance to Rous sarcoma virus in chickens. J. Natl. Cancer Inst. 27: