GENETICAL STUDIES ON SERUM ALKALINE PHOSPHATASE ISOZYME IN THE JAPANESE YOSHIZANE MAEDA, TSUTOMU HASHIGUCHI

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1 JAPAN. J. GENETICS Vol. 47. No. 3: (1972) GENETICAL STUDIES ON SERUM ALKALINE PHOSPHATASE ISOZYME IN THE JAPANESE QUAIL YOSHIZANE MAEDA, TSUTOMU HASHIGUCHI AND MANJIRO TAKETOMI Laboratory of Animal Breeding, Faculty of Agriculture, Kagoshima University, Kagoshima 890 Received January 12, 1972 The combination of zone electrophoresis with different enzyme staining methods has made it possible to study with greater efficiency electrophoretic variations of various enzymes (Hunter and Markert 1957). Markert and Miller (1959) have defined one of the different molecular form of an enzyme as isozyme. During the past few years, genetic control of isozyme has been described in various organisms. Genetic variations in alkaline phosphatase isozyme of animals were reported in man (Boyer 1961), cattle (Gahne 1963, 1967a, b), sheep (Rendel et al. 1964), chicken (Law and Munro 1965; Wilcox 1966; Tamaki and Tanabe 1970), pigeon (Brown and Manly 1970), silkworm (Yoshitake and Akiyama 1966) and Drosophila melanogaster (Beckman and Johnson 1964; Wallis and Fox 1968). Maeda et al. (1971) reported six phenotypes on alkaline phosphatase isozyme in the Japanese quail serum. In this study, the authors tried to clarify a mode of genetic control in respect of serum alkaline phosphatase isozyme in the Japanese quail, and at the same time, examined the phenotypic and gene frequencies in some populations of the Japanese quail. MATERIALS AND METHODS The Japanese quail (Coturnix coturnix japonica) used were obtained from a control population maintained at Kagoshima University, random bred population maintained at Saga University and Toyohashi population introduced from a fancier in Toyohashi city to Kagoshima University in At 8-10 weeks of age, the blood was taken from a wing vein with a syringe. After kept in a 15 C room for 6 hours, serum was poured from the coagulated blood and stored at -20 C until assayed. Within 6 days after collection, all samples were subjected to agar gel electrophoresis. Agar gel electrophoresis was carried out according to the method described by Ogita (1962) with the ascertainment that in the medium for enzyme separation were contained 1.5 g of agar and 4.0 g of polyvinylpyrolidone (Luviscol K 90 powder, molecular weight ca. 700,000) per 200 ml of phosphate buffer (ph 6.8, ionic strength 0.025). The electrode chamber contained veronal-sodium acetate buffer (ph 8.6, ionic strength 0.025). The electrophoresis was performed in an ice box at 5 C for about 2 hours at 1.0 ma / cm. After the electrophoresis, the gels were stained

2 166 Y. MAEDA, T. HASHIGUCHI AND M. TAKETOMI for alkaline phosphatase using the following stain solution: 100 ml Tris-HC1 buffer, ph mg Disodium a-naphthylphosphate (Nakarai Chemicals: Japan) 100 mg Fast Blue RR salt 10 drops of 10% solution of manganese chloride 10 drops of 10% solution of magnesium chloride 0.5 g polyvinylpyrolidone The gel plates, above mixture poured, were incubated for 6 hours at room ature. temper- RESULTS Description of serum alkaline phosphatase isozyme in the Japanese quail The results of detection of serum alkaline phosphatase isozyme of the quail by the agar gel electrophorsis are shown in Figs. 1 and 2. Japanese Fig. 1, Zymogram patterns of serum alkaline Japanese quail. phosphatase isozyme in the Fig, 2. Diagrammatic grams in the representation of serum alkaline Japanese quail. phosphatase zymo-

3 ALKALINE PHOSPHATASE ISOZYME IN THE JAPANESE QUAIL 167 Serum alkaline phosphatase of 8-10 week old quail was separated into two polymorphic regions, and it was found that there existed F-pattern appearing in the fast mobility region (Akp-1) and A, B and C pattern in the slow mobility region (Akp-2) on the anode end of the gel (Figs. 1 and 2). At the stage of sexual maturity, activity of F-pattern of the female was higher than that of the male (Maeda et al. 1971). In the Akp-2 region, there are six phenotypes according to the combination on A, B and C pattern : AA, AB, BB, BC, AC and CC (Fig. 2). Mode of genetic control of serum alkaline phosphatase in the Japanese quail The distribution of the six phenotypes AA, AB, BB, BC, AC and CC in 293 progenies from each possible kind of mating is shown in Table 1. Phenotypic frequencies of progeny from each mating are in good agreement with expected number from Mendelian law, without exception. These cross experiment data show that the variation can be explained by combination of three co-dominant allelic genes on one autosomal locus. If one designates the gene controlling the A pattern as Akp-2A, the one controlling the B pattern as Akp-2B, and one controlling the C pattern as Akp-2C, the genotypes corresponding to the phenotypes AA, AB, BB, BC, AC and CC will be Akp-2A/ Akp-2A, Akp % /Akp-2B, Akp-2B/Akp-2B, Akp-2B/Akp-2C, Akp-2A/Akp-2C and Akp-2C/ Akp-2C, respectively. Table 1. Distribution of alkaline phosphatase isozyme phenotypes in progeny from various matings in in Distribution of phenotypic and gene frequencies of serum alkaline phosphatase isozyme various populations The phenotypic and gene frequencies of three different populations are presented Table 2.

4 168 Y. MAEDA, T. HASHIGUCHI AND M. TAKETOMI Table 2. Phenotype and gene frequencies of serum alkaline several Japanese quail populations phosphatase isozyme in In all populations the observed numbers of each phenotype are in good agreement with the expected number from Hardy-Weinberg's equilibrium with gene frequencies. In Kagoshima and Saga populations, phenotypes BB, BC and CC show markedly high frequency among the six phenotypes. Phenotypes AB and AC in Kagoshima population and phenotype AA in Saga population were not found. All phenotypes, however, were found in Toyohashi population. In the total, although no statistically significant difference is observed as the result of a heterogeneity test between the observed number and the expected number (1.0>P>0.05), the frequency of occurrence of homozygous genotypes tends to be higher than that of the expected one estimated as Mendelian population. Whereas, the frequencies of heterozygous genotypes are lower than that of the expected one. In all populations, Akp-2A gene shows the lowest frequency of the three genes. Especially, Akp-2A gene frequency in Kagoshima and Saga population is very low, Akp-2A being in Kagoshima population and in Saga population. From comparison of gene frequency distribution, Kagoshima and Saga populations are similar to each other. But genotypic frequency distribution of Toyohashi population is different from that of these two populations. Gene frequencies of Akp-2A and Akp-2B in Toyohashi population are higher than those of Kagoshima and Saga populations. In case of the Akp-2C gene, the frequency of the Toyohashi population is lower than that of any other population. Gene frequencies in the total are : q Akp-2A, 0.081; q Akp-2B, 0.552; and q Akp-2C,

5 ALKALINE PHOSPHATASE ISOZYME IN THE, JAPANESE QUAIL 169 DISCUSSION The electrophoretical study of serum alkaline phosphatase in the Japanese quail has been reported by Savage et al. (1970) and Maeda et al. (1971). Savage et al. (1970), using acrylamide disc electrophoresis, found that an increase in the electrophoretic mobility of the slow serum alkaline phosphatase isozyme of the female was shown to be associated with the onset of egg production. In the previous report, the authors investigated the endocrine control of serum alkaline phosphatase isozyme of the Japanese quail and found that alkaline phosphatase activity at the immature stage concerned with thyroidal function and that F-pattern activity of the female at the adult stage is controlled by estrogen (Maeda et al. 1971). However, there are no reports on genetic control of serum alkaline phosphatase in the Japanese quail. Concerning the genetic variation of the avian alkaline phosphatase, there are reports on the chicken (Law et al. 1965; Wilcox 1966; Tamaki et al. 1970) and the pigeon (Brown and Manley 1969). In the chicken, zymograms of single plasma sample were observed to contain either slow or fast moving band. Fast and slow bands were to be controlled by dominant (Akp) and recessive (akp) allele, respectively. In the pigeon, using the acrylamide gel electrophoressis, four bands are seen in the anode side. From crossing experiment, these bands were controlled by co-dominant alleles. Serum alkaline phosphatase isozyme in the Japanese quail used in this study are controlled by three co-dominant alleles on one autosomal locus. In the comparison of gene frequencies, Akp-2B gene frequency is the highest (0.552) and Akp-2A is the lowest (0.081). In the total, the frequencies of occurrence of homozygous genotypes are higher than those of expected number and the frequencies of occurrence of heterozygous are lower than those of expected one. The reasons of these differences between Akp-2B gene frequency and Akp-2A gene frequency, and between observed number and expected number are unknown from only these data. For what kinds of fitness concern with these differences, the further research are required. Recently, authors found the polymorphism in Akp-1 region, we will report on this polymorphism in the next paper. SUMMARY By means of agar gel electrophoresis, serum alkaline phosphatase of the Japanese quail was separated into two polymorphic regions, and it was found that there existed F-pattern appearing in the fast mobility region (Akp-1) and A, B and C pattern in the slow mobility region (Akp-2) on the anode side. With respect to Akp-2 region, six phenotypes were observed : AA, AB, BB, BC, AC and CC. These phenotypes were shown to be controlled by three autosomal co-dominant alleles : Akp-2A, Akp-2B and Akp-2C. Gene frequencies in the total population were: q Akp-2A, 0.081; q Akp-2B, 0.552; q Akp-2C,

6 170 Y. MAEDA, T. HASHIGUCHI AND M. TAKETOMI LITERATURE CITED Beckman, L., and F. M. Johnson, 1964 Variations in larval alkaline phosphatase controlled by A$ alleles in Drosophila melanogaster. Genetics 49: Boyer, S. H., 1961 Alkaline phosphatase in human sera and placentae. Science 134: Brown, R. V., and J. H. Manley, Jr., 1970 Serum alkaline phosphatase inheritance in the pigeon. Anim. Blood Grps Biochem. Genet. 1: Gahne, B., 1963 Genetic variation of phosphatase in cattle serum. Nature 199: Gahne, B., 1967a Inherited high alkaline phosphatase activity in cattle serum. Hereditas 57: Gahne, B., 1967b Alkaline phosphatase isoenzyme in serum, seminal plasma and tissues of cattle. Hereditas 57: Hunter, R, L., and C. L. Markert, 1967 Histochemical demonstration of enzymes separated by zone electrophoresis in starch gels. Science 125: Law, G. R., and S. S. Munro, 1965 Inheritance of two alkaline phosphatase variants in fowl plasma. Science 149: Maeda, Y., T. Hashiguchi, and M. Taketomi,1971 Endocrine control of serum alkaline phosphatase isozyme in the Japanese quail, Coturnix coturnix japonica. Japan. Poultry Sci., 8: Markert, C. L., and F. Mcbller, 1959 Multiple forms of enzymes: tissue, ontogenetic and species specific patterns. Proc. Natl. Acad. Sci. 45: Ogita, Z, 1962 Genetico-biochemical analysis on the enzyme-activities in the house fly by agar gel electrophoresis. Japan. J. Genetics 37: Rendel, J., Aalund, 0., Freedland, R. A., and Moller, F., 1964 The relationship between the alkaline phosphatase polymorphism and blood-group 0 in sheep. Genetics 50: Savage, T. F., W. M. Collins, and E. C. Smith, 1970 Onset of egg production and its relationship to isoenzymes of serum alkaline phosphatase in Japanese quail. Poultry Sci., 49, Tamaki, Y., and Y. Tanabe, 1970 Genetic control of multiple molecular forms of the alkaline phosphatase in chicken plasma. Poultry Sci. 49: Wallis, B. B., and A. S. Fox, 1968 Genetic and development relation between two alkaline phosphatase in Drosophila melanogaster. Biochem. Genet. 2: Wilcox, F. H., 1966 A recessively inherited electrophoretic variant of alkaline phospatase in chicken serum. Genetics 53: Yoshitake, N., and A. Akiyama,1966 Genetic aspects on quantitative difference of the phosphatase activities in the silkworm, Bombyx mori L. SABCO J. 2: