Jun-ichi Asakawa

Two-dimensional gel studies of genetic variation in the plasma proteins of Amerindians and Japanese

SummaryGenetic variation has been studied in plasma samples from 107 Amerindian children and their parents, and 110 Japanese children and their parents by means of two-dimensional polyacrylamide gel electrophoresis. Twenty-three polypeptides were scored; the identity of nine of these is at present still unknown. Genetic variation was encountered in 11 of these polypeptides. We have previously reported that the index of heterozygosity was 6.2±0.7% for 20 “randomly selected”, silver stained polypeptides scored for genetic variation in Caucasoids (Rosenblum et al. 1983b). For technical reasons only 11 of these 20 polypeptides could be routinely scored in preparations from the Amerindian samples. For these 11 polypeptides, the indices of heterozygosity in the three populations were: Amerindians, 4.5±0.6%; Japanese, 5.7±0.7%; Caucasoids, 8.0±1.1%. Even with these relatively small numbers some striking ethnic differences as regards individual polypeptides are apparent.

Malignant tumor cell lines produce interleukin-1-like factor

SummarySixty-four malignant cell lines were examined for interleukin-1 (IL-1) activities in their conditioned medium using thymocyte and fibroblast proliferation assays. Sixteen cell lines showed high IL-1 activity. Comparison of these activities with human IL-1 showed similarity between some biological properties. However there was no correlation between cell origin and IL-1 activity. These results suggest the possibility that most malignant cells may produce an IL-1-like factor.

Electrophoretic variants of blood proteins in Japanese

SummaryA total of 15,387 individuals living in Hiroshima and Nagasaki, of whom 10,864 are unrelated, were examined for erythrocyte triosephosphate isomerase (TPI) by starch gel electrophoresis using TEMM buffer, pH 7.4. Four kinds of new variants, one having a cathodal migration and three having anodal migrations, were encountered in this population. These variants were further characterized by starch gel electrophoresis using tris-EDTA buffer, pH 9.3, and isoelectric focusing. An anodally migrating allozyme TPI 2HR1 exhibited markedly decreased enzyme activity, as evaluated by the staining intensity of the variant bands. The level of TPI activity in erythrocytes from this individual with the phenotype TPI 1-2HR1 was about 60% of the normal mean. Family studies confirmed the genetic nature of all the variants.

Heterozygosity and ethnic variation in Japanese platelet proteins

SummarySixty-two polypeptides visualized on silver-stained two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) preparations of platelets from 85 Japanese subjects, in total 5,252 spots, have been scored for genetic variation. Inherited variants of 11 of the polypeptides were recognized; the index of heterozygosity was 2.4%±0.2%. Thus far, 10 genetic polymorphisms identified by 2-D PAGE of plasma, erythrocytes, or platelets have been identified in both Japanese and Caucasian subjects. A comparison of allele frequencies reveals four significant ethnic differences. We also observed four polypeptides exhibiting a low frequency polymorphism in one group but not in the other, as well as three polymorphisms in Caucasians for which no counterpart polypeptide has thus far been recognized in the Japanese group and, vice versa, 11 such polymorphisms in Japanese. Although a similar comparison of 7 enzyme polymorphisms studied with one-dimensional electrophoresis (1-D E) in the same populations revealed a relatively higher number of significant ethnic differences, evidence is presented that this is due primarily to the greater number of 1-D E observations entering into the calculation. It is argued that this similarity in the frequency of ethnic differences among the polypeptides studied by 2-D PAGE and by 1-D E is further evidence that the proteins revealed by 2-D PAGE do not differ greatly in their response to the interplay of mutation, selection, and drift from those revealed by 1-D E studies of plasma proteins and erythrocyte enzymes.

Inherited electrophoretic variants detected in a Japanese population with two‐dimensional gels of erythrocyte lysates

Genetic variation has been studied in erythrocyte lysates from 100 Japanese children and their parents by means of two‐dimensional polyacrylamide gel electrophoresis. Fifty‐five polypeptides selected without respect to variability were considered suitable for scoring. Genetic variation was encountered in 14 of these polypeptides. Family data show that the segregation of 13 variants is consistent with an autosomal codominant mode of inheritance; the remaining variant exhibits a sex‐linked mode of inheritance. Of 8 presumably identical polypeptides found variable in Japanese and/or Caucasians, differences in the occurrence or allele frequencies of polymorphisms were recognized for four. Contrary to the experience of some investigators, the amount of variation and the ethnic differences we are encountering indicate that two‐dimensional polyacrylamide gel electrophoresis is a sensitive tool for the study of genetic events.

The Genetic Risk in Mice from Radiation: An Estimate of the Mutation Induction Rate per Genome

Restriction Landmark Genome Scanning (RLGS) is a method that uses end-labeled 32P NotI sites that are mostly associated with coding genes to visualizes thousands of DNA fragments as spots in two-dimensional autoradiograms. This approach allows direct detection of autosomal deletions as spots with half normal intensity. The method was applied to mouse offspring derived from spermatogonia exposed to 4 Gy of X rays. A genome-wide assessment of the mutation induction rate was estimated from the detected deletions. Examinations were made of 1,007 progeny (502 derived from control males and 505 from irradiated males) and 1,190 paternal and 1,240 maternal spots for each mouse. The results showed one deletion mutation in the unirradiated paternal genomes of 502 offspring (0.2%) and 5 deletions in the irradiated paternal genomes of 505 offspring (1%). The difference was marginally significant, with the deletion sizes ranged from 2–13 Mb. If the frequencies are taken at face value, the net increase was 0.8% after an exposure of 4 Gy, or 0.2% per Gy per individual if a linear dose response is assumed. Since the present RLGS analysis examined 1,190 NotI sites, while the mouse genome contains ∼25,000 genes, the genomic probability of any gene undergoing a deletion mutation would be 25× 0.2%, or 5% per Gy. Furthermore, since the present RLGS screened about 0.2% of the total genome, the probability of detecting a deletion anywhere in the total genome would be estimated to be 500 times 0.2% or 100% (i.e., 1 deletion per Gy). These results are discussed with reference to copy number variation in the human genome.

Origin of human triosephosphate isomerase isozymes: further evidence for the single structural locus hypothesis with Japanese variants.

SummaryFour electrophoretic variants of human erythrocyte triosephosphate isomerase (TPI) have been studied to investigate the origin of the multiple forms of human TPI, in particular the constitutive TPI-B isozyme and the cell division-associated TPI-A isozyme. The variant phenotype expressed by the constitutive TPI-B isozyme in both erythrocytes and peripheral lymphocytes was also expressed by the cell division-associated isozymes in mitogen-stimulated lymphocytes and hair root cells. These results strongly support the hypothesis of Decker and Mohrenweiser (1981) that TPI-B and TPI-A originated from the same structural gene. We also found that the isozyme e is different from TPI-A with respect to both its electrophoretic mobility and heat stability. This finding is in contrast to the recent conclusion of Yuan et al. (1981) that both the isozyme e and TPI-A are deamidation products of TPI-B.

Electrophoretic variants of blood proteins in Japanese I. Phosphoglucomutase-2 (PGM2)

SummaryA total of 17,126 individuals, of whom 11,833 are unrelated, living in Hiroshima and Nagasaki were examined for erythrocyte phosphoglucomutase-2 (PGM2) by starch gel electrophoresis using TEMM buffer, pH 7.4. Four kinds of hereditary rare variants were encountered, three detected in single families and the one remaining in 9 unrelated families. In addition, a fresh mutant whose main band migrated slightly cathodal to the d-band was detected in a male child in Nagasaki, whose parents were proximally exposed to the atomic bomb in that city. The results described here confirm our previous data that PGM2 variation is quite low among the Japanese.

Estimation of Mutation Induction Rates in AT-Rich Sequences Using a Genome Scanning Approach after X Irradiation of Mouse Spermatogonia

Abstract Asakawa, J., Nakamura, N., Katayama, H. and Cullings, H. M. Estimation of Mutation Induction Rates in AT-Rich Sequences Using a Genome Scanning Approach after X Irradiation of Mouse Spermatogonia. Radiat. Res. 168, 158–167 (2007). We have previously used NotI as the marker enzyme (recognizing GCGGCCGC) in a genome scanning approach for detection of mutations induced in mouse spermatogonia and estimated the mutation induction rate as about 0.7 × 10−5 per locus per Gy. To see whether different parts of the genome have different sensitivities for mutation induction, we used AflII (recognizing CTTAAG) as the marker enzyme in the present study. After the screening of 1,120 spots in each mouse offspring, we found five mutations among 92,655 spots from the unirradiated paternal genome, five mutations among 218,411 spots from the unirradiated maternal genome, and 13 mutations among 92,789 spots from 5 Gy-exposed paternal genome. Among the 23 mutations, 11 involved mouse satellite DNA sequences (AT-rich), and the remaining 12 mutations also involved AT-rich but non-satellite sequences. Both types of sequences were found as multiple, similar-sequence blocks in the genome. Counting each member of cluster mutations separately and excluding results on one hypermutable spot, the spontaneous mutation rates were estimated as 3.2 (± 1.9) × 10−5 and 2.3 (± 1.0) × 10−5 per locus per generation in the male and female genomes, respectively, and the mutation induction rate as 1.1 (± 1.2) × 10−5 per locus per Gy. The induction rate would be reduced to 0.9 × 10−5 per locus per Gy if satellite sequence mutations were excluded from this analysis. The results indicate that mutation induction rates do not largely differ between GC-rich and AT-rich regions: 1 × 10−5 per locus per Gy or less, which is close to 1.08 × 10−5 per locus per Gy, the current estimate for the mean mutation induction rate in mice.

Electrophoretic variants of blood proteins in Japanese VI. Transferrin

SummaryA multiplicity of transferrin variants have been detected in the course of the biochemical aspect of the study of the genetic effects of atomic bombs. Variants obtained from the studies of 19,770 individuals in Hiroshima and Nagasaki were compared by polyacrylamide slab gel electrophoresis using three kinds of buffer systems with different pH values and thin layer polyacrylamide gel isoelectric focusing. The variants were compared on the basis of their relative mobilities and isoelectric points; seven types of fast-moving variant (B-variant) and nine types of slow-moving variant (D-variant) were detected, involving a total of 154 and 273 individuals, respectively. All the variants were identified as genetic variants by family studies. No variant differend inaallele frequency between the two cities. The variants detected in this study were compared with variants detected in residents of Mie district (another Japanese population), Caucasoids, American blacks, and Amerindians. Six additional types of B-variant and four additional types of D-variant, which had not been detected in Hiroshima and Nagasaki, were identified.