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ABSTRACT: This paper presents the results of an electrophoretic survey of approximately 4000 individuals from the cities of Hiroshima and Nagasaki, Japan, for four serum proteins: albumin, ceruloplasmin, haptoglobin and transferrin. The haptoglobin gene frequencies obtained for the HP1-HP2 polymorphism are in agreement with earlier reports. Rare electrophoretic variants of albumin, ceruloplasmin and haptoglobin occur with frequencies of 2.48, 0.50 and 0.58 per 1000 determinations, respectively. The noteworthy finding of 8 distinct transferrin variants in these populations, with a combined frequency of 20.90 per 1000 determinations, is also presented. Four of these variants (DCh1, B1, B3 and Dhir2 which corresponds electrophoretically to D4) have been reported in other populations in Japan, but the other five have not previously been differentiated.
Annals of Human Genetics 10/2007; 40(4):407 - 418. · 2.57 Impact Factor
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ABSTRACT: In a previous starch-gel electrophoresis study of erythrocyte phosphoglucomutase-1 (PGM1) in 23,095 Japanese from Hiroshima and Nagasaki, we detected 14 types of rare variant alleles. To determine sequence differences in these rare alleles, cell lines were established from peripheral B-lymphocytes from 24 unrelated individuals in whom nine types of rare variants are presumed to exist on the basis of earlier electrophoresis studies. cDNAs reverse transcribed from mRNAs extracted from these cell lines were amplified by polymerase chain reaction and sequences determined. Amino acid substitution types were deduced from each cDNA sequence. Although two individuals were reported to have an identical electromorph (PGM1 4HR3), sequence analysis revealed that alleles encoding these electromorphs possessed different base substitutions, and one was renamed PGM1 4HR4. As the amino acid substitution of ten different variants could be deduced by cDNA sequence in this study, the effect of each amino acid substitution on enzyme activity could be precisely simulated. The secondary structure of each variant predicted by computer simulations revealed that very decreased activity observed on PGM1 4HR2 protein was caused by significant secondary structure change introduced by the amino acid substitution. On the basis of the crystal structure, the amino acid substitutions of the ten types of rare variants seem to be outside the active center of this enzyme.
Human Biology 11/2001; 73(5):755-62. · 1.31 Impact Factor
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ABSTRACT: The human haptoglobin (HP) HP*2 allele contains a 1.7-kilobase (kb) intragenic duplication that arose after a unique nonhomologous recombination between the prototype HP*1 alleles. During a genetic screening of 13,000 children of survivors exposed to atomic-bomb radiation and 10,000 children of unexposed persons, two children suspected of carrying de novo mutations at the haptoglobin locus were identified (one in each group). DNA analyses of single-cell-derived colonies of Epstein-Barr virus-transformed B cells revealed that the two children were mosaics comprising HP*2/HP*2 and HP*2/HP*1 cells at a ratio of approximately 3:1. We infer that the latter cells are caused by reversion of one HP*2 allele to HP*1 through an intramolecular homologous recombination between the duplicated segments of the Hp*2 allele that excised one of the segments. Because the mosaicism is substantial (approximately 25%), this recombination must have occurred in early embryogenesis. The frequency of finding these children and the extent of their mosaicisms corresponds to an HP*2 to HP*1 reversion rate of 8 x 10(-6) per cell during development. This leads to the prediction that the HP*1 allele also will be represented, although usually at a very low frequency, in any HP2-2 person. We tested this prediction by using PCR for a single individual and found the HP*1 allele at frequencies of 4 x 10(-6) and 3 x 10(-6) in somatic and sperm cells. The HP*1 allele was detected by PCR in all four other HP2-2 individuals, which supports the regular but rare occurrence somatically of homologous recombination within duplicated regions in humans, in agreement with previous observations in mouse and Drosophila.
Proceedings of the National Academy of Sciences 09/1999; 96(18):10314-9. · 9.68 Impact Factor
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Nature 10/1996; 383(6597):226. · 36.28 Impact Factor
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ABSTRACT: We have investigated the variation in human ribosomal DNA repeat units as revealed in two-dimensional electrophoretic separates of genomic restriction fragments that were end-labeled at NotI cleavage sites. The transcribed portion of the ribosomal DNA results in approximately 20 labeled fragments visible on each gel as multicopy spots. We have mapped these spots to the sequences responsible for their appearance on the gels, based on their migration positions and direct sequencing of spots, and describe several previously unreported sources of variation. By studying mother/father/child families we gained information on how much of the between-repeats variation is due to differences between and within repeat arrays on homologous chromosomes. Two instances in which a child exhibited more copies of a particular fragment than were present in the parents are described and hypothesized to be due to events such as multiple unequal sister-chromatid exchanges or gene conversions.
Genetics 10/1996; 144(1):307-16. · 4.01 Impact Factor
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ABSTRACT: Studies are under way for the detection of potential genetic effects of atomic bomb radiation at the DNA level in the children of survivors. In a pilot study, we have examined six minisatellites and five microsatellites in DNA derived from 100 families including 124 children. We detected a total of 28 mutations in three minisatellite loci. The mean mutation rates per locus per gamete in the six minisatellite loci were 1.5% for 65 exposed gametes for which mean parental gonadal dose was 1.9 Sv and 2.0% for 183 unexposed gametes. We detected four mutations in two tetranucleotide repeat sequences but no mutations in three trinucleotide repeat sequences. The mean mutation rate per locus per gamete was o% for the exposed gametes and 0.5% for the unexposed gametes in the five microsatellite loci. No significant differences in the mutation rates between the exposed and the unexposed gametes were detected in these repetitive sequences. Additional loci are being analyzed to increase the power of our study to observe a significant difference in the mutation rates at the 0.05 level of significance.
Environmental Health Perspectives 06/1996; 104 Suppl 3:511-9. · 7.04 Impact Factor
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ABSTRACT: The hemagglutination inhibition antibody titers against the JC and BK polyoma viruses (JCV and BKV, respectively) are significantly elevated in individuals exhibiting "rogue" cells among their cultured lymphocytes. However, the elevation is so much greater with respect to JCV that the BKV elevation could readily be explained by cross reactivity to the capsid protein of these two closely related viruses. The JCV exhibits high sequence homology with the simian papovavirus, simian virus 40 (SV40), and inoculation of human fetal brain cells with JCV produces polyploidy and chromosomal damage very similar to that produced by SV40. We suggest, by analogy with the effects of SV40, that these changes are due to the action of the viral large tumor antigen, a pluripotent DNA binding protein that acts in both transcription and replication. The implications of these findings for oncogenesis are briefly discussed.
Proceedings of the National Academy of Sciences 05/1996; 93(7):2690-5. · 9.68 Impact Factor
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Tanpakushitsu kakusan koso. Protein, nucleic acid, enzyme 05/1996; 41(5):556-60.
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ABSTRACT: In a pilot study to detect the potential effects of atomic bomb radiation on germ-line instability, we screened 64 children from 50 exposed families and 60 from 50 control families for mutations at six minisatellite loci by using Southern blot analysis with Pc-1, lambda TM-18, ChdTC-15, p lambda 3, lambda MS-1, and CEB-1 probes. In the exposed families, one or both parents received a radiation dose > 0.01 Sv. Among the 64 children, only one child had parents who were both exposed. Thus, of a total of 128 gametes that produced the 64 children, 65 gametes were derived from exposed parents and 63 were from unexposed parents, the latter being included in a group of 183 unexposed gametes used for calculating mutation rates. The average parental gonadal dose for the 65 gametes was 1.9 Sv. We detected a total of 28 mutations at the p lambda g3, lambda MS-1, and CEB-1 loci, but no mutations at the Pc-1, lambda TM-18, and ChdTC-15 loci. We detected 6 mutations in 390 alleles of the 65 exposed gametes and 22 mutations in 1098 alleles of the 183 gametes from the unexposed parents. The mean mutation rate per locus per gamete in these six minisatellite loci was 1.5% in the exposed parents and 2.0% in the unexposed parents. We observed no significant difference in mutation rates in the children of the exposed and the unexposed parents (P = .37, Fisher's exact probability test).
The American Journal of Human Genetics 12/1995; 57(6):1275-83. · 10.60 Impact Factor
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ABSTRACT: There is a continuing need for more efficient methods to examine human (and other) populations for altered germinal and somatic cell mutation rates. To this end, we have explored the potential usefulness of two-dimensional (2-D) electrophoresis of human DNA fragments obtained from restriction-enzyme-digested genomic DNA, using samples from father/mother/child trios. On a single 2-D DNA preparation, approximately 2000 DNA fragments varying in size from 1.0 to 5.0 kbp in the first dimension and 0.3 to 2.0 kbp in the second dimension are visualized. To enter into a genetic analysis of quantitative variation, these fragments must exhibit positional and quantitative stability. With respect to the latter, if spots that are the product of two homologous DNA fragments are to be distinguished with the requisite accuracy from spots that are the product of only one fragment, the coefficient of variation of spot intensity should be approximately < or = 0.12. At present, 482 of the spots in our preparations meet these standards. In an examination of preparations based on three Japanese mother/father/child trios, 43 of these 482 spots were found to exhibit variations that segregated within families according to Mendelian principles. Additionally, of the 2000 spots, 1114 (of which the aforementioned 482 are a subset) were deemed appropriate for the study of qualitative variation. A total of 142 variable spots were identified; the heterozygosity index for these DNA fragments was 4.4%. The genetic nature of the additional variants was again established by their segregation according to Mendelian principles. We have established the feasibility of cloning fragments from such gels and determining their nucleotide sequence. This technology should be highly efficient in monitoring for mutation resulting in loss/gain/rearrangement events in DNA fragments distributed throughout the genome.
Electrophoresis 02/1995; 16(2):241-52. · 3.30 Impact Factor
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ABSTRACT: We have investigated the extent to which restriction fragment length polymorphism can be detected by two-dimensional electrophoresis of end-labeled genomic restriction fragments. Genomic DNA was digested with NotI and EcoRV and labeled at the NotI recognition site before first-dimension electrophoresis in disk gels. DNA in the disk gels was further digested in situ with HinfI prior to second-dimension electrophoresis, yielding patterns in which approximately 2000 end-labeled fragments were simultaneously visualized. On the basis of studies of 6 mother/father/child trios, a group of 184 fragments was organized into 85 polymorphic systems in which all allelic fragments were detectable in the 2-D patterns. Another 206 fragments varied as to their presence among individuals, but their relatedness to other fragments was not established. Our data indicate that a large number of DNA polymorphisms can be simultaneously scored in 2-D separations of genomic DNA fragments.
Genomics 02/1995; 25(2):345-53. · 3.02 Impact Factor
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ABSTRACT: The continuing efforts to evaluate specific human populations for altered germinal mutation rates would profit from more efficient and more specific approaches than those of the past. To this end, we have explored the potential usefulness of two-dimensional electrophoresis of DNA fragments obtained from restriction-enzyme-digested genomic DNA. This permits the analysis, on a single preparation, of approximately 2000 DNA fragments varying in size from 1.0 to 5.0 kb in the first dimension and from 0.3 to 2.0 kb in the second dimension. To enter into a genetic analysis, these fragments must exhibit positional and quantitative stability. With respect to the latter, if spots that are the product of two homologous DNA fragments are to be distinguished with the requisite accuracy from spots that are the product of only one fragment, the coefficient of variation of spot intensity should be approximately < or = 0.12. At present, 482 of the spots in our preparations meet these standards. In an examination of preparations based on three Japanese mother/father/child trios, 43 of these 482 spots were found to exhibit variation that segregated within families according to Mendelian principles. We have established the feasibility of cloning a variant fragment from such gels and establishing its nucleotide sequence. This technology should be highly efficient in monitoring for mutations resulting in loss/gain/rearrangement events in DNA fragments distributed throughout the genome.
Proceedings of the National Academy of Sciences 10/1994; 91(19):9052-6. · 9.68 Impact Factor
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Tanpakushitsu kakusan koso. Protein, nucleic acid, enzyme 01/1994; 38(16):2723-7.
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ABSTRACT: In the course of feasibility studies to examine the efficiencies and practicalities of various techniques for screening for genetic variations, the human coagulation factor IX (F9) genes of 63 Japanese families were examined by PCR-denaturing gradient gel electrophoresis (PCR-DGGE). Four target sequences with lengths of 983-2,891 bp from the F9 genes of 126 unrelated individuals from Hiroshima and their 100 children were amplified by PCR, digested with restriction enzymes to approximately 500-bp fragments, and examined by DGGE--a total of 6,724 bp being examined per individual. GC-rich sequences (GC-clamps) of 40 bp were attached to both ends of the target sequences, as far as was feasible. Eleven types of new nucleotide substitutions were detected in the population, none of which produced RFLPs or caused hemophilia B. By examining two target sequences in a single lane, approximately 8,000 bp in a diploid individual could be examined. This approach is very effective for the detection of variations in DNA and is applicable to large-scale population studies.
The American Journal of Human Genetics 02/1993; 52(1):167-75. · 10.60 Impact Factor
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ABSTRACT: We have developed a technique to detect accurately heterozygous carriers of a deletion. Specific target sequences were amplified by the polymerase chain reaction (PCR), and the products subsequently were analyzed by high-performance liquid chromatography. Examples from four loci demonstrated that 24-27 cycles of amplification for a single-copy DNA, based on 50 ng of genomic DNA, results in excellent quantitation that readily permits the detection of heterozygous carriers of a deletion. We have demonstrated that triplex PCR (three targets in a single PCR) entails no loss of precision. We also have demonstrated that this method can accurately differentiate the heterozygous carriers of a deletion from normal individuals in four family studies, three for Duchenne muscular dystrophy patients and one for a hemophilia B patient.
Proceedings of the National Academy of Sciences 11/1992; 89(19):9126-30. · 9.68 Impact Factor
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ABSTRACT: The ATTTT repeat polymorphism located approximately 1,400 base pairs (bp) upstream from the beta-globin structural gene was analyzed by denaturing gradient gel electrophoresis (DGGE) of RNA:DNA duplexes. A study of 81 unrelated Japanese from Hiroshima revealed a sequence heteromorphism in this site. The alleles with five and six repeats of the ATTTT unit, which have been reported, were found in polymorphic proportions. Two unreported alleles were also detected, the first, in two persons, characterized by seven repeats and the other, in a single person, having an A-to-G nucleotide substitution in the fifth repeat.
Human Genetics 07/1991; 87(2):219-20. · 5.07 Impact Factor
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ABSTRACT: The applicability of ribonuclease cleavage at mismatches in RNA:DNA duplexes (RNase cleavage method) for determining nucleotide variant rates has been examined in a Japanese population. DNA segments of various lengths obtained from 4 different regions of a normal and 3 thalassemic cloned human beta-globin genes were inserted into transcription vectors. Sense and antisense RNA probes uniformly labeled with 32P were prepared. When RNA probes of 771 nucleotides (nt) or less were hybridized with cloned DNAs and the resulting duplexes were treated with a mixture of RNases A and T1, the length of products agreed with theoretical values. Twelve possible mismatches were examined. Since both sense and antisense probes were used, uncleavable mismatches such as G:T and G:G which were made from one combination of RNA and DNA strands could be converted to the cleavable C:A and C:C mismatches, respectively, by using the opposite combination. Deletions and insertions of 1 (G), 4 (TTCT), 5 (ATTTT) and 10 (ATTTTATTTT) nt were easily detected. A polymorphic substitution of T to C at position 666 of the second intervening sequence (IVS2-666) of the beta-globin gene was detected using genomic DNAs from cell lines established from the peripheral B lymphocytes of 59 unrelated Japanese from Hiroshima or those amplified by polymerase chain reaction (PCR). The frequency of the gene with C at the IVS2-666 (allele C) was 0.48 and that of the gene with T (allele T) was 0.52. The associations of the 2 alleles were in agreement with Hardy-Weinberg proportions. No contradiction to Mendelian inheritance was observed in the results obtained from 11 family studies. Two new polymorphic substitutions of C to A and A to T were detected at nucleotide positions 1789 and 1945 from the capping site, respectively, using genomic DNAs amplified by PCR. The feasibility of the RNase cleavage method combined with PCR for large-scale screening of variation in chromosomal DNA is discussed.
Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis 09/1990; 231(2):219-31. · 2.85 Impact Factor
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ABSTRACT: The data collected in Hiroshima and Nagasaki during the past 40 years on the children of survivors of the atomic bombings and on the children of a suitable control population are analyzed on the basis of the newly revised estimates of radiation doses. No statistically significant effects emerge with respect to eight different indicators. Since, however, it may confidently be assumed some mutations were induced, we have taken the data at face value and calculated the minimal gametic doubling doses of acute radiation for the individual indicators at various probability levels. An effort has also been made to calculate the most probable doubling dose for the indicators combined. The latter value is between 1.7 and 2.2 Sv. It is suggested the appropriate figure for chronic radiation would be between 3.4 and 4.5 Sv. These estimates suggest humans are less sensitive to the genetic effects of radiation than has been assumed on the basis of past extrapolations from experiments with mice.
The American Journal of Human Genetics 07/1990; 46(6):1053-72. · 10.60 Impact Factor
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Progress in clinical and biological research 02/1990; 340C:197-206.
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ABSTRACT: Since 1946 a continuous effort to evaluate the potential genetic effects of the atomic bombs has been sustained. Observations on children born in Hiroshima and Nagasaki include sex ratio, congenital malformations, stillbirths, survival of liveborn infants, chromosomal abnormalities (sex chromosomal abnormalities and balanced chromosomal rearrangements), mutations altering protein structure or activity, and physical growth and development. There are no statistically significant differences between the children of parents who received increased amounts of radiation at the time of the bombings and those whose parents did not. However, the difference between the two sets of children is consistent with the hypothesis of a genetic effect of the exposure, but its magnitude suggests humans are not as sensitive to the genetic effects of radiation as projected from the mouse paradigm.
Genome 02/1989; 31(2):853-9. · 1.65 Impact Factor
