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Microsatellite polymorphism due to differences in CT dinucleotide repeats was demonstrated in intron 14 of the canine BRCA1 gene. Genotype analysis of 103 unrelated dogs from 30 different breeds detected the presence of five alleles, including 10 of the expected 15 genotypes. Gene frequencies were biased and all alleles with the exception of one were below 0.1. This polymorphism, which occurs at the intron of canine BRCA1 should prove to be a useful marker for detecting the loss of heterozygosity (LOH). One of the more notable findings of the present study was the detection of homozygotes of rare alleles. This finding identified an accumulation of rare alleles in specific canine breeds and demonstrated the usefulness of this characteristic for the biological study of dog evolution.

Background: The studies on the association of deletion/ insertion (D/I) polymorphism of angiotensin converting enzyme (ACE) gene with blood pressure and hypertension reported contradictory results. Because there was no population-based study in Japan, we examine the hypothesized association in a cross-sectional sample of a Japanese cohort., Methods and Results: The blood pressure of 464 men and 876 women aged 40-80 years was measured, and their DNA was analyzed for ACE D/I genotypes. The prevalence of the D allele was 38.7 and 39.2% in men and women, respectively (overall 39%). There was a tendency for higher covariate (age, body mass index, albuminuria, hematocrit, alcohol consumption, smoking, diabetes mellitus, ischemic heart disease and antihypertensive medication) adjusted mean levels of diastolic blood pressure for the DD genotype in men but not in women. However, this tendency disappeared after dichotomization of blood pressure into diagnostic categories (normotension and hypertension). Results did not differ when the subjects were divided into two age groups (< or = 59 and > or = 60 years). Covariate-adjusted odds ratios for hypertension for presence of the D allele were close to the null value of one. ACE genetic variation accounted for only 0.1 and 0.7% of the inter-individual variation in systolic and diastolic blood pressure in men. These estimates were 0.2 and 0.1%, respectively, in women., Conclusion: Although there is a tendency of higher diastolic blood pressure in men with DD genotypes, there is no convincing evidence that ACE genotypes are associated with hypertension in this Japanese population.

The Duffy blood group antigen has been characterized by its roles on red blood cells: as a receptor for the malarial parasites and as a promiscuous receptor for chemokine superfamily. Recently, the Duffy blood group associated glycoprotein D (gpFy) cDNA has been cloned (Chaudhuri et al: Proc Natl Acad Sci USA 90:10793, 1993). In this report we describe the organization of genomic DNA coding for the gpFy and elucidate the molecular nature of Fya/b polymorphisms. By a Southern blotting analysis probed with gpFy cDNA, gpFy gene was shown to be composed of three DNA fragments; 1.1-kb Sac I, 1.9-kb EcoRI, and their intervening 47-bp fragments. We cloned the 1.1-kb Sac I and 1.9-kb EcoRI fragments by inverted polymerase chain reaction (IPCR) procedure. The promoter region of the gpFy gene was cloned by IPCR of 1.1-kb Sac I fragment and the 3' flanking sequence was cloned by IPCR of 1.9 kb EcoRI fragment. The both IPCR products contained on both side the known gpFy cDNA sequence without introns, as expected. Although no TATA or CCAAT boxes are present in the promoter sequence, several transcription factor binding site motifs are contained, including AP-1, HNF-5, TCF-1, ApoE B2, W-element, H-APF-1, and Sp-1. The 3' flanking region has two additional polyadenylation signals, other than that used in the cDNA, and also has an indirect and a direct repeat sequence clustered with the 5' flanking region. These facts indicate a possibility that the gpFy gene has been evolved by multiple retrotransposition events. By comparing the coding area of the gpFy gene in 28 Duffy-positive individuals, we elucidated that one base change that results in an amino acid substitution [GA-T(Asp44)-->GGT(Gly)] is in accordance with the Fya/Fyb polymorphism. This fact proves that the gpFy cDNA and its gene described in this report encode the Duffy blood group system.

To determine which Rh-polypeptide genes are related to which Rh (rhesus) antigens, genomic DNAs prepared from donors of ten kinds of different Rh phenotypes were analysed by Southern hybridization method using two probes for the 5' moiety and 3' moiety of an Rh-polypeptide cDNA. A polymorphism for the Rh blood-group system was observed with BamHI, HindIII, PstI and EcoRI restriction endonucleases between genomic DNAs from the RhD-positive and RhD-negative individuals as has previously been reported. Further study showed an additional genomic polymorphism. The new polymorphism was detected between the RhC/c phenotypes unrelated to the RhD/d phenotypes with RsaI restriction endonuclease using the probe for the 5' moiety of Rh polypeptide cDNA. A 1.9 kb RasI restriction fragment was missing in the genomes from individuals with the RhC phenotypes. However, this fragment was present in the genomes of Rhc phenotypes and in its half dose in the genomes of RhCc phenotypes. These results clearly demonstrate a new polymorphism between the RhC/c phenotypes regardless of the RhD-positive and RhD-negative phenotypes, and it is predicted that the Rh polypeptide genes encoding RhD, RhC/c and RhE/e antigens are different.

Mitochondrial DNA (mtDNA) polymorphism was studied in 21 Jindo dogs inhabiting Jin Island off the Korean peninsula. The polymorphism was analyzed with 10 restriction endonucleases that recognize six base pairs. The sizes of the mtDNA fragments produced by digestion using each endonucleases were separated by agarose gel electrophoresis, and the polymorphisms were detected with Japanese mongrel dog mtDNA as a probe. The mtDNA polymorphism in Jindo dogs was observed with four restriction endonucleases, Apa I, EcoR V, Hinc II, and Sty I. However, no polymorphism was detected with BamH I, Bgl II, EcoR I, Hind III, Pst I, or Xba I. The observed restriction endonuclease morphs were classified into 4 types of distinct cleavage patterns. The average number of nucleotide substitutions per nucleotide site in Jindodogs was estimated to be 0.0086. By UPG phylogenetic analysis, the 4 mtDNA types showed only one cluster. This suggests that Jindo dogs have not diverged from the other cluster up to the present and the species is considerably pure.

Genetic variants of salivary alpha-amylase were studied using isoelectric focusing in a pH gradient of 6-8 and silver staining methods. Five phenotypes which were tentatively named Amy1 N, Amy1 SN, Amy1 V1N, Amy1 V2SN and Amy1 V3N were detected. The phenotype frequencies in 371 unrelated Japanese were: Amy1 N = 94.33, Amy1 SN = 2.43, Amy1 V1N = 0.54, Amy1 V2SN = 0.27 and Amy1 V3N = 2.43%, respectively. Although variant bands of Amy1 V1N were detected by immunoblotting method using anti-human salivary amylase, those bands were not stained by starch-iodine method for the detection of amylase activity. This suggested that a mutation was occurred in the region of amylase molecule which showed the activity. Amy1 V2SN comprised the production of three Amy1 genes and indicated the duplication of Amy1 gene. The individuals showing Amy1 V3N phenotype accorded with those showing amylase variant by PAGE.

A genetic polymorphism of a human platelet polypeptide with a molecular weight of 28 kD detected by two-dimensional electrophoresis was investigated in family and population studies, and cell distribution. The 28-kD polypeptide showed autosomal codominant inheritance of two alleles. The gene frequencies of the two alleles were 0.925 and 0.075, respectively. The 28-kD polypeptide was observed in lymphocytes, neutrophils, eosinophils and monocytes, in addition to platelets. This polypeptide showed good reproducibility in electrophoresis, and appears to be useful as a genetic marker of the human genome in gene mapping and pedigree analysis.

We described a new genetic polymorphism of human platelet polypeptide, detected by two-dimensional polyacrylamide gel electrophoresis followed by silver-staining. The polymorphism was tentatively designated thrombocyte B (ThB) with a molecular weight of 34 kDa and isoelectric point of 4.7-4.8. In this polypeptide, three different electrophoretic types (1-1, 1-2, 2-2) were identified. Family and population studies indicate that the three phenotypes of the polypeptide are determined by two common alleles at a single autosomal locus. In a Japanese population, the gene frequency of ThB 1/ThB 2 was 0.74/0.26. The ThB polypeptide was not found in other blood cells and decreased or disappeared during the preparation or storage of platelets.

In 218 parotid saliva samples collected at random from a Japanese population, two phenotypes were observed by SDS polyacrylamide gel electrophoresis. Inheritance was controlled by a dominant allele at an autosomal locus. The frequencies of the genes determining these phenotypes were for the Japanese population studied: Ph+ = 0.026 +/- 0.008, Ph- = 0.974 +/- 0.008.

Nine salivary polymorphic systems (Pa, Pb, Pr, Db, PmF, PIF, Ph, Amy1 and s-AcP) were examined using parotid and whole saliva from random Japanese individuals. The gene frequencies obtained were: Pa+ = 0.221, Pb1 = 1.000 Pr1 = 0.741, Db+ = 0.033, PIF+ = 0.715, Ph+ = 0.029, Amyv1 = 0.013 and s-AcPA = 0.217, respectively.

In 195 parotid saliva samples collected at random from a Japanese population, two phenotypes were observed by electrophoresis in acid-urea starch gels. The protein showing polymorphisms was detected in the middle zone between Pa and Pb, and was tentatively designated Pm. Inheritance was controlled by a dominant allele at an autosomal locus. The frequencies of the genes determining these phenotypes were, for the Japanese population studied, Pm+=.38+/-.03, Pm-=.62+/-.03.

The distributions of the phenotype and gene frequencies of nine polymorphic systems in human parotid and whole saliva were studied in the Japanese population of the eastern part of Japan and compared with those of populations in other areas of Japan and other ethnic groups. The gene frequencies were: Pa+ = 0.214; Pb1 = 1.000; Pr1 = 0.753; Db+ = 0.035; PmF+ = 0.394; Ph+ = 0.028; PIF+ = 0.733; Amy1V = 0.011, and s-AcpA = 0.217.

Phenotype and gene frequencies of PIF (parotid isoelectric focusing variant) were determined in a series of individuals from eastern Japan. Among 422 unrelated individuals examined, 391 (92.66%) of PIF+ and 31 (7.34%) of PIF- phenotypes were observed; the gene frequencies were PIF+ = 0.729 and PIF- = 0.271.

Genetic polymorphism of the human parotid salivary acid phosphatase (s-AcP) in the Japanese population is described. The use of polyacrylamide gel isoelectric focusing electrophoresis with the pH range of 4.0-6.5 enabled us to discern three variant patterns controlled by two codominant alleles at the single autosomal locus. The two alleles were designated s-AcP:A and s-AcP:a, and the gene frequencies calculated from 183 Japanese subjects were s-AcP:A = 0.2268 +/- 0.022, s-AcP:a = 0.7732 +/- 0.022, respectively. The distribution of phenotypes fitted the Hardy-Weinberg equilibrium.

In 332 samples of human parotid saliva collected at random from a Japanese population, the genetic polymorphism of salivary alpha-amylase was detected by isoelectric focusing electrophoresis in a pH range of 5.2-7.2 polyacrylamide gel followed by silver staining. This polymorphism, that was tentatively designated Amy1 S, consisted of extra three isozymes of a normal pattern (Amy1 N) and isoelectric points of these three isozymes were 5.5, 5.8 and 6.1, respectively. The inheritance was controlled by a dominant allele at an autosomal locus. The frequency of the genes determining these phenotypes were studied as follows: Amy1 S = 0.014 +/- 0.004, Amy1 N = 0.986 +/- 0.004.