Shuichi Tsuchida

The two polymorphic alleles of esterase D (ESD), ESD*5 and ESD*7, are specific to Europeans and Asians, respectively. In this study the molecular basis was characterized: ESD*5, arising from ESD*1, has a G to A transition, resulting in Gly(257)(GGT) --> Asp(GAT); and ESD*7, originating from ESD*2, has an A to G transition, resulting in ASP(231)(GAT) --> Gly(GGT). Glycine is also involved in the common ESD*1/ESD*2 polymorphism [Gly(190)(GGA) --> Glu(GAA)]. Haplotype analysis using a few novel intragenic polymorphisms showed strong associations among polymorphic sites, suggesting that recombination has been less frequent in the human ESD gene, although it spans about 25 kb from exon 1 to exon 10. A marked difference was observed in the distribution of haplotype frequencies between Germans and Japanese.

Severe hydronephrosis and hydroureter associated with bilateral ureteral ectopia was diagnosed in a 9-month-old, female Siberian Husky. Surgical transplantation of the ectopic ureters into the urinary bladder was performed. Intravenous urography revealed a marked decrease in the size of hydronephrosis and hydroureter after the surgery. After surgery, intermittent urinary incontinence continued, but was resolved 9 months after the surgery.

We present the fourth known case of endometrial carcinoma, and the second case of endometrial small-cell carcinoma, to be associated with paraneoplastic retinopathy. Initial symptoms were decreased visual acuity and a narrowing of the visual field. Endometrial carcinoma was diagnosed several months later. An antibody to 34-kDa bovine retinal antigen was detected in the patient's serum. Thus, autoimmunity was suspected as the cause of the retinopathy. In patients with endometrial carcinoma with visual disturbance of unknown cause, paraneoplastic retinopathy should be suspected. (C) 1998 Academic Press.

A guanine-adenine substitution was observed in exon 5 of the human transferrin (TF) gene. The nucleotide change led to an AvaI digestion site. Analysis of the segregation of the AvaI polymorphism and serum TF phenotypes indicated that an intragenic recombination occurred between the AvaI polymorphic site and the mutation site in the TF gene which determines the two common TF alleles, TF*C1 and TF*C2.

(1) The polymerase chain reaction (PCR) was used to amplify Rh-related cDNAs from erythroid cells cultured by the selective two-phase liquid culture system for human erythroid progenitors in peripheral blood. Two Rh polypeptide cDNAs have been isolated from the PCR products and tentatively designated RhPI cDNA and RhPII cDNA. Both cDNA clones have an open reading frame composed of 1251 nucleotides. The RhPI cDNA clone shows a single nucleotide substitution with no amino acid substitution compared with the published sequence. The RhPII cDNA clone differs from the above by 41 nucleotide substitutions in the open reading frame, resulting in 31 amino acid substitutions. Besides these cDNA clones, eleven and five truncated isoforms of the RhPI and RhPII cDNAs, have been isolated, respectively. (2) The promoter region of the Duffy gene was cloned by IPCR of 1.1 kb SacI fragment and the 3' flanking sequence was cloned by IPCR of 1.9 kb EcoRI fragment. The IPCR products contained the known Duffy cDNA sequence without introns. By comparing the coding area of the Duffy gene in 28 Duffy positive individuals, we elucidated that one base change that results in an amino acid substitution (GAT(Asp(44))-->GGT(Gly)) is in accordance with the Fy(a)/Fy(b) polymorphism. This fact proves that the Duffy cDNA and its gene encode the Duffy blood group system. (3) Two common alleles in Esterase D (EsD) polymorphism, EsD1 and EsD2 were characterized by the substitution of one amino acid (Gly-Glu) caused by the point mutation of one nucleotide (G-A). The point mutation between cDNAs of EsD1 and EsD2 alleles was detectable as restriction fragment length polymorphism (RFLP) using SspI. The RFLP makes it possible to determine the EsD phenotypes using DNA samples from forensic materials without EsD enzymatic activity. (3) The authors report studies on 19 pairs of donors and recipients in bone marrow transplantation. A broad range of genetic markers at 42 gene loci, including one DNA marker 11 red blood cell markers, five human lymphocyte antigen types. 12 serum protein markers, five red cell enzyme markers, and eight salivary markers was evaluated before and after BMT over about 2 months. As a result. 11 out of 42 gene loci of genetic markers in recipients were transformed into the donor type.

We have analyzed the esterase D (EsD) polymorphism at the nucleic acid level. Two common alleles, EsD1 and EsD2, are characterized by the substitution of one amino acid (Gly-to-Glu), which is caused by the point mutation of one nucleotide (G-to-A). Individuals exhibiting the EsD1 and EsD 2 phenotypes are homozygotes for EsD 1 and EsD 2 cDNAs, respectively. Individuals showing the EsD 2-1 phenotype have two kinds of cDNAs, viz., EsD 1 and EsD 2. The point mutation difference between the cDNAs of the EsD1 and EsD2 alleles results in a different SspI digestion site. A restriction fragment length polymorphism caused by this difference with respect to the SspI digestion site makes it possible to determine the EsD phenotype using DNA samples extracted from forensic materials with no EsD enzymatic activity.

A new method for the detection of mitochondrial (mt)DNA polymorphism by agarose gel electrophoresis is presented. In this method, after mtDNA digestions with endonucleases, the mtDNA fragments are labeled directly with photoactivatable biotin in the digestion buffer, separated by agarose gel electrophoresis, transferred to a nylon membrane and detected enzymatically using streptavidin-alkaline phosphatase, Nitroblue tetrazolium (NBT) and 5-bromo-4-chloro-3-indolylphosphate (BCIP). This method is useful for the detection of mtDNA digestion patterns by agarose gel electrophoresis, especially when enough purified mtDNA cannot be obtained and the radioisotope cannot be handled because of several restrictions.

Mitochondrial DNA (mtDNA) polymorphism was studied in 20 mongrel dogs using 14 restriction enzymes. The polymorphism was observed in the cleavage patterns of Apa I, EcoR I, EcoR V, Hinc II and Sty I. Three morphs using EcoR I and Apa I and two morphs using EcoR V, Hinc II and Sty I were found. However, no polymorphism was detected in the cleavage patterns of BamH I, Bgl II, Hae II, Hind III, Pst I, Sal I, Sca I, Stu I and Xba I. The examined dogs were classified in seven types using five restriction endonucleases which recognized six nucleotide sequences. The value of nucleotide diversity was estimated to be 0.0055. A phylogenetic tree constructed by genetic distances among seven restriction types showed at least two clusters of mtDNA.

Genetic variants of salivary α-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.