Identification of missense mutation (G365R) of the butyrylcholinesterase (BCHE) gene in a Japanese patient with familial cholinesterasemia.

A point mutation which caused a silent phenotype of human serum butyrylcholinesterase (BChE) was identified in the genomic DNA of a 57-year-old Japanese woman who visited our hospital because of pneumonia. The propositus exhibited an unusually low level of BChE activity, whereas her son and daughter had an intermediate level. Immunologically, there was an absence of BChE protein in the propositus's serum. DNA sequence analysis of the propositus demonstrated a point mutation at codon 365 (GGA-CGA), resulting in a Gly-Arg substitution. A family study showed her son and daughter to have the same mutation.

Gene analysis of genomic DNA from stored serum by polymerase chain reaction: identification of three missense mutations in patients with cholinesterasemia and ABO genotyping.

We established a method to determine the butyrylcholinesterase genotype associated with a BCHE deficiency directly using multiple PCR from stored serum, which was stored at -70 degrees C for more than 30 years. PCR products from sera of six propositi were used for DNA sequence analysis. All of these BChE variants were characterized by a single nucleotide substitution. Four of them were homozygotes and demonstrated a C-->T single nucleotide point mutation at codon 100 from CCA (Pro) to TCA (Ser). The fifth case was a heterozygote of this mutation. The remaining one was a compound heterozygote showing a T-->C transition mutation at codon 203 from TCA (Ser) to CCA (Pro) and a G-->C transversion mutation at codon 365 from GGA (Gly) to CGA (Arg). Furthermore we developed a method to determine the ABO genotype from the same serum. These results indicated that serum is useful as a starting material for amplification of genomic DNA when fresh blood samples are not available.

[Missense mutations of the butyrylcholinesterase gene in six Japanese patients with low cholinesterasemia: genetic analysis using sera stored in a freezer].

Six serum samples with no detectable butyrylcholinesterase (BCHE) activity had been stored at -70 degrees C for more than 10 years. These sera were used for amplification of BCHE gene using polymerase chain reaction (PCR) and for nucleotide sequence analysis. Five of them demonstrated a C-->T transition at codon 100 (CCA-->TCA), resulting in a Pro-->Ser substitution. The other one was a compound heterozygote as revealed a T-->C transition mutation at codon 203 from TCA (Ser) to CCA (Pro) and G-->C transversion at codon 365 from GGA (Gly) to CGA (Arg). These results showed sera stored in a freezer could be used as a starting material for amplification of genomic DNA when it is not possible to obtain fresh blood samples.

Analysis of the origin and development of hatching gland cells by transplantation of the embryonic shield in the fish, Oryzias latipes.

Hatching gland cells of the medaka, Oryzias latipes, have been observed to differentiate from the anterior end of the hypoblast, which seems to first involute at the onset of gastrulation. These results suggest that the hatching gland cells of medaka originate from the embryonic shield, the putative organizer of this fish. The present study investigated whether hatching gland cells really originate from the embryonic shield in the medaka. Transplantation experiments with embryonic shield and in situ hybridization detection of hatching enzyme gene expression as a sign of terminal differentiation of the gland cells were carried out. The analysis was performed according to the following processes. First, identification and functional characterization of the embryonic shield region were made by determining the expression of medaka goosecoid gene and its organizer activity. Second, it was confirmed that the embryonic shield had an organizer activity, inducing a secondary embryo, and that the developmental patterns of hatching gland cells in primary and secondary embryos were identical. Finally, the hatching gland cells as identified by hatching enzyme gene expression were found to coincide with the dye-labeled progeny cells of the transplanted embryonic shield. In conclusion, it was determined that hatching gland cells were derived from the embryonic shield that functioned as the organizer in medaka.

Characterization and expression of embryonic globin in the rainbow trout, Oncorhynchus mykiss: intra-embryonic initiation of erythropoiesis.

When fractionated by reverse-phase high performance liquid chromatography (HPLC), the embryonic hemoglobin of the rainbow trout, Oncorhynchus mykiss, consisted of eight globins different from adult globins in terms of retention time. Amino acid sequences of the N-terminal regions of some globins were determined. In addition, four cDNA clones for embryonic globins from 10-day embryos were isolated (at 15 degrees C), sequenced and the amino acid sequences predicted. In comparison with the sequences of previously characterized globins, they corresponded to two alpha-type and two beta-type globins and therefore were named em.alpha-1, em.alpha-2, em.beta-1 and em.beta-2. The N-terminal 36 amino acids of one (E2) of the embryonic globins isolated by HPLC were identical to those of the sequence deduced from a cDNA, em.beta-2. The phylogenetic relationship between the embryonic globins and other globins previously reported was discussed. The present study is the first demonstration of amino acid sequences of embryonic globins in a teleost. To understand the initiation of erythropoiesis in the early development of the rainbow trout, histochemistry using o-dianisidine/hydrogen peroxide, immunohistochemistry using an antibody against embryonic hemoglobin, and northern blotting and whole embryo in situ hybridization using antisense RNA probe for em.beta-2 were performed. Embryonic globin mRNA, globin and hemoglobin appeared first in the anterior part of the intermediate cell mass (ICM) located in the median line beneath the notochord of embryos 6-7 days after fertilization at 15 degrees C (Vernier's stages 16-20). Shortly after that, the expression signal extended to the posterior part of the ICM and spread out laterally to blood islands on the posterior yolk sac. Thus, the initiation of erythropoiesis in the early embryo of rainbow trout is intraembryonic.

Formation of mature egg envelope subunit proteins from their precursors (choriogenins) in the fish, Oryzias latipes: loss of partial C-terminal sequences of the choriogenins.

The inner layer of egg envelope of the medaka, Oryzias latipes, comprises two major groups of glycoprotein subunits, ZI-1,2 and ZI-3. Their precursor proteins, choriogenin H (Chg H) and choriogenin L (Chg L), respectively, are synthesized in spawning female liver. In the present study, the primary structures of the precursors and the corresponding mature subunits were compared by peptide mapping and amino acid sequencing to find what difference in their molecular structures is relevant to the assembly of the soluble precursors into the insoluble inner layer. The primary structures of the solubilized subunits were mostly identical to those of the respective precursors, but they lacked C-terminal partial sequences that their precursors possessed, namely, ZI-1,2 subunit was shorter than Chg H by 34 amino acid residues and ZI-3 was shorter than Chg L by 27 residues. In addition, a consensus amino acid sequence, Arg-Lys-X-Arg, was found at the putative cleavage sites in the C-terminal region of the precursors. It is conjectured that the truncation of the precursor proteins is prerequisite for formation of mature chorion subunit proteins and their assembly into chorion.

First case of missense mutation (LDH-H:R171P) in exon 4 of the lactate dehydrogenase gene detected in a Japanese patient.

Complete deficiency of lactate dehydrogenase (LDH) subunit H was identified in a 41-year-old woman with paralysis of her left lower limb. The propositus had extremely low LDH activity and five of her family members had levels of LDH activity that ranged from lower than normal to normal level. A transversion mutation at codon 171 (CGC-->CCC), resulting in an Arg-->Pro substitution was identified in her DNA sequence. A new NruI restriction site was introduced into the polymerase chain reaction (PCR) product by PCR-primer introduced restriction analysis (PCR-PIRA) using a specific mismatched primer. Digestion with NruI revealed that the propositus and her mother were, respectively, homozygous and heterozygous for this mutation.

Enzyme responsible for egg envelope (chorion) hardening in fish: purification and partial characterization of two transglutaminases associated with their substrate, unfertilized egg chorion, of the rainbow trout, Oncorhynchus mykiss.

From our previous studies, we have suggested that "egg envelope (chorion) hardening-enzyme" (first proposed by Zotin [J. Embriol. Exp. Morph. 6, 546-568 (1958)]) in the rainbow trout, Oncorhynchus mykiss, is transglutaminase (TGase), and that it coexists with its substrate, the unfertilized egg chorion, and forms epsilon-(gamma-glutamyl)lysine cross-links between the chorion proteins. In the present study, we extracted the TGase activity from the isolated chorions by homogenization with isotonic saline (143 mM NaCl-10 mM Tris.HCl, pH 7.2) and fractionated the extract by Toyopearl HW55S gel filtration with the isotonic saline containing 5 mM CaCl2 and 5 mM 2-mercaptoethanol (2-ME). One peak of TGase activity (P2) was obtained. When the eluates were dialyzed against 5 mM CaCl2/5 mM 2-ME/10 mM Tris.HCl (pH 7.2), another peak of the activity (P1) appeared. P1 TGase activity, which becomes apparent in a medium of low ionic strength, is involved in acceleration of the chorion hardening after egg activation in fresh water, so-called water activation. We purified the two TGases, P1 and P2, by SP-Sepharose, Q-Sepharose, and TSK-gel column chromatography. The molecular mass of the native form of P1 TGase was estimated as 103 kDa by Toyopearl HW55S gel filtration and as 100 kDa by the TSK-gel filtration. SDS-PAGE analysis showed that it consisted of heterogeneous 86- and 76-kDa proteins. However, these proteins closely resembled each other in amino acid composition, which was characterized by high content of Thr, Gly, and Pro residues as compared with P2 TGase. In contrast, the P2 TGase was isolated as a homogeneous 76-kDa protein and characterized by high content of Glx (Glu/Gln) and His residues. Neither of the chorion TGases of rainbow trout, P1 and P2, was similar to the liver-type TGase of red sea bream or the tissue-type TGase of chum salmon in amino acid composition. Examination of susceptibility to various inhibitors, reactivation by CaCl2, pH dependency, and activity of the polymerization of chorion proteins suggested that the P1 and P2 TGases were essentially similar to each other in enzymatic properties.

Identification of a point mutation associated with a silent phenotype of human serum butyrylcholinesterase--a case of familial cholinesterasemia.

A point mutation which caused a silent phenotype of human serum butyrylcholinesterase (BChE) was identified in the DNA of a 47-year-old Japanese woman who visited our hospital complaining of hypertension. The propositus exhibited an unusually low level of BChE activity, whereas her younger sister and her daughter had intermediate levels of BChE activity and her elder sister a normal level. Immunologically, the amount of BChE protein in the serum of the propositus was normal. DNA sequence analysis of the propositus identified a point mutation at codon 199 (GCA --> GTA), resulting in a Ala --> Val substitution. This alteration is one downstream codon from the catalytic active site (Ser, 198). A family study showed her younger sister and her daughter to have the same mutation.

The third egg envelope subunit in fish: cDNA cloning and analysis, and gene expression.

The inner layer of the egg envelope of a teleost fish, the medaka, Oryzias latipes, consists of two major subunit groups, ZI-1,2 and ZI-3. On SDS-PAGE, the ZI-1,2 group presents three glycoprotein bands that were considered to be composed of a common polypeptide moiety derived from their precursor, choriogenin H (Chg H). ZI-3 is a single glycoprotein derived from the precursor, choriogenin L (Chg L). In the present study, a fraction of a novel subunit protein was found in the V8 protease digest of ZI-1.2 that was partially purified from oocyte envelopes. This protein fraction was not present in the purified precursor, Chg H. By RT-PCR employing the primers based on the amino acid sequence of this fraction, a cDNA for the novel subunit was amplified, and a full-length clone of the cDNA was obtained by screening a cDNA library constructed from the spawning female liver. The clone consisted of 2025 b.p. and contained an open reading frame encoding the novel protein of 634 amino acids. This protein included Pro-X-Y repeat sequences in two-fifths of the whole length from its N-terminus. Northern blot analysis revealed that the gene expression for this protein occurred in the liver but not in the ovary of spawning female fish. This protein is considered as the third major subunit of the inner layer of the egg envelope of medaka.

Nonsense mutation in exon 2 of the butyrylcholinesterase gene: a case of familial cholinesterasemia.

A point mutation that causes a silent phenotype for human serum butyrylcholinesterase (BChE) was proved by DNA analyses of a 64-year-old Japanese female who visited the hospital because of a common cold. The propositus and her two siblings showed extremely low BChE activity, but other family members (six individuals) manifested from intermediate to normal values of BChE activity. An immunological method revealed that the propositus and her two siblings showed absence of the BChE protein in serum. DNA sequence analysis of the propositus identified a point mutation at codon 400 (TGC-->TGA), resulting in the production of a stop codon. This alteration exists upstream of the Cys571 of the subunit, which forms a disulfide bridge with the Cys571 of another partner subunit.

Species-dependent migration of fish hatching gland cells that express astacin-like proteases in common [corrected].

Two constituent proteases of the hatching enzyme of the medaka (Oryzias latipes), choriolysin H (HCE) and choriolysin L (LCE), belong to the astacin protease family. Astacin family proteases have a consensus amino acid sequence of HExxHxxGFxHExxRxDR motif in their active site region. In addition, HCE and LCE have a consensus sequence, SIMHYGR, in the downstream of the active site. Oligonucleotide primers were constructed that corresponded to the above-mentioned amino acid sequences and polymerase chain reactions were performed in zebrafish (Brachydanio rerio) and masu salmon (Oncorynchus masou) embryos. Using the amplified fragments as probes, two full-length cDNA were isolated from each cDNA library of the zebrafish and the masu salmon. The predicted amino acid sequences of the cDNA were similar to that of the medaka enzymes, more similar to HCE than to LCE, and it was conjectured that hatching enzymes of zebrafish and masu salmon also belonged to the astacin protease family. The final location of hatching gland cells in the three fish species: medaka, zebrafish and masu salmon, is different. The hatching gland cells of medaka are finally located in the epithelium of the pharyngeal cavity, those of zebrafish are in the epidermis of the yolk sac, and those of masu salmon are both in the epithelium of the pharyngeal cavity and the lateral epidermis of the head. However, in the present study, it was found that the hatching gland cells of zebrafish and masu salmon originated from the anterior end of the hypoblast, the Polster, as did those of medaka by in situ hybridization. It was clarified, therefore, that such difference in the final location of hatching gland cells among these species resulted from the difference in the migratory route of the hatching gland cells after the Polster region.

Cloning of cDNA and estrogen-induced hepatic gene expression for choriogenin H, a precursor protein of the fish egg envelope (chorion).

A cDNA for choriogenin H (Chg H; formerly high-molecular weight spawning female-specific substances, or H-SF), a precursor protein of the inner layer subunits of egg envelope (chorion) of the teleost fish, Oryzias latipes, was cloned and analyzed. The clone consisted of 1913 bp and contained an open reading frame encoding a signal peptide of 22 aa and Chg H protein of 569 aa. The Chg protein possessed three potential N-glycosylation sites and Pro-X-Y repeat sequences in the first two-fifths of the N terminus. There were amino acid sequence similarities between Chg H and a gene product expressed in the liver of female winter flounder during vitellogenesis. Moreover, the amino acid sequence of Chg H is similar to that of ZP2 rather than ZP3 of zona pellucida of some mammals. Northern blot analysis indicated that gene expression for Chg H occurred only in the livers of spawning female fish and 17beta-estradiol-treated male fish, but not in the ovary of the spawning female fish. Gene expression for Chg H and Chg L (formerly low-molecular weight spawning female-specific substance, or L-SF) was induced and increased in parallel in the male fish liver after 17beta-estradiol treatment.

Purification and partial characterization of 76 kDa transglutaminase in the egg envelope (chorion) of rainbow trout, Oncorhynchus mykiss.

Transglutaminase (TGase), responsible for crosslinking between proteins, is known to be localized exclusively in the egg envelope (chorion) of rainbow trout, Oncorhynchus mykiss, and probably participates in the post-fertilization chorion hardening. We purified the TGase from unfertilized egg chorions by sequential chromatography using SP-Sepharose, Q-Sepharose, and TSK-gel G3000SWXL columns. The purified enzyme was a monomeric protein having the molecular mass of 76 kDa. It promoted incorporation of monodansyl-cadaverine into chorion protein and catalyzed the polymerization of chorion subunit proteins. The effect of various reagents suggested that the chorion TGase is a Ca2+-dependent SH-enzyme similar to the well-characterized TGases of various animals. The highest activity was observed at pH 6.0. The amines examined in the present study inhibited the TGase activity of the purified enzyme. However, they did not necessarily cause effective inhibition of its activity. These properties of the chorion TGase were essentially consistent with our previous observations on polymerization of chorion proteins, resulting in chorion hardening. We compared the amino acid composition of the purified TGase with those of the previously characterized TGases of fishes, such as chum salmon and red sea bream. The results suggest that the chorion 76 kDa TGase is not homologous with those liver TGases in terms of amino acid composition.

Genetic analysis of a Japanese patient with butyrylcholinesterase deficiency.

A patient (64-year-old, male) with familial cholinesterasemia caused by BChE deficiency was studied. DNA sequence analysis of all exons identified a point mutation, an A-->G transition at codon 128, resulting in a Tyr-->Cys substitution. The propositus showed extremely low BChE activity, but his other family members (three individuals) showed from intermediate to normal BChE activity. An immunological method revealed the absence of BChE protein in serum of the propositus. Both PCR primer introduced restriction analysis (PCR-PIRA) and sequence analysis revealed all three family members to be heterozygotes for this mutation.

Evaluation of solubilizing methods of the egg envelope of the fish, Oryzias latipes, and partial determination of amino acid sequence of its subunit protein, ZI-3.

The inner layer of most teleostean egg envelopes, especially those after hardening, is almost insoluble in ordinary solvent, and therefore the inner layer of only the unhardened egg envelope has been subjected to solubilization with some potent solvents. We comparatively evaluated the methods of solubilization of the inner layer of egg envelope of medaka, Oryzias latipes, with SDS, urea and guanidium chloride (GuHCI). Analysis of the solubilized samples by SDS-polyacrylamide gel electrophoresis, comparison of their amino acid compositions or peptide maps using high-performance liquid chromatography and partial determination of their amino acid sequences showed that SDS and GuHCI were appropriate for solubilization and characterization of the envelope. Urea solubilization resulted in some artificial modifications of lysine and/or cysteine residues of envelope proteins. Partial determination of amino acid sequence of a subunit, ZI-3, isolated from the SDS-or GuHCI-solubilized envelope strongly suggested the identity of the envelope subunit, ZI-3, and its precursor, L-SF.

Different exon-intron organizations of the genes for two astacin-like proteases, high choriolytic enzyme (choriolysin H) and low choriolytic enzyme (choriolysin L), the constituents of the fish hatching enzyme.

The hatching enzyme of the teleost, Oryzias latipes, is composed of two proteases, high choriolytic enzyme (choriolysin H, HCE) and low choriolytic enzyme (choriolysin L, LCE), which are similar in some enzymological characteristics and protein structure (55% identity in amino acid sequence) and belong to the astacin family. Two isoforms of HCE are detected. In the present study, the genes for HCE and LCE were isolated from the genomic library constructed from DNA of the inbred drR strain fish. In contrast to the close similarity of the enzymes, there was a marked difference in their gene organization. The LCE gene was a single copy gene and composed of eight exons interrupted by seven introns. The HCE genes were multicopy genes and lacked introns. In the haploid genome of the drR strain fish, there are eight HCE genes, seven of which were cloned. Each HCE gene was identified as that for either of the two isoforms of HCE. 5' flanking regions of the LCE gene and the HCE genes had consensus TATA box sequences, but not CAT box nor GC box sequences. The big difference in the exon-intron organization between the HCE genes and the LCE gene is discussed from an evolutionary viewpoint.

Temporal and spatial patterns of gene expression for the hatching enzyme in the teleost embryo, Oryzias latipes.

The hatching enzyme of the medaka, Oryzias latipes, consists of two proteases, high choriolytic enzyme (HCE) and low choriolytic enzyme (LCE). They are synthesized and accumulated in the same unicellular hatching glands and are secreted from them at the end of embryonic development to digest the egg envelope. Recently, these enzymes were purified, and their cDNA clones were isolated. In the present study, we examined temporal and spatial patterns of expression of the hatching enzyme genes during embryogenesis using cDNAs for HCE and LCE as probes. According to Northern blotting analysis, the expression of both genes started at the same time (stage 21-22 embryos: brain differentiation and lens formation) and the patterns of expression changed in parallel during development. In situ hybridization to whole embryo and the sections revealed that the expression of the HCE genes was detected first in the anterior end of the hypoblast layer in stage 16-17 (late gastrula) embryos. Distinct signals of the HCE gene expression were then detected in a group of cells located at the front of the head rudiment of embryos at stage 18-19 (1 somite). Treatment of the embryos with retinoic acid, which is known to affect the anterior differentiation of embryos, suppressed the hatching gland cell differentiation in accordance with the result of in situ hybridization. In stage 22 embryos, the HCE-positive cells dispersed in an ectodermal layer under the forebrain and optic vesicles. Thereafter, the hatching gland cells expressing the HCE mRNA were aligned along the branchial arches and finally rearranged to the inner wall of the pharyngeal cavity, following a marked elongation of the lower jaw. The results of in situ hybridization to whole embryos at consecutive developmental stages demonstrated that the hatching gland cells located at the most anterior portion of the hypoblast migrated posteriorward to endoderm (pharyngeal endoderm) by way of ectoderm, while they were expressing mRNA for the hatching enzyme. Retinoic acid treatment of embryos gave rise to aberrations in the final location of the hatching gland cells probably by disturbing their migration. Moreover, the number of hatching gland cells increased markedly during their migration. This fact strongly suggested a concurrence of gene expression and mitosis of a gland cell and/or a successive initiation of gene expression in maturing gland cells during migration.

[Determination of gene mutation of silent serum cholinesterase and its epidemiologic characters in the Japanese].

We detected 121 individuals with silent type of serum cholinesterase from 36 families in Japan. DNA analysis totaling 37 members of eleven blood unrelated families were carried out by four useful methods, namely, 1) PCR-SSCP analysis, 2) dot blot hybridization (DBH) with the use of synthetic oligonucleotide probe, 3) restriction endonuclease analysis (REA) and 4) direct sequencing analysis. Their mutations were classified into four groups, namely, 1) a G-->C transversion at codon 365, 2) a frameshift mutation with insertion of an extra A at codon 315, 3) an A-->G transition at codon 128 and 4) a C-->A transition at codon 400. The three procedures including (PCR-SSCP, DBH, REA) without the use of radio labeled materials (non-RI) are recommendable for the analyses. However, the direct sequencing analysis of bases with RI might be, at present, necessary for the final identification.

Cloning of cDNAs for the precursor protein of a low-molecular-weight subunit of the inner layer of the egg envelope (chorion) of the fish Oryzias latipes.

cDNA clones for L-SF, the precursor of a low-molecular-weight subunit (ZI-3) of the inner layer of the Oryzias latipes egg envelope were isolated from Lambda ZAP cDNA libraries constructed from the poly(A)+ RNA of the liver of spawning female fish and estrogen-treated male fish. Among them, a clone, L-SF41, is 1473 bp long and contains an open reading frame encoding a signal peptide of 19 amino acids and L-SF protein of 420 amino acids. L-SF protein seems to be glycosylated, judging from the result of the glycanase digestion. L-SF protein contains a domain similar to ZP-domains in ZP3 of some mammalian species. Northern blot analysis employing XhoI-SmaI fragments of the cloned cDNA as probes revealed that expression of the L-SF gene occurred exclusively in the livers of spawning female fish and estrogen-treated male fish and that there was no mRNA encoding L-SF in the ovary of the spawning female fish.