Heterogeneity of mRNA coding for Caenorhabditis elegans coronin-like protein.

We analyzed transcripts coding for the nematode Caenorhabditis elegans coronin, which had been identified by the genome project of C. elegans. We found that the gene coding for the C. elegans coronin has an alternatively spliced exon containing an alternative 5' splice site in the 3'-region. Moreover, two exons are internally cleaved by a mechanism different from the conventional splicing rules. In consequence, the gene produces five kinds of transcripts.

Cloning and sequencing of the gene for a Tetrahymena fimbrin-like protein.

Tetrahymena F-actin-binding protein, which induces bundling of Tetrahymena F-actin, was localized to a division furrow during cytokinesis. We report here the cloning and characterization of the gene and cDNA of a Tetrahymena F-actin-binding protein. The cDNA encodes a protein comprising 579 deduced amino acids with a calculated molecular mass of 65.9 kDa. The predicted amino acid sequence shares 37.7, 41.8, and 39% identity with the sequences of yeast fimbrin, Arabidopsis thaliana fimbrin, and Dictyostelium discoideum plastin, respectively. The Tetrahymena F-actin-binding protein also shares two actin-binding domains previously identified in the fimbrin/plastin family, but lacks the EF-hand Ca2+-binding motif, suggesting that this protein is a novel-fimbrin-like protein in Tetrahymena. Moreover, we cloned a genomic DNA encoding the Tetrahymena fimbrin-like protein and performed Southern and Northern hybridizations. The results indicate that the genomic DNA possesses 9 introns and that both the gene and transcript of Tetrahymena fimbrin-like protein are single. Thus, we suggest that Tetrahymena fimbrin-like protein localizes to the division furrow and probably cross-links actin filaments in a Ca(2+)-insensitive manner during cytokinesis.

Heterogeneity of chicken slow skeletal muscle troponin T mRNA.

The troponin T (TnT) transcripts in chicken slow skeletal muscle were characterized by S1 nuclease mapping and nucleotide sequencing of cDNA produced by RT-PCR and 5'-RACE. We found two kinds of transcripts in the 5'-region, one having the codon for alanine (position 135-137), C (258), and A (262) and the other lacking the codon and having T (258) and G (262) instead of C and A. In the 3'-region, we found four single base substitutions at 703 (T or C), 774 or T), 797 or T), and 827 (G or A). Four of the six substitutions lead to amino acid changes in chicken sTnT isoforms. We determined the genomic structure of the 3'-region of the chicken sTnT gene. The region includes 7 exons corresponding to position 249-891 of the chicken sTnT cDNA and no alternative exon, showing that the 3'-heterogeneity in sTnT transcripts was due to allelic variation. J. Exp. Zool. 286:149-156, 2000.

Tissue-specific distribution of breast-muscle-type and leg-muscle-type troponin T isoforms in birds.

In order to show the tissue-specific distribution of troponin T (TnT) isoforms in avian skeletal muscles, their expression was examined by electrophoresis of the breast and leg muscles of seven avian species and immunoblotting with the antiserum against fast skeletal muscle TnT. It has been reported in the chicken that breast-muscle-type (B-type) and leg-muscle-type (L-type) TnT isoforms are expressed specifically in the adult breast and leg muscles, respectively. Their differential expression patterns were confirmed in all birds examined in this study. The expression of a segment encoded by the exon x series of TnT was also examined by immunoblotting with the antiserum against a synthetic peptide derived from the exon x3 sequence, because the segment has been shown to be included exclusively in the B-type, but not in the L-type TnT. The expression of the segment was found only in the breast muscle, but not in the leg muscle of all birds examined. TnT cDNA sequences from the duck breast and leg muscles were determined and showed that only B-type TnT had an exon x-related sequence, suggesting that the expression of B-type TnT containing the exon x-derived segment is conserved consistently in the birds.

The structure of the avian fast skeletal muscle troponin T gene: seven novel tandem-arranged exons in the exon x region.

To elucidate the mechanism that produces enormous molecular diversity in troponin T (TnT) of fast skeletal muscle, we determined the 5'-half genomic sequence of the chicken fast muscle TnT gene. The sequence of ca. 16 kb included seven exons (exons 1, 2, 3, 4, w, 5, and 6), which have been reported previously and presumed by sequencing TnT cDNAs. Additionally we found six 15 nt and one 18 nt sequences in the region between exons 5 and 6 (i.e. the exon x region). They were encompassed by consensus splice donor and acceptor sites and preceded by putative branch sites, and designated herein as exons xa to xg. Our result shows that the sequence derived from exons x1, x2, and x3, the exons presumed previously by cDNA sequencing, is actually encoded by the seven exons xa to xg, establishing the precise gene structure in the exon x region. Based on our data, together with that on the 3'-half genomic sequence of the quail fast muscle TnT gene, we conclude that the avian fast skeletal muscle TnT gene includes 27 exons, 16 of which are alternatively spliced.

A sudden death from the rupture of a small intestine firstly suspected of acute pancreatitis.

A 51-year-old drunken male was carried to a hospital with acute abdominal pain and was suspected of acute pancreatitis. The patient was treated with fasting, electrolyte transfusion, and anodyne, but took a sudden turn for the worse and died in 16 hours. In the judicial autopsy, rupture of a small intestine was detected. As the police investigated, he had been kicked in the abdomen by an assailant before coming to the hospital. The cause of death was diagnosed to be acute peritonitis due to the rupture of a small intestine. Several problems were pointed out on medical examinations and treatments of this case.

Regulation of troponin T gene expression in chicken fast skeletal muscle: involvement of an M-CAT-like element distinct from the standard M-CAT.

Troponin T (TnT), like other myofibrillar proteins, is expressed as various isoforms in different muscle fibers and/or at different development stages. A recent study suggested the expression pattern of chicken fast TnT isoforms is fixed in a given cell lineage. In the present study, we isolated genomic clones of the chicken fast TnT gene to carry out molecular analysis of its expression mechanism. One of the clones, pWETNTa, contained the 5' upstream region and approximately 20 kb downstream from exon 1. We constructed promoter/upstream segments of the chicken fast TnT gene linked to the bacterial chloramphenicol acetyltransferase (CAT) gene and tested the regulatory function of the 5' upstream region by transient transfection of the gene constructs into muscle cells. We showed that a DNA segment between -264 and -44 bp from the most 5' transcriptional initiation site, which has an MEF2, and M-CAT-like element, a CArG box and two E boxes, was essential for the expression of the fast TnT gene. Furthermore, mutation of the M-CAT-like element in the segment resulted in the most serious reduction in the fast TnT promoter activity. The results suggested that the M-CAT-like element plays an important role in transcriptional regulation of the fast TnT gene. The M-CAT-like element is very similar to the M-CAT element, but in electrophoretic mobility shift assay, the factor(s) that bound to this motif was found to be different from the M-CAT binding factor (MCBF).

Cloning of chicken slow muscle troponin T and its sequence comparison with that of human.

A full-length cDNA coding for chicken slow muscle troponin T (TnT) was for the first time isolated from a cDNA library of 10-day-old embryos, using an RT-PCR product of chicken slow muscle TnT. It showed about 60% homology for chicken fast and slow muscle TnTs and 75.2% for human slow muscle TnT. The 16 amino acid sequence found in the carboxyl terminus of human slow muscle TnT was absent in the chicken slow muscle TnT. The 5E-5A-7E sequence found in the amino terminal region of chicken slow muscle TnT was partly similar to the counterpart of human slow muscle TnT, but not to those of chicken fast and cardiac muscle TnTs. With this report of chicken slow muscle TnT, cDNA information on chicken TnTs of all three striated muscles was completed following those of human TnTs.

Sudden death due presumably to internal use of methamphetamine.

A 26-year-old male was found naked and excited in the backyard of his neighbor's house. He was carried to a nearby hospital, and returned home with his family, but took a sudden turn for the worse and died. In a judicial autopsy, the ethanol concentration of blood was found to be 0.58 milligrams, and methamphetamine (MA) was detected in his blood by thin-layer chromatography. The concentration of MA in his blood was 4.38 mumol/dl, higher than the fatal level. The amount of MA in his stomach was 5.8 mg (34.58 mumol/100 g), indicating that he ingested MA by internal use. Among the autopsy cases of acute MA poisoning reported in Japan, hyperesthesia is known to last 1-3 h before death, whether the administration is by intravenous injection or orally. But the present case is quite unusual, as the death followed 6 h or more of hyperesthesia. This was attributed to the patient's combined intake of alcohol with MA, as it is known to decrease the mortality in mice.

A difference in the proteins found in young adults of inbred strains of Drosophila melanogaster which correlates with genetically-determined, long or short life span.

One-dimensional electrophoresis was performed on extracts of flies collected from across all ages. Protein gel patterns were compared for two strains of Drosophila melanogaster with distinctly long and short adult life spans that result from different alleles of longevity genes. An inter-strain difference was observed in the changes in protein pattern in the 77 kDa region in period of day 0-5 after emerging. We propose that the protein involved is a product of autosomal longevity alleles A1 and A2 at the Jm A locus and is related to development of longevity potentials in the preimaginal stage.

Purification and characterization of a protein associated with genetically-determined longevity difference in Drosophila melanogaster.

Earlier studies have shown a correlation between the presence of a 77 kDa protein in the proteins extracted from young adult Drosophila melanogaster (D.m.) and the autosomal longevity allele. A2 at the JmA locus. In this study, a 77 kDa protein has been isolated from pupae of D.m. of a long-lived strain of genotype A2A2, and was purified by DEAE chromatography, ConA column chromatography, and two cycles of gel filtration. The purified protein has a molecular weight of 76,600 (by SDS-PAGE), an isoelectric point of pH 6.5, and molar extinction coefficient A(280(1%) = 18.3. It is a glycoprotein containing 3.3% hexose. Supplementing the food of D.m. with the purified protein at 5 x 10(-4) micrograms/ml, beginning at day 5 after emergence, resulted in an increase in the survival rate and maximal life span of both short-lived and long-lived strains of D.m.

Cytoplasmic influence on the expression of nuclear genes affecting life span in Drosophila melanogaster.

In earlier studies we have found that the difference between short and long life spans of two inbred strains of Drosophila melanogaster is controlled by nuclear major genes. The present study has revealed a cytoplasmic factor that influences the expression of the nuclear longevity genes. The factor shows a typical maternal inheritance and is considered to be an extranuclear gene, such as mitochondrial DNA (chondriome). This paper marks the discovery of two basic forms of inheritance, nuclear and extra-nuclear, in the genetics of life span of D. melanogaster. These findings suggest that further studies, including genetic engineering, on longevity and aging might enable direct manipulation of these characters.

Relationship between genotypes of longevity genes and developmental speed in Drosophila melanogaster.

Hatching time (the period between egg-laying and hatching) and emerging time were surveyed and their relationship with the adult life span was investigated. A relationship between emerging time and adult life span was clearly evident: early emergers were often long-lived. This relation is considered to have a genetic basis because all the larvae in a group were bred in the same culture bottle. Thus, the longevity genes involved also appear to have control over the rate of development. No significant relation was observed between hatching time and adult life span or between hatching time and emerging time. These results suggest that the function of the longevity genes begins at the larval or pupal stage before emergence, and that adult life spans differentiate at this time.

A case of death due to dragging by a car: establishment of a homicide because of conscious negligence.

A drunken pedestrian (a 19-year-old male) lying on the street was hit by a car at night and dragged to a distance of about 600 meters. He was taken to a hospital soon but died there from brain injuries. His head and back were strongly and widely grazed against the road surface. Major parts of the left side of his skull and the brain were scraped off. The assailant thought that he had to escape from the place, because he was on probation because of established crime of drunken driving and drug abuse. As he continued to drive even after noticing that his car was dragging the victim, realizing that he might die as a consequence, a case of homicide due to conscious negligence was established.

Influence of temperature on the inheritance of adult life span in Drosophila melanogaster.

Mating experiments were performed at 27 degrees C, 22 degrees C, and 17 degrees C, to investigate the inheritance of adult life span of highly inbred strains of Drosophila melanogaster. Effects of temperature difference were quantitatively analyzed at the genotypic level. In the temperature range of 17-27 degrees C the autosomal longevity alleles, A1 and A2, exerted major effects. Their effects produced longer life spans as the temperature decreased. The sex-linked longevity alleles, X1 and X2, played a secondary role in influencing life span, and they displayed different effects at different temperatures. Each genotype showed correspondence to the life span expected from the combinations of these longevity genes at the respective temperatures. The genetic loci controlling life span in these inbred strains were named JmA and JmX from "Ju-myo" which means life span or longevity in Japanese.

Flow cytometric analysis of the Rh1 (Rho, D) antigen activity on red cells: various Rh blood group phenotypes including Du variants.

Rh1 (Rho, D) antigen activity has been analyzed by the use of the indirect immunofluorescence flow cytometry (FCM), and the Rh blood group genotypes were able to be successfully determined from the intensity of fluorescence detected in flow cytometry using the anti-D IgG that was fractionated in a Protein A Sepharose CL-4B column as the primary antibody. The relative amount of the fluorescein isothiocyanate (FITC) bound to the D (R1R1, CDe/CDe), the high grade Du (R2r',cDE/Cde), the low grade Du (K1r, CDue/cde), and the d (rr, cde/cde) red cells was estimated from the mean fluorescent intensity. The FITC-binding activity of the high grade Du and low grade Du was 83% and 21% that of D. The antigen-antibody complex density profile was analyzed by using the FITC-conjugated protein-A in place of the second antibody. Compared with the found results using anti-human globulin as the second antibody, this method was less sensitive but it still was able to demonstrate the different degrees of fluorescence according to the Rh genotypes. The present FCM method is both simple and useful for (1) measuring the relative amount of antigens, (2) for detecting the dosage effect and (3) for deferminins the blood group genotypes.

Mode of inheritance of major genes controlling life span differences between two inbred strains of Drosophila melanogaster.

Although life span is generally considered to be under polygenic control, we obtained experimental evidence of Mendelian genes exerting major effects on life span differences between two inbred strains of Drosophila melanogaster. Our data indicate two loci with major effects, one being autosomal and the other sex-linked. The alleles at the autosomal locus are designated A1 and A2, the latter producing longer life. Heterozygotes, A1A2, exhibit over-dominance. The alleles at the sex-linked locus, designated X1 and X2, produce life-extending effects. X1 revealed a dosage effect, causing homozygous females to live longer than hemizygous males; X2 showed no dosage effect. The identified genes are considered to control the period of activity of many genes maintaining life.

Detection of ABO blood group-active glycolipids extracted from red cell membrane and heat hematoma using TLC-immunostaining.

Glycolipids extracted from groups A, B, and O erythrocytes were developed on thin-layer plates; their ABO blood group antigenicities were detected by immunostaining method using avidin-biotin-complex (ABC). Among series of glycolipids of different flow rates, antigen-specific staining was observed in five bands from group A1 erythrocytes, four bands from group B, and two bands from group O. Monoclonal anti-A, -B, and -H antibodies specifically stained glycolipids from A1, B, and O erythrocytes, respectively. ABO blood grouping was possible from 5 g of epidural heat hematoma of a charred body by this method. ABC immunostaining on thin-layer chromatography is a useful and reliable method for ABO blood grouping in forensic practice.

A case of paternity testing influenced by the silent allele of Rh erythrocyte groups.

A paternity test is presented in which a father and his two children possessed an extremely rare amorphic gene R-29 (r,---). One of the children was determined to be illegitimate at the first trial as her Rh phenotype was R2R2(ccDEE) and the father's phenotype was R1R1(CCDee). At the Court of Appeal, however, the rare Rh gene r(---) was shown to be inherited from the father to the appellant child through extended tests including her brother whose phenotype was also R2R2(ccDEE). She was acknowledged to be legitimate.