Origin of Nicotiana tabacum detected by primary structure of fraction I protein.

Nicotiana tabacum is believed to have arisen after hybridization of Nicotiana sylvestris with a species in the Tomentosae section of the genus Nicotiana. Recent biochemical experiments have confirmed the conclusions from previous cytogenetic studies that N. sylvestris was the maternal parent and have indicated that Nicotiana tomentosiformis was the paternal parent. However, these studies did not take into account the possibility that a new species of Nicotiana, called K-12, discovered in South America in 1968, could also have been one of the parents. Fraction I proteins were purified from N. tabacum and its putative progenitors, and separated into large and small subunits. Chymotryptic peptides of each subunit were analyzed by ion exchange column chromatography with a gradient elution system. Among 38 resolved peaks of the large subunits, 2 peaks were found to be different among the putative species. Since only N. sylvestris showed an identical chromatogram with N. tabacum, N. sylvestris was concluded to be the maternal progenitor, as the genetic information for the large subunit of Fraction I protein was known to be inherited by the cytoplasmic mode. On the other hand, the small subunit of Fraction I protein is inherited by the Mendelian mode and therefore N. tabacum, an allopolyploid, could be expected to contain two types of small subunits, one derived from N. sylvestris and the other from a paternal progenitor. N. sylvestris lacks two of the 25 chymotryptic peptides of the small subunit of N. tabacum. Among 3 putative paternal progenitors, these two peaks appeared only in N. tomentosiformis, but not in Nicotiana otophora or K-12. Thus, N. tomentosiformis was concluded to be a paternal progenitor of N. tabacum. The conclusion was verified by comparing chymotryptic peptides of small subunits from three amphidiploids of N. sylvestris crossed with N. tomentosiformis, N. sylvestris crossed with N. otophora snd N. sylvestris crossed with K-12. The analytical results showed that only the progeny of N. sylvestris crossed with N. tomentosiformis contained the same small subunits as N. tabacium.     

Genetics of neurofibromatosis 1 in Japan: mutation rate and paternal age effect

Summary We have performed formal genetic studies on 26 patients (14 males, 12 females) with neurofibromatosis 1 (von Recklinghausen's disease, NF1) in Japan. Family studies of 74 members of 18 kindreds revealed that 50% of the cases were caused by a new mutation; the mutation rate was assumed to be 7.3–10.5 × 10^-5. A tendency of paternal age effect, which was not accounted for by the maternal age effect, was observed, but live-birth order had no significant effect. Genetic linkage of neurofibromatosis 1 to the NF1 gene or the genetic marker in the pericentric region of chromosome 17 was established in 3 informative families.     

Possible involvement of clathrin in neuritogenesis induced by a protease inhibitor (benzyloxycarbonyl-Leu-Leu-Leu-aldehyde) in PC12 cells.

Our previous reports showed that benzyloxycarbonyl (Z)-Leu-Leu-Leu-al (ZLLLal) induces neurite outgrowth in PC12 cells, and that 33-, 35-, and 180-kDa proteins from PC12 cells elute specifically from a Leu-Leu-Leu-al (LLLal)-coupled affinity column. Several lines of evidence suggest that the 33-, 35-, and 180-kDa proteins are components of clathrin, well-known for its role in endocytosis. Separation of clathrin into its heavy and light chains showed that the clathrin heavy chains have the ability to bind to a LLLal affinity column directly. Furthermore, ZLLLal enhances the rate of polymerization of clathrin triskelion to the coat structure. ZLLL-COOH does not cause neurite outgrowth in PC12 cells, and has no effect on the rate of clathrin polymerization. On immunocytochemical analysis of PC12 cells with an anti-clathrin heavy chain antibody, enhanced staining of the clathrin heavy chain was observed concomitantly with neurite outgrowth initiated by ZLLLal, but not by NGF. This study provides new insights into both the role of the clathrin molecule and the regulatory mechanism of neurite outgrowth.     

Detection of pre-mRNA splicing in vitro by an RNA-templated fluorogenic reaction

RNA splicing is an important target for basic research of disease mechanisms and for drug discovery. Here, we report a new method for analysis of the in vitro RNA splicing process that produces fluorescence using a reduction-triggered fluorescence (RETF) probe. The fluorescence signal is produced only when the two probes bind side-by-side with a specific RNA target. Precursor messenger RNA and mature messenger RNA originating from the chicken δ-crystallin (CDC) gene were successfully discriminated in solution using an RETF probe with the assistance of helper oligonucleotide strands. Also, we successfully applied RETF probes to the detection of emerging mature mRNA in an in vitro splicing process.     

Tissue and plasma globotriaosylsphingosine could be a biomarker for assessing enzyme replacement therapy for Fabry disease

Fabry disease is a genetic disease caused by a deficiency of α-galactosidase A (GLA), which leads to systemic accumulation of glycolipids, predominantly globotriaosylceramide (Gb3). With the introduction and spread of enzyme replacement therapy (ERT) with recombinant GLAs for this disease, a useful biomarker for assessing the response to ERT is strongly required. We measured the tissue level of lyso-globotriaosylsphingosine (lyso-Gb3) in Fabry mice by means of high performance liquid chromatography, and compared it with the Gb3 level. The results revealed a marked increase in the lyso-Gb3 level in most tissues of Fabry mice, and which decreased after the administration of a recombinant GLA as in the case of Gb3, which is usually used as a biomarker of Fabry disease. The response was more impressive for lyso-Gb3 compared with for Gb3, especially in kidney tissues, in which a defect significantly influences the morbidity and mortality in patients with this disease. The plasma level of lyso-Gb3 also decreased after the injection of the enzyme, and it was well related to the degradation of tissue lyso-Gb3. Thus, lyso-Gb3 is expected to be a useful new biomarker for assessing the response to ERT for Fabry disease.     

Deoxyribonucleoproteins of herring sperm nuclei. I. Chemical composition

The chemical composition of deoxyribonucleoproteins from herring sperm nuclei was analyzed and the results are summarized as follows: 1. Chemical analysis of nuclear proteins and nucleic acids revealed that arginine/P molar ratio in herring sperm nuclei is unity but the ratio of arginine residues in protamine to phosphorus in the total DNA is 0.86. 2. The deoxyribonucleoproteins were isolated and their composition showed that about 14% of the total DNA in herring sperm nuclei is free from protamine and is bound with nonprotamine proteins in the weight ratio of nonprotamine proteins to DNA of 0.25-0.30. The remaining 86% of the total DNA is combined mainly with protamine and a small amount of nonprotamine proteins; the weight ratios of protamine and nonprotamine proteins to DNA are 0.75 and 0.08, respectively. In the latter complex, the molar ratio of arginine residues in protamine to phosphorus in DNA is unity.     

Tissue distribution of an endogenous inhibitor of calcium-activated neutral protease and age-related changes in its activity in rats

1. The distribution of an endogenous inhibitor of calcium-activated neutral protease (CANP) and age-related changes in its activity were studied in male and female rats of different ages by a fluorometric assay on tissue extracts after heat treatment. 2. Ubiquitous distribution of CANP inhibitor in brain, cardiac muscle, lung, spleen, liver, skeletal muscle, kidney and testis and its abundance in spleen, liver and kidney was demonstrated. 3. Comparison in terms of units/ml of crude extracts showed that the level of CANP inhibitor exceeded that of CANP in most tissues and that the relative content of CANP inhibitor to mCANP and microCANP differed greatly among tissues. 4. Sex and species differences in CANP inhibitor activity in each tissue were of little significance. 5. Changes in CANP inhibitor during aging from 6 to 12 months was not obvious but senescent rats showed a tendency toward increased inhibitor activity. This increase was especially evident in the testis.     

Coordinated control of a designed trans-acting ligase ribozyme by a loop-receptor interaction

We previously developed a synthetic cis-acting RNA ligase ribozyme with 3′-5′ joining activity termed “DSL” (designed and selected ligase). DSL was easily transformed into a trans-acting form because of its highly modular architecture. In this study, we investigated the modular properties and turnover capabilities of a trans-acting DSL, tDSL-1/GUAA. tDSL-1/GUAA exhibited remarkably high activity compared with the parental cis-acting DSL, and it attained a high turnover number. Taken together, the results indicate that a loop-receptor interaction plays a significant role in determining the activity of the trans-acting ribozyme and in its ability to perform multiple turnovers of the reaction.     

Changes in Total and Polar Lipids and Their Fatty Acid Composition in Tobacco Leaves during Growth and Senescence

Total lipids, total fatty acids and most polar lipids of tobaccoleaves increased and decreased almost concomitantly with changesin chlorophyll during leaf development and senescence. In individualpolar lipids, marked changes were observed in compounds associatedwith chloroplast membranes, i.e., monogalactosyldiglyceride(MGDG), digalactosyldiglyceride and sulfoquinovosyldiglyceride.Phosphatidylglycerol (PG) was the first to decrease during leafdvelopment. Green leaves contained a considerable amount ofhexadecatrienoic acid (16: 3) in MGDG, which suggests that tobaccobelongs to 16: 3- plants. The proportion of linolenic acid infour chloroplast lipids was lower in senescent leaves than ingreen leaves. Similar phenomena were also observed in 16: 3of MGDG and in hexadecenoic acid of PG. (Received April 27, 1981; Accepted July 11, 1981)