Molecular cloning and characterization of a rice dehydroascorbate reductase.

Plant dehydroascorbate reductase (DHAR), which re-reduces oxidized ascorbate to maintain an appropriate level of ascorbate, is very important, but no gene or cDNA for plant DHAR has been cloned yet. Here, we describe a cDNA for a rice glutathione-dependent DHAR (designated DHAR1). A recombinant Dhar1p produced in Escherichia coli was functional. The expression sequence tag database suggests that Dhar1p homologs exist in various plants. Furthermore, the rice Dhar1p has a low similarity to rat DHAR, although the rice enzyme has a considerably higher specific activity than the mammalian one. The mRNA level of DHAR1, the protein level of Dhar1p and the DHAR activity in rice seedlings were elevated by high temperature, suggesting the protection role of DHAR at high temperature.

Development of sleep spindles in young children and adolescents.

The development of sleep spindles was studied quantitatively in 32 healthy subjects between the ages of 4 and 24 years. The peak frequency distribution of the spindles showed a bimodal pattern with 11.0 to 12.75 Hz in the frontal area and 12.5 to 14.5 Hz in the centroparietal area. The two types of spindle activity showed different courses of maturation. The peak frequency of the centroparietal spindles gradually increased linearly with age, whereas the frontal spindles abruptly increased in frequency during early adolescence. Regarding the power spectra, while centroparietal spindles showed little change in power from 4 to 24 years of age, frontal spindles decreased remarkably in power and became stable at about 13 years of age. The two types of spindles and the difference in their development may suggest the existence of different generators or a topographical difference during maturation in the thalamocortical network. The frontal spindle activity could be a good indicator to evaluate CNS maturation in young children and adolescents.

Molecular cloning and characterization of a cDNA for an iron-superoxide dismutase in rice (Oryza sativa L.).

We have isolated a cDNA encoding Fe-SOD from rice (Oryza sativa L.). The deduced amino acid sequence consists of a polypeptide with 255 amino acids, including a putative transit peptide (40 a.a.) in amino-terminal residues. This sequence is similar to the known plant Fe-SODs but not classified in the group of known Fe-SODs. The metal analysis and SOD assays of the partial purified recombinant protein expressed in E. coli showed that this cDNA encodes an iron-containing SOD. However this SOD activity was not inhibited by the treatment with hydrogen peroxide, which was expected to inhibit known Fe-SOD activity. mRNA of rice Fe-SOD was detected in all vegetative tissues examined, being especially abundant in calli, and strongly increased by light induction. These results suggested that this cDNA encodes rice Fe-SOD, which is apparently distinct from known plant Fe-SODs.

Molecular cloning and characterization of a cysteine-rich 16.6-kDa prolamin in rice seeds.

An alcohol-soluble storage protein, a 16.6-kDa prolamin found in rice seeds, was purified from both the total protein body and purified type I protein body fractions. The partial amino acid sequences of three tryptic peptides generated from the purified polypeptide were analyzed. A part of the 16.6-kDa prolamin cDNA was amplified from developing seed mRNA by the reverse transcribed polymerase chain reaction using an oligo (dT) primer and a primer which was synthesized based on the partial amino acid sequence. The amplified product was used to isolate the full-length cDNA clone (lambda RP16) from a developing seed cDNA library. The cDNA has an open reading frame encoding a hydrophobic polypeptide of 149 amino acids. The polypeptide was rich in glutamine (20.0%), cysteine (10.0%), and methionine (6.9%). The cysteine content was higher than those of most other rice storage proteins. Messenger RNA of the 16.6-kDa prolamin was detected in seeds, but not in other aerial tissues.

Differential gene expressions of rice superoxide dismutase isoforms to oxidative and environmental stresses.

Active oxygen species (AOSs) are produced under stress conditions of plant cells. Superoxide dismutase (SOD) catalyzes the first step in the AOS scavenging system. The responses of SOD genes to environmental stresses were analyzed in rice seedlings by the treatments of drought, salinity and chilling. The expressions of abscisic acid (ABA)-inducible genes, Mn-SOD gene (sodA1) and one of the cytosolic Cu/Zn-SOD genes (sodCc2), were strongly induced by the treatment of drought and salinity. While Fe-SOD gene (sodB) and the other cytosolic Cu/Zn-SOD gene (sodCc1) were also induced by ABA. However the mRNA level of sodB was decreased by drought treatment, and sodCc1 gene was not induced by drought and salinity treatments. Plastidic Cu/Zn-SOD gene (sodCp) quickly responded to salinity treatment in the light but not in the dark. In the treatment with hydrogen peroxide, sodCp gene was strongly induced shortly after the treatment. These results suggested that phytohormone and AOSs are associated with the regulation of SOD genes under environmental stresses.

[The influence of interferon therapy on CNS function--from the aspects of quantitative EEG and biogenic amines].

Various CNS side effects have been reported since interferon (IFN) was introduced for the treatment of chronic active hepatitis C (CAHC) patients. Most reports of EEG changes after IFN therapy were inspective, and there is no report of quantitative EEG analysis. In this study, changes in quantitative EEG and biogenic amines after IFN therapy were studied to enable identification of CNS side effects early in CAHC patients. Before and 7 days after IFN therapy, EEG records and plasma and urinary amines were examined in 36 CAHC patients (46.9 +/- 12.3 years, 29 men and 7 women) who were hospitalized for the IFN therapy. After IFN therapy, no notable change in biogenic amines was recognized. On EEG, 13 patients (39.4%) showed increased slow wave activities and 2 patients (6.1%) showed paroxysmal discharges after IFN therapy. On quantitative EEG, the patients showed significantly increased absolute power in slow alpha, theta and delta bands and decreased absolute power in fast beta band (paired-T test). After IFN therapy, 4 of 36 patients developed psychiatric disorders; 2 patients developed depressive symptoms and 2 other patients developed manic states. One depressive patient and one manic patient had 6 Hz spike and slow waves before IFN therapy. On quantitative EEG, the other manic patient had shown significantly increased absolute power in slow alpha and decreased power in fast alpha and beta bands, and the other depressive patient had shown significantly increased absolute power in fast theta band and decreased power in fast beta band before the development of the psychiatric disorders. These results suggest that the changes of quantitative EEG, between before and 7 days after IFN therapy, can be useful in assessing the risk for the development of psychiatric symptoms induced by IFN therapy. It also suggests that patients with slight EEG abnormality such as a 6 Hz spike and slow waves before IFN therapy need careful observation.

Gene cloning and expression of cytosolic glutathione reductase in rice (Oryza sativa L.).

We have isolated a cDNA (RGRC2) encoding glutathione reductase (GR) from rice (Oryza sativa L.). The comparison of deduced amino acid sequences from RGRC2 and other plant GR cDNAs indicated that RGRC2 encodes a putative cytosolic isoform. The recombinant RGRC2 protein had enzymatic properties comparable to those of GR from rice embryo. Subcellular fractionation showed that the RGRC2 protein is localized primarily in cytosol. mRNA and protein of RGRC2 were observed mainly in roots and calli but little in leaf tissues. Southern blot analysis showed that the RGRC2 gene exists as a single copy gene. Here, we have also isolated a genomic clone completely corresponding to RGRC2. The RGRC2 gene is split into 16 exons spread about 7.4 kb of chromosomal DNA, with coding sequence beginning in the 2nd exon and ending in the 16th exon. From the presence of two ABA-responsive elements in the 5'-flanking region of RGRC2, we examined the expression in rice seedlings treated with ABA and the ABA-related environmental stresses, chilling, drought and salinity. The expression of RGRC2 was strongly induced by all these treatments. We suggest that the expression of the rice cytosolic GR gene is regulated via ABA-mediated signal transduction pathway under environmental stresses.

Purification and characterization of glutaredoxin (thioltransferase) from rice (Oryza sativa L.).

We purified and characterized glutaredoxin (thioltransferase), which catalyzes thiol/disulfide exchange reaction, for the first time in plants. The purification procedure employed an immunoabsorbent, antiglutaredoxin-Sepharose. Glutaredoxin was purified about 2,200-fold from rice bran and it appeared to be homogeneous on SDS-PAGE. MALDI-TOF mass spectrometry revealed that the protein has a molecular mass of 11,097.9 Da. Rice glutaredoxin consists of 105 amino acid residues, containing the tetrapeptide -Cys-Phe-Pro (Tyr)-Cys-, which constitutes the active site of Escherichia coli and mammalian glutaredoxins. Inactivation assay also indicated that cysteine residues are responsible for enzyme activity. Kinetic analyses revealed that the enzyme did not exhibit normal Michaelis-Menten kinetics. The enzyme has an optimum pH of about 8.7 with 2-hydroxyethyl disulfide as a substrate. In addition, rice glutaredoxin has dehydroascorbate reductase activity, like mammalian glutaredoxin.

Structure of the rice glutaredoxin (thioltransferase) gene.

We have isolated the gene encoding a glutaredoxin in rice (Oryza sativa L.) and determined the nucleotide (nt) sequence of about a 4.2 kb long. The cloned gene (gRASC8) was found to contain four exons interrupted by three introns. The first exon begins the ATG translation start codon and the four exons code for a protein composed of 112 amino acids. The tetrapeptide -Cys-Pro-Phe-Cys- [-Cys-Pro-Phe(Tyr)-Cys-] which constitutes an active site of Escherichia coli and mammalian glutaredoxins, was conserved. The nt sequence contained consensus TATA and CAAT boxes, and two polyadenylation signals. Southern blot analysis of rice genomic DNA suggests that there are two copies of the glutaredoxin genes in rice.

Molecular cloning and characterization of a cDNA for plastidic copper/zinc-superoxide dismutase in rice (Oryza sativa L.).

A cDNA encoding plastidic Cu/Zn-SOD was isolated from rice. The deduced amino acid sequence included a transit peptide in the amino-terminal residues and showed a high similarity to those of plastidic Cu/Zn-SODs from other plants. The level of expression of this transcript was high in green leaves and differed from that of other rice SOD mRNAs in various tissues.

A case of isolated growth hormone (GH) deficiency with compound heterozygous abnormality at the GH-1 gene locus.

We report a Japanese boy with IGHD who is a compound heterozygote at the GH-1 gene locus. The patient and his mother were heterozygous for a 6.7 kb deletion of the GH-1 gene. A T-->C transition at position -123, an A-->G transition at position -6 and an A-->T transition at position -1 in the GH-1 promoter region and the addition of AGAA at base 250 in intron I were observed in one allele of the patient and his father. These results demonstrate that familial IGHD is a heterogeneous disease that perturbs different steps in the expression of the GH-1 gene.

[Developmental characteristics of frontal spindle and centro-parietal spindle].

The frequency and power of EEG sleep spindles during sleep stage 2 were analysed in 56 subjects between the age of 3 to 25 years in order to define the maturational pattern of two types of spindles. Although 24 of the 56 subjects were medicated with a single anticonvulsant, there was no remarkable difference in spindle maturation patterns between the subjects who received monotherapy and unmedicated subjects. The frontal spindles matured differently than centro-parietal spindles. The frequency of centro-parietal spindles developed gradually with age, whereas frontal spindles matured with a rather sudden increase of frequency during puberty. Although the power of centro-parietal spindles showed little changes with age, the power of frontal spindles declined remarkably during the first decade and settled down to a constant level after puberty. The developmental characteristics of these two kinds of spindles may reflect a difference in synchronicity of the thalamo-cortical pathway maturation including the inhibitory system. Both centro-parietal spindles and frontal spindles tended to develop slightly earlier in females than in males. The lag of spindle maturation in males might implicate developmental procedures in females. Separate observation of these two types of spindles is necessary for better understanding of sleep spindles as an indicator of central nervous system development.

Cloning of the rice seed alpha-globulin-encoding gene: sequence similarity of the 5'-flanking region to those of the genes encoding wheat high-molecular-weight glutenin and barley D hordein.

A genomic clone encoding the rice endosperm major globulin (alpha-globulin) with an apparent molecular mass of 26 kDa was isolated, and its nucleotide (nt) sequence and transcription start point (tsp) were determined. The tsp was identical to that of the gene encoding the wheat high-molecular-weight (HMW) glutenin subunit. The consensus '-300 element' and an A + T-rich sequence exist upstream from the TATA box in the 5'-flanking region. A nt sequence of about 130 bp in the 5'-flanking region was found to be markedly homologous to those of the genes encoding the wheat HMW glutenin subunit and barley D hordein.

Cloning and sequencing of a rice gene encoding the 13-kDa prolamin polypeptide.

A gene encoding the 13-kDa prolamin polypeptide was isolated from a rice genomic library (lambdaEMBL3) and the nucleotide sequence of an about 3-kbp EcoRI fragment was analyzed. The cloned gene (NRP33) codes for a protein composed of 156 amino acids, including a signal peptide of 19 amino acid residues and no intron is present in the genomic clone. The nucleotide sequence contains consensus TATA and CAAT boxes, and two polyadenylation signals. In addition, there are two conserved sequences named the -- 300 element and 10 consecutive repeats of the trinucleotide ATT in the 5' noncoding sequence.

Cloning and sequence analysis of a cDNA encoding rice glutaredoxin.

A full-length cDNA clone (RASC8) encoding glutaredoxin (thioltransferase) was isolated from a cDNA library of an aleurone layer prepared from a developing seed of rice (Oryza sativa L.). RASC8, 568bp in length, contained an ATG codon and two possible polyadenylation signals, and encoded 112 amino acid residues. Cys-Pro-Phe-Cys, which is the active site and a highly conserved sequence among thioltransferases, was found in the deduced amino acid sequence. RASC8 was introduced into an expression vector pMALc2 and the translated product possessed thioltransferase activity.

Amplification of ten deletion-rich exons of the dystrophin gene by polymerase chain reaction shows deletions in 36 of 90 Japanese families with Duchenne muscular dystrophy.

We analyzed DNA samples taken from 95 Duchenne muscular dystrophy (DMD) patients belonging to 90 different families in Japan using the polymerase chain reaction. Ten different regions at the 5' end or in the central region of the dystrophin cDNA gene that were previously shown to be prone to deletion were selected for amplification and analysis. Patients in 36 of the 90 families (40%) had deletions in at least one of these segments of the gene. Identical deletions were detected in the dystrophin gene of patients from the same family. The deletions were heterogeneous in size and location. One patient had deletions in 7 of the 10 amplified regions, while 19 patients from 18 families had a deletion in only one of the regions studied. Deletions at the 5' end were generally larger and more heterogeneous than those in the central region of the gene. One third of deletions had their proximal end breakpoints between exons 44 and 45. This region seems to be particularly vulnerable to gene breakage in DMD patients.

Exon skipping during splicing of dystrophin mRNA precursor due to an intraexon deletion in the dystrophin gene of Duchenne muscular dystrophy kobe.

Recent molecular studies have shown that in a patient with Duchenne muscular dystrophy (DMD) Kobe, the size of exon 19 of the dystrophin gene was reduced to 36 bp due to the deletion of 52 bp out of 88 bp of the exon. The consensus sequences at the 5' and 3' splice sites of exon 19 were unaltered (Matsuo, M., et al. 1990. Biochem. Biophys. Res. Commun. 170:963-967). To further elucidate the molecular nature of the defect, we examined the primary structure of cytoplasmic dystrophin mRNA of the DMD Kobe patient across the junctions of exons 18, 19, and 20 by gel electrophoresis and sequencing of polymerase chain reaction-amplified cDNA. The mRNA coding for dystrophin was reverse transcribed using random primers, and the cDNA was then enzymatically amplified in vitro. The targeted fragment was smaller than expected from the genomic DNA analysis. By sequencing of the amplified product, we found that exon 18 was joined directly to exon 20, so that exon 19 was completely absent, suggesting that this exon was skipped during processing of the dystrophin mRNA precursor. All other bases in the amplified product were unaltered. Therefore, the data strongly suggest that the internal exon deletion generates an abnormally spliced mRNA in which the sequence of exon 18 is joined to the sequence of exon 20. We propose that the deletion is responsible for abnormal processing of the DMD Kobe allele. This finding has important implications regarding the determinants of a functional splice site.

Screening of gene deletions by polymerase chain reaction in Japanese patients with Duchenne muscular dystrophy.

Gene deletions were screened in 49 Japanese Duchenne muscular dystrophy patients from 43 families, using the polymerase chain reaction. Enzymatic amplification was carried out on six regions prone to deletion. Fifteen of 43 families (33%) had gene deletions in at least one of the six regions. This frequency was almost the same as that previously reported in Caucasians. The mid-part of the dystrophin gene was the location most frequently deleted. The frequency of deletion of the region encompassing exon 45 was higher in Japanese families (18.4%) than in Caucasians.