Fumio Hishinuma

Functional correlation of fetal and adult forms of glycine receptors with developmental changes in inhibitory synaptic receptor channels

Functional maturation of the nicotinic acetylcholine receptor is executed by its gamma-to-epsilon subunit switching. The glycine receptor also has fetal (alpha 2) and adult (alpha 1) isoforms. However, whether subunit switching is responsible for developmental changes in glycine receptor function is not known. We recorded single-channel currents from homomeric glycine receptors expressed in Xenopus oocytes with cRNAs encoding the alpha 2 or alpha 1 subunits and compared them with those recorded from native glycine receptors in rat spinal neurons at various ontogenic periods. The mean channel life times of the alpha 1 and mature glycine receptors were equally short, whereas both the alpha 2 and fetal receptors showed a significantly longer open time. Consistent with these results, the decay time of the glycinergic inhibitory postsynaptic currents (IPSCs) in spinal neurons became shorter during postnatal development. We conclude that developmental switching of alpha subunits may accelerate the kinetics of IPSCs.

Cloning of a glycine receptor subtype expressed in rat brain and spinal cord during a specific period of neuronal development

Complementary (c) DNAs encoding a glycine receptor (GlyR) isomer were cloned from libraries constructed in λZAPII with poly (A)+ RNA of neonatal rat spinal cord. Northern blot analysis revealed that RNA hybridized to the cloned cDNA is detectable only for a period of late embryonic/ early postnatal stage of the spinal cord. Moreover, other central nervous tissues, such as hippocampus and cerebral cortex, in the infant rats are also rich in this message. The ‘neonatal (N) GlyR’ has 71% homology to that of another GlyR isoform in which adult rad cord is rich (AGlyR) Injection of a single RNA transcribed from the NGlyr‐cDNA into Xenopus oocyte induced functional formation of glycine‐gated Cl− channels, however, its pharmacological property differed from that of AGlyR.

Integration of heterologous genes into the chromosome of Saccharomyces cerevisiae using a delta sequence of yeast retrotransposon Ty

SummaryDistribution of a delta (δ) sequence of the Ty element on a chromosome of the yeast Saccharomyces cerevisiae was analysed by pulsed-field gel electrophoresis. More than 100 copies of the δ sequence were non-randomly distributed on the chromosome. Using the δ sequence as a recombination site, mouse α-amylase and human β-endorphin genes were introduced into the chromosomal DNA. The integration occurred on a particular chromosome in each case and the copy number was estimated as three to five. It was also found that single- or multi-copy integration occured at a single or multiple sites on the particular chromosome. The integrants secreted α-amylase and β-endorphin by three-to fivefold compared with single-copy integrants. This type of integration was mitotically stable over a period of 50 generations under non-selective conditions.

Increase in activin βA mRNA in rat hippocampus during long-term potentiation

We have used mRNA differential display to isolate genes that are induced by neural activity in rat hippocampus. One of these encodes activin βA subunit. Convulsive seizure caused by kainate significantly induced the expression of activin βA mRNA. Furthermore high frequency stimulation (HFS) of perforant pathway, which produced a persistent long‐term potentiation (LTP) (> 10 h), caused a marked increase at 3 h in the level of activin βA mRNA at the dentate gyrus of urethane‐anesthetized rat. The increase was NMDA receptor‐dependent. By contrast the level of inhibin α mRNA was not changed following the induction of LTP. The results suggest a role for activin in maintenance of neural plasticity in the adult brain.

Structure and molecular analysis of RGR1, a gene required for glucose repression of Saccharomyces cerevisiae.

An RGR1 gene product is required to repress expression of glucose-regulated genes in Saccharomyces cerevisiae. The abnormal morphology of rgr1 cells was studied. Scanning and transmission electron microscopic observations revealed that the cell wall of the daughter cell remained attached to that of mother cell. We cloned the RGR1 gene by complementation and showed that the cloned DNA was tightly linked to the chromosomal RGR1 locus. The cloned RGR1 gene suppressed all of the phenotypes caused by the mutation and encoded a 3.6-kilobase poly(A)+ RNA. The RGR1 gene is located on chromosome XII, as determined by pulsed-field gel electrophoresis, and we mapped rgr1 between gal2 and pep3 by genetic analysis. rgr1 was shown to be a new locus. We also determined the nucleotide sequence of RGR1, which was predicted to encode a 123-kilodalton protein. The null mutation resulted in lethality, indicating that the RGR1 gene is essential for growth. On the other hand, a carboxy-terminal deletion of the gene caused phenotypes similar to but more severe than those caused by the original mutation. The amount of reserve carbohydrates was reduced in rgr1 cells. Possible functions of the RGR1 product are discussed.

Expression of β-nerve growth factor mRNA in rat glioma cells and astrocytes from rat brain

A 50‐base synthetic oligodeoxynucleotide complementary to a portion of mouse nerve growth factor (NGF) mRNA was used as a probe for analysis of the expression of NGF gene. Northern blot analysis showed the presence of a major 1.3 kb transcript, which was identical in size to mouse NGF mRNA, in both C6Bu1 cells and rat astrocytes cultured from newborn rat brain. Further, the rearrangement of DNA sequence in and around the NGF gene locus of C6Bu1 cells was not detected by Southern blot analysis. These results indicate the expression of NGF mRNA in both C6Bu1 cells and astrocytes from rat brain, suggesting that astrocytes may produce NGF protein in the rat brain, especially in developing rat brain.

Isolation of Extrachromosomal Deoxyribonucleic Acids from Extremely Thermophilic Bacteria

Eight strains of thermophilic bacteria were examined for the presence of covalently closed circular deoxyribonucleic acid molecules by caesium chloride-ethidium bromide density gradient centrifugation. Four of the eight strains tested, Thermus flavus BS1, AT61, AT62 and Thermus thermophilus HB8 carried covalently closed circular DNA molecules. Thermus flavus BS1 haboured two species of plasmids with molecular weights of 6.1 X 10(6) and 17.0 X 10(6) as determined by electron microscopy. Thermus thermophilus HB8, T. flavus AT61 and T. flavus AT62 carried plasmids with molecular weights of 6.2 X 10(6), 6.6 X 10(6) and 6.6 X 10(6), respectively. Plasmids from T. flavus AT61 and AT62 were indistinguishable in their electrophoretic patterns in agarose or acrylamide gel after digestion with various restriction endonucleases. This is the first evidence for the presence of plasmids in extremely thermophilic bacteria, though their functions are unknown.

Genome organization of the linear plasmid, pSKL, isolated from Saccharomyces kluyveri

SummaryWe have determined the complete nucleotide sequence of the linear DNA plasmid, pSKL, isolated from Saccharomyces kluyveri. Sequence analysis showed that pSKL has a high (A+T) content of 71.7%, and that there are 10 open reading frames (ORFs) larger than 250 nucleotides. All 10 ORFs were shown to be transcribed in S. kluyveri cells by S1 nuclease mapping analysis. The localization of ORFs, direction of transcription, and the predicted amino acid sequences of each ORF were quite similar to that of pGKL2, one of the killer plasmids found in Kluyveromyces lactis. The amino acid sequences of the largest two ORFs (ORF2 and ORF6) have homology with several DNA polymerases and RNA polymerases, respectively.

Control of tryptophan synthetase amplified by varying the numbers of composite plasmids in Escherichia coli cells

Using pSC101, RSF1010, RSF2124 and RP4 plasmids as vectors and bacteriophage lambdatrpD-A60-3 DNA as a source of the Escherichia coli whole tryptophan operon, composite plasmids of pSC101-trp, RSF1010-trp, RSF2124-trp and RP4-trp were constructed in vitro with EcoRI restriction endonuclease and DNA ligase. Each composite plasmid could be maintained stably in E. coli cells. The copy number of pSC101-trp, RSF1010-trp, RSF2124-trp and RP4-trp were 4.2, 11.2, 11.9 and 1.6 per chromosome respectively. The tryptophan synthetase activities in cells containing pSC101-trp, RSF1010-trp, RSF2124-trp aand RP4-trp plasmid were found to be 2.1, 6.0, 5.0 and 2.5 times compared with the level in chromosomal trp+ cells when they were grown in a minimal medium. By partial derepression with indolylacrylic acid, the enzyme levels were elevated to 10.1, 16.3, 15.3, 12.3 times, respectively, that of the control cells. The tryptophan synthetase activities did not increase in proportion to the copy number of the plasmids, but were strongly affected by the repression system of host cells.

Estrogen can regulate the cell cycle in the early G1 phase of yeast by increasing the amount of adenylate cyclase mRNA

The effects of beta-estradiol (estrogen; a minor component of yeast cells) on S. cerevisiae cells in the G0 and G1 phases were examined. Results showed that estrogen stimulated the recovery of growth from G0 arrest induced by nutrient limitation or ts mutation of cdc35 (adenylate cyclase) in the early G1 phase, and inhibited entry into the resting G0 phase by increasing the intracellular cAMP level. However, estrogen had no effect on late G1 arrest induced by the alpha factor or ts mutation of cdc36. Estrogen was found to lead to higher steady-state levels of adenylate cyclase mRNA but not to affect the expression of the RAS1 and RAS2 genes, although these can also alter the intracellular cAMP level. These results suggest that estrogen influences the cell cycle of yeast in the early G1 phase by controlling the level of cAMP through the increase of adenylate cyclase mRNA.