Yuji Imai

Molecular Cloning of a Novel Polypeptide, DP5, Induced during Programmed Neuronal Death

To study the molecular mechanisms underlying neuronal programmed cell death (PCD), we performed differential display screening for genes, the expression of which was induced during PCD in the sympathetic neuron culture model deprived of NGF. We cloned a gene encoding a novel polypeptide (DP5) which consisted of 92 amino acids. DP5 polypeptide had no homology with any other known protein and contained no motif that would indicate its putative biochemical functions. DP5 mRNA levels peaked at 15 h after nerve growth factor withdrawal, concurrent with the time at which neurons were committed to die. The induction of DP5 gene expression was blocked when cell death was rescued by treatment with cycloheximide, KCl, or the cyclic AMP analogue CPTcAMP. Overexpression of the full-length DP5 in cultured sympathetic neurons was in itself sufficient to induce apoptosis. These results suggest that DP5 plays a role in programmed neuronal death.

Molecular cloning of novel leucine-rich repeat proteins and their expression in the developing mouse nervous system

It is well established that leucine-rich repeat (LRR) proteins such as connectin, slit, chaoptin, and Toll have pivotal roles in neuronal development in Drosophila as cell adhesion molecules. However, to date, little information concerning mammalian LRR proteins has been reported. In the present study, we sought LRR proteins of the mouse brain, based on the assumption that fundamental mechanisms are conserved between different species. We screened a neonatal mouse brain cDNA library with a human partial cDNA encoding LRR protein as a probe. We obtained two independent cDNAs encoding LRR proteins, designated NLRR-1 and NLRR-2 (Neuronal Leucine-Rich Repeat proteins). We analyzed the whole sequence of NLRR-1 and partial sequence of NLRR-2. Sequence analysis showed that these two clones are about 60% homologous to each other, and that NLRR-1 protein is a transmembrane protein. Northern blot analysis and in situ hybridization histochemistry showed that both NLRR-1 and NLRR-2 mRNAs were expressed primarily in the central nervous system (CNS); NLRR-1 mRNA was also detected in the non-neuronal tissues such as cartilage, while NLRR-2 mRNA expression was confined to the CNS at all developmental stages. These results suggest that there is at least one LRR protein family in the mouse and that these molecules may play significant but distinct roles in neural development and in the adult nervous system.

Cloning of a gene, YT521, for a novel RNA splicing-related protein induced by hypoxia/reoxygenation

To elucidate the role of astrocytes in the stress response of the central nervous system to ischemia, early gene expression was examined in rat cultured astrocytes after the exposure to hypoxia/reoxygenation, and we have previously cloned a novel RNA binding protein, RA301, from the reoxygenated astrocytes. Furthermore, we have now cloned a new gene for RA301 binding protein, termed YT521, by a yeast two-hybrid screening technique to explore RA301 functions. The YT521 cDNA is about 3200 bp long with an open reading frame encoding 712 amino acids. This amino acid sequence contains arginine-aspartic acid-glutamic acid rich region and glutamic acid rich one, and has a low degree of homology with RNA binding proteins such as U1-70k. Northern blot analysis revealed that YT521 mRNA expression was up-regulated in reoxygenated astrocytes. Induction of YT521 mRNA was mediated by endogenously generated reactive oxygen species, as it was suppressed by treatment of the cells with diphenyl iodonium which blocks oxygen-free radical formation by astrocytes. These expression patterns resembled those of RA301 mRNA. Far Western blot analysis showed that YT521 protein was not only interacting with RA301 protein, but also with SC35 and SF2, both of which are splicing factors. These results suggest that YT521 is a novel candidate for RNA splicing-related protein.

Cloning and expression of a neural differentiation-associated gene, p205, in the embryonal carcinoma cell line P19 and in the developing mouse.

Mouse P19 embryonal carcinoma cells can be reproducibly differentiated into neurons and glial cells upon treatment with high concentration of retinoic acid (RA). In order to understand the molecular mechanisms that control early neural differentiation, we screened a cDNA library made from 24-h RA-treated P19 cells with subtracted cDNA probes. One clone was positive in the secondary screening and was designated as p205. This clone (1.1 kb) has an open reading frame of 317 amino acids with homology to G-protein beta subunit. This protein sequence was identical to chicken and human genes previously identified as a major histocompatibility complex-associated gene. The complete conservation of its amino acid sequence between mouse, human and chicken provides strong evidence that the p205 protein fulfills a fundamental function. Developmental Northern blot analysis revealed that a p205 mRNA is expressed at high levels in the embryonic mouse brain, decreasing as development proceeds. In situ hybridization revealed that p205 mRNA is strongly and ubiquitously expressed in the embryonic and early postnatal mouse brain. This expression decreased during postnatal development and was localized in the dentate gyrus, habenula, piriform cortex, paraventricular nucleus of the hypothalamus and supraoptic nucleus of the adult brain. These results suggest that this protein plays an important role in the developing brain and neuronal differentiation.

Improvement of a proline-producing strain of Serratia marcescens by subcloning of a mutant allele of the proline gene

Abstract A hybrid plasmid pY1333 bearing a mutant allele, dpr-1 , which is responsible for proline overproduction, was not stable in a proline-hyperproducing strain SP187 during subcultures. A 7 kb fragment covering over a part of dpr-1 allele DNA and a segment for spectinomycin resistance of mini-F plasmid vector pKP1154 tended to be deleted in the hybrid plasmid pY1333. We subcloned the dpr-1 allele into an improved plasmid vector pKPT1124 in which the segment for spectinomycin resistance of plasmid vector pKP1154 was substituted for a segment for kanamycin resistance of a broad-host-range vector pKT240, resulting in an improved hybrid plasmid pY1350. Strain SP187 carrying the hybrid plasmid pY1350 was quite stable during cultivation and produced 75 mg of L-proline per ml. This amount was 1.5 times larger than that of plasmid-free host strain.

Stability and instability of a cloned mutant allele of the proline gene in Serratia marcescens. Use of a mini-F plasmid in the proline-overproducing mutant

Abstract For the purpose of improving proline production by a Serratia marcescens mutant, a mutant allele of the proline gene, dpr-1, that is responsible for proline overproduction, was cloned from the mutant genomic DNA into a mini-F plasmid. Furthermore, the dpr-1 allele was subcloned into plasmids pLG339 and pBR322 to investigate the effect of copy number of the hybrid plasmids on proline productivity and the plasmid stability. Strains carrying the dpr-1 hybrid plasmids grew more slowly than the control strain, indicating that the hybrid plasmids were deleterious to host cells. The dpr-1 alleles cloned on pBR322 and pLG339 were genetically unstable in the wild-type and the proline-overproducing strain, respectively. On the other hand, the allele cloned on the mini-F plasmid with the lowest copy number was considerably stable even in the proline-overproducing mutant. The mutant carrying the cloned dpr-1 on the mini-F plasmid produced 62 mg ml−1 of proline, which was approximately 10% more than the yield of the control strain.

Expressed-sequence-tag approach to identify differentially expressed genes following peripheral nerve axotomy

Gene expression profiles in the rat hypoglossal nucleus after axotomy were demonstrated using expressed-sequence-tag (EST) approach. To demonstrate the gene-expression profiles after axotomy, nerve-transected hypoglossal nuclei were dissected and collected from about 1000 rats, with which a cDNA library was constructed. More than 750 clones were sub-cloned and sequenced from the library. The clones which hit frequently are likely to be associated with mitochondrial respiratory chain, cytoskeletal protein and protein synthesis. One hundred three clones from among the sequenced clones were further processed for histological screening using unilateral-hypoglossal nerve-transected brain sections by in situ hybridization histochemistry. In situ hybridization study revealed that 26% of clones examined showed upregulated expression of mRNA in response to axotomy. They included genes encoding proteins associated with glucose, lipid and protein metabolism, cytoskeleton, neurotransmission and immune reaction. The present EST analysis may have an advantage in targeting genes which are associated with nerve injury with a good efficacy, as compared with other methods such as differential display and subtraction.