Yoichi Kato

Substrate recognition and selectivity of plant glycerol-3-phosphate acyltransferases (GPATs) from Cucurbita moscata and Spinacea oleracea.

Stromal glycerol-3-phosphate acyltransferases (GPAT) are responsible for the selective incorporation of saturated and unsaturated fatty-acyl chains into chloroplast membranes, which is an important determinant of a plants ability to tolerate chilling temperatures. The molecular mechanisms of plant chilling tolerance were elucidated by creating chimeric GPATs between squash (Cucurbita moscata, chilling-sensitive) and spinach (Spinacea oleracea, chilling-tolerant) and the results were interpreted using structural information on squash GPAT determined by X-ray crystallography at 1.55 A resolution. Enzymatic analysis of the chimeric GPATs showed that the chimeric GPATs containing the spinach region from residues 128 to 187 prefer the 18:1 unsaturated fatty acid rather than 16:0 saturated fatty acid. Structure analysis suggests that the size and character of the cavity that is formed from this region determines the specific recognition of acyl chains.

Post-meiotic expression of the mouse dihydropyrimidinase-related protein 3 (DRP-3) gene during spermiogenesis

The dihydropyrimidinase‐related protein (DRP) family, originally identified in humans by their homology to dihydropyrimidinase, contains at least four members. Genes of this family, and their counterparts in other mammals and chickens, are expressed mainly in fetal and neonatal brain, suggesting that the encoded proteins have a physiological role in the development of the central nervous system. In addition, the DRP‐3 gene is expressed in testis as a shorter mRNA than the brain form. As a first step in understanding the extra‐neuronal function of DRP‐3, the structure and expression of testis DRP‐3 were examined. Testis DRP‐3 cDNA showed the same sequence as brain DRP‐3 cDNA, except for the 5′‐terminal end, which encodes a 5′‐untranslated region and the 11 N‐terminal amino acid residues, indicating that the two forms of DRP‐3 mRNA were transcribed from a single copy gene. Northern blotting analysis detected DRP‐3 mRNA in 30‐, 40‐ and 70‐day‐old, but not in 10‐ and 20‐day‐old testes. In situ hybridization analysis indicated that the expression of DRP‐3 in testis is restricted to post‐meiotic round spermatids. This is the first report of the expression of DRP genes in extra‐neuronal cells. Mol. Reprod. Dev. 51:105–111, 1998.

Fourth component of Xenopus laevis complement: cDNA cloning and linkage analysis of the frog MHC

ComplementC4 shows extensive structural and functional similarity to complementC3, hence these components are believed to have originated by gene duplication from a common ancestor. Although to dateC3 cDNA clones have been isolated from all major classes of extant vertebrates includingXenopus, C4 cDNA clones have been isolated from mammalian species only. We describe here the molecular cloning and structural analysis ofXenopus C4 cDNA. The cDNA sequence encoding the thioester region ofXenopus C4 was amplified by reverse transcriptase-polymerase chain reaction usingXenopus liver mRNA as a template, and then used to screen a liver cDNA library. The amino acid sequence ofXenopus C4 deduced from a clone containing the entire protein-coding sequence showed 39%, 30%, 25%, and 20% overall identity with those of human C4, C3, C5, and α2-macroglobulin, respectively. The predicted amino acid sequence consisted of a 22-residue putative signal peptide, a 634-residue β chain, a 732-residue α chain, and a 287-residue γ chain. Of 30 cysteine residues, 27 were found in exactly the same positions as in humanC4. Genomic Southern blotting analysis indicated thatC4 is a single copy gene inXenopus and is part of the frog MHC cluster. These results clearly demonstrate thatC3/C4 gene duplication and linkage between theC4 gene and the major histocompatibility complex predate mammalian/amphibian divergence.

Duplication of the MHC-linked Xenopus complement factor B gene

We have previously reported the molecular cloning of the mammalian major histocompatibility complex (MHC) class III gene, complement factor B (Bf) from Xenopus laevis, and linkage of the gene to the frog MHC. Here, we estimated the copy number of the Xenopus Bf gene by genomic Southern blotting analysis and demonstrated that Xenopus laevis has two copies of the Bf gene. Both genes co-segregated with the MHC-linked HSP70 genes among 19 offspring of an f/r × f/r cross, indicating a close linkage of the two Bf genes to the frog MHC. Both genes are transcribed and contain open reading frames. When compared with the previously determined cDNA sequence (Xenopus Bf A), the predicted amino acid sequence of the second cDNA species (Xenopus Bf B) shows 82% overall identity. Polymerase chain reaction analysis indicated that all of the partially inbred frogs with the f, r, g, and j MHC haplotypes, as well as 12 outbred frogs tested have both Bf genes, suggesting that the duplicated Bf genes are stable genetic traits in Xenopus laevis.

Differential expression of dihydropyrimidinase-related protein genes in developing and adult enteric nervous system

Abstractu2002Dihydropyrimidinase-related proteins (DRPs) are involved in axonal outgrowth and pathfinding. However, little is known about their significance in the enteric nervous system (ENS), the largest and most complex division of the peripheral nervous system. Using in situ hybridization (ISH) and northern blotting, we examined mRNA expression of DRP-1–4 transcripts in the developing and adult mouse digestive tract and in the adult human colon. ISH detected the mouse DRP-3 transcript in the developing ENS on embryonic day (E)12 and at the later stages as well as in the adult intestine. Mouse DRP-1 and -2 transcripts appeared at E14. DRP-2 transcript was also detected in the adult intestine although DRP-1 expression was lower in the adult. DRP-4 gene was not expressed in the ENS during development or adulthood whereas the signal was apparent in the developing and adult central nervous system (CNS). The DRP expression pattern in the human colon was similar to that of the mouse large intestine. Northern blot analysis showed that DRPs were differentially expressed in the mouse and human intestines, supporting the results of ISH. These data suggest that DRPs play a role not only in the CNS but also in the ENS.