京都大学 / 0048 / 新制・課程博士 / 博士(農学) / 甲第18323号 / 農博第2048号 / 新制||農||1021(附属図書館) / 学位論文||H26||N4830(農学部図書室) / 31181 / 京都大学大学院農学研究科農学専攻 / (主査)教授 奥本 裕, 教授 白岩 立彦, 教授 冨永 達 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DFAM

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第21664号 / 医博第4470号 / 新制||医||1035(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 髙橋 良輔, 教授 浅野 雅秀, 教授 影山 龍一郎 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Rag1-/- mutant zebrafish lack lymphocytes and were used to study the basis of acquired protective immunity in the absence of lymphocytes to the intracellular bacterium Edwardsiella ictaluri. This study morphologically identified and quantified lymphocyte like cells (LLCs) present in the liver, kidney and spleen of these fish. LLCs included Natural Killer (NK) cells and non-specific cytotoxic cells (NCCs) and were discriminated by size, and by the presence of cytoplasmic granules. The antibodies anti-NITR9, anti-NCCRP-1 (5C6) and anti-MPEG-1 were used to evaluate these cell populations by flow cytometry. Gene expression profiles in these tissues were evaluated after the Rag1-/- mutants were intra coelomically injected with the toll like receptor (TLR)-2 ligand, β glucan, TLR3 ligand, Poly I:C, or TLR 7/8 ligand, R848. The genes interferon y (infγ), expressed by activated NK cells and macrophages, tumor necrosis factor α (tnfα), expressed by activated macrophages, myxovirus resistance (mx) expressed by cells induced by IFNα, T-cell transcription factor (t-bet) expressed by NK cells and novel immune type-receptor 9 (nitr-9) expressed by NK cells were evaluated. The TLR ligands induced different patterns of expression and stimulated both macrophages and NK cells. Then fish were vaccinated with an attenuated mutant of E. ictaluri (RE33®) with or without the TLR ligands then challenged with WT E. ictaluri to evaluate protection. RE33® alone and each TLR ligand alone provided protection. Coministration of β glucan and RE33® or R848 and RE33® resulted in survival higher than RE33® alone showing an adjuvant effect. Tissue specific gene expression of ifnγ, t-bet, nitr9, NK cell lysin a (nkla), nklb, nklc and nkld were correlated to protection. The final component of this study was the development of tools to discriminate NK cell populations and evaluate the contribution of macrophages. Rag1-/- zebrafish were modified to express cherry red in lymphocyte like cells using the Lymphocyte specific tyrosine kinase (lck) promotor. Also, rag1-/- zebrafish were modified so that the gene encoding the proto-oncogene serine/threonine-protein kinase that is involved in macrophage training (raf1) is disrupted. This study indicated that the acquired protection in the absence of lymphocytes likely involves NK cells with possible contribution by macrophages.

A temperature-sensitive, protein synthesis-defective mutant of Escherichia coli exhibiting an altered ribosomal protein L22 has been investigated. The temperature-sensitive mutation was mapped to the rplV gene for protein L22. The genes from the wild type and mutant strains were amplified by the polymerase chain reaction and the products were sequenced. A cytosine to thymine transition at position 22 of the coding sequence was found in the mutant DNA, predicting an arginine to cysteine alteration in the protein. A single cysteine residue was found in the isolated mutant protein. This amino acid change accounts for the altered mobility of the mutant protein in two-dimensional gels and during reversed-phase HPLC. The temperature-sensitive phenotype was fully complemented by a plasmid carrying the wild type L22 gene. Ribosomes from the complemented cells showed only wild type protein L22 by two dimensional gel analysis and were as heat-resistant as control ribosomes in a translation assay. The point mutation in the L22 gene is uniquely responsible for the temperature-sensitivity of this strain.

Enterococci were once thought to be harmless, commensal organisms that colonize the gastrointestinal tract of humans and other mammals. In the last 30 years, however, concern has grown in the clinical setting over two particular species, Enterococcus faecalis and Enterococcus faecium, which are frequently found to be the etiologic agents of nosocomial infections. Aminoacyl-phosphatidylglycerol synthases (aaPGSs) are integral membrane proteins that add amino acids to phosphatidylglycerol (PG) in the cellular envelope of bacteria. Addition of amino acids to PG confers resistance to various therapeutic antimicrobial agents, and contributes to evasion of the host immune response in a number of clinically relevant microorganisms. E. faecium possesses two distinct aaPGSs: aaPGS1 and aaPGS2. In addition, another gene coding for a putative hydrolase (pHyd) is located in the same operon as aaPGS2, and has no known function. To investigate the physiological relevance of aa-PG formation, and the function of aaPGS1, aaPGS2, and pHyd in E. faecium, we generated individual knockouts of these genes using a markerless deletion strategy. Deletion of aaPGS1 did not noticeably alter lipid aminoacylation, whereas deletion of aaPGS2 led to a loss of aa-PG synthesis. Deletion of pHyd also led to a loss of lipid aminoacylation; however, additional experiments are needed to verify that expression of aaPGS2 (which resides just downstream in the same operon) is unaffected in the pHyd-deletion strain. Development of the mutant strains described here will enable us to investigate additional phenotypes associated with these genes, and to determine whether aa-PG formation contributes to antibiotic resistance in E. faecium as in several other pathogenic microorganisms.

Cotton (Gossypium spp.) is the world’s leading textile fiber crop, and an important source of oil and protein. Insufficient candidate gene derived-markers suitable for genetic mapping and limited information on genes that control economically important traits are the major impediments to the genetic improvement of Upland cotton (G. hirsutum L.). The objectives of this study were to develop a SNP marker discovery strategy in tetraploid cotton species, SNP characterization and marker development from fiber initiation and elongation related genes, chromosomal assignment of these genes by SNP marker-based deletion analysis or linkage mapping, and genetic and molecular analysis of mutants affecting cotton fiber development. Phylogenetic grouping and comparision to At- and Dt-genome putative ancestral diploid species of allotetraploid cotton facilitated differentiation between genome specific polymorphisms (GSPs) and marker-suitable locus-specific polymorphisms (LSPs). By employing this strategry, a total of 222 and 108 SNPs were identified and the average frequency of SNP was 2.35% and 1.30% in six EXPANSIN A genes and six MYB genes, respectively. Both gene families showed independent and incongruent evolution in the two subgenomes and a faster evolution rate in Dt-genome than that in At-genome. SNPs were concordantly mapped to different chromsomes, which confirmed their value as candidate gene marker and indicated the reliability of SNP discovery stragey. QTL mapping by two F2 populations developed from fiber mutants detected major QTL which explain 62.8-87.1% of the phenotypic variation for lint percentage or lint index in the vicinity of BNL3482-138 on chromosome 26. Single marker regression analyses indicated STV79-108, which was located to the long arm of chromosome 12 (the known location of N1 and perhaps n2 loci), also had significant association (R2 % value 15.4-30.6) with lint percentage, lint index, embryo protein percentage and micronaire. Additional QTL and significant markers associated with other seed and fiber traits were detected on different chromosomes. Inheritance analysis indicated that both genetic models N1N1n2n2 and n2n2li3lisub>3 could lead to the fiberless phenotype. The observation of fuzzless-short lint phenotype indicated fiber initiation and elongation were controlled by different mechanisms. The penetrance of Li2 gene expression was observed in this study.

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