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内容記述 |
The human genome is composed of long-range GC% mosaic structures predicted to be related to chromosome bands. DNA replication timing, gene density, and repetitive sequence density have been connected to the chromosome band zones and the long-range GC% mosaic domains. The human MHC spans approximately 4 megabase-pair (Mbp) on the short arm of chromosome 6, and is composed of Mbp-level mosaic domains of GC%, and a boundary of the GC% mosaic domains exists in the junction area between MHC classes II and III. DNA replication timing during S phase is correlated cytogenetically with chromosome band zones, and thus the band boundaries have been predicted to contain a switch point for DNA replication timing. One aim of this thesis study was to determine the precise DNA replication timing for MHC classes II and III, focusing on the junction area. He showed that the replication timing changes precisely in the GC% boundary region with a 2-hour difference of the timing during S phase, supporting the prediction that the GC% boundary is a chromosome band boundary. It was supposed that the replication fork movement terminates (pauses) or significantly slows down in the switch region, which contains dense Alu clusters and a long polypurine / polypyrimidine tract with the triple-helix forming potential.<br /> The second aim was to clarify the global GC% distribution in and around the entire 4 Mbp of the human MHC region. He conducted a long-range chromosome walk of about 2.1 Mbp, and found the MHC and its surrounding region to be composed of five long-range GC% mosaic domains disclosing three new GC% mosaic boundaries. One boundary corresponds to the junction between the MHC class II and the centromeric non-MHC region, the second does to the junction between the MHC classes III and I, and the third does to the junction between the class I and the telomeric non-MHC region. It was thus shown that the GC% boundaries correspond to boundaries of functional domains of the respective genome region. In the case of the junction between the MHC class I and the telomeric non-MHC, the non-MHC region was evidently AT-rich, and the telomeric probe was located on 6p22.1 (G/Q) but close to 6p21.3 (R) by a standard fluorescence in situ hybridization (FISH) onto prometaphase chromosomes. Since the main body of the MHC region is present on 6p21.3, the walked region was shown to contain the boundary between 6p21.3 and 6p22.1. Characteristic structures in the GC% transition regions were disclosed.<br /> The third aim was to clarify the distribution of genes in the walked genome region, in order to understand the chromosome bands and GC% mosaic structures on functional and evolutionary views. First, he determined sequences of the terminal portions of the cloned fragments of individual cosmids which cover the telomeric portion of the MHC class I and the adjacent non-MHC. Because of the high density of the cosmids covering the individual regions, portions of each of various genes were found in a series or the consecutive cosmids. Nine genes correspond to those being previously mapped in this region and nine genes were newly identified in this study; SMT3B-like gene, GABAB receptor gene, mas-related gene, RASH-like gene, TRE17-like gene and MEA11-like gene, in the direction from the class I to the non-MHC, and three olfactory genes. Sequence similarity of some of these genes with those on chromosomes 7, 11, and 17, was observed. This set of chromosomes is distinct from the three chromosomes 1, 9 and 19, on which a wide range of genes with sequence similarities with those on 6p21.3 were present, showing that a possible boundary of the genome multiplication during evolution and/or of the genome rearrangement after the multiplication is located near the telomeric edge of the MHC class I. Elucidation of this type of boundaries and their correlation with boundaries of GC% mosaic domains and of DNA replication timings, should give knowledge of evolutionary processes and mechanisms to establish the present-day human and mammalian genomes.<br /> Among the newly identified genes in this region, he thought that GABR-B is the most interesting gene to be studied and determined its complete genomic sequence. The gamma-aminobutyric acid (GABA) is the most abundant and widely distributed inhibitory neurotransmitter present in the central nervous system. Two mRNA forms of the rat metabotropic gamma-aminobutyric acid receptor (GABAB receptor) have been characterized and predicted to be produced by alternative splicing. He determined a complete sequence of the human GABAB receptor gene. The pairwise alignment of this genomic sequence with that of the known rat GABAB receptor cDNAs showed that the human gene spans 31 kilobases and is composed of 23 exons. The sequence was the first example of the genomic sequence for the GABAB receptor, and thus the first study to characterize the structure for regulating transcription. The two isoforms of the GABAB receptor were found to be generated by alternative usage of promoters, rather than by alternative splicing. In both promoter regions, CpG islands and several potential transcription regulatory sites were identified. A cAMP response element (CRE) was present only in the promoter for the shorter isoform. Based on the fact that the GABAB receptor negatively regulates the CRE binding protein (CREB) - mediated transcriptions in the central nervous system, he proposed that the activation of GABAB receptor can differentiate the relative expression of its two isoforms through alternative usage of the promoters with and without a CRE, via the CRE- and CREB-mediated regulatory system. This may modulate synaptic transmission. An informative polymorphic marker (CA)n was found 1 kb upstream of the 5'-UTR of the GABAB receptor gene, and used to analyze 36 different HLA haplotypes. |
要旨・審査要旨 / Abstract, Screening Result (361.0 kB)
