Atsushi Kohda

Monoallelic expresion of the odourant receptor gene and axonal projection of olfactory sensory neurones

We have previously generated transgenic mice carrying the murine odourant receptor gene, MOR28, tagged with lacZ. In this animal, the endogenous MOR28 is differently tagged with GFP. It was found that the transgenic and endogenous MOR28 genes are expressed in a mutually exclusive manner and that the two sets of olfactory sensory neurones (OSNs), each expressing either the transgenic or the endogenous MOR28, project their axons to separate glomeruli.

Replication Timing Properties within the Mouse Distal Chromosome 7 Imprinting Cluster

Genomic imprinting is characterized by allele-specific expression of genes within chromosomal domains. Here we show, using fluorescence in situ hybridization (FISH) analysis, that the large chromosomal domain of the mouse distal chromosome 7 imprinting cluster, approximately 1 Mb in length between p57Kip2 and H19 genes, replicates asynchronously between the two alleles during S-phase. At the telomeric side of this domain, we found a transition from asynchronous replication at the imprinted p57Kip2 gene to synchronous replication at the Nap2 gene. Two-color FISH suggested that the paternal allele of this whole domain replicates earlier than its maternal allele. Treatment of the cells with a histone deacetylase inhibitor abolished this allele-specific feature accompanied with accelerated replication of the later-replicating allele at a domain level. Allele-specific asynchronous replication was observed even in ES cells. These results suggest that this imprinting cluster consists of a large replication domain which is already found at the early stage in development.

DNA Fluorescence in situ Hybridization in Paramecium: Telomere Localization in Macronucleus

Abstract In an attempt to develop a technique of fluorescence in situ hybridization (FISH) to detect DNA in Paramecium, we examined three different DNA probes, total genomic DNA, genomic DNA encoding C5 phagosomal membrane antigen, and telomere, prepared from P. multimicronucleatum. In accordance with the conventional method, total genomic DNA probe was denatured at 75–80°C for 2 min, and the cells were denatured at 75, 80, 85, or 90°C for 5 or 10 min. The homogeneous hybridization signal with the total genomic DNA probe was obtained at 85°C for 10 min, or at 90°C for 5 min or 10 min. This condition was applied for the smaller DNA probe, C5 (1. 3 kb, the size close to detection limits), in which the expected tiny signals throughout the macronuclear nucleoplasm was observed. However, the condition was not successful for the telomeric DNA probe. The hybridization signals of telomeric DNA were only detected when both cells and probes were denatured simultaneously in a same denaturation buffer. In the case of the simultaneously denatured samples, the preservation of the nuclear morphology was relatively poor, however, the signals of the telomeric DNA probe were observed in the periphery of the macronucleus. As a negative control, an irrelevant 40 kb human cosmid probe was examined by both conventional and simultaneous denaturation methods, and none of the hybridization signal was observed with this probe. These results suggest that the current methods allow us to follow localization of the specific sequences within the macronuclear compartment.