Tada-aki Hori

R-banding and nonisotopic in situ hybridization: precise localization of the human type II collagen gene (COL2A1)

SummaryA new mapping system, based on nonisotopic in situ hybridization combined with fluorescent staining of replicated prometaphase R-bands, is described. Replication of the bands is achieved by treatment of thymidinesynchronized cells with bromodeoxyuridine. The human COL2A1 gene was mapped to band 12q13.11–q13.12 in this manner, to illustrate the potential of the technique for improving the precision of chromosomal mapping and physical ordering of genes.

Chromosome mapping of the human cytidine-5'-triphosphate synthetase (CTPS) gene to band 1p34.1-p34.3 by fluorescence in situ hybridization

SummaryThe human cytidine-5′-triphosphate synthetase (CTPS) gene was mapped by a direct mapping system combined with fluorescence in situ hybridization and replicated prometaphase R-bands. By high-resolution banding analysis, the signals were localized to band 34.1–34.3 of the short arm of chromosome 1; 1p34.1–p34.3. Simple procedures for the detection of R-bands are described.

Altered frequency of initiation sites of DNA replication in Werner's syndrome cells

SummaryDNA replication of cultured fibroblasts of early passage derived from Werners syndrome (adult progeria) patients and from normal subjects were compared by DNA fiber autoradiography. The frequency of replication initiation was decreased in Werners syndrome cells derived from five patients compared with that in normal cells derived from three persons of different ages. The rate of DNA chain elongation did not differ between Werners syndrome cells and normal cells.

Mapping of the MYC gene to band 8q24.12→q24.13 by R-banding and distal to fra(8)(q24.11), FRA8E, by fluorescence in situ hybridization

The MYC gene was mapped to R-banded human prometaphase chromosomes and to chromosomes expressing fra(8)(q24.11) by fluorescence in situ hybridization. By high-resolution banding analysis, the fluorescent signals were localized to R-positive band q24.12----q24.13 of the long arm of chromosome 8. Furthermore, the signals were localized near the middle part, q24.12----q24.13, of the distal portion of fra(8)(q24.11) expression. Thus, the precise localization of MYC was to the subband 8q24.12----q24.13.

Regional assignment of the human thymidylate synthase (TS) gene to chromosome band 18p11.32 by nonisotopic in situ hybridization

SummaryThe human thymidylate synthase (TS) gene was regionally assigned to chromosome band 18p11.32 by nonisotopic in situ hybridization using biotinylated cDNA (1.1kb insert) and genomic DNA (6.8kb insert) probes of the human gene. There have been two provisional assignments for the TS gene to 18pter-q12 and 18q21-qter. The present result confirmed the first of these and further localized the TS gene to the telomeric region of the short arm of chromosome 18. The TS gene appears to be a novel telomeric anchor point for the construction of both physical and genetic linkage maps of human chromosome 18.

Autoradiographic Studies of DNA Replication in Werner’s Syndrome Cells

We have compared cultured fibroblasts of early passage derived from patients with the Werner syndrome and from normal subjects in several aspects of cell cycle time and DNA replication. The average cycle time was prolonged in Werners syndrome cells compared with normal cells because of changes in the duration of S phase. The durations of G1 and G2 were unchanged. In addition, the labeling index was lower in Werners syndrome cells, suggesting that there are two cell-cycle abnormalities in Werners syndrome cells. The cause of the prolongation of S phase was investigated by DNA fiber autoradiography and alkaline sucrose density gradient sedimentation. The rate of DNA chain elongation was not different in Werners syndrome cells from that in normal cells, but the frequency of replication initiation was decreased in Werners syndrome cells.

High incidence of sister chromatid exchanges and chromatid interchanges in the conditions of lowered activity of poly(ADP-ribose)polymerase

Abstract The effect of lowering the activity of poly(ADP-ribose)polymerase on chromosome stability has been examined. Chinese hamster ovary cells, CHO-Kl grown in a nicotinamide-free medium exhibited an increased frequency of sister chromatid exchanges in a time-dependent manner. Furthermore, addition of m-aminobenzamide which is known to be a strong inhibitor of poly(ADP-ribose)polymerase caused a manyfold increase in the frequency of both sister chromatid exchanges and non-sister chromatid interchanges. These results suggest that appropriate levels of NAD and the activity of poly(ADP-ribose)polymerase are required for maintaining chromosome stability.

Isolation, tissue expression, and chromosomal assignment of human RGS5, a novel G-protein signaling regulator gene

AbstractThe regulator of G-protein signaling (RGS) proteins have recently been identified as signal transduction molecules which have structural homology to SST2 of Saccharomyces cerevisiae and EGL-10 of Caenorhabditis elegans. Multiple genes homologous to SST2 are present in higher eukaryotes, and the group of these genes is termed the RGS family. RGS proteins are involved in the regulation of heterotrimeric G-proteins by acting as GTPase-activators. A putative new member of the RGS family was isolated from a neuroblastoma cDNA library. The amino acid sequence deduced from the cDNA possessed all consensus motifs of the RGS domain and showed closest homology to mouse RGS5 (90% identical), indicating that it was human RGS5 (hRGS5). The messenger RNA of hRGS5 was abundantly expressed in heart, lung, skeletal muscle, and small intestine, and at low levels in brain, placenta, liver, colon, and leukocytes. The chromosome localization of the gene in the 1q23 region was determined by a monochromosomal hybrid panel and a radiation hybrid panel.

Cloning, expression analysis, and chromosomal localization of HIP1R, an isolog of huntingtin interacting protein (HIP1)

AbstractHuntington disease (HD) is an inherited neurodegenerative disorder which is associated with CAG expansion in the coding region of the gene for huntingtin protein. Recently, a huntingtin interacting protein, HIP1, was isolated by the yeast two-hybrid system. Here we report the isolation of a cDNA clone for HIP1R (huntingtin interacting protein-1 related), which encodes a predicted protein product sharing a striking homology with HIP1. RT-PCR analysis showed that the messenger RNA was ubiquitously expressed in various human tissues. Based on PCR-assisted analysis of a radiation hybrid panel and fluorescence in situ hybridization, HIP1R was localized to the q24 region of chromosome 12.

Population cytogenetics of rare fragile sites in Japan

SummaryA population cytogenetic study of three groups of rare fragile sites defined in Human Gene Mapping 8 (HGM8, Berger et al. 1985) has been conducted using peripheral blood lymphocytes of healthy Japanese subjects. We have examined 1,022 blood donors for folate-sensitive and bromodeoxyuridine (BrdU)-requiring, and 845 for distamycin A-inducible fragile sites. Out of 17 rare autosomal fragile sites defined in HGM8, the following six were identified in Japan; folate-sensitive fra(2)(q11), fra(11)(q13) and fra(11)(q23), distamycin A-inducible fra(16)(q22) and fra(17)(p12), and BrdU-requiring fra(10)(q25). The incidences of distamycin A-inducible fra(16)(q22) (1.42%) and fra(17)(p12) (3.08%) were considerably higher than those of the other sites in Japan. Furthermore, a folate-sensitive fra(17)(p12) and a distamycin A-inducible fra(8)(q24.1) have been newly found in the present study. Their incidences were 0.10% (1/1,022) and 0.71% (6/845), respectively. Since the expression of this fra(17)(p12) was induced by fluorodeoxyuridine, supressed by thymidine, but not induced by distamycin A, it can be classified as a folate-sensitive site. The expression of the new distamycin A-inducible fra(8)(q24.1) was also enhanced by treatment with Hoechst 33258, berenil and 4′,6-diamidino-2-phenylindole (DAPI). This fragile site fulfils all four classical criteria suggested by Sutherland (1979) and also new criteria for a rare fragile site defined in HGM8 (Berger et al. 1985).