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Biomedical subjects

R Kominami

Publications and source records attributed to R Kominami.

At least 55 records · Page 3Linked to original sources
PubMed1997

Binding of mammalian ribosomal protein complex P0.P1.P2 and protein L12 to the GTPase-associated domain of 28 S ribosomal RNA and effect on the accessibility to anti-28 S RNA autoantibody.

We have investigated binding of rat ribosomal proteins to the "GTPase domain" of 28 S rRNA and its effect on accessibility to the anti-28 S autoantibody, which recognizes a unique tertiary structure of this RNA domain. Ribosomal protein L12 and P protein complex (P complex) consisting of P0, P1, and P2 both bound to the GTPase domain of rat 28 S rRNA in a buffer containing Mg2. Chemical footprinting analysis of their binding sites revealed that the P complex mainly protected a conserved internal loop region comprising residues 1855-1861 and 1920-1922, whereas L12 protected an adjacent helix region encompassing residues 1867-1878 and 1887-1899. These sites are close to but distinct from the binding site for anti-28 S antibody determined previously. The bindings of P complex and L12 increased the anti-28 S accessibility, as revealed by gel retardation and quantitative immunoprecipitation analyses. In a Mg2+-eliminated condition, the RNA failed to bind to either anti-28 S or L12 but assembled into a complex under their coexistence. However, the RNA retained a property of binding to the P complex even in the absence of Mg2+, and this binding conferred high anti-28 S accessibility. These results indicated that the bindings of the P complex and L12 to their respective sites influenced the GTPase domain to increase the accessibility to anti-28 S. A possible RNA conformation adjusted by the protein bindings is discussed.

Animals↗
PubMed1997

Telomerase activity and metastasis: expansion of cells having higher telomerase activity within culture lines and tumor tissues.

Tumor cells with metastatic potential may have a high telomerase activity that augments telomeric DNA repeats, allowing the cells to escape from the inhibition of cell proliferation due to shortened telomeres. We examined the expression level of telomerase activity using the telomeric repeat amplification protocol among a series of cell lines obtained by repeated transplantation of a mouse fibrosarcoma. The lines could be grouped into three; one has no metastatic potential, and the other two show metastatic abilities after intravenous or subcutaneous injection. Comparison of their telomerase activity indicated that more malignant lines had higher activity. A similar relation was seen in metastatic nodules formed through clonal expansion from the heterogeneous population of inoculated cells; clonality was monitored in terms of variable patterns of subtelomeric repeats. The results suggest that a high level of telomerase activity may not be requisite for metastasis, but may confer a propensity to dominate in a tumor tissue.

Animals↗
PubMed1996

A novel activity of HMG domains: promotion of the triple-stranded complex formation between DNA containing (GGA/TCC)11 and d(GGA)11 oligonucleotides.

The high mobility group protein (HMG)-box is a DNA-binding domain found in many proteins that bind preferentially to DNA of irregular structures in a sequence-independent manner and can bend the DNA. We show here that GST-fusion proteins of HMG domains from HMG1 and HMG2 promote a triple-stranded complex formation between DNA containing the (GGA/TCC)11 repeat and oligonucleotides of d(GGA)11 probably due to G:G base pairing. The activity is to reduce association time and requirements of Mg2+ and oligonucleotide concentrations. The HMG box of SRY, the protein determining male-sex differentiation, also has the activity, suggesting that it is not restricted to the HMG-box domains derived from HMG1/2 but is common to those from other members of the HMG-box family of proteins. Interestingly, the box-AB and box-B of HMG1 bend DNA containing the repeat, but SRY fails to bend in a circularization assay. The difference suggests that the two activities of association-promotion and DNA bending are distinct. These results suggest that the HMG-box domain has a novel activity of promoting the association between GGA repeats which might be involved in higher-order architecture of chromatin.

Base Composition↗
PubMed1996

A novel clonality assay for the mouse: application to hepatocellular carcinomas induced with diethylnitrosamine.

A polymerase chain reaction-based clonality assay was developed for mouse tumors and cellular proliferations of the mouse. This assay was based on a polymorphism of the phosphoglucokinase-1 (Pgk-1) gene on the X chromosome between two different mouse subspecies and the different methylation patterns of active and inactive X chromosomes. All 15 tumor cell lines examined showed one of the two allelic bands on gel electrophoresis, which is consistent with the theory that tumor cell lines are monoclonally derived. This suggests that the Pgk-1 system is useful for clonality studies that will give insight into cancer development. With this method, nine hepatocellular carcinomas were examined, and eight showed monoallelic patterns. The remaining tumor exhibited a biallelic pattern, which is suggestive of polyclonal origin; however, other possibilities are discussed.

Animals↗
PubMed1996

The presence/absence polymorphism and evolution of the p53 pseudogene in the genus Mus.

Distribution of the p53 pseudogene within the house mouse species (genus Mus) was studied with polymerase chain reaction for 37 individuals that were caught at different localities. Pseudogene-specific fragments were detected in some, but not all, individuals of Mus musculus subspecies regardless of locality and type of subspecies. In addition, 3 of 7 individuals belonging to different Mus species carried the pseudogene in their genomes. These results show the existence of an interspecific presence/absence polymorphism of the p53 pseudogene in mice. Sequence analysis of 11 amplified 0.3-kb fragments suggested that the pseudogene originated in an ancestral mouse about 7 million years ago. Thus alleles with and without the p53 pseudogene have persisted through the mice speciation. The evolutionary rate for the p53 functional gene was also estimated to be about 3.3 x 10(-9) per nucleotide site per year.

Animals↗
PubMed1996

Regionalized expression of the Dbx family homeobox genes in the embryonic CNS of the mouse.

Here we report the identification of a novel homeobox gene family Dbx in mouse, which consists of Dbx and Dbx2. The two genes share similar structural organization and are encoded by different chromosomes. The predicted Dbx and Dbx2 proteins share 85% identity in their homeodomain amino acid sequences, but otherwise showed no significant similarity. Characterization of the expression of these two genes in the embryos suggested their role in the development of the CNS. In the forebrain, Dbx is expressed in various regions, while Dbx2 showed a more restricted pattern of expression. In the midbrain, the expression domains of Dbx and Dbx2 overlap along the dorso-lateral wall of the ventricle. In the hindbrain and spinal cord, both genes are expressed in the boundary separating the basal and alar plates, which seems to correspond to the sulcus limitans. Expression of the Dbx/Dbx2 genes is restricted to the ventricular region of the embryonic CNS except for that of Dbx in the septum of the telencephalon. Together these observations indicate possible participation of the members of the Dbx family in regionalization of the CNS. While the expression of Dbx was restricted to the CNS, Dbx2 was also expressed in some of the mesenchymal cells, such as limb buds and tooth germs.

Amino Acid Sequence↗
PubMed1996

Formation of a triple-stranded DNA between d(GGA:TCC) repeats and d(GGA) repeat oligonucleotides.

Incubation of the 162-bp duplex containing d(GGA:TCC)11 repeats with d(GGA)11 and d(GGT)11 oligonucleotides, but not with d(TCC)11, gave a mobility-shifted band in polyacrylamide gel electrophoresis, suggestive of the triplex formation. The two conformers, however, showed difference in binding affinity and structure. The complex formed with d(GGT)11 exhibited patterns protected from DNase I digestion and from modifications with osmium tetroxide and dimethyl sulfate. The patterns were conformed to a proposed triplex model. In contrast, the complex with d(GGA)11 was sensitive to DNase I and to osmium tetroxide, which is reactive to pyrimidine bases of single-stranded DNA. These results suggest that the d(GGA:TCC)11 and d(GGA)11 triple-stranded complex forms a D-loop-like structure. By considering the ability of d(GGA)11 oligonucleotides to form a parallel homoduplex, the structure may be one in which the GGA-strand of the duplex pairs with d(GGA)11 in parallel, with the TCC-strand being looped out.

Alkylating Agents↗
PubMed1996

P53 gene deficiency does not enhance instability of mouse minisatellites in somatic cells of normal tissues.

The effect of p53-deficiency on somatic mutation of minisatellites in normal tissues was examined using p53-deficient (-/-) mice. In total, 248 mice consisting of three different genotypes, +/+, +/ -and -/-, were obtained and DNA from their embryos was probed with two minisatellites, Pc-1 and Pc-2. The somatic mutation was detected by Southern blot hybridization as the presence of a third nonparental band reflecting mosaicism in tissues. Mutation frequency of Pc-1 for (+/+) and (+/-) was 1.3% and 1.4%, respectively, which is consistent with previous studies. On the other hand, none of the mice lacking the p53 gene (-/-) exhibited mutation. The Pc-2 probe did not show any somatic mutation in the three groups. These results suggest that the p53 deficiency does not enhance the genomic instability of the minisatellite loci in normal somatic cells.

Animals↗
PubMed1996

Induction of a germline mutation at a hypervariable mouse minisatellite locus by 252Cf radiation.

Male C3H/HeN mice were exposed to 252Cf radiation and mated with unirradiated C57BL/6N females. F1 mice were analyzed for germline mutation at the paternally derived C3H/HeN allele of a hypervariable minisatellite locus, Ms6hm. This locus exhibited a high frequency of length change mutation spontaneously, and the mutation frequency of the paternally derived C3H/He allele in F1 mice born to unirradiated males was 8.4%. Exposure of male mice to 252Cf radiation resulted in even higher frequency of germline mutation. The spermatid stage germ cells were most sensitive to neutrons, and the mutation frequencies of the paternal allele were elevated to 18%, 26% and 24% for 0.35, 0.7 and 1.02 Gy of 252Cf radiation, respectively. Spermatozoa and spermatogonia stages were less sensitive and the mutation frequencies for 1.02 Gy of 252Cf radiation were 16% and 19%, respectively. The 252Cf radiation consisted of 35% gamma-rays and 65% neutrons. Assuming that these two radiations act additively, RBE of 252Cf neutrons for the induction of minisatellite mutation was calculated to be 5.9 for spermatozoa stage irradiation, 2.6 for spermatid stage irradiation and 6.5 for spermatogonia irradiation.

Alleles↗
PubMed1995

A base substitution within the GTPase-associated domain of mammalian 28 S ribosomal RNA causes high thiostrepton accessibility.

A molecular basis for the insensitivity of eukaryotic ribosomes to the antibiotic thiostrepton was investigated using synthetic 100-nucleotide-long fragments covering the GTPase domain of 23/28 S rRNA. Filter binding assay showed no detectable binding of the rat RNA to thiostrepton, but the binding capacity was markedly increased by base substitution of G1878 to A at the position corresponding to 1067 of Escherichia coli 23 S rRNA. The association constant (K alpha) for the rat A 1878 mutant was 0.60 x 10(6) M-1, which was comparable with that of the E. coli RNA (K alpha = 1.1 x 10(6) M-1). This suggests that the eukaryotic G 1878 participates in the resistance for thiostrepton. On the other hand, the RNA fragments of the two species had a similar binding capacity for E. coli ribosomal protein L11 and its mammalian homologue L12. Gel electrophoresis under a high ionic condition, however, revealed a difference between the two proteins. E. coli L11 formed stable complexes with both the E. coli RNA and the rat A 1878 mutant RNA in the presence of thiostrepton, while rat L12 failed to exhibit such complex formation. This suggests that the eukaryotic L12 protein may also be an element giving the resistance for thiostrepton. These results are discussed in terms of preserved three-dimensional conformation of the RNA backbone between prokaryotes and higher eukaryotes.

Adenosine↗
PubMed1995

Association of minisatellite instability with c-myc amplification and K-ras mutation in methylcholanthrene-induced mouse sarcomas.

Instability of microsatellite sequences are frequently found in human tumors. In addition, minisatellite sequences, another group of highly unstable sequences, serve as sensitive markers of genetic instability. We have studied minisatellite instability in methylcholanthrene-induced mouse sarcomas. These sarcomas frequently carry the amplified c-myc gene. Seven sarcomas without the amplification and seven others with the amplification were selected randomly. Regardless of the state of the c-myc gene amplification, these sarcomas exhibited a varying degree of transplantability in syngeneic mice. The hypervariable mouse minisatellite locus Ms6hm was found to be highly unstable, specifically among sarcomas with the amplified c-myc gene. However, chromosome instability, as analyzed by micronucleus assay, was observed similarly for two groups of sarcomas. In addition, transversion of G to C and A to T was detected at the K-ras gene in four of the seven sarcomas with the amplified c-myc gene, and these mutations are thought to be induced directly by methylcholanthrene. Thus, concomitant occurrence was observed for three seemingly unrelated mutations, amplification of the c-myc locus, point mutation of the K-ras gene, and instability at the hypervariable mouse minisatellite locus. The present study indicates a possible involvement of K-ras mutation and c-myc amplification in induction of genetic instability in methylcholanthrene-induced mouse sarcomas.

Animals↗
PubMed1995

Formation of a parallel-stranded DNA homoduplex by d(GGA) repeat oligonucleotides.

The GGA9-H molecules consisting of a double helical stretch followed by a single-stranded 3'-terminal overhang of nine GGA sequence repeats exhibited a gel mobility-shifted band in a concentration-dependent manner, suggestive of the intermolecular complex formation. The position of the shifted band in a gel was almost identical to that of the Y-shaped dimer marker of the same molecular weight that had the two double-helices at one side. This suggests that GGA9-H dimerizes in a parallel orientation without the formation of four-stranded hairpin structure. Since the GGA9-H homoduplex was stably formed at pH 4, 7 and 9, the formation does not require protonation or deprotonation of the N1 position of adenines. Neither does it require the N7 group of guanines responsible for Hoogsteen base pairing from the methylation interference and modification studies. Modification of the N7 group of guanines with dimethyl sulfate (DMS) did not inhibit the association and also the N7 group in the homoduplex was not protected from DMS. On the other hand, the GAA9-H having the G to A base substitution did not show such an association with either GGA9-H or GAA9-H. These results suggest that the homoduplex formation may be due to G.G base pairing through non-Hoogsteen hydrogen bonds.

Base Composition↗
PubMed1995

Frequent loss of heterozygosity on chromosome 4 in diethylnitrosamine-induced C3H/MSM mouse hepatocellular carcinomas in culture.

Genetic changes, in particular the loss of heterozygosity (LOH) and the presence of c-Ha-ras codon 61 point mutations, were investigated in diethylnitrosamine-induced hepatocellular carcinomas (HCCs) in C3H/MSM F1 mice. (MSM are wild mice.) LOH analysis of 48 primary tumors with microsatellite probes covering at least one proximal and one distal site of each autosome revealed no obvious positive results for LOH. Analysis of 23 cell lines established from seven of these HCCs, however, showed LOH on chromosome 4 in all (seven of seven), even in early passages (G2-G3). With regard to other chromosomes, LOH was observed only rarely on chromosomes 16 and 19. These allelotype features were maintained in later passages (G11-G14), with only a few additional occurrences of LOH appearing on chromosomes 1, 6, and 8. Extensive analyses with multiple microsatellite probes from chromosome 4 and with 52 cell lines established from 24 HCCs of 18 mice revealed LOH in 22 of the tumors (92%), with the shortest region about 10 cM distal to the alpha-interferon gene. No c-Ha-ras oncogene activation in codon 61 was observed. These data indicate that loss of tumor suppressor genes on chromosome 4 may play an important role in mouse hepatocarcinogenesis in progression in vivo or in immortalization in vitro or both.

Alleles↗
PubMed1995

Induction of karyotype instability in a murine tumor cell line by quercetin, 2-amino-1-methyl-6 phenylimidazo[4,5-b]pyridine, and okadaic acid, as revealed by transmission distortion of the inactive X chromosome.

Quercetin, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), and okadaic acid are found in various foods and have been shown to have mutagenic or promoter-like activity. The effects of these three compounds on the transmission of the inactive X chromosome were examined in MST-C6 murine tumor cells, which were derived from hybrid F1 mice from matings between C57BL/6 and MSM mice. Polymerase chain reaction analysis using polymorphic markers on the X chromosome detected transmission distortion of the inactive X chromosome due to nondisjunction as a copy-number imbalance in allelic bands. The cells exposed to all three chemicals (but not untreated cells) exhibited such imbalances at high frequencies under exposure conditions similar to those in previous experiments in which tumor progression and recombination were observed. The cells also showed increased frequencies of tumor formation when subcutaneously injected. These results suggest that the three chemicals are capable of inducing transmission distortion of the inactive X chromosome and that such activity may be a causative factor in promoting the tumorigenicity of MST-C6 cells.

Animals↗
PubMed1995

A new strategy of gene trapping in ES cells using 3'RACE.

"Gene trapping" in embryonic stem (ES) cells is a novel approach to identify a series of genes in mammals concomitant with the production of the corresponding mutant mice. However, this approach is currently unable to identify genes that are not expressed in ES cells. Here we describe a strategy to identify gene trapping clones which is not based on expression of a reporter gene. It uses the neor gene which lacks a polyadenylation signal and has a splice donor signal. Expression of the neor gene as fusion transcripts with the 3' end containing the polyadenylation signal of tagged genes allows the identification of these clones by 3' rapid amplification of the cDNA end in undifferentiated ES cells, even if the genes are not expressed in ES cells. Amplification was observed in about 25% of G418-resistant clones. Sequence analyses suggested the amplifications represent gene trapping events. The feasibility of this approach was further assessed by analysing one clone, PAT-12, in detail.

Amino Acid Sequence↗