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R Kominami

Publications and source records attributed to R Kominami.

At least 91 records · Page 5Linked to original sources
PubMed1993

Transcription of mouse ribosomal RNA gene with inactive extracts is activated by NAD+ in vitro.

S100 extract prepared from rapidly growing mouse FM3A cells (approx. 5 x 10(5) cells/ml) transcribed ribosomal RNA gene (rDNA) much more actively in vitro than that from stationary phase cells (1-2 x 10(6) cells/ml). When the inactive S100 extract was preincubated with NAD+, rDNA transcriptional activity was restored almost to the level of the active extract. The extract activated with NAD+ exhibited a gel-shift band in the gel mobility shift assay and enhancement of protection of the sequence between -44 and -8 nt from the initiation site from exonuclease III digestion. Such an extract labeled with [32P]NAD+ was analyzed by immunoprecipitation with anti-RNA polymerase I (pol I) antibody; a protein with M(r) 130 kDa was detected. In contrast, the polypeptide was hardly labeled in the active extract. 3-Aminobenzamide, a specific inhibitor of poly ADP-ribosylation, did not inhibit the activation by NAD+. These results suggest that the activation by NAD+ is due to enhancement of the formation of initiation complex by mono ADP-ribosylation of the second-largest subunit (130 kDa) of pol I.

Animals↗
PubMed1993

Two mouse hypervariable minisatellites: chromosomal location and simultaneous mutation.

Pc-1 and Pc-2 hypervariable minisatellites of the mouse show a structural similarity and hence have been compared in terms of chromosomal location and mutability. Polymorphisms of the Pc-2 locus detected by analyses of Southern blotting and polymerase chain reaction were used for typing 61 backcross mice that were obtained by mating (C57BL/6 x MSM)F1 females to MSM males. Segregation patterns indicated that the Pc-2 locus maps to the 60 cM region of chromosome 6, while that of Pc-1 maps to chromosome 4. Simultaneous mutation of the two loci was examined in 101 F1 mice. Pc-1 and Pc-2 loci showed mutation rates of 14.4 and 5.0% per gamete, respectively. Of the nine mice with a Pc-2 mutation, four carried a Pc-1 mutation at the same maternally- or paternally-derived allele. The rate of comutation was 44% (4/9), higher than the 12.2% (24/197) of Pc-1 mutation alone. This suggests that the germline mutation of the two hypervariable minisatellites may result from a common mechanism.

Alleles↗
PubMed1993

Telomere change and loss of heterozygosity of mouse primary tumors and cell lines.

Changes in the number of telomere repeat arrays were examined in mouse tumor cells. Telomeres that function for the protection of chromosomes were detected as bands and a smear by pulsed field gel electrophoresis and gel-hybridization using (TTAGGG)4, as a probe. Of eight primary tumors induced in F1 mice between C57BL/6 and C3H/He and between C57BL/6 and MSM, three showed telomere alteration, two having extra bands and one having lost several telomere bands. The others exhibited patterns similar to those of normal tissues. However, the change was detected in all four cell lines that were established from one of the tumors. One cell line was further cloned and examined. Two of the nine clones differed in the telomere pattern. The telomere change was also observed in two other cell lines, FM3A cells and nontransformed BALB3T3 cells. These results suggest that telomeres are highly mutable in tumor cells and cultured cell lines. Three of the tumors and one cell line were analyzed for loss of heterozygosity with 51 microsatellite probes covering all 19 autosomes. Also, karyotype analysis of the cell line was performed. No allelic loss was seen and chromosomal abnormality was rare, although aneuploidy and imbalance in chromosomal number were observed. Possible involvement of the telomere changes observed here in chromosome impairment is discussed.

Animals↗
PubMed1992

A single-stranded DNA binding protein from mouse tumor cells specifically recognizes the C-rich strand of the (AGG:CCT)n repeats that can alter DNA conformation.

A protein that binds to a synthetic oligonucleotide of (CCT)12 has been purified from Ehrlich ascites tumor cells by a (CCT)12 affinity chromatography. The protein (p70) has an apparent molecular mass of 70 kDa, as assayed by Southwestern analysis. A competition experiment revealed that p70 binds to (CCT)12, (CCCT)8 and (CCTCCCT)6, but not to (CTT)12, (CT)16 and (CCTGCCT)6, suggesting that p70 has a sequence-specificity. The complementary (AGG)12 and the double stranded DNA did not show the binding. It is also confirmed by S1 nuclease analysis that the (AGG:CCT)12 duplex takes a single-stranded conformation in the absence of the protein. This raises a possibility that the duplex forms two single-stranded loops in chromosomes, the C-rich strand being bound to p70. Structural analysis of the resulting (AGG)12 strand by non-denaturing polyacrylamide gel electrophoresis demonstrated the presence of slower and faster migrated conformers in a neutral pH buffer containing 50 mM NaCl at 5 degrees C. The ratio was dependent on the DNA concentration. Both conformers disappeared in the absence of NaCl. This suggests that (AGG)12 can form intra- and inter-molecular complexes by non-Watson-Crick, guanine:guanine base-pairing. The possible biological function of the (AGG:CCT)n duplex and the p70 is discussed.

Animals↗
PubMed1992

Direct evidence for interaction of the conserved GTPase domain within 28 S RNA with mammalian ribosomal acidic phosphoproteins and L12.

A complex consisting of the acidic phosphoproteins P0, P1, and P2 (P proteins), L12, and RNA fragments was isolated from rat liver ribosomes after treatment with RNase T1 in the presence of EDTA. The complex was reactive with the anti-28 S RNA antibody specific for the highly conserved "GTPase domain" within 28 S rRNA. This suggests an association of these proteins with the RNA domain. To characterize this complex, the P proteins and L12 were isolated and tested for their binding specificity to the RNA by RNase T1 protection and gel retardation assays. Protein L12 and the P protein complex (P complex) both bound to rat 28 S rRNA and protected sequences comprising residues 1859-1921 and 1838-1936, respectively. The sequences overlap each other and lie in the GTPase domain. An in vitro transcript covering residues 1841-1936 of the 28 S rRNA as well as the protected RNA fragments also showed an ability to bind to the P complex and L12, and the binding was cooperative. RNA sequence elements within residues 1841-1936 required for protein binding were defined using site-directed mutagenesis. A unique internal loop including residues 1858 and 1859 and a distinct subregion comprising residues 1867-1914 in this domain were necessary for the binding of the P complex and L12, respectively. These results indicate that P proteins and L12 bind to restricted sites in the GTPase domain and that the complex constitutes the GTPase-related functional site in mammalian ribosomes.

Animals↗
PubMed1992

A 35-kDa protein binding to a cytosine-rich strand of hypervariable minisatellite DNA.

A minisatellite-binding protein, Msbp-4, with a molecular mass of 35 kDa has been purified from mouse tumor cells that binds to hypervariable Pc-1 and Pc-2 minisatellites. The binding is much more efficient than that to genetically stable minisatellite homologues. As assayed by Southwestern analysis, Msbp-4 favors multiple copies of the Pc-2 repeat sequence GGCAGGA and requires the cytosine-rich single strand for the binding. The activity is also present in extracts from mouse testis but not from liver. The phosphatase treatment revealed that Msbp-4 is phosphorylated and may have a regulatory function, because dephosphorylation affects the activity and specificity of the binding. Sequence preference is demonstrated by a competition experiment using single-base substitution mutants. Thus, the binding properties of Msbp-4 observed here lead to an implication that the protein-DNA complexes result in formation of a single-stranded DNA loop of the G-rich strand in the minisatellite which may enhance the ability of the minisatellite to undergo recombination.

Animals↗
PubMed1992

Somatic mutation during metastasis of a mouse fibrosarcoma line detected by DNA fingerprint analysis.

Metastatic nodules were examined by DNA fingerprint analysis. The probes used, Pc-1 and Pc-2, detect mutations as shifts in bands of the minisatellite loci which are dispersed among chromosomes. Four clonal lines of a fibrosarcoma from an F1 mouse (C57BL/Ka x C3H/He) were selected for various metastatic potentials upon inoculation into syngeneic mice. These four lines exhibited many extra bands resulting from recombination and/or DNA slippage, indicating accumulation of mutations during the successive passages in mice. One of the four, a 505 cell line which had been passaged extensively in vitro and consisted of a heterogenous population, was inoculated into thirteen syngeneic mice, and gave rise to six lung metastatic nodules in two mice. All the nodules showed band-patterns distinct from one another, although nodules within a given mouse tended to show similar patterns. When a genetically tagged 505-05-01 clone was analyzed, three of nine metastatic nodules obtained also revealed new bands. These results strongly suggest that somatic mutations occur at a high frequency during metastasis, providing direct evidence of genetic instability of the tumor cells.

Animals↗
PubMed1992

Telomere elongation frequently observed during tumor metastasis.

Changes in the number of telomere repeat arrays have been examined during metastasis of two mouse tumor cell lines. Telomeres were detected as bands and a smear by pulsed-field gel electrophoresis and gel-hybridization using (TTAGGG)4 as a probe. Very long size variants of telomeres were frequently observed in metastatic nodules. This suggests that at least some of the tumor cells have an ability to elongate telomeres. This elongation may compensate for the continuous loss of telomere repeats due to cell divisions, which would eventually lead to cell death.

Animals↗
PubMed1992

Lack of allelic preference in amplification and loss of the c-myc oncogene in methylcholanthrene-induced mouse sarcomas.

Sarcomas were induced in F1 mice between C57BL/6N and C3H/He strains by subcutaneous injection of methylcholanthrene. The c-myc oncogene was found to be amplified in 16 cases among 43 sarcomas of C57BL/6N x C3H/He mice and 1 case among 5 sarcomas of the reciprocal cross. The origin of the amplified allele was determined by the polymerase chain reaction single strand conformation polymorphism analysis. Among the 17 sarcomas, only one had both of the alleles amplified. The rest of the tumors carried the amplified c-myc allele coming either from C57BL/6N (9 cases) or from C3H/He (8 cases). These results indicate that the c-myc allele is amplified randomly in methylcholanthrene-induced mouse sarcomas irrespective of its origin, such as paternal or maternal allele and C57BL/6N or C3H/He allele. In addition to these changes, the unamplified c-myc oncogene was found to be lost in 12 cases out of the 17 sarcomas with the amplification.

Alleles↗
PubMed1992

Compensatory changes in silver-stainability of nucleolar organizer regions in mice.

Silver-stainability of nucleolar organizer regions (NORs) that contain genes for ribosomal RNA (rDNA) was investigated using two mouse strains, BALB/cCrSlc and MOA, and their hybrid progeny. The patterns of segregation of the rDNA clusters were analyzed in terms of chromosomal C-banding and by use of a polymorphic probe for the variable region in backcrossed N2 and N3 individuals. The results indicate that the intensity of Ag-NOR staining is stably inherited in most of the rDNA clusters, irrespective of different genetic backgrounds. In some clusters, such as those on chromosome 12 of BALB/cCrSlc, a modulation of the intensity is observed. This modulation seems to be due to compensatory activation via a change in the number of actively transcribed genes. The change from silver-negative to silver-positive staining of the NOR of chromosome 12 of BALB/cCrSlc was correlated with demethylation of the genes.

Animals↗
PubMed1992

Ribosomal ribonucleic acid (rRNA) gene typing for species identification.

Deoxyribonucleic acid (DNA) typing of ribosomal ribonucleic acid (rRNA) genes was performed with a polymerase chain reaction (PCR) assay for species identification. A variable region of the 28S ribosomal RNA gene was amplified with primers complementary to flanking sequences phylogenetically well conserved. The products of twelve animal DNAs (human, Japanese monkey, dog, cattle, pig, cat, rabbit, mouse, rat, chicken, frog, and fish) were separated by polyacrylamide gel electrophoresis, each revealing a few bands ranging from 150 to 100 base pairs. The band patterns obtained from each DNA sample differed in number and size, which indicates the applicability of the method to species identification. Samples containing either as little as 1 pg of DNA or degraded DNA of 0.2 to 0.5 kb in length were able to give detectable bands. Postmortem human tissue DNAs were tested as an example. They showed a pattern identical to the human control one, which was distinct from those of the other animals examined.

Animals↗
PubMed1991

A novel spermidine-dependent endoribonuclease activity caused by RNA-protein complex in mouse FM3A cell extracts.

We have found a novel spermidine-dependent endoribonuclease activity in mouse FM3A cell extracts. This endoribonuclease cleaves RNA substrates containing a sequence CCCCCGGUUUGU in its middle. This activity is lost either by heat- or micrococcal nuclease-pretreatment. When heat-pretreated extracts and micrococcal nuclease-pretreated ones are mixed, the activity is restored, suggesting that this activity requires both RNA and protein components. Testing the restoration of the lost endoribonuclease activity in micrococcal nuclease-pretreated extracts by addition of fractionated cellular RNAs, we identified an approximately 65 nucleotide RNA required for this endoribonuclease activity.

Animals↗
PubMed1991

Structure of the core promoter of human and mouse ribosomal RNA gene. Asymmetry of species-specific transcription.

In vitro transcription of the ribosomal RNA gene (rDNA) shows a remarkable species specificity such that human and mouse rDNA cannot use heterologous extracts of each other. The region that is responsible for this specificity has been studied using human-mouse chimeric genes and characteristic structures of both core promoters are presented. When the mouse sequence is substituted by the corresponding human sequence from upstream, the promoter activity in the mouse extract begins to decline at nucleotide -32 or -30, decreasing gradually and is lost completely at -19. A similar gradual decrease was noted for the 3' side substitution, which started at nucleotide -14 and was completed when up to the nucleotide -22 mouse position was replaced by the corresponding sequence from human. Thus, in the mouse rDNA core promoter, the sequence that is involved in species specificity resides only in a stretch encompassing the non-conserved region between the distal conserved sequence (DCS) and the proximal conserved sequence (PCS), plus two altered nucleotides in the PCS. When human rDNA is transcribed with human cell extract, the mouse sequence cannot substitute for the human sequence within the region from nucleotide -43 to +17 without affecting promoter activity significantly. This asymmetry of species specificity is due to the presence of nucleotides -43, +1 and +17, which are sensitive to change in only the human core promoter. The difference in the 5' border is ascribed to the species specificity of a transcription factor TFID, which recognizes this region. But the large difference of the 3' border is apparently due to another factor, possibly RNA polymerase I itself, because this region is not recognized by TFID in either human or mouse. Mammalian rDNA core promoter appears to consist of a tandem mosaic in which three evolutionarily conserved sequences alternate with non-conserved sequences having certain functionally important nucleotides. Not only non-conserved sequences and non-conserved nucleotides in conserved sequences, but also the spacings between the three conserved regions, play a crucial role in species specificity.

Animals↗