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Biomedical subjects

R Kominami

Publications and source records attributed to R Kominami.

At least 109 records · Page 6Linked to original sources
PubMed1991

A human autoantibody specific for a unique conserved region of 28 S ribosomal RNA inhibits the interaction of elongation factors 1 alpha and 2 with ribosomes.

An autoantibody reactive with a conserved sequence of 28 S rRNA (anti-28 S) was identified in serum from a patient with systemic lupus erythematosus. Anti-28 S protected a unique 59-nucleotide fragment synthesized in vitro against RNase T1 digestion. RNA sequence analysis revealed that it corresponded to residues 1944-2002 in human 28 S rRNA and 1767-1825 in mouse 28 S rRNA. These sequences are identical and highly conserved throughout all known eukaryotic 28 S rRNAs. In addition, this fragment is homologous to residues 1052-1110 of Escherichia coli 23 S rRNA that lies within the GTP hydrolysis center of the 50 S ribosomal subunit. Anti-28 S and its Fab fragments strongly inhibited poly(U)-directed polyphenylalanine synthesis, but had no effect on ribosomal peptidyltransferase activity. This effect resulted from inhibition of the binding of elongation factors EF-1 alpha and EF-2 to ribosomes and of the associated GTP hydrolysis. The inhibitory effect was almost completely suppressed by preincubation of anti-28 S with 28 S rRNA or in vitro synthesized RNA fragments containing the immunoreactive region. These results show that the immunoreactive conserved region of 28 S rRNA participates in the interaction of ribosomes with the two elongation factors in protein synthesis.

Animals↗
PubMed1991

Genotype-restricted lymphoproliferation in autoimmune lpr mice.

Transfer of bone marrow (BM) from autoimmunity-prone mice homozygous for the new lymphoproliferation mutation (lprcg) caused systemic lymphoproliferation in irradiated lprcg/lprcg recipients but not in irradiated +/+ recipient (J. Exp. Med. 1990. 171:519; Eur. J. Immunol. 1991.21: 63). It was thus hypothesized that the lprcg gene expresses its function at lymph nodes (LN) to provide anomalous lprcg/lprcg lymphoid cells with the environment where they can accumulate. This was confirmed by LN transplantation and BM transfer studies. In the LN transplantation study lprcg/lprcg LN grafts with or without in vitro irradiation swelled and lprcg/+ LN grafts were slightly hyperplastic or apparently normal; however, whereas +/+ LN grafts atrophied in lprcg/lprcg recipients, they were all histologically normal in +/+ and lprcg/+ recipients. Irradiation of lprcg/lprcg LN grafts significantly retarded their swelling in lprcg/lprcg recipients. In the BM transfer study lprcg/lprcg BM cells caused systemic lymphoproliferation in lpr/lpr and gld/+, lprcg/+ recipients and sporadic LN swelling in lprcg/+ recipients but LN atrophy in gld/gld recipients. In the study using both techniques in combination, lpr/lpr LN grafts swelled but gld/gld LN grafts atrophied in lprcg/lprcg BM----+/+ chimeras. All the swollen LN contained Thy-1+CD4-CD8 lymphoid cells or "double-negative (DN)" T cells characteristic of the lpr disease. Analysis of DNA restriction fragment length polymorphism demonstrated that lprcg/lprcg DN cells derived from lprcg/lprcg BM cells accumulated in lpr/lpr LN and gld/+, lprcg/+LN. The following conclusions have been drawn: (a) the lprcg gene determines the ability of lprcg/lprcg DN cell to accumulate in LN; (b) this genetic trait is not totally recessive differing from lymphoproliferation; (c) lpr/lpr LN and gld/+, lprcg/+ LN are equivalent to lprcg/lprcg LN in the receptivity of lprcg/lprcg DN cell accumulation thus supporting the allelism of lpr with lprcg and the complementation between gld and lprcg (J. Exp. Med. 1990. 171:519), respectively; (d) the ability of lprcg/lprcg LN to accumulate DN cells is partially resistant to irradiation; (e) lprcg/lprcg DN cells may cause atrophy of gld/gld LN and +/+ LN and (f) the gld and lpr genes are different from each other in the phenotype expressed at the LN site.

Animals↗
PubMed1991

Existence of host-related DNA sequences in the schistosome genome.

DNA sequences homologous to the mouse intracisternal A particle and endogenous type C retrovirus were detected in the DNAs of Schistosoma japonicum adults and S. mansoni eggs. Furthermore, other kinds of repetitive sequences in the host genome such as mouse type 1 Alu sequence (B1), mouse type 2 Alu sequence (B2) and mo-2 sequence, a mouse mini-satellite, were also detected in the DNAs from adults and eggs of S. japonicum and eggs of S. mansoni. Almost all of the sequences described above were absent in the DNAs of S. mansoni adults. The DNA fingerprints of schistosomes, using the mo-2 sequence, were indistinguishable from each other and resembled those of their murine hosts. Moreover, the mo-2 sequence was hypermethylated in the DNAs of schistosomes and its amount was variable in them. These facts indicate that host-related sequences are actually present in schistosomes and that the mo-2 repetitive sequence exists probably in extra-chromosome.

Animals↗
PubMed1991

Quercetin induces recombinational mutations in cultured cells as detected by DNA fingerprinting.

Quercetin, a flavonoid, is found in many fruits and vegetables. This drug was previously shown to affect the metastatic potential of mouse tumor cells. Mutagenicity of quercetin was examined by means of DNA fingerprint analysis using the Pc-1 probe that efficiently detects mutations due to recombination. Treatment of BMT-11 and FM3A tumor cells with 55 microM quercetin resulted in gain and loss of bands in the fingerprints in both cell lines. The frequencies of the clones having undergone mutation were 3/11 and 6/26, respectively. This suggests that quercetin is mutagenic and induces recombination. This result seems to provide a molecular basis for the phenotypic variations of BMT-11 tumor cells induced by quercetin.

Animals↗
PubMed1991

Analysis of transforming genes in indirectly induced radiogenic thymomas in mice.

The expression of oncogenes was studied in 12 types of 178 mouse tumors induced by radiations and chemicals. DNA was analyzed in tumors in which the overexpression of oncogenes was noted. Amplification of the myc oncogene was found in chemically induced sarcomas, but not in sarcomas induced by radiation. Activation of oncogenes by small mutations and the inactivation of tumor suppressor genes has to be taken in account in the radiation induction of mouse tumors. We therefore made further analyses of radiogenic thymomas. Loss of heterozygocity was revealed in directly induced thymomas by the deletions of allele specific minisatellite bands. Analysis of a hypervariable minisatellite locus also revealed that these thymoma cells suffered high recombinogenic activity during tumorigenesis. In addition, transfection of cellular DNA to normal Golden hamster cells identified the activated K-ras oncogene in the directly induced radiogenic thymomas. Indirectly induced radiogenic thymomas were tested similarly. Transformed cells from secondary transfection experiment were positive for the mouse-specific repetitious sequences, but devoid of mouse ras oncogenes. Indirectly induced radiogenic thymomas originate from unirradiated normal thymus cells transplanted in irradiated hosts. The spontaneous activation of oncogenes yet to be identified may therefore be involved in the development of this tumor.

Animals↗
PubMed1991

Autoantibodies against ribosomal proteins found with high frequency in patients with systemic lupus erythematosus with active disease.

Antibodies to ribosomal protein components were examined in sera from 89 patients with active systemic lupus erythematosus (SLE). The anti-P, anti-S10 and anti-L12 antibodies were detected in 37, 28 and 2 patients, respectively. They were not detected in patients with various autoimmune diseases other than SLE or in healthy donors, which indicates that antiribosomal protein antibodies in sera are specific for patients with SLE. These antibodies are prevalent in patients with SLE and should provide good markers for the diagnosis of this disease.

Adolescent↗
PubMed1990

Autoantibodies specific for the 20-KDal ribosomal large subunit protein L12.

New antibodies reactive with a 20 KDal ribosomal protein of the large subunit were found in sera from two of eighty patients with systemic lupus erythematosus. This antigenic protein was identified as L12 by the mobility in two-dimensional gel electrophoresis. Both sera also contained anti-P activity against three acidic phosphoproteins (P proteins), but this activity was completely inhibited by preincubation with the isolated P proteins. Therefore, these anti-L12 can be useful for studying the function of L12 in protein synthesis.

Animals↗
PubMed1990

A GGCAGG motif in minisatellites affecting their germline instability.

Mouse and human genomes contain hypervariable DNA regions consisting of tandem repeats of a short sequence referred to as minisatellites. This variation is thought to arise through processes such as unequal crossover or replication slippage. A mo-1 minisatellite probe comprising a 14-base pair repeat sequence reveals many polymorphic fragments even in DNA of BALB/c sublines. Oligonucleotide probes with single base substitution in the mo-1 have been synthesized and used for assessing sequence involved in generation of polymorphisms. The results indicate that the loci containing mo-1 homologues with mutation in the GGCAGG sequence are monomorphic despite the other mutants showing polymorphism. Reciprocally, locus-specific polymorphic clones, Pc-1 and Pc-2, have been isolated with hybridization to mo-1, and both are shown to contain repeated sequence comprising the GGCAGG sequence. They reveal high mutation rates of 8.8% and 3.3% per gamete, respectively. These results strongly suggest that the motif contributes to the germline instability of minisatellites.

Animals↗
PubMed1990

Sequence-specific binding of a transcription factor TFID to the promoter region of mouse ribosomal RNA gene.

The binding sites of TFID, a species-dependent transcription factor, on the mouse ribosomal RNA gene (rDNA) have been analyzed by DNase I footprinting using partially purified TFID. The region -12 to -140 spanning over the upper half of the core promoter (-12 to -40) and an upstream sequence (-40 to -140) was protected. Human fraction D could not protect corresponding regions of mouse rDNA indicating that the protection was indeed caused by TFID. This was corroborated by a competition experiment using point mutants having different affinities to TFID. The analysis with deletion mutants of upstream sequence together with the competition data indicates that the binding of TFID to the core sequence is required for the binding of TFID or some co-purified proteins to the upstream sequence, while the presence of upstream sequence stabilizes the TFID binding to the core sequence. The pattern of protection of the upstream sequence suggests that at least a part of the upstream binding does not require a specific DNA sequence there but rather is caused by protein-protein interaction involving TFID bound with the core sequence. These protection patterns did not change with TFID highly purified by sequence-specific DNA affinity chromatography.

Animals↗
PubMed1990

Monoclonal antibodies against acidic phosphoproteins P0, P1, and P2 of eukaryotic ribosomes as functional probes.

Mice were immunized against ribosomal acidic proteins P1 and P2 from Artemia salina, and three kinds of monoclonal antibodies were isolated. One recognized P0 in addition to both P1 and P2 (anti-P). The other two recognized either P1 (anti-P1) or P2 (anti-P2) specifically and did not recognize P0. The anti-P antibody, but not anti-P1 or anti-P2, recognized a 22-amino acid peptide corresponding to the carboxyl-terminal sequence common to P1 and P2. This antibody, but not the others, inhibited poly(U)-directed polyphenylalanine synthesis. The anti-P1 bound to ribosomes but failed to inhibit polyphenylalanine synthesis: the anti-P2 did not bind to ribosomes at all. The anti-P and its Fab fragments inhibited the elongation step of protein synthesis, namely, the binding of elongation factors 1 alpha and 2 to ribosomes as well as their ribosome-coupled GTPase activities. Anti-P had little effect on the nonenzymatic phenylalanyl-tRNA binding to ribosomes and on peptidyltransferase activity. These results suggest the functional importance of the homologous carboxyl-terminal region of the three P proteins for the interaction of the ribosome with the two elongation factors. The epitope of anti-P1 must reside in a region of the protein which is not directly involved in its function.

Animals↗
PubMed1990

A sensitive assay for detecting mutations resulting from unequal homologous recombination without phenotypic selection.

Naturally occurring mutations are composed of a large number of mutations of many types that include mutations resulting from unequal homologus recombination between repetitive elements. The present paper describes a sensitive method for detecting such mutations without phenotypic selection. This system utilizes a tandemly arranged Vr repetitive sequence comprising 6000 copies in the mouse genome that is present in the spacer of the ribosomal RNA gene. For HincII digests of FM3A cell DNA, the Vr probe provides 4 major bands of 2.7 kb, 1.7 kb, 1.6 kb and 1.35 kb and several minor bands. Newly induced mutations due to unequal homologous recombination are observed as disappearance of the minor bands and appearance of extra bands. With this method a spontaneous mutation was detected in 14 cell clones randomly isolated after 60 days of continuous growth. Exposure to 4 micrograms/ml of N-methyl-N'-nitro-N-nitrosoguanidine for 2 h revealed 4 mutations in 11 clones examined. Culturing the cells after treatment in the presence of 12-O-tetradecanoylphorbol 13-acetate enhanced the frequency, yielding 6 mutations in 5 clones. The assay can skip phenotypic selection prior to analysis of DNA changes and hence provides a direct method for monitoring mutations resulting from homologous recombination in non-biased cell populations.

Animals↗
PubMed1989

Transcription of human ribosomal DNA may terminate at multiple sites.

The termination of human pre-rRNA transcription has been investigated. The most abundant possible termination site was detected 360 bp downstream from the 28S gene, in front of the first SalI box of the rDNA spacer. This site, however, is partially bypassed during transcription, and three additional termination points were detected inside the heterogeneous region of the rDNA spacer. Later sites were mapped about 930, 1030 and 1110 bp downstream from the 3' end of the 28S rRNA gene. The authors suggest that the T clusters and pyrimidine-rich regions play an important role in the termination processes. They either may influence the efficiency of the SalI boxes in terminating the synthesis of pre-rRNAs or may serve as independent signals for the fail-safe termination of readthrough transcripts. In both cases transcription of human rDNA ceases at multiple sites.

Animals↗
PubMed1989

The characterization of human male-specific 5.7 kb DNA(pKY-1) and its application to fetal sex-determination probe.

A male-specific 5.7 kb DNA(pKY-1) was cloned by screening human genomic libraries. Subsequent to structural characterization, a possibility of its application for prenatal diagnosis probe was tested. The results obtained were as follows: 1) the pKY-1 was specifically hybridized with human Y-chromosome specific repetitive 3.5 kb, but not with female DNA and 2.1 kb, known to be another human Y-specific repetitive-DNA. 2) HaeIII, EcoRI and their double digestion did not produce the Y-specific 3.5 kb fragments from pKY-1. 3) Although the digestion of pKY-1 with BamHI liberated 0.55 kb, ClaI clove remained 5.10 kb into 2.40 and 2.7 kb fragments, while their hybridizabilities with the 3.5 kb DNA were identically conserved. These data suggest that 5.7 kb fragment (pKY-1) should be proposed as a member of Y-chromosome-specific repetitive DNA family in human genome. Thus, this cloned DNA will be not only a useful probe for human fetal sex-determination but also an effective tool for analysis of Y-chromosome with its structure and repetitive DNA.

Blotting, Southern↗
PubMed1989

Structural determinant of the species-specific transcription of the mouse rRNA gene promoter.

Mammalian ribosomal DNA (rDNA) transcription has a certain species specificity such that, both in vivo and in vitro, human rDNA cannot be transcribed by mouse machinery and vice versa. This is due to a species-dependent transcription factor, TFID (Y. Mishima, I. Financsek, R. Kominami, and M. Muramatsu, Nucleic Acids Res. 10:6659-6670, 1982). On the basis of the information obtained from 5' and 3' substitution mutants, we prepared a chimeric gene in which the mouse sequence from positions -32 to -14 was inserted into the corresponding location of the human rDNA promoter. The chimeric gene could be transcribed by mouse extracts nearly as efficiently as the wild-type mouse promoter. The chimeric gene could also sequester transcription factor TFID at an efficiency similar to that for the mouse promoter. Partially purified mouse TFID that could not protect the human rDNA promoter against DNase I produced a clear footprint on this chimeric gene that was similar to that on mouse rDNA promoter. The basic structure of the mouse rDNA core promoter is discussed in relation to the interaction with TFID.

Animals↗
PubMed1989

Individual identification from semen by the deoxyribonucleic acid (DNA) fingerprint technique.

For individual identification from semen, the deoxyribonucleic acid (DNA) fingerprint technique was used. In a blind trial, we succeeded in determining the semen donors among several volunteers comparing the DNA fingerprints of the blood and semen samples, respectively. Thereafter, we examined semen in a condom left beside a naked female dead body. The DNA fingerprint of the semen was recognized to be identical to that of the blood from a suspected man arrested later. This is the first report that the DNA fingerprint technique was practically used in a criminal investigation in Japan.

DNA↗
PubMed1989

DNA fingerprinting by means of a nonradioactive probe of sulfonated DNA.

A DNA fingerprinting technique that uses a cold sulfonated DNA probe is described. DNA was extracted from blood samples of a family, who were subjected to DNA fingerprinting. The restriction fragments that were hybridized with the probe were colorimetrically detected by an enzyme immunoassay. For DNA detection and labeling a commercially available kit was employed. The sensitivity of this fingerprint detection method has been proven comparable to detection by the radioactive method, and hence is easier to use in conventional laboratories.

DNA↗