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Biomedical subjects

R Kominami

Publications and source records attributed to R Kominami.

143 records · Page 8Linked to original sources
PubMed1982

Fractionation and reconstitution of factors required for accurate transcription of mammalian ribosomal RNA genes: identification of a species-dependent initiation factor.

Mouse and human cell extracts (S100) can support an accurate and efficient transcription initiation on homologous ribosomal RNA gene (rDNA) templates. The cell extracts were fractionated with the aid of a phosphocellulose column into four fractions (termed A, B, C and D), including one containing a major part of the RNA polymerase I activity. Various reconstitution experiments indicate that fraction D is an absolute requirement for the correct and efficient transcription initiation by RNA polymerase I on both mouse and human genes. Fraction B effectively suppresses random initiation on these templates. Fraction A appears to further enhance the transcription which takes place with fractions C and D. Although fractions A, B and C are interchangeable between mouse and human extracts, fraction D is not; i.e. initiation of transcription required the presence of a homologous fraction D for both templates. The factor(s) in fraction D, however, is not literally species-specific, since mouse D fraction is capable of supporting accurate transcription initiation on a rat rDNA template in the presence of all the other fractions from human cell extract under the conditions where human D fraction is unable to support it. We conclude from these experiments that a species-dependent factor in fraction D plays an important role in the initiation of rDNA transcription in each animal species.

Animals↗
PubMed1982

Cloning and determination of the transcription termination site of ribosomal RNA gene of the mouse.

A Eco RI 6.6 kb DNA fragment containing the 3'-end of 28S ribosomal RNA gene of the mouse was detected by Southern blot hybridization, and cloned in a lambda-phage vector. The site of transcription termination and the processed 3'-end of 28S RNA were determined on the cloned fragment and the surrounding nucleotide sequence determined. The 3'-terminal nucleotides of mouse 28S RNA are similar to those of yeast, Drosophila and Xenopus although the homology was lost drastically beyond the 3'-end of 28S RNA. 45S precursor RNA terminated at 30 nucleotides downstream from the 3'-end of 28S RNA gene. A structure of a dyad symmetry with a loop was found immediately prior to the termination site of 45S RNA. The rDNA termination site thus shares some common features with termination sites recognized by other RNA polymerases.

Animals↗
PubMed1981

In vitro transcription of a cloned mouse ribosomal RNA gene.

An in vitro transcription system which utilizes cloned mouse ribosomal RNA gene (rDNA) fragments and a mouse cell extract has been developed. RNA polymerases I is apparently responsible for this transcription as evidenced by the complete resistance to a high concentration (200 micrograms/ml) of alpha-amanitin. Run-off products obtained with three different truncated rDNA fragments indicated that RNA was transcribed from a unique site of rDNA. The S1 nuclease protection mapping of the in vitro product and of in vivo 45S RNA confirmed this site, indicating that, in this in vitro system, transcription of rDNA started from the same site as in vivo. This site is located at several hundred nucleotides upstream from the putative initiation site reported by us (1) and by others (2). Some sequence homology surrounding this region was noted among mouse, Xenopus laevis and Drosophila melanogaster. The data also suggest that some processing of the primary transcript occurs in this in vitro system.

Animals↗
PubMed1981

Organization of ribosomal RNA gene repeats of the mouse.

The organization of the ribosomal RNA (rRNA) genes of the mouse was determined by Southern blot hybridization using cloned rDNA fragments as probes, which could encompass the entire spacer region between two rRNA gene regions. The rRNA genes are organized into tandem repeats of nearly uniform length of about 44 kb. The heterogeneity detected in the nontranscribed spacer appears to be caused by its sequence rather than its length difference. At least three kinds of repetitive sequences are present in the non-transcribed spacer region; two of them are located 13 kb upstream from the 5'-end of 18S RNA gene and the other located 1 to 4 kb downstream from the 3'-end of 28S RNA gene.

Animals↗
PubMed1981

Identification of unintegrated forms of Kirsten murine sarcoma viral DNA and restriction endonuclease cleavage map of linear DNA.

We detected unintegrated linear 7.0-kilobase pair DNA and covalently closed circular DNA species in NIH3T3 cells recently infected with Kirsten murine sarcoma virus. Using the linear DNA, we constructed a restriction endonuclease cleavage map and compared it with the map of Harvey murine sarcoma virus. The restriction endonuclease maps of two segments, one 1.2 kilobase pairs (SmaI site) to 3.7 kilobase pairs (HindIII site) from the right end (corresponding to the viral 3' side) and the other 0.5 kilobase pair (SmaI and KpnI sites) to 0.9 kilobase pair (KpnI site) from the left end, were identical in the two virus types.

Animals↗
PubMed1980

The nucleotide sequence of the putative transcription initiation site of a cloned ribosomal RNA gene of the mouse.

Approximately one kilobase pairs surrounding and upstream the transcription initiation site of a cloned ribosomal DNA (rDNA) of the mouse were sequenced. The putative transcription initiation site was determined by two independent methods: one nuclease S1 protection and the other reverse transcriptase elongation mapping using isolated 45S ribosomal RNA precursor (45S RNA) and appropriate restriction fragments of rDNA. Both methods gave an identical result; 45S RNA had a structure starting from ACTCTTAG---. Characteristically, mouse rDNA had many T clusters (greater than or equal to 5) upstream the initiation site, the longest being 21 consecutive T's. A pentadecanucleotide, TGCCTCCCGAGTGCA, appeared twice within 260 nucleotides upstream the putative initiation site. No such characteristic sequences were found downstream this site. Little similarity was found in the upstream of the transcription initiation site between the mouse, Xenopus laevis and Saccharomyces cerevisiae rDNA.

Animals↗
PubMed1980

Cloning and determination of a putative promoter region of a mouse ribosomal deoxyribonucleic acid fragment.

An endonuclease EcoRI digest of mouse DNA was subjected to molecular cloning, after partial purification with respect to the ribosomal RNA sequence, using lambda gtWES x lambda B with an in vitro packaging technique. Twelve positive clones were obtained from approximately 2 X 10(4) plaques. One of the clones transferred to the plasmid pBR322 (PMrEL-1) was about 14.9 kb long, hybridizing only with 18S rRNA but not with 28S rRNA. Hybridization of restriction fragments and electron microscopic studies of the R-loop confirmed that this fragment carried about half of the 18S rRNA sequences at one end, suggesting that it contained the initiation site for the 45S preribosomal RNA (pre-rRNA). S1-nuclease protection mapping with hybrids between restriction fragments of the cloned DNA and the 45S pre-rRNA indicated that at least major transcription of the 45S RNA started at a site approximately 4.0 kb upstream from the 5' end of the 18S rRNA. This was confirmed by electron microscopic observations of these hybrids.

Animals↗
PubMed1980

3'-terminal processing of ribosomal RNA precursors in mammalian cells.

The 3'-terminal structures of ribosomal 28S RNA and its precursors from rat and mouse were analyzed by means of periodate oxidation followed by reduction with 3H-borohydride. 3'-terminal labeled nucleoside derivatives produced by RNase T2 digestion were determined by thin-layer chromatography and oligonucleotides generated by RNase T1 digestion were analyzed by DEAE-Sephadex chromatography. In the rat, the major 3'-terminal sequences of ribosomal 28S RNA, nucleolar 28S, 32S, 41S, and 45S RNAs were YGUoh, GZ2Uoh, GZ12Uoh, GZ2Uoh, and GZ7Goh, respectively, whereas in the mouse corresponding sequences were YGUoh, GZ1,2, or 3Uoh, Goh, Uoh and GZ 13Uoh, respectively. (Y: pyrimidine nucleoside, Z: any nucleoside other than guanosine) These results suggest that a "transcribed spacer" sequence is present at the 3'-terminus of the 45S pre-ribosomal RNA, which is gradually removed during the steps of processing.

Animals↗
PubMed1980

Conserved sequence related to the 3'-terminal region of retrovirus RNA'S In normal cellular DNAs.

The nucleotide sequences related to the 3'-terminal protion of retrovirus genomic RNA have been detected in the DNA of animals, including humans. The DNA complementary to the 400 to 700 nucleotides from the 3'-terminal end of retrovirus RNA (cDNA3'), which contains the enriched conserved region, was hybridized with DNA from a variety of animal cells. Under the conditions of annealing in 0.72 M NaCl at 67 degrees C and hydroxyapatite chromatography at 55 degrees C, 20 to 50% of the radioactivity of the cDNA3' prepared from two retroviruses, a murine Rauscher virus (RLV) and a baboon virus (M7), annealed with normal cellular DNA of animals, including human tissue. The thermal denaturation profile revealed considerable mismatching between the duplex of the cDNA3' and human DNA, cDNA3' of retroviruses is most homologous to cellular DNA of the host species of origin and is less homologous to cellular DNA of species that are distant in the phylogeny of the host species. The conservation and evolution of nucleotide sequences related to the 3' end of retrovirus genomes in animal DNAs, including humans, suggest that the sequences may have important functions.

Animals↗
PubMed1979

In vitro synthesis of a 5S RNA precursor by isolated nuclei of rat liver and HeLa cells.

Isolated rat liver nuclei were incubated under appropriate conditions in the presence of 0.5 micrograms/ml alpha-amanitin and an RNAase inhibitor prepared from cytosol fraction, together with alpha-32P-UTP or alpha-32P-CTP and three other nucleoside triphosphates. RNA extracted by an SDS-hot phenol procedure was fractionated with sucrose density gradient centrifugation followed by acrylamide gel electrophoresis. Fingerprint analysis of the in vitro synthesized "5S" RNA, which was slightly larger than mature 5S RNA on gel electrophoresis, showed that it contained all the sequences of mature 5S RNA except for the oligonucleotide at the 3' end. Instead, it contained two additional spots which were not present in mature 5S RNA. Analysis of the extra spots revealed that they were derived from the 3' end of the in vitro synthesized "5S RNA, which were sequenced tentatively as -CUUGAUGCUUoh (extra sequence underlined). The 5' end of the product was (p)pGU--. Isolated HeLa cell nuclei synthesized similar sized "5S" RNA under the same conditions. We conclude from these results that in isolated nuclei of these mammalian cells RNA polymerase III starts transcription of 5S RNA gene at the same site as the 5' end of mature 5S RNA, proceeds toward the 3' direction and stops at a site probably 8 nucleotides downstream from the 3' end of mature 5S RNA. Experiments with a short pulse and with various "chases" have demonstrated the presence of a short-lived precursor 5S RNA which is similar in size and sequence to in vitro "5S" RNA, suggesting that 5S RNA is synthesized in vivo as a longer precursor molecular as demonstrated in this in vitro system, and is rapidly processed into mature 5S RNA.

Animals↗
PubMed1979

Conserved region of mammalian retrovirus RNA.

The viral RNAs of various mammalian retroviruses contain highly conserved sequences close to their 3' ends. This was demonstrated by interviral molecular hybridization between fractionated viral complementary DNA (cDNA) and RNA. cDNA near the 3' end (cDNA(3')) from a rat virus (RPL strain) was fractionated by size and mixed with mouse virus RNA (Rauscher leukemia virus). No hybridization occurred with total cDNA (cDNA(total)), in agreement with previous results, but a cross-reacting sequence was found with the fractionated cDNA(3'). The sequences between 50 to 400 nucleotides from the 3' terminus of heteropolymeric RNA were most hybridizable. The rat viral cDNA(3') hybridized with mouse virus RNA more extensively than with RNA of remotely related retroviruses. The related viral sequence of the rodent viruses (mouse and rat) showed as much divergence in heteroduplex thermal denaturation profiles as did the unique sequence DNA of these two rodents. This suggests that over a period of time, rodent viruses have preserved a sequence with changes correlated to phylogenetic distance of hosts. The cross-reacting sequence of replication-competent retroviruses was conserved even in the genome of the replication-defective sarcoma virus and was also located in these genomes near the 3' end of 30S RNA. A fraction of RD114 cDNA(3'), corresponding to the conserved region, cross-hybridized extensively with RNA of a baboon endogenous virus (M7). Fractions of similar size prepared from cDNA(3') of MPMV, a primate type D virus, hybridized with M7 RNA to a lesser extent. Hybridization was not observed between Mason-Pfizer monkey virus and M7 if total cDNA's were incubated with viral RNAs. The degree of cross-reaction of the shared sequence appeared to be influenced by viral ancestral relatedness and host cell phylogenetic relationships. Thus, the strikingly high extent of cross-reaction at the conserved region between rodent viruses and simian sarcoma virus and between baboon virus and RD114 virus may reflect ancestral relatedness of the viruses. Slight cross-reaction at the site between type B and C viruses of rodents (mouse mammary tumor virus and RPL virus, 58-2T) or type C and D viruses of primates (M7, RD114, and Mason-Pfizer monkey virus) may have arisen at the conserved region through a mechanism that depends more on the phylogenetic relatedness of the host cells than on the viral type or origin. Determining the sequence of the conserved region may help elucidate this mechanism. The conserved sequences in retroviruses described here may be an important functional unit for the life cycle of many retroviruses.

Animals↗
PubMed1977

Heterogeneity of 5' -termini of nucleolar 45S, 32S and 28S RNA in mouse hepatoma.

The 5'-termini of nucleolar 45S, 32S and 28S RNA's were analyzed by means of thin layer chromatography and Dowex-1 colum chromatography. 45S RNA did not bear a triphosphate at the 5'-terminus, but various monophosphates are found. 5'-termini of 32S and 28S RNA's were also heterogeneous. These results indicate that 45S molecules as isolated with the conventional procedure may not contains the primary transcript of the ribosomal gene, but a collection of large precursors with different degrees of processing at the 5'-terminus. The processing of the primary transcript may thus involve some unknown trimming processes at the 5'-terminus before the first major cleavage takes place.

Animals↗
PubMed1977

Effect of cycloheximide on the synthesis and processing of 5S RNA in HeLa cells.

The synthesis and incorporation into ribosomes of 5S RNA were investigated under conditions of protein synthesis inhibition by cycloheximide. Within 1 h after addition of cycloheximide to the culture medium, the synthesis of 5S RNA in HeLa cells, as measured by the pulse labeling of total 5S RNA, was inhibited by 25-30 per cent, but the degree of inhibition did not further increase for several hours. The synthesis of 4S RNA was less markedly inhibited, especially at earlier time periods. Although the appearance of labeled 5S RNA in cytoplasmic ribosomoes after chase was inhibited by approximately 60% or more this presumably reflects the inhibition of 60S ribosomal subunit formation by cycloheximide. Some utilization of preformed 5S RNA for new ribosomes was suggested after resumption of protein synthesis. In confirmation of the results of Kumar and Wu (1), a low dose of actinomycin D inhibited the appearance of 28S RNA in the cytoplasm rather specifically, whereas cycloheximide showed an apparently opposite differential effect.

Cycloheximide↗
PubMed1976

Conservation of the 5'-terminal nucleotide sequences of ribosomal 18-S RNA in eukaryotes. Differential evolution of large and small ribosomal RNA.

The 5'-terminal nucleotide sequences of ribosomal 18-S and 28-S RNA of seven species of eukaryotes including three mammals, one bird, one amphibian, one echinoderm and one slime mold, were analyzed either by means of terminal phosphorylation of RNA with polynucleotide kinase of by fingerprint analysis of uniformly labeled RNA. The following conclusions were obtained. 1. The 5'-terminal sequences of the 18-S RNA of the mouse, chicken and Dictyostelium discoideum were pUpApCp(Cp,Up)Gp---, suggesting strongly that all the eukaryotes had this same sequence at the 5'-terminus. Preliminary analysis of the 5'-termini of the 18-S RNA from human, rat, Xenopus and sea urchin cells revealed the same pUp(Np)Gp--- type 5'-terminal structure, supporting the above hypothesis. 2. The 5'-terminal sequences of the 28-S RNA of the human, rat, mouse and chicken cells were all pCpGp---, whereas those of the lower animals such as Xenopus, sea urchin and Dictyostelium were different.

Alkaline Phosphatase↗
PubMedDate not supplied

Re-evaluation of hydatidiform mole by DNA fingerprint method: the discrepancy in the diagnoses by pathological finding and the DNA fingerprint method.

Ten cases of hydatidiform mole (HM) were analysed by the DNA fingerprint method. DNA samples were prepared from HM tissue of HM and maternal and paternal bloods, followed by digestion with HaeIII restriction endonuclease, applied to agarose gel, and then transferred to a nitrocellulose filter. The filter was hybridized using mini-satellite DNA as a probe. A case of partial HM diagnosed by histological findings was revealed to be complete HM using DNA fingerprinting. The case was suggested to be a twin pregnancy with a cHM and an abortion, because the DNA fingerprint of hydropic tissue showed a paternal pattern and that of the non-hydropic tissue did not show such a pattern. DNA fingerprinting was considered to be useful in distinguishing complete HM from partial HM.

Adult↗