Masami Muramatsu

The nucleotide sequence of human fibroblast interferon cDNA

DNA synthesized by in vitro reverse transcription of the interferon mRNA has been cloned and amplified as recombinant DNA, TpIF319-13 (Taniguchi et al., 1979). The nucleotide sequence of this IF cDNA which consists of 770 bp (excluding the A:T tails) has been determined. The data reported predict the hitherto unknown amino acid sequence of human fibroblast interferon and its putative signal peptide.

Isolation of nucleoli from rat liver in the presence of magnesium ions

A procedure for the isolation of nucleoli from rat liver in the presence of Mg2+ ions was developed. Addition of 0.05 mM MgCl2 to the 0.34 M sucrose in which isolated nuclei were suspended for sonication was found to protect nucleoli when nuclei prepared with 2.3 M sucrose containing 10 mM MgCl2 were disrupted completely by sonication. The nucleolar preparation possesses almost identical properties with those of nucleoli prepared by the conventional Ca2+-method in the following points: (1) morphological characteristics as revealed by light and electron microscopy; (2) RNADNA ratio of around 1.3; (3) sedimentation profiles and labeling patterns of nucleolar RNA; (4) polyacrylamide gel electrophoresis of acetic acid soluble proteins. Furthermore, Mg2+-nucleoli showed a higher RNA synthetic activity than Ca2+-nucleoli. In addition, this method has proved to be advantageous in the case of regenerating liver in which nuclei and nucleoli are somewhat more fragile to sonication.

Drosophila melanogaster has different ribosomal RNA sequences on X and Y chromosomes

Abstract The primary structures of ribosomal RNAs transcribed from the nucleolus organizers on X and Y chromosomes of Drosophila melanogaster were compared by RNase T 1 fingerprints made with two different systems; i.e. homochromatography on DEAE-cellulose, and polyethyleneimine-cellulose thin-layer chromatography. Ribosomal RNA derived from the X-linked nucleolus organizer was obtained from a strain producing only female larvae and ribosomal RNA derived from the Y-linked nucleolus organizer was isolated from a mutant lacking the X-linked nucleolus organizer. No difference was detected between the fingerprints of 28 S RNA from these animals. In 18 S RNA, however, one oligonucleotide showed a remarkable difference in mobility. The structure of the X-linked organizer-specific oligonucleotide was 5′ U-C-U-U-U-U-U-U-C-C-U-A-U-G 3′, and that of the Y-linked organizer-specific oligonucleotide was 5′ U-C-U-C-U-U-U-U-C-C-U-A-U-G 3′, indicating one base substitution (U a3 C) between them. The absence of 5′-temninal phosphate in this oligonucleotide and available sequence data also suggest that these oligonucleotides did not come from either the 5′ or 3′ terminus of 18 S RNA. D. simulans , whose Y chromosome has no nucleolus organizer (Ritossa & Atwood, 1966), showed an 18 S RNA fingerprint having only the X-linked organizer-specific oligonucleotide. We conclude from these results that in Drosophila the ribosomal RNA gene sequences are different for the two nucleolus organizers located on the X and Y chromosomes. The implications of those findings concerning the parallel evolution of these genes are discussed.

Nucleolar DNA-dependent RNA polymerase from rat liver. 1. Purification and subunit structure.

DNA-dependent RNA polymerase I (or A) was purified from rat liver nucleoli. DNA was effectively removed from the solubilized enzyme with a defined concentration of polyethyleneglycol. The enzyme was purified further with successive DEAE-Sephadex and phosphocellulose column chromatography followed by glycerol gradient centrifugation. The procedure yielded an electrophoretically homogeneous enzyme with a specific activity 400 times that of the nucleolar extracts. The recovery of the activity was approximately 20%. The RNA polymerase I eluted as a single peak from DEAE-Sephadex was separated into two distinct peaks by a phosphocellulose column. The first peak eluting at about 0.12 M ammonium sulfate was designated as RNA polymerase IA and the second peak eluting at about 0.18 M as RNA polymerase IB. In normal rat liver nucleoli IA enzyme comprised approximately 20% of the total RNA polymerase I activity and the IB enzyme comprised approximately 80%. On sodium dodecyl sulfate polyacrylamide gel electrophoresis, enzyme IB contained five subunits with molecular weights of 195000 (a), 130000 (b), 65000 (c), 40000 (d), and 19000 (e) at nearly equimolar amounts. The calculated molecular weight of the enzyme (449000) agreed well with that predicted from the sedimentation coefficient of the enzyme. Enzyme IA contained identical subunits except that subunit c was absent. Preliminary studies could not demonstrate any significant differences in template specificity between IA and IB enzyme.

Novel repetitive sequence families showing size and frequency polymorphism in the genomes of mice

A middle repetitive sequence, PR1, originally found in mouse rDNA appeared as satellite-like bands when EcoRI and BglII digests of genomic DNA were subjected to Southern blot hybridization using PR1 as probe. The copy number and sizes of PR1-related satellite-like bands, designated as PR1 families, differed remarkably among the subspecies and laboratory strains of mice when the EcoRI digests of genomic DNAs were compared. These bands were not detected in rat and human DNAs. A unit of PR1 sequence was determined by examining cloned EcoRI 3.5 kb (kb, 10(3) bases) fragment and 6.6 kb rDNA by cross-hybridization and sequence analysis: 3.5 kb and 6.6 kb DNAs are composed of homologous PR1 regions and the flanking non-homologous sequences. The results indicate that amplification of different sequences containing PR1 has occurred in different subspecies and strains of mice, and that the segments of satellite-like bands are likely to have been created by recombination of the PR1 sequence with other DNA segments before amplification. The chromosomal distribution of the 3.5 kb PR1 family was studied by back-crossing the female F1 between BALB/c and DDD/1 to male DDD/1. The segregation data strongly suggest that most, if not all, of this family are located on a single chromosome. The stability of these PR1 families in the genomes of cultured cells of a given strain was also examined. An extra band homologous to PR1 appeared in their genomes, but was not detected in other tissues, indicating that some PR1 families may change even during cell propagation.

In vitro effect of cycloheximide on the nucleolar and extranucleolar nuclear RNA polymerases of rat liver

Summary Cycloheximide, which has recently been reported to specifically inhibit RNA polymerase I of water mold Blastocladiella emersonii ,, did not inhibit appreciably RNA polymerase I of rat liver in vitro . This finding indicates that cycloheximide does not serve as a specific inhibitor of nucleolar RNA polymerase molecule of rat liver.

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.

Chapter 10 Isolation of Nuclei and Nucleoli1

Publisher Summary This chapter reviews critically some theoretical and practical aspects of isolation of nuclei and nucleoli.. The isolation of nuclei and nucleoli from various cells will become a more important approach to the analysis of functions of theorganelles as the molecular biology of higher organisms progresses. The replication of DNA as the gene substance and the transcription of genetic information into RNA are carried out in the nucleus. The transfer of genetic information through the nuclear membrane must also take place before specific proteins are synthesized in the cytoplasm. Determination, isolation, and characterization of essential substances in the nucleus require isolation of pure nuclei, especially when similar substances exist in the cytoplasm as well. Isolation procedures for nuclei may vary not only with the tissues or cells under study but also with the component or substance in question. For example, the citric acid procedure cannot be used when isolation of histones is a subsequent objective. Sucrose solutions at different concentrations and with various additions are most frequently used for isolation of nuclei as well as other subcellular components because this neutral, nonionic substance is not only relatively inert, but also can provide both desired tonicity and density according to the experimental purpose.