Takumi Hatayama

Common antigenicity of mouse 42°C-specific heat-shock protein with mouse hsp 105

Abstract Mammalian cells incubated at 42°C synthesize a specific heat-shock protein at 42°C (42°C-hsp) that is not induced by heat-shock at 45°C or by other stresses that induce major heat shock proteins (Hatayama et al. (1986) Biochem. Biophys. Res. Commun. 137, 957–963). Antibody raised against a heat-shock protein with molecular weight of 105,000 (hsp 105) purified from mouse FM 3A cells cross-reacted to the 42°C-hsp of the same cells. The antibody reacted only weakly to hsp 105 and 42°C-hsp of human HeLa cells. These results suggested that hsp 105 and 42°C-hsp have the same antigenic determinant, and that 42°C-hsp may have a structure similar to that of hsp 105.

Induction of mRNAs for heat shock proteins in livers of rats after ischemia and partial hepatectomy.

SummaryWhen the body temperature of rats is elevated to 42°C, four heat shock proteins, with the molecular weights of 70000, 71000, 85000, and 100000 (hsp 70, hsp 71, hsp 85, and hsp 100, respectively), are induced in various tissues of rats (Fujio et al., J Biochem 101, 181–187, 1987). Heat shock proteins are induced by various stresses other than heat in varieties of cultured cells, so we studied whether heat shock proteins are induced in intact rats by different treatments. Analysis of the translation products of poly(A) + RNA isolated from the livers of rats recovering from ischemia of the liver showed that mRNAs for hsp 70, hsp 71, and hsp 85 were induced. These hsp-mRNAs were also induced in the livers of rats 6 h after a partial hepatectomy, and had returned to control levels 24 h after the surgery. These results suggested that heat shock proteins have not only the function of protection against various stresses but also physiological functions in the normal growth and development of animals.

HeLa cells synthesize a specific heat shock protein upon exposure to heat shock at 42°C but not at 45°C

Abstract Upon exposure to heat shock, HeLa cells synthesize a small set of proteins having the molecular weights of 70,000, 73,000, 78,000, 85,000, 92,000, and 105,000. In addition to these proteins, we found an unusual heat shock protein induced by heat shock at 42°C, but not at 45°C. The 42°C-specific protein, the molecular weight of which was 90,000, was not produced in control cells and the induction of the protein was completely inhibited by actinomycin D. The protein was not induced by other treatments that induced most heat shock proteins. Thus, this 42°C-specific protein seems to have a peculiar induction mechanism and a specific function in the cells.

Affinity labeling of the ribonucleic acid component adjacent to the peptidyl recognition center of peptidyl transferase in Escherichia coli ribosomes.

N-Iodacetylphenylalanyl-tRNA was used as an affinity label for localizing the RNA components intimately related to the peptidyl transferase activity of Escherichia coli ribosomesmthis analogue could specifically alkylate a unique nucleotide chain of 23-S RNA. The alkylation was strongly enhanced by poly(U), and was dependent on the presence of both 50- and 30-S subunits; Chloramphenicol inhibited the reaction, wheras blasticidin S stimulated it. The alkylated RNA base was found to be adenine. The nucleotide chain attacked by N-iodoacetylphenylalanyl-tRNA seemed to be localized at or near to the peptidyl recognition center of peptidyl transferase.

Transcribing chromatin is not preferentially enriched with acetylated histones

Chromatin fragments of the RNA polymerase II‐transcriptional complex were purified from the micrococcal nuclease digest of rat liver nuclei in the presence of n‐butyrate, a potent histone deacetylase inhibitor. Polyacrylamide gel electrophoretic analysis in Triton acid—urea revealed that the extent of histone acetylation of the complex did not differ markedly from that of the total chromatin.

Action of neocarzinostatin on cell nuclei: Release of specific chromatin

Abstract Neocarzinostatin solubilized chromatin from rat liver nuclei in the presence of 2-mercaptoethanol with little increase in acid-soluble materials. When DNA from the chromatins was electrophoresed on neutral and alkaline agarose gels, a series of bands with a multiple of a monomeric nucleosomal unit was observed, and the ratio of double-strand to single-strand breaks of DNA in chromatin was high (1:7) as compared with findings in the case of purified DNA (1:30). Hybridization analysis of the DNAs with cDNA of liver polysomal poly(A)+mRNA revealed that neocarzinostatin solubilized a specific chromatin and which differed from the active chromatin which was preferentially excised by micrococcal nuclease.

Partial characterization of RNA polymerase II complex released by micrococcal nuclease digestion of rat liver nuclei

Two forms of RNA polymerase II were released from rat liver chromatin by micrococcal nuclease digestion of the nuclei. One from behaved like a free RNA polymerase II and the other like a complex with other nuclear components. Both forms of RNA polymerase II activity were recovered in the 0.16 M NaCl-soluble fraction of the nuclear digest, and the complexed from the RNA polymerase II could transcribe its endogenous template under conditions permitting only of elongation of the RNA synthesis. The RNA polymerase II complex was further purified by gel filtration chromatography and column electrophoresis. Analysis of protein and DNA of the partially purified complex suggested that the RNA polymerase II was bound to mono- or dinucleosomes carrying some characteristic nonhistone proteins. Furthermore, in experiments on tissues from starved rats, the two forms of RNA polymerase II were found to originate from different functional states of the chromatin-bound enzyme in vivo.

Mode of inhibition of DNA replication in neocarzinostatin-treated HeLa cells

The effect of antitumor antibiotic neocarzinostatin on DNA replication in HeLa cells was studied by pulse-labeling of DNA with [3H]thymidine and sedimentation analysis of the DNA with alkaline sucrose gradients. The drug, which produced DNA damage, primarily inhibited the replicon initiation in the cells at low doses (less than or equal to 0.1 microgram/ml), and at high doses (greater than or equal to 0.5 microgram/ml) inhibited the DNA chain elongation. An analysis of the number of single-strand breaks of parental DNA, induced by neocarzinostatin, indicated that inhibition of the initiation occurred with introduction of single-strand breaks of less than 1.5 . 10(4)/cell, while inhibition of the elongation occurred with introduction of single-strand breaks of more than 7.5 . 10(4)/cell. Assuming that the relative molecular mass of DNA/HeLa cell was about 10(13) Da, the target size of DNA for inhibition of replicon initiation was calculated to be about 10(9) Da, such being close to an average size of loop DNA in the cell and for inhibition of chain elongation, 1-2 . 10(8) Da which was of the same order of magnitude as the size of replicons. Recovery of inhibited DNA replication by neocarzinostatin occurred during post-incubation of the cells and seemed to correlate with the degree of rejoining of the single-strand breaks of parental DNA. Caffeine and theophylline enhanced the recovery of the inhibited replicon initiation, but did not aid in the repair of the breaks in parental DNA.

Two species of chromatin-RNA polymerase II complex released from rat liver nuclei by nuclease digestion.

Abstract To investigate the structural organization of transcribing chromatin, rat liver nuclei were washed thoroughly to remove the free RNA polymerase and then digested with micrococcal nuclease or DNAase I. After extensive digestion with the nuclease in the presence of 0.1 to 0.25 m m -divalent cation, two species of the chromatin-RNA polymerase II complexes were separated by subsequent chromatography on a column of Bio-Gel A-15m (peak 1 and peak 2). The peak 1 complexes, recovered at the oligonucleosomal region, were RNA polymerase II-nucleosome monomer or dimer complexes. These complexes were further characterized by the possession of factors that enhanced chromatin transcription, and seemed to associate with ribonucleoprotein particles. The peak 2 complexes, eluted at the position of purified RNA polymerase II molecules, contained endogenous template, but were apparently not organized in the nucleosomal structure. The RNA polymerase II molecules in both complexes were found to be associated with nascent RNA synthesized in vivo . When the digestion with micrococcal nuclease was performed in the presence of 3 m m -MgCl 2 , peak 2 was recovered as a relatively large chromatin (S 2 -chromatin), while peak 1 was not extracted and remained in the residual chromatin (P 2 -chromatin). Analysis of RNA transcripts synthesized from these chromatins indicated that the two forms of the enzyme transcribed different DNA sequences, and both of the products were found in the polysomal poly(A) + messenger RNA. These lines of evidence suggest that there are separate RNA polymerase II transcriptional systems, which transcribe different DNA sequences of the genome.

Identification of the 30 S protein adjacent to peptidyl transferase catalytic center of Escherichia coli ribosomes.

Iodoacetylphenylalanyl-tRNAPhe was used as an affinity label to localize the ribosomal components involved in the peptidyl transferase catalytic center of Escherichia coli ribosomes. When labeling was carried out at pH 5.0, the affinity label could specifically label the ribosomal components which comprise the catalytic center. Analysis of ribosomal proteins which had reacted with the affinity label revealed that a 30 S subunit protein, S 20, was located at or near to the ribosomal binding site of the 3′-terminus of aminoacyl- or peptidyl-tRNA.