Hideya Endo

Gene affecting longevity of messenger RNA: A mutant ofEscherichia coli with altered mRNA stability

Summarywe have screened 897 temperature sensitive growth mutants ofE. coli for mutant strains showing longer mRNA half-life. The fate of pulse-labelled RNA was examined at 42° C after cessation of RNA synthesis and with prior exposure to nonpermissive temperature (42° C). Eight stains showed altered turn-over of RNA (presumably mRNA), and further analysis on mutant strain JE15144 indicated that the stability of pulse-labeled RNA as well as of tryptophan (trp) mRNA increased four to seven fold over its parental strain at 42° C. At 4 min or 10 min after addition of rifampicin, some 70 to 80% of polyribosome in the growing cells could still be conserved in JE15144 cultured at the nonpermissive temperature while little, if any, polyribosomes remained in its parental strain (PA3092) under the same condition. Two generation times were required for complete stoppage of growth of this mutant strain after shifting to 42° C, and protein synthesis continued at a significant, but slightly reduced, rate at 42° C. However, functional decay of mRNA in the mutant strain, with respect to the capacity for producing peptides, appeared to be similar to the parent strain, with half-lives of 3.5 min in PA3092 and 4.7 min in JE15144.

Reversal of heparin inhibition of nuclear protein kinase NII by polyamines and histones

Evidence is accumulating to suggest that the phosphorylation of histone and non&stone chromosomal proteins plays a key role in the regulation of the eukaryotic gene expression. Many of the non-h&tone chromosomal proteins are phosphoproteins and their phosphorylation is catalysed by protein kinases including both cyclic AMP-dependent and -independent protein kinases [l-3]. Two cyclic AMP-independent nuclear protein kinases, NI and NII [4], have been purified from rat liver nuclei and characterized [5,6]_ These two kinases, involved mainly in the phosphorylat~on of non-histone chromosomal proteins, are not regulated by direct acting effecters [7]. Cytosol cyclic AMP-independent protein kinases from rabbit reticulocytes and bovine adrenal cortex were found to be inhibited by glycosaminoglycan [8,9]. We now report that nuclear protein kinase NII is also selectively inhibited by heparin, a glycosa~noglycan, when either casein or phosvitin is used as the substrate, while no inhibition occurs with histone. Histone is rather effective in the recovery of the enzyme activity inhibited by heparin. A similar release of inhibiton by heparin was seen with spermine and spermidine, but not with putrescine. Analysis with heparin-Sepharose affinity chromatography suggested that polyamines exert the effect without ~fluencing the binding between heparin and the enzyme.

Cloning and characterization of a single-stranded DNA binding protein that specifically recognizes deoxycytidine stretch

We previously identified a G-rich silencer element involved in negative regulation of catalase gene expression in some hepatoma cells (Mol. Cell. Biol., (1992), 12, 2525-2533). To study a nuclear binding protein for this element, we screened cDNA libraries from a rat ascites hepatoma cell line by binding with a synthetic oligonucleotide probe and obtained several clones. One of them, designated SW, was studied in detail. A clone (SW2) of this series contained a near full length cDNA encoding a putative peptide with 463 amino acid residues. We isolated this peptide as a fusion protein. It was found that the protein strongly bound to the C-stretch of the DNA sequence in a single strand specific fashion, but absolutely did not to G-rich sequence. The protein bound weakly to the corresponding double-stranded DNA as well as to C-rich RNA sequence. This protein, though not the expected one, was found to be a novel protein whose DNA binding domain was located on the region containing at least 75 amino acid residues of the carboxyl terminus. A proline rich region was also observed in the middle part of the protein. Northern blot profiles indicated extensive and slight expression of both 2.0 kb and 2.7 kb mRNA species in some hepatoma cell lines and in the rat liver, respectively.

Potentiation of 5-Fluorouracil, Chromomycin A3, and Bleomycin by Amphotericin B or Polymyxin B in Transformed Fibroblastic Cells

By using cultured, transformed fibroblastic cells, antitumor agents including cytosine arabinoside, thio-tepa, nitrogen mustard N-oxide, mitomycin C, chromomycin A3, 5-fluorouracil, daunomycin, vinblastine, vincristine, and bleomycin A2 were screened for the potentiation of their activity by amphotericin B or polymyxin B. Among them, the action of 5-fluorouracil and chromomycin A3 on ribonucleic acid synthesis was potentiated in their action by amphotericin B, not by polymyxin B. Bleomycin A2 was potentiated in its action on deoxyribonucleic acid and ribonucleic acid synthesis only by polymyxin B.

Isolation and characterization of amphotericin B-resistant cell lines in Chinese hamster cells

Amphotericin B is a polyene macrolide antibiotic which interacts specifically with sterols in mammalian cell membranes. Amphotericin B-resistant (AMBr) lines of stable phenotype have been isolated from cultured Chinese hamster (V79) cells. Three AMBr clones (AMBr-1, -2 and -3) isolated independently after treatment with nitrosoguanidine were resistant to greater than or equal to microgram/ml of the antibiotic, while DNA synthesis as well as the colony-forming ability of the parental V79 cells was blocked by greater than 80% of control in the presence of 20--50 microgram/ml amphotericin B. The AMBr cell line also exhibited increased resistance to other polyene macrolide antibiotics such as nystatin and pentamycin. Other agents, however, such as cytosine arabinoside or ricin, blocked DNA synthesis in AMBr cells to the same extent as in V79 cells. The amphotericin B resistance phenotype was stably retained even after AMBr cells were cultured in the absence of the drug for over 200 generations. The control of free cholesterol or its esters was significantly decreased in all three resistant clones. Furthermore, cholesterol synthesis from acetate as well as mevalonate was partly defective in AMBr cells, compared with that in V79 cells.

FUNCTIONAL ANALYSIS OF MULTIPLE PROMOTERS OF THE RAT INSULIN-LIKE GROWTH FACTOR II GENE

We have characterized the multiple promoters of the rat insulin-like growth factor II (rIGFII) gene by in vivo transient expression assay using a series of deletion mutant templates. Among the four promoters (P1, P2, P3 and P6), two (P2 and P3) showed relatively strong promoter activities compared with the other two. One of the four promoters, P2, was further characterized by gel band-shift and footprinting analysis using HeLa cell nuclear extract, showing two retarded bands and at least one protected sequence stretch. The results indicated that P2 has a very simple structure like P3, and consists of no more than 141 base-pairs (bp) including a TATA box and two GC core hexanucleotides. Promoter strength shown by in vivo transient expression in different cell types failed to explain the differential employment of P2 and P3 in these cells, suggesting the involvement of other regulatory mechanisms that might operate only in the native state.

Mutagenesis of bacteriophage T4 by a carcinogen, 4-nitroquinoline 1-oxide

Abstract The mutagenic behaviour of a carcinogen, 4-nitroquinoline 1-oxide (4NQO), was investigated in bacteriophage T4. 4NQO was mutagenic for intracellular but not for extracellular phages. Mutations induced in the r II region of T4 by treatment of intracellular phages with 4NQO were classified according to their susceptibilities to induced reversion by 2-aminopurine, 5-bromodeoxyuridine, hydroxylamine, proflavine, and 4NQO. More than half the mutants were of the transition type revertible with the base analogues, but nearly all of these failed to respond to hydroxylamine mutagenesis. These were therefore classified as mutants of a transition class presumably carrying an AT base pair at the mutant site. None of the induced mutants was capable of reverting with 4NQO or proflavine. These results suggest that the guanine-hydroxymethylcytosine base pair of phage T4DNA is the major mutagenic target in the induction of transition mutations by 4NQO.

The identity of the aldolases isolated from rat muscle and primary hepatoma.

Abstract Muscle type aldolase 2 was purified in crystalline form from the primary tumor in rat liver induced by 3′-methyl-4-dimethylaminoazobenzene (3′-Me-DAB), and its properties were compared with those of aldolase from normal rat muscle and liver. Aldolases from muscle and primary hepatoma were found to be identical but markedly different from liver aldolase in electrophoretic mobility on cellulose acetate and isoelectric fractionation profile. No significant differences were detected in amino acid composition and catalytic properties of muscle and hepatoma enzymes determined by the following parameters; Km and Vmax values for fructose-1,6-diphosphate (FDP) and fructose-1-phosphate (F1P), FDP F 1 P activity ratio, and inhibition by adenosine triphosphate (ATP), which were in remarkable contrast to those of liver aldolase. In immunological properties hepatoma aldolase was identical with muscle aldolase but different from that of normal liver.

Methylguanidine, a Naturally Occurring Compound showing Mutagenicity after Nitrosation in Gastric Juice

Druckrey1 and Sander2 suggested that N-nitroso compounds may be formed intragastrically by reaction of nitrites with secondary amines or alkylureas present in ingested foods and that these natural compounds may hence be significantly carcinogenic in man. Sugimura et al.3–5 found that the mutagen, N-methyl-N′-nitro-N-nitrosoguanidine (MNNG)6,7 induced a high frequency of tumours in the glandular region of the rodent and dog stomach on oral application. MNNG is synthesized by nitrosation of N-methyl-N′-nitroguanidine (MNG) and the reaction occurs not only in strongly acidic conditions8 but also in human gastric juice (to be published). Therefore ‘spontaneous’ human gastric cancers may be at least partly caused by MNNG-like compounds formed after intragastric nitrosation of chemicals structurally related to MNG. We examined the mutagenicity of naturally occurring guanidines after treatment with nitrite in real and in simulated human gastric juice and found that methylguanidine, a compound present in several foods, is converted into a potent mutagen after nitrosation in gastric juice.

Potentiation of fusidic acid and lentinan effects upon normal and transformed fibroblastic cells by amphotericin B

Abstract Amphotericin B potentiated effects of fusidic acid and lentinan on macromolecular synthesis in cultured fibroblastic cells, which were resistant to each agent used alone.