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In its recommendations for the protection of people living in long-term contaminated territories after a nuclear accident, the International Commission on Radiological Protection (ICRP) emphasizes the effectiveness of directly involving the affected population and local professionals in the management of the situation, and the responsibility of authorities at both national and local levels to create the conditions and provide the means favouring the involvement and empowerment of the population. In this perspective, ICRP initiated in the fall 2011 a dialogue between representatives of the Fukushima Prefecture, local professionals, local communities and representatives of Belarusian, Norwegian and French organisations to find ways to respond to the challenges of the long-term rehabilitation of the living conditions in the territories affected by the Fukushima nuclear power plant. After a first series of twelve “ICRP Dialogue” meetings between 2011 and 2015 closed by an international workshop, a group of local stakeholders took over and organized a new series of eight “Fukushima Dialogue” meetings between 2016 and 2018. The article gives an overview of the dialogue meetings initiated by ICRP in Japan after the Fukushima nuclear accident.

Current knowledge of stem cell characteristics, maintenance and renewal, evolution with age, location in ‘niches’, and radiosensitivity to acute and protracted exposures is reviewed regarding haematopoietic tissue, mammary gland, thyroid, digestive tract, lung, skin, and bone. The identity of the target cells for carcinogenesis continues to point to the more primitive and mostly quiescent stem cell population (able to accumulate the protracted sequence of mutations necessary to result in malignancy), and, in a few tissues, to daughter progenitor cells. Several biological processes could contribute to the protection of stem cells from mutation accumulation: (1) accurate DNA repair; (2) rapid induced death of injured stem cells; (3) retention of the intact parental strand during divisions in some tissues so that mutations are passed to the daughter differentiating cells; and (4) stem cell competition, whereby undamaged stem cells outcompete damaged stem cells for residence in the vital niche. DNA repair mainly operates within a few days of irradiation, while stem cell replications and competition require weeks or many months depending on the tissue type. This foundation is used to provide a biological insight to protection issues including the linear-non-threshold and relative risk models, differences in cancer risk between tissues, dose-rate effects, and changes in the risk of radiation carcinogenesis by age at exposure and attained age.

Twelve high schools in Japan (of which six are in Fukushima Prefecture), four in France, eight in Poland and two in Belarus cooperated in the measurement and comparison of individual external doses in 2014. In total 216 high-school students and teachers participated in the study. Each participant wore an electronic personal dosimeter 'D-shuttle' for two weeks, and kept a journal of his/her whereabouts and activities. The distributions of annual external doses estimated for each region overlap with each other, demonstrating that the personal external individual doses in locations where residence is currently allowed in Fukushima Prefecture and in Belarus are well within the range of estimated annual doses due to the terrestrial background radiation level of other regions/countries. © 2016 IOP Publishing Ltd.

In interphase cells of fission yeast, the spindle pole body (SPB) is thought to be connected with chromosomal centromeres by an as yet unknown mechanism that spans the nuclear membrane. To elucidate this mechanism, we performed two-hybrid screens for proteins that interact with Kms1 and Sad1, which are constitutive membrane-bound components of the SPB that interact with each other. Seven and 26 genes were identified whose products potentially interact with Kms1 and Sad1, respectively. With the exception of Dlc1 (a homolog of the 14-kDa dynein light chain), all of the Kms1 interactors also interacted with Sad1. Among the genes identified were the previously known genes rhp9 +/ crb2 +, cut6 +, ags1 +/ mok1 +, gst3 +, kms2 +, and sid4 +. The products of kms2 + and sid4 + localize to the SPB. The novel genes were characterized by constructing disruption mutations and by localization of the gene products. Two of them, putative homologues of budding yeast UFE1 (which encodes a t-SNARE) and SFH1 (an essential component of a chromatin-remodeling complex), were essential for viability. Two further genes, which were only conditionally essential, genetically interact with sad1 + . One of these was named sif1 + (for Sad1-interacting factor) and is required for proper septum formation at high temperature. Cells in which this gene was overexpressed displayed a wee -like phenotype. The product of the other gene, apm1 +, is very similar to the medium chain of an adaptor protein complex in clathrin-coated vesicles. Apm1 appears to be required for SPB separation and spindle formation, and tended to accumulate at the SPB when it was overproduced. It was functionally distinct from its homologues Apm2 and Apm4. Other novel genes identified in this study included one for a nucleoporin and genes encoding novel membrane-bound proteins that were genetically related to Sad1. We found that none of the newly identified genes tested were necessary for centromere/telomere clustering.

We have isolated a fission yeast karyogamy mutant, tht1, in which nuclear congression and the association of two spindle pole bodies occurs but the subsequent fusion of nuclear envelopes is blocked. The tht1 mutation does not prevent meiosis, so cells execute meiosis with two unfused nuclei, leading to the production of aberrant asci. The tht1+ gene was cloned and sequenced. Predicted amino acid sequence has no significant homology to previously known proteins but strongly suggests that it is a type I membrane protein. The tht1+ gene is dispensable for vegetative growth and expressed only in conjugating cells. Tht1p is a glycoprotein susceptible to endoglycosilase H digestion. Site- directed mutagenesis showed that the N-glycosylation site, as well as the COOH-terminal region of Tht1p, is essential for its function. A protease protection assay indicated that the COOH terminus is cytoplasmic. Immunocytological analysis using a HA-tagged Tht1p suggested that the protein is localized in nuclear envelopes and in the ER during karyogamy and that its levels are reduced in cells containing fused nuclei.

To identify genes that contribute to the manifestation of rheumatoid arthritis we performed association studies via microsatellite analyses of immunorelevant loci (HLA-DRB, 5 T cell receptor loci, TNFa IL1, IL2, IL5R and CD40L). A total of 183 patients and 275 healthy controls were typed in terms of HLA and grouped according to the known predisposing HLA-DRB1 genes (DRB1*04; relative risk approx. 5; DRB1*01, relative risk approx. 2; a third group carried neither allele). Microsatellite polymorphisms characterizing the TCRBV6S3, CD3D, IL1A, IL2, and IL5R genes did not show significant associations with rheumatoid arthritis, whereas TCRBV6S1, TCRBV6S7, TNFa, and CD40L genes may influence relative protection or risk in certain groups of patients. Analysis of a microsatellite marker adjacent to the transcription element alpha (TEA) in the T cell receptor alpha delta complex indicates that in the cohort carrying neither the DRB1*04 nor the DRB1*01 allele the relative risk to acquire rheumatoid arthritis is increased (13) or decreased (0.07), depending on the inherited microsatellite allele adjacent to the TEA locus. Sequence analysis of the closely linked TEA region from patients and controls revealed a novel dimorphism. Only the newly identified TEA allele leads to binding of a nuclear protein that may be involved in the regulated expression of the TCRDA genes. Subsequent typing of rheumatoid arthritis patients and controls revealed, however, that the association of the microsatellite marker is largely independent of the TEA allele, confirming incomplete linkage in the 2 kb region of the TCRDA locus. These results are discussed in the context of hot spots of recombination in this genomic region and other linked candidate sequences that predispose to develop rheumatoid arthritis.

To identify the genes required to sustain aneuploid viability, we screened a deletion library of non-essential genes in the fission yeast Schizosaccharomyces pombe, in which most types of aneuploidy are eventually lethal to the cell. Aneuploids remain viable for a period of time and can form colonies by reducing the extent of the aneuploidy. We hypothesized that a reduction in colony formation efficiency could be used to screen for gene deletions that compromise aneuploid viability. Deletion mutants were used to measure the effects on the viability of spores derived from triploid meiosis and from a chromosome instability mutant. We found that the CCR4-NOT complex, an evolutionarily conserved general regulator of mRNA turnover, and other related factors, including poly(A)-specific nuclease for mRNA decay, are involved in aneuploid viability. Defective mutations in CCR4-NOT complex components in the distantly related yeast Saccharomyces cerevisiae also affected the viability of spores produced from triploid cells, suggesting that this complex has a conserved role in aneuploids. In addition, our findings suggest that the genes required for homologous recombination repair are important for aneuploid viability., Author Summary Aneuploidy is a major cause of abortive development and is implicated in tumorigenesis in humans. Recent studies revealed that the increased need for protein degradation might account for the detrimental effects of aneuploidy on a cell. Here, we investigated the genetic systems responsible for aneuploid viability. Using a collection of gene deletions in fission yeast, we isolated mutants that affect aneuploid viability. We found that an evolutionarily conserved transcription regulator, the CCR4-NOT complex, and its related factors are required for aneuploid viability, suggesting that regulation of mRNA turnover is required to tolerate aneuploidy. In addition, homologous recombination repair is important for aneuploid viability.

Inhibitory effects of neomycin administration (10-80 mg/kg body weight/day, s.c. for 7 days) on several enzyme activities of kidney and duodenal mucosa were compared between male rats and mice. In Wistar rat kidney, tubular brush border Mg(2+)-dependent, HCO3(-)-stimulated ATPase activity was inhibited by neomycin in a dose-dependent manner, while microsomal Mg(2+)-Na(+)-K(+)-ATPase activity and cytosol carbonic anhydrase (CA) activity were inhibited only by larger doses. In rat duodenal mucosa, Mg(2+)-HCO3(-)-ATPase and CA activities were also inhibited only by larger doses. Serum urea nitrogen (UN) concentration and urinary N-acetyl-beta-D-glucosaminidase (NAG) activity were increased in a dose-dependent manner. In ddY and C57black mice, however, all enzyme activities in kidney and duodenal mucosa were almost unaffected by any dose of neomycin and showed no changes in serum UN concentration and urinary NAG activity except for ddY mice in which NAG activity was only increased by the largest dose of neomycin. In light microscopic analysis, 80 mg neomycin/kg produced necrosis in the greater part of rat proximal tubuli with no changes in duodenal brush border. On the other hand, no histological changes were produced in the renal cortex or duodenal mucosa of mice by any dosage. In conclusion, there are organ-specific and species differences in the effects of neomycin between rats and mice.

Ad4BP, a zinc finger DNA-binding protein, was identified as a transcription factor regulating steroidogenic P-450 genes in a cAMP-dependent manner. Immunochemical and immunohistochemical studies with steroidogenic tissues, adrenal, ovary, and testis, were performed using the antiserum to Ad4BP. Ad4BP was expressed to the same extent in the three zones of the adrenal cortex. Immunohistochemical examination of ovarian follicle and corpus luteum showed the expression of Ad4BP. The granulosa and thecal cells, the two distinct types of the steroidogenic cells in the follicle, gave Ad4BP signals, which were stronger than in the latter cells than in the former. Immunoblot analyses of mature and regressed corpora lutea indicated a parallel expression of Ad4BP and side-chain cleavage P-450, and both proteins significantly decreased in the regressed tissues. Leydig cells surrounding seminiferous tubules gave clear immunostaining signals for Ad4BP. ELP, a mammalian counterpart of Drosophila FTZ-F1 detected in EC cells, and are isoforms transcribed from the same gene. The Ad4BP and ELP forms recognize same nucleotide sequences. Reverse transcriptase-polymerase chain reaction with specific primers for ELP revealed that steroidogenic tissues contained ELP as well as Ad4BP. The effects of the two proteins on the transcription of the CYP11B gene were compared using the expression vectors of Ad4BP and ELP. ELP did not activate transcription and showed a weak inhibitor effect on the Ad4BP-dependent transactivation of the CYP11B gene promoter when transfected simultaneously. A gel shift analysis using in vitro synthesized Ad4BP and ELP revealed that the binding activity of ELP is significantly weaker than that of Ad4BP.

Study of DNA methylation of the c-myc gene in liver tumors of BCF1 mice treated with various agents revealed frequent alteration from that in normal liver. The alteration, however, was complex and showed either an increase or a decrease of methylation to various degrees. On the other hand, the tumors obtained from middle-aged C3H/He mice, which develop liver tumors spontaneously at a high incidence rate starting in middle age, showed predominantly increases of methylation in the c-myc gene, while the large tumors found in older mice revealed decreases. The normal part of the liver showed a slight gradual age-dependent increase. These suggest that hypermethylation of the c-myc gene is common to aging and early tumor development in liver. Thus the alteration of DNA methylation seems to be a good clue to investigate why the tumor incidence rate increases rapidly as individuals grow older.

Carbonic anhydrase (CA) and Mg2+-dependent ATPase and Mg2+-dependent, HCO3−-dependent ATPase (Mg2+-HCO3−-ATPase) activities in rat duodenal mucosa and kidney cortex were examined with respect to thyroidal status. Administration of 50 and 150 μg thyroxine (T4)/kg per day s.c. for 7 days decreased duodenal cytosol CA activity to 66% of control with the former and 43% with the latter dose, while Mg2+-HCO3−-ATPase activity in brush borders of duodenal mucosa was increased to 116% of control by 150 μg T4/kg. CA and Mg2+-HCO3−-ATPase activities in the cytosol and brush border of kidney cortex did not change after administration of T4. Hypothyroidism induced by thyroidectomy for 2 and 4 weeks or administration of methimazole (2·5–20 mg/kg per day s.c. or peroral) for 2, 3 and 4 weeks all increased duodenal cytosol CA activity, to about 140% at 2 weeks and 153% at 4 weeks after thyroidectomy, and to about 136% after the oral administration of 10 mg methimazole/kg per day for 4 weeks, while brush border Mg2+-HCO3−-ATPase activity was decreased to 56% of control 4 weeks after thyroidectomy and to 74% after the s.c. administration of 20 mg methimazole/kg per day for 3 weeks. The increase in CA activity and the decrease in ATPase activity after thyroidectomy were restored to normal levels by replacement with T4. Neither enzyme activity in the kidney changed in hypothyroidism. Serum concentrations of T4 and cortisol-like material increased after administration of T4, and serum concentrations of T4, aldosterone and cortisol-like material all decreased in hypothyroidism. Correlations were observed between duodenal CA and Mg2+-HCO3−-ATPase activities and serum concentrations of T4 (P < 0·01). These results reveal that the decrease in CA activity and the increase in Mg2+-HCO3−-ATPase activity of duodenal mucosa in hyperthyroidism are reversed in hypothyroidism, while both enzyme activities in the kidney are unrelated to thyroidal status. Journal of Endocrinology (1990) 126, 119–129

Cloning and sequencing of the ure1 + gene of Schizosaccharomyces pombe indicated that it encodes the urease which had been biochemically identified. The fission yeast urease has a one-subunit structure like those from plants but different from bacterial ureases which are composed of two or three distinct subunits. Genetic analyses showed that the ure1 + gene product is actually involved in urea metabolism.

Transcription factors p53 and nuclear factor kappaB (NF-kappaB) have been implicated in apoptosis induced by DNA-damaging agents, but the relationship between these two factors at the molecular level is largely unknown. We have isolated apoptosis-resistant mutant sublines from a radiosensitive mouse lymphoma 3SB cell line that undergoes p53-depen-dent apoptosis after X-ray irradiation, and we have analyzed the NF-kappaB activity. Two of these apoptosis-resistant sublines expressed mutant p53 protein and exhibited a defect in the induction of cyclin-dependent kinase inhibitor p21 after X-ray irradiation. A decrease in the DNA binding activity of NF-kappaB was observed in the parental 3SB cells after exposure to X-rays, whereas the same activity was unaffected by radiation in the two mutant sublines. A similar down-regulation of NF-kappaB activity by X-rays was observed in thymocytes derived from p53 wild-type and heterozygous mice, but not in thymocytes from p53 homozygous knock-out mice. These results suggest that NF-kappaB inactivation is p53 dependent and is required for X-ray-induced apoptosis in thymic lymphoma cells and normal thymocytes.

Male F(1) hybrids between MSM mice carrying a deficient p53 allele and BALB/c mice were irradiated with gamma-rays, and 80 thymic lymphomas were obtained, 46 of which developed in mice carrying the deficient p53 allele. Because the Y chromosome contributes little to cellular function, the stability of the Y chromosome in the tumors was assessed by polymerase chain reaction by examining three genes: Smcy and Sry on the short arm and Sts in the pseudoautosomal region of the long arm of the Y chromosome. Twenty-one lymphomas had lost one or two genes, probably as a result of mitotic recombination or interstitial deletion, whereas no lymphomas had lost all three genes. The p53 status of the lymphomas was determined by genotyping and allelic loss analysis; 34 had retained two wild-type p53 alleles, suggesting normal function; 34 had lost both alleles, indicating loss of function; and the other 12 had at least one wild-type p53 allele, so their p53 status was unclear. Compilation of these data revealed that changes in the Y chromosome were detected in only two of the 34 lymphomas retaining functional p53 but in 18 of the 34 lymphomas lacking p53 function, suggesting that p53 deficiency leads to an increase in the accumulation of radiation-induced aberrant chromosomes. This is consistent with our previous result from analysis of the inactive X chromosome. In contrast, a decrease in the fidelity of mitotic transmission in p53-deficient lymphomas was not noted for the Y chromosome.

F1 offspring of male MSM male mice with a p53-deficient (knockout) allele and normal female BALB/c mice were backcrossed with MSM mice to produce N2 mice. Female F1 and N2 mice were irradiated with gamma-radiation, and thymic lymphomas were obtained from 69 F1 and 82 N2 mice heterozygous for X chromosome markers. Of these 151 mice, 91 carried a p53-deficient allele. These lymphomas were analyzed for allelic loss by using four marker loci distributed on X chromosome to assess the stability of the inactive X chromosome, which contributes little to cellular functions. Twenty lymphomas showed allelic loss of all four loci, suggesting loss of a whole inactive X chromosome due to mitotic nondisjunction, whereas 24 lost only a part of an X chromosome, as a result of somatic recombination. The p53 status of the lymphomas was determined by genotyping and allelic loss analysis: 53 had retained two wild-type p53 alleles, suggesting normal function; 69 had lost both alleles, indicating loss of function; and the remaining 29 had at least one wild-type p53 allele, so their p53 status was unclear. Compilation of these two data revealed one nondisjunction-type change and five recombination-type mutations on X chromosome in 53 lymphomas retaining functional p53. In contrast, 14 and 16 of these alterations, respectively, were observed in 69 lymphomas lacking p53 function. These results suggest that p53 loss significantly increases the accumulation of recombinant chromosomes and decreases the fidelity of mitotic chromosome transmission of the X chromosome in gamma-ray-induced lymphomas.

In the meiotic prophase nucleus of the fission yeast Schizosaccharomyces pombe, chromosomes are arranged in an oriented manner: telomeres cluster in close proximity to the spindle pole body (SPB), while centromeres form another cluster at some distance from the SPB. We have isolated a mutant, kms1, in which the structure of the meiotic prophase nucleus appears to be distorted. Using specific probes to localize the SPB and telomeres, multiple signals were observed in the mutant nuclei, in contrast to the case in wild-type. Genetic analysis showed that in the mutant, meiotic recombination frequency was reduced to about one-quarter of the wild-type level and meiotic segregation was impaired. This phenotype strongly suggests that the telomere-led rearrangement of chromosomal distribution that normally occurs in the fission yeast meiotic nucleus is an important prerequisite for the efficient pairing of homologous chromosomes. The kms1 mutant was also impaired in karyogamy, suggesting that the kms1+ gene is involved in SPB function. However, the kms1+ gene is dispensable for mitotic growth. The predicted amino acid sequence of the gene product shows no significant similarity to known proteins.

Acute promyelocytic leukemia (APL) has been characterized by 15;17 chromosomal translocation, which involves the retinoic acid receptor alpha (RARA) gene on chromosome 17 and the PML gene on chromosome 15. An extremely restricted region (ERR) of 50 bps within the second intron of the RARA gene was identified as the cluster region of breakpoints by sequencing analyses. ERR was tested by in vitro transfection-recombination assay, and was shown to be the recombination hot spot. In this study, presence of DNA binding proteins to the 148 bps DNA fragment which contains ERR was confirmed by gel-mobility shift analysis in the nuclear extract of NIH3T3 cells and human leukemia cell lines. Furthermore, in vitro study with the mouse sarcoma cell lines using the recombination reporter plasmid containing ERR showed that ERR might be involved in the homologous recombination in addition to the illegitimate recombination. The DNA binding proteins specific to ERR might play an important role in chromosome translocation.

Instability of microsatellite sequences are frequently found in human tumors. In addition, minisatellite sequences, another group of highly unstable sequences, serve as sensitive markers of genetic instability. We have studied minisatellite instability in methylcholanthrene-induced mouse sarcomas. These sarcomas frequently carry the amplified c-myc gene. Seven sarcomas without the amplification and seven others with the amplification were selected randomly. Regardless of the state of the c-myc gene amplification, these sarcomas exhibited a varying degree of transplantability in syngeneic mice. The hypervariable mouse minisatellite locus Ms6hm was found to be highly unstable, specifically among sarcomas with the amplified c-myc gene. However, chromosome instability, as analyzed by micronucleus assay, was observed similarly for two groups of sarcomas. In addition, transversion of G to C and A to T was detected at the K-ras gene in four of the seven sarcomas with the amplified c-myc gene, and these mutations are thought to be induced directly by methylcholanthrene. Thus, concomitant occurrence was observed for three seemingly unrelated mutations, amplification of the c-myc locus, point mutation of the K-ras gene, and instability at the hypervariable mouse minisatellite locus. The present study indicates a possible involvement of K-ras mutation and c-myc amplification in induction of genetic instability in methylcholanthrene-induced mouse sarcomas.

Analysis was made on the genomic structure, functions, and expression of the mouse ELP gene, which codes for the embryonal long terminal repeat binding protein. Extensive screening of the cDNA library of embryonal carcinoma cells (EC cells) identified four isoforms of ELP: ELP1 (the original ELP isolate), ELP2, ELP3, and Ad4BP/SF1. Analysis of the genomic sequences revealed that these ELP isoforms were generated by alternative promoter usage and differential splicing. The mRNAs of isoforms initiated at four transcription start sites distributed on three exons. Sequence analysis of the four isoforms identified three polypeptides. The N-terminal portion of ELP1 and ELP2 was longer than ELP3, and Ad4BP/SF1 by 77 aa. The DNA-binding domain and region II were shared by all four isoforms. The C-terminal portion shared by ELP2, ELP3, and Ad4BP/SF1 was 131 aa in length, and that specific to ELP1 was 57 aa in length. The ELP3 and Ad4BP/SF1 isoforms were identical for the coding sequence, but the two differed at the 5' noncoding region. Region II and III domains of nuclear receptors were thought to be involved in ligand-binding and transcriptional activation. ELP1, which lacked region III, functioned as a repressor. The isoforms carrying intact region II and region III functioned as transactivators. Expression of the four isoforms was studied in mouse tissues and in tissue culture cells by quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) analysis. Complex patterns of expression of these isoforms were observed in various tissues. All four ELP isoforms were expressed only in EC cells.

Dose-response of an induction of a germline mutation was studied at a hypervariable mouse minisatellite locus, Ms6hm, which consists of tandem repeats of a sequence motif GGGCA. Male C3H/HeN mice were exposed to various doses of 60Co gamma-ray and mated with unirradiated C57BL/6N female mice. Matings were done at various time after irradiation to assess the effects of radiation on spermatozoa, spermatids and spermatogonia. DNA samples of F1 offsprings were analysed by Southern blotting for the repeat length mutation at the Ms6hm locus. The mutation frequency per gamete of the paternal allele was 9.1% for the unirradiated control group. The spermatids stage was most sensitive to radiation and a statistically significant dose-response was observed. The mutation frequency of the paternal allele in F1 mice increased to 22% for 1 Gy, 28% for 2 Gy, and 28% for 3 Gy. The spermatogonia stage was less sensitive to radiation, and the mutation frequencies of the paternal allele were 14% for 2 Gy, and 16% for 3 Gy. The spermatozoa stage germ cells were also less sensitive and the frequency of mutation of the paternal allele increased to 14% for 3 Gy. However, these increases were statistically not significant. Possible mechanisms of radiation induction of germline mutation at the hypervariable minisatellite locus will be discussed.

A cell line derived from human lung cancer (AOI) was employed in the present study. A panel of cytokines were quantified by ELISA technique following cellular exposure to X-irradiation. Tremendous increase in the levels of both IL-1 alpha and IL-6 were observed, GM-CSF was also detected. A comparison of time kinetics of IL-1 alpha and IL-6 production was made with that of cell cycle progression which was determined by FCM BrdU/DNA bivariate analysis. No cell cycle specific changes were found. The biological implication of radiation-induced cytokine production was discussed.

Acute promyelocytic leukemia (APL) has been characterized by 15;17 chromosomal translocation, which involves the retinoic acid receptor alpha (RARA) gene on chromosome 17 and the PML gene on chromosome 15. The breakpoints have been mapped to three cluster regions in the PML gene, and to RARA gene intron 2. We have examined the distribution of breakpoints within RARA gene intron 2. An extremely restricted region (ERR) of 50 bp within RARA gene intron 2 was identified as the cluster region of breakpoints by polymerase chain reaction and sequence analysis of DNA from APL patients. To study experimentally the mechanism involved in the translocation, ERR was tested in NIH3T3 cells by in vitro transfection-recombination assay, in which target sequences were placed either downstream of the SV40 promoter or upstream of the neo gene. Cells were conferred resistance to G418 only when the promoter was fused to the neo gene by recombination of two target sequences during transfection. The molecular junctions were analysed in five clones, and all of them were shown to be confined within a 20 bp region in a 148 bp DNA fragment containing ERR. This suggests that ERR might be the illegitimate recombination hot spot in mammalian cells.

Pc-1 and Pc-2 hypervariable minisatellites of the mouse show a structural similarity and hence have been compared in terms of chromosomal location and mutability. Polymorphisms of the Pc-2 locus detected by analyses of Southern blotting and polymerase chain reaction were used for typing 61 backcross mice that were obtained by mating (C57BL/6 x MSM)F1 females to MSM males. Segregation patterns indicated that the Pc-2 locus maps to the 60 cM region of chromosome 6, while that of Pc-1 maps to chromosome 4. Simultaneous mutation of the two loci was examined in 101 F1 mice. Pc-1 and Pc-2 loci showed mutation rates of 14.4 and 5.0% per gamete, respectively. Of the nine mice with a Pc-2 mutation, four carried a Pc-1 mutation at the same maternally- or paternally-derived allele. The rate of comutation was 44% (4/9), higher than the 12.2% (24/197) of Pc-1 mutation alone. This suggests that the germline mutation of the two hypervariable minisatellites may result from a common mechanism.

Study of DNA methylation of the c-myc gene in liver tumors of BCF1 mice treated with various agents revealed frequent alteration from that in normal liver. The alteration, however, was complex and showed either an increase or a decrease of methylation to various degrees. On the other hand, the tumors obtained from middle-aged C3H/He mice, which develop liver tumors spontaneously at a high incidence rate starting in middle age, showed predominantly increases of methylation in the c-myc gene, while the large tumors found in older mice revealed decreases. The normal part of the liver showed a slight gradual age-dependent increase. These suggest that hypermethylation of the c-myc gene is common to aging and early tumor development in liver. Thus the alteration of DNA methylation seems to be a good clue to investigate why the tumor incidence rate increases rapidly as individuals grow older.

Treatment of mouse embryo fibroblasts with concentrations of 5-iododeoxyuridine (IUdR) too low to induce detectable replication of endogenous C-type viruses has been found to augment the susceptibility of permissive and nonpermissive cells to exogenous infection by murine leukemia viruses, without changing the kinetics of viral replication. No evidence could be obtained to support the hypothesis that this phenomenon results from induction of endogenous virus followed by complementation or recombination between endogenous and exogenous virus. Instead, the responsible mechanism seems likely to involve IUdR-induced DNA strand breakage, repair, and enhanced recombinational integration of the exogenous viral genome into cellular DNA.

The highly oncogenic erythroleukemia-inducing Friend mink cell focus-inducing (MCF) virus was molecularly cloned in phage lambda gtWES.lambda B, and the DNA sequences of the env gene and the long terminal repeat were determined. The nucleotide sequences of Friend MCF virus and Friend spleen focus-forming virus were quite homologous, supporting the hypothesis that Friend spleen focus-forming virus might be generated via Friend MCF virus from an ecotropic Friend virus mainly by some deletions. Despite their different pathogenicity, the nucleotide sequences of the env gene of Friend MCF virus and Moloney MCF virus were quite homologous, suggesting that the putative parent sequence for the generation of both MCF viruses and the recombinational mechanism for their generation might be the same. We compare the amino acid sequences in lymphoid leukemia-inducing ecotropic Moloney virus and Moloney MCF virus, and erythroblastic leukemia-inducing ecotropic Friend virus, Friend-MCF virus, and Friend spleen focus-forming virus. The Friend MCF virus long terminal repeat was found to be 550 base pairs long. This contained two copies of the 39-base-pair tandem repeat, whereas the spleen focus-forming virus genome contained a single copy of the same sequence.

5-Azacytidine was found to induce the expression of BALB:virus-1 and BALB:virus-2 from K-BALB cells and ecotropic endogenous virus from AKR2B cells. Efficiency of the induction was high and comparable to that by 5-bromodeoxyuridine. The level of methylcytosine in newly synthesized DNA was drastically decreased when K-BALB cells were treated with 5-azacytidine. There was an inverse relationship between the level of DNA modification and the frequency of virus expression.

The kinetics of intracellular viral antigen production in UC1-B, BALB3T3 and BL-5 cells (all Fv-1b) and of focus formation in UC1-B cells have been studied following infection with free RadLV∗, a naturally occurring, B-tropic murine leukemia virus adapted to continuous propagation in vitro, and with infective centers of BL-5(RadLV) cells chronically infected with RadLV∗. Whereas such infective centers induced focus formation in UC1-B cells with high efficiency within a single culture passage, the development of infective center activity in BL-5 cells freshly infected with RadLV∗ and the direct induction of focus formation in UC1-B cells infected with free RadLV∗ both required one or more subcultures and several rounds of cell replication. The end-point dilution titer was not detected until four to five subcultures and about 15 rounds of cell cycle replication had been completed. Viral antigen content, measured by indirect immunofluorescence (IF), followed a triphasic pattern with respect to time and viral input concentration. Yet, both the IF assay at 3 days and the focus formation assay at 7 days (one subculture) were characterized by single-hit kinetics, excluding the possibility that mutual complementation of defective RadLV∗ particles might account for the delayed expression of viral infection. When viral replication was related to the number of rounds of cell replication, rather than to elapsed time, no significant differences among the three cell lines were observed. As in previous studies with NB-tropic viruses, RadLV∗ failed to rescue a putative sarcoma virus from UC1-B cells. It is concluded that RadLV∗ is not defective and can replicate to high titer in several Fv-1b cell lines in the absence of a detectable murine sarcoma virus (MSV) genome. However, the kinetics of its replication suggest that certain expressions of infection may be dependent on the attainment of a threshold level of some viral product(s), the synthesis of which is dependent on sustained cell proliferation.

As a result of some comparative basic investigations between the so called long acting sulfa drugs such as sulfisomezole, sulfamethoxypyridazine, sulfaphenazol, and sulfadimethoxine and the short acting one such as sulfisomidine, the authors found the fact that the formers had much more excellent characteristics than the latter.These excellent characteristics, namely, were their maintenance of high concentration in blood and tissue due to the very slow urinary excretion and the low acetylation rate.From these data, when the authors payed attention to the therapy of chronic sinusitis hitherto using with sulfa drugs, it seemed to be expected that the long acting sulfa drugs could improve the effect of therapy in chronic sinusitis.However, as to excellent characteristics, there was little to choose among them, while each of them had the excellent characteristics itself respectively.On the other hand, concluding from investigations in vitro, although staphylococci were a little more sensible to the long acting sulfa drugs than the previous short acting ones, they were easily to get the resistance against the formers comparatively in early stage and the degree of resistance did not diminish in a long period of time. So, in these points, it was to be considerable that these long acting sulfa drugs have not been improved.

The use of DEAE-dextran and polybrene during infection of mouse embryo fibroblast cell lines of Fv-1bb origin with the radiation leukemia virus strikingly increased the proportion of cells in which virus-induced cytoplasmic antigens were detectable by indirect immunofluorescence and significantly accelerated the time course of their detection, thus making possible the development of a rapid, sensitive, and practical new assay based upon the percentage of immunofluorescence-positive cells. Direct comparisons with several other in vitro assays are presented. The assay was also tested successfully with Gross leukemia virus grown on cells of Fv-1nn origin, and should be applicable to other murine leukemia viruses and, perhaps with minor modifications, to other mammalian leukemia viruses as well.

A mutant of the fission yeast Schizosaccharomyces pombe grew normally in the mitotic cycle but produced two-spored asci in the meiosis cycle. These spores were diploid, and the segregation of centromere-linked markers in the dyads was mostly reductional. Only the first meiotic division appears to occur in this tws1 mutant, resulting in enclosure of diploid nuclei into spores.

SEVERAL rapid in vitro methods1–5 for the quantitative assay of murine lymphatic leukaemia viruses (MuLV) have replaced the once standard in vivo bioassay for leukaemogenicity6,7, which was slow, cumbersome, and relatively costly. The in vitro procedures, however, suffer from two limitations: first, some MuLV propagated serially in vitro may lose leukaemogenicity while retaining infectivity in vitro8, so the in vitro assays do not necessarily correlate with biological activity in vivo; second, some wild-type MuLV extracted from normal or neoplastic tissues in vivo seem to require a period of adaptation to in vitro growth conditions before they can be assayed reliably by in vitro methods9.

Ultraviolet irradiation gave rise to frequent curing of killer plasmids pGKl1 and pGK12 of Kluyveromyces lactis. Almost all of the nonkillers obtained lost both plasmids, but one of them lost only pGKl1. The disappearance of pGKl1 was accompanied by the simultaneous loss of the killer activity and of the resistance to the killer factor. A new plasmid, pGKl1S, was obtained, which arose from a deletion in the central region of pGKl1. Genetic analysis suggested that pGKl1S has the killer gene lost by the deletion and the resistance gene intact and that pGKl1S shares the same replication control with pGKl1.

Xenotropic murine type-C virus was induced after X-ray or UV light irradiation of K-Balb cells. The induction was markedly suppressed by postirradiation treatment of the cells with antipain, a protease inhibitor of microbial origin. Virus expression was unaffected when the cells were treated with halogenated pyrimidines.

The kinetics of infection by a B-tropic, an N-tropic, and an NB-tropic murine leukemia virus (MuLV) have been studied on the cells of several mouse strains of permissive, restrictive, and F 1 hybrid genotype. The titration curves reveal that host cell restriction is measured by two independent parameters: (1) the slope of the curve (“hitness”); and (2) the level of sensitivity at comparable multiplicities of infection. Once established, infection is propagated from cell to cell at similar rates in both permissive and restrictive cell cultures. Anomalous results were obtained with the cells of strain NZB mice. Rat cells propagated these MuLV with 1-hit kinetics at low levels of sensitivity.

The carcinogenic effect of estrogens, diethylstilbestrol (DES) and 17 beta-estradiol (E2), and its modification by N-nitrosobutylurea (NBU) were studied in female W/Fu rats. Multiple mammary tumors (MT) of medullary carcinoma type developed at a high rate following prolonged treatment with estrogens. All MTs were located adjacent to the nipple and were slow-growing. The induction rate, multiplicity and size of estrogen-induced MTs were not influenced by pretreatment with a small amount of NBU, which alone did not induce any tumor. Ten of 12 rats (82%) receiving combined treatment with NBU and DES developed hepatic tumors (HT), while no rats in other treatment groups developed HT. All HTs were multiple nodules of various sizes bulging from the liver surface, and were considered to be neoplastic nodules. A high frequency of HT development was unexpected, because independent treatment with NBU or DES alone did not induce HT in female rats. It appears that DES played a role as a carcinogen, inducing MT and pituitary tumor (PT) through its estrogenic potency (like natural estrogen, E2), while it also acted as a promoter or co-carcinogen in the induction of HT through its pharmacologic effects. These findings may be relevant to an increased frequency of liver neoplasm among women taking oral contraceptives containing synthetic estrogens.

The direct inactivation of avian and murine oncoviruses by γ rays was examined using 60 Co as a γ-ray source. The inactivation of murine leukemia virus (M-MuLV) followed single-hit kinetics with a target size comparable to the molecular weight of its genomic RNA. The subgroup D Schmidt-Ruppin strain of avian sarcoma virus (SR-RSV D) was more resistant to γ rays than M-MuLV and showed multihit inactivation kinetics with an extrapolation number of 5. The two viruses showed similar uv-inactivation kinetics, as previously reported, and were about 40–60 times more resistant to uv light than vesicular stomatitis virus. The reason for the difference in the effects of γ rays on MuLV and SR-RSV D was not elucidated. The genomic RNA of the SR-RSV D strain was degraded by γ irradiation faster than its infectivity, but viral clones isolated from the foci formed after γ irradiation had a complete genome. These results suggest that SR-RSV D has a strong repair function, possibly connected with reverse transcriptase activity.

Conversion from restrictive (two-hit) to permissive (one-hit) kinetics was observed when N- or B-tropic murine leukemia viruses were titrated on mouse embryo fibroblasts of nonpermissive Fv-1 genotype that had previously been nonproductively infected with the same virus at an average multiplicity of one. The effect was transient, disappearing within about 24 hr after the first infection, and was abrogated by exposure of the first virus preparation to increasing doses of ultraviolet light, with a D 37 inactivation dose of 2550 ergs/mm 2 , about three times that for inactivation of viral replication. Prior infection of nonpermissive mouse cells with the NZB xenotropic virus did not alter their restrictive response to later infection with ecotropic viruses. Low multiplicity infection of NIH 3T3 cells with a B-tropic virus, followed by treatment with iodoeoxyuridine, failed to induce productive infection by endogenous or exogenous virus. Cells of F 1 hybrid Fv-1 genotype, which are restrictive for both N- and B-tropic viruses, were converted to permissiveness for virus of either tropism after low multiplicity infection with virus of the opposite tropism. No evidence of NB-tropic recombinant progeny could be detected under these experimental conditions. The implications of these experimental observations with respect to the mechanism of restriction governed by the Fv-1 gene are discussed.

Bovine leukemia virus (BLV) propagated in a cell clone of fetal lamb kidney origin was transmitted by cell contact to different mammalian cells including human cells. The transmission of the BLV genome was effectively achieved by cocultivation of mitomycin-C-killed, virus-producing cells of the cell clone with recipient cells. In particular, human cells of neural origin were highly susceptible to BLV infection, while some other cells were resistant. The transmission of the BVL genome from virus-nonproducing cells failed which suggests the existence of virus specific receptors on the cells. The donor cells contained three integrated BLV proviruses. In recipient cells only one provirus was found. The majority of cells contained both unintegrated and integrated BLV provirus. In the cells containing the transmitted BLV, the viral genome was expressed to its protein products. The results indirectly suggest that retroviruses with similar properties could cause various neural diseases in man.

The Moloney leukemia virus (M-MuLV) genome was introduced into undifferentiated teratocarcinoma cells by transfection of a plasmid with the virus genome linked to pSV2neo, which carries a bacterial drug resistance gene, neo, or by cotransfection with pSV2neo. In the resulting cells, the M-MuLV genome remained hypomethylated, but its expression was suppressed in cells in an undifferentiated state. The pattern of DNA methylation of the viral genome remained unchanged when the cells were induced to differentiate into epithelial tissues. However, spontaneous M-MuLV expression was detected with differentiation of the cells. To determine to what extent the viral long terminal repeat (LTR) was responsible for this suppression in undifferentiated cells, I constructed plasmids in which neo was placed under the control of the promoter sequence of the dihydrofolate reductase gene or the M-MuLV LTR, and compared the biological activities of the plasmids in Ltk- cells and in undifferentiated teratocarcinoma cells. In Ltk- cells, these plasmids were highly efficient in making the cells resistant to selection by G418. However, in undifferentiated teratocarcinoma cells, the M-MuLV LTR promoted neo gene expression at only 10% of the expected efficiency, as compared with the expression of the neo gene under the control of the simian virus to or dihydrofolate reductase promoter. Thus, the mechanisms of gene regulation are not the same in undifferentiated and differentiated teratocarcinoma cells.