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Sample records for increase hprt mutant

  1. Chromosome instability of HPRT-mutant subclones induced by ionising radiation of various LET.

    PubMed

    Govorun, R D; Koshlan, I V; Koshlan, N A; Krasavin, E A; Shmakova, N L

    2002-01-01

    The induction of HPRT-mutations and survival of Chinese hamster cells (line B11ii-FAF28, clone 431) were studied after irradiation by 4He and 12C-ions of various LET (20-360 keV/micrometers), produced by the U-200 heavy ion accelerator. The RBE increases with LET up to the maximum at 100-200 keV/micrometers and then decreases. Cytogenetic analysis was performed on the HPRT-mutant subclones selected from unirradiated Chinese hamster V-79 cells and from HPRT-mutant subclones that arose after exposure to gamma-rays, 1 GeV protons and 14N-ions (LET-77 keV/micrometers), produced by the synchrophasotron and the U-400M heavy ion accelerator. Slow growing mutant subclones were observed. The cytogenetic properties of individual clones were highly heterogeneous and chromosome instability was observed in both spontaneous and radiation-induced mutants. Chromosome instability was highest among spontaneous mutants and decreased with increasing LET.

  2. Chromosome instability of HPRT-mutant subclones induced by ionising radiation of various let

    NASA Astrophysics Data System (ADS)

    Govorun, R. D.; Koshlan, I. V.; Koshlan, N. A.; Krasavin, E. A.; Shmakova, N. L.

    The induction of HPRT-mutations and survival of Chinese hamster cells (line B11ii-FAF28, clone 431) were studied after irradiation by 4He and 12C-ions of various LET (20 - 360 keV/μm), produced by the U-200 heavy ion accelerator. The RBE increases with LET up to the maximum at 100-200 keV/μm and then decreases. Cytogenetic analysis was performed on the HPRT-mutant subclones selected from unirradiated Chinese hamster V-79 cells and from HPRT- mutant subclones that arose after exposure to γ-rays, 1GeV protons and 14N-ions (LET - 77 keV/μm), produced by the synchrophasotron and the U-400M heavy ion accelerator. Slow growing mutant subclones were observed. The cytogenetic properties of individual clones were highly heterogeneous and chromosome instability was observed in both spontaneous and radiation-induced mutants. Chromosome instability was highest among spontaneous mutants and decreased with increasing LET.

  3. Hypomutability in Fanconi anemia cells is associated with increased deletion frequency at the HPRT locus

    SciTech Connect

    Papadopoulo, D.; Guillouf, C.; Moustacchi, E. ); Mohrenweiser, H. )

    1990-11-01

    Fanconi anemia (FA) is an inherited human disorder associated with a predisposition to cancer and characterized by anomalies in the processing of DNA cross-links and certain monoadducts. The authors reported previously that the frequency of psoralen-photoinduced mutations at the HPRT locus is lower in FA cells than in normal cells. This hypomutability is shown here to be associated with an increased frequency of deletions in the HPRT gene when either a mixture of cross-links and monoadducts or monoadducts alone are induced. Molecular analysis of mutants in the HPRT gene was carried out. In normal cells the majority of spontaneous and induced mutants are point mutations whereas in FA deletion mutations predominate. In that case a majority of mutants were found to lack individual exons or small clusters of exons whereas in normal cells large (complete or major gene loss) and small deletions are almost equally represented. Thus they propose that the FA defect lies in a mutagenic pathway that, in normal cells, involves by passing lesions and subsequent gap filling by a recombinational process during replication.

  4. DNA lesion and Hprt mutant frequency in rat lymphocytes and V79 Chinese hamster lung cells exposed to cadmium.

    PubMed

    Jianhua, Zhou; Lian, Xue; Shuanlai, Zheng; Juan, Du; Shuanxi, Yang

    2006-03-01

    Cadmium is a potential carcinogenic environmental and occupational pollutant. A wide variety of mutagens have been shown to cause DNA damage, but it is not yet clear whether the DNA damage is relative to inducement of mutations. DNA damage and the formation of mutations at the hypoxanthine guanine phosphoribosyl trans ferase (HPRT) induced by cadmium chloride (CdCl(2)) were investigated with rat lymphocytes and V79 Chinese hamster lung cells. The hprt mutant frequency (MF) assay was used as the method to measure gene mutation in the rat lymphocytes and V79 cells exposed to CdCl(2), and comet assay analysis was performed to detect DNA lesion and repair in CdCl(2)-induced V79 cells. The results showed that CdCl(2) treatment caused a strong genotoxic effect and a marginal effect on the frequency of gene mutations. The hprt mutant frequencies in the rat lymphocytes and V79 cells exposed to CdCl(2) were statistically higher than those of the negative control. There was statistical significance in TL, TD and percentage of comet cell with tails. CdCl(2) treatment can induce DNA single-strand breaks. There was a dose-dependent increase between CdCl(2) and DNA lesion. After cells were treated with CdCl(2) and hydrogen peroxide (H(2)O(2)), the TL and TD declined with repair time increasing, which indicated that DNA damages were repaired gradually. However, DNA repair with treatment of CdCl(2) was slower than that of H(2)O(2) in V79 cells, which suggests that CdCl(2) affected DNA repair of damaged cells. The study also showed that the hprt MF and comet assay can be used for genotoxicity testing of heavy metals. DNA damage detected with the comet assay may be relative to mutagenesis.

  5. Frequencies of HPRT mutants and micronuclei in lymphocytes of cancer patients under chemotherapy: a prospective study.

    PubMed

    Tates, A D; van Dam, F J; Natarajan, A T; Zwinderman, A H; Osanto, S

    1994-05-01

    Fifteen cancer patients, including 10 testicular carcinoma patients, were treated with several types of combination chemotherapy. Blood samples were collected before, during and after chemotherapy. Subsequently, lymphocytes were analyzed for frequencies of HPRT mutants (MF) and micronuclei (MNF). Significantly elevated MFs were detected in eight patients. Mean expression time (+/- SD) for mutations was 98 +/- 54 days (range: 42-172 days). In some patients, enhanced MFs persisted for a period of 430-490 days after cessation of chemotherapy. In five patients MNFs were increased 2-6-fold and the enhancement was fairly persistent. Ifosfamide and cyclophosphamide appeared to be the most mutagenic and clastogenic constituents of the chemotherapy, while evidence for adverse effects of adriamycin, 4-epi-adriamycin and bleomycin was equivocal. Results indicate that the clinical use of mutagenic drugs must be weighed against the risks of persistent genetic damage and secondary malignancies in cured patients and their potential offspring. Further studies are necessary to determine the true risks and incidence of such abnormalities following chemotherapy for curable forms of cancer.

  6. The molecular nature of spontaneous mutations at the hprt locus in the radiosensitive CHO mutant xrs-5.

    PubMed

    Schwartz, J L; Porter, R C; Hsie, A W

    1996-03-26

    The radiosensitive mutant xrs-5, a derivative of the Chinese hamster ovary (CHO) K1 cell, is defective in DNA double-strand break rejoining ability and in V(D)J recombination. The radiosensitivity and defective repair phenotype are complemented by the 80-kDa subunit of the Ku protein. We determined the nature of the mutations that develop spontaneously at the hprt locus in this cell line using both multiplex PCR deletion screening and DNA sequencing. Ninety-two independent spontaneous mutants were analyzed and the results were compared to the mutation spectrum of 64 previously analyzed hprt spontaneous mutants isolated from the parental CHO-K1 cell line. More than 50% of the spontaneous xrs-5 mutants had lost one or more exons while less than 25% of spontaneous CHO-K1 mutants had lost one or more exons. Most of the deletions in xrs-5 cells involved the loss of multiple exons while single exon deletions predominated in CHO-K1. There was also a nonrandom distribution of breakpoints in both CHO-K1 and xrs-5. Most of the deletion breakpoints were 3' to exon 9, around exons 4-6, or near exon 1. Although the frequency of base substitutions was lower in xrs-5, the spectrum of base substitutions was qualitatively similar to that of CHO-K1. There was no significant difference in the spontaneous mutant frequency in xrs-5 and CHO-K1. The results suggest that in certain regions of the hprt gene, base alterations can be converted to large deletions, and that alterations in the Ku protein complex can influence this process.

  7. Somatic mutant frequency at the HPRT locus in children associated with a pediatric cancer cluster linked to exposure to two superfund sites.

    PubMed

    Vacek, Pamela M; Messier, Terri; Rivers, Jami; Sullivan, Linda; O'Neill, J Patrick; Finette, Barry A

    2005-05-01

    The somatic mutant frequency (Mf) of the hypoxanthine phosphoribosyl transferase (HPRT) gene has been widely used as a biomarker for the genotoxic effects of exposure but few studies have found an association with environmental exposures. We measured background Mfs in 49 current and former residents of Dover Township, New Jersey, who were exposed during childhood to industrially contaminated drinking water. The exposed subjects were the siblings of children who developed cancer after residing in Dover Township, where the incidence of childhood cancer has been elevated since 1979. Mfs from this exposed group were compared to Mfs in 43 age-matched, presumably unexposed residents of neighboring communities with no known water contamination and no increased cancer incidence. Statistical comparisons were based on the natural logarithm of Mf (lnMF). The mean Mf for the exposed group did not differ significantly from the unexposed group (3.90 x 10(-6) vs. 5.06 x 10(-6); P = 0.135), but unselected cloning efficiencies were higher in the exposed group (0.55 vs. 0.45; P = 0.005). After adjustment for cloning efficiency, lnMf values were very similar in both groups and age-related increases were comparable to those previously observed in healthy children. The results suggest that HPRT Mf may not be a sensitive biomarker for the genotoxic effects of environmental exposures in children, particularly when substantial time has elapsed since exposure. Copyright 2005 Wiley-Liss, Inc

  8. Frequency of Hprt mutant lymphocytes and micronucleated erythrocytes in p53-haplodeficient mice treated perinatally with AZT and AZT in combination with 3TC.

    PubMed

    Dobrovolsky, Vasily N; Shaddock, Joseph G; Mittelstaedt, Roberta A; Bishop, Michelle E; Lewis, Sherry M; Lee, Fei W; Aidoo, Anane; Leakey, Julian E A; Dunnick, June K; Heflich, Robert H

    2007-01-01

    Azidothymidine (AZT) is a nucleoside reverse transcriptase inhibitor (NRTI) that is used for reducing mother-to-child transmission of human immunodeficiency virus I. Combinations of AZT and 3'-thiacytidine (3TC) are even more effective than AZT alone. AZT, however, is a mutagen and carcinogen in rodent models and 3TC can increase the genotoxicity of AZT. Since p53 plays a key role in human and mouse tumorigenesis, p53-haplodeficient mice are currently being evaluated as a model for assessing the carcinogenicity of perinatal exposure to NRTIs. In the present study, male C57BL/6 p53(+/+) and p53(-/-) mice were mated with C3H p53(+/+) females; the pregnant females were treated on gestation day 12 through parturition with 40, 80, and 160 mg/kg of AZT or a combination of 160 mg/kg AZT and 100 mg/kg 3TC (AZT-3TC); the p53(+/+) and p53(+/-) offspring were treated daily after birth through postnatal day (PND) 28. The frequencies of micronucleated reticulocytes (MN-RETs) and micronucleated normochromatic erythrocytes (MN-NCEs) were determined on PND1, PND10, and PND28; the frequency of Hprt mutant lymphocytes was measured on PND28. The frequencies of MN-RETs and MN-NCEs were increased in treated animals at all time points; there were no differences in the responses of p53(+/+) and p53(+/-) animals treated with identical doses of NRTIs. After correction for clonal expansion, both AZT and AZT-3TC treatments induced small but significant increases in the frequency of Hprt mutant lymphocytes in p53(+/-) mice, but not in p53(+/+) mice. The data indicate that p53 haplodeficiency affects the genotoxicity of NRTIs; thus, p53(+/-) mice may be a sensitive model for evaluating the carcinogenicity of perinatal exposure to NRTIs. (c) 2006 Wiley-Liss, Inc.

  9. hprt mutant lymphocyte frequencies in workers at a 1,3-butadiene production plant.

    PubMed Central

    Ward, J B; Ammenheuser, M M; Bechtold, W E; Whorton, E B; Legator, M S

    1994-01-01

    1,3-Butadiene is a major industrial chemical that has been shown to be a carcinogen at multiple sites in mice and rats at concentrations as low as 6.25 ppm. Occupational exposures have been reduced in response to these findings, but it may not be possible to determine by using traditional epidemiological methods, whether current exposure levels are adequate for protection of worker health. However, it is possible to evaluate the biological significance of exposure to genotoxic chemicals at the time of exposure by measuring levels of genetic damage in exposed populations. We have conducted a pilot study to evaluate the effects of butadiene exposure on the frequencies of lymphocytes containing mutations at the hypoxanthine-guanine phosphoribosyl transferase (hprt) locus in workers in a butadiene production plant. At the same time, urine specimens from the same individuals were collected and evaluated for the presence of butadiene-specific metabolites. Eight workers from areas of the plant where the highest exposures to butadiene occur were compared to five workers from plant areas where butadiene exposures were low. In addition, six subjects with no occupational exposure to butadiene were also studied as outside controls. All of the subjects were nonsmokers. An air sampling survey conducted for 6 months, and ending about 3 months before the study, indicated that average butadiene levels in the air of the high-exposure areas were about 3.5 +/- 7.5 ppm. They were 0.03 +/- 0.03 ppm in the low-exposure areas. Peripheral blood lymphocytes from the subjects were assayed using an autoradiographic test for hprt mutations.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:7698091

  10. Bilirubin UDP-Glucuronosyltransferase 1A1 (UGT1A1) Gene Promoter Polymorphisms and HPRT, Glycophorin A, and Micronuclei Mutant Frequencies in Human Blood

    SciTech Connect

    Grant, D; Hall, I J; Eastmond, D; Jones, I M; Bell, D A

    2004-10-06

    A dinucleotide repeat polymorphism (5-, 6-, 7-, or 8-TA units) has been identified within the promoter region of UDP-glucuronosyltransferase 1A1 gene (UGT1A1). The 7-TA repeat allele has been associated with elevated serum bilirubin levels that cause a mild hyperbilirubinemia (Gilbert's syndrome). Studies suggest that promoter transcriptional activity of UGT1A1 is inversely related to the number of TA repeats and that unconjugated bilirubin concentration increases directly with the number of TA repeat elements. Because bilirubin is a known antioxidant, we hypothesized that UGT1A1 repeats associated with higher bilirubin may be protective against oxidative damage. We examined the effect of UGT1A1 genotype on somatic mutant frequency in the hypoxanthine-guanine phosphoribosyl-transferase (HPRT) gene in human lymphocytes and the glycophorin A (GPA) gene of red blood cells (both N0, NN mutants), and the frequency of lymphocyte micronuclei (both kinetochore (K) positive or micronuclei K negative) in 101 healthy smoking and nonsmoking individuals. As hypothesized, genotypes containing 7-TA and 8-TA displayed marginally lower GPA{_}NN mutant frequency relative to 5/5, 5/6, 6/6 genotypes (p<0.05). In contrast, our analysis showed that lower expressing UGT1A1 alleles (7-TA and 8-TA) were associated with modestly increased HPRT mutation frequency (p<0.05) while the same low expression genotypes were not significantly associated with micronuclei frequencies (K-positive or K-negative) when compared to high expression genotypes (5-TA and 6-TA). We found weak evidence that UGT1A1 genotypes containing 7-TA and 8-TA were associated with increased GPA{_}N0 mutant frequency relative to 5/5, 5/6, 6/6 genotypes (p<0.05). These data suggest that UGT1A1 genotype may modulate somatic mutation of some types, in some cell lineages, by a mechanism not involving bilirubin antioxidant activity. More detailed studies examining UGT1A1 promoter variation, oxidant/antioxidant balance and genetic

  11. Mutagenicity monitoring following battlefield exposures: Longitudinal study of HPRT mutations in Gulf War I veterans exposed to depleted uranium.

    PubMed

    Albertini, Richard J; Vacek, Pamela M; Carter, Elizabeth W; Nicklas, Janice A; Squibb, Katherine S; Gucer, Patricia W; Engelhardt, Susan M; McDiarmid, Melissa A

    2015-08-01

    A total of 70 military Veterans have been monitored for HPRT T-cell mutations in five separate studies at 2-year intervals over an 8-year period. Systemic depleted uranium (DU) levels were measured at the time of each study by determining urinary uranium (uU) excretion. Each HPRT study included 30-40 Veterans, several with retained DU-containing shrapnel. Forty-nine Veterans were evaluated in multiple studies, including 14 who were in all five studies. This permitted a characterization of the HPRT mutation assay over time to assess the effects of age, smoking and non-selected cloning efficiencies, as well as the inter- and intra-individual variability across time points. Molecular analyses identified the HPRT mutation and T-cell receptor (TCR) gene rearrangement in 1,377 mutant isolates. An unexpected finding was that in vivo clones of HPRT mutant T-cells were present in some Veterans, and could persist over several years of the study. The calculated HPRT mutant frequencies (MFs) were repeatedly elevated in replicate studies in three outlier Veterans with elevated urinary uranium excretion levels. However, these three outlier Veterans also harbored large and persistent in vivo HPRT mutant T-cell clones, each of which was represented by a single founder mutation. Correction for in vivo clonality allowed calculation of HPRT T-cell mutation frequencies (MutFs). Despite earlier reports of DU associated increases in HPRT MFs in some Veterans, the results presented here demonstrate that HPRT mutations are not increased by systemic DU exposure. Additional battlefield exposures were also evaluated for associations with HPRT mutations and none were found.

  12. Multiplex polymerase chain reaction-based deletion analysis of spontaneous, gamma ray- and alpha-induced hprt mutants of CHO-K1 cells.

    PubMed

    Schwartz, J L; Rotmensch, J; Sun, J; An, J; Xu, Z; Yu, Y; Hsie, A W

    1994-11-01

    Independent Chinese hamster ovary (CHO)-K1 cell mutants at the hypoxanthine-guanine phosphoribosyltransferase (hprt) locus were isolated from untreated, 60Co gamma ray- and 212Bi alpha-exposed cells and the genetic changes underlying the mutation determined by multiplex polymerase chain reaction (PCR)-based exon deletion analysis. In the 71 spontaneous mutants analyzed, 77.5% of the clones showed no change in exon number or size, 15.5% showed a loss of a single exon, 4.2% showed a loss of 2-8 exons, and 2.8% showed loss of all nine hprt exons (total gene deletion). Exposure to 6 Gy of gamma rays, which reduced survival levels to 10%, produced a significantly different deletion spectrum that was shifted toward deletions with 45% of the 20 mutants analyzed showing a loss of a single exon and 30% showing a loss of all nine exons. Exposure to 2 Gy alpha radiation from 212Bi, a 220Rn daughter, a dose which also reduced survival levels to about 10%, resulted in a deletion spectrum similar to the gamma-ray spectrum in that more than 75% of the 49 mutants analyzed were deletions. The alpha spectrum, however, was significantly different from both the spontaneous and gamma spectra with 55.1% of the alpha mutants showing a loss of all nine exons, 10.2% showing loss of a single exon, and 14.3% showing loss of 2-8 exons. Thus, alpha-radiation appears to produce larger intragenic deletions than gamma radiation. The results suggest that intragenic deletion size should be considered when low- and high linear energy transfer (LET) mutation spectra are compared.

  13. Polymerase chain reaction-deletion analysis of spontaneous, gamma ray-, and alpha-induced hprt mutants of CHO-K1 cells.

    SciTech Connect

    Schwartz, J. L.; Rotmensch, J.; Sun, J.; An, J.; Xu, Z.; Yu, Y.; Hsie, A. W.; Center for Mechanistic Biology and Biotechnology; Univ. of Chicago; Univ. of Texas Medical Branch

    1994-01-01

    Independent Chinese hamster ovary (CHO)-K1 cell mutants at the hypoxanthine-guanine phosphoribosyltransferase (hprt) locus were isolated from untreated, {sup 60}Co {gamma} ray-and {sup 212}Bi {alpha}-exposed cells and the genetic changes underlying the mutation determined by multiplex polymerase chain reaction (PCR)-based exon deletion analysis. In the 71 spontaneous mutants analyzed, 77.5% of the clones showed no change in exon number or size, 15.5% showed a loss of a single exon, 4.2% showed a loss of 2-8 exons, and 2.8% showed loss of all nine hprt exons (total gene deletion). Exposure to 6 Gy of {gamma} rays, which reduced survival levels to 10%, produced a significantly different deletion spectrum that was shifted toward deletions with 45% of the 20 mutants analyzed showing a loss of a single exon and 30% showing a loss of all nine exons. Exposure to 2 Gy {alpha} radiation from 212Bi, a 220Rn daughter, a dose which also reduced survival levels to about 10%, resulted in a deletion spectrum similar to the {gamma}-ray spectrum in that more than 75% of the 49 mutants analyzed were deletions. The {alpha} spectrum, however, was significantly different from both the spontaneous and {gamma} spectra with 55.1% of the {alpha} mutants showing a loss of all nine exons, 10.2% showing loss of a single exon, and 14.3% showing loss of 2-8 exons. Thus, {alpha}-radiation appears to produce larger intragenic deletions than {gamma} radiation. The results suggest that intragenic deletion size should be considered when low- and high linear energy transfer (LET) mutation spectra are compared.

  14. Measurement of chromosomal aberrations, sister chromatid exchange, hprt mutations, and DNA adducts in peripheral lymphocytes of human populations at increased risk for cancer

    PubMed Central

    Jacobson-Kram, David; Albertini, Richard J.; Branda, Richard F.; Falta, Michael T.; Iype, P. Thomas; Kolodner, Ken; Liou, Saou-Hsing; McDiarmid, Melissa A.; Morris, Marcia; Nicklas, Janice A.; O'Neill, J. Patrick; Poirier, Miriam C.; Putman, Donald; Strickland, Paul T.; Williams, Jerry R.; Xiao, Shuqin

    1993-01-01

    Using a multidisciplinary approach, we have measured various indicators of DNA damage in peripheral lymphocytes of human populations potentially at increased risk for cancer. Sister chromatid exchanges (SCE) and polycyclic aromatic hydrocarbon (PAH)–DNA adducts were evaluated in a group of firefighters; chromosomal aberrations and hprt mutations were evaluated in a group of cancer patients undergoing radioimmunoglobulin therapy (RIT); SCE and acrolein-modified DNA were measured in cancer chemotherapy patients and in pharmacists preparing chemotherapy prescriptions; and SCE and PAH–DNA adducts are being measured in U.S. army troops stationed in Kuwait. Our results indicate that both SCE and PAH–DNA adduct levels were not elevated in firefighters, but that other factors such as smoking status and race were risk factors for increased SCE and PAH–DNA adducts. RIT was found to increase background rates of chromosome-type aberrations and frequencies of hprt mutations and there was a strong correlation between levels of therapy-induced chromosome damage sustained in vivo and in vitro sensitivity to radiation-induced chromosome damage. Peripheral blood lymphocytes of cancer patients treated with cyclophosphamide showed higher levels of SCE and had a higher incidence of acrolein adducts in DNA. Lymphocytes from pharmacists preparing antineoplastic drugs were found to acquire increased in vitro sensitivity to SCE induction by phosphoramide mustard with increased lifetime duration of drug handling. A prospective, longitudinal study was performed to identify environmental factors that modulate genetic damage in breast cancer patients. Women with benign breast masses and no apparent disease served as controls. Mutant frequency, cloning efficiency, and chromosomal aberration frequency did not differ significantly among the three groups. The results described and the data being gathered on troops stationed in Kuwait suggest that all the methodologies described can be

  15. Influence of sex, smoking and age on human hprt mutation frequencies and spectra.

    PubMed Central

    Curry, J; Karnaoukhova, L; Guenette, G C; Glickman, B W

    1999-01-01

    Examination of the literature for hprt mutant frequencies from peripheral T cells yielded data from 1194 human subjects. Relationships between mutant frequency, age, sex, and smoking were examined, and the kinetics were described. Mutant frequency increases rapidly with age until about age 15. Afterward, the rate of increase falls such that after age 53, the hprt mutant frequency is largely stabilized. Sex had no effect on mutant frequency. Cigarette smoking increased mean mutant frequency compared to nonsmokers, but did not alter age vs. mutant frequency relationships. An hprt in vivo mutant database containing 795 human hprt mutants from 342 individuals was prepared. No difference in mutational spectra was observed comparing smokers to nonsmokers, confirming previous reports. Sex affected the frequency of deletions (>1 bp) that are recovered more than twice as frequently in females (P = 0. 008) compared to males. There is no indication of a significant shift in mutational spectra with age for individuals older than 19 yr, with the exception of A:T --> C:G transversions. These events are recovered more frequently in older individuals. PMID:10388825

  16. Cellular and molecular analyses of hprt mutation in human hepatocyte L02 cells after exposure to carbon ions

    NASA Astrophysics Data System (ADS)

    Li, Qiang; He, Jing; Jin, Xiao-Dong; Gong, Li; Li, Sha

    Mutations play an important role in carcinogenesis. The quantitative evaluation of mutation induction by heavy charged particles helps us to delineate the risks of space radiation on astronauts, as well as the risks of heavy ions on patients during tumor therapy. Hprt mutation assay, which has been used as a biological dosimeter, is an ideal gene mutation test in mammalian cells in vitro. In order to provide basic data and evidence for the risk assessment of heavy ions, the relationships between hprt mutation induction and radiation dose in human hepatocyte L02 cells were investigated for highand low-LET carbon ions and X-rays. Moreover, the carbon ion induced hprt mutation spectrum was analyzed. In our study, human hepatocyte L02 cells were irradiated with carbon ions with LET of 30keV/µm and X-rays (0.2keV/µm), respectively. The survival fraction of L02 cells was measured by means of colony-forming assay. The mutation frequency was detected by measuring 6-thioguanine-resistant clones after 10 days of incubation at the presence of 15mg/L 6-TG. To obtain the mutation spectrum, 9 10 mutant cell clones at each dose were randomly selected from the 6-TG containing medium, and were further cultured and analyzed. The deletion patterns of the 9 exons of hprt gene were analyzed with multiplex polymerase chain reactions (multiplex PCR). Our results show that the number of mutants per 106 surviving cells increased with increasing the radiation dose for both the irradiations, and the mutation frequency increased up to 1Gy while reduced with increasing dose further. Partial deletion was the most dominant deletion pattern in the hprt mutant cells, and with the increase of dose, hprt genes tended to have more total deletions and less point deletions. It can be inferred that human hepatocyte L02 cells are more radiosensitive to high-LET carbon ions than to low-LET X-rays, and carbon ions are more effective in inducing hprt mutation in L02 cells. It has been also found that the

  17. Creation of Mice Bearing a Partial Duplication of HPRT Gene Marked with a GFP Gene and Detection of Revertant Cells In Situ as GFP-Positive Somatic Cells

    PubMed Central

    Noda, Asao; Suemori, Hirofumi; Hirai, Yuko; Hamasaki, Kanya; Kodama, Yoshiaki; Mitani, Hiroshi; Landes, Reid D.; Nakamura, Nori

    2015-01-01

    It is becoming clear that apparently normal somatic cells accumulate mutations. Such accumulations or propagations of mutant cells are thought to be related to certain diseases such as cancer. To better understand the nature of somatic mutations, we developed a mouse model that enables in vivo detection of rare genetically altered cells via GFP positive cells. The mouse model carries a partial duplication of 3’ portion of X-chromosomal HPRT gene and a GFP gene at the end of the last exon. In addition, although HPRT gene expression was thought ubiquitous, the expression level was found insufficient in vivo to make the revertant cells detectable by GFP positivity. To overcome the problem, we replaced the natural HPRT-gene promoter with a CAG promoter. In such animals, termed HPRT-dup-GFP mouse, losing one duplicated segment by crossover between the two sister chromatids or within a single molecule of DNA reactivates gene function, producing hybrid HPRT-GFP proteins which, in turn, cause the revertant cells to be detected as GFP-positive cells in various tissues. Frequencies of green mutant cells were measured using fixed and frozen sections (liver and pancreas), fixed whole mount (small intestine), or by means of flow cytometry (unfixed splenocytes). The results showed that the frequencies varied extensively among individuals as well as among tissues. X-ray exposure (3 Gy) increased the frequency moderately (~2 times) in the liver and small intestine. Further, in two animals out of 278 examined, some solid tissues showed too many GFP-positive cells to score (termed extreme jackpot mutation). Present results illustrated a complex nature of somatic mutations occurring in vivo. While the HPRT-dup-GFP mouse may have a potential for detecting tissue-specific environmental mutagens, large inter-individual variations of mutant cell frequency cause the results unstable and hence have to be reduced. This future challenge will likely involve lowering the background mutation

  18. HPRT-Deficiency Dysregulates cAMP-PKA Signaling and Phosphodiesterase 10A Expression: Mechanistic Insight and Potential Target for Lesch-Nyhan Disease?

    PubMed Central

    Guibinga, Ghiabe-Henri; Murray, Fiona; Barron, Nikki

    2013-01-01

    Lesch-Nyhan Disease (LND) is the result of mutations in the X-linked gene encoding the purine metabolic enzyme, hypoxanthine guanine phosphoribosyl transferase (HPRT). LND gives rise to severe neurological anomalies including mental retardation, dystonia, chorea, pyramidal signs and a compulsive and aggressive behavior to self injure. The neurological phenotype in LND has been shown to reflect aberrant dopaminergic signaling in the basal ganglia, however there are little data correlating the defect in purine metabolism to the neural-related abnormalities. In the present studies, we find that HPRT-deficient neuronal cell lines have reduced CREB (cAMP response element-binding protein) expression and intracellular cyclic AMP (cAMP), which correlates with attenuated CREB-dependent transcriptional activity and a reduced phosphorylation of protein kinase A (PKA) substrates such as synapsin (p-syn I). Of interest, we found increased expression of phosphodiesterase 10A (PDE10A) in HPRT-deficient cell lines and that the PDE10 inhibitor papaverine and PDE10A siRNA restored cAMP/PKA signaling. Furthermore, reconstitution of HPRT expression in mutant cells partly increased cAMP signaling synapsin phosphorylation. In conclusion, our data show that HPRT-deficiency alters cAMP/PKA signaling pathway, which is in part due to the increased of PDE10A expression and activity. These findings suggest a mechanistic insight into the possible causes of LND and highlight PDE10A as a possible therapeutic target for this intractable neurological disease. PMID:23691025

  19. HPRT-deficiency dysregulates cAMP-PKA signaling and phosphodiesterase 10A expression: mechanistic insight and potential target for Lesch-Nyhan Disease?

    PubMed

    Guibinga, Ghiabe-Henri; Murray, Fiona; Barron, Nikki

    2013-01-01

    Lesch-Nyhan Disease (LND) is the result of mutations in the X-linked gene encoding the purine metabolic enzyme, hypoxanthine guanine phosphoribosyl transferase (HPRT). LND gives rise to severe neurological anomalies including mental retardation, dystonia, chorea, pyramidal signs and a compulsive and aggressive behavior to self injure. The neurological phenotype in LND has been shown to reflect aberrant dopaminergic signaling in the basal ganglia, however there are little data correlating the defect in purine metabolism to the neural-related abnormalities. In the present studies, we find that HPRT-deficient neuronal cell lines have reduced CREB (cAMP response element-binding protein) expression and intracellular cyclic AMP (cAMP), which correlates with attenuated CREB-dependent transcriptional activity and a reduced phosphorylation of protein kinase A (PKA) substrates such as synapsin (p-syn I). Of interest, we found increased expression of phosphodiesterase 10A (PDE10A) in HPRT-deficient cell lines and that the PDE10 inhibitor papaverine and PDE10A siRNA restored cAMP/PKA signaling. Furthermore, reconstitution of HPRT expression in mutant cells partly increased cAMP signaling synapsin phosphorylation. In conclusion, our data show that HPRT-deficiency alters cAMP/PKA signaling pathway, which is in part due to the increased of PDE10A expression and activity. These findings suggest a mechanistic insight into the possible causes of LND and highlight PDE10A as a possible therapeutic target for this intractable neurological disease.

  20. Molecular characterization of X-ray-induced mutations at the HPRT locus in plateau-phase Chinese hamster ovary cells.

    PubMed

    Morgan, T L; Fleck, E W; Poston, K A; Denovan, B A; Newman, C N; Rossiter, B J; Miller, J H

    1990-10-01

    CHO-K1 cells were irradiated in plateau phase to determine the effect of dose, dose fractionation, and delayed replating on the type, location and frequency of mutations induced by 250 kVp X-rays at the hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus. Independent HPRT-deficient cell lines were isolated from each group for Southern blot analysis using a hamster HPRT cDNA probe. When compared with irradiation with 4 Gy and immediate replating, dose fractionation (2 Gy + 24 h + 2 Gy) the entire gene. Since an increase in survival was noted under these conditions, these data suggest that repair of sublethal and potentially lethal damage acts equally on all premutagenic lesions, regardless of type or location. Differences in the mutation spectrum were noted when cells were irradiated at 2 Gy and replated immediately. The location of the deletion breakpoints was determined in 15 mutants showing partial loss of the HPRT locus. In 12 of these cell lines one or both of the breakpoints appeared to be located near the center of the gene, indicating a nonrandom distribution of mutations. These results indicate that damage induced by ionizing radiation results in a nonrandom distribution of genetic damage, suggesting that certain regions of the genome may be acutely sensitive to the mutagenic effects of ionizing radiation.

  1. Molecular characterisation of camptothecin-induced mutations at the hprt locus in Chinese hamster cells.

    PubMed

    Balestrieri, E; Zanier, R; Degrassi, F

    2001-05-09

    The capacity of the topoisomerase I inhibitor camptothecin (CPT) to induce single locus mutations at the hypoxanthine-guanine phosphoribosyltransferase (hprt) gene and the DNA changes underlying induced mutations were analysed in Chinese hamster ovary cells. Camptothecin treatments increased hprt mutations up to 50-fold over the spontaneous levels at highly cytotoxic doses. Genomic DNA was isolated from 6-thioguanine resistant clones and subjected to multiplex PCR to screen for gross alterations in the gene structure. The molecular analysis revealed that deletion mutants represented 80% of the analysed clones, including total hprt deletion, multiple and single exon deletions. Furthermore, a fraction of the analysed clones showed deletions of more than one exon that were characterised by the absence of non-contiguous exons. These data show that single locus mutations induced by camptothecin are characterised by large deletions or complex rearrangements rather than single base substitutions and suggest that the recombinational repair of camptothecin-induced strand breaks at replication fork may be involved in the generations of these alterations at the chromatin structure level.

  2. A Saccharomyces cerevisiae mutant with increased virulence.

    PubMed

    Wheeler, Robert T; Kupiec, Martin; Magnelli, Paula; Abeijon, Claudia; Fink, Gerald R

    2003-03-04

    Saccharomyces cerevisiae, bakers' yeast, is not a pathogen in healthy individuals, but is increasingly isolated from immunocompromised patients. The more frequent isolation of S. cerevisiae clinically raises a number of questions concerning the origin, survival, and virulence of this organism in human hosts. Here we compare the virulence of a human isolate, a strain isolated from decaying fruit, and a common laboratory strain in a mouse infection model. We find that the plant isolate is lethal in mice, whereas the laboratory strain is avirulent. A knockout of the SSD1 gene, which alters the composition and cell wall architecture of the yeast cell surface, causes both the clinical and plant isolates to be more virulent in the mouse model of infection. The hypervirulent ssd1 Delta/ssd1 Delta yeast strain is a more potent elicitor of proinflammatory cytokines from macrophages in vitro. Our data suggest that the increased virulence of the mutant strains is a consequence of unique surface characteristics that overstimulate the proinflammatory response.

  3. Molecular characterization and structure analysis of HPRT in a Chinese patient with Lesch-Nyhan disease.

    PubMed

    Jian, Wei-Xia; Peng, Wen-Hui; Li, Hai-Ling; Feng, Qi-Wen; Wang, Wei-Xing; Su, Qing

    2013-01-01

    Lesch-Nyhan disease (LND) is caused by deficiency of hypoxanthine guanine phosphoribosyltransferase (HPRT). The aim of the present study is to characterize the molecular deficiency of a clinical diagnosed Chinese patient with attenuated variant of LND. The coding region and the intron-exon boundaries of HPRT1 gene were sequenced by standard methods, and HPRT activity was assayed by HPLC method. Structure analysis was performed to estimate the consequence of the mutant of HPRT1 gene. A new mutation c.245T>G (p.Ile82Ser) was identified in this patient, and heterozygous mutation was found in the patient's mother. The activity of HPRT in the patient was completely undetectable. Structure study indicates that the mutation of p.Ile82Ser may lead to loss of hydrophobic side chain and disrupt its normal conformation of HPRT protein. It is helpful for diagnosis of LND that sequencing analysis of HPRT1 gene is performed in male infant and juvenile with hyperuricaemia and neurologic dysfunction in Chinese.

  4. Molecular analyses of in vivo hprt mutations in human T-lymphocytes: IV. Studies in newborns

    SciTech Connect

    McGinniss, M.J.; Nicklas, J.A.; Albertini, R.J. )

    1989-01-01

    In order to characterize in vivo gene mutations that occur during fetal development, molecular analyses were undertaken of mutant 6-thioguanine resistant T-lymphocytes isolated from placental cord blood samples of 13 normal male newborns. These mutant T-cells were studied to define hypoxanthine-guanine phosphoribosyltransferase (hprt) gene structural alterations and to determine T-cell receptor (TCR) gene rearrangement patterns. Structural hprt alterations, as shown by Southern blot analyses, occurred in 85% of these mutant clones. These alterations consisted mostly of deletion of exons 2 and 3. These findings contrast with the 10-20% of gross structural alterations occurring randomly across the entire gene previously reported for T-cell mutants isolated from normal young adults. Iterative analyses of hprt structural alterations and TCR gene rearrangement patterns show that approximately one-third of the newborn derived mutants may have originated as pre- or intrathymic hprt mutations. This too contrasts with previous findings in adults where the background in vivo hprt mutations appeared to originate in postthymic T-lymphocytes.

  5. Altered membrane NTPase activity in Lesch-Nyhan disease fibroblasts: comparison with HPRT knockout mice and HPRT-deficient cell lines.

    PubMed

    Pinto, Cibele S; Jinnah, Hyder A; Shirley, Thomas L; Nyhan, William L; Seifert, Roland

    2005-06-01

    Lesch-Nyhan disease (LND) is a rare disorder caused by a defect of an enzyme in the purine salvage pathway, hypoxanthine phosphoribosyl transferase (HPRT). It is still unknown how the metabolic defect translates into the complex neuropsychiatric phenotype characterized by self-injurious behavior, dystonia and mental retardation. There are abnormalities in purine and pyrimidine nucleotide content in HPRT-deficient cells. We hypothesized that altered nucleotide concentrations in HPRT deficiency change G-protein-mediated signal transduction. Therefore, our original study aim was to examine the high-affinity GTPase activity of G-proteins in membranes from primary human skin and immortalized mouse skin fibroblasts, rat B103 neuroblastoma cells and mouse Neuro-2a neuroblastoma cells. Unexpectedly, in membranes from human fibroblasts, B103- and Neuro-2a cells, V(max) of low-affinity nucleoside 5'-triphosphatase (NTPase) activities was decreased up to 7-fold in HPRT deficiency. In contrast, in membranes from mouse fibroblasts, HPRT deficiency increased NTPase activity up to 4-fold. The various systems analyzed differed from each other in terms of K(m) values for NTPs, absolute V(max) values and K(i) values for nucleoside 5'-[beta,gamma-imido]triphosphates. Our data show that altered membrane NTPase activity is a biochemical hallmark of HPRT deficiency, but species and cell-type differences have to be considered. Thus, future studies on biochemical changes in LND should be conducted in parallel in several HPRT-deficient systems.

  6. Mutational spectrum of ICR-191 at the hprt locus in human lymphoblastoid cells.

    PubMed

    Taft, S A; Liber, H L; Skopek, T R

    1994-01-01

    Human TK6 lymphoblasts were treated with the acridine derivative ICR-191, and mutants at the hprt locus were isolated. Mutant hprt cDNA was reverse-transcribed from mRNA, amplified by polymerase chain reaction (PCR), and sequenced. Additions of single G:C base pairs (+1 frameshift mutations) in repetitive G:C sequences were found in 82% (32/39) of the mutants. Sixteen of the +1 frameshifts analyzed were located in a single sequence of six consecutive guanine bases in exon 3. The remaining +1 frameshifts occurred at six different GGG sequences (14 mutants) and a single GGGG sequence (2 mutants) in other hprt exons. The repetitive guanine sequences that underwent frameshift mutagenesis were located in both the transcribed and nontranscribed strands of hprt. No single base deletions (-1 frameshift mutations) were observed. Base substitutions were observed in 13% (5/39) of the clones analyzed and occurred at both G:C and A:T bases. Loss of exon 4 from the cDNA was also observed in 5% (2/39) of the mutants. Hprt mutants containing seven consecutive guanines (produced from a +1 frameshift in a GGGGGG sequence) were treated with ICR-191 and wild-type revertants selected in CHAT medium. Revertants were recovered at a frequency of approximately 10(-7) and contained the wild-type sequence (GGGGGG) in all clones analyzed. The observed frequency of ICR-191-induced-1 frameshift reversion in the GGGGGGG sequence was approximately 500-fold lower than the estimated frequency of +1 frameshifts observed in the wild-type GGGGGG sequence following the same ICR-191 treatment. These results suggest that ICR-191 produces predominantly +1 frameshift mutations at the hprt locus in human cells.

  7. Altered Turnover of Hypoxanthine Phosphoribosyltransferase in Erythroid Cells of Mice Expressing Hprt a and Hprt b Alleles

    PubMed Central

    Johnson, Gerald G.; Chapman, Verne M.

    1987-01-01

    We have previously shown that mice expressing Hprt a allele(s) have erythrocyte hypoxanthine phosphoribosyltransferase (HPRT) levels that are approximately 25-fold (Mus musculus castaneus) and 70-fold ( Mus spretus) higher than in mice that express the Hprt b allele (Mus musculus domesticus; C57BI/6J; C3H/HeHa), and that these differences in erythrocyte HPRT levels are due to differences in the turnover rates of the HPRT A and B proteins as reticulocytes mature to erythrocytes. We show here that: (1) the taxonomic subgroups of the genus Mus are essentially monomorphic for the occurrence of either the Hprt a or the Hprt b allele, with Hprt a being common in the aboriginal species (M. spretus, Mus hortulanus and Mus abbotti) and in several commensal species (Mus musculus musculus, M. m. castaneus, Mus musculus molossinus), while Hprt b is common in feral M. m. domesticus populations as well as in all inbred strains of mice tested; (2) in all these diverse Mus subgroups there is a strict association of Hprt a with high and Hprt b with low levels of erythrocyte HPRT; and, (3) the association between the occurrence of the Hprt a allele and elevated erythrocyte HPRT levels is retained following repeated backcrosses of wild-derived Hprt a allele(s) into the genetic background of inbred strains of mice with the Hprt b allele. Collectively, these observations indicate that the elevated and low levels of erythrocyte HPRT are specified by differences in the Hprt a and b structural genes. Since evidence indicates that Hprt a and b encode HPRT proteins which differ in primary structure, we infer that the structure of HPRT is an important factor in determining its sensitivity to turnover in mouse erythroid cells. Hprt a and b may provide a useful system of "normal" allelic gene products for identifying factors that participate in protein turnover during mouse reticulocyte maturation. PMID:3609725

  8. Identification of mutant monoclonal antibodies with increased antigen binding.

    PubMed

    Pollock, R R; French, D L; Gefter, M L; Scharff, M D

    1988-04-01

    Sib selection and an ELISA have been used to isolate hybridoma subclones producing mutant antibodies that bind antigen better than the parental monoclonal antibody. Such mutants arise spontaneously in culture at frequencies of 2.5-5 X 10(-5). The sequences of the heavy and light chain variable regions of the mutant antibodies are identical to that of the parent and the Ka values of the mutants and the parent are the same. The increase in binding is associated with abnormalities of the constant region polypeptide and probably reflect changes in avidity of these antibodies.

  9. Molecular structural analysis of HPRT mutations induced by thermal and epithermal neutrons in Chinese hamster ovary cells.

    PubMed

    Kinashi, Y; Sakurai, Y; Masunaga, S; Suzuki, M; Takagaki, M; Akaboshi, M; Ono, K

    2000-09-01

    Chinese hamster ovary (CHO) cells were exposed to thermal and epithermal neutrons, and the occurrence of mutations at the HPRT locus was investigated. The Kyoto University Research Reactor (KUR), which has been improved for use in neutron capture therapy, was the neutron source. Neutron energy spectra ranging from nearly pure thermal to epithermal can be chosen using the spectrum shifters and thermal neutron filters. To determine mutant frequency and cell survival, cells were irradiated with thermal and epithermal neutrons under three conditions: thermal neutron mode, mixed mode with thermal and epithermal neutrons, and epithermal neutron mode. The mutagenicity was different among the three irradiation modes, with the epithermal neutrons showing a mutation frequency about 5-fold that of the thermal neutrons and about 1.5-fold that of the mixed mode. In the thermal neutron and mixed mode, boron did not significantly increase the frequency of the mutants at the same dose. Therefore, the effect of boron as used in boron neutron capture therapy (BNCT) is quantitatively minimal in terms of mutation induction. Over 300 independent neutron-induced mutant clones were isolated from 12 experiments. The molecular structure of HPRT mutations was determined by analysis of all nine exons by multiplex polymerase chain reaction. In the thermal neutron and mixed modes, total and partial deletions were dominant and the fraction of total deletions was increased in the presence of boron. In the epithermal neutron mode, more than half of the mutations observed were total deletions. Our results suggest that there are clear differences between thermal and epithermal neutron beams in their mutagenicity and in the structural pattern of the mutants that they induce. Mapping of deletion breakpoints of 173 partial-deletion mutants showed that regions of introns 3-4, 7/8-9 and 9-0 are sensitive to the induction of mutants by neutron irradiation.

  10. Lesch-Nyhan syndrome: mRNA expression of HPRT in patients with enzyme proven deficiency of HPRT and normal HPRT coding region of the DNA.

    PubMed

    Nguyen, Khue Vu; Naviaux, Robert K; Paik, Kacie K; Nyhan, William L

    2012-08-01

    Inherited mutation of the purine salvage enzyme, hypoxanthine guanine phosphoribosyltransferase (HPRT) gives rise to Lesch-Nyhan syndrome (LNS) or Lesch-Nyhan variants (LNV). We report a case of two LNS affected members of a family with deficiency of activity of HPRT in intact cultured fibroblasts in whom mutation could not be found in the HPRT coding sequence but there was markedly decreased HPRT expression of mRNA. Published by Elsevier Inc.

  11. The renal phenotype of allopurinol-treated HPRT-deficient mouse

    PubMed Central

    Zarattini, Paola; Clai, Milan; Corbelli, Alessandro; Carraro, Michele; Marchetti, Marialaura; Ronda, Luca; Paredi, Gianluca; Rastaldi, Maria Pia; Percudani, Riccardo

    2017-01-01

    Excess of uric acid is mainly treated with xanthine oxidase (XO) inhibitors, also called uricostatics because they block the conversion of hypoxanthine and xanthine into urate. Normally, accumulation of upstream metabolites is prevented by the hypoxanthine-guanine phosphoribosyltransferase (HPRT) enzyme. The recycling pathway, however, is impaired in the presence of HPRT deficiency, as observed in Lesch-Nyhan disease. To gain insights into the consequences of purine accumulation with HPRT deficiency, we investigated the effects of the XO inhibitor allopurinol in Hprt-lacking (HPRT-/-) mice. Allopurinol was administered in the drinking water of E12-E14 pregnant mothers at dosages of 150 or 75 μg/ml, and mice sacrificed after weaning. The drug was well tolerated by wild-type animals and heterozygous HPRT+/- mice. Instead, a profound alteration of the renal function was observed in the HPRT-/- model. Increased hypoxanthine and xanthine concentrations were found in the blood. The kidneys showed a yellowish appearance, diffuse interstitial nephritis, with dilated tubules, inflammatory and fibrotic changes of the interstitium. There were numerous xanthine tubular crystals, as determined by HPLC analysis. Oil red O staining demonstrated lipid accumulation in the same location of xanthine deposits. mRNA analysis showed increased expression of adipogenesis-related molecules as well as profibrotic and proinflammatory pathways. Immunostaining showed numerous monocyte-macrophages and overexpression of alpha-smooth muscle actin in the tubulointerstitium. In vitro, addition of xanthine to tubular cells caused diffuse oil red O positivity and modification of the cell phenotype, with loss of epithelial features and appearance of mesenchymal characteristics, similarly to what was observed in vivo. Our results indicate that in the absence of HPRT, blockade of XO by allopurinol causes rapidly developing renal failure due to xanthine deposition within the mouse kidney. Xanthine seems

  12. Distinguishing potential sources of genotoxic exposure via HPRT mutations.

    PubMed

    Molholt, B; Finette, B A

    2000-01-01

    We utilize T-cell HPRT mutations to monitor exposure to environmental mutagens in siblings of children who have developed cancer at a persistently high rate in Toms River, New Jersey, U.S.A. A preliminary epidemiological study has found a statistically-significant association between drinking public water (by pregnant mother or infant) and subsequent risk for childhood cancer. Three potential sources of mutagenic exposures in Toms River may have increased the rate of carcinogenic initiation significantly in children: 1. Benzidine-based, other azo dye and anthraquinone dye wastes released by Ciba-Geigy, 2. Styrene-acrylonitrile (SAN) trimer and other plastic wastes of Union Carbide, and 3. Radium-224, present in unusually high concentrations in the Cohansey aquifer. Specific patterns of HPRT mutations are utilized to distinguish these various potential sources of carcinogenic exposures in the drinking water of families with childhood cancer and to differentiate chemically or radiologically induced cancers from those which occur spontaneously.

  13. Mutant E. coli strain with increased succinic acid production

    DOEpatents

    Donnelly, Mark; Millard, Cynthia S.; Stols, Lucy

    2001-09-25

    A method for isolating succinic acid producing bacteria is provided comprising increasing the biomass of an organism which lacks the ability to catabolize pyruvate, and then subjecting the biomass to glucose-rich medium in an anaerobic environment to enable pyruvate-catabolizing mutants to grow. The invention also provides for a mutant that produces high amounts of succinic acid, which has been derived from a parent which lacked the genes for pyruvate formate lyase and lactate dehydrogenase, and which belongs to the E.coli Group of Bacteria.

  14. Mutant E. coli strain with increased succinic acid production

    DOEpatents

    Donnelly, M.; Millard, C.S.; Stols, L.

    1998-06-23

    A method for isolating succinic acid producing bacteria is provided comprising increasing the biomass of an organism which lacks the ability to catabolize pyruvate, and then subjecting the biomass to glucose-rich medium in an anaerobic environment to enable pyruvate-catabolizing mutants to grow. The invention also provides for a mutant that produces high amounts of succinic acid, which as been derived from a parent which lacked the genes for pyruvate formate lyase and lactate dehydrogenase, and which belongs to the E.coli Group of Bacteria. 2 figs.

  15. Mutant E. coli strain with increased succinic acid production

    DOEpatents

    Donnelly, Mark; Millard, Cynthia S.; Stols, Lucy

    1998-01-01

    A method for isolating succinic acid producing bacteria is provided comprising increasing the biomass of an organism which lacks the ability to catabolize pyruvate, and then subjecting the biomass to glucose-rich medium in an anaerobic environment to enable pyruvate-catabolizing mutants to grow. The invention also provides for a mutant that produces high amounts of succinic acid, which as been derived from a parent which lacked the genes for pyruvate formate lyase and lactate dehydrogenase, and which belongs to the E.coli Group of Bacteria.

  16. Mutant E. coli strain with increased succinic acid production

    DOEpatents

    Donnelly, Mark; Millard, Cynthia S.; Stols, Lucy

    2002-01-01

    A method for isolating succinic acid producing bacteria is provided comprising increasing the biomass of an organism which lacks the ability to catabolize pyruvate, and then subjecting the biomass to glucose-rich medium in an anaerobic environment to enable pyruvate-catabolizing mutants to grow. The invention also provides for a mutant that produces high amounts of succinic acid, which has been derived from a parent which lacked the genes for pyruvate formate lyase and lactate dehydrogenase, and which belongs to the E.coli Group of Bacteria.

  17. A model of sensitivity: 1,3-butadiene increases mutant frequencies and genomic damage in mice lacking a functional microsomal epoxide hydrolase gene.

    PubMed

    Wickliffe, Jeffrey K; Ammenheuser, Marinel M; Salazar, James J; Abdel-Rahman, Sherif Z; Hastings-Smith, Darlene A; Postlethwait, Edward M; Lloyd, R Stephen; Ward, Jonathan B

    2003-01-01

    The specific role that polymorphisms in xenobiotic metabolizing enzymes play in modulating sensitivity to 1,3-butadiene (BD) genotoxicity has been relatively unexplored. The enzyme microsomal epoxide hydrolase (mEH) is important in detoxifying the mutagenic epoxides of BD (butadiene monoepoxide [BDO], butadiene diepoxide [BDO(2)]). Polymorphisms in the human mEH gene appear to affect the function of the enzyme. We exposed mice with normal mEH activity (WT) and knockout mice without mEH activity (KO) to 20 ppm BD (inhalation) or 30 mg/kg BDO(2) (intraperitoneal [IP] injection). We then compared Hprt mutant frequencies (MFs) among these groups. KO mice exposed to BD exhibited a significant (P < 0.05) 12.4-fold increase in MF over controls and a significant 5.4-fold increase in MF over exposed WT mice. Additionally, KO mice exposed to BDO(2) exhibited a significant 4.5-fold increase in MF over controls and a significant 1.7-fold increase in MF over exposed WT mice. We also compared genomic damage in WT and KO mice (comet tail moment) following IP exposure to 3 mg/kg and 30 mg/kg BDO(2). KO mice exposed to 3 mg/kg exhibited significantly more DNA damage than controls (7.5-12.1-fold increase) and exposed WT mice (3 mg/kg; 4.8-fold increase). KO mice exposed to 30 mg/kg BDO(2) exhibited significantly more DNA damage than all other groups (2.3-27.9-fold increase). Correlation analysis indicated that a significant, positive relationship (r(2) = 0.92) exists between comet-measured damage and Hprt MFs. The lack of mEH activity increases the genetic sensitivity of mice exposed to BD and BDO(2). This model should facilitate a mechanistic understanding of the observed variation in human genetic sensitivity following exposure to BD. Copyright 2003 Wiley-Liss, Inc.

  18. Molecular nature of spontaneous mutations at the hypoxanthine-guanine phosphoribosyltransferase (hprt) locus in Chinese hamster ovary cells.

    PubMed

    Xu, Z; Yu, Y; Schwartz, J L; Meltz, M L; Hsie, A W

    1995-01-01

    The hypoxanthine-guanine phosphoribosyltransferase (hprt) locus has been widely used as a selectable genetic marker for studies of mammalian cell mutagenesis. We report here the spontaneous mutation spectrum at the hprt locus in 64 independently isolated mutants of Chinese hamster ovary (CHO) cells. All nine hprt exons were simultaneously analyzed via multiplex polymerase chain reaction (PCR) for rapid detection of gene deletions or insertions. Structural point mutations were identified by direct sequence analysis of the PCR amplified cDNA. The molecular nature of RNA splicing errors and insertions was analyzed by solid-phase direct exon sequencing. Single base substitutions were found in 24 mutants (38%), of which 21 were missense and 3 were nonsense mutations. Transversions were about twice as frequent as transitions. Fifteen mutants (23%) had deletions involving either intragenic small fragments (2), single exons (9), or multiple exons (4). The majority of deletion breakpoints (71%) were located in regions surrounding exons 4, 5, and 6. RNA splicing mutations were observed in 15 mutants (23%) and affected exons 3-8; most (6/15) resulted in the loss of exon 7. Two insertion mutants, one with a 209 bp insert in exon 4 and the other with a 88 bp insert accompanied by a 24 bp deletion in exon 6, represent novel mutations reported for the first time in spontaneous mutants of the mammalian hprt gene.

  19. Increased Anxiety in Offspring Reared by Circadian Clock Mutant Mice

    PubMed Central

    Koizumi, Hiroko; Kurabayashi, Nobuhiro; Watanabe, Yuto; Sanada, Kamon

    2013-01-01

    The maternal care that offspring receive from their mothers early in life influences the offspring’s development of emotional behavior in adulthood. Here we found that offspring reared by circadian clock-impaired mice show elevated anxiety-related behavior. Clock mutant mice harboring a mutation in Clock, a key component of the molecular circadian clock, display altered daily patterns of nursing behavior that is fragmented during the light period, instead of long bouts of nursing behavior in wild-type mice. Adult wild-type offspring fostered by Clock mutant mice exhibit increased anxiety-related behavior. This is coupled with reduced levels of brain serotonin at postnatal day 14, whose homeostasis during the early postnatal period is critical for normal emotional behavior in adulthood. Together, disruption of the circadian clock in mothers has an adverse impact on establishing normal anxiety levels in offspring, which may increase their risk of developing anxiety disorders. PMID:23776596

  20. Molecular analysis of mutations in the human HPRT gene.

    PubMed

    Keohavong, Phouthone; Xi, Liqiang; Grant, Stephen G

    2014-01-01

    The HPRT assay uses incorporation of toxic nucleotide analogues to select for cells lacking the purine scavenger enzyme hypoxanthine-guanine phosphoribosyl transferase. A major advantage of this assay is the ability to isolate mutant cells and determine the molecular basis for their functional deficiency. Many types of analyses have been performed at this locus: the current protocol involves generation of a cDNA and multiplex PCR of each exon, including the intron/exon junctions, followed by direct sequencing of the products. This analysis detects point mutations, small deletions and insertions within the gene, mutations affecting RNA splicing, and products of illegitimate V(D)J recombination within the gene. Establishment of and comparisons with mutational spectra hold the promise of identifying exposures to mutation-inducing genotoxicants from their distinctive pattern of gene-specific DNA damage at this easily analyzed reporter gene.

  1. The organization of the human HPRT gene.

    PubMed Central

    Kim, S H; Moores, J C; David, D; Respess, J G; Jolly, D J; Friedmann, T

    1986-01-01

    The organization of the X-linked gene for human hypoxanthine phosphoribosyltransferase (HPRT, EC 2.4.2.8.) has been determined by a combination of restriction endonuclease mapping, heteroduplex analysis and DNA sequence analysis of overlapping genomic clones. The entire gene is 42 kilobases in length and split into 9 exons. The sizes of the 7 internal exons and the exon-intron boundaries are identical to those of mouse HPRT gene. The 5' end of the gene lacks the prototypical 5' transcriptional regulatory sequence elements but contains extremely GC-rich sequences and five GC hexanucleotide motifs (5'-GGCGGG-3'). These structural features are very similar to those found in the mouse HPRT gene and to some of the regulatory signals common to a class of constitutively expressed "housekeeping" genes. Several transcriptional start sites have been identified by nuclease protection studies. Extensive sequence homology between the mouse and human genes is found in the 3' non-coding portion of the gene. Images PMID:3008106

  2. Analysis of HeLa cell hypoxanthine phosphoribosyltransferase mutants and revertants by two-dimensional polyacrylamide gel electrophoresis: evidence for silent gene activation.

    PubMed Central

    Milman, G; Lee, E; Ghangas, G S; McLaughlin, J R; George, M

    1976-01-01

    The spot corresponding to hypoxanthine phosphoribosyltransferase (HPRT; IMP:pyrophosphate phosphoribosyltransferase, EC 2.4.2.8) has been identified in two-dimensional polyacrylamide gels of HeLa cell extracts. This spot is absent in gels of 24 HPRT dificient mutants. A missense mutant displays a new HPRT spot at the same molecular weight but different isoelectric focusing position. Five independently isolated revertants of the missense mutant display spots corresponding to both the wild-type and mutant proteins indicating that they synthesize HPRT from two separate genes. If the missense protein is synthesized from a mutated form of the initially active HPRT gene, then wild-type HPRT protein in the revertants must be snythesized from a newly activated but prevously silent wild-type gene. The newly activated gene in the revertants of the missense mutation appears unstable producing a high frequency of spontaneous HPRT mutants. Images PMID:63948

  3. Reduced levels of dopamine and altered metabolism in brains of HPRT knock-out rats: a new rodent model of Lesch-Nyhan Disease.

    PubMed

    Meek, Stephen; Thomson, Alison J; Sutherland, Linda; Sharp, Matthew G F; Thomson, Julie; Bishop, Valerie; Meddle, Simone L; Gloaguen, Yoann; Weidt, Stefan; Singh-Dolt, Karamjit; Buehr, Mia; Brown, Helen K; Gill, Andrew C; Burdon, Tom

    2016-05-17

    Lesch-Nyhan disease (LND) is a severe neurological disorder caused by loss-of-function mutations in the gene encoding hypoxanthine phosphoribosyltransferase (HPRT), an enzyme required for efficient recycling of purine nucleotides. Although this biochemical defect reconfigures purine metabolism and leads to elevated levels of the breakdown product urea, it remains unclear exactly how loss of HPRT activity disrupts brain function. As the rat is the preferred rodent experimental model for studying neurobiology and diseases of the brain, we used genetically-modified embryonic stem cells to generate an HPRT knock-out rat. Male HPRT-deficient rats were viable, fertile and displayed normal caged behaviour. However, metabolomic analysis revealed changes in brain biochemistry consistent with disruption of purine recycling and nucleotide metabolism. Broader changes in brain biochemistry were also indicated by increased levels of the core metabolite citrate and reduced levels of lipids and fatty acids. Targeted MS/MS analysis identified reduced levels of dopamine in the brains of HPRT-deficient animals, consistent with deficits noted previously in human LND patients and HPRT knock-out mice. The HPRT-deficient rat therefore provides a new experimental platform for future investigation of how HPRT activity and disruption of purine metabolism affects neural function and behaviour.

  4. Reduced levels of dopamine and altered metabolism in brains of HPRT knock-out rats: a new rodent model of Lesch-Nyhan Disease

    PubMed Central

    Meek, Stephen; Thomson, Alison J.; Sutherland, Linda; Sharp, Matthew G. F.; Thomson, Julie; Bishop, Valerie; Meddle, Simone L.; Gloaguen, Yoann; Weidt, Stefan; Singh-Dolt, Karamjit; Buehr, Mia; Brown, Helen K.; Gill, Andrew C.; Burdon, Tom

    2016-01-01

    Lesch-Nyhan disease (LND) is a severe neurological disorder caused by loss-of-function mutations in the gene encoding hypoxanthine phosphoribosyltransferase (HPRT), an enzyme required for efficient recycling of purine nucleotides. Although this biochemical defect reconfigures purine metabolism and leads to elevated levels of the breakdown product urea, it remains unclear exactly how loss of HPRT activity disrupts brain function. As the rat is the preferred rodent experimental model for studying neurobiology and diseases of the brain, we used genetically-modified embryonic stem cells to generate an HPRT knock-out rat. Male HPRT-deficient rats were viable, fertile and displayed normal caged behaviour. However, metabolomic analysis revealed changes in brain biochemistry consistent with disruption of purine recycling and nucleotide metabolism. Broader changes in brain biochemistry were also indicated by increased levels of the core metabolite citrate and reduced levels of lipids and fatty acids. Targeted MS/MS analysis identified reduced levels of dopamine in the brains of HPRT-deficient animals, consistent with deficits noted previously in human LND patients and HPRT knock-out mice. The HPRT-deficient rat therefore provides a new experimental platform for future investigation of how HPRT activity and disruption of purine metabolism affects neural function and behaviour. PMID:27185277

  5. Molecular analysis of hprt mutations induced by chromium picolinate in CHO AA8 cells.

    PubMed

    Coryell, Virginia H; Stearns, Diane M

    2006-11-07

    Chromium picolinate (CrPic) is a popular dietary supplement, marketed to the public for weight loss, bodybuilding, and control of blood sugar. Recommendations for long-term use at high dosages have led to questions regarding its safety. Previous studies have reported that CrPic can cause chromosomal aberrations and mutations. The purpose of the current work was to compare the mutagenicity of CrPic as a suspension in acetone versus a solution in DMSO, and to characterize the hprt mutations induced by CrPic in CHO AA8 cells. Treatments of 2% acetone or 2% DMSO alone produced no significant increase in 6-thioguanine (6-TG)-resistant mutants after 48 h exposures. Mutants resistant to 6-TG were generated by exposing cells for 48 h to 80 microg/cm(2) CrPic in acetone or to 1.0mM CrPic in DMSO. CrPic in acetone produced an average induced mutation frequency (MF) of 56 per 10(6) surviving cells relative to acetone solvent. CrPic in acetone was 3.5-fold more mutagenic than CrPic in DMSO, which produced an MF of 16.2. Characterization of 61 total mutations in 48 mutants generated from exposure to CrPic in acetone showed that base substitutions comprised 33% of the mutations, with transversions being predominant; deletions made up 62% of the mutations, with one-exon deletions predominating; and 1-4 bp insertions made up 5% of the characterized mutations. CrPic induced a statistically greater number of deletions and a statistically smaller number of base substitutions than have been measured in spontaneously generated mutants. These data confirm previous studies showing that CrPic is mutagenic, and support the contention that further study is needed to verify the safety of CrPic for human consumption.

  6. A Mutant Gene That Increases Gibberellin Production in Brassica1

    PubMed Central

    Rood, Stewart B.; Williams, Paul H.; Pearce, David; Murofushi, Noboru; Mander, Lewis N.; Pharis, Richard P.

    1990-01-01

    A single gene mutant (elongated internode [ein/ein]) with accelerated shoot elongation was identified from a rapid cycling line of Brassica rapa. Relative to normal plants, mutant plants had slightly accelerated floral development, greater stem dry weights, and particularly, increased internode and inflorescence elongation. The application of the triazole plant growth retardant, paclobutrazol, inhibited shoot elongation, returning ein to a more normal phenotype. Conversely, exogenous gibberellin A3 (GA3) can convert normal genotypes to a phenotype resembling ein. The content of endogenous GA1 and GA3 were estimated by gas chromatography-selected ion monitoring using [2H]GA1, as a quantitative internal standard and at day 14 were 1.5- and 12.1-fold higher per stem, respectively, in ein than in normal plants, although GA concentrations were more similar. The endogenous levels of GA20 and GA1, and the rate of GA19 metabolism were simultaneously analyzed at day 7 by feeding [2H2]GA19 and measuring metabolites [2H2]GA20 and [2H2]GA1 and endogenous GA20 and GA1, with [2H5]GA20 and [2H5]GA1 as quantitative internal standards. Levels of GA1 and GA20 were 4.6- and 12.9-fold higher, respectively, and conversions to GA20 and GA1 were 8.3 and 1.3 times faster in ein than normal plants. Confirming the enhanced rate of GA1 biosynthesis in ein, the conversion of [3H]GA20 to [3H]GA1 was also faster in ein than in the normal genotype. Thus, the ein allele results in accelerated GA1 biosynthesis and an elevated content of endogenous GAs, including the dihydroxylated GAs A1 and A3. The enhanced GA production probably underlies the accelerated shoot growth and development, and particularly, the increased shoot elongation. Images Figure 1 PMID:16667574

  7. Increased volatile anesthetic requirement in short-sleeping Drosophila mutants

    PubMed Central

    Weber, Bernd; Schaper, Christian; Bushey, Daniel; Rohlfs, Marko; Steinfath, Markus; Tononi, Giulio; Cirelli, Chiara; Scholz, Jens; Bein, Berthold

    2009-01-01

    Background Anesthesia and sleep share physiological and behavioral similarities. The anesthetic requirement of the recently identified Drosophila mutant minisleeper and other Drosophila mutants was investigated. Methods Sleep and wakefulness were determined by measuring activity of individual wild-type and mutant flies. Based on the response of the flies at different concentrations of the volatile anesthetics isoflurane and sevoflurane, concentration-response curves were generated and EC50 values were calculated. Results The average amount of daily sleep in wild-type Drosophila (n=64) was 965 ±15 minutes and 1022 ± 29 in na[har38] p>0.05; n=32) (mean ± SEM, all p compared to wild-type and other shaker alleles). Shmns flies slept 584 ±13 minutes (n=64, p<0.01), Sh102 412 ± 22 minutes (n=32, p<0.01) and Sh120 782 ± 25 minutes (n=32, p<0.01). The EC50 values for isoflurane were 0.706 (95% confidence interval 0.649 to 0.764, n=661) and for sevoflurane 1.298 (1.180 to 1.416, n=522) in wild-type Drosophila, 1.599 (1.527 to 1.671, n=308) and 2.329 (2.177 to 2.482, n=282) in Sh102, 1.306 (1.212 to 1.400, n=393) and 2.013 (1.868 to 2.158, n=550) in Shmns, 0.957 (0.860 to 1.054, n=297) and 1.619 (1.508 to 1.731, n=386) in Sh120, and 0.6154 (0.581 to 0.649, n=360; p<0.05) and 0.9339 (0.823 to 1.041, n= 274) in na[har38], respectively (all p<0.01). Conclusions A single-gene mutation in Drosophila that causes an extreme reduction in daily sleep is responsible for a significant increase in the requirement of volatile anesthetics. This suggests that a single gene mutation affects both sleep behavior and anesthesia and sedation. PMID:19164958

  8. A mutant gene that increases gibberellin production in Brassica

    SciTech Connect

    Rood, S.B. ); Williams, P.H. ); Pearce, D.; Pharis, R.P. ); Murofushi, Noboru ); Mander, L.N. )

    1990-07-01

    A single gene mutant (elongated internode (ein/ein)) with accelerated shoot elongation was identified from a rapid cycling line of Brassica rapa. Relative to normal plants, mutant plants had slightly accelerated floral development, greater stem dry weights, and particularly, increased internode and inflorescence elongation. The application of the triazole plant growth retardant, paclobutrazol, inhibited shoot elongation, returning ein to a more normal phenotype. Conversely, exogenous gibberellin A{sub 3} (GA{sub 3}) can convert normal genotypes to a phenotype resembling ein. The content of endogenous GA{sub 1} and GA{sub 3} were estimated by gas chromatography-selected ion monitoring using ({sup 2}H)GA{sub 1} as a quantitative internal standard and at day 14 were 1.5- and 12.1-fold higher per stem, respectively, in ein than in normal plants, although GA concentrations were more similar. The endogenous levels of GA{sub 20} and GA{sub 1}, and the rate of GA{sub 19} metabolism were simultaneously analyzed. Levels of GA{sub 1} and GA{sub 20} were 4.6- and 12.9-fold higher, respectively, and conversions to GA{sub 20} and GA{sub 1} were 8.3 and 1.3 times faster in ein than normal plants. Confirming the enhanced rate of GA{sub 1} biosynthesis in ein, the conversion of ({sup 3}H)GA{sub 20} to ({sup 3}H) GA{sub 1} was also faster in ein than in the normal genotype. Thus, the ein allele results in accelerated GA{sub 1} biosynthesis and an elevated content of endogenous GAs, including the dihydroxylated GAs A{sub 1} and A{sub 3}.

  9. Molecular analysis and comparison of radiation-induced large deletions of the HPRT locus in primary human skin fibroblasts

    NASA Technical Reports Server (NTRS)

    Yamada, Y.; Park, M. S.; Okinaka, R. T.; Chen, D. J.

    1996-01-01

    Genetic alterations in gamma-ray- and alpha-particle-induced HPRT mutants were examined by multiplex polymerase chain reaction (PCR) analysis. A total of 39-63% of gamma-ray-induced and 31-57% of alpha-particle-induced mutants had partial or total deletions of the HPRT gene. The proportion of these deletion events was dependent on radiation dose, and at the resolution limits employed there were no significant differences between the spectra induced by equitoxic doses of alpha particles (0.2-0.4 Gy) and gamma rays (3 Gy). The molecular nature of the deletions was analyzed by the use of sequence tagged site (STS) primers and PCR amplification as a "probe" for specific regions of the human X chromosome within the Xq26 region. These STSs were closely linked and spanned regions approximately 1.7 Mbp from the telomeric side and 1.7 Mbp from the centromeric side of the HPRT gene. These markers include: DXS53, 299R, DXS79, yH3L, 3/19, PR1, PR25, H2, yH3R, 1/44, 1/67, 1/1, DXS86, D8C6, DXS10 and DXS144. STS analyses indicated that the maximum size of total deletions in radiation-induced HPRT mutants can be greater than 2.7 Mbp and deletion size appears to be dependent on radiation dose. There were no apparent differences in the sizes of the deletions induced by alpha particles or gamma rays. On the other hand, deletions containing portions of the HPRT gene were observed to be 800 kbp or less, and the pattern of the partial deletion induced by alpha particles appeared to be different from that induced by gamma rays.

  10. Molecular analysis and comparison of radiation-induced large deletions of the HPRT locus in primary human skin fibroblasts

    NASA Technical Reports Server (NTRS)

    Yamada, Y.; Park, M. S.; Okinaka, R. T.; Chen, D. J.

    1996-01-01

    Genetic alterations in gamma-ray- and alpha-particle-induced HPRT mutants were examined by multiplex polymerase chain reaction (PCR) analysis. A total of 39-63% of gamma-ray-induced and 31-57% of alpha-particle-induced mutants had partial or total deletions of the HPRT gene. The proportion of these deletion events was dependent on radiation dose, and at the resolution limits employed there were no significant differences between the spectra induced by equitoxic doses of alpha particles (0.2-0.4 Gy) and gamma rays (3 Gy). The molecular nature of the deletions was analyzed by the use of sequence tagged site (STS) primers and PCR amplification as a "probe" for specific regions of the human X chromosome within the Xq26 region. These STSs were closely linked and spanned regions approximately 1.7 Mbp from the telomeric side and 1.7 Mbp from the centromeric side of the HPRT gene. These markers include: DXS53, 299R, DXS79, yH3L, 3/19, PR1, PR25, H2, yH3R, 1/44, 1/67, 1/1, DXS86, D8C6, DXS10 and DXS144. STS analyses indicated that the maximum size of total deletions in radiation-induced HPRT mutants can be greater than 2.7 Mbp and deletion size appears to be dependent on radiation dose. There were no apparent differences in the sizes of the deletions induced by alpha particles or gamma rays. On the other hand, deletions containing portions of the HPRT gene were observed to be 800 kbp or less, and the pattern of the partial deletion induced by alpha particles appeared to be different from that induced by gamma rays.

  11. Zymomonas mobilis mutants with an increased rate of alcohol production

    SciTech Connect

    Osman, Y.A.; Ingram, L.O.

    1987-07-01

    Two new derivatives of Zymomonas mobilis CP4 were isolated from enrichment cultures after 18 months of serial transfer. These new strains were selected for the ability to grow and produce ethanol rapidly on transfer into fresh broth containing ethanol and allyl alcohol. Ethanol production by these strains was examined in batch fermentations under three sets of conditions. Both new derivatives were found to be superior to the parent strain CP4 with respect to the speed and completeness of glucose conversion to ethanol. The best of these, strain YO2, produced 9.5% ethanol (by weight; 11.9% by volume) after 17.4 h compared with 31.8 h for the parent strain CP4. The addition of 1 mM magnesium sulfate improved ethanol production in all three strains. Two factors contributed to the decrease in fermentation time required by the mutants: more rapid growth with minimal lag on subculturing and the retention of higher rates of ethanol production as fermentation proceeded. Alcohol dehydrogenase isozymes were altered in both new strains and no longer catalyzed the oxidation of allyl alcohol into the toxic product acrolein. This loss of allyl alcohol-oxidizing capacity is proposed as a primary factor contributing to increased allyl alcohol resistance, although it is likely that other mutations affecting glycolysis also contribute to the improvement in ethanol production.

  12. Delayed effects of accelerated heavy ions on the induction of HPRT mutations in V79 hamster cells.

    PubMed

    Bláha, Pavel; Koshlan, Nataliya A; Koshlan, Igor V; Petrova, Daria V; Bogdanova, Yulia V; Govorun, Raisa D; Múčka, Viliam; Krasavin, Evgeny A

    2017-10-01

    Fundamental research on the harmful effects of ionizing radiation on living cells continues to be of great interest. Recently, priority has been given to the study of high-charge and high-energy (HZE) ions that comprise a substantial part of the galactic cosmic ray (GCR) spectra that would be encountered during long-term space flights. Moreover, predictions of the delayed genetic effects of high linear energy transfer (LET) exposure is becoming more important as heavy ion therapy use is increasing. This work focuses mainly on the basic research on the delayed effects of HZE ions on V79 Chinese hamster cells, with emphasis on the induction of HPRT mutations after prolonged expression times (ET). The research was conducted under various irradiation conditions with accelerated ions (18)O (E=35.2MeV/n), (20)Ne (E=47.7MeV/n and 51.8MeV/n), and (11)B (E=32.4MeV/n), with LET in the range from 49 to 149 keV/μm and with (60)Co γ-rays. The HPRT mutant fractions (MF) were detected in irradiated cells in regular intervals during every cell culture recultivation (every 3days) up to approximately 40days (70-80 generations) after irradiation. The MF maximum was reached at different ET depending on ionizing radiation characteristics. The position of the maximum was shifting towards longer ET with increasing LET. We speculate that the delayed mutations are created de novo and that they are the manifestation of genomic instability. Although the exact mechanisms involved in genomic instability initiation are yet to be identified, we hypothesize that differences in induction of delayed mutations by radiations with various LET values are related to variations in energy deposition along the particle track. A dose dependence of mutation yield is discussed as well. Copyright © 2017 Elsevier B.V. All rights reserved.

  13. New Infestin-4 Mutants with Increased Selectivity against Factor XIIa

    PubMed Central

    Vuimo, Tatiana A.; Surov, Stepan S.; Ovsepyan, Ruzanna A.; Korneeva, Vera A.; Vorobiev, Ivan I.; Orlova, Nadezhda A.; Minakhin, Leonid; Kuznedelov, Konstantin; Severinov, Konstantin V.; Ataullakhanov, Fazoil I.; Panteleev, Mikhail A.

    2015-01-01

    Factor XIIa (fXIIa) is a serine protease that triggers the coagulation contact pathway and plays a role in thrombosis. Because it interferes with coagulation testing, the need to inhibit fXIIa exists in many cases. Infestin-4 (Inf4) is a Kazal-type inhibitor of fXIIa. Its specificity for fXIIa can be enhanced by point mutations in the protease-binding loop. We attempted to adapt Inf4 for the selective repression of the contact pathway under various in vitro conditions, e.g., during blood collection and in ‘global’ assays of tissue factor (TF)-dependent coagulation. First, we designed a set of new Inf4 mutants that, in contrast to wt-Inf4, had stabilized canonical conformations during molecular dynamics simulation. Off-target activities against factor Xa (fXa), plasmin, and other coagulation proteases were either reduced or eliminated in these recombinant mutants, as demonstrated by chromogenic assays. Interactions with fXIIa and fXa were also analyzed using protein-protein docking. Next, Mutant B, one of the most potent mutants (its Ki for fXIIa is 0.7 nM) was tested in plasma. At concentrations 5–20 μM, this mutant delayed the contact-activated generation of thrombin, as well as clotting in thromboelastography and thrombodynamics assays. In these assays, Mutant B did not affect coagulation initiated by TF, thus demonstrating sufficient selectivity and its potential practical significance as a reagent for coagulation diagnostics. PMID:26670620

  14. Elevated frequencies of hypoxanthine phosphoribosyltransferase lymphocyte mutants are detected in Russian liquidators 6 to 10 years after exposure to radiation from the Chernobyl nuclear power plant accident.

    PubMed

    Thomas, C B; Nelson, D O; Pleshanov, P; Vorobstova, I; Tureva, L; Jensen, R; Jones, I M

    1999-02-02

    This study was conducted to determine whether the frequency of hypoxanthine phosphoribosyltransferase (HPRT) deficient lymphocyte mutants would detect an effect of radiation exposure in a population of Russians who were exposed to low levels of radiation while working in 1986 and 1987 as liquidators cleaning up after the Chernobyl nuclear power reactor accident. The HPRT lymphocyte cloning assay was performed on peripheral blood lymphocytes collected between 1992 and 1996 from 142 liquidators and 66 Russian controls, and between 1989 and 1993 from 231 American controls. Russian and American controls were not significantly different for either cloning efficiency or mutant frequency (MF); inclusion of both sets of controls in the analysis increased the ability to detect a Chernobyl exposure effect in the liquidators. After adjusting for age and smoking, the results revealed no significant difference in cloning efficiency of Chernobyl liquidators relative to Russian controls but a significant, 24% increase in liquidator HPRT mutant frequency over Russian controls (90% confidence interval was 7% to 45% increase). The analytical method also accounted for differences in precision of the individual estimates of log CE and log MF and accommodated for outliers. The increase in HPRT mutant frequency of liquidators is an attribute of the exposed population as a whole rather than of individuals. These results demonstrate that, under appropriate circumstances, the HPRT specific locus mutation assay of peripheral blood lymphocytes can be used to detect a semi-acute, low dose radiation exposure of a population, even 6 to 10 years after the exposure. Copyright 1999 Elsevier Science B.V.

  15. Azetidine-2-carboxylic acid resistant mutants of Arabidopsis thaliana with increased salt tolerance

    SciTech Connect

    Lehle, F.R.; Murphy, M.A.; Khan, R.A. )

    1989-04-01

    Nineteen mutant Arabidopsis families resistant to the proline analog azetidine-2-carboxylic acid (ACA) were characterized in terms of NaCl tolerance and proline content. Mutants were selected from about 64,000 progeny of about 16,000 self-pollinated Columbia parents which had been mutated with ethyl methane sulfonate during seed imbibition. Selections were performed during seed germination on aseptic agar medium containing 0.2 to 0.25 mM ACA. Nineteen mutant families, 12 clearly independent, retained resistance to ACA in the M{sub 4} generation. Based on germination on 150 mM NaCl, 13 of the mutant families were more tolerant than the wild type. Two mutants of intermediate resistance to ACA were markedly more salt tolerant than the others. Four mutant families appeared to overproduce proline. Of these, only 3 showed slight increases in salt tolerance.

  16. Comparison of hprt variant frequencies and chromosome aberration frequencies in lymphocytes from radiotherapy and chemotherapy patients: A prospective study

    SciTech Connect

    Ammenheuser, M.M.; Au, W.W.; Whorton, E.B. Jr.; Belli, J.A.; Ward, J.B. Jr. )

    1991-01-01

    The autoradiographic 6-thioguanine-resistant mutant lymphocyte assay and a chromosome aberration assay were used to determine the time-course of appearance and persistence of elevated frequencies of hprt variants and dicentric chromosomes in patients receiving x-irradiation therapy. The hprt mutation assays were done with frozen/thawed lymphocytes isolated from aliquots of the same blood samples used for the chromosome aberration assays. Five multiple sclerosis patients were also studied before and at 2 and 4 wk intervals after treatment with monthly i.v. doses of 750 mg/m{sup 2} of cyclophosphamide (CP). There were no significant elevations in chromosome aberrations at these post-treatment sample times. The results demonstrate the complementary nature of these two human monitoring assays and emphasize the importance of careful selection of optimal sampling times.

  17. Expression of Maternally and Embryonically Derived Hypoxanthine Phosphoribosyl Transferase (Hprt) Activity in Mouse Eggs and Early Embryos

    PubMed Central

    Kratzer, Paul G.

    1983-01-01

    X-chromosome activity in early mouse development has been studied by a gene dosage method that involves measuring the activity level of the X-linked enzyme hypoxanthine phosphoribosyl transferase (HPRT) in single eggs and embryos from XO females and from females heterozygous for In(X)1H, a paracentric inversion of the X chromosome. The HPRT activity in oocytes increased threefold over a 24-hr period beginning after ovulation. Afterward, the activity plateaued in unfertilized eggs but continued to increase for at least 66 hr in presumed OY embryos. Both before and after ovulation, the level of activity in unfertilized eggs from In(X)/X females was twice that from XO females, and the distributions of activity in eggs for both sets of females remained unimodal. Beginning with the two-cell stage, distributions of activity for embryos from In(X)/X females were trimodal, which is evidence for embryonic activity. It is proposed that activation of a maternal mRNA or proenzyme is responsible for the HPRT activity increase in oocytes and early embryos and is supplemented by dosage-dependent activity of the embryonic Hprt gene as early as the two-cell stage. PMID:6618165

  18. Analysis of solvent control and 1-nitrosopyrene-induced chinese hamster ovary cell mutants by southern and northern blots and the polymerase chain reaction

    SciTech Connect

    Newton, R.K.; Mittelstaedt, R.A.; Heflich, R.H. )

    1992-01-01

    1-Nitrosopyrene, a metabolite of the tumorigenic environmental pollutant 1-nitropyrene, is a potent mutagen at the hprt locus in Chinese hamster ovary (CHO) cells. A single DNA adduct, N-(deoxyguanosin-8-yl)-l-aminopyrene, is produced in CHO cells treated with 1-nitrosopyrene, and this adduct is found in rats and mice exposed to 1-nitropyrene. In this study, the structure of the hprt gene and the structure and amount of hprt mRNA were analyzed in 43 CHO cell mutants (16 isolated from solvent control cultures and 27 isolated from 1-nitrosopyrene-treated cultures). PstI- and BamHI-digested DNA from the mutants were subjected to Southern blot analysis using a hamster hprt cDNA probe. None of the 1-nitrosopyrene-induced mutants and only one of the control mutants displayed hybridization patterns that were different from the parent CHO cells. Northern blot analysis revealed that two control mutants had truncated hprt mRNAs, while 56% of the control mutants and 78% of the induced mutants had reduced levels of hprt mRNA. Using polymerase chain reaction amplification of cDNA synthesized from RNA, the hprt protein-coding region could be amplified from 23 of the 1-nitrosopyrene-induced mutants and 11 of the control mutants. The amplification products from 3 of the control mutants and 5 of the induced mutants were smaller than that found with RNA from parental CHO cells. These results indicate that the mutagenic DNA damage produced by 1-nitrosopyrene in CHO cells does not cause major structural alterations in the hprt gene and suggest that 1-nitrosopyrene acts as a point mutagen. A large number of both control and 1-nitrosopyrene-induced mutants exhibited a marked reduction in hprt mRNA concentration or possessed truncated mRNA hprt protein coding sequences. These alterations may contribute to the 6-thioguanine-resistant phenotype.

  19. Increased riboflavin production from activated bleaching earth by a mutant strain of Ashbya gossypii.

    PubMed

    Tajima, Satoshi; Itoh, Yoko; Sugimoto, Takashi; Kato, Tatsuya; Park, Enoch Y

    2009-10-01

    The production of riboflavin from vegetable oil was increased using a mutant strain of Ashbya gossypii. This mutant was generated by treating the wild-type strain with N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). Riboflavin production was 10-fold higher in the mutant compared to the wild-type strain. The specific intracellular catalase activity after 3 d of culture was 6-fold higher in the mutant than in the wild-type strain. For the mutant, riboflavin production in the presence of 40 mM hydrogen peroxide was 16% less than that in the absence of hydrogen peroxide, whereas it was 56% less for the wild-type strain. The isocitrate lyase (ICL) activity of the mutant was 0.26 mU/mg of protein during the active riboflavin production phase, which was 2.6-fold higher than the wild-type strain. These data indicate that the mutant utilizes the carbon flux from the TCA cycle to the glyoxylate cycle more efficiently than the wild-type strain, resulting in enhanced riboflavin production. This novel mutant has the potential to be of use for industrial-scale riboflavin production from waste-activated bleaching earth (ABE), thereby transforming a useless material into a valuable bioproduct.

  20. TATA-Binding Protein Mutants That Increase Transcription from Enhancerless and Repressed Promoters In Vivo

    PubMed Central

    Geisberg, Joseph V.; Struhl, Kevin

    2000-01-01

    Using a genetic screen, we isolated three TATA-binding protein (TBP) mutants that increase transcription from promoters that are repressed by the Cyc8-Tup1 or Sin3-Rpd3 corepressors or that lack an enhancer element, but not from an equivalently weak promoter with a mutated TATA element. Increased transcription is observed when the TBP mutants are expressed at low levels in the presence of wild-type TBP. These TBP mutants are unable to support cell viability, and they are toxic in strains lacking Rpd3 histone deacetylase or when expressed at higher levels. Although these mutants do not detectably bind TATA elements in vitro, genetic and chromatin immunoprecipitation experiments indicate that they act directly at promoters and do not increase transcription by titration of a negative regulatory factor(s). The TBP mutants are mildly defective for associating with promoters responding to moderate or strong activators; in addition, they are severely defective for RNA polymerase (Pol) III but not Pol I transcription. These results suggest that, with respect to Pol II transcription, the TBP mutants specifically increase expression from core promoters. Biochemical analysis indicates that the TBP mutants are unaffected for TFIID complex formation, dimerization, and interactions with either the general negative regulator NC2 or the N-terminal inhibitory domain of TAF130. We speculate that these TBP mutants have an unusual structure that allows them to preferentially access TATA elements in chromatin templates. These TBP mutants define a criterion by which promoters repressed by Cyc8-Tup1 or Sin3-Rpd3 resemble enhancerless, but not TATA-defective, promoters; hence, they support the idea that these corepressors inhibit the function of activator proteins rather than the Pol II machinery. PMID:10669725

  1. Increased expression of the maize immunoglobulin binding protein homolog b-70 in three zein regulatory mutants.

    PubMed Central

    Boston, R S; Fontes, E B; Shank, B B; Wrobel, R L

    1991-01-01

    Plants carrying floury-2, Defective endosperm-B30, or Mucronate mutations overproduce b-70, a maize homolog of the mammalian immunoglobulin binding protein. During endosperm development in these mutants, levels of both b-70 protein and RNA increase dramatically between 14 days and 20 days after pollination. At later stages, b-70 RNA levels decline while protein levels remain high. The increase in b-70 RNA levels is endosperm specific and dependent on gene dosage in the floury-2 mutant. In all three mutants, the increases in b-70 RNA and protein levels are inversely proportional to changes in zein synthesis. Although b-70 polypeptides can be extracted from purified protein bodies, they carry a carboxy-terminal endoplasmic reticulum retention signal, HDEL. We propose that induction of b-70 in these mutants is a cellular response to abnormally folded or improperly assembled storage proteins and probably reflects its role as a polypeptide chain binding protein. PMID:1840924

  2. Increased sensitivity to salt stress in tocopherol-deficient Arabidopsis mutants growing in a hydroponic system.

    PubMed

    Ellouzi, Hasna; Hamed, Karim Ben; Cela, Jana; Müller, Maren; Abdelly, Chedly; Munné-Bosch, Sergi

    2013-02-01

    Recent studies suggest that tocopherols could play physiological roles in salt tolerance but the mechanisms are still unknown. In this study, we analyzed changes in growth, mineral and oxidative status in vte1 and vte4 Arabidopsis thaliana mutants exposed to salt stress. vte1 and vte4 mutants lack α-tocopherol, but only the vte1 mutant is additionally deficient in γ-tocopherol. Results showed that a deficiency in vitamin E leads to reduced growth and increased oxidative stress in hydroponically-grown plants. This effect was observed at early stages, not only in rosettes but also in roots. The vte1 mutant was more sensitive to salt-induced oxidative stress than the wild type and the vte4 mutant. Salt sensitivity was associated with (i) high contents of Na(+), (ii) reduced efficiency of PSII photochemistry (Fv/Fm ratio) and (iii) more pronounced oxidative stress as indicated by increased hydrogen peroxide and malondialdeyde levels. The vte 4 mutant, which accumulates γ- instead of α-tocopherol showed an intermediate sensitivity to salt stress between the wild type and the vte1 mutant. Contents of abscisic acid, jasmonic acid and the ethylene precursor, 1-aminocyclopropane-1-carboxylic acid were higher in the vte1 mutant than the vte4 mutant and wild type. It is concluded that vitamin E-deficient plants show an increased sensitivity to salt stress both in rosettes and roots, therefore indicating the positive role of tocopherols in stress tolerance, not only by minimizing oxidative stress, but also controlling Na(+)/K(+) homeostasis and hormonal balance.

  3. Increased sensitivity to salt stress in tocopherol-deficient Arabidopsis mutants growing in a hydroponic system

    PubMed Central

    Ellouzi, Hasna; Hamed, Karim Ben; Cela, Jana; Müller, Maren; Abdelly, Chedly; Munné-Bosch, Sergi

    2013-01-01

    Recent studies suggest that tocopherols could play physiological roles in salt tolerance but the mechanisms are still unknown. In this study, we analyzed changes in growth, mineral and oxidative status in vte1 and vte4 Arabidopsis thaliana mutants exposed to salt stress. vte1 and vte4 mutants lack α-tocopherol, but only the vte1 mutant is additionally deficient in γ-tocopherol. Results showed that a deficiency in vitamin E leads to reduced growth and increased oxidative stress in hydroponically-grown plants. This effect was observed at early stages, not only in rosettes but also in roots. The vte1 mutant was more sensitive to salt-induced oxidative stress than the wild type and the vte4 mutant. Salt sensitivity was associated with (i) high contents of Na+, (ii) reduced efficiency of PSII photochemistry (Fv/Fm ratio) and (iii) more pronounced oxidative stress as indicated by increased hydrogen peroxide and malondialdeyde levels. The vte 4 mutant, which accumulates γ- instead of α-tocopherol showed an intermediate sensitivity to salt stress between the wild type and the vte1 mutant. Contents of abscisic acid, jasmonic acid and the ethylene precursor, 1-aminocyclopropane-1-carboxylic acid were higher in the vte1 mutant than the vte4 mutant and wild type. It is concluded that vitamin E-deficient plants show an increased sensitivity to salt stress both in rosettes and roots, therefore indicating the positive role of tocopherols in stress tolerance, not only by minimizing oxidative stress, but also controlling Na+/K+ homeostasis and hormonal balance. PMID:23299430

  4. Altered histamine neurotransmission in HPRT-deficient mice.

    PubMed

    Tschirner, Sarah K; Gutzki, Frank; Kaever, Volkhard; Seifert, Roland; Schneider, Erich H

    2015-11-16

    Lesch-Nyhan syndrome (LNS) is an X-chromosomal disorder with congenital deficiency of the purine salvage enzyme hypoxanthine-guanine phosphoribosyltransferase (HPRT) as underlying defect. We determined the concentrations of dopamine, histamine and their metabolites in brains of HPRT knockout mice, which serve as an animal model for LNS, and compared the results to those obtained from wild-type controls. Analyses were performed by high performance liquid chromatography (HPLC)-coupled tandem mass spectrometry (MS/MS). Besides a decrease of dopamine and 3-methoxytyramine (3-MT) concentrations in the cerebral hemisphere, HPRT-deficient mice also exhibited significantly reduced 1-methylhistamine (1-MH) and 1-methylimidazole-4-acetic acid (1-MI4AA) concentrations in the brain hemisphere and medulla. Moreover, the amount of 1-MI4AA was significantly decreased in the cerebellum. Our findings show that neuronal perturbations caused by HPRT deficiency are not restricted to the dopamine system but also affect histaminergic neurotransmission. These new insights into the brain metabolism of an LNS mouse model may help to find new therapeutic strategies to improve the quality of life of LNS patients.

  5. Hypoxanthine-guanine phosophoribosyltransferase (HPRT) deficiency: Lesch-Nyhan syndrome

    PubMed Central

    Torres, Rosa J; Puig, Juan G

    2007-01-01

    Deficiency of hypoxanthine-guanine phosphoribosyltransferase (HPRT) activity is an inborn error of purine metabolism associated with uric acid overproduction and a continuum spectrum of neurological manifestations depending on the degree of the enzymatic deficiency. The prevalence is estimated at 1/380,000 live births in Canada, and 1/235,000 live births in Spain. Uric acid overproduction is present inall HPRT-deficient patients and is associated with lithiasis and gout. Neurological manifestations include severe action dystonia, choreoathetosis, ballismus, cognitive and attention deficit, and self-injurious behaviour. The most severe forms are known as Lesch-Nyhan syndrome (patients are normal at birth and diagnosis can be accomplished when psychomotor delay becomes apparent). Partial HPRT-deficient patients present these symptoms with a different intensity, and in the least severe forms symptoms may be unapparent. Megaloblastic anaemia is also associated with the disease. Inheritance of HPRT deficiency is X-linked recessive, thus males are generally affected and heterozygous female are carriers (usually asymptomatic). Human HPRT is encoded by a single structural gene on the long arm of the X chromosome at Xq26. To date, more than 300 disease-associated mutations in the HPRT1 gene have been identified. The diagnosis is based on clinical and biochemical findings (hyperuricemia and hyperuricosuria associated with psychomotor delay), and enzymatic (HPRT activity determination in haemolysate, intact erythrocytes or fibroblasts) and molecular tests. Molecular diagnosis allows faster and more accurate carrier and prenatal diagnosis. Prenatal diagnosis can be performed with amniotic cells obtained by amniocentesis at about 15–18 weeks' gestation, or chorionic villus cells obtained at about 10–12 weeks' gestation. Uric acid overproduction can be managed by allopurinol treatment. Doses must be carefully adjusted to avoid xanthine lithiasis. The lack of precise

  6. Hypoxanthine-guanine phosophoribosyltransferase (HPRT) deficiency: Lesch-Nyhan syndrome.

    PubMed

    Torres, Rosa J; Puig, Juan G

    2007-12-08

    Deficiency of hypoxanthine-guanine phosphoribosyltransferase (HPRT) activity is an inborn error of purine metabolism associated with uric acid overproduction and a continuum spectrum of neurological manifestations depending on the degree of the enzymatic deficiency. The prevalence is estimated at 1/380,000 live births in Canada, and 1/235,000 live births in Spain. Uric acid overproduction is present inall HPRT-deficient patients and is associated with lithiasis and gout. Neurological manifestations include severe action dystonia, choreoathetosis, ballismus, cognitive and attention deficit, and self-injurious behaviour. The most severe forms are known as Lesch-Nyhan syndrome (patients are normal at birth and diagnosis can be accomplished when psychomotor delay becomes apparent). Partial HPRT-deficient patients present these symptoms with a different intensity, and in the least severe forms symptoms may be unapparent. Megaloblastic anaemia is also associated with the disease. Inheritance of HPRT deficiency is X-linked recessive, thus males are generally affected and heterozygous female are carriers (usually asymptomatic). Human HPRT is encoded by a single structural gene on the long arm of the X chromosome at Xq26. To date, more than 300 disease-associated mutations in the HPRT1 gene have been identified. The diagnosis is based on clinical and biochemical findings (hyperuricemia and hyperuricosuria associated with psychomotor delay), and enzymatic (HPRT activity determination in haemolysate, intact erythrocytes or fibroblasts) and molecular tests. Molecular diagnosis allows faster and more accurate carrier and prenatal diagnosis. Prenatal diagnosis can be performed with amniotic cells obtained by amniocentesis at about 15-18 weeks' gestation, or chorionic villus cells obtained at about 10-12 weeks' gestation. Uric acid overproduction can be managed by allopurinol treatment. Doses must be carefully adjusted to avoid xanthine lithiasis. The lack of precise

  7. Increased Steady-State Mutant Huntingtin mRNA in Huntington's Disease Brain.

    PubMed

    Liu, Wanzhao; Chaurette, Joanna; Pfister, Edith L; Kennington, Lori A; Chase, Kathryn O; Bullock, Jocelyn; Vonsattel, Jean Paul G; Faull, Richard L M; Macdonald, Douglas; DiFiglia, Marian; Zamore, Phillip D; Aronin, Neil

    2013-01-01

    Huntington's disease is caused by expansion of CAG trinucleotide repeats in the first exon of the huntingtin gene, which is essential for both development and neurogenesis. Huntington's disease is autosomal dominant. The normal allele contains 6 to 35 CAG triplets (average, 18) and the mutant, disease-causing allele contains >36 CAG triplets (average, 42). We examined 279 postmortem brain samples, including 148 HD and 131 non-HD controls. A total of 108 samples from 87 HD patients that are heterozygous at SNP rs362307, with a normal allele (18 to 27 CAG repeats) and a mutant allele (39 to 73 CAG repeats) were used to measure relative abundance of mutant and wild-type huntingtin mRNA. We used allele-specific, quantitative RT-PCR based on SNP heterozygosity to estimate the relative amount of mutant versus normal huntingtin mRNA in postmortem brain samples from patients with Huntington's disease. In the cortex and striatum, the amount of mRNA from the mutant allele exceeds that from the normal allele in 75% of patients. In the cerebellum, no significant difference between the two alleles was evident. Brain tissues from non-HD controls show no significant difference between two alleles of huntingtin mRNAs. Allelic differences were more pronounced at early neuropathological grades (grades 1 and 2) than at late grades (grades 3 and 4). More mutant HTT than normal could arise from increased transcription of mutant HTT allele, or decreased clearance of mutant HTT mRNA, or both. An implication is that equimolar silencing of both alleles would increase the mutant HTT to normal HTT ratio.

  8. Dwarfism and increased adiposity in the gh1 mutant zebrafish vizzini.

    PubMed

    McMenamin, Sarah K; Minchin, James E N; Gordon, Tiffany N; Rawls, John F; Parichy, David M

    2013-04-01

    Somatic growth and adipogenesis are closely associated with the development of obesity in humans. In this study, we identify a zebrafish mutant, vizzini, that exhibits both a severe defect in somatic growth and increased accumulation of adipose tissue. Positional cloning of vizzini revealed a premature stop codon in gh1. Although the effects of GH are largely through igfs in mammals, we found no decrease in the expression of igf transcripts in gh1 mutants during larval development. As development progressed, however, we found overall growth to be progressively retarded and the attainment of specific developmental stages to occur at abnormally small body sizes relative to wild type. Moreover, both subcutaneous (sc) and visceral adipose tissues underwent precocious development in vizzini mutants, and at maturity, the sizes of different fat deposits were greatly expanded relative to wild type. In vivo confocal imaging of sc adipose tissue (SAT) expansion revealed that vizzini mutants exhibit extreme enlargement of adipocyte lipid droplets without a corresponding increase in lipid droplet number. These findings suggest that GH1 signaling restricts SAT hypertrophy in zebrafish. Finally, nutrient deprivation of vizzini mutants revealed that SAT mobilization was greatly diminished during caloric restriction, further implicating GH1 signaling in adipose tissue homeostasis. Overall, the zebrafish gh1 mutant, vizzini, exhibits decreased somatic growth, increased adipose tissue accumulation, and disrupted adipose plasticity after nutrient deprivation and represents a novel model to investigate the in vivo dynamics of vertebrate obesity.

  9. Deficiency of the purine metabolic gene HPRT dysregulates microRNA-17 family cluster and guanine-based cellular functions: a role for EPAC in Lesch-Nyhan syndrome

    PubMed Central

    Guibinga, Ghiabe-Henri; Murray, Fiona; Barron, Nikki; Pandori, William; Hrustanovic, Gorjan

    2013-01-01

    Lesch-Nyhan syndrome (LNS) is a neurodevelopmental disorder caused by mutations in the gene encoding the purine metabolic enzyme hypoxanthine-guanine phosphoribosyltransferase (HPRT). A series of motor, cognitive and neurobehavioral anomalies characterize this disease phenotype, which is still poorly understood. The clinical manifestations of this syndrome are believed to be the consequences of deficiencies in neurodevelopmental pathways that lead to disordered brain function. We have used microRNA array and gene ontology analysis to evaluate the gene expression of differentiating HPRT-deficient human neuron-like cell lines. We set out to identify dysregulated genes implicated in purine-based cellular functions. Our approach was based on the premise that HPRT deficiency affects preeminently the expression and the function of purine-based molecular complexes, such as guanine nucleotide exchange factors (GEFs) and small GTPases. We found that several microRNAs from the miR-17 family cluster and genes encoding GEF are dysregulated in HPRT deficiency. Most notably, our data show that the expression of the exchange protein activated by cAMP (EPAC) is blunted in HPRT-deficient human neuron-like cell lines and fibroblast cells from LNS patients, and is altered in the cortex, striatum and midbrain of HPRT knockout mouse. We also show a marked impairment in the activation of small GTPase RAP1 in the HPRT-deficient cells, as well as differences in cytoskeleton dynamics that lead to increased motility for HPRT-deficient neuron-like cell lines relative to control. We propose that the alterations in EPAC/RAP1 signaling and cell migration in HPRT deficiency are crucial for neuro-developmental events that may contribute to the neurological dysfunctions in LNS. PMID:23804752

  10. Deficiency of the purine metabolic gene HPRT dysregulates microRNA-17 family cluster and guanine-based cellular functions: a role for EPAC in Lesch-Nyhan syndrome.

    PubMed

    Guibinga, Ghiabe-Henri; Murray, Fiona; Barron, Nikki; Pandori, William; Hrustanovic, Gorjan

    2013-11-15

    Lesch-Nyhan syndrome (LNS) is a neurodevelopmental disorder caused by mutations in the gene encoding the purine metabolic enzyme hypoxanthine-guanine phosphoribosyltransferase (HPRT). A series of motor, cognitive and neurobehavioral anomalies characterize this disease phenotype, which is still poorly understood. The clinical manifestations of this syndrome are believed to be the consequences of deficiencies in neurodevelopmental pathways that lead to disordered brain function. We have used microRNA array and gene ontology analysis to evaluate the gene expression of differentiating HPRT-deficient human neuron-like cell lines. We set out to identify dysregulated genes implicated in purine-based cellular functions. Our approach was based on the premise that HPRT deficiency affects preeminently the expression and the function of purine-based molecular complexes, such as guanine nucleotide exchange factors (GEFs) and small GTPases. We found that several microRNAs from the miR-17 family cluster and genes encoding GEF are dysregulated in HPRT deficiency. Most notably, our data show that the expression of the exchange protein activated by cAMP (EPAC) is blunted in HPRT-deficient human neuron-like cell lines and fibroblast cells from LNS patients, and is altered in the cortex, striatum and midbrain of HPRT knockout mouse. We also show a marked impairment in the activation of small GTPase RAP1 in the HPRT-deficient cells, as well as differences in cytoskeleton dynamics that lead to increased motility for HPRT-deficient neuron-like cell lines relative to control. We propose that the alterations in EPAC/RAP1 signaling and cell migration in HPRT deficiency are crucial for neuro-developmental events that may contribute to the neurological dysfunctions in LNS.

  11. Lack of DNA damage induction by okadaic acid, a marine toxin, in the CHO-Hprt and the in vitro UDS assays.

    PubMed

    Le Hégarat, Ludovic; Nesslany, Fabrice; Mourot, Annick; Marzin, Daniel; Fessard, Valérie

    2004-12-12

    Okadaic acid (OA) is a marine toxin produced by dinoflagellates and responsible for human intoxications. OA is a specific inhibitor of serine/threonine protein phosphatases PP1 and PP2A and a potent tumor promoter in mouse skin and rat glandular stomach. In a previous study, we demonstrated that OA induced aneuploidy in CHO-K1 cells using the cytokinesis-block micronucleus (CBMN) assay coupled to FISH and concluded that OA was not a direct mutagen. As some previous in vitro mutagenicity studies had given positive results with OA, we decided to perform two additional in vitro mutagenicity assays in accordance with the OECD guidelines: (i) the CHO/Hprt test, which provides end points about locus-specific gene mutation; (ii) the in vitro unscheduled DNA synthesis (UDS) assay in rat hepatocytes, which measures [(3)H]thymidine incorporation into DNA undergoing excision repair. In the CHO/Hprt assay, there was no significant increase in the number of mutants for doses ranging from 5 to 5000 nM in the presence or absence of rat liver S9 fraction. In the in vitro UDS assay, OA did not induce primary DNA damages in rat hepatocytes following 18 h exposure at concentrations between 1.32 and 100 nM. As OA could affect the DNA repair systems via the inhibition of protein phosphatases, its effects on the repair kinetic of 2AAF-induced DNA damage were also investigated with the UDS assay. The results showed that OA did not interact with the DNA-repair process involved in in vitro UDS in rat hepatocytes. We concluded that OA failed to induce direct DNA damage but acted principally by altering the chromosome number, which could contribute to its carcinogenic effect.

  12. Ionizing radiation-induced mutant frequencies increase transiently in male germ cells of older mice.

    PubMed

    Xu, Guogang; McMahan, C Alex; Hildreth, Kim; Garcia, Rebecca A; Herbert, Damon C; Walter, Christi A

    2012-05-15

    Spontaneous mutant frequency in the male germline increases with age, thereby increasing the risk of siring offspring with genetic disorders. In the present study we investigated the effect of age on ionizing radiation-induced male germline mutagenesis. lacI transgenic mice were treated with ionizing radiation at 4-, 15- and 26-month-old, and mutant frequencies were determined for pachytene spermatocytes and round spermatids at 15 days or 49 days after ionizing radiation treatment. Cells collected 15 days after treatment were derivatives of irradiated differentiating spermatogenic cells while cells collected 49 days later were derivatives of spermatogonial stem cells. The results showed that (1) spontaneous mutant frequency increased in spermatogenic cells recovered from nonirradiated old mice (26-months-old), particularly in the round spermatids; (2) mutant frequencies were significantly increased in round spermatids obtained from middle-aged mice (15-months-old) and old age mice (26-months-old) at 15 and 49 days after irradiation compared to the sham-treated old mice; and (3) pachytene spermatocytes obtained from 15- or 26-month-old mice displayed a significantly increased mutant frequency at 15 days post irradiation. This study indicates that age modulates the mutagenic response to ionizing radiation in the male germline.

  13. WR-2721 protects against cytoxan-induced hprt mutagenesis without affecting therapeutic effectiveness

    SciTech Connect

    Kataoka, Yasushi; Perrin, J.; Hunter, N.; Milas, L.; Grdina, D. ||

    1995-12-31

    The radioprotector S-2-(3-aminopropylamino)ethylphosphorothioic acid (WR-2721) was evaluated for its ability to protect against cytoxan-induced mutagenesis at the hypoxanthine-guanine phosphoribosyl transferase (hprt) locus in mouse splenocytes under conditions that would not interfere with the therapeutic effectiveness of cytoxan in the treatment of fibrosarcoma lung tumors. Mutations at the hprt locus increase in frequency as a function of the dose of cytoxan used. With a spontaneous mutation frequency in C3H mice of 1.5 {times} 10{sup {minus}6}, mutation frequencies increased from 6.2 {times} 10{sup {minus}6} to 2.0 {times} 10{sup {minus}5} as the dose of cytoxan increased from 50 to 200 mg/kg. C3H male mice were injected in their tail veins with 3.5 {times} 10{sup 5} viable fibrosarcoma (FSa) cells. This protocol gave rise to an average of 68 tumor colonies per mouse. Four days following injection animals were treated with cytoxan at a dose of 100 mg/kg, which gave rise to significant tumor cell killing and a reduction in tumor colony number to less than an average of one per animal. WR-2721 at a concentration of 100 mg/kg did not affect on cytoxan`s therapeutic effectiveness. However, a 100 mg/kg dose of WR-2721 was effective in reducing the cytoxan induced hprt mutation frequency in mice from 160 to 35 per 10{sup 5} viable cells regardless of whether it was administered 30 min before or 2 h following cytoxan treatment.

  14. Molecular characterization of a deletion in the HPRT1 gene in a patient with Lesch-Nyhan syndrome.

    PubMed

    Taniguchi, A; Yamada, Y; Hakoda, M; Sekita, C; Kawamoto, M; Kaneko, H; Yamanaka, H

    2011-12-01

    Lesch-Nyhan syndrome is caused by a deficiency of hypoxanthine phosphoribosyltransferase (HPRT) encoded by HPRT1. About 20% of patients have a deletion of HPRT1 and large deletions of HPRT1 are not always fully characterized at the molecular level. Here, we report on a case of Lesch-Nyhan syndrome with a 33-kb deletion involving exon 1 of HPRT1. This novel mutation is caused by a nonhomologous recombination between different classes of interspersed repetitive DNA.

  15. Identification of An Arsenic Tolerant Double Mutant With a Thiol-Mediated Component And Increased Arsenic Tolerance in PhyA Mutants

    SciTech Connect

    Sung, D.Y.; Lee, D.; Harris, H.; Raab, A.; Feldmann, J.; Meharg, A.; Kumabe, B.; Komives, E.A.; Schroeder, J.I.; /SLAC, SSRL /Sydney U. /Aberdeen U. /UC, San Diego

    2007-04-06

    A genetic screen was performed to isolate mutants showing increased arsenic tolerance using an Arabidopsis thaliana population of activation tagged lines. The most arsenic-resistant mutant shows increased arsenate and arsenite tolerance. Genetic analyses of the mutant indicate that the mutant contains two loci that contribute to arsenic tolerance, designated ars4 and ars5. The ars4ars5 double mutant contains a single T-DNA insertion, ars4, which co-segregates with arsenic tolerance and is inserted in the Phytochrome A (PHYA) gene, strongly reducing the expression of PHYA. When grown under far-red light conditions ars4ars5 shows the same elongated hypocotyl phenotype as the previously described strong phyA-211 allele. Three independent phyA alleles, ars4, phyA-211 and a new T-DNA insertion allele (phyA-t) show increased tolerance to arsenate, although to a lesser degree than the ars4ars5 double mutant. Analyses of the ars5 single mutant show that ars5 exhibits stronger arsenic tolerance than ars4, and that ars5 is not linked to ars4. Arsenic tolerance assays with phyB-9 and phot1/phot2 mutants show that these photoreceptor mutants do not exhibit phyA-like arsenic tolerance. Fluorescence HPLC analyses show that elevated levels of phytochelatins were not detected in ars4, ars5 or ars4ars5, however increases in the thiols cysteine, gamma-glutamylcysteine and glutathione were observed. Compared with wild type, the total thiol levels in ars4, ars5 and ars4ars5 mutants were increased up to 80% with combined buthionine sulfoximine and arsenic treatments, suggesting the enhancement of mechanisms that mediate thiol synthesis in the mutants. The presented findings show that PHYA negatively regulates a pathway conferring arsenic tolerance, and that an enhanced thiol synthesis mechanism contributes to the arsenic tolerance of ars4ars5.

  16. Elevated Mutagenesis Does Not Explain the Increased Frequency of Antibiotic Resistant Mutants in Starved Aging Colonies

    PubMed Central

    Katz, Sophia; Hershberg, Ruth

    2013-01-01

    The frequency of mutants resistant to the antibiotic rifampicin has been shown to increase in aging (starved), compared to young colonies of Eschierchia coli. These increases in resistance frequency occur in the absence of any antibiotic exposure, and similar increases have also been observed in response to additional growth limiting conditions. Understanding the causes of such increases in the frequency of resistance is important for understanding the dynamics of antibiotic resistance emergence and spread. Increased frequency of rifampicin resistant mutants in aging colonies is cited widely as evidence of stress-induced mutagenesis (SIM), a mechanism thought to allow bacteria to increase mutation rates upon exposure to growth-limiting stresses. At the same time it has been demonstrated that some rifampicin resistant mutants are relatively fitter in aging compared to young colonies, indicating that natural selection may also contribute to increased frequency of rifampicin resistance in aging colonies. Here, we demonstrate that the frequency of mutants resistant to both rifampicin and an additional antibiotic (nalidixic-acid) significantly increases in aging compared to young colonies of a lab strain of Escherichia coli. We then use whole genome sequencing to demonstrate conclusively that SIM cannot explain the observed magnitude of increased frequency of resistance to these two antibiotics. We further demonstrate that, as was previously shown for rifampicin resistance mutations, mutations conferring nalidixic acid resistance can also increase fitness in aging compared to young colonies. Our results show that increases in the frequency of antibiotic resistant mutants in aging colonies cannot be seen as evidence of SIM. Furthermore, they demonstrate that natural selection likely contributes to increases in the frequency of certain antibiotic resistance mutations, even when no selection is exerted due to the presence of antibiotics. PMID:24244205

  17. Increasing the Triacylglycerol Content in Dunaliella tertiolecta through Isolation of Starch-Deficient Mutants.

    PubMed

    Sirikhachornkit, Anchalee; Vuttipongchaikij, Supachai; Suttangkakul, Anongpat; Yokthongwattana, Kittisak; Juntawong, Piyada; Pokethitiyook, Prayad; Kangvansaichol, Kunn; Meetam, Metha

    2016-05-28

    The production cost of biodiesel from microalgae is still not competitive, compared with that of petroleum fuels. The genetic improvement of microalgal strains to increase triacylglycerol (TAG) accumulation is one way to reduce production costs. One of the most promising approaches is the isolation of starch-deficient mutants, which have been reported to successfully increase TAG yields. To date, such a stable mutant is not available in an oleaginous marine microalga, despite several advantages of using marine species for biodiesel production. Algae in the genus Dunaliella are known to tolerate high salt concentration and other environmental stresses. In addition, the cultivation processes for large-scale outdoor commercialization have been well established for this genus. In this study, Dunaliella tertiolecta was used to screen for starch-deficient mutants, using an iodine vapor-staining method. Four out of 20,016 UV-mutagenized strains showed a substantial reduction of starch content. A significantly higher TAG content, up to 3-fold of the wild-type level, was observed in three of the mutants upon induction by nitrogen depletion. The carotenoid production and growth characteristics of these mutants, under both normal and oxidative stress conditions, were not compromised, suggesting that these processes are not necessarily affected by starch deficiency. The results from this work open up new possibilities for exploring Dunaliella for biodiesel production.

  18. Increased Activation of Hereditary Pancreatitis-associated Human Cationic Trypsinogen Mutants in Presence of Chymotrypsin C*

    PubMed Central

    Szabó, András; Sahin-Tóth, Miklós

    2012-01-01

    Mutations in human cationic trypsinogen (PRSS1) cause autosomal dominant hereditary pancreatitis. Increased intrapancreatic autoactivation of trypsinogen mutants has been hypothesized to initiate the disease. Autoactivation of cationic trypsinogen is proteolytically regulated by chymotrypsin C (CTRC), which mitigates the development of trypsin activity by promoting degradation of both trypsinogen and trypsin. Paradoxically, CTRC also increases the rate of autoactivation by processing the trypsinogen activation peptide to a shorter form. The aim of this study was to investigate the effect of CTRC on the autoactivation of clinically relevant trypsinogen mutants. We found that in the presence of CTRC, trypsinogen mutants associated with classic hereditary pancreatitis (N29I, N29T, V39A, R122C, and R122H) autoactivated at increased rates and reached markedly higher active trypsin levels compared with wild-type cationic trypsinogen. The A16V mutant, known for its variable disease penetrance, exhibited a smaller increase in autoactivation. The mechanistic basis of increased activation was mutation-specific and involved resistance to degradation (N29I, N29T, V39A, R122C, and R122H) and/or increased N-terminal processing by CTRC (A16V and N29I). These observations indicate that hereditary pancreatitis is caused by CTRC-dependent dysregulation of cationic trypsinogen autoactivation, which results in elevated trypsin levels in the pancreas. PMID:22539344

  19. MicroRNA-mediated dysregulation of neural developmental genes in HPRT deficiency: clues for Lesch-Nyhan disease?

    PubMed

    Guibinga, Ghiabe-Henri; Hrustanovic, Gorjan; Bouic, Kathryn; Jinnah, Hyder A; Friedmann, Theodore

    2012-02-01

    Mutations in the gene encoding the purine biosynthetic enzyme hypoxanthine-guanine phosphoribosyltransferase (HPRT) cause the intractable neurodevelopmental Lesch-Nyhan disease (LND) associated with aberrant development of brain dopamine pathways. In the current study, we have identified an increased expression of the microRNA miR181a in HPRT-deficient human dopaminergic SH-SY5Y neuroblastoma cells. Among the genes potentially regulated by miR181a are several known to be required for neural development, including Engrailed1 (En1), Engrailed2 (En2), Lmx1a and Brn2. We demonstrate that these genes are down-regulated in HPRT-deficient SH-SY5Y cells and that over-expression of miR181a significantly reduces endogenous expression of these genes and inhibits translation of luciferase plasmids bearing the En1/2 or Lmx1a 3'UTR miRNA-binding elements. Conversely, inhibition of miR181a increases the expression of these genes and enhances translation of luciferase constructs bearing the En1/2 and Lmx1a 3'UTR miRNA-binding sequences. We also demonstrate that key neurodevelopmental genes (e.g. Nurr1, Pitx3, Wnt1 and Mash1) known to be functional partners of Lmx1a and Brn2 are also markedly down-regulated in SH-SY5Y cells over-expressing miR181a and in HPRT-deficient cells. Our findings in SH-SY5Y cells demonstrate that HPRT deficiency is accompanied by dysregulation of some of the important pathways that regulate the development of dopaminergic neurons and dopamine pathways and that this defect is associated with and possibly due at least partly to aberrant expression of miR181a. Because aberrant expression of miR181a is not as apparent in HPRT-deficient LND fibroblasts, the relevance of the SH-SY5Y neuroblastoma cells to human disease remains to be proven. Nevertheless, we propose that these pleiotropic neurodevelopment effects of miR181a may play a role in the pathogenesis of LND.

  20. MicroRNA-mediated dysregulation of neural developmental genes in HPRT deficiency: clues for Lesch–Nyhan disease?

    PubMed Central

    Guibinga, Ghiabe-Henri; Hrustanovic, Gorjan; Bouic, Kathryn; Jinnah, Hyder A.; Friedmann, Theodore

    2012-01-01

    Mutations in the gene encoding the purine biosynthetic enzyme hypoxanthine–guanine phosphoribosyltransferase (HPRT) cause the intractable neurodevelopmental Lesch–Nyhan disease (LND) associated with aberrant development of brain dopamine pathways. In the current study, we have identified an increased expression of the microRNA miR181a in HPRT-deficient human dopaminergic SH-SY5Y neuroblastoma cells. Among the genes potentially regulated by miR181a are several known to be required for neural development, including Engrailed1 (En1), Engrailed2 (En2), Lmx1a and Brn2. We demonstrate that these genes are down-regulated in HPRT-deficient SH-SY5Y cells and that over-expression of miR181a significantly reduces endogenous expression of these genes and inhibits translation of luciferase plasmids bearing the En1/2 or Lmx1a 3′UTR miRNA-binding elements. Conversely, inhibition of miR181a increases the expression of these genes and enhances translation of luciferase constructs bearing the En1/2 and Lmx1a 3′UTR miRNA-binding sequences. We also demonstrate that key neurodevelopmental genes (e.g. Nurr1, Pitx3, Wnt1 and Mash1) known to be functional partners of Lmx1a and Brn2 are also markedly down-regulated in SH-SY5Y cells over-expressing miR181a and in HPRT-deficient cells. Our findings in SH-SY5Y cells demonstrate that HPRT deficiency is accompanied by dysregulation of some of the important pathways that regulate the development of dopaminergic neurons and dopamine pathways and that this defect is associated with and possibly due at least partly to aberrant expression of miR181a. Because aberrant expression of miR181a is not as apparent in HPRT-deficient LND fibroblasts, the relevance of the SH-SY5Y neuroblastoma cells to human disease remains to be proven. Nevertheless, we propose that these pleiotropic neurodevelopment effects of miR181a may play a role in the pathogenesis of LND. PMID:22042773

  1. Mutant IDH1-driven cellular transformation increases RAD51-mediated homologous recombination and temozolomide resistance.

    PubMed

    Ohba, Shigeo; Mukherjee, Joydeep; See, Wendy L; Pieper, Russell O

    2014-09-01

    Isocitrate dehydrogenase 1 (IDH1) mutations occur in most lower grade glioma and not only drive gliomagenesis but are also associated with longer patient survival and improved response to temozolomide. To investigate the possible causative relationship between these events, we introduced wild-type (WT) or mutant IDH1 into immortalized, untransformed human astrocytes, then monitored transformation status and temozolomide response. Temozolomide-sensitive parental cells exhibited DNA damage (γ-H2AX foci) and a prolonged G2 cell-cycle arrest beginning three days after temozolomide (100 μmol/L, 3 hours) exposure and persisting for more than four days. The same cells transformed by expression of mutant IDH1 exhibited a comparable degree of DNA damage and cell-cycle arrest, but both events resolved significantly faster in association with increased, rather than decreased, clonogenic survival. The increases in DNA damage processing, cell-cycle progression, and clonogenicity were unique to cells transformed by mutant IDH1, and were not noted in cells transformed by WT IDH1 or an oncogenic form (V12H) of Ras. Similarly, these effects were not noted following introduction of mutant IDH1 into Ras-transformed cells or established glioma cells. They were, however, associated with increased homologous recombination (HR) and could be reversed by the genetic or pharmacologic suppression of the HR DNA repair protein RAD51. These results show that mutant IDH1 drives a unique set of transformative events that indirectly enhance HR and facilitate repair of temozolomide-induced DNA damage and temozolomide resistance. The results also suggest that inhibitors of HR may be a viable means to enhance temozolomide response in IDH1-mutant glioma.

  2. Identification of potential molecular markers of ionizing radiation-induced mutations at the hprt locus in CHO cells

    SciTech Connect

    Schwartz, J.L.; Sun, J.; Porter, R.C.

    1995-11-01

    Using multiplex polymerase chain reaction-based exon deletion analysis, we have analyzed mutations at the hprt locus from independent CHO cell mutants isolated from untreated, {sup 60}Co x-ray-, and {sup 212}Bi-exposed CHO-K1 cello and its radiation-sensitive derivative, xrs-5. In the 71 spontaneous CHO-K1 mutants analyzed, 78% showed no change in exon number or size, 20% showed loss of 1-8 exons (partial deletion), and 3% showed loss of all nine hprt exons (total deletion). Exposure of CHO-K1 cells to 6 Gy of {gamma} rays (10% survival) produced 45% of the 20 mutants analyzed showing partial deletion, and 30% showing total deletion. Exposure to an equitoxic dose of a radiation from {sup 212}Bi, a {sup 220}Rn daughter, resulted in a spectrum similar to the {gamma}-ray spectrum in that more than 75% of the 49 mutants analyzed were deletions. The {alpha}-radiation, however, tended to produce larger intragenic deletions that {gamma} radiation. Of the 87 spontaneous xrs-5 mutants analyzed for deletions 44% showed partial deletion, and 14% showed total deletion. Exposure to {alpha} radiation (10% survival) resulted in a deletion spectrum similar to that seen in CHO-K1 cells. Of the 49 mutants analyzed, 43% showed no change in exon number or size, 16% showed partial deletion, and 41% showed total deletion. While the defect in xrs-5 has a profound effect on spontaneous mutation spectra, it does not appear to affect {alpha}-induced mutation spectra.

  3. Effects of 2.45 GHz electromagnetic fields with a wide range of SARs on bacterial and HPRT gene mutations.

    PubMed

    Koyama, Shin; Takashima, Yoshio; Sakurai, Tomonori; Suzuki, Yukihisa; Taki, Masao; Miyakoshi, Junji

    2007-01-01

    Present day use of mobile phones is ubiquitous. This causes some concern for human health due to exposure to high-frequency electromagnetic fields (HFEMF) from mobile phones. Consequently, we have examined the effects of 2.45 GHz electromagnetic fields on bacterial mutations and the hypoxanthine-guanine phosphoribosyl transferase (HPRT) gene mutations. Using the Ames test, bacteria were exposed to HFEMF for 30 min at specific absorption rates (SARs) from 5 to 200 W/kg. In all strains, there was no significant difference in the frequency of revertant colonies between sham exposure and HFEMF-exposed groups. In examination of mutations of the HPRT gene, Chinese hamster ovary (CHO)-K1 cells were exposed to HFEMF for 2 h at SARs from 5 to 200 W/kg. We detected a combination effect of simultaneous exposure to HFEMF and bleomycin at the respective SARs. A statistically significant difference was observed between the cells exposed to HFEMF at the SAR of 200 W/kg. Cells treated with the combination of HFEMF at SARs from 50 to 200 W/kg and bleomycin exhibited increased HPRT mutations. As the exposure to HFEMF induced an increase in temperature, these increases of mutation frequency may be a result of activation of bleomycin by heat. We consider that the increase of mutation frequency may be due to a thermal effect.

  4. Bladder dysfunction in a new mutant mouse model with increased superoxide--lack of nitric oxide?

    PubMed

    Soler, Roberto; Füllhase, Claudius; Lu, Baisong; Bishop, Colin E; Andersson, Karl-Erik

    2010-02-01

    Nitric oxide mediates urethral smooth muscle relaxation and may also be involved in detrusor activity control. Mice with mutation in the Immp2l gene have high superoxide ion levels and a consequent decrease in the bioavailable amount of nitric oxide. We studied bladder function in this mouse model. Young male mutants at ages 4 to 6 months, old female mutants at age 18 months and healthy WT age matched controls were used. The detrusor contractile response to carbachol and electrical field stimulation was tested in isolated detrusor strips in organ baths. In vivo bladder function was evaluated by cystometry in conscious animals. Young male mutants had significantly lower micturition and higher post-void residual volume than WT controls. They had pronounced voiding difficulty and strained when initiating micturition. Detrusor contractile responses to carbachol and electrical field stimulation were similar in mutant and WT mice. Old female mutant mice had lower bladder capacity and micturition volume, and higher micturition frequency and bladder-to-body weight ratio than WT controls. In the in vitro study detrusor strips from mutants showed a lower maximum response to carbachol. Mice with mutation in the Immp2l gene have bladder dysfunction, mainly characterized by emptying abnormalities in young males and increased detrusor activity in old females. Detrusor function was preserved in young males and impaired in old females. These animals are a natural model of oxidative stress with low bioavailable nitric oxide. Thus, they are interesting tools in which to evaluate the role of these conditions on bladder dysfunction. Copyright 2010 American Urological Association. Published by Elsevier Inc. All rights reserved.

  5. Bladder Dysfunction in a New Mutant Mouse Model With Increased Superoxide—Lack of Nitric Oxide?

    PubMed Central

    Soler, Roberto; Füllhase, Claudius; Lu, Baisong; Bishop, Colin E.; Andersson, Karl-Erik

    2013-01-01

    Purpose Nitric oxide mediates urethral smooth muscle relaxation and may also be involved in detrusor activity control. Mice with mutation in the Immp2l gene have high superoxide ion levels and a consequent decrease in the bioavailable amount of nitric oxide. We studied bladder function in this mouse model. Material and Methods Young male mutants at ages 4 to 6 months, old female mutants at age 18 months and healthy WT age matched controls were used. The detrusor contractile response to carbachol and electrical field stimulation was tested in isolated detrusor strips in organ baths. In vivo bladder function was evaluated by cystometry in conscious animals. Results Young male mutants had significantly lower micturition and higher post-void residual volume than WT controls. They had pronounced voiding difficulty and strained when initiating micturition. Detrusor contractile responses to carbachol and electrical field stimulation were similar in mutant and WT mice. Old female mutant mice had lower bladder capacity and micturition volume, and higher micturition frequency and bladder-to-body weight ratio than WT controls. In the in vitro study detrusor strips from mutants showed a lower maximum response to carbachol. Conclusions Mice with mutation in the Immp2l gene have bladder dysfunction, mainly characterized by emptying abnormalities in young males and increased detrusor activity in old females. Detrusor function was preserved in young males and impaired in old females. These animals are a natural model of oxidative stress with low bioavailable nitric oxide. Thus, they are interesting tools in which to evaluate the role of these conditions on bladder dysfunction. PMID:20022053

  6. C. elegans feeding defective mutants have shorter body lengths and increased autophagy.

    PubMed

    Mörck, Catarina; Pilon, Marc

    2006-08-03

    Mutations that cause feeding defects in the nematode C. elegans are known to increase life span. Here we show that feeding defective mutants also have a second general trait in common, namely that they are small. Our measurements of the body lengths of a variety of feeding defective mutants, or of a variety of double mutants affecting other pathways that regulate body length in C. elegans, i.e. the DBL-1/TGFbeta, TAX-6/calcineurin and the SMA-1/betaH-spectrin pathways, indicate that food uptake acts as a separate pathway regulating body length. In early stages, before eating begins, feeding defective worms have no defect in body length or, in some cases, have only slightly smaller body length compared to wild-type. A significant difference in body length is first noticeable at later larval stages, a difference that probably correlates with increasing starvation. We also show that autophagy is induced and that the quantity of fat is decreased in starved worms. Our results indicate that the long-term starvation seen in feeding-defective C. elegans mutants activates autophagy, and leads to depletion of fat deposits, small cell size and small body size.

  7. The spe-10 mutant has longer life and increased stress resistance.

    PubMed

    Cypser, J R; Johnson, T E

    1999-01-01

    We investigated the life span of spe-10 mutant nematodes. We also tested resistance of spe-10 mutants to ultraviolet (UV) light, heat, and paraquat and examined the relationship between resistance to UV light and the fertility defect of these animals. The spe-10 mutation significantly increased mean life span. Additionally, the mutation significantly increased resistance to both UV light and to heat. Resistance to paraquat was not significantly different from that of wild-type, nor were any dauers formed at 27 degrees C. No significant correlation was found between the UV resistance and the fertility defect, nor was the UV resistance attributable to a hormetic effect. These results reinforce the importance of stress resistance in specifying increased life span and indirectly suggest that this fertility defect is not a direct cause of life span extension.

  8. Increased proximal bifurcation of CA1 pyramidal apical dendrites in sema3A mutant mice.

    PubMed

    Nakamura, Fumio; Ugajin, Kozue; Yamashita, Naoya; Okada, Takako; Uchida, Yutaka; Taniguchi, Masahiko; Ohshima, Toshio; Goshima, Yoshio

    2009-10-10

    Semaphorin-3A (Sema3A) is an attractive guidance molecule for cortical apical dendrites. To elucidate the role of Sema3A in hippocampal dendritic formation, we examined the Sema3A expression pattern in the perinatal hippocampal formation and analyzed hippocampal dendrites of the brains from young adult sema3A mutant mice. Sema3A protein was predominantly expressed in the hippocampal plate and the inner marginal zone at the initial period of apical dendritic growth. Neuropilin-1 and plexin-A, the receptor components for Sema3A, were also localized in the same regions. The Golgi impregnation method revealed that in wildtype mice more than 90% of hippocampal CA1 pyramidal neurons extended a single trunk or apical trunks bifurcated in stratum radiatum. Seven percent of the pyramidal neurons showed proximal bifurcation of apical trunks in stratum pyramidale or at the border of the stratum pyramidale and stratum radiatum. In sema3A mutant mice, proximally bifurcated apical dendrites were increased to 32%, while the single apical dendritic pyramidal neurons were decreased. We designate this phenotype in sema3A mutant mice as "proximal bifurcation." In the dissociated culture system, approximately half of the hippocampal neurons from wildtype mice resembled pyramidal neurons, which possess a long, thick, and tapered dendrite. In contrast, only 30% of the neurons from sema3A mutants exhibited pyramidal-like morphology. Proximal bifurcation of CA1 pyramidal neurons was also increased in the mutant mice of p35, an activator of cyclin-dependent kinase 5 (Cdk5). Thus, Sema3A may facilitate the initial growth of CA1 apical dendrites via the activation of p35/Cdk5, which may in turn signal hippocampal development.

  9. Cooperating JAK1 and JAK3 mutants increase resistance to JAK inhibitors.

    PubMed

    Springuel, Lorraine; Hornakova, Tekla; Losdyck, Elisabeth; Lambert, Fanny; Leroy, Emilie; Constantinescu, Stefan N; Flex, Elisabetta; Tartaglia, Marco; Knoops, Laurent; Renauld, Jean-Christophe

    2014-12-18

    The acquisition of growth signal self-sufficiency is 1 of the hallmarks of cancer. We previously reported that the murine interleukin-9-dependent TS1 cell line gives rise to growth factor-independent clones with constitutive activation of the Janus kinase (JAK)- signal transducer and activator of transcription (STAT) pathway. Here, we show that this transforming event results from activating mutations either in JAK1, JAK3, or in both kinases. Transient and stable expression of JAK1 and/or JAK3 mutants showed that each mutant induces STAT activation and that their coexpression further increases this activation. The proliferation of growth factor-independent TS1 clones can be efficiently blocked by JAK inhibitors such as ruxolitinib or CMP6 in short-term assays. However, resistant clones occur upon long-term culture in the presence of inhibitors. Surprisingly, resistance to CMP6 was not caused by the acquisition of secondary mutations in the adenosine triphosphate-binding pocket of the JAK mutant. Indeed, cells that originally showed a JAK1-activating mutation became resistant to inhibitors by acquiring another activating mutation in JAK3, whereas cells that originally showed a JAK3-activating mutation became resistant to inhibitors by acquiring another activating mutation in JAK1. These observations underline the cooperation between JAK1 and JAK3 mutants in T-cell transformation and represent a new mechanism of acquisition of resistance against JAK inhibitors.

  10. Mutant APH(2″)-IIa Enzymes with Increased Activity against Amikacin and Isepamicin▿

    PubMed Central

    Toth, Marta; Frase, Hilary; Chow, Joseph W.; Smith, Clyde; Vakulenko, Sergei B.

    2010-01-01

    Directed evolution by random PCR mutagenesis of the gene for the aminoglycoside 2″-IIa phosphotransferase generated R92H/D268N and N196D/D268N mutant enzymes, resulting in elevated levels of resistance to amikacin and isepamicin but not to other aminoglycoside antibiotics. Increases in the activities of the mutant phosphotransferases for isepamicin are the result of decreases in Km values, while improved catalytic efficiency for amikacin is the result of both a decrease in Km values and an increase in turnover of the antibiotic. Enzymes with R92H, D268N, and D268N single amino acid substitutions did not result in elevated MICs for aminoglycosides. PMID:20145089

  11. ZEB1 expression is increased in IDH1-mutant lower-grade gliomas.

    PubMed

    Nesvick, Cody L; Zhang, Chao; Edwards, Nancy A; Montgomery, Blake K; Lee, Michaela; Yang, Chunzhang; Wang, Herui; Zhu, Dongwang; Heiss, John D; Merrill, Marsha J; Ray-Chaudhury, Abhik; Zhuang, Zhengping

    2016-10-01

    Transcription factors that induce epithelial-mesenchymal transition (EMT) promote invasion, chemoresistance and a stem-cell phenotype in epithelial tumors, but their roles in central nervous system tumors are not well-understood. We hypothesized these transcription factors have a functional impact in grades II-III gliomas. Using the National Cancer Institute (NCI) Repository for Molecular Brain Neoplasia Data (REMBRANDT) and the Cancer Genome Atlas (TCGA) Lower-Grade Glioma (LGG) data, we determined the impact of EMT-promoting transcription factors (EMT-TFs) on overall survival in grades II-III gliomas, compared their expression across common genetic subtypes and subsequently validated these findings in a set of 31 tumors using quantitative real-time polymerase chain reaction (PCR) and immunohistochemistry. Increased expression of the gene coding for the transcriptional repressor Zinc Finger E box-binding Homeobox 1 (ZEB1) was associated with a significant increase in overall survival (OS) on Kaplan-Meier analysis. Genetic subtype analysis revealed that ZEB1 expression was relatively increased in IDH1/2-mutant gliomas, and IDH1/2-mutant gliomas expressed significantly lower levels of many ZEB1 transcriptional targets. Similarly, IDH1/2-mutant tumors expressed significantly higher levels of targets of microRNA 200C (MIR200C), a key regulator of ZEB1. In a validation study, ZEB1 mRNA was significantly increased in IDH1-mutant grades II-III gliomas, and ZEB1 protein expression was more pronounced in these tumors. Our findings demonstrate a novel relationship between IDH1/2 mutations and expression of ZEB1 and its transcriptional targets. Therapy targeting ZEB1-associated pathways may represent a novel therapeutic avenue for this class of tumors.

  12. Increased photoproduction of hydrogen by non-autotrophic mutants of Rhodopseudomonas capsulata.

    PubMed Central

    Willison, J C; Madern, D; Vignais, P M

    1984-01-01

    Non-autotrophic ( Aut -) mutants of Rhodopseudomonas capsulata B10 were tested for their efficiency of nitrogenase-mediated H2 production. Three of these mutants ( IR3 , IR4 and IR5 ) showed an increase stoichiometry of H2 production, mediated by nitrogenase, from certain organic substrates. For example, in a medium containing 7 mM-L-glutamate as nitrogen source, strain IR4 produced 10-20% more H2 than did the wild type with DL-lactate or L-malate as major carbon source, 20-50% more H2 with DL-malate, and up to 70% more with D-malate. Strain IR4 was deficient in 'uptake' hydrogenase activity as measured by H2-dependent reduction of Methylene Blue or Benzyl Viologen. However, this observation did not explain the increased efficiency of H2 production, since H2 uptake (H2 recycling) was undetectable in cells of the wild type. Instead, increased H2 production by the mutant appeared to be due to an improved conversion of organic substrates to H2 and CO2, presumably due to an altered carbon metabolism. The metabolism of D-malate by different strains was studied. An NAD+-dependent D-malic enzyme was synthesized constitutively by the wild type, and showed a Km for D-malate of 3 mM. The activity of this enzyme was approx. 50% higher in strain IR4 than in the wild type, and the mutant also grew twice as fast as the wild type with D-malate as sole carbon source. PMID:6146310

  13. Developmental increase of asynchronic glutamate release from hippocampal synapses in mutant taiep rat.

    PubMed

    Fuenzalida, Marco; Aliaga, Esteban; Olivares, Virginia; Roncagliolo, Manuel; Bonansco, Christian

    2009-06-01

    During development, regulation of the strength of synaptic transmission plays a central role in the formation of mammalian brain circuitries. In taiep rat, a neurological mutant with severe reactive astrogliosis and demyelination, we have described alterations in the synaptic transmission in central neurons, characterized by asynchronous excitatory postsynaptic currents ((ASYN)EPSCs), because of delayed neurotransmitter release. This hippocampal synaptic dysfunction has been described in juvenile mutants, concomitantly with the appearance of their main glial alterations. However, it is unknown whether this abnormal synaptic activity is correlated with some alterations of synaptic maturation during the postnatal development. Using intracellular electrophysiological recordings and immunohistochemistry assays, we studied the maturation of CA3-CA1 synapses in taiep rats. In taiep, the number of (ASYN)EPSCs evoked by conventional stimulation of Schaffer collaterals increases with age (P7-P30) and can be evoked by stimulation of single fiber. The amplitude and frequency of spontaneous EPSC (sEPSC) increased during the postnatal development in both control and taiep rats. However, in taiep, the increase of sEPSC frequency was significantly higher than in the control rats. The frequency of miniature EPSC (mEPSC) increased over the studied age range, without differences between taiep and control rats. In both control and taiep groups, the synaptophysin immunostaining (SYP-IR) in the stratum radiatum of CA1 region was significantly lower in the juvenile (P30) than in the neonatal (P10) rats, suggesting that synaptic pruning is normally occurring in taiep, even when SYP-IR was higher in taiep than control in both ages studied. These results suggest that, in taiep mutants, the asynchronic transmission is due to a dysfunction in the glutamate release mechanisms that progressively increases during development, which is not attributable to the existence of aberrant synaptic

  14. Transfection of rat embryo cells with mutant p53 increases the intrinsic radiation resistance

    SciTech Connect

    Pardo, F.S.; Su, M.; Gerweck, L.; Schmidt, E.V.; Borek, C.; Preffer, F.; Dombkowski, D.

    1994-11-01

    Dominant oncogenic sequences have been shown to modulate the intrinsic radiation sensitivity of cells of both human and murine tumor cell lines. Whether transfection with candidate tumor-suppressor genes can modulate intrinsic radiation sensitivity is unknown. The data presented here demonstrate that transfection of rat embryo cells with a mutant p53 allele can increase the intrinsic radiation resistance of cells in vitro. First, transfection with mutant p53 resulted in transformed cellular morphology. Second, the transfected clone and the corresponding pooled population of transfected clones were more resistant to ionizing radiation in vitro. Last, analyses of the parameters of cell kinetics suggested that the radiobiological effects were unlikely to be due to altered parameters of cell kinetics at the time of irradiation, suggesting that mutant p53 altered the intrinsic radiation resistance of transfected cells by a more direct mechanism. Further experimentation will be necessary to develop a mechanistic approach for the study of these alterations. 29 refs., 3 figs., 2 tabs.

  15. Increased Nitrogenase-Dependent H2 Photoproduction by hup Mutants of Rhodospirillum rubrum

    PubMed Central

    Kern, Monika; Klipp, Werner; Klemme, Jobst-Heinrich

    1994-01-01

    Transposon Tn5 mutagenesis was used to isolate mutants of Rhodospirillum rubrum which lack uptake hydrogenase (Hup) activity. Three Tn5 insertions mapped at different positions within the same 13-kb EcoRI fragment (fragment E1). Hybridization experiments revealed homology to the structural hydrogenase genes hupSLM from Rhodobacter capsulatus and hupSL from Bradyrhizobium japonicum in a 3.8-kb EcoRI-ClaI subfragment of fragment E1. It is suggested that this region contains at least some of the structural genes encoding the nickel-dependent uptake hydrogenase of R. rubrum. At a distance of about 4.5 kb from the fragment homologous to hupSLM, a region with homology to a DNA fragment carrying hypDE and hoxXA from B. japonicum was identified. Stable insertion and deletion mutations were generated in vitro and introduced into R. rubrum by homogenotization. In comparison with the wild type, the resulting hup mutants showed increased nitrogenase-dependent H2 photoproduction. However, a mutation in a structural hup gene did not result in maximum H2 production rates, indicating that the capacity to recycle H2 was not completely lost. Highest H2 production rates were obtained with a mutant carrying an insertion in a nonstructural hup-specific sequence and with a deletion mutant affected in both structural and nonstructural hup genes. Thus, besides the known Hup activity, a second, previously unknown Hup activity seems to be involved in H2 recycling. A single regulatory or accessory gene might be responsible for both enzymes. In contrast to the nickel-dependent uptake hydrogenase, the second Hup activity seems to be resistant to the metal chelator EDTA. Images PMID:16349271

  16. Mutant frequency of radiotherapy technicians appears to be associated with recent dose of ionizing radiation

    SciTech Connect

    Messing, K.; Ferraris, J.; Bradley, W.E.; Swartz, J.; Seifert, A.M. )

    1989-10-01

    The frequency of hypoxanthine phosphoribosyl transferase (HPRT) mutants among peripheral T-lymphocytes of radiotherapy technicians primarily exposed to 60Co was measured by the T-cell cloning method. Mutant frequencies of these technicians in 1984 and 1986 were significantly higher than those of physiotherapy technicians who worked in a neighboring service, and correlated significantly with thermoluminescence dosimeter readings recorded during the 6 mo preceding mutant frequency determination. Correlations decreased when related to dose recorded over longer time intervals. HPRT mutant frequency determination in peripheral lymphocytes is a good measure of recently received biologically effective radiation dose in an occupationally exposed population.

  17. LET and ion-species dependence for mutation induction and mutation spectrum on hprt locus in normal human fibroblasts.

    PubMed

    Tsuruoka, Chizuru; Suzuki, Masao; Fujitaka, Kazunobu

    2004-11-01

    We have been studying LET and ion species dependence of RBE in mutation frequency and mutation spectrum of deletion pattern of exons in hprt locus. Normal human skin fibroblasts were irradiated with heavy-ion beams, such as carbon- (290 MeV/u and 135 MeV/u), neon- (230 MeV/u and 400 MeV/u), silicon- (490 MeV/u) and iron- (500 MeV/u) ion beams, generated by Heavy Ion Medical Accelerator in Chiba (HIMAC) at national Institute of Radiological Sciences (NIRS). Mutation induction in hprt locus was detected to measure 6-thioguanine resistant colonies and deletion spectrum of exons was analyzed by multiplex PCR. The LET-RBE curves of mutation induction for carbon- and neon-ion beams showed a peak around 75 keV/micrometers and 155 keV/micrometers, respectively. On the other hand, there observed no clear peak for silicon-ion beams. The deletion spectrum of exons was different in induced mutants among different ion species. These results suggested that quantitative and qualitative difference in mutation occurred when using different ion species even if similar LET values.

  18. Isolation and characterization of a mutant of Pseudomonas aureofaciens ATCC 15926 with an increased capacity for synthesis of pyrrolnitrin.

    PubMed

    Salcher, O; Lingens, F

    1980-06-01

    A mutant having a 30-fold increased ability to synthesize pyrrolnitrin was isolated from Pseudomonas aureofaciens ATCC 15926 after mutagenesis with N-methyl-N'-nitro-N-nitrosoguanidine. The mutant strain also differed from the parent strain in pigmentation and in its inability to catabolize anthranilic acid and the benzene moiety of tryptophan and kynurenine.

  19. Carrier and prenatal diagnosis of Lesch-Nyhan disease due to a defect in HPRT gene expression regulation.

    PubMed

    Torres, Rosa J; Garcia, Marta G; Puig, Juan G

    2012-12-15

    Lesch-Nyhan disease (LND) is caused by lack of hypoxanthine-guanine phosphoribosyltransferase (HPRT) activity. Mutations in HPRT1 gene show variability in type and location within the gene, and in certain patients the HPRT coding sequence is normal and the molecular defect cannot be found. These patients presented a decreased HPRT1 expression of unknown cause. This is the first report of a carrier and prenatal diagnosis of LND due to a defect in HPRT gene expression regulation. Copyright © 2012 Elsevier B.V. All rights reserved.

  20. Isolation of Streptomyces globisporus and Blakeslea trispora mutants with increased carotenoid content.

    PubMed

    Matselyukh, B P; Matselyukh, D Ya; Golembiovska, S L; Polishchuk, L V; Lavrinchuk, V Ya

    2013-01-01

    Hyperpigmented mutants of Streptomyces globisporus 1912-Hp7 and Blakeslea trispora 18(+), 184(-) were isolated by action of hydrogen peroxide and nitrosoguanidine, correspondingly, from initial strains S. globisporus 1912-4Lcp and B. trispora 72(-), 198(+). The carotenoids of dry biomass of obtained strains, rubbed thoroughly with glass powder by a pestle in porcelain mortar were extracted by acetone and purified by TLC. Identification of the individual carotenoids was performed by means of HPLC and LC/MS spectrometry. It was shown that strain S. globisporus 1912-4Crt produced beta-carotene/lycopene (6.91/3.24 mg/L), mutants 1912-4Lcp and 1912-7Hp synthesized only lycopene (26.05 and 50.9 mg/L, respectively), and strains B. trispora 18(+) and 184(-)-beta-carotene (6.2% in dry biomass or more 2.5 g/L) without illumination in shake flasks. It is the first example of high constitutive production of the carotenoids by the representative of genus Streptomyces without photoinduction or increased synthesis of sigma factor The improved strains of B. trispora 18(+) and 184(-) can be used for biotechnological production of beta-carotene in industrial conditions.

  1. Increased actin polymerization and stabilization interferes with neuronal function and survival in the AMPKγ mutant Loechrig.

    PubMed

    Cook, Mandy; Bolkan, Bonnie J; Kretzschmar, Doris

    2014-01-01

    loechrig (loe) mutant flies are characterized by progressive neuronal degeneration, behavioral deficits, and early death. The mutation is due to a P-element insertion in the gene for the γ-subunit of the trimeric AMP-activated protein kinase (AMPK) complex, whereby the insertion affects only one of several alternative transcripts encoding a unique neuronal isoform. AMPK is a cellular energy sensor that regulates a plethora of signaling pathways, including cholesterol and isoprenoid synthesis via its downstream target hydroxy-methylglutaryl (HMG)-CoA reductase. We recently showed that loe interferes with isoprenoid synthesis and increases the prenylation and thereby activation of RhoA. During development, RhoA plays an important role in neuronal outgrowth by activating a signaling cascade that regulates actin dynamics. Here we show that the effect of loe/AMPKγ on RhoA prenylation leads to a hyperactivation of this signaling pathway, causing increased phosphorylation of the actin depolymerizating factor cofilin and accumulation of filamentous actin. Furthermore, our results show that the resulting cytoskeletal changes in loe interfere with neuronal growth and disrupt axonal integrity. Surprisingly, these phenotypes were enhanced by expressing the Slingshot (SSH) phosphatase, which during development promotes actin depolymerization by dephosphorylating cofilin. However, our studies suggest that in the adult SSH promotes actin polymerization, supporting in vitro studies using human SSH1 that suggested that SSH can also stabilize and bundle filamentous actin. Together with the observed increase in SSH levels in the loe mutant, our experiments suggest that in mature neurons SSH may function as a stabilization factor for filamentous actin instead of promoting actin depolymerization.

  2. Sucralose Increases Antimicrobial Resistance and Stimulates Recovery of Escherichia coli Mutants.

    PubMed

    Qu, Yilin; Li, Rongyan; Jiang, Mingshan; Wang, Xiuhong

    2017-07-01

    Because of heavy use of antimicrobials, antimicrobial resistance in bacteria has become of great concern. The effect of some widely used food additives such as sucralose on bacteria in the gut and the environment has also drawn increasing attention. In this study, we investigated the interaction between antimicrobials and sucralose impacting antimicrobial resistance and mutation of Escherichia coli (E. coli). To examine antimicrobial resistance and mutation frequency, different subinhibitory concentrations of sucralose were added to cultures of E.coli BW25113 that were then treated with antimicrobials, oxolinic acid, or moxifloxacin. Then the E.coli were assayed for bacterial survival and recovery of mutants resistant to an unrelated antimicrobial, rifampicin. Pre-treatment of E.coli BW25113 with 1/2 minimal inhibitory concentration (MIC) of sucralose increased the survival rate in oxolinic acid or moxifloxacin. A 1/3 MIC of sucralose increased rifampicin-resistant mutation rate of E.coli BW25113 after 72 h, while rifampicin-resistant mutation rate was increased when co-treated with 1/8 MIC, 1/4 MIC, 1/3 MIC sucralose, and oxolinic acid after 24 h. Sucralose can increase the antimicrobial resistance and mutation frequency of E.coli to some antimicrobials.

  3. Increased tolerance to salt stress in OPDA-deficient rice ALLENE OXIDE CYCLASE mutants is linked to an increased ROS-scavenging activity

    PubMed Central

    Hazman, Mohamed; Hause, Bettina; Eiche, Elisabeth; Nick, Peter; Riemann, Michael

    2015-01-01

    Salinity stress represents a global constraint for rice, the most important staple food worldwide. Therefore the role of the central stress signal jasmonate for the salt response was analysed in rice comparing the responses to salt stress for two jasmonic acid (JA) biosynthesis rice mutants (cpm2 and hebiba) impaired in the function of ALLENE OXIDE CYCLASE (AOC) and their wild type. The aoc mutants were less sensitive to salt stress. Interestingly, both mutants accumulated smaller amounts of Na+ ions in their leaves, and showed better scavenging of reactive oxygen species (ROS) under salt stress. Leaves of the wild type and JA mutants accumulated similar levels of abscisic acid (ABA) under stress conditions, and the levels of JA and its amino acid conjugate, JA–isoleucine (JA-Ile), showed only subtle alterations in the wild type. In contrast, the wild type responded to salt stress by strong induction of the JA precursor 12-oxophytodienoic acid (OPDA), which was not observed in the mutants. Transcript levels of representative salinity-induced genes were induced less in the JA mutants. The absence of 12-OPDA in the mutants correlated not only with a generally increased ROS-scavenging activity, but also with the higher activity of specific enzymes in the antioxidative pathway, such as glutathione S-transferase, and fewer symptoms of damage as, for example, indicated by lower levels of malondialdehyde. The data are interpreted in a model where the absence of OPDA enhanced the antioxidative power in mutant leaves. PMID:25873666

  4. Increased tolerance to salt stress in OPDA-deficient rice ALLENE OXIDE CYCLASE mutants is linked to an increased ROS-scavenging activity.

    PubMed

    Hazman, Mohamed; Hause, Bettina; Eiche, Elisabeth; Nick, Peter; Riemann, Michael

    2015-06-01

    Salinity stress represents a global constraint for rice, the most important staple food worldwide. Therefore the role of the central stress signal jasmonate for the salt response was analysed in rice comparing the responses to salt stress for two jasmonic acid (JA) biosynthesis rice mutants (cpm2 and hebiba) impaired in the function of ALLENE OXIDE CYCLASE (AOC) and their wild type. The aoc mutants were less sensitive to salt stress. Interestingly, both mutants accumulated smaller amounts of Na(+) ions in their leaves, and showed better scavenging of reactive oxygen species (ROS) under salt stress. Leaves of the wild type and JA mutants accumulated similar levels of abscisic acid (ABA) under stress conditions, and the levels of JA and its amino acid conjugate, JA-isoleucine (JA-Ile), showed only subtle alterations in the wild type. In contrast, the wild type responded to salt stress by strong induction of the JA precursor 12-oxophytodienoic acid (OPDA), which was not observed in the mutants. Transcript levels of representative salinity-induced genes were induced less in the JA mutants. The absence of 12-OPDA in the mutants correlated not only with a generally increased ROS-scavenging activity, but also with the higher activity of specific enzymes in the antioxidative pathway, such as glutathione S-transferase, and fewer symptoms of damage as, for example, indicated by lower levels of malondialdehyde. The data are interpreted in a model where the absence of OPDA enhanced the antioxidative power in mutant leaves.

  5. Identification of Proteus mirabilis Mutants with Increased Sensitivity to Antimicrobial Peptides

    PubMed Central

    McCoy, Andrea J.; Liu, Hongjian; Falla, Timothy J.; Gunn, John S.

    2001-01-01

    Antimicrobial peptides (APs) are important components of the innate defenses of animals, plants, and microorganisms. However, some bacterial pathogens are resistant to the action of APs. For example, Proteus mirabilis is highly resistant to the action of APs, such as polymyxin B (PM), protegrin, and the synthetic protegrin analog IB-367. To better understand this resistance, a transposon mutagenesis approach was used to generate P. mirabilis mutants sensitive to APs. Four unique PM-sensitive mutants of P. mirabilis were identified (these mutants were >2 to >128 times more sensitive than the wild type). Two of these mutants were also sensitive to IB-367 (16 and 128 times more sensitive than the wild type). Lipopolysaccharide (LPS) profiles of the PM- and protegrin-sensitive mutants demonstrated marked differences in both the lipid A and O-antigen regions, while the PM-sensitive mutants appeared to have alterations of either lipid A or O antigen. Matrix-assisted laser desorption ionization–time of flight mass spectrometry analysis of the wild-type and PM-sensitive mutant lipid A showed species with one or two aminoarabinose groups, while lipid A from the PM- and protegrin-sensitive mutants was devoid of aminoarabinose. When the mutants were streaked on an agar-containing medium, the swarming motility of the PM- and protegrin-sensitive mutants was completely inhibited and the swarming motility of the mutants sensitive to only PM was markedly decreased. DNA sequence analysis of the mutagenized loci revealed similarities to an O-acetyltransferase (PM and protegrin sensitive) and ATP synthase and sap loci (PM sensitive). These data further support the role of LPS modifications as an elaborate mechanism in the resistance of certain bacterial species to APs and suggest that LPS surface charge alterations may play a role in P. mirabilis swarming motility. PMID:11408219

  6. Spaceflight results in increase of thick filament but not thin filament proteins in the paramyosin mutant of Caenorhabditis elegans

    NASA Astrophysics Data System (ADS)

    Adachi, R.; Takaya, T.; Kuriyama, K.; Higashibata, A.; Ishioka, N.; Kagawa, H.

    We have investigated the effect of microgravity during spaceflight on body-wall muscle fiber size and muscle proteins in the paramyosin mutant of Caenorhabditis elegans. Both mutant and wild-type strains were subjected to 10 days of microgravity during spaceflight and compared to ground control groups. No significant change in muscle fiber size or quantity of the protein was observed in wild-type worms; where as atrophy of body-wall muscle and an increase in thick filament proteins were observed in the paramyosin mutant unc-15(e73) animals after spaceflight. We conclude that the mutant with abnormal muscle responded to microgravity by increasing the total amount of muscle protein in order to compensate for the loss of muscle function.

  7. Increased Lipid Accumulation in the Chlamydomonas reinhardtii sta7-10 Starchless Isoamylase Mutant and Increased Carbohydrate Synthesis in Complemented Strains ▿

    PubMed Central

    Work, Victoria H.; Radakovits, Randor; Jinkerson, Robert E.; Meuser, Jonathan E.; Elliott, Lee G.; Vinyard, David J.; Laurens, Lieve M. L.; Dismukes, G. Charles; Posewitz, Matthew C.

    2010-01-01

    The accumulation of bioenergy carriers was assessed in two starchless mutants of Chlamydomonas reinhardtii (the sta6 [ADP-glucose pyrophosphorylase] and sta7-10 [isoamylase] mutants), a control strain (CC124), and two complemented strains of the sta7-10 mutant. The results indicate that the genetic blockage of starch synthesis in the sta6 and sta7-10 mutants increases the accumulation of lipids on a cellular basis during nitrogen deprivation relative to that in the CC124 control as determined by conversion to fatty acid methyl esters. However, this increased level of lipid accumulation is energetically insufficient to completely offset the loss of cellular starch that is synthesized by CC124 during nitrogen deprivation. We therefore investigated acetate utilization and O2 evolution to obtain further insights into the physiological adjustments utilized by the two starchless mutants in the absence of starch synthesis. The results demonstrate that both starchless mutants metabolize less acetate and have more severely attenuated levels of photosynthetic O2 evolution than CC124, indicating that a decrease in overall anabolic processes is a significant physiological response in the starchless mutants during nitrogen deprivation. Interestingly, two independent sta7-10:STA7 complemented strains exhibited significantly greater quantities of cellular starch and lipid than CC124 during acclimation to nitrogen deprivation. Moreover, the complemented strains synthesized significant quantities of starch even when cultured in nutrient-replete medium. PMID:20562225

  8. Increased lipid accumulation in the Chlamydomonas reinhardtii sta7-10 starchless isoamylase mutant and increased carbohydrate synthesis in complemented strains.

    PubMed

    Work, Victoria H; Radakovits, Randor; Jinkerson, Robert E; Meuser, Jonathan E; Elliott, Lee G; Vinyard, David J; Laurens, Lieve M L; Dismukes, G Charles; Posewitz, Matthew C

    2010-08-01

    The accumulation of bioenergy carriers was assessed in two starchless mutants of Chlamydomonas reinhardtii (the sta6 [ADP-glucose pyrophosphorylase] and sta7-10 [isoamylase] mutants), a control strain (CC124), and two complemented strains of the sta7-10 mutant. The results indicate that the genetic blockage of starch synthesis in the sta6 and sta7-10 mutants increases the accumulation of lipids on a cellular basis during nitrogen deprivation relative to that in the CC124 control as determined by conversion to fatty acid methyl esters. However, this increased level of lipid accumulation is energetically insufficient to completely offset the loss of cellular starch that is synthesized by CC124 during nitrogen deprivation. We therefore investigated acetate utilization and O(2) evolution to obtain further insights into the physiological adjustments utilized by the two starchless mutants in the absence of starch synthesis. The results demonstrate that both starchless mutants metabolize less acetate and have more severely attenuated levels of photosynthetic O(2) evolution than CC124, indicating that a decrease in overall anabolic processes is a significant physiological response in the starchless mutants during nitrogen deprivation. Interestingly, two independent sta7-10:STA7 complemented strains exhibited significantly greater quantities of cellular starch and lipid than CC124 during acclimation to nitrogen deprivation. Moreover, the complemented strains synthesized significant quantities of starch even when cultured in nutrient-replete medium.

  9. Isolation and characterization of chromosome-gain and increase-in-ploidy mutants in yeast.

    PubMed

    Chan, C S; Botstein, D

    1993-11-01

    We have developed a colony papillation assay for monitoring the copy number of genetically marked chromosomes II and III in Saccharomyces cerevisiae. The unique feature of this assay is that it allows detection of a gain of the marked chromosomes even if there is a gain of the entire set of chromosomes (increase-in-ploidy). This assay was used to screen for chromosome-gain or increase-in-ploidy mutants. Five complementation groups have been defined for recessive mutations that confer an increase-in-ploidy (ipl) phenotype, which, in each case, cosegregates with a temperature-sensitive growth phenotype. Four new alleles of CDC31, which is required for spindle pole body duplication, were also recovered from this screen. Temperature-shift experiments with ipl1 cells show that they suffer severe nondisjunction at 37 degrees. Similar experiments with ipl2 cells show that they gain entire sets of chromosomes and become arrested as unbudded cells at 37 degrees. Molecular cloning and genetic mapping show that IPL1 is a newly identified gene, whereas IPL2 is allelic to BEM2, which is required for normal bud growth.

  10. Isolation and Characterization of Chromosome-Gain and Increase-in-Ploidy Mutants in Yeast

    PubMed Central

    Chan, CSM.; Botstein, D.

    1993-01-01

    We have developed a colony papillation assay for monitoring the copy number of genetically marked chromosomes II and III in Saccharomyces cerevisiae. The unique feature of this assay is that it allows detection of a gain of the marked chromosomes even if there is a gain of the entire set of chromosomes (increase-in-ploidy). This assay was used to screen for chromosome-gain or increase-in-ploidy mutants. Five complementation groups have been defined for recessive mutations that confer an increase-in-ploidy (ipl) phenotype, which, in each case, cosegregates with a temperature-sensitive growth phenotype. Four new alleles of CDC31, which is required for spindle pole body duplication, were also recovered from this screen. Temperature-shift experiments with ipl1 cells show that they suffer severe nondisjunction at 37°. Similar experiments with ipl2 cells show that they gain entire sets of chromosomes and become arrested as unbudded cells at 37°. Molecular cloning and genetic mapping show that IPL1 is a newly identified gene, whereas IPL2 is allelic to BEM2, which is required for normal bud growth. PMID:8293973

  11. An increased replication fidelity mutant of foot-and-mouth disease virus retains fitness in vitro and virulence in vivo.

    PubMed

    Zeng, Jianxiong; Wang, Haiwei; Xie, Xiaochun; Yang, Decheng; Zhou, Guohui; Yu, Li

    2013-10-01

    In a screen for RNA mutagen-resistant foot-and-mouth disease virus (FMDV) strains, we isolated an FMDV mutant with RNA-dependent RNA polymerase (RdRp) R84H substitution. This mutant, selected under the mutagenic pressure of 5-fluorouracil (5-FU), is resistant not only to 5-FU but also to other two RNA mutagens, 5-azacytidine and ribavirin, suggesting that the RdRp R84H mutant is a high fidelity variant. Subsequently, the increased fidelity of this mutant was verified through analysis of mutation frequency, which revealed a 1.4-fold enhancement in RdRp fidelity compared with the wild-type virus. Further studies indicated that the R84H mutant exhibited slightly increased fitness in vitro, and its virulence was not reduced in suckling mice. These results indicated that an increase in RdRp fidelity does not always correlate with reduced virus fitness and virus attenuation. Thus, this isolated R84H mutant provides a new platform to examine the evolutionary dynamics of fidelity-changing RNA viruses, such as mutagen resistance, fitness and virulence.

  12. Increased mitochondrial biogenesis preserves intestinal stem cell homeostasis and contributes to longevity in Indy mutant flies.

    PubMed

    Rogers, Ryan P; Rogina, Blanka

    2014-04-01

    The Drosophila Indy (I'm Not Dead Yet) gene encodes a plasma membrane transporter of Krebs cycle intermediates, with robust expression in tissues associated with metabolism. Reduced INDY alters metabolism and extends longevity in a manner similar to caloric restriction (CR); however, little is known about the tissue specific physiological effects of INDY reduction. Here we focused on the effects of INDY reduction in the Drosophila midgut due to the importance of intestinal tissue homeostasis in healthy aging and longevity. The expression of Indy mRNA in the midgut changes in response to aging and nutrition. Genetic reduction of Indy expression increases midgut expression of the mitochondrial regulator spargel/dPGC-1, which is accompanied by increased mitochondrial biogenesis and reduced reactive oxygen species (ROS). These physiological changes in the Indy mutant midgut preserve intestinal stem cell (ISC) homeostasis and are associated with healthy aging. Genetic studies confirm that dPGC-1 mediates the regulatory effects of INDY, as illustrated by lack of longevity extension and ISC homeostasis in flies with mutations in both Indy and dPGC1. Our data suggest INDY may be a physiological regulator that modulates intermediary metabolism in response to changes in nutrient availability and organismal needs by modulating dPGC-1.

  13. Expression of OsCAS (Calcium-Sensing Receptor) in an Arabidopsis Mutant Increases Drought Tolerance.

    PubMed

    Zhao, Xin; Xu, Mengmeng; Wei, Rongrong; Liu, Yang

    2015-01-01

    The calcium-sensing receptor (CaS), which is localized in the chloroplasts, is a crucial regulator of extracellular calcium-induced stomatal closure in Arabidopsis. It has homologs in Oryza sativa and other plants. These sequences all have a rhodanese-like protein domain, which has been demonstrated to be associated with specific stress conditions. In this study, we cloned the Oryza sativa calcium-sensing receptor gene (OsCAS) and demonstrated that OsCAS could sense an increase of extracellular Ca2+ concentration and mediate an increase in cytosolic Ca2+ concentration. The OsCAS gene was transformed into an Arabidopsis CaS knockout mutant (Salk) and overexpressed in the transgenic plants. OsCAS promoted stomatal closure. We screened homozygous transgenic Arabidopsis plants and determined physiological indices such as the oxidative damage biomarker malondialdehyde (MDA), relative membrane permeability (RMP), proline content, and chlorophyll fluorescence parameters, after 21 days of drought treatment. Our results revealed lower RMP and MDA contents and a higher Proline content in transgenic Arabidopsis plants after drought stress, whereas the opposite was observed in Salk plants. With respect to chlorophyll fluorescence, the electron transport rate and effective PSII quantum yield decreased in all lines under drought stress; however, in the transgenic plants these two parameters changed fewer and were higher than those in wild-type and Salk plants. The quantum yield of regulated energy dissipation and nonregulated energy dissipation in PSII were higher in Salk plants, whereas these values were lower in the transgenic plants than in the wild type under drought stress. The above results suggest that the transgenic plants showed better resistance to drought stress by decreasing damage to the cell membrane, increasing the amount of osmoprotectants, and maintaining a relatively high photosynthetic capacity. In conclusion, OsCAS is an extracellular calcium-sensing receptor

  14. Increased thymus- and decreased parathyroid-fated organ domains in Splotch mutant embryos

    PubMed Central

    Griffith, Ann V.; Cardenas, Kim; Carter, Carla; Gordon, Julie; Iberg, Aimee; Engleka, Kurt; Epstein, Jonathan A.; Manley, Nancy R.; Richie, Ellen R.

    2009-01-01

    Embryos that are homozygous for Splotch, a null allele of Pax3, have a severe neural crest cell (NCC) deficiency that generates a complex phenotype including spina bifida, exencephaly and cardiac outflow tract abnormalities. Contrary to the widely held perception that thymus aplasia or hypoplasia is a characteristic feature of Pax3Sp/Sp embryos, we find that thymic rudiments are larger and parathyroid rudiments are smaller in E11.5–12.5 Pax3Sp/Sp compared to Pax3+/+ embryos. The thymus originates from bilateral third pharyngeal pouch primordia containing endodermal progenitors of both thymus and parathyroid glands. Analyses of Foxn1 and Gcm2 expression revealed a dorsal shift in the border between parathyroid- and thymus-fated domains at E11.5, with no change in the overall cellularity or volume of each shared primordium. The border shift increases the allocation of third pouch progenitors to the thymus domain and correspondingly decreases allocation to the parathyroid domain. Initial patterning in the E10.5 pouch was normal suggesting that the observed change in the location of the organ domain interface arises during border refinement between E10.5 and E11.5. Given the well-characterized NCC defects in Splotch mutants, these findings implicate NCCs in regulating patterning of third pouch endoderm into thymus- versus parathyroid-specified domains, and suggest that organ size is determined in part by the number of progenitor cells specified to a given fate. PMID:19135046

  15. Characterization of a zebra mutant of rice with increased susceptibility to light stress.

    PubMed

    Kusumi, K; Komori, H; Satoh, H; Iba, K

    2000-02-01

    The rice zebra mutant TCM248 is a single recessive mutant. This mutant develops transverse-striped leaves with green and white sectors under alternate light/dark growth conditions. Mutants that were grown under a higher light intensity during the light period showed a more intense striped phenotype. The white tissues contained abnormal chloroplasts with few internal membrane structures, while the green tissues in the mutants contained normal chloroplasts. The white tissue contained only trace amounts of Chls and carotenoids, and mRNA accumulation of nuclear genes encoding chloroplast proteins (rbcS, cab) was strongly suppressed compared to that in the wild type plants. A series of growth condition shift experiments demonstrated that the mutant displayed the striped phenotype only if it was exposed to the alternate light/dark growth conditions during a limited stage of early leaf development. These data suggest that the zebra gene is involved in the acquisition of photoprotective capacity of the plants and that this gene functions at an early stage of chloroplast differentiation.

  16. ZFIN, the Zebrafish Model Organism Database: increased support for mutants and transgenics.

    PubMed

    Howe, Douglas G; Bradford, Yvonne M; Conlin, Tom; Eagle, Anne E; Fashena, David; Frazer, Ken; Knight, Jonathan; Mani, Prita; Martin, Ryan; Moxon, Sierra A Taylor; Paddock, Holly; Pich, Christian; Ramachandran, Sridhar; Ruef, Barbara J; Ruzicka, Leyla; Schaper, Kevin; Shao, Xiang; Singer, Amy; Sprunger, Brock; Van Slyke, Ceri E; Westerfield, Monte

    2013-01-01

    ZFIN, the Zebrafish Model Organism Database (http://zfin.org), is the central resource for zebrafish genetic, genomic, phenotypic and developmental data. ZFIN curators manually curate and integrate comprehensive data involving zebrafish genes, mutants, transgenics, phenotypes, genotypes, gene expressions, morpholinos, antibodies, anatomical structures and publications. Integrated views of these data, as well as data gathered through collaborations and data exchanges, are provided through a wide selection of web-based search forms. Among the vertebrate model organisms, zebrafish are uniquely well suited for rapid and targeted generation of mutant lines. The recent rapid production of mutants and transgenic zebrafish is making management of data associated with these resources particularly important to the research community. Here, we describe recent enhancements to ZFIN aimed at improving our support for mutant and transgenic lines, including (i) enhanced mutant/transgenic search functionality; (ii) more expressive phenotype curation methods; (iii) new downloads files and archival data access; (iv) incorporation of new data loads from laboratories undertaking large-scale generation of mutant or transgenic lines and (v) new GBrowse tracks for transgenic insertions, genes with antibodies and morpholinos.

  17. ZFIN, the Zebrafish Model Organism Database: increased support for mutants and transgenics

    PubMed Central

    Howe, Douglas G.; Bradford, Yvonne M.; Conlin, Tom; Eagle, Anne E.; Fashena, David; Frazer, Ken; Knight, Jonathan; Mani, Prita; Martin, Ryan; Moxon, Sierra A. Taylor; Paddock, Holly; Pich, Christian; Ramachandran, Sridhar; Ruef, Barbara J.; Ruzicka, Leyla; Schaper, Kevin; Shao, Xiang; Singer, Amy; Sprunger, Brock; Van Slyke, Ceri E.; Westerfield, Monte

    2013-01-01

    ZFIN, the Zebrafish Model Organism Database (http://zfin.org), is the central resource for zebrafish genetic, genomic, phenotypic and developmental data. ZFIN curators manually curate and integrate comprehensive data involving zebrafish genes, mutants, transgenics, phenotypes, genotypes, gene expressions, morpholinos, antibodies, anatomical structures and publications. Integrated views of these data, as well as data gathered through collaborations and data exchanges, are provided through a wide selection of web-based search forms. Among the vertebrate model organisms, zebrafish are uniquely well suited for rapid and targeted generation of mutant lines. The recent rapid production of mutants and transgenic zebrafish is making management of data associated with these resources particularly important to the research community. Here, we describe recent enhancements to ZFIN aimed at improving our support for mutant and transgenic lines, including (i) enhanced mutant/transgenic search functionality; (ii) more expressive phenotype curation methods; (iii) new downloads files and archival data access; (iv) incorporation of new data loads from laboratories undertaking large-scale generation of mutant or transgenic lines and (v) new GBrowse tracks for transgenic insertions, genes with antibodies and morpholinos. PMID:23074187

  18. Altered myocardial metabolic adaptation to increased fatty acid availability in cardiomyocyte-specific CLOCK mutant mice.

    PubMed

    Peliciari-Garcia, Rodrigo A; Goel, Mehak; Aristorenas, Jonathan A; Shah, Krishna; He, Lan; Yang, Qinglin; Shalev, Anath; Bailey, Shannon M; Prabhu, Sumanth D; Chatham, John C; Gamble, Karen L; Young, Martin E

    2016-10-01

    A mismatch between fatty acid availability and utilization leads to cellular/organ dysfunction during cardiometabolic disease states (e.g., obesity, diabetes mellitus). This can precipitate cardiac dysfunction. The heart adapts to increased fatty acid availability at transcriptional, translational, post-translational and metabolic levels, thereby attenuating cardiomyopathy development. We have previously reported that the cardiomyocyte circadian clock regulates transcriptional responsiveness of the heart to acute increases in fatty acid availability (e.g., short-term fasting). The purpose of the present study was to investigate whether the cardiomyocyte circadian clock plays a role in adaptation of the heart to chronic elevations in fatty acid availability. Fatty acid availability was increased in cardiomyocyte-specific CLOCK mutant (CCM) and wild-type (WT) littermate mice for 9weeks in time-of-day-independent (streptozotocin (STZ) induced diabetes) and dependent (high fat diet meal feeding) manners. Indices of myocardial metabolic adaptation (e.g., substrate reliance perturbations) to STZ-induced diabetes and high fat meal feeding were found to be dependent on genotype. Various transcriptional and post-translational mechanisms were investigated, revealing that Cte1 mRNA induction in the heart during STZ-induced diabetes is attenuated in CCM hearts. At the functional level, time-of-day-dependent high fat meal feeding tended to influence cardiac function to a greater extent in WT versus CCM mice. Collectively, these data suggest that CLOCK (a circadian clock component) is important for metabolic adaption of the heart to prolonged elevations in fatty acid availability. This article is part of a Special Issue entitled: Heart Lipid Metabolism edited by G.D. Lopaschuk. Copyright © 2015 Elsevier B.V. All rights reserved.

  19. Mice lacking components of adaptive immunity show increased Brucella abortus virB mutant colonization.

    PubMed

    Rolán, Hortensia García; Tsolis, Renée M

    2007-06-01

    The Brucella abortus type IV secretion system (T4SS), encoded by the virB genes, is essential for survival in mononuclear phagocytes in vitro. In the mouse model, a B. abortus virB mutant was initially able to colonize the spleen at the level of the wild type for approximately 3 to 5 days, which coincided with the development of adaptive immunity. To investigate the relationship between survival in macrophages cultivated in vitro and persistence in tissues in vivo, we tested the ability of mutant mice lacking components of adaptive immunity to eliminate the virB mutant from the spleen during a mixed infection with the B. abortus wild type. Ifng(-/-) or beta(2)m(-/-) mice were able to clear the virB mutant to the same degree as control mice. However, spleens of Rag1(-/-) mice and Igh6(-/-) mice were more highly colonized by the virB mutant than control mice after 14 to 21 days, suggesting that, in these mice, there is not an absolute requirement for the T4SS to mediate persistence of B. abortus in the spleen. Macrophages isolated from Igh6(-/-) mice killed the virB mutant to the same extent as macrophages from control mice, showing that the reduced ability of these mice to clear the virB mutant from the spleen does not correlate with diminished macrophage function in vitro. These results show that in the murine model host, the T4SS is required for persistence beyond 3 to 5 days after infection and suggest that the T4SS may contribute to evasion of adaptive immune mechanisms by B. abortus.

  20. Increased apoptosis and hypomyelination in cerebral white matter of macular mutant mouse brain.

    PubMed

    Takikita, Shoichi; Takano, Tomoyuki; Narita, Tsutomu; Maruo, Yoshihiro

    2015-09-01

    Hypomyelination in developing brain is often accompanied by congenital metabolic disorders. Menkes kinky hair disease is an X-linked neurodegenerative disease of impaired copper transport, resulting from a mutation of the Menkes disease gene, a transmembrane copper-transporting p-type ATPase gene (ATP7A). In a macular mutant mouse model, the murine ortholog of Menkes gene (mottled gene) is mutated, and widespread neurodegeneration and subsequent death are observed. Although some biochemical analysis of myelin protein in macular mouse has been reported, detailed histological study of myelination in this mouse model is currently lacking. Since myelin abnormality is one of the neuropathologic findings of human Menkes disease, in this study early myelination in macular mouse brain was evaluated by immunohistochemistry. Two-week-old macular mice and normal littermates were perfused with 4% paraformaldehyde. Immunohistochemical staining of paraffin embedded and vibratome sections was performed using antibodies against either CNPase, cleaved caspase-3 or O4 (marker of immature oligodendrocytes). This staining showed that cerebral myelination in macular mouse was generally hypoplastic and that hypomyelination was remarkable in internal capsule, corpus callosum, and cingulate cortex. In addition, an increased number of cleaved caspase-3 positive cells were observed in corpus callosum and internal capsule. Copper deficiency induced by low copper diet has been reported to induce oligodendrocyte dysfunction and leads to hypomyelination in this mouse model. Taken together, hypomyelination observed in this study in a mouse model of Menkes disease is assumed to be induced by increased apoptosis of immature oligodendrocytes in developing cerebrum, through deficient intracellular copper metabolism.

  1. Increased apoptosis and hypomyelination in cerebral white matter of macular mutant mouse brain

    PubMed Central

    Takikita, Shoichi; Takano, Tomoyuki; Narita, Tsutomu; Maruo, Yoshihiro

    2015-01-01

    Hypomyelination in developing brain is often accompanied by congenital metabolic disorders. Menkes kinky hair disease is an X-linked neurodegenerative disease of impaired copper transport, resulting from a mutation of the Menkes disease gene, a transmembrane copper-transporting p-type ATPase gene (ATP7A). In a macular mutant mouse model, the murine ortholog of Menkes gene (mottled gene) is mutated, and widespread neurodegeneration and subsequent death are observed. Although some biochemical analysis of myelin protein in macular mouse has been reported, detailed histological study of myelination in this mouse model is currently lacking. Since myelin abnormality is one of the neuropathologic findings of human Menkes disease, in this study early myelination in macular mouse brain was evaluated by immunohistochemistry. Two-week-old macular mice and normal littermates were perfused with 4% paraformaldehyde. Immunohistochemical staining of paraffin embedded and vibratome sections was performed using antibodies against either CNPase, cleaved caspase-3 or O4 (marker of immature oligodendrocytes). This staining showed that cerebral myelination in macular mouse was generally hypoplastic and that hypomyelination was remarkable in internal capsule, corpus callosum, and cingulate cortex. In addition, an increased number of cleaved caspase-3 positive cells were observed in corpus callosum and internal capsule. Copper deficiency induced by low copper diet has been reported to induce oligodendrocyte dysfunction and leads to hypomyelination in this mouse model. Taken together, hypomyelination observed in this study in a mouse model of Menkes disease is assumed to be induced by increased apoptosis of immature oligodendrocytes in developing cerebrum, through deficient intracellular copper metabolism. PMID:26937406

  2. Increased behavioral and neuronal responses to a hallucinogenic drug in PACAP heterozygous mutant mice.

    PubMed

    Hazama, Keisuke; Hayata-Takano, Atsuko; Uetsuki, Kazuki; Kasai, Atsushi; Encho, Naoki; Shintani, Norihito; Nagayasu, Kazuki; Hashimoto, Ryota; Reglodi, Dora; Miyakawa, Tsuyoshi; Nakazawa, Takanobu; Baba, Akemichi; Hashimoto, Hitoshi

    2014-01-01

    Accumulating evidence from human genetic studies implicates the pituitary adenylate cyclase-activating polypeptide (PACAP) gene as a risk factor for psychiatric disorders, including schizophrenia and stress-related diseases. Mice with homozygous disruption of the PACAP gene display profound behavioral and neurological abnormalities that are ameliorated with the atypical antipsychotic and dopamine D2 and serotonin (5-HT)2 antagonist risperidone and the 5-HT2 receptor antagonist ritanserin; however, the underlying mechanisms remain unknown. Here, we investigated if PACAP heterozygous mutant (PACAP(+/-)) mice, which appear behaviorally normal, are vulnerable to aversive stimuli. PACAP(+/-) mice were administered a 5-HT2 receptor agonist, (±)-2,5-dimethoxy-4-iodoamphetamine (DOI), a hallucinogenic drug, and their responses were compared with the littermate wild-type mice. After DOI injection, PACAP(+/-) mice showed increased head-twitch responses, while their behavior was normal after saline. DOI induced deficits in sensorimotor gating, as determined by prepulse inhibition, specifically in PACAP(+/-) mice. However, other 5-HT2 receptor-dependent responses, such as corticosterone release and hypothermia, were similarly observed in PACAP(+/-) and wild-type mice. c-Fos expression analysis, performed in various brain regions, revealed that the DOI-induced increase in the number of c-Fos-positive cells was more pronounced in 5-HT2A receptor-negative cells in the somatosensory cortex in PACAP(+/-) mice compared with wild-type mice. These results indicate that PACAP(+/-) mice exhibit specific vulnerability to DOI-induced deficits in cortical sensory function, such as exaggerated head-twitch responses and sensorimotor gating deficits. Our findings provide insight into the neural mechanisms underlying impaired behavioral responses in which 5-HT2 receptors are implicated.

  3. Increased Behavioral and Neuronal Responses to a Hallucinogenic Drug in PACAP Heterozygous Mutant Mice

    PubMed Central

    Kasai, Atsushi; Encho, Naoki; Shintani, Norihito; Nagayasu, Kazuki; Hashimoto, Ryota; Reglodi, Dora; Miyakawa, Tsuyoshi; Nakazawa, Takanobu; Baba, Akemichi; Hashimoto, Hitoshi

    2014-01-01

    Accumulating evidence from human genetic studies implicates the pituitary adenylate cyclase-activating polypeptide (PACAP) gene as a risk factor for psychiatric disorders, including schizophrenia and stress-related diseases. Mice with homozygous disruption of the PACAP gene display profound behavioral and neurological abnormalities that are ameliorated with the atypical antipsychotic and dopamine D2 and serotonin (5-HT)2 antagonist risperidone and the 5-HT2 receptor antagonist ritanserin; however, the underlying mechanisms remain unknown. Here, we investigated if PACAP heterozygous mutant (PACAP+/−) mice, which appear behaviorally normal, are vulnerable to aversive stimuli. PACAP+/− mice were administered a 5-HT2 receptor agonist, (±)-2,5-dimethoxy-4-iodoamphetamine (DOI), a hallucinogenic drug, and their responses were compared with the littermate wild-type mice. After DOI injection, PACAP+/− mice showed increased head-twitch responses, while their behavior was normal after saline. DOI induced deficits in sensorimotor gating, as determined by prepulse inhibition, specifically in PACAP+/− mice. However, other 5-HT2 receptor-dependent responses, such as corticosterone release and hypothermia, were similarly observed in PACAP+/− and wild-type mice. c-Fos expression analysis, performed in various brain regions, revealed that the DOI-induced increase in the number of c-Fos-positive cells was more pronounced in 5-HT2A receptor-negative cells in the somatosensory cortex in PACAP+/− mice compared with wild-type mice. These results indicate that PACAP+/− mice exhibit specific vulnerability to DOI-induced deficits in cortical sensory function, such as exaggerated head-twitch responses and sensorimotor gating deficits. Our findings provide insight into the neural mechanisms underlying impaired behavioral responses in which 5-HT2 receptors are implicated. PMID:24586556

  4. Increased Outer Membrane Vesicle Formation in a Helicobacter pylori tolB Mutant.

    PubMed

    Turner, Lorinda; Praszkier, Judyta; Hutton, Melanie L; Steer, David; Ramm, Georg; Kaparakis-Liaskos, Maria; Ferrero, Richard L

    2015-08-01

    Multiple studies have established the importance of the tol-pal gene cluster in bacterial cell membrane integrity and outer membrane vesicle (OMV) formation in Escherichia coli. In contrast, the functions of Tol-Pal proteins in pathogenic organisms, including those of the Epsilonproteobacteria, remain poorly if at all defined. The aim of this study was to characterize the roles of two key components of the Tol-Pal system, TolB and Pal, in OMV formation in the pathogenic bacterium, Helicobacter pylori. H. pylori ΔtolB, Δpal and ΔtolBpal mutants, as well as complemented strains, were generated and assessed for changes in morphology and OMV production by scanning electron microscopy and enzyme-linked immunoassay (ELISA), respectively. The protein content and pro-inflammatory properties of OMVs were determined by mass spectroscopy and interleukin-8 (IL-8) ELISA on culture supernatants from OMV-stimulated cells, respectively. H. pylori ΔtolB and Δpal bacteria exhibited aberrant cell morphology and/or flagella biosynthesis. Importantly, the disruption of H. pylori tolB but not pal resulted in a significant increase in OMV production. The OMVs from H. pylori ΔtolB and Δpal bacteria harbored many of the major outer membrane and virulence proteins observed in wild-type (WT) OMVs. Interestingly, ΔtolB, Δpal and ΔtolBpal OMVs induced significantly higher levels of IL-8 production by host cells, compared with WT OMVs. This work demonstrates that TolB and Pal are important for membrane integrity in H. pylori. Moreover, it shows how H. pylori tolB-pal genes may be manipulated to develop "hypervesiculating" strains for vaccine purposes. © 2015 John Wiley & Sons Ltd.

  5. Structural and enzymatic analysis of soybean beta-amylase mutants with increased pH optimum.

    PubMed

    Hirata, Akira; Adachi, Motoyasu; Sekine, Atsushi; Kang, You-Na; Utsumi, Shigeru; Mikami, Bunzo

    2004-02-20

    Comparison of the architecture around the active site of soybean beta-amylase and Bacillus cereus beta-amylase showed that the hydrogen bond networks (Glu380-(Lys295-Met51) and Glu380-Asn340-Glu178) in soybean beta-amylase around the base catalytic residue, Glu380, seem to contribute to the lower pH optimum of soybean beta-amylase. To convert the pH optimum of soybean beta-amylase (pH 5.4) to that of the bacterial type enzyme (pH 6.7), three mutants of soybean beta-amylase, M51T, E178Y, and N340T, were constructed such that the hydrogen bond networks were removed by site-directed mutagenesis. The kinetic analysis showed that the pH optimum of all mutants shifted dramatically to a neutral pH (range, from 5.4 to 6.0-6.6). The Km values of the mutants were almost the same as that of soybean beta-amylase except in the case of M51T, while the Vmax values of all mutants were low compared with that of soybean beta-amylase. The crystal structure analysis of the wild type-maltose and mutant-maltose complexes showed that the direct hydrogen bond between Glu380 and Asn340 was completely disrupted in the mutants M51T, E178Y, and N340T. In the case of M51T, the hydrogen bond between Glu380 and Lys295 was also disrupted. These results indicated that the reduced pKa value of Glu380 is stabilized by the hydrogen bond network and is responsible for the lower pH optimum of soybean beta-amylase compared with that of the bacterial beta-amylase.

  6. Sheathless Mutant of Cyanobacterium Gloeothece sp. Strain PCC 6909 with Increased Capacity To Remove Copper Ions from Aqueous Solutions▿

    PubMed Central

    Micheletti, Ernesto; Pereira, Sara; Mannelli, Francesca; Moradas-Ferreira, Pedro; Tamagnini, Paula; De Philippis, Roberto

    2008-01-01

    The cyanobacterium Gloeothece sp. strain PCC 6909 and its sheathless mutant were tested for their abilities to remove copper ions from aqueous solutions, with the aim of defining the role of the various outermost polysaccharidic investments in the removal of the metal ions. Microscopy studies and chemical analyses revealed that, although the mutant does not possess a sheath, it releases large amounts of polysaccharidic material (released exocellular polysaccharides [RPS]) into the culture medium. The RPS of the wild type and the mutant are composed of the same 11 sugars, although they are present in different amounts, and the RPS of the mutant possesses a larger amount of acidic sugars and a smaller amount of deoxysugars than the wild type. Unexpectedly, whole cultures of the mutant were more effective in the removal of the heavy metal than the wild type (46.3 ± 3.1 and 26.7 ± 1.5 mg of Cu2+ removed per g of dry weight, respectively). Moreover, we demonstrated that the contribution of the sheath to the metal-removal capacity of the wild type is scarce and that the RPS of the mutant is more efficient in removing copper. This suggests that the metal ions are preferably bound to the cell wall and to RPS functional groups rather than to the sheath. Therefore, the increased copper binding efficiency observed with the sheathless mutant can be attributed to the release of a polysaccharide containing larger amounts and/or more accessible functional groups (e.g., carboxyl and amide groups). PMID:18326679

  7. Increased D-allose production by the R132E mutant of ribose-5-phosphate isomerase from Clostridium thermocellum.

    PubMed

    Yeom, Soo-Jin; Seo, Eun-Sun; Kim, Yeong-Su; Oh, Deok-Kun

    2011-03-01

    Ribose-5-phosphate isomerase from Clostridium thermocellum converted D-psicose to D-allose, which may be useful as a pharmaceutical compound, with no by-product. The 12 active-site residues, which were obtained by molecular modeling on the basis of the solved three-dimensional structure of the enzyme, were substituted individually with Ala. Among the 12 Ala-substituted mutants, only the R132A mutant exhibited an increase in D-psicose isomerization activity. The R132E mutant showed the highest activity when the residue at position 132 was substituted with Ala, Gln, Ile, Lys, Glu, or Asp. The maximal activity of the wild-type and R132E mutant enzymes for D-psicose was observed at pH 7.5 and 80°C. The half-lives of the wild-type enzyme at 60°C, 65°C, 70°C, 75°C, and 80°C were 11, 7.0, 4.2, 1.5, and 0.6 h, respectively, whereas those of the R132E mutant enzymes were 13, 8.2, 5.1, 3.1, and 0.9 h, respectively. The specific activity and catalytic efficiency (k(cat)/K(m)) of the R132E mutant for D-psicose were 1.4- and 1.5-fold higher than those of the wild-type enzyme, respectively. When the same amount of enzyme was used, the conversion yield of D-psicose to D-allose was 32% for the R132E mutant enzyme and 25% for the wild-type enzyme after 80 min.

  8. Increased Resistance of Complex I Mutants to Phytosphingosine-induced Programmed Cell Death*S⃞

    PubMed Central

    Castro, Ana; Lemos, Catarina; Falcão, Artur; Glass, N. Louise; Videira, Arnaldo

    2008-01-01

    We have studied the effects of phytosphingosine (PHS) on cells of the filamentous fungus Neurospora crassa. Highly reduced viability, impairment of asexual spore germination, DNA condensation and fragmentation, and production of reactive oxygen species were observed in conidia treated with the drug, suggesting that PHS induces an apoptosis-like death in this fungus. Interestingly, we found that complex I mutants are more resistant to PHS treatment than the wild type strain. This effect appears to be specific because it was not observed in mutants defective in other components of the mitochondrial respiratory chain, pointing to a particular involvement of complex I in cell death. The response of the mutant strains to PHS correlated with their response to hydrogen peroxide. The fact that complex I mutants generate fewer reactive oxygen species than the wild type strain when exposed to PHS likely explains the PHS-resistant phenotype. As compared with the wild type strain, we also found that a strain containing a deletion in the gene encoding an AIF (apoptosis-inducing factor)-like protein is more resistant to PHS and H2O2. In contrast, a strain containing a deletion in a gene encoding an AMID (AIF-homologous mitochondrion-associated inducer of death)-like polypeptide is more sensitive to both drugs. These results indicate that N. crassa has the potential to be a model organism to investigate the molecular basis of programmed cell death in eukaryotic species. PMID:18474589

  9. Increased resistance of complex I mutants to phytosphingosine-induced programmed cell death.

    PubMed

    Castro, Ana; Lemos, Catarina; Falcão, Artur; Glass, N Louise; Videira, Arnaldo

    2008-07-11

    We have studied the effects of phytosphingosine (PHS) on cells of the filamentous fungus Neurospora crassa. Highly reduced viability, impairment of asexual spore germination, DNA condensation and fragmentation, and production of reactive oxygen species were observed in conidia treated with the drug, suggesting that PHS induces an apoptosis-like death in this fungus. Interestingly, we found that complex I mutants are more resistant to PHS treatment than the wild type strain. This effect appears to be specific because it was not observed in mutants defective in other components of the mitochondrial respiratory chain, pointing to a particular involvement of complex I in cell death. The response of the mutant strains to PHS correlated with their response to hydrogen peroxide. The fact that complex I mutants generate fewer reactive oxygen species than the wild type strain when exposed to PHS likely explains the PHS-resistant phenotype. As compared with the wild type strain, we also found that a strain containing a deletion in the gene encoding an AIF (apoptosis-inducing factor)-like protein is more resistant to PHS and H2O2. In contrast, a strain containing a deletion in a gene encoding an AMID (AIF-homologous mitochondrion-associated inducer of death)-like polypeptide is more sensitive to both drugs. These results indicate that N. crassa has the potential to be a model organism to investigate the molecular basis of programmed cell death in eukaryotic species.

  10. Evaluating low lignin mutants of forage sorghum for increased conversion efficiency to sugars and ethanol

    USDA-ARS?s Scientific Manuscript database

    Reduced lignin near-isogenic lines of Atlas bmr-6, bmr-12, and bmr-6 bmr-12 forage sorghum (Sorghum biocolor (L.)) were evaluated as sources of biomass for conversion to sugars and ethanol. These mutants have the advantage of reduced lignin contents and high biomass yields. Field replicates of wil...

  11. A mutant human proinsulin is secreted from islets of Langerhans in increased amounts via an unregulated pathway.

    PubMed Central

    Carroll, R J; Hammer, R E; Chan, S J; Swift, H H; Rubenstein, A H; Steiner, D F

    1988-01-01

    A coding mutation in the human insulin gene (His-B10----Asp) is associated with familial hyperproinsulinemia. To model this syndrome, we have produced transgenic mice that express high levels of the mutant prohormone in their islets of Langerhans. Strain 24-6 mice, containing about 100 copies of the mutant gene, were normoglycemic but had marked increases of serum human proinsulin immunoreactive components. Biosynthetic studies on isolated islets revealed that approximately 65% of the proinsulin synthesized in these mice was the human mutant form. Unlike the normal endogenous mouse proinsulin, which was almost exclusively handled via a regulated secretory pathway, up to 15% of the human [Asp10]proinsulin was rapidly secreted after synthesis via an unregulated or constitutive pathway, and approximately 20% was degraded within the islet cells. The secreted human [Asp10]proinsulin was not processed proteolytically. However, the processing of the normal mouse and human mutant proinsulins within the islets from transgenic mice was not significantly impaired. These findings suggest that the hyperproinsulinemia of the patients is the result of the continuous secretion of unprocessed mutant prohormone from the islets via this alternative unregulated pathway. Images PMID:3057496

  12. Reduced hydroperoxidase (HPI and HPII) activity in the Deltafur mutant contributes to increased sensitivity to UVA radiation in Escherichia coli.

    PubMed

    Hoerter, James D; Arnold, Alan A; Ward, Christopher S; Sauer, Michael; Johnson, Steve; Fleming, Todd; Eisenstark, Abraham

    2005-05-13

    In Escherichia coli, Deltafur (ferric uptake regulator) mutants are hypersensitive to various oxidative agents, including UVA radiation (400-315 nm). Studies suggest that UVA radiation mediates its biological effects on bacteria via oxidative mechanisms that lead to reactive oxygen species, including the superoxide anion radical (O2.-), hydroxyl radical (HO.), hydrogen peroxide (H2O2) and singlet oxygen (1O2). There is accumulating evidence that Fur may play an important role in the defense against UVA radiation. In addition to regulating almost all genes directly involved in iron acquisition, Fur also regulates the expression of manganese and iron superoxide dismutase (MnSOD, FeSOD), key enzymes in the defense against oxygen toxicity in E. coli. In Deltafur mutants, there is a complete absence of FeSOD. Previous results suggest that the native iron chelating agent, enterobactin, which exists in increased levels in Deltafur mutants, is an endogenous chromophore for UVA, releasing Fe2+ into the cytoplasm to catalyze the production of highly reactive hydroxyl radicals. We now report that the hypersensitivity of Deltafur mutants to UVA irradiation is associated with reduced hydroperoxidase I (HPI) and hydroperoxidase II (HPII) activity, and is associated with a decrease in the transcription of katE and katG genes. The observed decrease in HPII activity in Deltafur mutants is also associated with reduced rpoS gene transcription. This study provides additional evidence that the Fur gene product, in addition to its known regulatory effect on the expression of SOD and iron uptake mechanisms, also regulates HPI and HPII activity levels in E. coli. An H2O2-inducible antioxidant defense system leading to an increase in HPI activity, is unaltered in Deltafur mutants.

  13. Identification of 17 independent mutations responsible for human hypoxanthine-guanine phosphoribosyltransferase (HPRT) deficiency.

    PubMed Central

    Davidson, B L; Tarlé, S A; Van Antwerp, M; Gibbs, D A; Watts, R W; Kelley, W N; Palella, T D

    1991-01-01

    Complete hypoxanthine-guanine phosphoribosyltransferase (HPRT) deficiency causes the Lesch-Nyhan syndrome, an X-linked, purine metabolism disorder manifested by hyperuricemia, hyperuricaciduria, and neurologic dysfunction. Partial HPRT deficiency causes hyperuricemia and gout. One requirement for understanding the molecular basis of HPRT deficiency is the determination of which amino acids in this salvage enzyme are necessary for structural or catalytic competence. In this study we have used the PCR coupled with direct sequencing to determine the nucleotide and subsequent amino acid changes in 22 subjects representing 17 unrelated kindreds from the United Kingdom. These mutations were confirmed by using either RNase mapping or Southern analyses. In addition, experiments were done to determine enzyme activity and electrophoretic mobility, and predictive paradigms were used to study the impact of these amino acid substitutions on secondary structure. Images Figure 2 Figure 3 Figure 4 PMID:2018042

  14. Early-Life Exposure to Benzo[a]pyrene Increases Mutant Frequency in Spermatogenic Cells in Adulthood

    PubMed Central

    Xu, Guogang; McMahan, C. Alex; Walter, Christi A.

    2014-01-01

    Children are vulnerable to environmental mutagens, and the developing germline could also be affected. However, little is known about whether exposure to environmental mutagens in childhood will result in increased germline mutations in subsequent adult life. In the present study, male transgenic lacI mice at different ages (7, 25 and 60 days old) were treated with a known environmental mutagen (benzo[a]pyrene, B[a]P) at different doses (0, 50, 200 or 300 mg/kg body weight). Mutant frequency was then determined in a meiotic cell type (pachytene spermatocyte), a post-meiotic cell type (round spermatid) and epididymal spermatozoa after at least one cycle of spermatogenesis. Our results show that 1) mice treated with B[a]P at 7 or 25 days old, both being pre-adult ages, had significantly increased mutant frequencies in all spermatogenic cell types tested when they were 60 days old; 2) spermatogenic cells from mice treated before puberty were more susceptible to B[a]P-associated mutagenesis compared to adult mice; and 3) unexpectedly, epididymal spermatozoa had the highest mutant frequency among the spermatogenic cell types tested. These data show that pre-adult exposure to B[a]P increases the male germline mutant frequency in young adulthood. The data demonstrate that exposure to environmental genotoxins at different life phases (e.g., pre-adult and adult) can have differential effects on reproductive health. PMID:24489914

  15. A Mutant Strain of a Surfactant-Producing Bacterium with Increased Emulsification Activity

    NASA Astrophysics Data System (ADS)

    Liu, Qingmei; Yao, Jianming; Pan, Renrui; Yu, Zengliang

    2005-06-01

    As reported in this paper, a strain of oil-degrading bacterium Sp-5-3 was determined to belong to Enterobacteriaceae, which would be useful for microbial enhanced oil recovery (MEOR). The aim of our study was to generate a mutant using low energy N+ beam implantation. With 10 keV of energy and 5.2 × 1014 N+/cm2 of dose - the optimum condition, a mutant, S-34, was obtained, which had nearly a 5-fold higher surface and a 13-fold higher of emulsification activity than the wild type. The surface activity was measured by two methods, namely, a surface tension measuring instrument and a recording of the repulsive circle of the oil film; the emulsification activity was scaled through measuring the separating time of the oil-fermentation mixture. The metabolic acid was determined as methane by means of gas chromatography.

  16. Increased phagocytosis of Mycobacterium marinum mutants defective in lipooligosaccharide production: a structure-activity relationship study.

    PubMed

    Alibaud, Laeticia; Pawelczyk, Jakub; Gannoun-Zaki, Laila; Singh, Vipul K; Rombouts, Yoann; Drancourt, Michel; Dziadek, Jaroslaw; Guérardel, Yann; Kremer, Laurent

    2014-01-03

    Mycobacterium marinum is a waterborne pathogen responsible for tuberculosis-like infections in ectotherms and is an occasional opportunistic human pathogen. In the environment, M. marinum also interacts with amoebae, which may serve as a natural reservoir for this microorganism. However, the description of mycobacterial determinants in the early interaction with macrophages or amoebae remains elusive. Lipooligosaccharides (LOSs) are cell surface-exposed glycolipids capable of modulating the host immune system, suggesting that they may be involved in the early interactions of M. marinum with macrophages. Herein, we addressed whether LOS composition affects the uptake of M. marinum by professional phagocytes. Mutants with various truncated LOS variants were generated, leading to the identification of several previously uncharacterized biosynthetic genes (wbbL2, MMAR_2321, and MMAR_2331). Biochemical and structural approaches allowed resolving the structures of LOS precursors accumulating in this set of mutants. These strains with structurally defined LOS profiles were then used to infect both macrophages and Acanthamoebae. An inverse correlation between LOS completeness and uptake of mycobacteria by phagocytes was found, allowing the proposal of three mutant classes: class I (papA4), devoid of LOS and highly efficiently phagocytosed; class II, accumulating only early LOS intermediates (wbbL2 and MMAR_2331) and efficiently phagocytosed but less than class I mutants; class III, lacking LOS-IV (losA, MMAR_2319, and MMAR_2321) and phagocytosed similarly to the control strain. These results indicate that phagocytosis is conditioned by the LOS pattern and that the LOS pathway used by M. marinum in macrophages is conserved during infection of amoebae.

  17. Mutation in the Human HPRT1 Gene and the Lesch-Nyhan Syndrome.

    PubMed

    Nguyen, Khue Vu; Nyhan, William L

    2016-08-02

    Lesch-Nyhan syndrome (LNS) is a rare X-linked inherited neurogenetic disorder of purine metabolism in which the enzyme, hypoxanthine-guanine phosphoribosyltransferase (HGprt) is defective. The authors report a novel mutation which led to HGprt-related neurological dysfunction (HND) in two brothers from the same family with a missense mutation in exon 6 of the coding region of the HPRT1 gene: c.437T>C, p.L146S. Molecular diagnosis discloses the genetic heterogeneity of the HPRT1 gene responsible for HGprt deficiency. It allows fast, accurate carrier detection and genetic counseling.

  18. HPRT deficiency in Spain: what have we learned in the past 30 years (1984-2013)?

    PubMed

    Torres, R J; Prior, C; Garcia, M G; Beltran, L M; Puig, J G

    2014-01-01

    Since 1984, we have diagnosed at the La Paz University Hospital, Madrid, Spain, 41 patients with hypoxanthine phosphoribosyltransferase (HPRT) activity deficiency. These patients belonged to 34 families. We have also performed molecular and enzymatic diagnosis in three patients from India, one from Belgium, and three from Colombia. About 1/3 of these patients were followed up at La Paz University Hospital at least every year. This fact has allowed us to examine the complete spectrum of HPRT deficiency as well as to perform a more accurate diagnosis and treatment. In the present review, we also summarized our studies on the basis of physiopathology of the neurological manifestation of Lesch Nyhan disease (LND).

  19. Kras mutations increase telomerase activity and targeting telomerase is a promising therapeutic strategy for Kras-mutant NSCLC

    PubMed Central

    Shi, Bowen; Zhang, Lianmin; Qian, Dong; Li, Chenguang; Zhang, Hua; Wang, Shengguang; Zhu, Jinfang; Gao, Liuwei; Zhang, Qiang; Jia, Bin; Hao, Ligang; Wang, Changli; Zhang, Bin

    2017-01-01

    As shortened telomeres inhibit tumor formation and prolong life span in a KrasG12D mouse lung cancer model, we investigated the implications of telomerase in Kras-mutant NSCLC. We found that Kras mutations increased TERT (telomerase reverse transcriptase) mRNA expression and telomerase activity and telomere length in both immortalized bronchial epithelial cells (BEAS-2B) and lung adenocarcinoma cells (Calu-3). MEK inhibition led to reduced TERT expression and telomerase activity. Furthermore, telomerase inhibitor BIBR1532 shortened telomere length and inhibited mutant Kras-induced long-term proliferation, colony formation and migration capabilities of BEAS-2B and Calu-3 cells. Importantly, BIBR1532 sensitized oncogenic Kras expressing Calu-3 cells to chemotherapeutic agents. The Calu-3-KrasG12D xenograft mouse model confirmed that BIBR1532 enhanced the antitumor efficacy of paclitaxel in vivo. In addition, higher TERT expression was seen in Kras-mutant NSCLC than that with wild-type Kras. Our data suggest that Kras mutations increase telomerase activity and telomere length by activating the RAS/MEK pathway, which contributes to an aggressive phenotype of NSCLC. Kras mutations-induced lung tumorigenesis and chemoresistance are attenuated by telomerase inhibition. Targeting telomerase/telomere may be a promising therapeutic strategy for patients with Kras-mutant NSCLC. PMID:27329725

  20. Murine startle mutant Nmf11 affects the structural stability of the glycine receptor and increases deactivation

    PubMed Central

    Wilkins, Megan E.; Caley, Alex; Gielen, Marc C.; Harvey, Robert J.

    2016-01-01

    Key points Hyperekplexia or startle disease is a serious neurological condition affecting newborn children and usually involves dysfunctional glycinergic neurotransmission.Glycine receptors (GlyRs) are major mediators of inhibition in the spinal cord and brainstem.A missense mutation, replacing asparagine (N) with lysine (K), at position 46 in the GlyR α1 subunit induced hyperekplexia following a reduction in the potency of the transmitter glycine; this resulted from a rapid deactivation of the agonist current at mutant GlyRs.These effects of N46K were rescued by mutating a juxtaposed residue, N61 on binding Loop D, suggesting these two asparagines may interact.Asparagine 46 is considered to be important for the structural stability of the subunit interface and glycine binding site, and its mutation represents a new mechanism by which GlyR dysfunction induces startle disease. Abstract Dysfunctional glycinergic inhibitory transmission underlies the debilitating neurological condition, hyperekplexia, which is characterised by exaggerated startle reflexes, muscle hypertonia and apnoea. Here we investigated the N46K missense mutation in the GlyR α1 subunit gene found in the ethylnitrosourea (ENU) murine mutant, Nmf11, which causes reduced body size, evoked tremor, seizures, muscle stiffness, and morbidity by postnatal day 21. Introducing the N46K mutation into recombinant GlyR α1 homomeric receptors, expressed in HEK cells, reduced the potencies of glycine, β‐alanine and taurine by 9‐, 6‐ and 3‐fold respectively, and that of the competitive antagonist strychnine by 15‐fold. Replacing N46 with hydrophobic, charged or polar residues revealed that the amide moiety of asparagine was crucial for GlyR activation. Co‐mutating N61, located on a neighbouring β loop to N46, rescued the wild‐type phenotype depending on the amino acid charge. Single‐channel recording identified that burst length for the N46K mutant was reduced and fast agonist application

  1. A Caenorhabditis elegans mutant lacking functional nicotinamide nucleotide transhydrogenase displays increased sensitivity to oxidative stress.

    PubMed

    Arkblad, Eva L; Tuck, Simon; Pestov, Nikolay B; Dmitriev, Ruslan I; Kostina, Maria B; Stenvall, Jörgen; Tranberg, Mattias; Rydström, Jan

    2005-06-01

    Proton-translocating mitochondrial nicotinamide nucleotide transhydrogenase (NNT) was investigated regarding its physiological role in Caenorhabditis elegans. NNT catalyzes the reduction of NADP(+) by NADH driven by the electrochemical proton gradient, Deltap, and is thus a potentially important source of mitochondrial NADPH. Mitochondrial detoxification of reactive oxygen species (ROS) by glutathione-dependent peroxidases depends on NADPH for regeneration of reduced glutathione. Transhydrogenase may therefore be directly involved in the defense against oxidative stress. nnt-1 deletion mutants of C. elegans, nnt-1(sv34), were isolated and shown to grow essentially as wild type under normal laboratory conditions, but with a strongly lowered GSH/GSSG ratio. Under conditions of oxidative stress, caused by the superoxide-generating agent methyl viologen, growth of worms lacking nnt-1 activity was severely impaired. A similar result was obtained by using RNAi. Reintroducing nnt-1 in the nnt-1(sv34) knockout mutant led to a partial rescue of growth under oxidative stress conditions. These results provide evidence for the first time that nnt-1 is important in the defense against mitochondrial oxidative stress.

  2. Nuclear hormone receptor DHR96 mediates the resistance to xenobiotics but not the increased lifespan of insulin-mutant Drosophila

    PubMed Central

    Afschar, Sonita; Toivonen, Janne M.; Tain, Luke Stephen; Wieser, Daniela; Finlayson, Andrew John; Driege, Yasmine; Alic, Nazif; Emran, Sahar; Stinn, Julia; Froehlich, Jenny; Piper, Matthew D.; Partridge, Linda

    2016-01-01

    Lifespan of laboratory animals can be increased by genetic, pharmacological, and dietary interventions. Increased expression of genes involved in xenobiotic metabolism, together with resistance to xenobiotics, are frequent correlates of lifespan extension in the nematode worm Caenorhabditis elegans, the fruit fly Drosophila, and mice. The Green Theory of Aging suggests that this association is causal, with the ability of cells to rid themselves of lipophilic toxins limiting normal lifespan. To test this idea, we experimentally increased resistance of Drosophila to the xenobiotic dichlordiphenyltrichlorethan (DDT), by artificial selection or by transgenic expression of a gene encoding a cytochrome P450. Although both interventions increased DDT resistance, neither increased lifespan. Furthermore, dietary restriction increased lifespan without increasing xenobiotic resistance, confirming that the two traits can be uncoupled. Reduced activity of the insulin/Igf signaling (IIS) pathway increases resistance to xenobiotics and extends lifespan in Drosophila, and can also increase longevity in C. elegans, mice, and possibly humans. We identified a nuclear hormone receptor, DHR96, as an essential mediator of the increased xenobiotic resistance of IIS mutant flies. However, the IIS mutants remained long-lived in the absence of DHR96 and the xenobiotic resistance that it conferred. Thus, in Drosophila IIS mutants, increased xenobiotic resistance and enhanced longevity are not causally connected. The frequent co-occurrence of the two traits may instead have evolved because, in nature, lowered IIS can signal the presence of pathogens. It will be important to determine whether enhanced xenobiotic metabolism is also a correlated, rather than a causal, trait in long-lived mice. PMID:26787908

  3. Nuclear hormone receptor DHR96 mediates the resistance to xenobiotics but not the increased lifespan of insulin-mutant Drosophila.

    PubMed

    Afschar, Sonita; Toivonen, Janne M; Hoffmann, Julia Marianne; Tain, Luke Stephen; Wieser, Daniela; Finlayson, Andrew John; Driege, Yasmine; Alic, Nazif; Emran, Sahar; Stinn, Julia; Froehlich, Jenny; Piper, Matthew D; Partridge, Linda

    2016-02-02

    Lifespan of laboratory animals can be increased by genetic, pharmacological, and dietary interventions. Increased expression of genes involved in xenobiotic metabolism, together with resistance to xenobiotics, are frequent correlates of lifespan extension in the nematode worm Caenorhabditis elegans, the fruit fly Drosophila, and mice. The Green Theory of Aging suggests that this association is causal, with the ability of cells to rid themselves of lipophilic toxins limiting normal lifespan. To test this idea, we experimentally increased resistance of Drosophila to the xenobiotic dichlordiphenyltrichlorethan (DDT), by artificial selection or by transgenic expression of a gene encoding a cytochrome P450. Although both interventions increased DDT resistance, neither increased lifespan. Furthermore, dietary restriction increased lifespan without increasing xenobiotic resistance, confirming that the two traits can be uncoupled. Reduced activity of the insulin/Igf signaling (IIS) pathway increases resistance to xenobiotics and extends lifespan in Drosophila, and can also increase longevity in C. elegans, mice, and possibly humans. We identified a nuclear hormone receptor, DHR96, as an essential mediator of the increased xenobiotic resistance of IIS mutant flies. However, the IIS mutants remained long-lived in the absence of DHR96 and the xenobiotic resistance that it conferred. Thus, in Drosophila IIS mutants, increased xenobiotic resistance and enhanced longevity are not causally connected. The frequent co-occurrence of the two traits may instead have evolved because, in nature, lowered IIS can signal the presence of pathogens. It will be important to determine whether enhanced xenobiotic metabolism is also a correlated, rather than a causal, trait in long-lived mice.

  4. Hypersensitivity to mutation and sister-chromatid-exchange induction in CHO cell mutants defective in incising DNA containing UV lesions

    SciTech Connect

    Thompson, L.H.; Brookman, K.W.; Dillehay, L.E.; Mooney, C.L.; Carrano, A.V.

    1982-01-01

    Five UV-sensitive mutant strains of CHO cells representing different genetic complementation groups were analyzed for their ability to perform the incision step of nucleotide excision repair after UV exposure. The assay utilized inhibitors of DNA synthesis to accumulate the short-lived strand breaks resulting from repair incisions. After 6 J/m/sup 2/, each of the mutants showed < 10% of the incision rate of the parental AA8 cells. After 50 J/m/sup 2/, the rate in AA8 was similar to that at 6 J/m/sup 2/, but the rates in the mutants were significantly higher (approx. 20% of the rate of AA8). Thus by this incision assay the mutants were phenotypically indistinguishable. Each of the mutants were hypersensitive to mutation induction at both the hprt and aprt loci by a factor of 10, and in the one strain tested ouabain resistance was induced sevenfold more efficiently than in AA8 cells. Sister chromatid exchange was also induced with sevenfold increased efficiency in the two mutant strains examined. Thus, here CHO mutants resemble xeroderma pigmentosum cells in terms of their incision defects and their hypersensitivity to DNA damage by UV.

  5. Efficient identification of inhibitors targeting the closed active site conformation of the HPRT from Trypanosoma cruzi.

    PubMed

    Freymann, D M; Wenck, M A; Engel, J C; Feng, J; Focia, P J; Eakin, A E; Craig, S P

    2000-12-01

    Currently, only two drugs are recommended for treatment of infection with Trypanosoma cruzi, the etiologic agent of Chagas' disease. These compounds kill the trypomastigote forms of the parasite circulating in the bloodstream, but are relatively ineffective against the intracellular stage of the parasite life cycle. Neither drug is approved by the FDA for use in the US. The hypoxanthine phosphoribosyltransferase (HPRT) from T. cruzi is a possible new target for antiparasite chemotherapy. The crystal structure of the HPRT in a conformation approximating the transition state reveals a closed active site that provides a well-defined target for computational structure-based drug discovery. A flexible ligand docking program incorporating a desolvation correction was used to screen the Available Chemicals Directory for inhibitors targeted to the closed conformation of the trypanosomal HPRT. Of 22 potential inhibitors identified, acquired and tested, 16 yielded K(i)'s between 0.5 and 17 microM versus the substrate phosphoribosylpyrophosphate. Surprisingly, three of eight compounds tested were effective in inhibiting the growth of parasites in infected mammalian cells. This structure-based docking method provided a remarkably efficient path for the identification of inhibitors targeting the closed conformation of the trypanosomal HPRT. The inhibition constants of the lead inhibitors identified are unusually favorable, and the trypanostatic activity of three of the compounds in cell culture suggests that they may provide useful starting points for drug design for the treatment of Chagas' disease.

  6. Phosphoproteomic Analyses of NRAS(G12) and NRAS(Q61) Mutant Melanocytes Reveal Increased CK2α Kinase Levels in NRAS(Q61) Mutant Cells.

    PubMed

    Posch, Christian; Sanlorenzo, Martina; Vujic, Igor; Oses-Prieto, Juan A; Cholewa, Brian D; Kim, Sarasa T; Ma, Jeffrey; Lai, Kevin; Zekhtser, Mitchell; Esteve-Puig, Rosaura; Green, Gary; Chand, Shreya; Burlingame, Alma L; Panzer-Grümayer, Renate; Rappersberger, Klemens; Ortiz-Urda, Susana

    2016-10-01

    In melanoma, mutant and thereby constantly active neuroblastoma rat sarcoma (NRAS) affects 15-20% of tumors, contributing to tumor initiation, growth, invasion, and metastasis. Recent therapeutic approaches aim to mimic RAS extinction by interfering with critical signaling pathways downstream of the mutant protein. This study investigates the phosphoproteome of primary human melanocytes bearing mutations in the two hot spots of NRAS, NRAS(G12) and NRAS(Q61). Stable isotope labeling by amino acids in cell culture followed by mass spectrometry identified 14,155 spectra of 3,371 unique phosphopeptides mapping to 1,159 proteins (false discovery rate < 2%). Data revealed pronounced PI3K/AKT signaling in NRAS(G12V) mutant cells and pronounced mitogen-activated protein kinase (MAPK) signaling in NRAS(Q61L) variants. Computer-based prediction models for kinases involved, revealed that CK2α is significantly overrepresented in primary human melanocytes bearing NRAS(Q61L) mutations. Similar differences were found in human NRAS(Q61) mutant melanoma cell lines that were also more sensitive to pharmacologic CK2α inhibition compared with NRAS(G12) mutant cells. Furthermore, CK2α levels were pronounced in patient samples of NRAS(Q61) mutant melanoma at the mRNA and protein level. The preclinical findings of this study reveal that codon 12 and 61 mutant NRAS cells have distinct signaling characteristics that could allow for the development of more effective, mutation-specific treatment modalities.

  7. Harbouring public good mutants within a pathogen population can increase both fitness and virulence.

    PubMed

    Lindsay, Richard J; Kershaw, Michael J; Pawlowska, Bogna J; Talbot, Nicholas J; Gudelj, Ivana

    2016-12-28

    Existing theory, empirical, clinical and field research all predict that reducing the virulence of individuals within a pathogen population will reduce the overall virulence, rendering disease less severe. Here, we show that this seemingly successful disease management strategy can fail with devastating consequences for infected hosts. We deploy cooperation theory and a novel synthetic system involving the rice blast fungus Magnaporthe oryzae. In vivo infections of rice demonstrate that M. oryzae virulence is enhanced, quite paradoxically, when a public good mutant is present in a population of high-virulence pathogens. We reason that during infection, the fungus engages in multiple cooperative acts to exploit host resources. We establish a multi-trait cooperation model which suggests that the observed failure of the virulence reduction strategy is caused by the interference between different social traits. Multi-trait cooperative interactions are widespread, so we caution against the indiscriminant application of anti-virulence therapy as a disease-management strategy.

  8. Increased prevalence of mutant null alleles that cause hereditary fructose intolerance in the American population.

    PubMed

    Coffee, Erin M; Yerkes, Laura; Ewen, Elizabeth P; Zee, Tiffany; Tolan, Dean R

    2010-02-01

    Mutations in the aldolase B gene (ALDOB) impairing enzyme activity toward fructose-1-phosphate cleavage cause hereditary fructose intolerance (HFI). Diagnosis of the disease is possible by identifying known mutant ALDOB alleles in suspected patients; however, the frequencies of mutant alleles can differ by population. Here, 153 American HFI patients with 268 independent alleles were analyzed to identify the prevalence of seven known HFI-causing alleles (A149P, A174D, N334K, Delta4E4, R59Op, A337V, and L256P) in this population. Allele-specific oligonucleotide hybridization analysis was performed on polymerase chain reaction (PCR)-amplified genomic DNA from these patients. In the American population, the missense mutations A149P and A174D are the two most common alleles, with frequencies of 44% and 9%, respectively. In addition, the nonsense mutations Delta4E4 and R59Op are the next most common alleles, with each having a frequency of 4%. Together, the frequencies of all seven alleles make up 65% of HFI-causing alleles in this population. Worldwide, these same alleles make up 82% of HFI-causing mutations. This difference indicates that screening for common HFI alleles is more difficult in the American population. Nevertheless, a genetic screen for diagnosing HFI in America can be improved by including all seven alleles studied here. Lastly, identification of HFI patients presenting with classic symptoms and who have homozygous null genotypes indicates that aldolase B is not required for proper development or metabolic maintenance.

  9. Increased prevalence of mutant null alleles that cause hereditary fructose intolerance in the American population

    PubMed Central

    Coffee, Erin M.; Yerkes, Laura; Ewen, Elizabeth P.; Zee, Tiffany

    2010-01-01

    Mutations in the aldolase B gene (ALDOB) impairing enzyme activity toward fructose-1-phosphate cleavage cause hereditary fructose intolerance (HFI). Diagnosis of the disease is possible by identifying known mutant ALDOB alleles in suspected patients; however, the frequencies of mutant alleles can differ by population. Here, 153 American HFI patients with 268 independent alleles were analyzed to identify the prevalence of seven known HFI-causing alleles (A149P, A174D, N334K, Δ4E4, R59Op, A337V, and L256P) in this population. Allele-specific oligonucleotide hybridization analysis was performed on polymerase chain reaction (PCR)-amplified genomic DNA from these patients. In the American population, the missense mutations A149P and A174D are the two most common alleles, with frequencies of 44% and 9%, respectively. In addition, the nonsense mutations Δ4E4 and R59Op are the next most common alleles, with each having a frequency of 4%. Together, the frequencies of all seven alleles make up 65% of HFI-causing alleles in this population. Worldwide, these same alleles make up 82% of HFI-causing mutations. This difference indicates that screening for common HFI alleles is more difficult in the American population. Nevertheless, a genetic screen for diagnosing HFI in America can be improved by including all seven alleles studied here. Lastly, identification of HFI patients presenting with classic symptoms and who have homozygous null genotypes indicates that aldolase B is not required for proper development or metabolic maintenance. PMID:20033295

  10. Harbouring public good mutants within a pathogen population can increase both fitness and virulence

    PubMed Central

    Lindsay, Richard J; Kershaw, Michael J; Pawlowska, Bogna J; Talbot, Nicholas J; Gudelj, Ivana

    2016-01-01

    Existing theory, empirical, clinical and field research all predict that reducing the virulence of individuals within a pathogen population will reduce the overall virulence, rendering disease less severe. Here, we show that this seemingly successful disease management strategy can fail with devastating consequences for infected hosts. We deploy cooperation theory and a novel synthetic system involving the rice blast fungus Magnaporthe oryzae. In vivo infections of rice demonstrate that M. oryzae virulence is enhanced, quite paradoxically, when a public good mutant is present in a population of high-virulence pathogens. We reason that during infection, the fungus engages in multiple cooperative acts to exploit host resources. We establish a multi-trait cooperation model which suggests that the observed failure of the virulence reduction strategy is caused by the interference between different social traits. Multi-trait cooperative interactions are widespread, so we caution against the indiscriminant application of anti-virulence therapy as a disease-management strategy. DOI: http://dx.doi.org/10.7554/eLife.18678.001 PMID:28029337

  11. Increase of multidrug efflux pump expression in fluoroquinolone-resistant Salmonella mutants induced by ciprofloxacin selective pressure.

    PubMed

    Kang, Hyun-Wol; Woo, Gun-Jo

    2014-10-01

    Multidrug-resistant foodborne pathogens are a leading public health concern, as antimicrobial resistance can lead to therapeutic failure. In this study, a ciprofloxacin-susceptible Salmonella Istanbul (Sal10-FC-KU12) was isolated from chicken meat obtained from a market in Korea to induce ciprofloxacin-resistant mutants (SalML, SalMM, and SalMH). Minimum inhibitory concentrations (MICs) of 12 antibiotics were measured in the presence or absence of an efflux pump inhibitor. Expression levels of efflux pump-related genes (acrB, acrF, marA, ramA, rob, and soxS) were determined using quantitative reverse transcription polymerase chain reaction (qRT-PCR). Elevated MICs for the derived mutants were shown to result from the action of the efflux pump, with increased expression of marA, ramA, and acrB compared with the wild-type strain. The results of this study suggest that continued use of ciprofloxacin might induce the emergence of Salmonella mutants resistant not only to fluoroquinolones, but also to several other classes of antimicrobials. Copyright © 2014 Elsevier Ltd. All rights reserved.

  12. dhm1, an Arabidopsis mutant with increased sensitivity to alkamides shows tumorous shoot development and enhanced lateral root formation.

    PubMed

    Pelagio-Flores, Ramón; Ortiz-Castro, Randy; López-Bucio, José

    2013-04-01

    The control of cell division by growth regulators is critical to proper shoot and root development. Alkamides belong to a class of small lipid amides involved in plant morphogenetic processes, from which N-isobutyl decanamide is one of the most active compounds identified. This work describes the isolation and characterization of an N-isobutyl decanamide-hypersensitive (dhm1) mutant of Arabidopsis (Arabidopsis thaliana). dhm1 seedlings grown in vitro develop disorganized tumorous tissue in petioles, leaves and stems. N-isobutyl decanamide treatment exacerbates the dhm1 phenotype resulting in widespread production of callus-like structures in the mutant. Together with these morphological alterations in shoot, dhm1 seedlings sustained increased lateral root formation and greater sensitivity to alkamides in the inhibition of primary root growth. The mutants also show reduced etiolation when grown in darkness. When grown in soil, adult dhm1 plants were characterized by reduced plant size, and decreased fertility. Genetic analysis indicated that the mutant phenotype segregates as a single recessive Mendelian trait. Developmental alterations in dhm1 were related to an enhanced expression of the cell division marker CycB1-uidA both in the shoot and root system, which correlated with altered expression of auxin and cytokinin responsive gene markers. Pharmacological inhibition of auxin transport decreased LR formation in WT and dhm1 seedlings in a similar manner, indicating that auxin transport is involved in the dhm1 root phenotype. These data show an important role of alkamide signaling in cell proliferation and plant architecture remodeling likely acting through the DHM1 protein.

  13. Mutagenicity of the racemic mixtures of butadiene monoepoxide and butadiene diepoxide at the Hprt locus of T-lymphocytes following inhalation exposures of female mice and rats.

    PubMed

    Meng, Q; Henderson, R F; Walker, D M; Bauer, M J; Reilly, A A; Walker, V E

    1999-08-11

    The purpose of this study was to determine if Hprt mutant frequency (Mf) data from rodents exposed directly to individual epoxy metabolites of 1,3-butadiene (BD) can be used to identify the relative significance of each intermediate in the mutagenicity of BD in mice vs. rats. To this end, the relative contributions of the racemic mixtures of BD monoepoxide (BDO) and BD diepoxide (BDO(2)) to BD-induced mutagenicity was investigated by exposing mice and rats to selected concentrations of BDO and BDO(2) (i.e., 2.5 and 4.0 ppm, respectively) and comparing the mutagenic potency of each intermediate to that of BD (at 62.5 ppm) when comparable blood levels of metabolites are achieved (in the mouse). Female B6C3F1 mice and F344 rats (4-5 weeks old) were exposed to rac-BDO (0, 2.5, or 25 ppm) or (+/-)-BDO(2) (0, 2, 4 ppm) by inhalation for 4 weeks (6 h/day, 5 days/week), and then groups of control and exposed animals (n=3-12/group) were necropsied at multiple time points post-exposure for measuring Hprt Mfs in splenic lymphocytes (via the T-cell cloning assay) and estimating mutagenic potencies (represented by the difference in the areas under the mutant T-cell 'manifestation' curves of treated vs. control animals). The resulting Mf data, along with the extant metabolism data, suggest that at lower BD exposures (Hprt Mfs over time in T-cells of exposed vs. control animals, as used in this study, can be valuable for predicting the potential role

  14. Methylation of the mouse hprt gene differs on the active and inactive X chromosomes.

    PubMed Central

    Lock, L F; Melton, D W; Caskey, C T; Martin, G R

    1986-01-01

    It has been proposed that DNA methylation is involved in the mechanism of X inactivation, the process by which equivalence of levels of X-linked gene products is achieved in female (XX) and male (XY) mammals. In this study, Southern blots of female and male DNA digested with methylation-sensitive restriction endonucleases and hybridized to various portions of the cloned mouse hprt gene were compared, and sites within the mouse hprt gene were identified that are differentially methylated in female and male cells. The extent to which these sites are methylated when carried on the active and inactive X chromosomes was directly determined in a similar analysis of DNA from clonal cell lines established from a female embryo derived from a mating of two species of mouse, Mus musculus and Mus caroli. The results revealed two regions of differential methylation in the mouse hprt gene. One region, in the first intron of the gene, includes four sites that are completely unmethylated when carried on the active X and extensively methylated when carried on the inactive X. These same sites are extensively demethylated in hprt genes reactivated either spontaneously or after 5-azacytidine treatment. The second region includes several sites in the 3' 20kilobases of the gene extending from exon 3 to exon 9 that show the converse pattern; i.e., they are completely methylated when carried on the active X and completely unmethylated when carried on the inactive X. At least one of these sites does not become methylated after reactivation of the gene. The results of this study, together with the results of previous studies by others of the human hprt gene, indicate that these regions of differential methylation on the active and inactive X are conserved between mammalian species. Furthermore, the data described here are consistent with the idea that at least the sites in the 5' region of the gene play a role in the X inactivation phenomenon and regulation of expression of the mouse hprt

  15. A low, adaptive dose of gamma-rays reduced the number and altered the spectrum of S1- mutants in human-hamster hybrid AL cells

    NASA Technical Reports Server (NTRS)

    Ueno, A. M.; Vannais, D. B.; Gustafson, D. L.; Wong, J. C.; Waldren, C. A.

    1996-01-01

    We examined the effects of a low, adaptive dose of 137Cs-gamma-irradiation (0.04 Gy) on the number and kinds of mutants induced in AL human-hamster hybrid cells by a later challenge dose of 4 Gy. The yield of S1- mutants was significantly less (by 53%) after exposure to both the adaptive and challenge doses compared to the challenge dose alone. The yield of hprt- mutants was similarly decreased. Incubation with cycloheximide (CX) or 3-aminobenzamide largely negated the decrease in mutant yield. The adaptive dose did not perturb the cell cycle, was not cytotoxic, and did not of itself increase the mutant yield above background. The adaptive dose did, however, alter the spectrum of S1- mutants from populations exposed only to the adaptive dose, as well as affecting the spectrum of S1- mutants generated by the challenge dose. The major change in both cases was a significant increase in the proportion of complex mutations compared to small mutations and simple deletions.

  16. A low, adaptive dose of gamma-rays reduced the number and altered the spectrum of S1- mutants in human-hamster hybrid AL cells

    NASA Technical Reports Server (NTRS)

    Ueno, A. M.; Vannais, D. B.; Gustafson, D. L.; Wong, J. C.; Waldren, C. A.

    1996-01-01

    We examined the effects of a low, adaptive dose of 137Cs-gamma-irradiation (0.04 Gy) on the number and kinds of mutants induced in AL human-hamster hybrid cells by a later challenge dose of 4 Gy. The yield of S1- mutants was significantly less (by 53%) after exposure to both the adaptive and challenge doses compared to the challenge dose alone. The yield of hprt- mutants was similarly decreased. Incubation with cycloheximide (CX) or 3-aminobenzamide largely negated the decrease in mutant yield. The adaptive dose did not perturb the cell cycle, was not cytotoxic, and did not of itself increase the mutant yield above background. The adaptive dose did, however, alter the spectrum of S1- mutants from populations exposed only to the adaptive dose, as well as affecting the spectrum of S1- mutants generated by the challenge dose. The major change in both cases was a significant increase in the proportion of complex mutations compared to small mutations and simple deletions.

  17. Trace amount of satellite RNA associated with tobacco ringspot virus: increase stimulated by nonaccumulating satellite RNA mutants.

    PubMed

    Passmore, B K; van Tol, H; Buzayan, J M; Stabinsky, D; Bruening, G

    1995-06-01

    The small satellite RNA of tobacco ringspot virus (sTRSV RNA) is dependent on tobacco ringspot virus (TRSV) for replication and encapsidation. sTRSV RNA has appeared during serial passage of certain TRSV strains in some hosts. Co-inoculation of bean with TRSV and either of two related, nonaccumulating mutants of sTRSV RNA induced the appearance of sTRSV RNA in a single passage (van Tol et al., 1991, Virology 180, 23-30). The sTRSV RNA obtained after serial passage and after co-inoculation have the same nucleotide sequence, designated the endogenous sequence. The endogenous sTRSV RNA nucleotide sequence differs from that of each of the nonaccumulating sTRSV RNA at three positions. In order to detect possible trace amounts of endogenous satellite RNA in virion RNA preparations, RNA from two TRSV isolates was subjected to reverse transcription and polymerase chain reaction of the transcript (RT-PCR), using primers with sTRSV RNA terminal sequences. The yield of RT-PCR product suggests that the virion RNA preparations contained approximately 0.1 fg of sTRSV RNA per microgram of virion RNA. The nucleotide sequence of the RT-PCR product corresponded to that of the endogenous sTRSV RNA. The endogenous sTRSV RNA of TRSV inocula appears to be latent, being maintained in very small amounts during serial passage of TRSV in some hosts but capable of dramatic increase during serial passage in other hosts or when TRSV was co-inoculated with either of two specific sTRSV RNA mutants. Ten other nonaccumulating sTRSV RNA mutants did not induce a detected increase in sTRSV RNA.

  18. A single nucleotide change in mutY increases the emergence of antibiotic-resistant Campylobacter jejuni mutants

    PubMed Central

    Dai, Lei; Muraoka, Wayne T.; Wu, Zuowei; Sahin, Orhan; Zhang, Qijing

    2015-01-01

    Objectives Mutator strains play an important role in the emergence of antibiotic-resistant bacteria. Campylobacter jejuni is a leading cause of foodborne illnesses worldwide and is increasingly resistant to clinically important antibiotics. The objective of this study was to identify the genetic basis that contributes to a mutator phenotype in Campylobacter and determine the role of this phenotype in the development of antibiotic resistance. Methods A C. jejuni isolate (named CMT) showing a mutator phenotype was subjected to WGS analysis. Comparative genomics, site-specific reversion and mutation, and gene knockout were conducted to prove the mutator effect was caused by a single nucleotide change in the mutY gene of C. jejuni. Results The C. jejuni CMT isolate showed ∼100-fold higher mutation frequency to ciprofloxacin than the WT strain. Under selection by ciprofloxacin, fluoroquinolone-resistant mutants emerged readily from the CMT isolate. WGS identified a single nucleotide change (G595→T) in the mutY gene of the CMT isolate. Further experiments using defined mutant constructs proved its specific role in elevating mutation frequencies. The mutY point mutation also led to an ∼700-fold increase in the emergence of ampicillin-resistant mutants, indicating its broader impact on antibiotic resistance. Structural modelling suggested the G595→T mutation probably affects the catalytic domain of MutY and consequently abolishes the anti-mutator function of this DNA repair protein. Conclusions The G595→T mutation in mutY abolishes its anti-mutator function and confers a mutator phenotype in Campylobacter, promoting the emergence of antibiotic-resistant Campylobacter. PMID:26169557

  19. Qualitatively and quantitatively similar effects of active and passive maternal tobacco smoke exposure on in utero mutagenesis at the HPRT locus.

    PubMed

    Grant, Stephen G

    2005-06-29

    Induced mutagenesis in utero is likely to have life-long repercussions for the exposed fetus, affecting survival, birth weight and susceptibility to both childhood and adult-onset diseases, such as cancer. In the general population, such exposures are likely to be a consequence of the lifestyle choices of the parents, with exposure to tobacco smoke one of the most pervasive and easily documented. Previous studies attempting to establish a direct link between active smoking and levels of somatic mutation have largely discounted the effects of passive or secondary exposure, and have produced contradictory results. Data from three studies of possible smoking effects on in utero mutagenesis at the HPRT locus were compiled and reanalyzed, alone and in combination. Where possible, passive exposure to environmental tobacco smoke was considered as a separate category of exposure, rather than being included in the non-smoking controls. Molecular spectra from these studies were reanalyzed after adjustment for reported mutation frequencies from the individual studies and the entire data set. A series of related studies on mutation at the X-linked HPRT locus in human newborn cord blood samples has led to the novel conclusion that only passive maternal exposure to tobacco mutagens has a significant effect on the developing baby. We performed a pooled analysis of the complete data from these studies, at the levels of both induced mutation frequency and the resulting mutational spectrum. Our analysis reveals a more commonsensical, yet no less cautionary result: both active maternal smoking and secondary maternal exposure produce quantitatively and qualitatively indistinguishable increases in fetal HPRT mutation. Further, it appears that this effect is not perceptibly ameliorated if the mother adjusts her behavior (i.e. stops smoking) when pregnancy is confirmed, although this conclusion may also be affected by continued passive exposure.

  20. Generation of Hprt-disrupted rat through mouse←rat ES chimeras

    PubMed Central

    Isotani, Ayako; Yamagata, Kazuo; Okabe, Masaru; Ikawa, Masahito

    2016-01-01

    We established rat embryonic stem (ES) cell lines from a double transgenic rat line which harbours CAG-GFP for ubiquitous expression of GFP in somatic cells and Acr3-EGFP for expression in sperm (green body and green sperm: GBGS rat). By injecting the GBGS rat ES cells into mouse blastocysts and transplanting them into pseudopregnant mice, rat spermatozoa were produced in mouse←rat ES chimeras. Rat spermatozoa from the chimeric testis were able to fertilize eggs by testicular sperm extraction combined with intracytoplasmic sperm injection (TESE-ICSI). In the present paper, we disrupted rat hypoxanthine-guanine phosphoribosyl transferase (Hprt) gene in ES cells and produced a Hprt-disrupted rat line using the mouse←rat ES chimera system. The mouse←rat ES chimera system demonstrated the dual advantages of space conservation and a clear indication of germ line transmission in knockout rat production. PMID:27062982

  1. Mutant strains of Spirulina (Arthrospira) platensis to increase the efficiency of micro-ecological life support systems

    NASA Astrophysics Data System (ADS)

    Brown, Igor

    The European Micro-Ecological Life Support System Alternative (MELiSSA) is an advanced idea for organizing a bioregenerative system for long term space flights and extraterrestrial settlements (Hendrickx, De Wever et al., 2005). Despite the hostility of both lunar and Martian environments to unprotected life, it seems possible to cultivate photosynthetic bacteria using closed bioreactors illuminated and heated by solar energy. Such reactors might be employed in critical processes, e.g. air revitalization, foodcaloric and protein source, as well as an immunomodulators production. The MELiSSA team suggested cyanobacterium Spirulina as most appropriate agent to revitalize air and produce a simple "fast" food. This is right suggestion because Spirulina was recently shown to be an oxygenic organism with the highest level of O2 production per unit mass (Ananyev et al., 2005). Chemical composition of Spirulina includes proteins (55Aiming to make Spirulina cultivation in life support systems like MELiSSA more efficient, we selected Spirulina mutant strains with increased fraction of methionine in the biomass of this cyanobacterium and compared the effect of parental wild strain of Spirulina and its mutants on the tendency of such experimental illnesses as radiationinduced lesions and hemolythic anemia. Results: It was found that mutant strains 198B and 27G contain higher quantities of total protein, essential amino acids, c-phycocyanin, allophycocyanin and chlorophyll a than parental wild strain of S. platensis. The strain 198B is also characterized with increased content of carotenoids. Revealed biochemical peculiarities of mutant strains suggest that these strains can serve as an additional source of essential amino acids as well as phycobiliproteins and carotenoids for the astronauts. Feeding animals suffering from radiation-induced lesions, c-phycocyanin, extracted from strain 27G, led to a correction in deficient dehydrogenase activity and energy-rich phosphate levels

  2. Characterization of a JAZ7 activation-tagged Arabidopsis mutant with increased susceptibility to the fungal pathogen Fusarium oxysporum

    PubMed Central

    Thatcher, Louise F.; Cevik, Volkan; Grant, Murray; Zhai, Bing; Jones, Jonathan D.G.; Manners, John M.; Kazan, Kemal

    2016-01-01

    In Arabidopsis, jasmonate (JA)-signaling plays a key role in mediating Fusarium oxysporum disease outcome. However, the roles of JASMONATE ZIM-domain (JAZ) proteins that repress JA-signaling have not been characterized in host resistance or susceptibility to this pathogen. Here, we found most JAZ genes are induced following F. oxysporum challenge, and screening T-DNA insertion lines in Arabidopsis JAZ family members identified a highly disease-susceptible JAZ7 mutant (jaz7-1D). This mutant exhibited constitutive JAZ7 expression and conferred increased JA-sensitivity, suggesting activation of JA-signaling. Unlike jaz7 loss-of-function alleles, jaz7-1D also had enhanced JA-responsive gene expression, altered development and increased susceptibility to the bacterial pathogen Pst DC3000 that also disrupts host JA-responses. We also demonstrate that JAZ7 interacts with transcription factors functioning as activators (MYC3, MYC4) or repressors (JAM1) of JA-signaling and contains a functional EAR repressor motif mediating transcriptional repression via the co-repressor TOPLESS (TPL). We propose through direct TPL recruitment, in wild-type plants JAZ7 functions as a repressor within the JA-response network and that in jaz7-1D plants, misregulated ectopic JAZ7 expression hyper-activates JA-signaling in part by disturbing finely-tuned COI1-JAZ-TPL-TF complexes. PMID:26896849

  3. Characterization of a JAZ7 activation-tagged Arabidopsis mutant with increased susceptibility to the fungal pathogen Fusarium oxysporum.

    PubMed

    Thatcher, Louise F; Cevik, Volkan; Grant, Murray; Zhai, Bing; Jones, Jonathan D G; Manners, John M; Kazan, Kemal

    2016-04-01

    In Arabidopsis, jasmonate (JA)-signaling plays a key role in mediating Fusarium oxysporum disease outcome. However, the roles of JASMONATE ZIM-domain (JAZ) proteins that repress JA-signaling have not been characterized in host resistance or susceptibility to this pathogen. Here, we found most JAZ genes are induced following F. oxysporum challenge, and screening T-DNA insertion lines in Arabidopsis JAZ family members identified a highly disease-susceptible JAZ7 mutant (jaz7-1D). This mutant exhibited constitutive JAZ7 expression and conferred increased JA-sensitivity, suggesting activation of JA-signaling. Unlike jaz7 loss-of-function alleles, jaz7-1D also had enhanced JA-responsive gene expression, altered development and increased susceptibility to the bacterial pathogen PstDC3000 that also disrupts host JA-responses. We also demonstrate that JAZ7 interacts with transcription factors functioning as activators (MYC3, MYC4) or repressors (JAM1) of JA-signaling and contains a functional EAR repressor motif mediating transcriptional repression via the co-repressor TOPLESS (TPL). We propose through direct TPL recruitment, in wild-type plants JAZ7 functions as a repressor within the JA-response network and that in jaz7-1D plants, misregulated ectopic JAZ7 expression hyper-activates JA-signaling in part by disturbing finely-tuned COI1-JAZ-TPL-TF complexes.

  4. Cyclic loading increases friction and changes cartilage surface integrity in lubricin-mutant mouse knees

    PubMed Central

    Drewniak, Elizabeth I; Jay, Gregory D; Fleming, Braden C; Zhang, Ling; Warman, Matthew L; Crisco, Joseph J

    2012-01-01

    Objective To investigate the effects of lubricin gene dosage and cyclic loading on whole joint coefficient of friction and articular cartilage surface integrity in mouse knee joints. Methods Joints from mice with 2 (Prg4+/+), 1 (Prg4+/−), or no (Prg4−/−) functioning lubricin alleles were subjected to 26 hours of cyclic loading using a custom-built pendulum. Coefficient of friction values were measured at multiple time points. Contralateral control joints were left unloaded. Following testing, joints were examined for histologic evidence of damage and cell viability. Results At baseline, the coefficient of friction values in Prg4−/− mice were significantly higher than those in Prg4+/+ and Prg4+/− mice (P < 0.001). Cyclic loading continuously increased the coefficient of friction in Prg4−/− mouse joints. In contrast, Prg4+/− and Prg4+/+ mouse joints had no coefficient of friction increases during the first 4 hours of loading. After 26 hours of loading, joints from all genotypes had increased coefficient of friction values compared to baseline and unloaded controls. Significantly greater increases occurred in Prg4−/− and Prg4+/− mouse joints compared to Prg4+/+ mouse joints. The coefficient of friction values were not significantly associated with histologic evidence of damage or loss of cell viability. Conclusion Our findings indicate that mice lacking lubricin have increased baseline coefficient of friction values and are not protected against further increases caused by loading. Prg4+/− mice are indistinguishable from Prg4+/+ mice at baseline, but have significantly greater coefficient of friction values following 26 hours of loading. Lubricin dosage affects joint properties during loading, and may have clinical implications in patients for whom injury or illness alters lubricin abundance. PMID:21905020

  5. Cyclic loading increases friction and changes cartilage surface integrity in lubricin-mutant mouse knees.

    PubMed

    Drewniak, Elizabeth I; Jay, Gregory D; Fleming, Braden C; Zhang, Ling; Warman, Matthew L; Crisco, Joseph J

    2012-02-01

    To investigate the effects of lubricin gene dosage and cyclic loading on whole joint coefficient of friction and articular cartilage surface integrity in mouse knee joints. Joints from mice with 2 (Prg4(+/+)), 1 (Prg4(+/-)), or no (Prg4(-/-)) functioning lubricin alleles were subjected to 26 hours of cyclic loading using a custom-built pendulum. Coefficient of friction values were measured at multiple time points. Contralateral control joints were left unloaded. Following testing, joints were examined for histologic evidence of damage and cell viability. At baseline, the coefficient of friction values in Prg4(-/-) mice were significantly higher than those in Prg4(+/+) and Prg4(+/-) mice (P < 0.001). Cyclic loading continuously increased the coefficient of friction in Prg4(-/-) mouse joints. In contrast, Prg4(+/-) and Prg4(+/+) mouse joints had no coefficient of friction increases during the first 4 hours of loading. After 26 hours of loading, joints from all genotypes had increased coefficient of friction values compared to baseline and unloaded controls. Significantly greater increases occurred in Prg4(-/-) and Prg4(+/-) mouse joints compared to Prg4(+/+) mouse joints. The coefficient of friction values were not significantly associated with histologic evidence of damage or loss of cell viability. Our findings indicate that mice lacking lubricin have increased baseline coefficient of friction values and are not protected against further increases caused by loading. Prg4(+/-) mice are indistinguishable from Prg4(+/+) mice at baseline, but have significantly greater coefficient of friction values following 26 hours of loading. Lubricin dosage affects joint properties during loading, and may have clinical implications in patients for whom injury or illness alters lubricin abundance. Copyright © 2012 by the American College of Rheumatology.

  6. [Heavy metal cation-induced increase in the antimicrobial activity of gramicidin S. Increased sensitivity of metal-resistant mutants of Escherichia coli B to the antibiotic].

    PubMed

    Kuzovnikova, T A; Fedorov, Iu I

    1990-04-01

    Gramicidin S response of metal resistant mutants of E. coli B and the effect of concentrations of Cu2+, Ag+, Co2+ and Cd2+ on the growth and sensitivity of E. coli B to cationic antibiotics, i.e. gramicidin S2+ and streptomycin2+, were studied. It was shown that the metal-cumulating mutants of E. coli B with two different mechanisms of cross resistance to Cu2+, Cd2+ and Ag+ had higher sensitivity to gramicidin S than the initial wild type strain of E. coli B. It was found that in the threshold or higher doses the salts of Cu, Ag, Co and Cd increased the gramicidin S antimicrobial action on actively metabolizing cells of E. coli B. Analysis of the experimental data as well as the literature ones suggested that the synergic action of gramicidin S and the heavy metals stemmed from an increase in the cationic conductivity of the cytoplasma membrane modified by the metals in the threshold doses which induced an increase in the transport and accumulation of the cations in the bacterial cells by the electric field gradient (with the negative sign inside). Withdrawal of Ca2+ and Mg2+ from the E. coli outer structures into the cytoplasm impaired the barrier properties of the outer membrane and promoted binding of the gramicidin S cations to the liberated anionic groups of the E. coli outer structures and potentiation of the gramicidin S antimicrobial activity as was shown in our experiments.

  7. Lesch-Nyhan syndrome in an Indian family with novel mutation in the HPRT1 gene.

    PubMed

    Sharma, Suvasini; Jiménez, Rosa Torres; Aneja, Satinder; Garcia, Marta G; Sethi, Gulshan R

    2012-11-01

    The authors report two brothers who presented with motor delay and stiffness. The elder boy had auto-mutilation of lips and fingers. Serum uric acid was elevated in both the children. Both the boys had undetectable hypoxanthine-guanine phosphoribosyl transferase activity in hemolysate, confirming the diagnosis of Lesch-Nyhan syndrome. Molecular genetic testing revealed a new mutation in the HPRT1 gene.

  8. Alzheimer's disease shares gene expression aberrations with purinergic dysregulation of HPRT deficiency (Lesch-Nyhan disease).

    PubMed

    Kang, Tae Hyuk; Friedmann, Theodore

    2015-03-17

    Transcriptomic studies of murine D3 embryonic stem (ES) cells deficient in the purinergic biosynthetic function hypoxanthine guanine phosphoribosyltransferase (HPRT) and undergoing dopaminergic neuronal differentiation has demonstrated a marked shift from neuronal to glial gene expression and aberrant expression of multiple genes also known to be aberrantly expressed in Alzheimer's and other CNS disorders. Such genetic dysregulations may indicate some shared pathogenic metabolic mechanisms in diverse CNS diseases. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

  9. Increased mutant frequency and altered mutation spectrum of the lacI transgene in Wilson disease rats with hepatitis.

    PubMed

    Sone, H; Li, Y J; Ishizuka, M; Aoki, Y; Nagao, M

    2000-09-15

    The mutant strain Long-Evans Cinnamon (LEC) rat, which accumulates copper in the liver because of a mutation in the Atp7b gene, encoding a copper-ATPase, is a model of Wilson disease. It spontaneously develops hepatitis, and subsequently hepatocellular carcinoma and cholangiofibrosis. Excess intracellular copper has been thought to induce DNA damage through reactive oxygen species produced by Cu (II)/Cu (I) redox cycling, and also by direct interaction with DNA. We have developed lacI transgenic Wilson disease (WND-B) rats by mating LEC with Big Blue F344 rats carrying a lambda shuttle vector harboring the lacI gene. lacI mutations of the livers of C-B heterozygous (Atp7b w/m, lacI) and WND-B homozygous (Atp7b m/m, lacI) rats at 6, 24, and 40 weeks of ages were analyzed. Mutant frequencies in the WND-B rats were 2.0 +/- 0.7 x 10(-5), 5.3 +/- 0.9 x 10(-5), and 5.3 +/- 1.0 x 10(-5), respectively, significantly higher than those of C-B rats. Nucleotide sequence analysis revealed that the frequency of deletion mutations of more than two nucleotides were much higher, 15% in WND-B rats, but only 2% in C-B rats. In addition, the average size of deletion was larger in the former. Loss of oligonucleotide-repeat units was specific and relatively frequent in WND-B rats. This type of mutation might be implicated in the induction of DNA strand scissions by reactive oxygen species. These findings suggest that the increase in mutant frequencies and/or the specific type of mutation according to copper accumulation play a crucial role in hepatocarcinogenesis in LEC rats.

  10. Chronic hepatitis, hepatocyte fragility, and increased soluble phosphoglycokeratins in transgenic mice expressing a keratin 18 conserved arginine mutant

    PubMed Central

    1995-01-01

    The two major intermediate filament proteins in glandular epithelia are keratin polypeptides 8 and 18 (K8/18). To evaluate the function and potential disease association of K18, we examined the effects of mutating a highly conserved arginine (arg89) of K18. Expression of K18 arg89-->his/cys and its normal K8 partner in cultured cells resulted in punctate staining as compared with the typical filaments obtained after expression of wild-type K8/18. Generation of transgenic mice expressing human K18 arg89-->cys resulted in marked disruption of liver and pancreas keratin filament networks. The most prominent histologic abnormalities were liver inflammation and necrosis that appeared at a young age in association with hepatocyte fragility and serum transaminase elevation. These effects were caused by the mutation since transgenic mice expressing wild-type human K18 showed a normal phenotype. A relative increase in the phosphorylation and glycosylation of detergent solubilized K8/18 was also noted in vitro and in transgenic animals that express mutant K18. Our results indicate that the highly conserved arg plays an important role in glandular keratin organization and tissue fragility as already described for epidermal keratins. Phosphorylation and glycosylation alterations in the arg mutant keratins may account for some of the potential changes in the cellular function of these proteins. Mice expressing mutant K18 provide a novel animal model for human chronic hepatitis, and for studying the tissue specific function(s) of K8/18. PMID:8522591

  11. Prenatal diagnosis based on HPRT1 gene mutation in a Lesch-Nyhan family.

    PubMed

    Liu, N; Zhuo, Z-H; Wang, H-L; Kong, X-D; Shi, H-R; Wu, Q-H; Jiang, M

    2015-01-01

    We explored the feasibility of applying gene diagnosis in prenatal diagnosis by analysis of hypoxanthine-guanine phosphoribosyltransferase-1 (HPRT1) gene mutation in a Chinese Lesch-Nyhan family. A homozygous mutation of p.R170X (c.508C>T) in HPRT1 gene was detected in the proband, and a heterozygous mutation of p.R170X was detected in his mother. This mutation failed to be found in the 50 unrelated healthy individuals. Prenatal diagnosis indicated that the foetus was male and also carried p.R170X (c.508C>T) mutation, same as the proband. Parents of the foetus decided termination of pregnancy, and the result of gene analysis for the aborted tissue was consistent with that of prenatal diagnosis. We can see that Lesch-Nyhan syndrome (LNS) is caused by non-sense mutation p.R170X(c.508C>T)in HPRT1 gene in this family. Prenatal gene diagnosis is a valid strategy to prevent LNS because it can avoid the birth of LNS foetuses.

  12. Preferential repair and strand-specific repair of benzo[a]pyrene diol epoxide adducts in the HPRT gene of diploid human fibroblasts.

    PubMed Central

    Chen, R H; Maher, V M; Brouwer, J; van de Putte, P; McCormick, J J

    1992-01-01

    If excision repair-proficient human cells are allowed time for repair before onset of S phase, the premutagenic lesions formed by (+/-)-7 beta,8 alpha-dihydroxy-9 alpha,10 alpha-epoxy- 7,8,9,10-tetrahydrobenzo[a]pyrene (benzo[a]pyrene diol epoxide, BPDE) are lost from the transcribed strand of the hypoxanthine (guanine) phosphoribosyltransferase (HPRT) gene faster than from the nontranscribed strand. No change in strand distribution is seen with repair-deficient cells. These results suggest strand-specific repair of BPDE-induced DNA damage in human cells. To test this, we measured the initial number of BPDE adducts formed in each strand of the actively transcribed HPRT gene and the rate of repair, using UvrABC excinuclease in conjunction with Southern hybridization and strand-specific probes. We also measured the rate of loss of BPDE adducts from the inactive 754 locus. The frequencies of adducts formed by exposure to BPDE (1.0 or 1.2 microM) in either strand of a 20-kilobase fragment that lies entirely within the transcription unit of the HPRT gene were similar; the frequency in the 14-kilobase 754 fragment was approximately 20% lower. The rates of repair in the two strands of the HPRT fragment differed significantly. Within 7 hr after treatment with 1.2 microM BPDE, 53% of the adducts had been removed from the transcribed strand, but only 26% from the nontranscribed strand; after 20 hr, these values were 87% and 58%, respectively. In contrast, only approximately 14% of the BPDE adducts were lost from the 754 locus in 20 hr, a value even lower than the rate of loss from the overall genome (i.e., 38%). These results demonstrate strand-specific and preferential repair of BPDE adducts in human cells. They suggest that the heterogeneous repair of BPDE adducts in the human genome cannot be accounted for merely by the greatly increased rate of the repair specific to the transcribed strand of the active genes, and they point to a role for the chromatin structure. Images

  13. Yeast Cytosine Deaminase Mutants with Increased Thermostability Impart Sensitivity to 5-Fluorocytosine

    PubMed Central

    Stolworthy, Tiffany S.; Korkegian, Aaron M.; Willmon, Candice L.; Ardiani, Andressa; Cundiff, Jennifer; Stoddard, Barry L.; Black, Margaret E.

    2008-01-01

    SUMMARY Prodrug gene therapy (PGT) is a treatment strategy in which tumor cells are transfected with a 'suicide' gene that encodes a metabolic enzyme capable of converting a nontoxic prodrug into a potent cytotoxin. One of the most promising PGT enzymes is cytosine deaminase (CD), a microbial salvage enzyme that converts cytosine to uracil. CD also converts 5-fluorocytosine (5FC) to 5-fluorouracil (5FU), an inhibitor of DNA synthesis and RNA function. Over 150 studies of cytosine deaminase-mediated PGT applications have been reported since 2000, all using wild-type enzymes. However, various forms of cytosine deaminase are limited by inefficient turnover of 5FC and/or limited thermostability. In a previous study we stabilized and extended the half-life of yeast cytosine deaminase (yCD) by repacking of its hydrophobic core at several positions distant from the active site. Here we report that random mutagenesis of residues selected based on alignment with similar enzymes, followed by selection for enhanced sensitization to 5FC, also produces an enzyme variant (yCD-D92E) with elevated Tm values and increased activity half-life. The new mutation is located at the enzyme's dimer interface, indicating that independent mutational pathways can lead to an increase in the temperature that induces protein unfolding and aggregation in thermal denaturation experiments measured by circular dichroism spectroscopy, and an increase in the half-life of enzyme activity at physiological temperature, as well as more subtle effect on enzyme kinetics. Each independently derived set of mutations significantly improves the enzyme's performance in PGT assays both in cell culture and in animal models. PMID:18291415

  14. Yeast cytosine deaminase mutants with increased thermostability impart sensitivity to 5-fluorocytosine.

    PubMed

    Stolworthy, Tiffany S; Korkegian, Aaron M; Willmon, Candice L; Ardiani, Andressa; Cundiff, Jennifer; Stoddard, Barry L; Black, Margaret E

    2008-03-28

    Prodrug gene therapy (PGT) is a treatment strategy in which tumor cells are transfected with a 'suicide' gene that encodes a metabolic enzyme capable of converting a nontoxic prodrug into a potent cytotoxin. One of the most promising PGT enzymes is cytosine deaminase (CD), a microbial salvage enzyme that converts cytosine to uracil. CD also converts 5-fluorocytosine (5FC) to 5-fluorouracil, an inhibitor of DNA synthesis and RNA function. Over 150 studies of CD-mediated PGT applications have been reported since 2000, all using wild-type enzymes. However, various forms of CD are limited by inefficient turnover of 5FC and/or limited thermostability. In a previous study, we stabilized and extended the half-life of yeast CD (yCD) by repacking of its hydrophobic core at several positions distant from the active site. Here we report that random mutagenesis of residues selected based on alignment with similar enzymes, followed by selection for enhanced sensitization to 5FC, also produces an enzyme variant (yCD-D92E) with elevated T(m) values and increased activity half-life. The new mutation is located at the enzyme's dimer interface, indicating that independent mutational pathways can lead to an increase in stability, as well as a more subtle effect on enzyme kinetics. Each independently derived set of mutations significantly improves the enzyme's performance in PGT assays both in cell culture and in animal models.

  15. LLC-PK sub 1 mutant with increased Na sup + -H sup + exchange and decreased sensitivity to amiloride

    SciTech Connect

    Haggerty, J.G.; Agarwal, N.; Cragoe, E.J. Jr.; Adelberg, E.A.; Slayman, C.W. Merck Sharp Dohme Research Laboratories, West Point, PA )

    1988-10-01

    LLC-PK{sub 1} cells contain a well-characterized Na{sup +}-H{sup +} antiporter that is sensitive to ethylisopropylamiloride (EIPA) in the submicromolar range. Using a modification of the method of Franchi et al., the authors have selected mutants that can recover from an acid load in the presence of EIPA. One such mutant, designated PKE20, has been studied in detail. The maximal velocity (V{sub max}) for the Na{sup +}-H{sup +} antiporter, assayed as EIPA-sensitive {sup 22}Na{sup +} uptake, has increased from 44 to 106 nmol{center dot}min{sup {minus}1}{center dot}10{sup 6} cells{sup {minus}1} in PKE20. No detactable change has occurred in the K{sub m} for Na{sup +} or in the dependence of Na{sup +} uptake on intracellular pH. However, the PKE20 antiporter exhibits a greatly decreased sensitivity to amiloride and its derivatives, with drops in inhibitory potency ranging from 25-fold (amiloride) to 100-fold (EIPA). The mutation is specific for the antiporter; measurements of Na{sup +}-K{sup +} pump and Na{sup +}-dependent amino acid uptake show only small changes, which appear to result from minor antiporter-induced alterations in internal Na{sup +} concentration. PKE20 cells should prove useful in experiments to identify and isolate the antiporter protein.

  16. Terminal oxidase mutants of the cyanobacterium Synechocystis sp. PCC 6803 show increased electrogenic activity in biological photo-voltaic systems.

    PubMed

    Bradley, Robert W; Bombelli, Paolo; Lea-Smith, David J; Howe, Christopher J

    2013-08-28

    Biological photo-voltaic systems are a type of microbial fuel cell employing photosynthetic microbes at the anode, enabling the direct transduction of light energy to electrical power. Unlike the anaerobic bacteria found in conventional microbial fuel cells that use metals in the environment as terminal electron acceptors, oxygenic photosynthetic organisms are poorly adapted for electron transfer out of the cell. Mutant strains of the cyanobacterium Synechocystis sp. PCC 6803 were created in which all combinations of the three respiratory terminal oxidase complexes had been inactivated. These strains were screened for the ability to reduce the membrane-impermeable soluble electron acceptor ferricyanide, and the results were compared to the performance of the mutants in a biological photo-voltaic system. Deletion of the two thylakoid-localised terminal oxidases, the bd-quinol oxidase and cytochrome c oxidase resulted in a 16-fold increase in ferricyanide reduction rate in the dark compared to the wild-type. A further improvement to a 24-fold increase was seen upon deletion of the remaining "alternative respiratory terminal oxidase". These increases were reflected in the peak power generated in the biological photo-voltaic systems. Inactivation of all three terminal oxidase complexes resulted in a substantial redirection of reducing power; in the dark the equivalent of 10% of the respiratory electron flux was channelled to ferricyanide, compared to less than 0.2% in the wild-type. Only minor improvements in ferricyanide reduction rates over the wild-type were seen in illuminated conditions, where carbon dioxide is preferentially used as an electron sink. This study demonstrates the potential for optimising photosynthetic microbes for direct electrical current production.

  17. Increased longevity of some C. elegans mitochondrial mutants explained by activation of an alternative energy-producing pathway.

    PubMed

    Gallo, Marco; Park, Donha; Riddle, Donald L

    2011-10-01

    The Caenorhabditis elegans misc-1 gene encodes a mitochondrial carrier with a role in oxidative stress response. The knock-out mutant has no lifespan phenotype and fails to upregulate the gei-7-mediated glyoxylate shunt, an extra-mitochondrial pathway of energy production. We show that gei-7 is required for the longevity of the mitochondrial mutant clk-1. Our data suggest that only mitochondrial mutants that upregulate gei-7 can achieve longevity. Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.

  18. Data supporting the design and evaluation of a universal primer pair for pseudogene-free amplification of HPRT1 in real-time PCR.

    PubMed

    Valadan, Reza; Hedayatizadeh-Omran, Akbar; Alhosseini-Abyazani, Mahdyieh Naghavi; Amjadi, Omolbanin; Rafiei, Alireza; Tehrani, Mohsen; Alizadeh-Navaei, Reza

    2015-09-01

    Hypoxanthine-guanine phosphoribosyltransferase 1 (HPRT1) is a common housekeeping gene for sample normalization in the quantitative reverse transcriptase polymerase chain (qRT-PCR). However, co-amplification of HPRT1 pseudogenes may affect accurate results obtained in qRT-PCR. We designed a primer pair (HPSF) for pseudogene-free amplification of HPRT1 in qRT-PCR [1]. We showed specific amplification of HPRT1 mRNA in some common laboratory cell lines, including HeLa, NIH/3T3, CHO, BHK, COS-7 and VERO. This article provides data supporting the presence and location of HPRT1 pseudogenes within human and mouse genome, and the strategies used for designing primers that avoid the co-amplification of contaminating pseudogenes in qRT-PCR. In silico analysis of human genome showed three homologous sequences for HPRT1 on chromosomes 4, 5 and 11. The mRNA sequence of HPRT1 was aligned with the pseudogenes, and the primers were designed toward 5' end of HPRT1 mRNA that was only specific to HPRT1 mRNA not to the pseudogenes. The standard curve plot generated by HPSF primers showed the correlation coefficient of 0.999 and the reaction efficiency of 99.5%. Our findings suggest that HPSF primers can be recommended as a candidate primer pair for accurate and reproducible qRT-PCR assays.

  19. Mild Lesch-Nyhan Disease in a Boy with a Null Mutation in HPRT1: An Exception to the Known Genotype-Phenotype Correlation.

    PubMed

    Bayat, Allan; Christensen, Mette; Wibrand, Flemming; Duno, Morten; Lund, Allan

    2015-01-01

    Hypoxanthine-guanine phosphoribosyltransferase (HPRT) deficiency results in a continuous spectrum of clinical phenotypes though all include overproduction of uric acid with hyperuricaemia, urate nephrolithiasis and gout. HPRT1 mutations that result in very low or no HPRT enzyme activities are generally associated with the classic Lesch-Nyhan disease (LND) phenotype with intellectual disability, motor handicap and self-injurious behaviour. Mutations that permit a higher residual HPRT activity are seen in some patients with the milder LND variant phenotypes with varying degrees of cognitive, motor handicap and maladaptive behaviour without recurrent self-injury. We present a boy with a LND variant phenotype due to a deletion of exon 5 of HPRT1 predicted to fully abolish HPRT activity. Metabolic analysis confirms lack of significant residual enzyme activity. The boy, currently age 10, presented with hyperuricaemia, hypotonia, developmental delay and extrapyramidal and pyramidal involvement. He has never shown any signs of self-injurious or maladaptive behaviour. This boy is one of the rare cases with a suspected null mutation in HPRT1 that associates with a milder than expected phenotype with lack of self-injurious behaviour. Key Clinical Message HPRT1 mutations that result in very low or no hypoxanthine-guanine phosphoribosyltransferase enzyme activities are generally associated with the classic Lesch-Nyhan disease. This report presents one of the rare cases with a null mutation in the HPRT1 gene that associates with a milder than expected phenotype with lack of self-injurious behaviour.

  20. Data supporting the design and evaluation of a universal primer pair for pseudogene-free amplification of HPRT1 in real-time PCR

    PubMed Central

    Valadan, Reza; Hedayatizadeh-Omran, Akbar; Alhosseini-Abyazani, Mahdyieh Naghavi; Amjadi, Omolbanin; Rafiei, Alireza; Tehrani, Mohsen; Alizadeh-Navaei, Reza

    2015-01-01

    Hypoxanthine-guanine phosphoribosyltransferase 1 (HPRT1) is a common housekeeping gene for sample normalization in the quantitative reverse transcriptase polymerase chain (qRT-PCR). However, co-amplification of HPRT1 pseudogenes may affect accurate results obtained in qRT-PCR. We designed a primer pair (HPSF) for pseudogene-free amplification of HPRT1 in qRT-PCR [1]. We showed specific amplification of HPRT1 mRNA in some common laboratory cell lines, including HeLa, NIH/3T3, CHO, BHK, COS-7 and VERO. This article provides data supporting the presence and location of HPRT1 pseudogenes within human and mouse genome, and the strategies used for designing primers that avoid the co-amplification of contaminating pseudogenes in qRT-PCR. In silico analysis of human genome showed three homologous sequences for HPRT1 on chromosomes 4, 5 and 11. The mRNA sequence of HPRT1 was aligned with the pseudogenes, and the primers were designed toward 5′ end of HPRT1 mRNA that was only specific to HPRT1 mRNA not to the pseudogenes. The standard curve plot generated by HPSF primers showed the correlation coefficient of 0.999 and the reaction efficiency of 99.5%. Our findings suggest that HPSF primers can be recommended as a candidate primer pair for accurate and reproducible qRT-PCR assays. PMID:26217821

  1. Gene expression and mRNA editing of serotonin receptor 2C in brains of HPRT gene knock-out mice, an animal model of Lesch-Nyhan disease

    PubMed Central

    Bertelli, Matteo; Alushi, Brunilda; Veicsteinas, Arsenio; Jinnah, H.A.; Micheli, Vanna

    2016-01-01

    Lesch-Nyhan disease (LND), a genetic disorder associated with motor and psychiatric disturbance and self-injurious behaviour (SIB) is caused by a complete deficiency of hypoxanthine-guanine phosphoribosyltransferase (HPRT). The connection between enzyme deficiency and neurological involvement is still unclear. Evidence exists for a role of basal ganglia dysfunction with decreased dopamine and excess serotonin striatal content. In this study, we investigate the role of serotonin receptor 2C (HTR2C) in the brains of HPRT gene knock-out mice, a model of LND. HTR2C expression is analyzed by real-time polymerase chain reaction (PCR) using SYBR-green detection methods. The percentage of edited HTR2C mRNA was determined by direct sequencing of amplification products of the region containing the editing sites. We found a 55% increase in the expression of HTR2C gene but no significant difference in mRNA editing levels between knock-out and control mice. The above alteration found in HPRT-deficient mice is similar to those found in other animal models used to study aggressive and self-injurious behaviour. PMID:19473847

  2. Combined preconditioning and in vivo chemoselection with 6-thioguanine alone achieves highly efficient reconstitution of normal hematopoiesis with HPRT-deficient bone marrow

    PubMed Central

    Hacke, Katrin; Szakmary, Akos; Cuddihy, Andrew R; Rozengurt, Nora; Lemp, Nathan A; Aubrecht, Jiri; Lawson, Gregory W; Rao, Nagesh P; Crooks, Gay M; Schiestl, Robert H.; Kasahara, Noriyuki

    2014-01-01

    Purine analogs such as 6-thioguanine (6TG) cause myelotoxicity upon conversion into nucleotides by hypoxanthine-guanine phosphoribosyltransferase (HPRT). Here we have developed a novel and highly efficient strategy employing 6TG as a single agent for both conditioning and in vivo chemoselection of HPRT-deficient HSC. The dose-response and time course of 6TG myelotoxicity were first compared in HPRT-wild type mice and HPRT-deficient transgenic mice. Dosage and schedule parameters were optimized to employ 6TG for myelo-suppressive conditioning, immediately followed by in vivo chemoselection of HPRT-deficient transgenic donor bone marrow (BM) transplanted into syngeneic HPRT-wild type recipients. At appropriate doses, 6TG induced selective myelotoxicity without any adverse effects on extra-hematopoietic tissues in HPRT-wild type mice, while HSC deficient in HPRT activity were highly resistant to its cytotoxic effects. Combined 6TG conditioning and post transplant chemoselection consistently achieved ~95% engraftment of HPRT-deficient donor BM, with low overall toxicity. Long-term reconstitution of immunophenotypically normal BM was achieved in both primary and secondary recipients. Our results provide proof-of-concept that single-agent 6TG can be used both for myelo-suppressive conditioning without requiring irradiation, and for in vivo chemoselection of HPRT-deficient donor cells. Our results show that by applying the myelosuppressive effects of 6TG both before (as conditioning) and after transplantation (as chemoselection), highly efficient engraftment of HPRT-deficient hematopoietic stem cells can be achieved. PMID:22001673

  3. Inhibition of erythrocyte phosphoribosyltransferases (APRT and HPRT) by Pb2+: a potential mechanism of lead toxicity.

    PubMed

    Baranowska-Bosiacka, I; Dziedziejko, V; Safranow, K; Gutowska, I; Marchlewicz, M; Dołegowska, B; Rać, M E; Wiszniewska, B; Chlubek, D

    2009-05-02

    Many reports show that red blood cells of people exposed to lead have a decreased ATP concentration, decreased adenylate energy charge value and many metabolic and morphological abnormalities. Since the synthesis of nucleotides in erythrocytes occurs only through salvage pathways, we hypothesized that a decrease in nucleotide concentrations may be caused by lead-induced inhibition of erythrocyte phosphoribosyltransferases: adenine APRT (EC 2.4.2.7) and hypoxanthine-guanine HPRT (EC 2.4.2.8). These enzymes enable the reutilization of purine bases (adenine, guanine, hypoxanthine) converting them to mononucleotides (AMP, GMP, IMP), substrates for the synthesis of high-energy nucleotides. To confirm the hypothesis two experiments were performed: (i) in vitro, using a lysate of human erythrocytes incubated (5, 10, 30min) with lead ions (100microM, 10microM, 1microM, 500nM, 100nM lead acetate) and 100microM sodium acetate for the control, (ii) in vivo, using a lysate of rat erythrocytes taken from rats chronically exposed to lead (0.1% lead acetate in drinking water for 9 months, resulting in whole blood lead concentration 7microg/dL). The activities of APRT and HPRT were determined using HPLC method, which allowed concurrent determination of the activity of both enzymes in erythrocyte lysates. We have shown that, lead ions: (i) moderately inhibit both phosphoribosyltransferases in erythrocytes, this influence being detectable even at very low concentrations (ii) participate in hemolysis, the intensity of which negatively correlates with the activity of phosphoribosyltransferases. Our results indicate the necessity of further research on the role of lead-induced APRT and HPRT inhibition as one of the mechanisms of lead toxicity.

  4. Loss of cargo binding in the human myosin VI deafness mutant (R1166X) leads to increased actin filament binding

    PubMed Central

    Arden, Susan D.; Tumbarello, David A.; Butt, Tariq; Kendrick-Jones, John; Buss, Folma

    2016-01-01

    Mutations in myosin VI have been associated with autosomal-recessive (DFNB37) and autosomal-dominant (DFNA22) deafness in humans. Here, we characterise an myosin VI nonsense mutation (R1166X) that was identified in a family with hereditary hearing loss in Pakistan. This mutation leads to the deletion of the C-terminal 120 amino acids of the myosin VI cargo-binding domain, which includes the WWY-binding motif for the adaptor proteins LMTK2, Tom1 as well as Dab2. Interestingly, compromising myosin VI vesicle-binding ability by expressing myosin VI with the R1166X mutation or with single point mutations in the adaptor-binding sites leads to increased F-actin binding of this myosin in vitro and in vivo. As our results highlight the importance of cargo attachment for regulating actin binding to the motor domain, we perform a detailed characterisation of adaptor protein binding and identify single amino acids within myosin VI required for binding to cargo adaptors. We not only show that the adaptor proteins can directly interact with the cargo-binding tail of myosin VI, but our in vitro studies also suggest that multiple adaptor proteins can bind simultaneously to non-overlapping sites in the myosin VI tail. In conclusion, our characterisation of the human myosin VI deafness mutant (R1166X) suggests that defects in cargo binding may leave myosin VI in a primed/activated state with an increased actin-binding ability. PMID:27474411

  5. In comparison with nitrate nutrition, ammonium nutrition increases growth of the frostbite1 Arabidopsis mutant.

    PubMed

    Podgórska, Anna; Ostaszewska, Monika; Gardeström, Per; Rasmusson, Allan G; Szal, Bożena

    2015-01-01

    Ammonium nutrition inhibits the growth of many plant species, including Arabidopsis thaliana. The toxicity of ammonium is associated with changes in the cellular redox state. The cellular oxidant/antioxidant balance is controlled by mitochondrial electron transport chain. In this study, we analysed the redox metabolism of frostbite1 (fro1) plants, which lack mitochondrial respiratory chain complex I. Surprisingly, the growth of fro1 plants increased under ammonium nutrition. Ammonium nutrition increased the reduction level of pyridine nucleotides in the leaves of wild-type plants, but not in the leaves of fro1 mutant plants. The observed higher activities of type II NADH dehydrogenases and cytochrome c oxidase in the mitochondrial electron transport chain may improve the energy metabolism of fro1 plants grown on ammonium. Additionally, the observed changes in reactive oxygen species (ROS) metabolism in the apoplast may be important for determining the growth of fro1 under ammonium nutrition. Moreover, bioinformatic analyses showed that the gene expression changes in fro1 plants significantly overlap with the changes previously observed in plants with a modified apoplastic pH. Overall, the results suggest a pronounced connection between the mitochondrial redox system and the apoplastic pH and ROS levels, which may modify cell wall plasticity and influence growth. © 2014 John Wiley & Sons Ltd.

  6. Cell homeostasis in a Leishmania major mutant overexpressing the spliced leader RNA is maintained by an increased proteolytic activity.

    PubMed

    Toledo, Juliano S; Ferreira, Tiago R; Defina, Tânia P A; Dossin, Fernando de M; Beattie, Kenneth A; Lamont, Douglas J; Cloutier, Serge; Papadopoulou, Barbara; Schenkman, Sergio; Cruz, Angela K

    2010-10-01

    Although several stage-specific genes have been identified in Leishmania, the molecular mechanisms governing developmental gene regulation in this organism are still not well understood. We have previously reported an attenuation of virulence in Leishmania major and L. braziliensis carrying extra-copies of the spliced leader RNA gene. Here, we surveyed the major differences in proteome and transcript expression profiles between the spliced leader RNA overexpressor and control lines using two-dimensional gel electrophoresis and differential display reverse transcription PCR, respectively. Thirty-nine genes related to stress response, cytoskeleton, proteolysis, cell cycle control and proliferation, energy generation, gene transcription, RNA processing and post-transcriptional regulation have abnormal patterns of expression in the spliced leader RNA overexpressor line. The evaluation of proteolytic pathways in the mutant revealed a selective increase of cysteine protease activity and an exacerbated ubiquitin-labeled protein population. Polysome profile analysis and measurement of cellular protein aggregates showed that protein translation in the spliced leader RNA overexpressor line is increased when compared to the control line. We found that L. major promastigotes maintain homeostasis in culture when challenged with a metabolic imbalance generated by spliced leader RNA surplus through modulation of intracellular proteolysis. However, this might interfere with a fine-tuned gene expression control necessary for the amastigote multiplication in the mammalian host.

  7. Increased vulnerability of hippocampal neurons from presenilin-1 mutant knock-in mice to amyloid beta-peptide toxicity: central roles of superoxide production and caspase activation.

    PubMed

    Guo, Q; Sebastian, L; Sopher, B L; Miller, M W; Ware, C B; Martin, G M; Mattson, M P

    1999-03-01

    Many cases of early-onset inherited Alzheimer's disease (AD) are caused by mutations in the presenilin-1 (PS1) gene. Overexpression of PS1 mutations in cultured PC12 cells increases their vulnerability to apoptosis-induced trophic factor withdrawal and oxidative insults. We now report that primary hippocampal neurons from PS1 mutant knock-in mice, which express the human PS1M146V mutation at normal levels, exhibit increased vulnerability to amyloid beta-peptide toxicity. The endangering action of mutant PS1 was associated with increased superoxide production, mitochondrial membrane depolarization, and caspase activation. The peroxynitrite-scavenging antioxidant uric acid and the caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone protected hippocampal neurons expressing mutant PS1 against cell death induced by amyloid beta-peptide. Increased oxidative stress may contribute to the pathogenic action of PS1 mutations, and antioxidants may counteract the adverse property of such AD-linked mutations.

  8. Determination of Activity of the Enzymes Hypoxanthine Phosphoribosyl Transferase (HPRT) and Adenine Phosphoribosyl Transferase (APRT) in Blood Spots on Filter Paper.

    PubMed

    Auler, Kasie; Broock, Robyn; Nyhan, William L

    2015-07-01

    Hypoxanthine-guanine phosphoribosyl-transferase (HPRT) deficiency is the cause of Lesch-Nyhan disease. Adenine phosphoribosyl-transferase (APRT) deficiency causes renal calculi. The activity of each enzyme is readily determined on spots of whole blood on filter paper. This unit describes a method for detecting deficiencies of HPRT and APRT. Copyright © 2015 John Wiley & Sons, Inc.

  9. Lesch-Nyhan Syndrome in a Family with a Deletion Followed by an Insertion within the HPRT1 Gene.

    PubMed

    Nguyen, Khue Vu; Nyhan, William L

    2015-01-01

    Lesch-Nyhan syndrome (LNS) is a rare X-linked inherited neurogenetic disorder of purine metabolism in which the enzyme, hypoxanthine-guanine phosphoribosyltransferase(HGprt) is defective. The authors report a novel mutation which led to LNS in a family with a deletion followed by an insertion (INDELS) via the serial replication slippage mechanism: c.428_432delTGCAGinsAGCAAA, p.Met143Lysfs*12 in exon 6 of HPRT1 gene. Molecular diagnosis discloses the genetic heterogeneity of HPRT1 gene responsible for HGprt deficiency. It allows fast, accurate carrier detection and genetic counseling.

  10. Quantitative dynamics and increased variability of segmentation gene expression in the Drosophila Krüppel and knirps mutants.

    PubMed

    Surkova, Svetlana; Golubkova, Elena; Manu; Panok, Lena; Mamon, Lyudmila; Reinitz, John; Samsonova, Maria

    2013-04-01

    Here we characterize the response of the Drosophila segmentation system to mutations in two gap genes, Kr and kni, in the form of single or double homozygotes and single heterozygotes. Segmentation gene expression in these genotypes was quantitatively monitored with cellular resolution in space and 6.5 to 13min resolution in time. As is the case with wild type, we found that gene expression domains in the posterior portion of the embryo shift to the anterior over time. In certain cases, such as the gt posterior domain in Kr mutants, the shifts are significantly larger than is seen in wild type embryos. We also investigated the effects of Kr and kni on the variability of gene expression. Mutations often produce variable phenotypes, and it is well known that the cuticular phenotype of Kr mutants is variable. We sought to understand the molecular basis of this effect. We find that throughout cycle 14A the relative levels of eve and ftz expression in stripes 2 and 3 are variable among individual embryos. Moreover, in Kr and kni mutants, unlike wild type, the variability in positioning of the posterior Hb domain and eve stripe 7 is not decreased or filtered with time. The posterior Gt domain in Kr mutants is highly variable at early times, but this variability decreases when this domain shifts in the anterior direction to the position of the neighboring Kni domain. In contrast to these findings, positional variability throughout the embryo does not decrease over time in double Kr;kni mutants. In heterozygotes the early expression patterns of segmentation genes resemble patterns seen in homozygous mutants but by the onset of gastrulation they become similar to the wild type patterns. Finally, we note that gene expression levels are reduced in Kr and kni mutant embryos and have a tendency to decrease over time. This is a surprising result in view of the role that mutual repression is thought to play in the gap gene system.

  11. High-LET Patterns of DSBs in DNA Loops, the HPRT Gene and Phosphorylation Foci

    NASA Technical Reports Server (NTRS)

    Ponomarev, Artem L.; Huff, Janice L.; Cucinotta, Francis A.

    2007-01-01

    We present new results obtained with our model based on the track structure and chromatin geometry that predicts the DSB spatial and genomic distributions in a cell nucleus with the full genome represented. The model generates stochastic patterns of DSBs in the physical space of the nucleus filled with the realistic configuration of human chromosomes. The model was re-used to find the distribution of DSBs in a physical volume corresponding to a visible phosphorylation focus believed to be associated with a DSB. The data shows whether there must more than one DSB per foci due to finite size of the visible focus, even if a single DSB is radiochemically responsible for the phosphorylation of DNA in its vicinity. The same model can predict patterns of closely located DSBs in a given gene, or in a DNA loop, one of the large-scale chromatin structures. We demonstrated for the example of the HPRT gene, how different sorts of radiation lead to proximity effect in DSB locations, which is important for modeling gene deletions. The spectrum of intron deletions and total gene deletions was simulated for the HPRT gene. The same proximity effect of DSBs in a loop can hinder DSB restitutions, as parts of the loop between DSBs is deleted with a higher likelihood. The distributions of DSBs and deletions of DNA in a loop are presented.

  12. High-LET Patterns of DSBs in DNA Loops, the HPRT Gene and Phosphorylation Foci

    NASA Technical Reports Server (NTRS)

    Ponomarev, Artem L.; Huff, Janice L.; Cucinotta, Francis A.

    2007-01-01

    We present new results obtained with our model based on the track structure and chromatin geometry that predicts the DSB spatial and genomic distributions in a cell nucleus with the full genome represented. The model generates stochastic patterns of DSBs in the physical space of the nucleus filled with the realistic configuration of human chromosomes. The model was re-used to find the distribution of DSBs in a physical volume corresponding to a visible phosphorylation focus believed to be associated with a DSB. The data shows whether there must more than one DSB per foci due to finite size of the visible focus, even if a single DSB is radiochemically responsible for the phosphorylation of DNA in its vicinity. The same model can predict patterns of closely located DSBs in a given gene, or in a DNA loop, one of the large-scale chromatin structures. We demonstrated for the example of the HPRT gene, how different sorts of radiation lead to proximity effect in DSB locations, which is important for modeling gene deletions. The spectrum of intron deletions and total gene deletions was simulated for the HPRT gene. The same proximity effect of DSBs in a loop can hinder DSB restitutions, as parts of the loop between DSBs is deleted with a higher likelihood. The distributions of DSBs and deletions of DNA in a loop are presented.

  13. Increased misfolding and truncation of tau in APP/PS1/tau transgenic mice compared to mutant tau mice.

    PubMed

    Héraud, Céline; Goufak, Doris; Ando, Kunie; Leroy, Karelle; Suain, Valérie; Yilmaz, Zehra; De Decker, Robert; Authelet, Michèle; Laporte, Vincent; Octave, Jean-Noël; Brion, Jean-Pierre

    2014-02-01

    Neurofibrillary degeneration in transgenic models of tauopathies has been observed to be enhanced when these models are crossed with transgenic models developing an Aβ pathology. The mechanisms leading to this enhanced tau pathology are not well understood. We have performed a detailed analysis of tau misprocessing in a new transgenic mouse model combining APP, PS1 and tau mutations (5xFAD×Tg30 mice) by comparison with littermates expressing only a FTD mutant tau (Tg30 mice). These 5xFAD×Tg30 mice showed a more severe deficient motor phenotype than Tg30 mice and developed with age a dramatically accelerated NFT load in the brain compared to Tg30 mice. Insoluble tau in 5xFAD×Tg30 mice compared to insoluble tau in Tg30 mice showed increased phosphorylation, enhanced misfolding and truncation changes mimicking more closely the post-translational changes characteristic of PHF-tau in Alzheimer's disease. Endogenous wild-type mouse tau was recruited at much higher levels in insoluble tau in 5xFAD×Tg30 than in Tg30 mice. Extracellular amyloid load, Aβ40 and Aβ42, β-CTFs and β-CTF phosphorylation levels were lower in 5xFAD×Tg30 mice than in 5xFAD mice. Despite this reduction of Aβ, a significant hippocampal neuronal loss was observed in 5xFAD×Tg30 but not in 5xFAD mice indicating its closer association with increased tau pathology. This 5xFAD×Tg30 model thus mimics more faithfully tau pathology and neuronal loss observed in AD and suggests that additional post-translational changes in tau and self-recruitment of endogenous tau drive the enhanced tau pathology developing in the presence of Aβ pathology. © 2013.

  14. Increased prevalence of a rare mutant of pandemic H1N1 influenza virus in a Eurasian region.

    PubMed

    Yang, Ting-Ting; Wang, Zhao-Guo; Li, Shan-Peng; Liu, Xiao-Lin; Yi, Ying; Yang, Yu; Yu, Ping; Chen, Ji-Ming

    2011-01-01

    In 2009, a novel swine-origin H1N1 influenza virus sparked an influenza pandemic. The emergence of mutations in the viral genome is therefore of ongoing concern. In this study, the hemagglutinin (HA) gene sequences of 3444 pandemic H1N1 influenza viruses reported to the GenBank database and the sequences of 48 pandemic H1N1 influenza viruses detected in the Chinese city of Qingdao were analyzed. Among the 3492 viruses, 101 carried a serine to proline substitution at position 128 (S128P) in the viral HA gene. All the 101 S128P mutants belonged to Clade 7 which has become dominant worldwide since the summer of 2009. Among the 3492 viruses, 1646 were collected before July 25, 2009, and none of these viruses carried the S128P mutation. Furthermore, after July 25, 2009, the prevalence of the S128P mutant was 33.56% (99/295) in a region of Eurasia including Russia, Mongolia, mainland China and South Korea, but only 0.11% (2/1846) in the rest of the world. The data suggested that the originally rare S128P mutant has become prevalent in the Eurasia region, indicating that the S128P mutant likely transmitted more efficiently than other strains of the virus. Therefore, it is of significance to observe whether the S128P mutant will be more dominant worldwide in the coming future and investigate the exact effects of the S128P mutation.

  15. Melanization of a meristematic mutant of Fonsecaea monophora increases tolerance to stress factors while no effects on antifungal susceptibility.

    PubMed

    Sun, Jiufeng; Zhang, Junmin; Najafzadeh, M J; Badali, Hamid; Li, Xiqing; Xi, Liyan; de Hoog, G S

    2011-11-01

    Melanin is a complex polymer, which is widely distributed in nature, and is known as an important virulence factor in opportunistic and pathogenic fungi. In this study, three melanin mutants of Fonsecaea monophora from a case of chromoblastomycosis were generated from a parent strain that lacked hyphal morphology but was meristematic instead. Two albino mutants, one of which (CBS 125187) produced secreted melanin and another (CBS 125149) lacked melanin, grew faster than a mutant with cell-wall-associated and secreted melanin (CBS 125188) and than the meristematic parent strain (CBS 122845) (P < 0.05). The albino strains were also more sensitive to low pH, high UV radiation, and oxidative stress (P < 0.05). However, susceptibility testing against eight antifungal agents showed no statistical difference (P > 0.05). The discovery of three melanin mutants of a single meristematic mutant provided an alternative way to study the role of cell-wall-associated and secreted melanins in the pathogenesis of black fungi.

  16. Stringent mating-type-regulated auxotrophy increases the accuracy of systematic genetic interaction screens with Saccharomyces cerevisiae mutant arrays.

    PubMed

    Singh, Indira; Pass, Rebecca; Togay, Sine Ozmen; Rodgers, John W; Hartman, John L

    2009-01-01

    A genomic collection of haploid Saccharomyces cerevisiae deletion strains provides a unique resource for systematic analysis of gene interactions. Double-mutant haploid strains can be constructed by the synthetic genetic array (SGA) method, wherein a query mutation is introduced by mating to mutant arrays, selection of diploid double mutants, induction of meiosis, and selection of recombinant haploid double-mutant progeny. The mechanism of haploid selection is mating-type-regulated auxotrophy (MRA), by which prototrophy is restricted to a particular haploid genotype generated only as a result of meiosis. MRA escape leads to false-negative genetic interaction results because postmeiotic haploids that are supposed to be under negative selection instead proliferate and mate, forming diploids that are heterozygous at interacting loci, masking phenotypes that would be observed in a pure haploid double-mutant culture. This work identified factors that reduce MRA escape, including insertion of terminator and repressor sequences upstream of the MRA cassette, deletion of silent mating-type loci, and utilization of alpha-type instead of a-type MRA. Modifications engineered to reduce haploid MRA escape reduced false negative results in SGA-type analysis, resulting in >95% sensitivity for detecting gene-gene interactions.

  17. Isolation and Characterization of Clostridium butyricum DSM 5431 Mutants with Increased Resistance to 1,3-Propanediol and Altered Production of Acids

    PubMed Central

    Abbad-Andaloussi, S.; Manginot-Durr, C.; Amine, J.; Petitdemange, E.; Petitdemange, H.

    1995-01-01

    Clostridium butyricum mutants were isolated from the parent strain DSM 5431 after mutagenesis with N-methyl-N(prm1)-nitro-N-nitrosoguanidine and two selection procedures: osmotic pressure and the proton suicide method. Isolated mutants were more resistant to glycerol and to 1,3-propanediol (1,3-PD) than was the wild type, and they produced more biomass. In batch culture on 62 g of glycerol per liter, the wild type produced more acetic acid than butyrate, with an acetate/butyrate ratio of 5.0, whereas the mutants produced almost the same quantities of both acids or more butyrate than acetate with acetate/butyrate ratios from 0.6 to 1.1. The total acid formation was higher in the wild-type strain. Results of analysis of key metabolic enzymatic activities were in accordance with the pattern of fermentation product formation: either the butyrate kinase activity increased or the acetate kinase activity decreased in cell extracts of the mutants. A decreased level of the hydrogenase and NADH-ferredoxin activities concomitant with an increase in ferredoxin-NAD(sup+) reductase activities supports the conclusion that the maximum percentage of NADH available and used for the formation of 1,3-PD was higher for the mutants (97 to 100%) than for the wild type (70%). In fed-batch culture, at the end of the fermentation (72 h for the wild-type strain and 80 to 85 h for the mutants), 44% more glycerol was consumed and 50% more 1,3-PD was produced by the mutants than by the wild-type strain. PMID:16535195

  18. Novel Escherichia coli RF1 mutants with decreased translation termination activity and increased sensitivity to the cytotoxic effect of the bacterial toxins Kid and RelE.

    PubMed

    Diago-Navarro, Elizabeth; Mora, Liliana; Buckingham, Richard H; Díaz-Orejas, Ramón; Lemonnier, Marc

    2009-01-01

    Novel mutations in prfA, the gene for the polypeptide release factor RF1 of Escherichia coli, were isolated using a positive genetic screen based on the parD (kis, kid) toxin-antitoxin system. This original approach allowed the direct selection of mutants with altered translational termination efficiency at UAG codons. The isolated prfA mutants displayed a approximately 10-fold decrease in UAG termination efficiency with no significant changes in RF1 stability in vivo. All three mutations, G121S, G301S and R303H, were situated close to the nonsense codon recognition site in RF1:ribosome complexes. The prfA mutants displayed increased sensitivity to the RelE toxin encoded by the relBE system of E. coli, thus providing in vivo support for the functional interaction between RF1 and RelE. The prfA mutants also showed increased sensitivity to the Kid toxin. Since this toxin can cleave RNA in a ribosome-independent manner, this result was not anticipated and provided first evidence for the involvement of RF1 in the pathway of Kid toxicity. The sensitivity of the prfA mutants to RelE and Kid was restored to normal levels upon overproduction of the wild-type RF1 protein. We discuss these results and their utility for the design of novel antibacterial strategies in the light of the recently reported structure of ribosome-bound RF1.

  19. Novel Escherichia coli RF1 mutants with decreased translation termination activity and increased sensitivity to the cytotoxic effect of the bacterial toxins Kid and RelE

    PubMed Central

    Diago-Navarro, Elizabeth; Mora, Liliana; Buckingham, Richard H; Díaz-Orejas, Ramón; Lemonnier, Marc

    2008-01-01

    Novel mutations in prfA, the gene for the polypeptide release factor RF1 of Escherichia coli, were isolated using a positive genetic screen based on the parD (kis, kid) toxin–antitoxin system. This original approach allowed the direct selection of mutants with altered translational termination efficiency at UAG codons. The isolated prfA mutants displayed a ∼10-fold decrease in UAG termination efficiency with no significant changes in RF1 stability in vivo. All three mutations, G121S, G301S and R303H, were situated close to the nonsense codon recognition site in RF1:ribosome complexes. The prfA mutants displayed increased sensitivity to the RelE toxin encoded by the relBE system of E. coli, thus providing in vivo support for the functional interaction between RF1 and RelE. The prfA mutants also showed increased sensitivity to the Kid toxin. Since this toxin can cleave RNA in a ribosome-independent manner, this result was not anticipated and provided first evidence for the involvement of RF1 in the pathway of Kid toxicity. The sensitivity of the prfA mutants to RelE and Kid was restored to normal levels upon overproduction of the wild-type RF1 protein. We discuss these results and their utility for the design of novel antibacterial strategies in the light of the recently reported structure of ribosome-bound RF1. PMID:19019162

  20. The lack of mitochondrial complex I in a CMSII mutant of Nicotiana sylvestris increases photorespiration through an increased internal resistance to CO2 diffusion.

    PubMed

    Priault, P; Tcherkez, G; Cornic, G; De Paepe, R; Naik, R; Ghashghaie, J; Streb, P

    2006-01-01

    The cytoplasmic male sterile II (CMSII) mutant lacking complex I of the mitochondrial electron transport chain has a lower photosynthetic activity but exhibits higher rates of excess electron transport than the wild type (WT) when grown at high light intensity. In order to examine the cause of the lower photosynthetic activity and to determine whether excess electrons are consumed by photorespiration, light, and intercellular CO(2), molar fraction (c(i)) response curves of carbon assimilation were measured at varying oxygen molar fractions. While oxygen is the major acceptor for excess electrons in CMSII and WT leaves, electron flux to photorespiration is favoured in the mutant as compared with the WT leaves. Isotopic mass spectrometry measurements showed that leaf internal conductance to CO(2) diffusion (g(m)) in mutant leaves was half that of WT leaves, thus decreasing the c(c) and favouring photorespiration in the mutant. The specificity factor of Rubisco did not differ significantly between both types of leaves. Furthermore, carbon assimilation as a function of electrons used for carboxylation processes/electrons used for oxygenation processes (J(C)/J(O)) and as a function of the calculated chloroplastic CO(2) molar fraction (c(c)) values was similar in WT and mutant leaves. Enhanced rates of photorespiration also explain the consumption of excess electrons in CMSII plants and agreed with potential ATP consumption. Furthermore, the lower initial Rubisco activity in CMSII as compared with WT leaves resulted from the lower c(c) in ambient air, since initial Rubisco activity on the basis of equal c(c) values was similar in WT and mutant leaves. The retarded growth and the lower photosynthetic activity of the mutant were largely overcome when plants were grown in high CO(2) concentrations, showing that limiting CO(2) supply for photosynthesis was a major cause of the lower growth rate and photosynthetic activity in CMSII.

  1. Analysis of triclosan-selected Salmonella enterica mutants of eight serovars revealed increased aminoglycoside susceptibility and reduced growth rates.

    PubMed

    Rensch, Ulrike; Klein, Guenter; Kehrenberg, Corinna

    2013-01-01

    The biocide triclosan (TRC) is used in a wide range of household, personal care, veterinary, industrial and medical products to control microbial growth. This extended use raises concerns about a possible association between the application of triclosan and the development of antibiotic resistance. In the present study we determined triclosan mutant prevention concentrations (MPC) for Salmonella enterica isolates of eight serovars and investigated selected mutants for their mechanisms mediating decreased susceptibility to triclosan. MPCTRC values were 8-64-fold higher than MIC values and ranged between 1-16 µg/ml. The frequencies at which mutants were selected varied between 1.3 x 10(-10)-9.9 x 10(-11). Even if MIC values of mutants decreased by 3-7 dilution steps in the presence of the efflux pump inhibitor Phe-Arg-β-naphtylamide, only minor changes were observed in the expression of genes encoding efflux components or regulators, indicating that neither the major multidrug efflux pump AcrAB-TolC nor AcrEF are up-regulated in triclosan-selected mutants. Nucleotide sequence comparisons confirmed the absence of alterations in the regulatory regions acrRA, soxRS, marORAB, acrSE and ramRA of selected mutants. Single bp and deduced Gly93→Val amino acid exchanges were present in fabI, the target gene of triclosan, starting from a concentration of 1 µg/ml TRC used for MPC determinations. The fabI genes were up to 12.4-fold up-regulated. Complementation experiments confirmed the contribution of Gly93→Val exchanges and fabI overexpression to decreased triclosan susceptibility. MIC values of mutants compared to parent strains were even equal or resulted in a more susceptible phenotype (1-2 dilution steps) for the aminoglycoside antibiotics kanamycin and gentamicin as well as for the biocide chlorhexidine. Growth rates of selected mutants were significantly lower and hence, might partly explain the rare occurrence of Salmonella field isolates exhibiting decreased

  2. Analysis of Triclosan-Selected Salmonella enterica Mutants of Eight Serovars Revealed Increased Aminoglycoside Susceptibility and Reduced Growth Rates

    PubMed Central

    Rensch, Ulrike; Klein, Guenter; Kehrenberg, Corinna

    2013-01-01

    The biocide triclosan (TRC) is used in a wide range of household, personal care, veterinary, industrial and medical products to control microbial growth. This extended use raises concerns about a possible association between the application of triclosan and the development of antibiotic resistance. In the present study we determined triclosan mutant prevention concentrations (MPC) for Salmonella enterica isolates of eight serovars and investigated selected mutants for their mechanisms mediating decreased susceptibility to triclosan. MPCTRC values were 8 - 64-fold higher than MIC values and ranged between 1 - 16 µg/ml. The frequencies at which mutants were selected varied between 1.3 x 10-10 - 9.9 x 10-11. Even if MIC values of mutants decreased by 3-7 dilution steps in the presence of the efflux pump inhibitor Phe-Arg-β-naphtylamide, only minor changes were observed in the expression of genes encoding efflux components or regulators, indicating that neither the major multidrug efflux pump AcrAB-TolC nor AcrEF are up-regulated in triclosan-selected mutants. Nucleotide sequence comparisons confirmed the absence of alterations in the regulatory regions acrRA, soxRS, marORAB, acrSE and ramRA of selected mutants. Single bp and deduced Gly93→Val amino acid exchanges were present in fabI, the target gene of triclosan, starting from a concentration of 1 µg/ml TRC used for MPC determinations. The fabI genes were up to 12.4-fold up-regulated. Complementation experiments confirmed the contribution of Gly93→Val exchanges and fabI overexpression to decreased triclosan susceptibility. MIC values of mutants compared to parent strains were even equal or resulted in a more susceptible phenotype (1-2 dilution steps) for the aminoglycoside antibiotics kanamycin and gentamicin as well as for the biocide chlorhexidine. Growth rates of selected mutants were significantly lower and hence, might partly explain the rare occurrence of Salmonella field isolates exhibiting decreased

  3. Intrinsic properties of lumbar motor neurones in the adult G127insTGGG superoxide dismutase-1 mutant mouse in vivo: evidence for increased persistent inward currents.

    PubMed

    Meehan, C F; Moldovan, M; Marklund, S L; Graffmo, K S; Nielsen, J B; Hultborn, H

    2010-12-01

    Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease characterized by a preferential loss of motor neurones. Previous publications using in vitro neonatal preparations suggest an increased excitability of motor neurones in various superoxide dismutase-1 (SOD1) mutant mice models of ALS which may contribute to excitotoxicity of the motor neurones. Using intracellular recording, we tested this hypothesis in vivo in the adult presymptomatic G127insTGGG (G127X) SOD1 mutant mouse model of ALS. At resting membrane potentials the basic intrinsic properties of lumbar motor neurones in the adult presymptomatic G127X mutant are not significantly different from those of wild type. However, at more depolarized membrane potentials, motor neurones in the G127X SOD1 mutants can sustain higher frequency firing, showing less spike frequency adaption (SFA) and with persistent inward currents (PICs) being activated at lower firing frequencies and being more pronounced. We demonstrated that, in vivo, at resting membrane potential, spinal motor neurones of the adult G127X mice do not show an increased excitability. However, when depolarized they show evidence of an increased PIC and less SFA which may contribute to excitotoxicity of these neurones as the disease progresses. © 2010 The Authors. Acta Physiologica © 2010 Scandinavian Physiological Society.

  4. High-throughput quantitative analysis with cell growth kinetic curves for low copy number mutant cells.

    PubMed

    Xing, James Z; Gabos, Stephan; Huang, Biao; Pan, Tianhong; Huang, Min; Chen, Jie

    2012-10-01

    The mutation rate in cells induced by environmental genotoxic hazards is very low and difficult to detect using traditional cell counting assays. The established genetic toxicity tests currently recognized by regulatory authorities, such as conventional Ames and hypoxanthine guanine phosphoribosyl-transferase (HPRT) assays, are not well suited for higher-throughput screening as they require large amounts of test compounds and are very time consuming. In this study, we developed a novel cell-based assay for quantitative analysis of low numbers of cell copies with HPRT mutation induced by an environmental mutagen. The HPRT gene mutant cells induced by the mutagen were selected by 6-thioguanine (6-TG) and the cell's kinetic growth curve monitored by a real-time cell electronic sensor (RT-CES) system. When a threshold is set at a certain cell index (CI) level, samples with different initial mutant cell copies take different amounts of time in order for their growth (or CI accumulation) to cross this threshold. The more cells that are initially seeded in the test well, the faster the cell accumulation and therefore the shorter the time required to cross this threshold. Therefore, the culture time period required to cross the threshold of each sample corresponds to the original number of cells in the sample. A mutant cell growth time threshold (MT) value of each sample can be calculated to predict the number of original mutant cells. For mutagenesis determination, the RT-CES assay displayed an equal sensitivity (p > 0.05) and coefficients of variation values with good correlation to conventional HPRT mutagenic assays. Most importantly, the RT-CES mutation assay has a higher throughput than conventional cellular assays.

  5. MUTANT FREQUENCY AND MUTATIONAL SPECTRA IN THETK AND HPRT GENES OF N-ETHYL-N-NITROSOUREA TREATED MOUSE LYMPHOMA CELLS

    EPA Science Inventory

    Abstract

    The mouse lymphoma assay (MLA) utilizing the Tk locus is widely used to identify chemical mutagens. The autosomal location of the Tk locus allows for the detection of a wide range of mutational events, from point mutations to chromosome alterations. However, the ...

  6. MUTANT FREQUENCY AND MUTATIONAL SPECTRA IN THETK AND HPRT GENES OF N-ETHYL-N-NITROSOUREA TREATED MOUSE LYMPHOMA CELLS

    EPA Science Inventory

    Abstract

    The mouse lymphoma assay (MLA) utilizing the Tk locus is widely used to identify chemical mutagens. The autosomal location of the Tk locus allows for the detection of a wide range of mutational events, from point mutations to chromosome alterations. However, the ...

  7. A combination of gene expression ranking and co-expression network analysis increases discovery rate in large-scale mutant screens for novel Arabidopsis thaliana abiotic stress genes.

    PubMed

    Ransbotyn, Vanessa; Yeger-Lotem, Esti; Basha, Omer; Acuna, Tania; Verduyn, Christoph; Gordon, Michal; Chalifa-Caspi, Vered; Hannah, Matthew A; Barak, Simon

    2015-05-01

    As challenges to food security increase, the demand for lead genes for improving crop production is growing. However, genetic screens of plant mutants typically yield very low frequencies of desired phenotypes. Here, we present a powerful computational approach for selecting candidate genes for screening insertion mutants. We combined ranking of Arabidopsis thaliana regulatory genes according to their expression in response to multiple abiotic stresses (Multiple Stress [MST] score), with stress-responsive RNA co-expression network analysis to select candidate multiple stress regulatory (MSTR) genes. Screening of 62 T-DNA insertion mutants defective in candidate MSTR genes, for abiotic stress germination phenotypes yielded a remarkable hit rate of up to 62%; this gene discovery rate is 48-fold greater than that of other large-scale insertional mutant screens. Moreover, the MST score of these genes could be used to prioritize them for screening. To evaluate the contribution of the co-expression analysis, we screened 64 additional mutant lines of MST-scored genes that did not appear in the RNA co-expression network. The screening of these MST-scored genes yielded a gene discovery rate of 36%, which is much higher than that of classic mutant screens but not as high as when picking candidate genes from the co-expression network. The MSTR co-expression network that we created, AraSTressRegNet is publicly available at http://netbio.bgu.ac.il/arnet. This systems biology-based screening approach combining gene ranking and network analysis could be generally applicable to enhancing identification of genes regulating additional processes in plants and other organisms provided that suitable transcriptome data are available.

  8. Galactosylated fucose epitopes in nematodes: increased expression in a Caenorhabditis mutant associated with altered lectin sensitivity and occurrence in parasitic species.

    PubMed

    Yan, Shi; Bleuler-Martinez, Silvia; Plaza, David Fernando; Künzler, Markus; Aebi, Markus; Joachim, Anja; Razzazi-Fazeli, Ebrahim; Jantsch, Verena; Geyer, Rudolf; Wilson, Iain B H; Paschinger, Katharina

    2012-08-17

    The modification of α1,6-linked fucose residues attached to the proximal (reducing-terminal) core N-acetylglucosamine residue of N-glycans by β1,4-linked galactose ("GalFuc" epitope) is a feature of a number of invertebrate species including the model nematode Caenorhabditis elegans. A pre-requisite for both core α1,6-fucosylation and β1,4-galactosylation is the presence of a nonreducing terminal N-acetylglucosamine; however, this residue is normally absent from the final glycan structure in invertebrates due to the action of specific hexosaminidases. Previously, we have identified two hexosaminidases (HEX-2 and HEX-3) in C. elegans, which process N-glycans. In the present study, we have prepared a hex-2;hex-3 double mutant, which possesses a radically altered N-glycomic profile. Whereas in the double mutant core α1,3-fucosylation of the proximal N-acetylglucosamine was abolished, the degree of galactosylation of core α1,6-fucose increased, and a novel Galα1,2Fucα1,3 moiety attached to the distal core N-acetylglucosamine residue was detected. Both galactosylated fucose moieties were also found in two parasitic nematodes, Ascaris suum and Oesophagostomum dentatum. As core modifications of N-glycans are known targets for fungal nematotoxic lectins, the sensitivity of the C. elegans double hexosaminidase mutant was assessed. Although this mutant displayed hypersensitivity to the GalFuc-binding lectin CGL2 and the N-acetylglucosamine-binding lectin XCL, the mutant was resistant to CCL2, which binds core α1,3-fucose. Thus, the use of C. elegans mutants aids the identification of novel N-glycan modifications and the definition of in vivo specificities of nematotoxic lectins with potential as anthelmintic agents.

  9. β-(1,3)-Glucan Unmasking in Some Candida albicans Mutants Correlates with Increases in Cell Wall Surface Roughness and Decreases in Cell Wall Elasticity.

    PubMed

    Hasim, Sahar; Allison, David P; Retterer, Scott T; Hopke, Alex; Wheeler, Robert T; Doktycz, Mitchel J; Reynolds, Todd B

    2017-01-01

    Candida albicans is among the most common human fungal pathogens, causing a broad range of infections, including life-threatening systemic infections. The cell wall of C. albicans is the interface between the fungus and the innate immune system. The cell wall is composed of an outer layer enriched in mannosylated glycoproteins (mannan) and an inner layer enriched in β-(1,3)-glucan and chitin. Detection of C. albicans by Dectin-1, a C-type signaling lectin specific for β-(1,3)-glucan, is important for the innate immune system to recognize systemic fungal infections. Increased exposure of β-(1,3)-glucan to the immune system occurs when the mannan layer is altered or removed in a process called unmasking. Nanoscale changes to the cell wall during unmasking were explored in live cells with atomic force microscopy (AFM). Two mutants, the cho1Δ/Δ and kre5Δ/Δ mutants, were selected as representatives that exhibit modest and strong unmasking, respectively. Comparisons of the cho1Δ/Δ and kre5Δ/Δ mutants to the wild type reveal morphological changes in their cell walls that correlate with decreases in cell wall elasticity. In addition, AFM tips functionalized with Dectin-1 revealed that the forces of binding of Dectin-1 to all of the strains were similar, but the frequency of binding was highest for the kre5Δ/Δ mutant, decreased for the cho1Δ/Δ mutant, and rare for the wild type. These data show that nanoscale changes in surface topology are correlated with increased Dectin-1 adhesion and decreased cell wall elasticity. AFM, using tips functionalized with immunologically relevant molecules, can map epitopes of the cell wall and increase our understanding of pathogen recognition by the immune system. Copyright © 2016 American Society for Microbiology.

  10. β-(1,3)-Glucan Unmasking in Some Candida albicans Mutants Correlates with Increases in Cell Wall Surface Roughness and Decreases in Cell Wall Elasticity

    SciTech Connect

    Hasim, Sahar; Allison, David P.; Retterer, Scott T.; Hopke, Alex; Wheeler, Robert T.; Doktycz, Mitchel J.; Reynolds, Todd B.

    2016-11-14

    Candida albicans is among the most common human fungal pathogens, causing a broad range of infections, including life-threatening systemic infections. The cell wall of C. albicans is the interface between the fungus and the innate immune system. The cell wall is composed of an outer layer enriched in mannosylated glycoproteins (mannan) and an inner layer enriched in β-(1,3)-glucan and chitin. Detection of C. albicans by Dectin-1, a C-type signaling lectin specific for β-(1,3)-glucan, is important for the innate immune system to recognize systemic fungal infections. Increased exposure of β-(1,3)-glucan to the immune system occurs when the mannan layer is altered or removed in a process called unmasking. Nanoscale changes to the cell wall during unmasking were explored in this paper in live cells with atomic force microscopy (AFM). Two mutants, the cho1Δ/Δ and kre5Δ/Δ mutants, were selected as representatives that exhibit modest and strong unmasking, respectively. Comparisons of the cho1Δ/Δ and kre5Δ/Δ mutants to the wild type reveal morphological changes in their cell walls that correlate with decreases in cell wall elasticity. In addition, AFM tips functionalized with Dectin-1 revealed that the forces of binding of Dectin-1 to all of the strains were similar, but the frequency of binding was highest for the kre5Δ/Δ mutant, decreased for the cho1Δ/Δ mutant, and rare for the wild type. These data show that nanoscale changes in surface topology are correlated with increased Dectin-1 adhesion and decreased cell wall elasticity. Finally, AFM, using tips functionalized with immunologically relevant molecules, can map epitopes of the cell wall and increase our understanding of pathogen recognition by the immune system.

  11. β-(1,3)-Glucan Unmasking in Some Candida albicans Mutants Correlates with Increases in Cell Wall Surface Roughness and Decreases in Cell Wall Elasticity

    DOE PAGES

    Hasim, Sahar; Allison, David P.; Retterer, Scott T.; ...

    2016-11-14

    Candida albicans is among the most common human fungal pathogens, causing a broad range of infections, including life-threatening systemic infections. The cell wall of C. albicans is the interface between the fungus and the innate immune system. The cell wall is composed of an outer layer enriched in mannosylated glycoproteins (mannan) and an inner layer enriched in β-(1,3)-glucan and chitin. Detection of C. albicans by Dectin-1, a C-type signaling lectin specific for β-(1,3)-glucan, is important for the innate immune system to recognize systemic fungal infections. Increased exposure of β-(1,3)-glucan to the immune system occurs when the mannan layer is alteredmore » or removed in a process called unmasking. Nanoscale changes to the cell wall during unmasking were explored in this paper in live cells with atomic force microscopy (AFM). Two mutants, the cho1Δ/Δ and kre5Δ/Δ mutants, were selected as representatives that exhibit modest and strong unmasking, respectively. Comparisons of the cho1Δ/Δ and kre5Δ/Δ mutants to the wild type reveal morphological changes in their cell walls that correlate with decreases in cell wall elasticity. In addition, AFM tips functionalized with Dectin-1 revealed that the forces of binding of Dectin-1 to all of the strains were similar, but the frequency of binding was highest for the kre5Δ/Δ mutant, decreased for the cho1Δ/Δ mutant, and rare for the wild type. These data show that nanoscale changes in surface topology are correlated with increased Dectin-1 adhesion and decreased cell wall elasticity. Finally, AFM, using tips functionalized with immunologically relevant molecules, can map epitopes of the cell wall and increase our understanding of pathogen recognition by the immune system.« less

  12. Analysis of mutant quantity and quality in human-hamster hybrid AL and AL-179 cells exposed to 137Cs-gamma or HZE-Fe ions

    NASA Technical Reports Server (NTRS)

    Waldren, C.; Vannais, D.; Drabek, R.; Gustafson, D.; Kraemer, S.; Lenarczyk, M.; Kronenberg, A.; Hei, T.; Ueno, A.; Chatterjee, A. (Principal Investigator)

    1998-01-01

    We measured the number of mutants and the kinds of mutations induced by 137Cs-gamma and by HZE-Fe (56Fe [600 MeV/amu, LET = 190 KeV/micrometer) in standard AL human hamster hybrid cells and in a new variant hybrid, AL-179. We found that HZE-Fe was more mutagenic than 137Cs-gamma per unit dose (about 1.6 fold), but was slightly less mutagenic per mean lethal dose, DO, at both the S1 and hprt- loci of AL cells. On the other hand, HZE-Fe induced about nine fold more complex S1- mutants than 137Cs-gamma rays, 28% vs 3%. 137Cs-gamma rays induced about twice as many S1- mutants and hprt-mutants in AL-179 as in AL cells, and about nine times more of the former were complex, and potentially unstable kinds of mutations.

  13. Analysis of mutant quantity and quality in human-hamster hybrid AL and AL-179 cells exposed to 137Cs-gamma or HZE-Fe ions

    NASA Technical Reports Server (NTRS)

    Waldren, C.; Vannais, D.; Drabek, R.; Gustafson, D.; Kraemer, S.; Lenarczyk, M.; Kronenberg, A.; Hei, T.; Ueno, A.; Chatterjee, A. (Principal Investigator)

    1998-01-01

    We measured the number of mutants and the kinds of mutations induced by 137Cs-gamma and by HZE-Fe (56Fe [600 MeV/amu, LET = 190 KeV/micrometer) in standard AL human hamster hybrid cells and in a new variant hybrid, AL-179. We found that HZE-Fe was more mutagenic than 137Cs-gamma per unit dose (about 1.6 fold), but was slightly less mutagenic per mean lethal dose, DO, at both the S1 and hprt- loci of AL cells. On the other hand, HZE-Fe induced about nine fold more complex S1- mutants than 137Cs-gamma rays, 28% vs 3%. 137Cs-gamma rays induced about twice as many S1- mutants and hprt-mutants in AL-179 as in AL cells, and about nine times more of the former were complex, and potentially unstable kinds of mutations.

  14. A transforming Src mutant increases the bioavailability of EGFR-ligands via stimulation of the cell surface metalloproteinase ADAM17

    PubMed Central

    Maretzky, Thorsten; Zhou, Wenhui; Huang, Xin-Yun; Blobel, Carl P.

    2012-01-01

    ADAM17 (a disintegrin and metalloproteinase 17) is a cell-surface metalloproteinase that regulates signaling via the epidermal growth factor receptor (EGFR) and has important roles in diseases such as cancer and rheumatoid arthritis. ADAM17 can be activated by stimulation of several tyrosine kinase receptors, raising questions about whether oncogenic tyrosine kinases could also enhance EGFR signaling and activation of ERK via stimulation of ADAM17. The main goal of this study was to evaluate the role of Src in activating ADAM17. We provide evidence that a constitutively active transforming form of Src, the E378G mutant, as well as v-Src enhance ADAM17-mediated shedding of the EGFR-ligand TGFα. Moreover, we demonstrate that constitutive shedding of TGFα can be reduced by inhibition of Src in several cell lines, including COS7, MCF7, PAE and HaCaT cells. Src(E378G)-stimulated shedding of TGFα is abolished in Adam17−/− cells, but can be rescued by wild type ADAM17 and a mutant ADAM17 lacking its cytoplasmic domain. These findings demonstrate that ADAM17 is the principal TGFα sheddase that is activated by Src in a manner that does not require the cytoplasmic domain of ADAM17. Finally, we show that stimulation of ADAM17 by Src(E378G) leads to enhanced paracrine signaling via release of EGFR-ligands into the culture supernatant. These results raise the possibility that activation of ADAM17 by oncogenic forms of Src can aid in promoting tumorigenesis by enhancing signaling via the EGFR and ERK in an autocrine and paracrine manner. Enhanced autocrine signaling could further activate tumor cells expressing oncogenic mutants of Src, whereas paracrine signaling could stimulate EGFR and ERK signaling in surrounding non-transformed cells such as stromal cells, thereby contributing to crosstalk between tumor cells and stromal cells. PMID:20871631

  15. Expression of the Brazil nut methionine-rich protein and mutants with increased methionine in transgenic potato.

    PubMed

    Tu, H M; Godfrey, L W; Sun, S S

    1998-07-01

    A cDNA encoding the methionine-rich (19 mol% Met) protein in Brazil nut was placed under the regulation of CaMV 35S promoter and nopaline synthase terminator and introduced into the potato cultivar Russet Burbank via Agrobacterium-mediated transformation. To further enhance the Met content in the transgenic plants, chimeric genes containing four mutant constructs, BoxIa (with 5 additional Met), BoxIIa (2 additional Met), BoxIaIIa (7 additional Met), and BoxIIa2 (7 additional Met), were also generated by sequence modifications of the cDNA and transferred into potato. Analysis of the microtubers and leaves of the transgenic potato plants revealed, in general, with the exception of the BoxIIa2, the presence of mRNA transcripts of the expected size and the correctly processed Met-rich 9 kDa subunit polypeptides. The expression levels in the leaves among the various constructs and individual transgenic plants varied between <0.01% and 0.2% of total protein. The corresponding expression in the tubers was usually 2- to 4-fold lower than in leaves. In the case of BoxIIa2, which contains two tandem repeats of the BoxIIa mutant sequence, a larger (10.5-11 kDa) polypeptide was detected. These findings demonstrated that it is feasible to exploit the variable region of the Brazil Nut 2S protein for enhanced Met contents and perhaps for other desirable properties.

  16. HST1 increases replicative lifespan of a sir2Δ mutant in the absence of PDE2 in Saccharomyces cerevisiae.

    PubMed

    Kang, Woo Kyu; Devare, Mayur; Kim, Jeong-Yoon

    2017-02-01

    Silent information regulator 2 (Sir2), which is the founding member of the sirtuin family of proteins, is a pro-longevity factor for replicative lifespan (RLS) in Saccharomyces cerevisiae. Sir2 is required for transcriptional silencing at mating type loci, telomeres, and rDNA loci. Sir2 also represses transcription of highly expressed growth-related genes, such as PMA1 and some ribosomal protein genes. Although the Sir2 paralogues Hst1, Hst2, Hst3, and Hst4 occur in S. cerevisiae, none of them could replace the transcriptional regulation of PMA1 by Sir2 in the wild type. In this study, we demonstrate that Hst1, the closest Sir2 paralogue, deacetylates the acetylated lysine 16 of histone H4 (H4K16Ac) and represses PMA1 transcription in the sir2Δ pde2Δ mutant. We further show that Hst1 plays a role in extending the RLS of the sir2Δ pde2Δ mutant. Collectively, our results suggest that Hst1 can substitute for Sir2 by deacetylating H4K16Ac only in the sir2Δ pde2Δ.

  17. Spontaneous nisin-resistant Listeria monocytogenes mutants with increased expression of a putative penicillin-binding protein and their sensitivity to various antibiotics.

    PubMed

    Gravesen, A; Sørensen, K; Aarestrup, F M; Knøchel, S

    2001-01-01

    A concern regarding the use of bacteriocins, as for example the lantibiotic nisin, for biopreservation of certain food products is the possibility of resistance development and potential cross-resistance to antibiotics in the target organism. The genetic basis for nisin resistance development is as yet unknown. We analyzed changes in gene expression following nisin resistance development in Listeria monocytogenes 412 by restriction fragment differential display. The mutant had increased expression of a protein with strong homology to the glycosyltransferase domain of high-molecular-weight penicillin-binding proteins (PBPs), a histidine protein kinase, a protein of unknown function, and ClpB (putative functions from homology). The three former proteins had increased expression in a total of six out of 10 independent mutants originating from five different wild-type strains, indicating a prevalent nisin resistance mechanism under the employed isolation conditions. Increased expression of the putative PBP may affect the cell wall composition and thereby alter the sensitivity to cell wall-targeting compounds. The mutants had an isolate-specific increase in sensitivity to different beta-lactams and a slight decrease in sensitivity to another lantibiotic, mersacidin. A model incorporating these observations is proposed based on current knowledge of nisin's mode of action.

  18. Ambroxol-induced rescue of defective glucocerebrosidase is associated with increased LIMP-2 and saposin C levels in GBA1 mutant Parkinson's disease cells.

    PubMed

    Ambrosi, Giulia; Ghezzi, Cristina; Zangaglia, Roberta; Levandis, Giovanna; Pacchetti, Claudio; Blandini, Fabio

    2015-10-01

    Heterozygous mutations in GBA1 gene, encoding for lysosomal enzyme glucocerebrosidase (GCase), are a major risk factor for sporadic Parkinson's disease (PD). Defective GCase has been reported in fibroblasts of GBA1-mutant PD patients and pharmacological chaperone ambroxol has been shown to correct such defect. To further explore this issue, we investigated GCase and elements supporting GCase function and trafficking in fibroblasts from sporadic PD patients--with or without heterozygous GBA1 mutations--and healthy subjects, in basal conditions and following in vitro exposure to ambroxol. We assessed protein levels of GCase, lysosomal integral membrane protein-2 (LIMP-2), which mediates GCase trafficking to lysosomes, GCase endogenous activator saposin (Sap) C and parkin, which is involved in degradation of defective GCase. We also measured activities of GCase and cathepsin D, which cleaves Sap C from precursor prosaposin. GCase activity was reduced in fibroblasts from GBA1-mutant patients and ambroxol corrected this defect. Ambroxol increased cathepsin D activity, GCase and Sap C protein levels in all groups, while LIMP-2 levels were increased only in GBA1-mutant PD fibroblasts. Parkin levels were slightly increased only in the PD group without GBA1 mutations and were not significantly modified by ambroxol. Our study confirms that GCase activity is deficient in fibroblasts of GBA1-mutant PD patients and that ambroxol corrects this defect. The drug increased Sap C and LIMP-2 protein levels, without interfering with parkin. These results confirm that chemical chaperone ambroxol modulates lysosomal markers, further highlighting targets that may be exploited for innovative PD therapeutic strategies. Copyright © 2015 Elsevier Inc. All rights reserved.

  19. FTY720/fingolimod increases NPC1 and NPC2 expression and reduces cholesterol and sphingolipid accumulation in Niemann-Pick type C mutant fibroblasts.

    PubMed

    Newton, Jason; Hait, Nitai C; Maceyka, Michael; Colaco, Alexandria; Maczis, Melissa; Wassif, Christopher A; Cougnoux, Antony; Porter, Forbes D; Milstien, Sheldon; Platt, Nicholas; Platt, Frances M; Spiegel, Sarah

    2017-04-01

    Niemann-Pick type C (NPC) disease is a fatal neurodegenerative disorder caused by mutations in NPC1 or NPC2 with decreased functions leading to lysosomal accumulation of cholesterol and sphingolipids. FTY720/fingolimod, used for treatment of multiple sclerosis, is phosphorylated by nuclear sphingosine kinase 2, and its active phosphorylated form (FTY720-P) is an inhibitor of class I histone deacetylases. In this study, administration of clinically relevant doses of FTY720 to mice increased expression of NPC1 and -2 in brain and liver and decreased cholesterol in an SphK2-dependent manner. FTY720 greatly increased expression of NPC1 and -2 in human NPC1 mutant fibroblasts that correlated with formation of FTY720-P and significantly reduced the accumulation of cholesterol and glycosphingolipids. In agreement with this finding, FTY720 pretreatment of human NPC1 mutant fibroblasts restored transport of the cholera toxin B subunit, which binds ganglioside GM1, to the Golgi apparatus. Together, these findings suggest that FTY720 administration can ameliorate cholesterol and sphingolipid storage and trafficking defects in NPC1 mutant fibroblasts. Because neurodegeneration is the main clinical feature of NPC disease, and FTY720 accumulates in the CNS and has several advantages over available histone deacetylase inhibitors now in clinical trials, our work provides a potential opportunity for treatment of this incurable disease.-Newton, J., Hait, N. C., Maceyka, M., Colaco, A., Maczis, M., Wassif, C. A., Cougnoux, A., Porter, F. D., Milstien, S., Platt, N., Platt, F. M., Spiegel, S. FTY720/fingolimod increases NPC1 and NPC2 expression and reduces cholesterol and sphingolipid accumulation in Niemann-Pick type C mutant fibroblasts.

  20. Lesch-Nyhan disease in two families from Chiloé Island with mutations in the HPRT1 gene.

    PubMed

    Nguyen, Khue Vu; Silva, Sebastian; Troncoso, Monica; Naviaux, Robert K; Nyhan, William L

    2017-07-03

    Lesch-Nyhan disease (LND) is a rare X-linked inherited neurogenetic disorder of purine metabolism in which the enzyme, hypoxanthine-guanine phosphoribosyltransferase (HGprt) is defective. The authors report two independent point mutations leading to splicing errors: IVS 2 +1G>A, c.134 +1G>A, and IVS 3 +1G>A, c.318 +1G>A in the hypoxanthine-phosphoribosyltransferase1 (HPRT1) gene which result in exclusion of exon 2 and exon 3 respectively, in the HGprt enzyme protein from different members of two Chiloé Island families. Molecular analysis has revealed the heterogeneity of genetic mutation of the HPRT1 gene responsible for the HGprt deficiency. It allows fast, accurate carrier detection and genetic counseling.

  1. Age-associated increase of spontaneous mutant frequency and molecular nature of mutation in newborn and old lacZ-transgenic mouse.

    PubMed

    Ono, T; Ikehata, H; Nakamura, S; Saito, Y; Hosoi, Y; Takai, Y; Yamada, S; Onodera, J; Yamamoto, K

    2000-02-14

    Accumulation of mutation has long been hypothesized to be a cause of aging and contribute to many of the degenerative diseases, which appear in the senescent phase of life. To test this hypothesis, age-associated changes in spontaneous mutation in different tissues of the body as well as the molecular nature of such changes should be examined. This kind of approach has become feasible only lately with a development of new transgenic mice suitable for mutation assay. Here, using one of these transgenic mice harboring lacZ gene, we have shown that the age-associated increase in spontaneous mutant frequency is common to all tissues examined; spleen, liver, heart, brain, skin and testis, while the rates of increase in mutant frequency differed among the tissues. DNA sequencing of the 496 lacZ mutants recovered from the tissues of newborn and old mice has revealed that spectra of mutations are similar at the two age points with G:C to A:T transition at CpG site being a predominant type of mutation. Furthermore, some mutations in old tissues are complex type and not found in tissues of newborn mice. These results suggest that similar mechanisms may be operating for mutation induction in fetal and postnatal aging process. In addition, the appearance of complex types of mutations in the old tissues suggests a unique cause for these mutations in aging tissues.

  2. The role of PK/PD parameters to avoid selection and increase of resistance: mutant prevention concentration.

    PubMed

    Blondeau, J M; Hansen, G; Metzler, K; Hedlin, P

    2004-06-01

    The continuing escalation of antimicrobial resistant human pathogens and the limited number of new antimicrobial agents under development has dictated that our knowledge on the emergence of resistance and any potential strategies to slow the rate at which resistance occurs is of paramount importance. Investigations with fluoroquinolones resulted in the mutant prevention concentration (MPC) concept which represents a novel in vitro measurement of fluoroquinolone potency. In essence, the MPC defines the antimicrobial drug concentration threshold that would require an organism to simultaneously possess two resistance mutations for growth in the presence of the drug. An alternative definition is the drug concentration that prevents the growth of first-step resistant mutants or the minimal inhibitory concentration of the most resistant organism present in the heterogeneous bacterial population when tested against > or =10(9) organisms. From in vitro investigations, the new fluoroquinolones (gatifloxacin, gemifloxacin, moxifloxacin) were all found to have lower MPC values than did levofloxacin against clinical isolates of Streptococcus pneumoniae. Ciprofloxacin was found to have lower MPC values than levofloxacin against clinical isolates of Pseudomonas aeruginosa. When MPC data is applied to achievable and sustainable serum drug concentrations in the body, estimation of the time the serum drug concentration exceed both MIC and MPC values can be determined. This data along with kill data allows for an estimate of the amount of time drug concentration needs to exceed MIC/MPC values to not only result in significant kill but also to minimize resistance development. To date, MPC measurements have been determined in in vitro microbiological and pharmacological models and animal and human data are being investigated. The data summarized in this overview detail resistance issues for P. aeruginosa, S. pneumoniae and other pathogens. Also presented is a summary of the MPC

  3. Interactions of abiraterone, eplerenone and prednisolone with wild-type and mutant androgen receptor: a rationale for increasing abiraterone exposure or combining with MDV3100

    PubMed Central

    Richards, Juliet; Lim, Ai Chiin; Hay, Colin W; Taylor, Angela E.; Wingate, Anna; Nowakowska, Karolina; Pezaro, Carmel; Carreira, Suzanne; Goodall, Jane; Arlt, Wiebke; McEwan, Iain J; de Bono, Johann S; Attard, Gerhardt

    2014-01-01

    Prostate cancer progression can be associated with androgen receptor (AR) mutations acquired following treatment with castration and/or an anti-androgen. Abiraterone, a rationally-designed inhibitor of CYP17A1 recently approved for the treatment of docetaxel-treated castration-resistant prostate cancer (CRPC), is often effective, but requires co-administration with glucocorticoids to curtail side effects. Here we hypothesized that progressive disease on abiraterone may occur secondary to glucocorticoid-induced activation of mutated AR. We found that prednisolone plasma levels in CRPC patients were sufficiently high to activate mutant AR. Mineralocorticoid receptor antagonists, such as spironoloactone and eplerenone that are used to treat side-effects related to mineralocorticoid excess, also bound to and activated signaling through both wild-type and mutant AR. Abiraterone inhibited in vitro proliferation and AR-regulated gene expression of AR-positive prostate cancer cells, which could be explained by AR antagonism in addition to inhibition of steroidogenesis. Interestingly, activation of mutant AR by eplerenone was inhibited by MDV3100, bicalutamide or greater concentrations of abiraterone. Therefore, an increase in abiraterone exposure above this threshold could reverse resistance secondary to activation of AR by residual ligands or co-administered drugs. Together, our findings provide a strong rationale for clinical evaluation of combined CYP17A1 inhibition and AR antagonism. PMID:22411952

  4. Elevated salicylic acid levels conferred by increased expression of ISOCHORISMATE SYNTHASE 1 contribute to hyperaccumulation of SUMO1 conjugates in the Arabidopsis mutant early in short days 4.

    PubMed

    Villajuana-Bonequi, Mitzi; Elrouby, Nabil; Nordström, Karl; Griebel, Thomas; Bachmair, Andreas; Coupland, George

    2014-07-01

    Post-translational modification of proteins by attachment of small ubiquitin-like modifier (SUMO) is essential for plant growth and development. Mutations in the SUMO protease early in short days 4 (ESD4) cause hyperaccumulation of conjugates formed between SUMO and its substrates, and phenotypically are associated with extreme early flowering and impaired growth. We performed a suppressor mutagenesis screen of esd4 and identified a series of mutants called suppressor of esd4 (sed), which delay flowering, enhance growth and reduce hyperaccumulation of SUMO conjugates. Genetic mapping and genome sequencing indicated that one of these mutations (sed111) is in the gene salicylic acid induction-deficient 2 (SID2), which encodes ISOCHORISMATE SYNTHASE I, an enzyme required for biosynthesis of salicylic acid (SA). Analyses showed that compared with wild-type plants, esd4 contains higher levels of SID2 mRNA and about threefold more SA, whereas sed111 contains lower SA levels. Other sed mutants also contain lower SA levels but are not mutant for SID2, although most reduce SID2 mRNA levels. Therefore, higher SA levels contribute to the small size, early flowering and elevated SUMO conjugate levels of esd4. Our results support previous data indicating that SUMO homeostasis influences SA biosynthesis in wild-type plants, and also demonstrate that elevated levels of SA strongly increase the abundance of SUMO conjugates.

  5. Isolation of an Escherichia coli K-12 mutant strain able to form biofilms on inert surfaces: involvement of a new ompR allele that increases curli expression.

    PubMed

    Vidal, O; Longin, R; Prigent-Combaret, C; Dorel, C; Hooreman, M; Lejeune, P

    1998-05-01

    Classical laboratory strains of Escherichia coli do not spontaneously colonize inert surfaces. However, when maintained in continuous culture for evolution studies or industrial processes, these strains usually generate adherent mutants which form a thick biofilm, visible with the naked eye, on the wall of the culture apparatus. Such a mutant was isolated to identify the genes and morphological structures involved in biofilm formation in the very well characterized E. coli K-12 context. This mutant acquired the ability to colonize hydrophilic (glass) and hydrophobic (polystyrene) surfaces and to form aggregation clumps. A single point mutation, resulting in the replacement of a leucine by an arginine residue at position 43 in the regulatory protein OmpR, was responsible for this phenotype. Observations by electron microscopy revealed the presence at the surfaces of the mutant bacteria of fibrillar structures looking like the particular fimbriae described by the Olsén group and designated curli (A. Olsén, A. Jonsson, and S. Normark, Nature 338:652-655, 1989). The production of curli (visualized by Congo red binding) and the expression of the csgA gene encoding curlin synthesis (monitored by coupling a reporter gene to its promoter) were significantly increased in the presence of the ompR allele described in this work. Transduction of knockout mutations in either csgA or ompR caused the loss of the adherence properties of several biofilm-forming E. coli strains, including all those which were isolated in this work from the wall of a continuous culture apparatus and two clinical strains isolated from patients with catheter-related infections. These results indicate that curli are morphological structures of major importance for inert surface colonization and biofilm formation and demonstrate that their synthesis is under the control of the EnvZ-OmpR two-component regulatory system.

  6. A Mutant Small Heat Shock Protein with Increased Thylakoid Association Provides an Elevated Resistance Against UV-B Damage in Synechocystis 6803*

    PubMed Central

    Balogi, Zsolt; Cheregi, Ottilia; Giese, Kim C.; Juhász, Kata; Vierling, Elizabeth; Vass, Imre; Vígh, László; Horváth, Ibolya

    2008-01-01

    Besides acting as molecular chaperones, the amphitropic small heat shock proteins (sHsps) are suggested to play an additional role in membrane quality control. We investigated sHsp membrane function in the model cyanobacterium Synechocystis sp. PPC 6803 using mutants of the single sHsp from this organism, Hsp17. We examined mutants in the N-terminal arm, L9P and Q16R, for altered interaction with thylakoid and lipid membranes and examined the effects of these mutations on thylakoid functions. These mutants are unusual in that they retain their oligomeric state and chaperone activity in vitro but fail to confer thermotolerance in vivo. We found that both mutant proteins had dramatically altered membrane/lipid interaction properties. Whereas L9P showed strongly reduced binding to thylakoid and model membranes, Q16R was almost exclusively membrane-associated, properties that may be the cause of reduced heat tolerance of cells carrying these mutations. Among the lipid classes tested, Q16R displayed the highest interaction with negatively charged SQDG. In Q16R cells a specific alteration of the thylakoid-embedded Photosystem II (PSII) complex was observed. Namely, the binding of plastoquinone and quinone analogue acceptors to the QB site was modified. In addition, the presence of Q16R dramatically reduced UV-B damage of PSII activity because of enhanced PSII repair. We suggest these effects occur at least partly because of increased interaction of Q16R with SQDG in the PSII complex. Our findings further support the model that membrane association is a functional property of sHsps and suggest sHsps as a possible biotechnological tool to enhance UV protection of photosynthetic organisms. PMID:18574246

  7. Control of grain protein contents through SEMIDWARF1 mutant alleles: sd1 increases the grain protein content in Dee-geo-woo-gen but not in Reimei.

    PubMed

    Terao, Tomio; Hirose, Tatsuro

    2015-06-01

    A new possibility for genetic control of the protein content of rice grains was suggested by the allele differences of the SEMIDWARF1 (SD1) mutation. Two quantitative trait loci-qPROT1 and qPROT12-were found on chromosomes 1 and 12, respectively, using backcrossed inbred lines of Sasanishiki/Habataki//Sasanishiki///Sasanishiki. One of them, qPROT1, increased almost all grain proteins instead of only certain proteins in the recessive Habataki allele. Fine mapping of qPROT1 revealed that two gene candidates-Os01g0883800 and Os01g0883900-were included in this region. Os01g0883800 encoded Gibberellin 20 oxidase 2 as well as SD1, the dwarf gene used in the so-called 'Green Revolution'. Mutant analyses as well as sequencing analysis using the semi-dwarf mutant cultivars Dee-geo-woo-gen and Calrose 76 revealed that the sd1 mutant showed significantly higher grain protein contents than their corresponding wild-type cultivars, strongly suggesting that the high protein contents were caused by sd1 mutation. However, the sd1 mutant Reimei did not have high grain protein contents. It is possible to control the grain protein content and column length separately by selecting for sd1 alleles. From this finding, the genetic control of grain protein content, as well as the column length of rice cultivars, might be possible. This ability might be useful to improve rice nutrition, particularly in areas where the introduction of semi-dwarf cultivars is not advanced.

  8. Arabidopsis AtDjA3 Null Mutant Shows Increased Sensitivity to Abscisic Acid, Salt, and Osmotic Stress in Germination and Post-germination Stages

    PubMed Central

    Salas-Muñoz, Silvia; Rodríguez-Hernández, Aída A.; Ortega-Amaro, Maria A.; Salazar-Badillo, Fatima B.; Jiménez-Bremont, Juan F.

    2016-01-01

    DnaJ proteins are essential co-chaperones involved in abiotic and biotic stress responses. Arabidopsis AtDjA3 gene encodes a molecular co-chaperone of 420 amino acids, which belongs to the J-protein family. In this study, we report the functional characterization of the AtDjA3 gene using the Arabidopsis knockout line designated j3 and the 35S::AtDjA3 overexpression lines. Loss of AtDjA3 function was associated with small seed production. In fact, j3 mutant seeds showed a reduction of 24% in seed weight compared to Col-0 seeds. Expression analysis showed that the AtDjA3 gene was modulated in response to NaCl, glucose, and abscisic acid (ABA). The j3 line had increased sensitivity to NaCl and glucose treatments in the germination and cotyledon development in comparison to parental Col-0. Furthermore, the j3 mutant line exhibited higher ABA sensitivity in comparison to parental Col-0 and 35S::AtDjA3 overexpression lines. In addition, we examined the expression of ABI3 gene, which is a central regulator in ABA signaling, in j3 mutant and 35S::AtDjA3 overexpression lines. Under 5 μM ABA treatment at 24 h, j3 mutant seedlings displayed higher ABI3 expression, whereas in 35S::AtDjA3 overexpression lines, ABI3 gene expression was repressed. Taken together, these results demonstrate that the AtDjA3 gene is involved in seed development and abiotic stress tolerance. PMID:26941772

  9. HPRT mutations in V79 Chinese hamster cells induced by accelerated Ni, Au and Pb ions.

    PubMed

    Stoll, U; Barth, B; Scheerer, N; Schneider, E; Kiefer, J

    1996-07-01

    Mutation induction by accelerated heavy ions to 6-TG resistance (HPRT system) in V79 Chinese hamster cells was investigated with Ni (6-630 Me V/u), Au (2.2, 8.7 Me V/u) and Pb ions (11.6-980 Me V/u) corresponding to a LET range between 180 and 12895 ke V/microns. Most experiments could only be performed once due to technical limitations using accelerator beam times. Survival curves were exponential, mutation induction curves linear with fluence. From their slopes inactivation- and mutation-induction cross-sections were derived. If they are plotted versus LET, single, ion-specific curves are obtained. It is shown that other parameters like ion energy and effective charge play an important role. In the case of Au and Pb ions the cross-sections follow a common line, since these ions have nearly the same atomic weight, so that they should have similar spatial ionization patterns in matter at the same energies. Calculated RBEs were higher for mutation induction than for killing for all LETs.

  10. A Trivalent Enzymatic System for Uricolytic Therapy of HPRT Deficiency and Lesch-Nyhan Disease.

    PubMed

    Ronda, Luca; Marchetti, Marialaura; Piano, Riccardo; Liuzzi, Anastasia; Corsini, Romina; Percudani, Riccardo; Bettati, Stefano

    2017-07-01

    Because of the evolutionary loss of the uricolytic pathway, humans accumulate poorly soluble urate as the final product of purine catabolism. Restoration of uricolysis through enzyme therapy is a promising treatment for severe hyperuricemia caused by deficiency of hypoxanthine-guanine phosphoribosyltransferase (HPRT). To this end, we studied the effect of PEG conjugation on the activity and stability of the enzymatic complement required for conversion of urate into the more soluble (S)-allantoin. We produced in recombinant form three zebrafish enzymes required in the uricolytic pathway. We carried out a systematic study of the effect of PEGylation on the function and stability of the three enzymes by varying PEG length, chemistry and degree of conjugation. We assayed in vitro the uricolytic activity of the PEGylated enzymatic triad. We defined conditions that allow PEGylated enzymes to retain native-like enzymatic activity even after lyophilization or prolonged storage. A combination of the three enzymes in an appropriate ratio allowed efficient conversion of urate to (S)-allantoin with no accumulation of intermediate metabolites. Pharmaceutical restoration of the uricolytic pathway is a viable approach for the treatment of severe hyperuricemia.

  11. Analysis of the HPRT1 gene in 35 Italian Lesch-Nyhan families: 45 patients and 77 potential female carriers.

    PubMed

    de Gemmis, Paola; Anesi, Laura; Lorenzetto, Elisa; Gioachini, Ilenia; Fortunati, Elisabetta; Zandonà, Gessica; Fanin, Erika; Fairbanks, Lynette; Andrighetto, Gilberto; Parmigiani, Pietro; Dolcetta, Diego; Nyhan, William L; Hladnik, Uros

    2010-10-13

    Lesch-Nyhan (LND) disease is an inborn error of purine metabolism which results from deficiency of the activity of hypoxanthine-guanine phosphoribosyltransferase (HPRT). In the classical form of the disease the activity of the enzyme is completely deficient and the patient has cognitive impairment, spasticity, dystonia and self-injurious behaviour, as well as elevated concentrations of uric acid in blood and urine that leads to consequences such as nephropathy, urinary tract calculi and tophaceous gout. There are disease variants without self-injurious behaviour. In these cases neurological manifestations may vary widely. The HPRT1 gene is located on the X chromosome in position Xq26-27.2, and mutations have been found in quite a large number of patients. Documenting our experience with the diagnosis of LND in 45 Italian patients from 35 nonrelated families and 77 females at risk of being carriers of the condition. Internal review. An institute devoted to the investigation and care of patients with rare diseases. In 94% of the LND families gDNA sequencing of the patients was informative while in 6% a cDNA study was required. For the carrier females gDNA sequencing was informative in 71% of the families, 23% required qPCR studies and 6% required segregation studies combined with enzymatic activity testing. Classical cDNA studies proved to be unreliable in carrier females as there is a significant risk of failure to detect the mutated allele. Four novel HPRT1 mutations were found: c.145C>T (p.Leu49Phe), c.112C>T (p.Pro38Ser), c.89_96dup8 (p.Glu33Argfs) and c.506dupC (p.Arg170Thrfs). In the diagnosis of LND it is very important to consider all the possible alterations of the HPRT1 gene when searching for mutations especially if no affected male is available. Biochemical assessment of the enzymatic activity of HPRT in an affected male is the ideal starting point for molecular analysis of the gene. Copyright © 2010 Elsevier B.V. All rights reserved.

  12. Cystogenesis in ARPKD results from increased apoptosis in collecting duct epithelial cells of Pkhd1 mutant kidneys

    SciTech Connect

    Hu, Bo; He, Xiusheng; Li, Ao; Qiu, Qingchao; Li, Cunxi; Liang, Dan; Zhao, Ping; Ma, Jie; Coffey, Robert J.; Zhan, Qimin; Wu, Guanqing

    2011-01-15

    Mutations in the PKHD1 gene result in autosomal recessive polycystic kidney disease (ARPKD) in humans. To determine the molecular mechanism of the cystogenesis in ARPKD, we recently generated a mouse model for ARPKD that carries a targeted mutation in the mouse orthologue of human PKHD1. The homozygous mutant mice display hepatorenal cysts whose phenotypes are similar to those of human ARPKD patients. By littermates of this mouse, we developed two immortalized renal collecting duct cell lines with Pkhd1 and two without. Under nonpermissive culture conditions, the Pkhd1{sup -/-} renal cells displayed aberrant cell-cell contacts and tubulomorphogenesis. The Pkhd1{sup -/-} cells also showed significantly reduced cell proliferation and elevated apoptosis. To validate this finding in vivo, we examined proliferation and apoptosis in the kidneys of Pkhd1{sup -/-} mice and their wildtype littermates. Using proliferation (PCNA and Histone-3) and apoptosis (TUNEL and caspase-3) markers, similar results were obtained in the Pkhd1{sup -/-} kidney tissues as in the cells. To identify the molecular basis of these findings, we analyzed the effect of Pkhd1 loss on multiple putative signaling regulators. We demonstrated that the loss of Pkhd1 disrupts multiple major phosphorylations of focal adhesion kinase (FAK), and these disruptions either inhibit the Ras/C-Raf pathways to suppress MEK/ERK activity and ultimately reduce cell proliferation, or suppress PDK1/AKT to upregulate Bax/caspase-9/caspase-3 and promote apoptosis. Our findings indicate that apoptosis may be a major player in the cyst formation in ARPKD, which may lead to new therapeutic strategies for human ARPKD.

  13. Compartmentalized self-replication under fast PCR cycling conditions yields Taq DNA polymerase mutants with increased DNA-binding affinity and blood resistance.

    PubMed

    Arezi, Bahram; McKinney, Nancy; Hansen, Connie; Cayouette, Michelle; Fox, Jeffrey; Chen, Keith; Lapira, Jennifer; Hamilton, Sarah; Hogrefe, Holly

    2014-01-01

    Faster-cycling PCR formulations, protocols, and instruments have been developed to address the need for increased throughput and shorter turn-around times for PCR-based assays. Although run times can be cut by up to 50%, shorter cycle times have been correlated with lower detection sensitivity and increased variability. To address these concerns, we applied Compartmentalized Self Replication (CSR) to evolve faster-cycling mutants of Taq DNA polymerase. After five rounds of selection using progressively shorter PCR extension times, individual mutations identified in the fastest-cycling clones were randomly combined using ligation-based multi-site mutagenesis. The best-performing combinatorial mutants exhibit 35- to 90-fold higher affinity (lower Kd ) for primed template and a moderate (2-fold) increase in extension rate compared to wild-type Taq. Further characterization revealed that CSR-selected mutations provide increased resistance to inhibitors, and most notably, enable direct amplification from up to 65% whole blood. We discuss the contribution of individual mutations to fast-cycling and blood-resistant phenotypes.

  14. High Production of 2,3-butanediol by a Mutant Strain of the Newly Isolated Klebsiella pneumoniae SRP2 with Increased Tolerance Towards Glycerol

    PubMed Central

    Rahman, Md. Shafiqur; Xu, Chunbao (Charles); Ma, Kesen; Nanda, Malaya; Qin, Wensheng

    2017-01-01

    Biodiesel, a renewable fuel produced by transesterification of animal fats and vegetable oils, generates about 10% (v/v) of crude glycerol as a core byproduct. The high volume of this non bio-degradable glycerol is becoming of a great environmental and economical concern due to its worldwide ever-growing surplus. Herein we report a high production of 2,3-butanediol (2,3-BD) from pure and biodiesel derived crude glycerol using a mutant K. pneumoniae SRM2 obtained from a newly isolated strain Klebsiella pneumoniae SRP2. The mutant strain SRM2 with standing high glycerol concentration (220 g L-1 of medium) could rapidly convert glycerol aerobically to 2,3-BD, a versatile product extensively used in chemical, pharmaceutical and fuel industries Our study revealed that an increased GDH activity led to a substantially enhanced production of 2,3-BD. The mutant strain exhibited 1.3-fold higher activity of GDH than that of parent strain (500.08 vs. 638.6 µmol min -1 mg -1 protein), yielding of 32.3 g L-1 and 77.5 g L-1 2,3-BD with glycerol in batch and fed-batch process respectively. However, in batch culture with crude glycerol, cell growth and glycerol consumption were expressively boosted, and 2,3-BD production was 27.7 g L-1 from 75.0 g/L crude glycerol. In this report, the optimal conditions for high production of 2,3-BD were defined in a completely aerobic process, and 0.59 g g-1 product yield of 2,3-BD was attained by the mutated strain K. pneumoniae SRM2, which is the highest amount obtained from batch biotransformation process of glycerol metabolism till today. These results indicated that our newly developed mutant can tolerate high concentration of glycerol, have a high glycerol utilization rate, and high product yield of 2,3-BD. It is demonstrated that the mutant strain K. pneumoniae SRM2 has an ability to produce fewer co-products at trace concentrations at higher glycerol concentrations, and could be a potential candidate for 2,3-DB production in an industrial

  15. Increased number of multi-oocyte follicles (MOFs) in juvenile p27Kip1 mutant mice: potential role of granulosa cells.

    PubMed

    Pérez-Sanz, J; Arluzea, J; Matorras, R; González-Santiago, N; Bilbao, J; Yeh, N; Barlas, A; Romin, Y; Manova-Todorova, K; Koff, A; de la Hoz, C

    2013-04-01

    Why are female mice that lack a functional p27 protein infertile? The absence of a functional p27 leads to a dramatic increase in the number of multi-oocyte follicles (MOFs) in juvenile female mice; p27 would promote the individualization of follicles favoring the development of fertile eggs. p27-/- female mice are infertile. p27 suppresses excessive follicular endowment and activation and promotes follicular atresia in mice. Ovaries from wild type (WT) and p27Kip1 mutant mice aged 2, 4 and 12 weeks were subjected to immunohistochemistry/immunofluorescence. The slides with whole organs serially sectioned were scanned and examined by image analysis. Compared with WT, p27Kip1 mutant pre-pubertal mice had a greater number of oocytes, a greater number of growing follicles and a greater number of MOFs. These differences were statistically significant (P < 0.05), particularly in the case of MOFs (P > 0.001). The unusually large number of MOFs in juvenile p27-deficient mice is a novel observation. In WT mice p27 protein remains present in the oocyte nucleus but gradually decreases in the ooplasm during follicular growth, while granulosa cells show dynamic, follicle stage-related changes. These results have been obtained in mice and they cannot be directly extrapolated to humans. The dramatic increase in the numbers of MOFs in juvenile p27 mutants has not been previously reported. The number of MOFs declines sharply as the mice become sexually mature, pointing to their negative selection. These findings open a new approach to the study of sterility. This study has been funded by the Basque Government, Dept. of Health grant 2007111063 and Dept. of Industry (Saiotek) grant S-PC11UN008. Jairo Perez-Sanz was the recipient of a grant from Fundación Jesús de Gangoiti Barrera. The authors have no conflicts of interest to declare.

  16. Salicylic acid deficiency in NahG transgenic lines and sid2 mutants increases seed yield in the annual plant Arabidopsis thaliana.

    PubMed

    Abreu, Maria Elizabeth; Munné-Bosch, Sergi

    2009-01-01

    Salicylic acid-deficient NahG transgenic lines and sid2 mutants were used to evaluate the role of this compound in the development of the short-lived, annual plant Arabidopsis thaliana, with a particular focus on the interplay between salicylic acid and other phytohormones. Low salicylic acid levels led to increased growth, as well as to smaller abscisic acid levels and reduced damage to PSII (as indicated by F(v)/F(m) ratios) during the reproductive stages in rosette leaves of NahG transgenic lines and sid2 mutants, compared with wild-type plants. Furthermore, salicylic acid deficiency highly influenced seed yield and composition. Seed production increased by 4.4-fold and 3.5-fold in NahG transgenic lines and sid2 mutants, respectively, compared to the wild type. Salicylic acid deficiency also improved seed composition in terms of antioxidant vitamin concentrations, seeds of salicylic acid-deficient plants showing higher levels of alpha- and gamma-tocopherol (vitamin E) and beta-carotene (pro-vitamin A) than seeds of wild-type plants. Seeds of salicylic acid-deficient plants also showed higher nitrogen concentrations than seeds of wild-type plants. It is concluded that (i) the sid2 gene, which encodes for isochorismate synthase, plays a central role in salicylic acid biosynthesis during plant development in A. thaliana, (ii) salicylic acid plays a role in the regulation of growth, senescence, and seed production, (iii) there is a cross-talk between salicylic acid and other phytohormones during plant development, and (iv) the concentrations of antioxidant vitamins in seeds may be influenced by the endogenous levels of salicylic acid in plants.

  17. Enhanced Photosynthesis and Growth in atquac1 Knockout Mutants Are Due to Altered Organic Acid Accumulation and an Increase in Both Stomatal and Mesophyll Conductance1

    PubMed Central

    Martins, Samuel C.V.; Daloso, Danilo M.; Martinoia, Enrico; Nunes-Nesi, Adriano; DaMatta, Fábio M.; Fernie, Alisdair R.; Araújo, Wagner L.

    2016-01-01

    Stomata control the exchange of CO2 and water vapor in land plants. Thus, whereas a constant supply of CO2 is required to maintain adequate rates of photosynthesis, the accompanying water losses must be tightly regulated to prevent dehydration and undesired metabolic changes. Accordingly, the uptake or release of ions and metabolites from guard cells is necessary to achieve normal stomatal function. The AtQUAC1, an R-type anion channel responsible for the release of malate from guard cells, is essential for efficient stomatal closure. Here, we demonstrate that mutant plants lacking AtQUAC1 accumulated higher levels of malate and fumarate. These mutant plants not only display slower stomatal closure in response to increased CO2 concentration and dark but are also characterized by improved mesophyll conductance. These responses were accompanied by increases in both photosynthesis and respiration rates, without affecting the activity of photosynthetic and respiratory enzymes and the expression of other transporter genes in guard cells, which ultimately led to improved growth. Collectively, our results highlight that the transport of organic acids plays a key role in plant cell metabolism and demonstrate that AtQUAC1 reduce diffusive limitations to photosynthesis, which, at least partially, explain the observed increments in growth under well-watered conditions. PMID:26542441

  18. Enhanced Photosynthesis and Growth in atquac1 Knockout Mutants Are Due to Altered Organic Acid Accumulation and an Increase in Both Stomatal and Mesophyll Conductance.

    PubMed

    Medeiros, David B; Martins, Samuel C V; Cavalcanti, João Henrique F; Daloso, Danilo M; Martinoia, Enrico; Nunes-Nesi, Adriano; DaMatta, Fábio M; Fernie, Alisdair R; Araújo, Wagner L

    2016-01-01

    Stomata control the exchange of CO2 and water vapor in land plants. Thus, whereas a constant supply of CO2 is required to maintain adequate rates of photosynthesis, the accompanying water losses must be tightly regulated to prevent dehydration and undesired metabolic changes. Accordingly, the uptake or release of ions and metabolites from guard cells is necessary to achieve normal stomatal function. The AtQUAC1, an R-type anion channel responsible for the release of malate from guard cells, is essential for efficient stomatal closure. Here, we demonstrate that mutant plants lacking AtQUAC1 accumulated higher levels of malate and fumarate. These mutant plants not only display slower stomatal closure in response to increased CO2 concentration and dark but are also characterized by improved mesophyll conductance. These responses were accompanied by increases in both photosynthesis and respiration rates, without affecting the activity of photosynthetic and respiratory enzymes and the expression of other transporter genes in guard cells, which ultimately led to improved growth. Collectively, our results highlight that the transport of organic acids plays a key role in plant cell metabolism and demonstrate that AtQUAC1 reduce diffusive limitations to photosynthesis, which, at least partially, explain the observed increments in growth under well-watered conditions. © 2016 American Society of Plant Biologists. All Rights Reserved.

  19. The Pharmacological Chaperone Isofagomine Increases Activity of the Gaucher Disease L444P Mutant Form of β-Glucosidase

    PubMed Central

    Khanna, Richie; Benjamin, Elfrida R.; Pellegrino, Lee; Schilling, Adriane; Rigat, Brigitte A.; Soska, Rebecca; Nafar, Hadis; Ranes, Brian E.; Feng, Jessie; Lun, Yi; Powe, Allan C.; Palling, David J.; Wustman, Brandon A.; Schiffmann, Raphael; Mahuran, Don J.; Lockhart, David J.; Valenzano, Kenneth J.

    2010-01-01

    SUMMARY Gaucher disease is caused by mutations in the gene that encodes the lysosomal enzyme acid β-glucosidase (GCase). We have shown previously that the small molecule pharmacological chaperone isofagomine (IFG) binds and stabilizes N370S GCase, resulting in increased lysosomal trafficking and cellular activity. In this study, we investigated the effect of IFG on L444P GCase. Incubation of Gaucher patient-derived lymphoblastoid cell lines (LCLs) or fibroblasts with IFG led to approximately 3.5- and 1.3-fold increases in L444P GCase activity, respectively, as measured in cell lysates. The effect in fibroblasts was increased approximately 2-fold using glycoprotein-enrichment, GCase-immunocapture, or by incubating cells overnight in IFG-free media prior to assay, methods designed to maximize GCase activity by reducing IFG carryover and inhibition in the enzymatic assay. IFG incubation also increased the lysosomal trafficking and in situ activity of L444P GCase in intact cells, as measured by reduction in endogenous glucosylceramide levels. Importantly, this reduction was seen only following three-day incubation in IFG-free media, underscoring the importance of IFG removal to restore lysosomal GCase activity. In mice expressing murine L444P GCase, oral administration of IFG resulted in significant increases (2- to 5-fold) in GCase activity in disease-relevant tissues, including brain. Additionally, eight-week IFG administration significantly lowered plasma chitin III and IgG levels, and 24-week administration significantly reduced spleen and liver weights. Taken together, these data suggest that IFG can increase the lysosomal activity of L444P GCase in cells and tissues. Moreover, IFG is orally available and distributes into multiple tissues, including brain, and may thus merit therapeutic evaluation for patients with neuronopathic and non-neuronopathic Gaucher disease. PMID:20148966

  20. V(D)J recombinase mediated inter-chromosomal HPRT alterations at cryptic recombination signal sequences in peripheral human T cells.

    PubMed

    Messier, Terri L; O'Neill, J Patrick; Finette, Barry A

    2006-08-01

    The V(D)J recombinase enzyme complex is responsible for the development of a diverse immune system by catalyzing intra-molecular rearrangements of immunoglobulin (Ig) and T cell receptor (TCR) genes at specific recombination signal sequences (RSSs). This enzyme complex has also been implicated in mediating pathologic and non-pathologic intra- and inter-molecular genomic rearrangements at cryptic (Psi) RSSs outside the immune system loci in lymphoid cells. We describe here two V(D)J recombinase mediated genomic rearrangements resulting in alterations at the HPRT locus in human T-cells. These are inter-chromosomal insertions in which DNA fragments are inserted at breakpoints generated by V(D)J recombinase cleavage at Psi RSS sites in the HPRT locus at Xq26. In the first, a TCR signal ended segment from chromosome 14q11 is inserted at a Psi RSS in intron 1 of the HPRT locus. In the second, a DNA fragment from 9q22 is integrated between the coding ends generated by a V(D)J recombinase mediated HPRT deletion. Identification of these in vivo V(D)J mediated inter-chromosomal insertions at Psi RSSs in the HPRT gene supports the accumulating evidence that V(D)J recombinase can mediate mutagenic rearrangements in humans with potential pathologic consequences.

  1. Genes encoding plant-specific class III peroxidases are responsible for increased cold tolerance of the brassinosteroid-insensitive 1 mutant.

    PubMed

    Kim, Beg Hab; Kim, Sun Young; Nam, Kyoung Hee

    2012-12-01

    We previously reported that one of the brassinosteroidinsensitive mutants, bri1-9, showed increased cold tolerance compared with both wild type and BRI1-overexpressing transgenic plants, despite its severe growth retardation. This increased tolerance in bri1-9 resulted from the constitutively high expression of stress-inducible genes under normal conditions. In this report, we focused on the genes encoding class III plant peroxidases (AtPrxs) because we found that, compared with wild type, bri1-9 plants contain higher levels of reactive oxygen species (ROS) that are not involved with the activation of NADPH oxidase and show an increased level of expression of a subset of genes encoding class III plant peroxidases. Treatment with a peroxidase inhibitor, salicylhydroxamic acid (SHAM), led to the reduction of cold resistance in bri1-9. Among 73 genes that encode AtPrxs in Arabidopsis, we selected four (AtPrx1, AtPrx22, AtPrx39, and AtPrx69) for further functional analyses in response to cold temperatures. T-DNA insertional knockout mutants showed increased sensitivity to cold stress as measured by leaf damage and ion leakage. In contrast, the overexpression of AtPrx22, AtPrx39, and AtPrx69 increased cold tolerance in the BRI1-GFP plants. Taken together, these results indicate that the appropriate expression of a particular subset of AtPrx genes and the resulting higher levels of ROS production are required for the cold tolerance.

  2. Genes Encoding Plant-Specific Class III Peroxidases Are Responsible for Increased Cold Tolerance of the brassinosteroid-insensitive 1 Mutant

    PubMed Central

    Kim, Beg Hab; Kim, Sun Young; Nam, Kyoung Hee

    2012-01-01

    We previously reported that one of the brassinosteroid-insensitive mutants, bri1-9, showed increased cold tolerance compared with both wild type and BRI1-overexpressing transgenic plants, despite its severe growth retardation. This increased tolerance in bri1-9 resulted from the constitutively high expression of stress-inducible genes under normal conditions. In this report, we focused on the genes encoding class III plant peroxidases (AtPrxs) because we found that, compared with wild type, bri1-9 plants contain higher levels of reactive oxygen species (ROS) that are not involved with the activation of NADPH oxidase and show an increased level of expression of a subset of genes encoding class III plant peroxidases. Treatment with a peroxidase inhibitor, salicylhydroxamic acid (SHAM), led to the reduction of cold resistance in bri1-9. Among 73 genes that encode AtPrxs in Arabidopsis, we selected four (AtPrx1, AtPrx22, AtPrx39, and AtPrx69) for further functional analyses in response to cold temperatures. T-DNA insertional knockout mutants showed increased sensitivity to cold stress as measured by leaf damage and ion leakage. In contrast, the overexpression of AtPrx22, AtPrx39, and AtPrx69 increased cold tolerance in the BRI1-GFP plants. Taken together, these results indicate that the appropriate expression of a particular subset of AtPrx genes and the resulting higher levels of ROS production are required for the cold tolerance. PMID:23180292

  3. Silencing the wild-type and mutant K-ras increases the resistance to 5-flurouracil in HCT-116 as a colorectal cancer cell line.

    PubMed

    Teimoori-Toolabi, Ladan; Hashemi, Saba; Azadmanesh, Kayhan; Eghbalpour, Farnaz; Safavifar, Farnaz; Khorramizadeh, Mohammad Reza

    2015-02-01

    Colon cancer is the second to third common cancer worldwide. Several efforts have been made to reveal the pathways responsible for drug resistance in this type of cancer. We aimed to investigate the effect of silencing both mutant and wild-type Kristen Rous sarcoma (k-ras) on the response of human colorectal tumor 116 (HCT-116) as a colon cancer cell line to the cytotoxic effect of 5-flurouracil (5-FU). One oligonucelotide against mutant k-ras (12th codon, namely 207) and two against wild-type k-ras (namely 535 and 689) were cloned into pSilencer neo2.1. The linearized vectors besides the negative control plasmid were stably transfected into HCT-116. The proliferation rates of these cells in different concentrations of 5-FU and the apoptosis rates of the cells after treatment with lethal doses of 5-FU were studied. Moreover, the cell cycle in these cells was also analyzed by staining the cells with propidium iodide. Stably transfected cells were named HCT207ks, HCT535ks, HCT689ks, and HCT-Sc (transfected with the negative control plasmid). Decreased expression of k-ras in HCT207ks, HCT535ks, and to a lesser extent in HCT689ks was proved by quantitative real-time PCR. Although in HCT207ks the cells were mostly in G0/G1 and G2/M phases, in HCT535ks and HCT689ks, the cells in the S phase were higher in comparison with nontransfected HCT-116. Lethal doses of 5-FU in HCT-116 and HCT-Sc were 2.5-3 and 3-3.5 µmol/l, whereas in HCT207ks, HCT535ks, and HCT689ks, they were 35-40, 37.5-40, and 22.5-25 µmol/l. In conclusion, silencing mutant and wild-type k-ras would increase the resistance of HCT-116 cell line as a model of colorectal cancer to 5-FU. The degree of resistance was related directly to the k-ras mRNA level. Therefore, both mutant and wild-type k-ras may play a role in sensitizing colorectal cancer cells to 5-FU as a common chemotherapeutic drug.

  4. An attenuated mutant of the Rv1747 ATP-binding cassette transporter of Mycobacterium tuberculosis and a mutant of its cognate kinase, PknF, show increased expression of the efflux pump-related iniBAC operon

    PubMed Central

    Spivey, Vicky L; Whalan, Rachael H; Hirst, Elizabeth M A; Smerdon, Stephen J; Buxton, Roger S

    2013-01-01

    The ATP-binding cassette transporter Rv1747 is required for the growth of Mycobacterium tuberculosis in mice and in macrophages. Its structure suggests it is an exporter. Rv1747 forms a two-gene operon with pknF coding for the serine/threonine protein kinase PknF, which positively modulates the function of the transporter. We show that deletion of Rv1747 or pknF results in a number of transcriptional changes which could be complemented by the wild type allele, most significantly up-regulation of the iniBAC genes. This operon is inducible by isoniazid and ethambutol and by a broad range of inhibitors of cell wall biosynthesis and is required for efflux pump functioning. However, neither the Rv1747 or pknF mutant showed increased susceptibility to a range of drugs and cell wall stress reagents including isoniazid and ethambutol, cell wall structure and cell division appear normal by electron microscopy, and no differences in lipoarabinomannan were found. Transcription from the pknF promoter was not induced by a range of stress reagents. We conclude that the loss of Rv1747 affects cell wall biosynthesis leading to the production of intermediates that cause induction of iniBAC transcription and implicates it in exporting a component of the cell wall, which is necessary for virulence. PMID:23915284

  5. Increased Postharvest Life of TomLox B Silenced Mutants of Tomato (Solanum lycopersicum) Var. TA234.

    PubMed

    León-García, Elizabeth; Vela-Gutiérrez, Gilber; Del Ángel-Coronel, Oscar A; Torres-Palacios, Cristobal; La Cruz-Medina, Javier De; Gómez-Lim, Miguel A; García, Hugo Sergio

    2017-09-16

    A healthy lifestyle includes fruits and vegetables consumption. Tomato is one of the most consumed vegetables, although it is susceptible to physical damage through postharvest handling, thus leading to important losses. Softening is an important variable during tomato ripening; excessive softening is undesirable and leads to postharvest losses. TomloxB plays an important role in ripening, mainly in the loss of cellular integrity caused by fatty acids released from the lipid matrix of membranes that initiate oxidative deterioration, which is in turn carried into senescence. In order to increase postharvest life, we produced transgenic tomato plants via Rhizobium radiobacter with tomato lipoxygenase B (TomloxB) antisense constructs under control of the cauliflower mosaic virus (CaMV) 35S promoter. Lipoxygenase activity and firmness were measured in tomato fruit and the fatty acids profile was determined. Transgenic fruits were maintained for 40 days at room temperature in optimal conditions, whereas wild type fruits remained in similar conditions for only six days. Firmness in pink and red stages was significantly lower in wild type fruits than in two transgenic lines. Linolenic acid was the most important fatty acid consumed by lipoxygenase in both turning and pink stages of ripening. Lipoxygenase activity was smaller in transformed fruits in comparison with the wild type. These results suggest that silencing the TomloxB gene promoted significant changes in the physiology of transformed tomatoes, being the increase in postharvest life the most important.

  6. Overexpression of mutant Ptch in rhabdomyosarcomas is associated with promoter hypomethylation and increased Gli1 and H3K4me3 occupancy.

    PubMed

    Nitzki, Frauke; Tolosa, Ezequiel J; Cuvelier, Nicole; Frommhold, Anke; Salinas-Riester, Gabriela; Johnsen, Steven A; Fernandez-Zapico, Martin E; Hahn, Heidi

    2015-04-20

    Mice with heterozygous loss of the tumor suppressor Patched1 (Ptch) develop rhabdomyosarcoma (RMS)-like tumors. However, Ptch transcripts are consistently overexpressed in these tumors. We have recently shown that the upregulated transcripts are derived from the mutated Ptch allele thus leading to the hypothesis that the wild-type allele is repressed during RMS development. Here we describe epigenetic changes taking place at the Ptch locus during RMS development. We showed a lower degree of DNA-methylation in methylation-sensitive CpG regions of the Ptch promoter in RMS compared to normal muscle from heterozygous Ptch animals. In agreement with these results, treatment of heterozygous Ptch mice with the DNA demethylating agent 5-aza-2-deoxycytidine (5-aza-dC) between embryonic days E9.5-E11.5 significantly accelerated RMS formation. Since Ptch promoter methylation occurs after/around E13.5, the window for RMS initiation during embryogenesis, these results provide additional evidence that Ptch promoter hypomethylation may contribute to RMS formation. We have also demonstrated increased trimethylation of histone H3 lysine 4 (H3K4me3) and preferential binding of Gli1, a known Ptch activator, to the mutant locus in RMS. Together, these findings support an alternative model for RMS formation in heterozygous Ptch mice including loss of methylation and concomitant occupancy by activating histone marks of mutant Ptch.

  7. Treatment with bexarotene, a compound that increases apolipoprotein-E, provides no cognitive benefit in mutant APP/PS1 mice

    PubMed Central

    2013-01-01

    Background Though the precise cause(s) of Alzheimer’s disease (AD) remain unknown, there is strong evidence that decreased clearance of β-amyloid (Aβ) from the brain can contribute to the disease. Therapeutic strategies to promote natural Aβ clearance mechanisms, such as the protein apolipoprotein-E (APOE), hold promise for the treatment of AD. The amount of APOE in the brain is regulated by nuclear receptors including retinoid X receptors (RXRs). Drugs that activate RXRs, including bexarotene, can increase APOE and ABCA1 production, and have been shown to decrease the Aβ burden and improve cognition in mouse models of Aβ amyloidosis. Although recent bexarotene studies failed to replicate the rapid clearance of Aβ from brains, behavioral and cognitive effects of this compound remain controversial. Findings In efforts to clarify these behavioral findings, mutant APP/PS1 mice were acutely dosed with bexarotene. While ABCA1 was upregulated in mutant APP/PS1 mice treated with bexarotene, this drug failed to attenuate Aβ plaques or cognitive deficits in these mice. Conclusions We recommend rigorous preclinical study to evaluate the mechanism and utility of such a compound for AD therapy. PMID:23764200

  8. Oral vaccination with a rough attenuated mutant of S. Infantis increases post-wean weight gain and prevents clinical signs of salmonellosis in S. Typhimurium challenged pigs.

    PubMed

    Foster, Neil; Richards, Luke; Higgins, John; Kanellos, Theo; Barrow, Paul

    2016-02-01

    We show that oral inoculation of 14 day old conventional piglets with a rough attenuated Salmonella enterica serovar Infantis 1326/28Ф(r) (serogroup C1), 24h prior to oral challenge with S. enterica serovar Typhimurium 4/74 (serogroup B), resulted in significant weight gain (~10%) measured at 14 days post-weaning (38 days of age). Two days after challenge the S. Typhimurium induced stunting and, in some cases loss, of villi but this was prevented by pre-inoculation with the S. Infantis strain. The clinical signs of disease associated with S. Typhimurium 4/74 challenge and faecal shedding were also significantly (P<0.05) reduced by pre-inoculation with the S. Infantis mutant. Pre-inoculation of pigs with the S. Infantis mutant also increased weight gain in pigs challenged with pathogenic Escherichia coli. However, Mycobacterium bovis BCG, an unrelated intracellular bacterium, did not protect against challenge with S. Typhimurium 4/74. Copyright © 2015 Elsevier Ltd. All rights reserved.

  9. A Porphyromonas gingivalis Mutant Defective in a Putative Glycosyltransferase Exhibits Defective Biosynthesis of the Polysaccharide Portions of Lipopolysaccharide, Decreased Gingipain Activities, Strong Autoaggregation, and Increased Biofilm Formation▿ †

    PubMed Central

    Yamaguchi, Mikiyo; Sato, Keiko; Yukitake, Hideharu; Noiri, Yuichiro; Ebisu, Shigeyuki; Nakayama, Koji

    2010-01-01

    The Gram-negative anaerobic bacterium Porphyromonas gingivalis is a major pathogen in periodontal disease, one of the biofilm-caused infectious diseases. The bacterium possesses potential virulence factors, including fimbriae, proteinases, hemagglutinin, lipopolysaccharide (LPS), and outer membrane vesicles, and some of these factors are associated with biofilm formation; however, the precise mechanism of biofilm formation is still unknown. Colonial pigmentation of the bacterium on blood agar plates is related to its virulence. In this study, we isolated a nonpigmented mutant that had an insertion mutation within the new gene PGN_1251 (gtfB) by screening a transposon insertion library. The gene shares homology with genes encoding glycosyltransferase 1 of several bacteria. The gtfB mutant was defective in biosynthesis of both LPSs containing O side chain polysaccharide (O-LPS) and anionic polysaccharide (A-LPS). The defect in the gene resulted in a complete loss of surface-associated gingipain proteinases, strong autoaggregation, and a marked increase in biofilm formation, suggesting that polysaccharide portions of LPSs influence attachment of gingipain proteinases to the cell surface, autoaggregation, and biofilm formation of P. gingivalis. PMID:20624909

  10. Reduced Sweetness of a Monellin (MNEI) Mutant Results from Increased Protein Flexibility and Disruption of a Distant Poly-(L-Proline) II Helix

    PubMed Central

    Templeton, Catherine M.; Ostovar pour, Saeideh; Hobbs, Jeanette R.; Blanch, Ewan W.; Munger, Steven D.

    2011-01-01

    Monellin is a highly potent sweet-tasting protein but relatively little is known about how it interacts with the sweet taste receptor. We determined X-ray crystal structures of 3 single-chain monellin (MNEI) proteins with alterations at 2 core residues (G16A, V37A, and G16A/V37A) that induce 2- to 10-fold reductions in sweetness relative to the wild-type protein. Surprisingly, no changes were observed in the global protein fold or the positions of surface amino acids important for MNEI sweetness that could explain these differences in protein activity. Differential scanning calorimetry showed that while the thermal stability of each mutant MNEI was reduced, the least sweet mutant, G16A-MNEI, was not the least stable protein. In contrast, solution spectroscopic measurements revealed that changes in protein flexibility and the C-terminal structure correlate directly with protein activity. G16A mutation-induced disorder in the protein core is propagated via changes to hydrophobic interactions that disrupt the formation and/or position of a critical C-terminal poly-(L-proline) II helix. These findings suggest that MNEI interaction with the sweet taste receptor is highly sensitive to the relative positions of key residues across its protein surface and that loss of sweetness in G16A-MNEI may result from an increased entropic cost of binding. PMID:21343241

  11. Short branched-chain C6 carboxylic acids result in increased growth, novel 'unnatural' fatty acids and increased membrane fluidity in a Listeria monocytogenes branched-chain fatty acid-deficient mutant.

    PubMed

    Sen, Suranjana; Sirobhushanam, Sirisha; Hantak, Michael P; Lawrence, Peter; Brenna, J Thomas; Gatto, Craig; Wilkinson, Brian J

    2015-10-01

    Listeria monocytogenes is a psychrotolerant food borne pathogen, responsible for the high fatality disease listeriosis, and expensive food product recalls. Branched-chain fatty acids (BCFAs) of the membrane play a critical role in providing appropriate membrane fluidity and optimum membrane biophysics. The fatty acid composition of a BCFA-deficient mutant is characterized by high amounts of straight-chain fatty acids and even-numbered iso fatty acids, in contrast to the parent strain where odd-numbered anteiso fatty acids predominate. The presence of 2-methylbutyrate (C5) stimulated growth of the mutant at 37°C and restored growth at 10°C along with the content of odd-numbered anteiso fatty acids. The C6 branched-chain carboxylic acids 2-ethylbutyrate and 2-methylpentanoate also stimulated growth to a similar extent as 2-methylbutyrate. However, 3-methylpentanoate was ineffective in rescuing growth. 2-Ethylbutyrate and 2-methylpentanoate led to novel major fatty acids in the lipid profile of the membrane that were identified as 12-ethyltetradecanoic acid and 12-methylpentadecanoic acid respectively. Membrane anisotropy studies indicated that growth of strain MOR401 in the presence of these precursors increased its membrane fluidity to levels of the wild type. Cells supplemented with 2-methylpentanoate or 2-ethylbutyrate at 10°C shortened the chain length of novel fatty acids, thus showing homeoviscous adaptation. These experiments use the mutant as a tool to modulate the membrane fatty acid compositions through synthetic precursor supplementation, and show how existing enzymes in L. monocytogenes adapt to exhibit non-native activity yielding unique 'unnatural' fatty acid molecules, which nevertheless possess the correct biophysical properties for proper membrane function in the BCFA-deficient mutant.

  12. Emerged HA and NA Mutants of the Pandemic Influenza H1N1 Viruses with Increasing Epidemiological Significance in Taipei and Kaohsiung, Taiwan, 2009–10

    PubMed Central

    Kao, Chuan-Liang; Chan, Ta-Chien; Tsai, Chu-Han; Chu, Kuan-Ying; Chuang, Shu-Fang; Lee, Chang-Chun; Li, Zheng-Rong Tiger; Wu, Ko-Wen; Chang, Luan-Yin; Shen, Yea-Huei; Huang, Li-Min; Lee, Ping-Ing; Yang, ChingLai; Compans, Richard; Rouse, Barry T.; King, Chwan-Chuen

    2012-01-01

    The 2009 influenza pandemic provided an opportunity to observe dynamic changes of the hemagglutinin (HA) and neuraminidase (NA) of pH1N1 strains that spread in two metropolitan areas -Taipei and Kaohsiung. We observed cumulative increases of amino acid substitutions of both HA and NA that were higher in the post–peak than in the pre-peak period of the epidemic. About 14.94% and 3.44% of 174 isolates had one and two amino acids changes, respective, in the four antigenic sites. One unique adaptive mutation of HA2 (E374K) was first detected three weeks before the epidemic peak. This mutation evolved through the epidemic, and finally emerged as the major circulated strain, with significantly higher frequency in the post-peak period than in the pre-peak (64.65% vs 9.28%, p<0.0001). E374K persisted until ten months post-nationwide vaccination without further antigenic changes (e.g. prior to the highest selective pressure). In public health measures, the epidemic peaked at seven weeks after oseltamivir treatment was initiated. The emerging E374K mutants spread before the first peak of school class suspension, extended their survival in high-density population areas before vaccination, dominated in the second wave of class suspension, and were fixed as herd immunity developed. The tempo-spatial spreading of E374K mutants was more concentrated during the post–peak (p = 0.000004) in seven districts with higher spatial clusters (p<0.001). This is the first study examining viral changes during the naïve phase of a pandemic of influenza through integrated virological/serological/clinical surveillance, tempo-spatial analysis, and intervention policies. The vaccination increased the percentage of E374K mutants (22.86% vs 72.34%, p<0.001) and significantly elevated the frequency of mutations in Sa antigenic site (2.36% vs 23.40%, p<0.001). Future pre-vaccination public health efforts should monitor amino acids of HA and NA of pandemic influenza viruses isolated at

  13. Transcriptomic analysis of Clostridium thermocellum Populus hydrolysate-tolerant mutant strain shows increased cellular efficiency in response to Populus hydrolysate compared to the wild type strain

    PubMed Central

    2014-01-01

    Background The thermophilic, anaerobic bacterium, Clostridium thermocellum is a model organism for consolidated processing due to its efficient fermentation of cellulose. Constituents of dilute acid pretreatment hydrolysate are known to inhibit C. thermocellum and other microorganisms. To evaluate the biological impact of this type of hydrolysate, a transcriptomic analysis of growth in hydrolysate-containing medium was conducted on 17.5% v/v Populus hydrolysate-tolerant mutant (PM) and wild type (WT) strains of C. thermocellum. Results In two levels of Populus hydrolysate medium (0% and 10% v/v), the PM showed both gene specific increases and decreases of gene expression compared to the wild-type strain. The PM had increased expression of genes in energy production and conversion, and amino acid transport and metabolism in both standard and 10% v/v Populus hydrolysate media. In particular, expression of the histidine metabolism increased up to 100 fold. In contrast, the PM decreased gene expression in cell division and sporulation (standard medium only), cell defense mechanisms, cell envelope, cell motility, and cellulosome in both media. The PM downregulated inorganic ion transport and metabolism in standard medium but upregulated it in the hydrolysate media when compared to the WT. The WT differentially expressed 1072 genes in response to the hydrolysate medium which included increased transcription of cell defense mechanisms, cell motility, and cellulosome, and decreased expression in cell envelope, amino acid transport and metabolism, inorganic ion transport and metabolism, and lipid metabolism, while the PM only differentially expressed 92 genes. The PM tolerates up to 17.5% v/v Populus hydrolysate and growth in it elicited 489 genes with differential expression, which included increased expression in energy production and conversion, cellulosome production, and inorganic ion transport and metabolism and decreased expression in transcription and cell

  14. Transcriptomic analysis of Clostridium thermocellum Populus hydrolysate-tolerant mutant strain shows increased cellular efficiency in response to Populus hydrolysate compared to the wild type strain.

    PubMed

    Linville, Jessica L; Rodriguez, Miguel; Brown, Steven D; Mielenz, Jonathan R; Cox, Chris D

    2014-08-16

    The thermophilic, anaerobic bacterium, Clostridium thermocellum is a model organism for consolidated processing due to its efficient fermentation of cellulose. Constituents of dilute acid pretreatment hydrolysate are known to inhibit C. thermocellum and other microorganisms. To evaluate the biological impact of this type of hydrolysate, a transcriptomic analysis of growth in hydrolysate-containing medium was conducted on 17.5% v/v Populus hydrolysate-tolerant mutant (PM) and wild type (WT) strains of C. thermocellum. In two levels of Populus hydrolysate medium (0% and 10% v/v), the PM showed both gene specific increases and decreases of gene expression compared to the wild-type strain. The PM had increased expression of genes in energy production and conversion, and amino acid transport and metabolism in both standard and 10% v/v Populus hydrolysate media. In particular, expression of the histidine metabolism increased up to 100 fold. In contrast, the PM decreased gene expression in cell division and sporulation (standard medium only), cell defense mechanisms, cell envelope, cell motility, and cellulosome in both media. The PM downregulated inorganic ion transport and metabolism in standard medium but upregulated it in the hydrolysate media when compared to the WT. The WT differentially expressed 1072 genes in response to the hydrolysate medium which included increased transcription of cell defense mechanisms, cell motility, and cellulosome, and decreased expression in cell envelope, amino acid transport and metabolism, inorganic ion transport and metabolism, and lipid metabolism, while the PM only differentially expressed 92 genes. The PM tolerates up to 17.5% v/v Populus hydrolysate and growth in it elicited 489 genes with differential expression, which included increased expression in energy production and conversion, cellulosome production, and inorganic ion transport and metabolism and decreased expression in transcription and cell defense mechanisms. These

  15. Expression of Camelina WRINKLED1 Isoforms Rescue the Seed Phenotype of the Arabidopsis wri1 Mutant and Increase the Triacylglycerol Content in Tobacco Leaves

    PubMed Central

    An, Dahee; Kim, Hyojin; Ju, Seulgi; Go, Young Sam; Kim, Hyun Uk; Suh, Mi Chung

    2017-01-01

    Triacylglycerol (TAG) is an energy-rich reserve in plant seeds that is composed of glycerol esters with three fatty acids. Since TAG can be used as a feedstock for the production of biofuels and bio-chemicals, producing TAGs in vegetative tissue is an alternative way of meeting the increasing demand for its usage. The WRINKLED1 (WRI1) gene is a well-established key transcriptional regulator involved in the upregulation of fatty acid biosynthesis in developing seeds. WRI1s from Arabidopsis and several other crops have been previously employed for increasing TAGs in seed and vegetative tissues. In the present study, we first identified three functional CsWRI1 genes (CsWRI1A. B, and C) from the Camelina oil crop and tested their ability to induce TAG synthesis in leaves. The amino acid sequences of CsWRI1s exhibited more than 90% identity with those of Arabidopsis WRI1. The transcript levels of the three CsWRI1 genes showed higher expression levels in developing seeds than in vegetative and floral tissues. When the CsWRI1A. B, or C was introduced into Arabidopsis wri1-3 loss-of-function mutant, the fatty acid content was restored to near wild-type levels and percentages of the wrinkled seeds were remarkably reduced in the transgenic lines relative to wri1-3 mutant line. In addition, the fluorescent signals of the enhanced yellow fluorescent protein (eYFP) fused to the CsWRI1 genes were observed in the nuclei of Nicotiana benthamiana leaf epidermal cells. Nile red staining indicated that the transient expression of CsWRI1A. B, or C caused an enhanced accumulation of oil bodies in N. benthamiana leaves. The levels of TAGs was higher by approximately 2.5- to 4.0-fold in N. benthamiana fresh leaves expressing CsWRI1 genes than in the control leaves. These results suggest that the three Camelina WRI1s can be used as key transcriptional regulators to increase fatty acids in biomass. PMID:28174580

  16. Radiation-induced total-deletion mutations in the human hprt gene: a biophysical model based on random walk interphase chromatin geometry

    NASA Technical Reports Server (NTRS)

    Wu, H.; Sachs, R. K.; Yang, T. C.

    1998-01-01

    PURPOSE: To develop a biophysical model that explains the sizes of radiation-induced hprt deletions. METHODS: Key assumptions: (1) Deletions are produced by two DSB that are closer than an interaction distance at the time of DSB induction; (2) Interphase chromatin is modelled by a biphasic random walk distribution; and (3) Misrejoining of DSB from two separate tracks dominates at low-LET and misrejoining of DSB from a single track dominates at high-LET. RESULTS: The size spectra for radiation-induced total deletions of the hprt gene are calculated. Comparing with the results of Yamada and coworkers for gamma-irradiated human fibroblasts the study finds that an interaction distance of 0.75 microm will fit both the absolute frequency and the size spectrum of the total deletions. It is also shown that high-LET radiations produce, relatively, more total deletions of sizes below 0.5 Mb. The model predicts an essential gene to be located between 2 and 3 Mb from the hprt locus towards the centromere. Using the same assumptions and parameters as for evaluating mutation frequencies, a frequency of intra-arm chromosome deletions is calculated that is in agreement with experimental data. CONCLUSIONS: Radiation-induced total-deletion mutations of the human hprt gene and intrachange chromosome aberrations share a common mechanism for their induction.

  17. Radiation-induced total-deletion mutations in the human hprt gene: a biophysical model based on random walk interphase chromatin geometry

    NASA Technical Reports Server (NTRS)

    Wu, H.; Sachs, R. K.; Yang, T. C.

    1998-01-01

    PURPOSE: To develop a biophysical model that explains the sizes of radiation-induced hprt deletions. METHODS: Key assumptions: (1) Deletions are produced by two DSB that are closer than an interaction distance at the time of DSB induction; (2) Interphase chromatin is modelled by a biphasic random walk distribution; and (3) Misrejoining of DSB from two separate tracks dominates at low-LET and misrejoining of DSB from a single track dominates at high-LET. RESULTS: The size spectra for radiation-induced total deletions of the hprt gene are calculated. Comparing with the results of Yamada and coworkers for gamma-irradiated human fibroblasts the study finds that an interaction distance of 0.75 microm will fit both the absolute frequency and the size spectrum of the total deletions. It is also shown that high-LET radiations produce, relatively, more total deletions of sizes below 0.5 Mb. The model predicts an essential gene to be located between 2 and 3 Mb from the hprt locus towards the centromere. Using the same assumptions and parameters as for evaluating mutation frequencies, a frequency of intra-arm chromosome deletions is calculated that is in agreement with experimental data. CONCLUSIONS: Radiation-induced total-deletion mutations of the human hprt gene and intrachange chromosome aberrations share a common mechanism for their induction.

  18. SEQUENCE ANALYSIS OF MUTATIONS INDUCED BY N-ETHYL-N-NITROSOUREA IN THE TK AND HPRT GENES OF MOUSE LYMPHOMA CELLS.

    EPA Science Inventory

    The mouse lymphoma assay is widely used to identify chemicals that are capable of inducing mutational damages. The Tk+/- gene located on an autosome in mouse lymphoma cells may recover a wider range of mutational events than the X-linked Hprt locus. However, chemical-induced muta...

  19. SEQUENCE ANALYSIS OF MUTATIONS INDUCED BY N-ETHYL-N-NITROSOUREA IN THE TK AND HPRT GENES OF MOUSE LYMPHOMA CELLS.

    EPA Science Inventory

    The mouse lymphoma assay is widely used to identify chemicals that are capable of inducing mutational damages. The Tk+/- gene located on an autosome in mouse lymphoma cells may recover a wider range of mutational events than the X-linked Hprt locus. However, chemical-induced muta...

  20. Cardiac malformations in Pdgfrα mutant embryos are associated with increased expression of WT1 and Nkx2.5 in the second heart field

    PubMed Central

    Bax, Noortje A.M.; Bleyl, Steven B.; Gallini, Radiosa; Wisse, Lambertus J.; Hunter, Jennifer; van Oorschot, Angelique A.M.; Mahtab, Edris A.F.; Lie-Venema, Heleen; Goumans, Marie-Jose; Betsholtz, Christer; Gittenberger-de Groot, Adriana C.

    2010-01-01

    Platelet-derived growth factor receptor alpha (Pdgfrα) identifies cardiac progenitor cells in the posterior part of the second heart field. We aim to elucidate the role of Pdgfrα in this region. Hearts of Pdgfrα deficient mouse embryos (E9.5–E14.5) showed cardiac malformations consisting of atrial and sinus venosus myocardium hypoplasia, including venous valves and sinoatrial node. In vivo staining for Nkx2.5, showed increased myocardial expression in Pdgfrα mutants, confirmed by Western blot analysis. Due to hypoplasia of the primary atrial septum, mesenchymal cap and dorsal mesenchymal protrusion, the atrioventricular septal complex failed to fuse. Impaired epicardial development and severe blebbing coincided with diminished migration of epicardium-derived cells and myocardial thinning, which could be linked to increased WT1 and altered α4-integrin expression. Our data provide novel insight in a possible role for Pdgfrα in transduction pathways that lead to repression of Nkx2.5 and WT1 during development of posterior heart field derived cardiac structures. PMID:20658695

  1. Increased immunity to cottontail rabbit papillomavirus infection in EIII/JC inbred rabbits after vaccination with a mutant E6 that correlates with spontaneous regression.

    PubMed

    Hu, Jiafen; Cladel, Nancy M; Christensen, Neil D

    2007-01-01

    Our previous studies showed that a progressive cottontail rabbit papillomavirus (CRPV) strain containing a single amino acid change in E6 (E6G252E) induced papilloma regression in EIII/JC inbred rabbits. This finding implied that the point mutation might cause an increase in the antigenicity of the mutant versus the wild-type E6. To test this hypothesis, groups of four EIII/JC inbred rabbits were immunized with wild-type CRPVE6, CRPVE6G252E, CRPV E5, or with vector alone. A gene gun delivery system was used to deliver the DNA vaccines. Two of four rabbits from both E6G252E- and wild-type E6-vaccinated groups were free of papillomas at week 12 after viral challenge. Significantly smaller papillomas were found on E6G252E-vaccinated rabbits than on E6-, E5-, and control vector-vaccinated rabbits (p = 0.01, unpaired Student t test) and these small papillomas regressed at week 20 after viral challenge. E5 vaccination failed to provide protection against viral challenge, and the mean papilloma size was also comparable to that of the control vector-vaccinated rabbits (p > 0.05, unpaired Student t test). We conclude that a single amino acid change in the CRPV E6 protein (G252E) increased protection against wild-type infectious CRPV.

  2. Increase in chitin as an essential response to defects in assembly of cell wall polymers in the ggp1delta mutant of Saccharomyces cerevisiae.

    PubMed Central

    Popolo, L; Gilardelli, D; Bonfante, P; Vai, M

    1997-01-01

    The GGP1/GAS1 gene codes for a glycosylphosphatidylinositol-anchored plasma membrane glycoprotein of Saccharomyces cerevisiae. The ggp1delta mutant shows morphogenetic defects which suggest changes in the cell wall matrix. In this work, we have investigated cell wall glucan levels and the increase of chitin in ggp1delta mutant cells. In these cells, the level of alkali-insoluble 1,6-beta-D-glucan was found to be 50% of that of wild-type cells and was responsible for the observed decrease in the total alkali-insoluble glucan. Moreover, the ratio of alkali-soluble to alkali-insoluble glucan almost doubled, suggesting a change in glucan solubility. The increase of chitin in ggp1delta cells was found to be essential since the chs3delta ggp1delta mutations determined a severe reduction in the growth rate and in cell viability. Electron microscopy analysis showed the loss of the typical structure of yeast cell walls. Furthermore, in the chs3delta ggp1delta cells, the level of alkali-insoluble glucan was 57% of that of wild-type cells and the alkali-soluble/alkali-insoluble glucan ratio was doubled. We tested the effect of inhibition of chitin synthesis also by a different approach. The ggp1delta cells were treated with nikkomycin Z, a well-known inhibitor of chitin synthesis, and showed a hypersensitivity to this drug. In addition, studies of genetic interactions with genes related to the construction of the cell wall indicate a synthetic lethal effect of the ggp1delta kre6delta and the ggp1delta pkc1delta combined mutations. Our data point to an involvement of the GGP1 gene product in the cross-links between cell wall glucans (1,3-beta-D-glucans with 1,6-beta-D-glucans and with chitin). Chitin is essential to compensate for the defects due to the lack of Ggp1p. Moreover, the activities of Ggp1p and Chs3p are essential to the formation of the organized structure of the cell wall in vegetative cells. PMID:8990299

  3. Increase in fruit size of a spontaneous mutant of 'Gala' apple (Malus x domestica Borkh.) is facilitated by altered cell production and enhanced cell size.

    PubMed

    Malladi, Anish; Hirst, Peter M

    2010-06-01

    Fruit size regulation was studied in the apple cultivar 'Gala' and a large fruit size spontaneous mutant of 'Gala', 'Grand Gala' (GG). GG fruits were 15% larger in diameter and 38% heavier than 'Gala' fruits, largely due to an increase in size of the fruit cortex. The mutation in GG altered growth prior to fruit set and during fruit development. Prior to fruit set, the carpel/floral-tube size was enhanced in GG and was associated with higher cell number, larger cell size, and increased ploidy through endoreduplication, an altered form of the cell cycle normally absent in apple. The data suggest that the mutation in GG promotes either cell production or endoreduplication in the carpel/floral-tube cells depending on their competence for division. Ploidy was not altered in GG leaves. During fruit growth, GG fruit cells exited cell production earlier, and with a DNA content of 4C suggesting G2 arrest. Cell size was higher in GG fruits during exit from cell production and at later stages of fruit growth. Final cell diameter in GG fruit cortex cells was 15% higher than that in 'Gala' indicating that enhanced fruit size in GG was facilitated by increased cell size. The normal progression of cell expansion in cells arrested in G2 may account for the increase in cell size. Quantitative RT-PCR analysis indicated higher MdCDKA1 expression and reduced MdCYCA2 expression during early fruit development in GG fruits. Together, the data indicate an important role for cell expansion in regulating apple fruit size.

  4. M.tuberculosis Mutants Lacking Oxygenated Mycolates Show Increased Immunogenicity and Protective Efficacy as Compared to M. bovis BCG Vaccine in an Experimental Mouse Model

    PubMed Central

    Hedhli, Dorsaf; Denis, Olivier; Barkan, Daniel; Daffé, Mamadou; Glickman, Michael S.; Huygen, Kris

    2013-01-01

    The existing vaccine against tuberculosis (M. bovis BCG) exerts some protection against the extrapulmonary forms of the disease, particularly in young children, but is not very effective against the pulmonary form of TB, which often results from the reactivation of a latent M. tuberculosis (M.tb)infection. Among the new approaches in TB vaccine development, live attenuated M.tb mutants are a promising new avenue. Here we report on the vaccine potential of two highly attenuated M.tb mutants, MGM1991 and M.tbhma::hyg (HMA), lacking all oxygenated mycolates in their cell wall. In C57BL/6 mice, stronger Th1 (IFN-γ, IL-2 and TNF-α) and IL-17 responses could be induced following subcutaneous vaccination with either of the two mutants, than following vaccination with M. bovis BCG. Significantly more mycobacteria specific IFN-γ producing CD4+ and particularly CD8+ T cells could be detected by intracellular cytokine staining in mice vaccinated with the M.tb mutants. Finally, vaccination with either of the two mutants conferred stronger protection against intratracheal M.tb challenge than vaccination with BCG, as indicated by reduced bacterial replication in lungs at 4 to 12 weeks after challenge. Protection against M. tb dissemination, as indicated by reduced bacterial numbers in spleen, was comparable for both mutants to protection conferred by BCG. PMID:24146869

  5. Molecular characterization of mutation and comparison of mutation profiles in the hprt gene of Chinese hamster ovary cells treated with benzo[a]pyrene trans-7,8-diol-anti-9,10-epoxide, 1-nitrobenzol[a]pyrene trans-7,8-diol-anti-9,10-epoxide, and 3-nitrobenzol[a]pyrene trans-7,8-diol-anti-9,10-epoxide

    SciTech Connect

    Zhan, D.J.; Heflich, R.H.; Fu, P.P.

    1996-12-31

    Both 1- and 3-nitrobenzol[a] pyrene (nitro-BaP) are environmental contaminants, potent mutagens in Salmonella, and moderate mutagens in Chinese hamster ovary (CHO) cells. The mutagenicity of their oxidized metabolites, trans-7,8-dihydroxy-anti-9, 10-epoxy-7,8,9,10-tetrahydro-1-nitrobenzol[a]pyrene (1-nitro-BaP-DE) and trans-7,8-dihydroxy-anti-9, 10-epoxy-7,8,9,10-tetrahydro-3-nitrobenzo[a]pyrene (3-nitro-BaP-DE), together with trans-7,8-dihydroxy-anti-9,10-epoxy-7,8,9,10-tetrahydrobenzol[a]pyren (BaP-DE), was determined in CHO-K1 cells, and the resulting mutations at the hprt locus were characterized by polymerase chain reaction (PCR) amplification of reverse-transcribed hprt mRNA, followed by DNA sequence analysis. The mutant frequencies, in mutants/10{sup 6} clonable cells, at 30 and 100 ng/ml, were BaP-DE, 248 and 456; 1-nitro-BaP-DE, 68 and 260; 3-nitro-BaP-DE, 81 and 232, respectively. In general, the three diolepoxides exhibited similar mutational spectra: (1) 64% (23/36 sequenced mutants) of BaP-DE, 53% (19/36) of 1-nitro-BaP-DE, and 64% (23/36) of 3-nitro-BaP-DE mutants resulted from simple base pair substitution, with the predominant mutation being G{r_arrow}T transversion: (2) 90%, 100%, and 100% of mutations at G:C had the mutated dG on the nontranscribed DNA strand; and (3) about one quarter of the mutants produced by each mutagen had one or more PCR products with partial or complete exon deletions. 61 refs., 1 fig., 7 tabs.

  6. Increased body temperature accelerates aggregation of the Leu-68-->Gln mutant cystatin C, the amyloid-forming protein in hereditary cystatin C amyloid angiopathy.

    PubMed Central

    Abrahamson, M; Grubb, A

    1994-01-01

    Hereditary cystatin C amyloid angiopathy is a dominantly inherited disorder, characterized by dementia, paralysis, and death from cerebral hemorrhage in early adult life. A variant of the cysteine proteinase inhibitor, cystatin C, is deposited as amyloid in the tissues of the patients and their spinal-fluid level of cystatin C is abnormally low. The disease-associated Leu-68-->Gln mutant (L68Q) cystatin C has been produced in an Escherichia coli expression system and isolated by use of denaturing buffers, immunosorption, and gel filtration. Parallel physicochemical and functional investigations of L68Q-cystatin C and wild-type cystatin C revealed that both proteins effectively inhibit the cysteine proteinase cathepsin B (equilibrium constants for dissociation, 0.4 and 0.5 nM, respectively) but differ considerably in their tendency to dimerize and form aggregates. While wild-type cystatin C is monomeric and functionally active even after prolonged storage at elevated temperatures, L68Q-cystatin C starts to dimerize and lose biological activity immediately after it is transferred to a nondenaturing buffer. The dimerization of L68Q-cystatin C is highly temperature-dependent, with a rise in incubation temperature from 37 to 40 degrees C resulting in a 150% increase in dimerization rate. The aggregation at physiological concentrations is likewise increased at 40 compared to 37 degrees C, by approximately 60%. These properties of L68Q-cystatin C have bearing upon our understanding of the pathophysiological process of hereditary cystatin C amyloid angiopathy. They might also be of clinical relevance, since medical intervention to abort febrile periods of carriers of the disease trait may reduce the in vivo formation of L68Q-cystatin C aggregates. Images PMID:8108423

  7. The crystal structure of the sevenfold mutant of barley beta-amylase with increased thermostability at 2.5 A resolution.

    PubMed

    Mikami, B; Yoon, H J; Yoshigi, N

    1999-01-22

    The three-dimensional structure of the sevenfold mutant of barley beta-amylase (BBA-7s) with increased thermostability was determined by X-ray crystallography. The enzyme was purified as a single component and crystallized by a hanging drop method in the presence of 14 % PEG 6000. The crystals belong to space group P43212 with cell dimensions a=b=72.11 A, c=250.51 A. The diffraction data up to 2.5 A were collected after soaking the crystal in 100 mM maltose with Rsym of 8.6 %. The structure was determined by a molecular replacement method using soybean beta-amylase (SBA) as a search model and refined to an R-factor of 18.7 %. The final model included 500 amino acid residues, 141 water molecules and three glucose residues, which were located at subsites 1-2 and 4 in the active site. The r.m.s. distance of 485 Calpha atoms between BBA-7s and SBA was 0.62 A. Out of the seven mutated amino acids, four (Ser295Ala, Ile297Val, Ser351Pro and Ala376Ser) were substitutions from the common residues with SBA to the thermostable forms. A comparison of the structures of BBA-7s and SBA indicated that the side-chain of Ser376 makes new hydrogen bonds to the main-chain of an adjacent beta-strand, and that the side-chains of Val297 reduce an unfavorable interaction between the side-chains of Ala314. The mutation of Ser295Ala breaks the hydrogen bond between Ser295 OG and Tyr195 OH, which seems to be the reason for the unoccupied glucose residue at subsite 3. The tandem mutations at 350-352 including substitutions to two Pro residues suggested the reduction of main-chain entropy in the unfolded structure of this solvent-exposed protruded loop.

  8. A herpes simplex virus type 1 mutant disrupted for microRNA H2 with increased neurovirulence and rate of reactivation.

    PubMed

    Jiang, Xianzhi; Brown, Don; Osorio, Nelson; Hsiang, Chinhui; Li, Lily; Chan, Lucas; BenMohamed, Lbachir; Wechsler, Steven L

    2015-04-01

    The herpes simplex virus type 1 (HSV-1) latency-associated transcript (LAT) encodes several microRNAs. One of these, miR-H2, overlaps and is antisense to the ICP0 gene and appears to decrease expression of the ICP0 protein. To determine if miR-H2 plays a role in the HSV-1 latency-reactivation cycle, we constructed a mutant, McK-ΔH2, in which this microRNA has been disrupted without altering the predicted amino acid sequence of ICP0. McK-ΔH2 produced increased amounts of ICP0. Although replication of McK-ΔH2 was similar to that of its wild-type (wt) McKrae parental virus in RS cells and mouse eyes, McK-ΔH2 was more neurovirulent in Swiss-Webster mice than McKrae based on the percent of mice that died from herpes encephalitis following ocular infection. In addition, using a mouse trigeminal ganglia (TG) explant model of induced reactivation, we show here for the first time that miR-H2 appears to play a role in modulating HSV-1 reactivation. Although the percent of TG from which virus reactivated by day 10 after explant was similar for McK-ΔH2, wt McKrae, and the marker-rescued virus McK-ΔH2Res, at earlier times, significantly more reactivation was seen with McK-ΔH2. Our results suggest that in the context of the virus, miR-H2 downregulates ICP0 and this moderates both HSV-1 neurovirulence and reactivation.

  9. Pre-thymic somatic mutation leads to high mutant frequency at hypoxanthine-guanine phosphoribosyltransferase gene

    SciTech Connect

    Jett, J.

    1994-12-01

    While characterizing the background mutation spectrum of the Hypoxathine-guanine phosphoribosyltransferase (HPRT) gene in a healthy population, an outlier with a high mutant frequency of thioguanine resistant lymphocytes was found. When studied at the age of 46, this individual had been smoking 60 cigarettes per day for 38 years. His mutant frequency was calculated at 3.6 and 4.2x10{sup {minus}4} for two sampling periods eight months apart. Sequencing analysis of the HPRT gene in his mutant thioguanine resistant T lymphocytes was done to find whether the cells had a high rate of mutation, or if the mutation was due to a single occurrence of mutation and, if so, when in the T lymphocyte development the mutation occurred. By T-cell receptor analysis it has been found that out of 35 thioguanine resistant clones there was no dominant gamma T cell receptor gene rearrangement. During my appointment in the Science & Engineering Research Semester, I found that 34 of those clones have the same base substitution of G{yields}T at cDNA position 197. Due to the consistent mutant frequency from both sampling periods and the varying T cell receptors, the high mutant frequency cannot be due to recent proliferation of a mature mutant T lymphocyte. From the TCR and DNA sequence analysis we conclude that the G{yields}T mutation must have occurred in a T lymphocyte precursor before thymic differentiation so that the thioguanine resistant clones share the same base substitution but not the same gamma T cell receptor gene.

  10. Mutations in Succinate Dehydrogenase Subunit C Increase Radiosensitivity and Bystander Responses

    NASA Astrophysics Data System (ADS)

    Zhou, Hongning; Hei, Tom K.

    Although radiation-induced bystander effect is well studied in the past decade, the precise mech-anisms are still unclear. It is likely that a combination of pathways involving both primary and secondary signaling processes is involved in producing a bystander effect. There is recent evidence that mitochondria play a critical role in bystander responses. Recently studies found that a mutation in succinate dehydrogenese subunit C (SDHC), an integral membrane protein in complex II of the electron transport chain, resulted in increased superoxide, oxidative stress, apoptosis, tumorigenesis, and genomic instability, indicating that SDHC play a critical role in maintaining mitochondrial function. In the present study, using Chinese hamster fibroblasts (B1 cells) and the mutants (B9 cells) containing a single base substitution that produced a premature stop codon resulting in a 33-amino acid COOH-terminal truncation of the SDHC protein, we found that B9 cells had an increase in intracellular superoxide content, nitric oxide species, and mitochondrial membrane potential when compared with wild type cells. After irradiated with a grade of doses of gamma rays, B9 cells show an increased radiosensitivity, especially at high doses. The HPRT- mutant yield after gamma-ray irradiation in B9 cells was significantly higher than that of B1 cells. A single, 3Gy dose of gamma-rays increased the background mutant level by more than 4 fold. In contrast, the mutant induction was less than 2 fold in B1 cells. In addition, B9 cells produced a higher bystander mutagenesis after alpha particle irradiation than the B1 cells. Furthermore, pretreated with carboxy-2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (c-PTIO), a nitric oxide scavenger, significantly decreased the bystander effect. Our findings demonstrate that a mutation in SDHC increases radiosensitivity in both directly irradiated cells and in neighboring bystander cells, and mito-chondrial function play an essential role in

  11. Increased Hydrolysis of Oximino-β-Lactams by CMY-107, a Tyr199Cys Mutant Form of CMY-2 Produced by Escherichia coli

    PubMed Central

    Vetouli, E. E.; Bozavoutoglou, E.; Lebessi, E.; Tzelepi, E.; Tzouvelekis, L. S.

    2015-01-01

    The cephalosporinase CMY-107, a Tyr199Cys mutant form of CMY-2 encoded by an IncI self-transferable plasmid carried by an Escherichia coli clinical strain, was characterized. The enzyme hydrolyzed oximino-cephalosporins and aztreonam more efficiently than CMY-2 did. PMID:26438499

  12. Increased Prevalence of EGFR-Mutant Lung Cancer in Women and in East Asian Populations: Analysis of Estrogen-Related Polymorphisms

    PubMed Central

    Bell, DaphneW.; Brannigan, BrianW.; Matsuo, Keitaro; Finkelstein, Dianne M.; Sordella, Raffaella; Settleman, Jeff; Mitsudomi, Tetsuya; Haber, Daniel A.

    2012-01-01

    Purpose Somatic mutations in the epidermal growth factor receptor (EGFR) gene occur in a subset of non – small-cell lung cancer (NSCLC) and are highly predictive of the clinical response to selective EGFR kinase inhibitors.The prevalence of EGFR-mutant NSCLC is appreciably higher in females than in males and in East Asian than in Caucasian populations. We hypothesized that these disparate frequencies may be attributable to underlying genetic modifiers. Given the coincident differences in sex and ethnic origin, we tested allozymatic variants of enzymes involved in estrogen biosynthesis and metabolism, encoded by polymorphic alleles known to differ in frequency between Caucasian and Asian populations, as modifying alleles. Experimental Design We genotyped nine polymorphisms in the CYP1A1, CYP17A1, CYP19, HSD17B1, COMT, GSTM1, and GSTT1 genes, in a series of 100 Japanese NSCLCs, selected for equal representation of EGFR wild-type (wt) and EGFR-mutant cases, as well as male and female cases. Associations between polymorphic variants and the EGFR genotype and sex of NSCLC cases were examined using Fisher’s exact test of significance. Results Only CYP1A1*2C showed a difference in allele frequency that approached statistical significance. Heterozygotes were underrepresented among EGFR-mutant cases compared with EGFR-wt cases (27% versus 47%, P = 0.08), with a concurrent trend toward overrepresentation of CYP1A1*2CIle/Ile homozygotes among EGFR-mutant cases as compared with EGFR-wt cases (69% versus 51%, P = 0.13). Conclusion Within the power of this study, our findings suggest that the selected polymorphic variants in the estrogen biosynthesis and metabolism pathways are unlikely to be major genetic modifiers of the prevalence of EGFR-mutant NSCLC. PMID:18593984

  13. Human HPRT1 gene and the Lesch-Nyhan disease: Substitution of alanine for glycine and inversely in the HGprt enzyme protein.

    PubMed

    Nguyen, Khue Vu; Naviaux, Robert K; Nyhan, William L

    2017-02-01

    Lesch-Nyhan disease (LND) is a rare X-linked inherited neurogenetic disorder of purine metabolism in which the enzyme, hypoxanthine-guanine phosphoribosyltransferase (HGprt) is defective. The authors report three novel independent mutations in the coding region of the HPRT1 gene from genomic DNA of (a) a carrier sister of two male patients with LND: c.569G>C, p.G190A in exon 8; and (b) two LND affected male patients unrelated to her who had two mutations: c.648delC, p.Y216X, and c.653C>G, p.A218G in exon 9. Molecular analysis reveals the heterogeneity of genetic mutation of the HPRT1 gene responsible for the HGprt deficiency. It allows fast, accurate detection of carriers and genetic counseling.

  14. ALDH16A1 is a novel non-catalytic enzyme that may be involved in the etiology of gout via protein–protein interactions with HPRT1

    PubMed Central

    Vasiliou, Vasilis; Sandoval, Monica; Backos, Donald S.; Jackson, Brian C.; Chen, Ying; Reigan, Philip; Lanaspa, Miguel A.; Johnson, Richard J.; Koppaka, Vindhya; Thompson, David C.

    2013-01-01

    Gout, a common form of inflammatory arthritis, is strongly associated with elevated uric acid concentrations in the blood (hyperuricemia). A recent study in Icelanders identified a rare missense single nucleotide polymorphism (SNP) in the ALDH16A1 gene, ALDH16A1*2, to be associated with gout and serum uric acid levels. ALDH16A1 is a novel and rather unique member of the ALDH superfamily in relation to its gene and protein structures. ALDH16 genes are present in fish, amphibians, protista, bacteria but absent from archaea, fungi and plants. In most mammalian species, two ALDH16A1 spliced variants (ALDH16A1, long form and ALDH16A1_v2, short form) have been identified and both are expressed in HepG-2, HK-2 and HK-293 human cell lines. The ALDH16 proteins contain two ALDH domains (as opposed to one in the other members of the superfamily), four transmembrane and one coiled-coil domains. The active site of ALDH16 proteins from bacterial, frog and lower animals contain the catalytically important cysteine residue (Cys-302); this residue is absent from the mammalian and fish orthologs. Molecular modeling predicts that both the short and long forms of human ALDH16A1 protein would lack catalytic activity but may interact with the hypoxanthine-guanine phosphoribosyltransferase (HPRT1) protein, a key enzyme involved in uric acid metabolism and gout. Interestingly, such protein-protein interactions with HPRT1 are predicted to be impaired for the long or short forms of ALDH16A1*2. These results lead to the intriguing possibility that association between ALDH16A1 and HPRT1 may be required for optimal HPRT activity with disruption of this interaction possibly contributing to the hyperuricemia seen in ALDH16A1*2 carriers. PMID:23348497

  15. Deficiency in the glycerol channel Fps1p confers increased freeze tolerance to yeast cells: application of the fps1delta mutant to frozen dough technology.

    PubMed

    Izawa, Shingo; Ikeda, Kayo; Maeta, Kazuhiro; Inoue, Yoshiharu

    2004-12-01

    Intracellular glycerol content affects the freeze-thaw stress tolerance of Saccharomyces cerevisiae. We have recently reported that intracellular-glycerol-enriched cells cultured in glycerol medium acquire tolerance to freeze stress and retain high leavening ability even in dough after frozen storage [Izawa et al. (2004) Appl Microbiol Biotechnol http://dx.doi.org/10.1007/s00253-004-1624-4]. A deletion mutant of the FPS1 gene, which encodes a glycerol channel, accumulates glycerol inside the cell without an exogenous supply of glycerol into the medium. We found that the fps1delta cells acquired tolerance to freeze stress and retained high leavening ability in dough after frozen storage for 7 days. These results suggest that the fps1delta mutant is a useful strain for developing better frozen-dough with a commercial advantage.

  16. LKB1/KRAS mutant lung cancers constitute a genetic subset of NSCLC with increased sensitivity to MAPK and mTOR signalling inhibition

    PubMed Central

    Mahoney, C L; Choudhury, B; Davies, H; Edkins, S; Greenman, C; Haaften, G van; Mironenko, T; Santarius, T; Stevens, C; Stratton, M R; Futreal, P A

    2009-01-01

    LKB1/STK11 is a multitasking tumour suppressor kinase. Germline inactivating mutations of the gene are responsible for the Peutz-Jeghers hereditary cancer syndrome. It is also somatically inactivated in approximately 30% of non-small-cell lung cancer (NSCLC). Here, we report that LKB1/KRAS mutant NSCLC cell lines are sensitive to the MEK inhibitor CI-1040 shown by a dose-dependent reduction in proliferation rate, whereas LKB1 and KRAS mutations alone do not confer similar sensitivity. We show that this subset of NSCLC is also sensitised to the mTOR inhibitor rapamycin. Importantly, the data suggest that LKB1/KRAS mutant NSCLCs are a genetically and functionally distinct subset and further suggest that this subset of lung cancers might afford an opportunity for exploitation of anti-MAPK/mTOR-targeted therapies. PMID:19165201

  17. K-Ras mutant fraction in A/J mouse lung increases as a function of benzo[a]pyrene dose

    EPA Science Inventory

    K-Ras mutant fraction (MF) was measured to examine the default assumption of low dose linearity in the benzo[a]pyrene (B[a]P) mutational response. Groups of ten male A/J mice (7-9 weeks-old) received a single i.p. injection of 0, 0.05, 0.5, 5, or 50 mg/kg B[a]P, and were sacrifi...

  18. K-Ras mutant fraction in A/J mouse lung increases as a function of benzo[a]pyrene dose

    EPA Science Inventory

    K-Ras mutant fraction (MF) was measured to examine the default assumption of low dose linearity in the benzo[a]pyrene (B[a]P) mutational response. Groups of ten male A/J mice (7-9 weeks-old) received a single i.p. injection of 0, 0.05, 0.5, 5, or 50 mg/kg B[a]P, and were sacrifi...

  19. Pigment pattern formation in the quail mutant of the silkworm, Bombyx mori: parallel increase of pteridine biosynthesis and pigmentation of melanin and ommochromes.

    PubMed

    Kato, Tomomi; Sawada, Hiroshi; Yamamoto, Takayuki; Mase, Keisuke; Nakagoshi, Motoko

    2006-08-01

    The larval pigment pattern in the silkworm, Bombyx mori, is formed by melanin, ommochromes and pteridines. During development all these pigments are synthesized autonomously, and possibly also with mutual interaction between them, to yield unique pigment patterns. In order to find the key trigger for such pigment pattern formation, developmental changes in pteridine biosynthesis were studied using the quail mutant (q/q), which has darker larval marks formed by melanin and an abundance of ommochromes in the integument. In the current study, emphasis has been placed on the analysis of GTP-cyclohydrolase I (GTP-CH I), which is a key enzyme for the biosynthesis of pteridines, during the development of the silkworm. Results of Northern blotting showed that in the quail mutant strong signals of GTP-CH I mRNA appeared around each period of ecdysis, while no such signals appeared in the background strain (+q/q) used. Also, both GTP-CH I activities and pteridine content were higher in the quail mutant compared with the background strain. These results strongly suggest that pteridine biosynthesis is closely linked to the formation of melanin and ommochromes. It is also suggested here that in the silkworm a recessive gene (q) may be involved in the regulation of its pigment pattern formation.

  20. [13C]Methionine NMR and metal-binding studies of recombinant human transferrin N-lobe and five methionine mutants: conformational changes and increased sensitivity to chloride.

    PubMed Central

    He, Q Y; Mason, A B; Tam, B M; MacGillivray, R T; Woodworth, R C

    1999-01-01

    The N-lobe of human serum transferrin (hTF/2N) and single point mutants in which each of the five methionine residues was individually mutated have been produced in a mammalian tissue-culture expression system. Since the five methionine residues are well distributed in the transferrin N-lobe, (13)C NMR of the [epsilon-(13)C]methionine-labelled proteins has been used to monitor conformational changes of the protein during metal binding. All five methionine residues have been assigned [Beatty, Cox, Frenkiel, Tam, Mason, MacGillivray, Sadler and Woodworth (1996) Biochemistry 35, 7635-7642]. The tentative two-dimensional NMR assignment for two of the five methionine residues, namely Met(26) and Met(109), has been corrected. A series of NMR spectra for the complexes of (13)C-Met-labelled hTF/2N with six different metal ions, Fe(III), Cu(II), Cr(III), Co(III), Ga(III) and In(III), demonstrate that the conformational change of the protein upon metal binding can be observed by means of the changes in the NMR chemical shifts associated with certain methionine residues, regardless of whether diamagnetic or paramagnetic metals are used. Changing any of the methionine residues should have minimal effects on transferrin function, since structural analysis shows that none of these residues contacts functional amino acids or has any obvious role in iron uptake or release. In fact, UV-visible spectra show little perturbation of the electronic spectra of any of the mutants. Nevertheless, the M109L mutant (Met(109)-->Leu) releases iron at half the rate of the wild-type N-lobe, and chloride shows a significantly greater retarding effect on the rate of iron release from all five mutants. All the methionine mutants (especially in the apo form) show a poor solubility in Hepes buffer lacking anions such as bicarbonate. These findings imply a more general effect of anion binding to surface residues than previously realized. PMID:10585877

  1. Mouse mutants from chemically mutagenized embryonic stem cells

    PubMed Central

    Munroe, Robert J.; Bergstrom, Rebecca A.; Zheng, Qing Yin; Libby, Brian; Smith, Richard; John, Simon W.M.; Schimenti, Kerry J.; Browning, Victoria L.; Schimenti, John C.

    2010-01-01

    The drive to characterize functions of human genes on a global scale has stimulated interest in large-scale generation of mouse mutants. Conventional germ-cell mutagenesis with N-ethyl-N-nitrosourea (ENU) is compromised by an inability to monitor mutation efficiency, strain1 and interlocus2 variation in mutation induction, and extensive husbandry requirements. To overcome these obstacles and develop new methods for generating mouse mutants, we devised protocols to generate germline chi-maeric mice from embryonic stem (ES) cells heavily mutagenized with ethylmethanesulphonate (EMS). Germline chimaeras were derived from cultures that underwent a mutation rate of up to 1 in 1,200 at the Hprt locus (encoding hypoxanthine guanine phosphoribosyl transferase). The spectrum of mutations induced by EMS and the frameshift mutagen ICR191 was consistent with that observed in other mammalian cells. Chimaeras derived from ES cells treated with EMS transmitted mutations affecting several processes, including limb development, hair growth, hearing and gametogenesis. This technology affords several advantages over traditional mutagenesis, including the ability to conduct shortened breeding schemes and to screen for mutant phenotypes directly in ES cells or their differentiated derivatives. PMID:10700192

  2. Mouse mutants from chemically mutagenized embryonic stem cells.

    PubMed

    Munroe, R J; Bergstrom, R A; Zheng, Q Y; Libby, B; Smith, R; John, S W; Schimenti, K J; Browning, V L; Schimenti, J C

    2000-03-01

    The drive to characterize functions of human genes on a global scale has stimulated interest in large-scale generation of mouse mutants. Conventional germ-cell mutagenesis with N-ethyl-N-nitrosourea (ENU) is compromised by an inability to monitor mutation efficiency, strain and interlocus variation in mutation induction, and extensive husbandry requirements. To overcome these obstacles and develop new methods for generating mouse mutants, we devised protocols to generate germline chimaeric mice from embryonic stem (ES) cells heavily mutagenized with ethylmethanesulphonate (EMS). Germline chimaeras were derived from cultures that underwent a mutation rate of up to 1 in 1,200 at the Hprt locus (encoding hypoxanthine guanine phosphoribosyl transferase). The spectrum of mutations induced by EMS and the frameshift mutagen ICR191 was consistent with that observed in other mammalian cells. Chimaeras derived from ES cells treated with EMS transmitted mutations affecting several processes, including limb development, hair growth, hearing and gametogenesis. This technology affords several advantages over traditional mutagenesis, including the ability to conduct shortened breeding schemes and to screen for mutant phenotypes directly in ES cells or their differentiated derivatives.

  3. Different Mutant/Wild-Type p53 Combinations Cause a Spectrum of Increased Invasive Potential in Nonmalignant Immortalized Human Mammary Epithelial Cells1

    PubMed Central

    Junk, Damian J; Vrba, Lukas; Watts, George S; Oshiro, Marc M; Martinez, Jesse D; Futscher, Bernard W

    2008-01-01

    Aberrations of p53 occur in most, if not all, human cancers. In breast cancer, p53 mutation is the most common genetic defect related to a single gene. Immortalized human mammary epithelial cells resemble the earliest forms of aberrant breast tissue growth but do not express many malignancy-associated phenotypes. We created a model of human mammary epithelial tumorigenesis by infecting hTERT-HME1 immortalized human mammary epithelial cells expressing wild-type p53 with four different mutant p53 constructs to determine the role of p53 mutation on the evolution of tumor phenotypes. We demonstrate that different mutant/wild-type p53 heterozygous models generate loss of function, dominant negative activity, and a spectrum of gain of function activities that induce varying degrees of invasive potential. We suggest that this model can be used to elucidate changes that occur in early stages of human mammary epithelial tumorigenesis. These changes may constitute novel biomarkers or reveal novel treatment modalities that could inhibit progression from primary to metastatic breast disease. PMID:18472962

  4. Mutant fatty acid desaturase

    DOEpatents

    Shanklin, John; Cahoon, Edgar B.

    2004-02-03

    The present invention relates to a method for producing mutants of a fatty acid desaturase having a substantially increased activity towards fatty acid substrates with chains containing fewer than 18 carbons relative to an unmutagenized precursor desaturase having an 18 carbon atom chain length substrate specificity. The method involves inducing one or more mutations in the nucleic acid sequence encoding the precursor desaturase, transforming the mutated sequence into an unsaturated fatty acid auxotroph cell such as MH13 E. coli, culturing the cells in the absence of supplemental unsaturated fatty acids, thereby selecting for recipient cells which have received and which express a mutant fatty acid desaturase with an elevated specificity for fatty acid substrates having chain lengths of less than 18 carbon atoms. A variety of mutants having 16 or fewer carbon atom chain length substrate specificities are produced by this method. Mutant desaturases produced by this method can be introduced via expression vectors into prokaryotic and eukaryotic cells and can also be used in the production of transgenic plants which may be used to produce specific fatty acid products.

  5. Combined ALK and MDM2 inhibition increases antitumor activity and overcomes resistance in human ALK mutant neuroblastoma cell lines and xenograft models.

    PubMed

    Wang, Hui Qin; Halilovic, Ensar; Li, Xiaoyan; Liang, Jinsheng; Cao, Yichen; Rakiec, Daniel P; Ruddy, David A; Jeay, Sebastien; Wuerthner, Jens U; Timple, Noelito; Kasibhatla, Shailaja; Li, Nanxin; Williams, Juliet A; Sellers, William R; Huang, Alan; Li, Fang

    2017-04-20

    The efficacy of ALK inhibitors in patients with ALK-mutant neuroblastoma is limited, highlighting the need to improve their effectiveness in these patients. To this end, we sought to develop a combination strategy to enhance the antitumor activity of ALK inhibitor monotherapy in human neuroblastoma cell lines and xenograft models expressing activated ALK. Herein, we report that combined inhibition of ALK and MDM2 induced a complementary set of anti-proliferative and pro-apoptotic proteins. Consequently, this combination treatment synergistically inhibited proliferation of TP53 wild-type neuroblastoma cells harboring ALK amplification or mutations in vitro, and resulted in complete and durable responses in neuroblastoma xenografts derived from these cells. We further demonstrate that concurrent inhibition of MDM2 and ALK was able to overcome ceritinib resistance conferred by MYCN upregulation in vitro and in vivo. Together, combined inhibition of ALK and MDM2 may provide an effective treatment for TP53 wild-type neuroblastoma with ALK aberrations.

  6. Acquired Resistance to the Mutant-Selective EGFR Inhibitor AZD9291 Is Associated with Increased Dependence on RAS Signaling in Preclinical Models.

    PubMed

    Eberlein, Catherine A; Stetson, Daniel; Markovets, Aleksandra A; Al-Kadhimi, Katherine J; Lai, Zhongwu; Fisher, Paul R; Meador, Catherine B; Spitzler, Paula; Ichihara, Eiki; Ross, Sarah J; Ahdesmaki, Miika J; Ahmed, Ambar; Ratcliffe, Laura E; O'Brien, Elizabeth L Christey; Barnes, Claire H; Brown, Henry; Smith, Paul D; Dry, Jonathan R; Beran, Garry; Thress, Kenneth S; Dougherty, Brian; Pao, William; Cross, Darren A E

    2015-06-15

    Resistance to targeted EGFR inhibitors is likely to develop in EGFR-mutant lung cancers. Early identification of innate or acquired resistance mechanisms to these agents is essential to direct development of future therapies. We describe the detection of heterogeneous mechanisms of resistance within populations of EGFR-mutant cells (PC9 and/or NCI-H1975) with acquired resistance to current and newly developed EGFR tyrosine kinase inhibitors, including AZD9291. We report the detection of NRAS mutations, including a novel E63K mutation, and a gain of copy number of WT NRAS or WT KRAS in cell populations resistant to gefitinib, afatinib, WZ4002, or AZD9291. Compared with parental cells, a number of resistant cell populations were more sensitive to inhibition by the MEK inhibitor selumetinib (AZD6244; ARRY-142886) when treated in combination with the originating EGFR inhibitor. In vitro, a combination of AZD9291 with selumetinib prevented emergence of resistance in PC9 cells and delayed resistance in NCI-H1975 cells. In vivo, concomitant dosing of AZD9291 with selumetinib caused regression of AZD9291-resistant tumors in an EGFRm/T790M transgenic model. Our data support the use of a combination of AZD9291 with a MEK inhibitor to delay or prevent resistance to AZD9291 in EGFRm and/or EGFRm/T790M tumors. Furthermore, these findings suggest that NRAS modifications in tumor samples from patients who have progressed on current or EGFR inhibitors in development may support subsequent treatment with a combination of EGFR and MEK inhibition. ©2015 American Association for Cancer Research.

  7. Acquired resistance to mutant-selective EGFR inhibitor AZD9291 is associated with increased dependence on RAS signaling in preclinical models

    PubMed Central

    Eberlein, Catherine A.; Stetson, Daniel; Markovets, Aleksandra A.; Al-Kadhimi, Katherine J.; Lai, Zhongwu; Fisher, Paul R.; Meador, Catherine B.; Spitzler, Paula; Ichihara, Eiki; Ross, Sarah J.; Ahdesmaki, Miika J.; Ahmed, Ambar; Ratcliffe, Laura E.; Christey O’Brien, Elizabeth L.; Barnes, Claire H.; Brown, Henry; Smith, Paul D.; Dry, Jonathan R.; Beran, Garry; Thress, Kenneth S.; Dougherty, Brian; Pao, William; Cross, Darren A. E.

    2015-01-01

    Resistance to targeted EGFR inhibitors is likely to develop in EGFR mutant lung cancers. Early identification of innate or acquired resistance mechanisms to these agents is essential to direct development of future therapies. We describe the detection of heterogeneous mechanisms of resistance within populations of EGFR mutant cells (PC9 and/or NCI-H1975) with acquired resistance to current and newly developed EGFR TKIs including AZD9291. We report the detection of NRAS mutations, including a novel E63K mutation, and a gain of copy number of WT NRAS or WT KRAS in cell populations resistant to gefitinib, afatinib, WZ4002 or AZD9291. Compared to parental cells, a number of resistant cell populations were more sensitive to inhibition by the MEK inhibitor selumetinib (AZD6244; ARRY-142886) when treated in combination with the originating EGFR inhibitor. In vitro, a combination of AZD9291 with selumetinib prevented emergence of resistance in PC9 cells and delayed resistance in NCI-H1975 cells. In vivo, concomitant dosing of AZD9291 with selumetinib caused regression of AZD9291-resistant tumours in an EGFRm/T790M transgenic model. Our data support the use of a combination of AZD9291 with a MEK inhibitor to delay or prevent resistance to AZD9291 in EGFRm and/or EGFRm/T790M tumours. Further, these findings suggest that NRAS modifications in tumour samples from patients who have progressed on current or EGFR inhibitors in development may support subsequent treatment with a combination of EGFR and MEK inhibition. PMID:25870145

  8. Streptavidin mutants

    SciTech Connect

    2000-02-08

    The present invention relates to streptavidin proteins and peptides having a altered physical properties such as an increased stability or increased or decreased affinity for binding biotin. The invention also relates to methods for the detection, identification, separation and isolation of targets using streptavidin proteins or peptides. Streptavidin with increased or reduced affinity allows for the use of the streptavidin-biotin coupling systems for detection and isolation systems wherein it is necessary to remove of one or the other of the binding partners. Such systems are useful for the purification of functional proteins and viable cells. The invention also relates to nucleic acids which encode these streptavidin proteins and peptides and to recombinant cells such as bacteria, yeast and mammalian cells which contain these nucleic acids.

  9. Streptavidin mutants

    DOEpatents

    Sano, Takeshi; Cantor, Charles R.; Vajda, Sandor; Reznik, Gabriel O.; Smith, Cassandra L.; Pandori, Mark W.

    2000-01-01

    The present invention relates to streptavidin proteins and peptides having a altered physical properties such as an increased stability or increased or decreased affinity for binding biotin. The invention also relates to methods for the detection, identification, separation and isolation of targets using streptavidin proteins or peptides. Streptavidin with increased or reduced affinity allows for the use of the streptavidin-biotin coupling systems for detection and isolation systems wherein it is necessary to remove of one or the other of the binding partners. Such systems are useful for the purification of functional proteins and viable cells. The invention also relates to nucleic acids which encode these streptavidin proteins and peptides and to recombinant cells such as bacteria, yeast and mammalian cells which contain these nucleic acids.

  10. Increased neurovirulence and reactivation of the herpes simplex virus type 1 latency associated transcript (LAT) negative mutant dLAT2903 with a disrupted LAT miR-H2

    PubMed Central

    Jiang, Xianzhi; Brown, Don; Osorio, Nelson; Hsiang, Chinhui; BenMohamed, Lbachir; Wechsler, Steven L.

    2015-01-01

    At least six microRNAs (miRNAs) appear to be encoded by the latency associated transcript (LAT) of herpes simplex virus type 1 (HSV-1). The gene for ICP0, an important immediate early (IE) viral protein, is antisense to, and overlaps with, the region of LAT from which miRNA H2 (miR-H2) is derived. We recently reported that a mutant (McK-ΔH2) disrupted for miR-H2 on the wild type HSV-1 strain McKrae genomic background has increased ICP0 expression, increased neurovirulence, and slightly more rapid reactivation. We report here that HSV-1 mutants deleted for the LAT promoter nonetheless make significant amounts of miR-H2 during lytic tissue culture infection, presumably via readthrough transcription from an upstream promoter. To determine if miR-H2 might also play a role in the HSV-1 latency-reactivation cycle of a LAT negative mutant, we constructed dLAT-ΔH2, in which miR-H2 is disrupted in dLAT2903 without altering the predicted amino acid sequence of the overlapping ICP0 open reading frame. Similar to McK-ΔH2, dLAT-ΔH2 expressed more ICP0, was more neurovirulent, and had increased reactivation in the mouse TG explant induced reactivation model of HSV-1 compared to its parental virus. Interestingly, although the increased reactivation of McK-ΔH2 compared to its parental wt virus was subtle and only detected at very early times after explant TG induced reactivation, the increased reactivation of dLAT-ΔH2 compared to its dLAT2903 parental virus appeared more robust and was significantly increased even at late times after induction. These results confirm that miR-H2 plays a role in modulating the HSV-1 reactivation phenotype. PMID:26069184

  11. Expression of val-12 mutant ras p21 in an IL-3-dependent murine myeloid cell line is associated with loss of serum-dependence and increases in membrane PIP2-specific phospholipase C activity.

    PubMed

    Rizzo, M T; Boswell, H S; English, D; Gabig, T G

    1991-01-01

    We previously showed that the proliferative response of a serum- and interleukin-3 (IL-3)-dependent murine myeloid cell line, NFS/N1-H7, was partially inhibited by pertussis toxin as a result of toxin-induced increased adenylate cyclase activity. In the present studies, we examined the role of the phosphoinositide cycle in the proliferative response of these cells and demonstrated that there was no change in PIP (phosphatidylinositol bisphosphate)-specific phospholipase C activity in response to IL-3 alone. However, serum caused a pertussis toxin-insensitive increase in PIP2-specific phospholipase C activity as reflected by decreased cellular levels of 32P-labelled PIP2. Proliferation of a subline selected from val-12-mutant H-ras-transfected NFS-H7 cells, clone E5, was insensitive to pertussis toxin, occurred in the absence of serum but remained serum-stimulatable and absolutely dependent on IL-3. This val-12 mutant ras-expressing cell line showed an increase in 32P-labelled PIP (phosphatidylinositol phosphate) in response to serum whereas the parent cell line did not. Membrane fractions from 32P-labelled ras-transfected cells displayed higher GTP gamma S-, GTP-, or F(-)-stimulated PIP2-specific phospholipase C activity compared to membranes from the parent cell line. Thus serum-dependence and adenylate cyclase-mediated pertussis toxin-sensitivity of the parent cell line was bypassed by val-12 mutant ras p21, possibly as a result of increased PIP2-specific phospholipase C activity.

  12. Molecular characterization of mutation and comparison of mutation profiles in the hprt gene of Chinese hamster ovary cells treated with benzo[a]pyrene trans-7,8-diol-anti-9,10-epoxide, 1-nitrobenzo[a]pyrene trans-7,8-diol-anti-9,10-epoxide, and 3-nitrobenzo[a]pyrene trans-7,8- diol-anti-9,10-epoxide.

    PubMed

    Zhan, D J; Heflich, R H; Fu, P P

    1996-01-01

    Both 1- and 3-nitrobenzo[a]pyrene (nitro-BaP) are environmental contaminants, potent mutagens in Salmonella, and moderate mutagens in Chinese hamster ovary (CHO) cells. The mutagenicity of their oxidized metabolites,trans-7,8-dihydroxy-anti-9,10-epoxy-7,8,9,10-epoxy -7,8,9,10-tetrahydro-1-nitrobenzo[a]pyrene (1-nitro-BaP-DE) and trans-7,8-dihydroxy-anti-9,10-epoxy-7,8,9,10-tetrahydro-3-nitrobenzo[a]- pyrene (3-nitro-BaPDE), together with trans-7,8-dihydroxy-anti-9, 10-ep- oxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BaP-DE), was determined in CHO-K1 cells, and the resulting mutations at the hprt locus were characterized by polymerase chain reaction (PCR) amplification of reverse-transcribed hprt mRNA, followed by DNA sequence analysis. The mutant frequencies, in mutants/10(6) clonable cells, at 30 and 100 ng/ml, were BaP-DE, 248 and 456; 1-nitro-BaP-DE, 68 and 260; 3-nitro-BaP-DE, 81 and 232, respectively. In general, the three diolepoxides exhibited similar mutational spectra: 1) 64% (23/36 sequenced mutants) of BaP-DE, 53% (19/36) of 1-nitro-BaP-DE, and 64% (23/36) of 3-nitro-BaP-DE mutants resulted from simple base pair substitution, with the predominant mutation being G-->T transversion; 2) 90%, 100%, and 100% of mutations at G:C had the mutated dG on the nontranscribed DNA strand; and 3) about one quarter of the mutants produced by each mutagen had one or more PCR products with partial or complete exon deletions. The mutagens induced few frameshifts or complex mutations. Among the differences in mutational specificity for the three diolepoxides, the proportion of substituted dGs with 3' purines was significant (P < 0.05) for BaP-DE (16/19, 84%) and 3-nitro-BaP-DE (17/20, 85%), but not significant for 1-nitro-BaP-DE-induced mutants (11/17, 65%, P > 0.05). Also, high proportions of BaP-DE and 3-nitro-BaP-DE base pair substitutions at G:C occurred in DNA sequence contexts of 5'-GG-3', 5'-GGA-3', and 5'-TGGA-3', while the proportions of 1-nitro-BaP-DE mutants in these

  13. Increased neurovirulence and reactivation of the herpes simplex virus type 1 latency-associated transcript (LAT)-negative mutant dLAT2903 with a disrupted LAT miR-H2.

    PubMed

    Jiang, Xianzhi; Brown, Don; Osorio, Nelson; Hsiang, Chinhui; BenMohamed, Lbachir; Wechsler, Steven L

    2016-02-01

    At least six microRNAs (miRNAs) appear to be encoded by the latency-associated transcript (LAT) of herpes simplex virus type 1 (HSV-1). The gene for ICP0, an important immediate early (IE) viral protein, is anti-sense to, and overlaps with, the region of LAT from which miRNA H2 (miR-H2) is derived. We recently reported that a mutant (McK-ΔH2) disrupted for miR-H2 on the wild-type HSV-1 strain McKrae genomic background has increased ICP0 expression, increased neurovirulence, and slightly more rapid reactivation. We report here that HSV-1 mutants deleted for the LAT promoter nonetheless make significant amounts of miR-H2 during lytic tissue culture infection, presumably via readthrough transcription from an upstream promoter. To determine if miR-H2 might also play a role in the HSV-1 latency/reactivation cycle of a LAT-negative mutant, we constructed dLAT-ΔH2, in which miR-H2 is disrupted in dLAT2903 without altering the predicted amino acid sequence of the overlapping ICP0 open reading frame. Similar to McK-ΔH2, dLAT-ΔH2 expressed more ICP0, was more neurovirulent, and had increased reactivation in the mouse TG explant-induced reactivation model of HSV-1 compared with its parental virus. Interestingly, although the increased reactivation of McK-ΔH2 compared with its parental wild-type (wt) virus was subtle and only detected at very early times after explant TG induced reactivation, the increased reactivation of dLAT-ΔH2 compared with its dLAT2903 parental virus appeared more robust and was significantly increased even at late times after induction. These results confirm that miR-H2 plays a role in modulating the HSV-1 reactivation phenotype.

  14. The pharmacological chaperone AT2220 increases the specific activity and lysosomal delivery of mutant acid alpha-glucosidase, and promotes glycogen reduction in a transgenic mouse model of Pompe disease.

    PubMed

    Khanna, Richie; Powe, Allan C; Lun, Yi; Soska, Rebecca; Feng, Jessie; Dhulipala, Rohini; Frascella, Michelle; Garcia, Anadina; Pellegrino, Lee J; Xu, Su; Brignol, Nastry; Toth, Matthew J; Do, Hung V; Lockhart, David J; Wustman, Brandon A; Valenzano, Kenneth J

    2014-01-01

    Pompe disease is an inherited lysosomal storage disorder that results from a deficiency in acid α-glucosidase (GAA) activity due to mutations in the GAA gene. Pompe disease is characterized by accumulation of lysosomal glycogen primarily in heart and skeletal muscles, which leads to progressive muscle weakness. We have shown previously that the small molecule pharmacological chaperone AT2220 (1-deoxynojirimycin hydrochloride, duvoglustat hydrochloride) binds and stabilizes wild-type as well as multiple mutant forms of GAA, and can lead to higher cellular levels of GAA. In this study, we examined the effect of AT2220 on mutant GAA, in vitro and in vivo, with a primary focus on the endoplasmic reticulum (ER)-retained P545L mutant form of human GAA (P545L GAA). AT2220 increased the specific activity of P545L GAA toward both natural (glycogen) and artificial substrates in vitro. Incubation with AT2220 also increased the ER export, lysosomal delivery, proteolytic processing, and stability of P545L GAA. In a new transgenic mouse model of Pompe disease that expresses human P545L on a Gaa knockout background (Tg/KO) and is characterized by reduced GAA activity and elevated glycogen levels in disease-relevant tissues, daily oral administration of AT2220 for 4 weeks resulted in significant and dose-dependent increases in mature lysosomal GAA isoforms and GAA activity in heart and skeletal muscles. Importantly, oral administration of AT2220 also resulted in significant glycogen reduction in disease-relevant tissues. Compared to daily administration, less-frequent AT2220 administration, including repeated cycles of 4 or 5 days with AT2220 followed by 3 or 2 days without drug, respectively, resulted in even greater glycogen reductions. Collectively, these data indicate that AT2220 increases the specific activity, trafficking, and lysosomal stability of P545L GAA, leads to increased levels of mature GAA in lysosomes, and promotes glycogen reduction in situ. As such, AT2220 may

  15. The Pharmacological Chaperone AT2220 Increases the Specific Activity and Lysosomal Delivery of Mutant Acid Alpha-Glucosidase, and Promotes Glycogen Reduction in a Transgenic Mouse Model of Pompe Disease

    PubMed Central

    Lun, Yi; Soska, Rebecca; Feng, Jessie; Dhulipala, Rohini; Frascella, Michelle; Garcia, Anadina; Pellegrino, Lee J.; Xu, Su; Brignol, Nastry; Toth, Matthew J.; Do, Hung V.; Lockhart, David J.; Wustman, Brandon A.; Valenzano, Kenneth J.

    2014-01-01

    Pompe disease is an inherited lysosomal storage disorder that results from a deficiency in acid α-glucosidase (GAA) activity due to mutations in the GAA gene. Pompe disease is characterized by accumulation of lysosomal glycogen primarily in heart and skeletal muscles, which leads to progressive muscle weakness. We have shown previously that the small molecule pharmacological chaperone AT2220 (1-deoxynojirimycin hydrochloride, duvoglustat hydrochloride) binds and stabilizes wild-type as well as multiple mutant forms of GAA, and can lead to higher cellular levels of GAA. In this study, we examined the effect of AT2220 on mutant GAA, in vitro and in vivo, with a primary focus on the endoplasmic reticulum (ER)-retained P545L mutant form of human GAA (P545L GAA). AT2220 increased the specific activity of P545L GAA toward both natural (glycogen) and artificial substrates in vitro. Incubation with AT2220 also increased the ER export, lysosomal delivery, proteolytic processing, and stability of P545L GAA. In a new transgenic mouse model of Pompe disease that expresses human P545L on a Gaa knockout background (Tg/KO) and is characterized by reduced GAA activity and elevated glycogen levels in disease-relevant tissues, daily oral administration of AT2220 for 4 weeks resulted in significant and dose-dependent increases in mature lysosomal GAA isoforms and GAA activity in heart and skeletal muscles. Importantly, oral administration of AT2220 also resulted in significant glycogen reduction in disease-relevant tissues. Compared to daily administration, less-frequent AT2220 administration, including repeated cycles of 4 or 5 days with AT2220 followed by 3 or 2 days without drug, respectively, resulted in even greater glycogen reductions. Collectively, these data indicate that AT2220 increases the specific activity, trafficking, and lysosomal stability of P545L GAA, leads to increased levels of mature GAA in lysosomes, and promotes glycogen reduction in situ. As such, AT2220 may

  16. Genetic and Diet-Induced Obesity Increased Intestinal Tumorigenesis in the Double Mutant Mouse Model Multiple Intestinal Neoplasia X Obese via Disturbed Glucose Regulation and Inflammation

    PubMed Central

    Hetland, Ragna Bogen

    2015-01-01

    We have studied how spontaneous or carcinogen-induced intestinal tumorigenesis was affected by genetic or diet-induced obesity in C57BL/6J-Apc Min/+ X C57BL/6J-Lep ob/+ mice. Obesity was induced by the obese (ob) mutation in the lep gene coding for the hormone leptin, or by a 45% fat diet. The effects of obesity were examined on spontaneous intestinal tumors caused by the multiple intestinal neoplasia (Min) mutation in the adenomatous polyposis coli (Apc) gene and on tumors induced by the dietary carcinogen 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP). F1 ob/ob (homozygous mutated) mice had increased body weight (bw) and number of spontaneous and PhIP-induced small intestinal tumors (in Apc Min/+ mice), versus ob/wt (heterozygous mutated) and wt/wt mice (homozygous wild-type). A 45% fat diet exacerbated bw and spontaneous tumor numbers versus 10% fat, but not PhIP-induced tumors. Except for bw, ob/wt and wt/wt were not significantly different. The obesity caused hyperglucosemia and insulinemia in ob/ob mice. A 45% fat diet further increased glucose, but not insulin. Inflammation was seen as increased TNFα levels in ob/ob mice. Thus the results implicate disturbed glucose regulation and inflammation as mechanisms involved in the association between obesity and intestinal tumorigenesis. Ob/ob mice had shorter lifespan than ob/wt and wt/wt mice. PMID:26347815

  17. G2019S LRRK2 mutant fibroblasts from Parkinson's disease patients show increased sensitivity to neurotoxin 1-methyl-4-phenylpyridinium dependent of autophagy.

    PubMed

    Yakhine-Diop, Sokhna M S; Bravo-San Pedro, José M; Gómez-Sánchez, Rubén; Pizarro-Estrella, Elisa; Rodríguez-Arribas, Mario; Climent, Vicente; Aiastui, Ana; López de Munain, Adolfo; Fuentes, José M; González-Polo, Rosa A

    2014-10-03

    Parkinson's disease (PD) is a neurodegenerative disorder of unknown etiology. It is considered as a multifactorial disease dependent on environmental and genetic factors. Deregulation in cell degradation has been related with a significant increase in cell damage, becoming a target for studies on the PD etiology. In the present study, we have characterized the parkinsonian toxin 1-methyl-4-phenylpyridinium ion (MPP(+))-induced damage in fibroblasts from Parkinson's patients with the mutation G2019S in leucine-rich repeat kinase 2 protein (LRRK2) and control individuals without this mutation. The results reveal that MPP(+) induces mTOR-dependent autophagy in fibroblasts. Moreover, the effects of caspase-dependent cell death to MPP(+) were higher in cells with the G2019S LRRK2 mutation, which showed basal levels of autophagy due to the G2019S LRRK2 mutation (mTOR-independent). The inhibition of autophagy by 3-methyladenine (3-MA) treatment reduces these sensitivity differences between both cell types, however, the inhibition of autophagosome-lysosome fusion by bafilomycin A1 (Baf A1) increases these differences. This data confirm the importance of the combination of genetic and environmental factors in the PD etiology. Thereby, the sensitivity to the same damage may be different in function of a genetic predisposition, reason why individuals with certain mutations can develop some early-onset diseases, such as individuals with G2019S LRRK2 mutation and PD. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

  18. Increase in IS256 transposition in invasive vancomycin heteroresistant Staphylococcus aureus isolate belonging to ST100 and its derived VISA mutants.

    PubMed

    Di Gregorio, Sabrina; Fernandez, Silvina; Perazzi, Beatriz; Bello, Natalia; Famiglietti, Angela; Mollerach, Marta

    2016-09-01

    In Staphylococcus aureus, transposition of IS256 has been described to play an important role in biofilm formation and antibiotic resistance. This study describes the molecular characterization of two clinical heterogeneous vancomycin-intermediate S. aureus (hVISA) isolates recovered from the same patient (before and after antibiotic treatment) and two VISA derivatives obtained by serial passages in the presence of vancomycin. Our results showed that antibiotic treatment (in vivo and in vitro) could enhance IS256 transposition, being responsible for the eventual loss of agr function. As far as we know this is the first study that reports the increase of IS256 transposition in isogenic strains after antibiotic treatment in a clinical setting. Copyright © 2016 Elsevier B.V. All rights reserved.

  19. Reduced early hypoxic/ischemic brain damage is associated with increased GLT-1 levels in mice expressing mutant (P301L) human tau

    PubMed Central

    Liao, Guanghong; Zhou, Miou; Cheung, Simon; Galeano, James; Nguyen, Nam; Baudry, Michel; Bi, Xiaoning

    2009-01-01

    Mutations in tau proteins are associated with a group of neurodegenerative diseases, termed tauopathies. To investigate whether over-expressing human tau with P301L mutation also affects stroke-induced brain damage, we performed hypoxia/ischemia (H/I) in young adult P301L tau transgenic mice. Surprisingly, brain infarct volume was significantly smaller in transgenic mice compared to wild-type mice 24 h after H/I induction. TUNEL staining also revealed less brain apoptosis in transgenic mice following H/I. H/I resulted in a significant increase in tau fragments generated by caspase activation and a marked decrease in tau phosphorylation at residue T231 in cortex of wild-type but not transgenic mice. Activation of calpain and caspase-3 following H/I was also reduced in transgenic compared to wild-type mice, as reflected by lower levels of the specific spectrin breakdown products generated by calpain or caspase-3. Finally, basal levels of the glial glutamate transporter, GLT-1, were higher in brains of transgenic as compared to wild-type mice. These results support the idea that enhanced levels of GLT-1 in transgenic mice are responsible for reducing H/I-induced brain damage by decreasing extracellular glutamate accumulation and subsequent calpain and caspase activation. PMID:18992725

  20. TT Mutant Homozygote of Kruppel-like Factor 5 Is a Key Factor for Increasing Basal Metabolic Rate and Resting Metabolic Rate in Korean Elementary School Children.

    PubMed

    Choi, Jung Ran; Kwon, In-Su; Kwon, Dae Young; Kim, Myung-Sunny; Lee, Myoungsook

    2013-12-01

    We investigated the contribution of genetic variations of KLF5 to basal metabolic rate (BMR) and resting metabolic rate (RMR) and the inhibition of obesity in Korean children. A variation of KLF5 (rs3782933) was genotyped in 62 Korean children. Using multiple linear regression analysis, we developed a model to predict BMR in children. We divided them into several groups; normal versus overweight by body mass index (BMI) and low BMR versus high BMR by BMR. There were no differences in the distributions of alleles and genotypes between each group. The genetic variation of KLF5 gene showed a significant correlation with several clinical factors, such as BMR, muscle, low-density lipoprotein cholesterol, and insulin. Children with the TT had significantly higher BMR than those with CC (p = 0.030). The highest muscle was observed in the children with TT compared with CC (p = 0.032). The insulin and C-peptide values were higher in children with TT than those with CC (p= 0.029 vs. p = 0.004, respectively). In linear regression analysis, BMI and muscle mass were correlated with BMR, whereas insulin and C-peptide were not associated with BMR. In the high-BMR group, we observed that higher muscle, fat mass, and C-peptide affect the increase of BMR in children with TT (p < 0.001, p < 0.001, and p = 0.018, respectively), while Rohrer's index could explain the usual decrease in BMR (adjust r(2) = 1.000, p < 0.001, respectively). We identified a novel association between TT of KLF5 rs3782933 and BMR in Korean children. We could make better use of the variation within KLF5 in a future clinical intervention study of obesity.

  1. Extreme Depletion of PIP3 Accompanies the Increased Life Span and Stress Tolerance of PI3K-null C. elegans Mutants

    PubMed Central

    Bharill, Puneet; Ayyadevara, Srinivas; Alla, Ramani; Shmookler Reis, Robert J.

    2013-01-01

    The regulation of animal longevity shows remarkable plasticity, in that a variety of genetic lesions are able to extend lifespan by as much as 10-fold. Such studies have implicated several key signaling pathways that must normally limit longevity, since their disruption prolongs life. Little is known, however, about the proximal effectors of aging on which these pathways are presumed to converge, and to date, no pharmacologic agents even approach the life-extending effects of genetic mutation. In the present study, we have sought to define the downstream consequences of age-1 nonsense mutations, which confer 10-fold life extension to the nematode Caenorhabditis elegans – the largest effect documented for any single mutation. Such mutations insert a premature stop codon upstream of the catalytic domain of the AGE-1/p110α subunit of class-I PI3K. As expected, we do not detect class-I PI3K (and based on our sensitivity, it constitutes <14% of wild-type levels), nor do we find any PI3K activity as judged by immunodetection of phosphorylated AKT, which strongly requires PIP3 for activation by upstream kinases, or immunodetection of its product, PIP3. In the latter case, the upper 95%-confidence limit for PIP3 is 1.4% of the wild-type level. We tested a variety of commercially available PI3K inhibitors, as well as three phosphatidylinositol analogs (PIAs) that are most active in inhibiting AKT activation, for effects on longevity and survival of oxidative stress. Of these, GDC-0941, PIA6, and PIA24 (each at 1 or 10 μM) extended lifespan by 7–14%, while PIAs 6, 12, and 24 (at 1 or 10 μM) increased survival time in 5 mM peroxide by 12–52%. These effects may have been conferred by insulinlike signaling, since a reporter regulated by the DAF-16/FOXO transcription factor, SOD-3::GFP, was stimulated by these PIAs in the same rank order (PIA24 > PIA6 > PIA12) as lifespan. A second reporter, PEPCK::GFP, was equally activated (∼40%) by all three. PMID

  2. ZNF75: Isolation of a cDNA clone of the KRAB zinc finger gene subfamily mapped in YACs 1 Mb telomeric of HPRT

    SciTech Connect

    Villa, A.; Zucchi, I.; Susani, L.; Morali, F.; Patrosso, C.; Frattini, A.; Lucchini, F.; Repetto, M.; Sacco, M.G.; Zoppe, M. )

    1993-11-01

    The authors have previously mapped a zinc finger genomic motif (ZNF75) to the Xq26 cytogenetic band by using a hybrid panel. Here, they report the isolation of the transcribed counterpart in a cDNA clone and its further localization. The cDNA clone, from a lung fibroblast library, is assembled from three exons, including a 289 amino acid (AA) long open reading frame containing a recently described motif, the Kruppel-associated box, 42 AA long, in exon 2. By comparison with other reported members of the subfamily, the exon-intron boundaries also appear to be very well conserved. Further analysis allowed mapping of this gene 1 Mb downstream from the HPRT gene in the published YAC contig that extends across Xq26. Two other motifs, 87 and 78% homologous to ZNF75 at the amino acid level, were identified by PCR on total human DNA, but map outside Xq24-qter. 25 refs., 4 figs.

  3. Increased Risk of Lung Cancer Associated with a Functionally Impaired Polymorphic Variant of the Human DNA Glycosylase NEIL2

    PubMed Central

    Dey, Sanjib; Maiti, Amit K; Hegde, Muralidhar L; Hegde, Pavana M; Boldogh, Istvan; Sarkar, Partha S; Abdel-Rahman, Sherif Z; Sarker, Altaf H.; Hang, Bo; Xie, Jingwu; Tomkinson, Alan E; Zhou, Mian; Shen, Binghui; Wang, Guanghai; Wu, Chen; Yu, Dianke; Lin, Dongxin; Cardenas, Victor; Hazra, Tapas K

    2012-01-01

    Human NEIL2, one of five oxidized base-specific DNA glycosylases, is unique in preferentially repairing oxidative damage in transcribed genes. Here we show that depletion of NEIL2 causes a 6- to 7-fold increase in spontaneous mutation frequency in the HPRT gene of the V79 Chinese hamster lung cell line. This prompted us to screen for NEIL2 variants in lung cancer patients’ genomic DNA. We identified several polymorphic variants, among which R103Q and R257L were frequently observed in lung cancer patients. We then characterized these variants biochemically, and observed a modest decrease in DNA glycosylase activity relative to the wild type (WT) only with the R257L mutant protein. However, in reconstituted repair assays containing WT NEIL2 or its R257L and R103Q variants together with other DNA base excision repair (BER) proteins (PNKP, Polβ, Lig IIIα and XRCC1) or using NEIL2-FLAG immunocomplexes, an ~ 5-fold decrease in repair was observed with the R257L variant compared to WT or R103Q NEIL2, apparently due to the R257L mutant’s lower affinity for other repair proteins, particularly Polβ. Notably, increased endogenous DNA damage was observed in NEIL2 variant (R257L)-expressing cells relative to WT cells. Taken together, our results suggest that the decreased DNA repair capacity of the R257L variant can induce mutations that lead to lung cancer development. PMID:22497777

  4. Stochastic properties of radiation-induced DSB: DSB distributions in large scale chromatin loops, the HPRT gene and within the visible volumes of DNA repair foci.

    PubMed

    Ponomarev, Artem L; Costes, Sylvain V; Cucinotta, Francis A

    2008-11-01

    We computed probabilities to have multiple double-strand breaks (DSB), which are produced in DNA on a regional scale, and not in close vicinity, in volumes matching the size of DNA damage foci, of a large chromatin loop, and in the physical volume of DNA containing the HPRT (human hypoxanthine phosphoribosyltransferase) locus. The model is based on a Monte Carlo description of DSB formation by heavy ions in the spatial context of the entire human genome contained within the cell nucleus, as well as at the gene sequence level. We showed that a finite physical volume corresponding to a visible DNA repair focus, believed to be associated with one DSB, can contain multiple DSB due to heavy ion track structure and the DNA supercoiled topography. A corrective distribution was introduced, which was a conditional probability to have excess DSB in a focus volume, given that there was already one present. The corrective distribution was calculated for 19.5 MeV/amu N ions, 3.77 MeV/amu alpha-particles, 1000 MeV/amu Fe ions, and X-rays. The corrected initial DSB yield from the experimental data on DNA repair foci was calculated. The DSB yield based on the corrective function converts the focus yield into the DSB yield, which is comparable with the DSB yield based on the earlier PFGE experiments. The distribution of DSB within the physical limits of the HPRT gene was analyzed by a similar method as well. This corrective procedure shows the applicability of the model and empowers the researcher with a tool to better analyze focus statistics. The model enables researchers to analyze the DSB yield based on focus statistics in real experimental situations that lack one-to-one focus-to-DSB correspondance.

  5. Saccharomyces cerevisiae aldolase mutants.

    PubMed Central

    Lobo, Z

    1984-01-01

    Six mutants lacking the glycolytic enzyme fructose 1,6-bisphosphate aldolase have been isolated in the yeast Saccharomyces cerevisiae by inositol starvation. The mutants grown on gluconeogenic substrates, such as glycerol or alcohol, and show growth inhibition by glucose and related sugars. The mutations are recessive, segregate as one gene in crosses, and fall in a single complementation group. All of the mutants synthesize an antigen cross-reacting to the antibody raised against yeast aldolase. The aldolase activity in various mutant alleles measured as fructose 1,6-bisphosphate cleavage is between 1 to 2% and as condensation of triose phosphates to fructose 1,6-bisphosphate is 2 to 5% that of the wild-type. The mutants accumulate fructose 1,6-bisphosphate from glucose during glycolysis and dihydroxyacetone phosphate during gluconeogenesis. This suggests that the aldolase activity is absent in vivo. PMID:6384192

  6. Increasing the electron-transfer ability of Cyanidioschyzon merolae ferredoxin by a one-point mutation – A high resolution and Fe-SAD phasing crystal structure analysis of the Asp58Asn mutant

    SciTech Connect

    Ueno, Yuko; Matsumoto, Takashi; Yamano, Akihito; Imai, Takeo; Morimoto, Yukio

    2013-07-12

    Highlights: •A single amino acid change on the ferredoxin surface affects electron transfer. •Precise positions of amide atoms were located utilizing no prior structural data. •Ultra high resolution and SAD phasing may be used for bias-free model building. -- Abstract: Cyanidioschyzon merolae (Cm) is a single cell red algae that grows in rather thermophilic (40–50 °C) and acidic (pH 1–3) conditions. Ferredoxin (Fd) was purified from this algae and characterized as a plant-type [2Fe–2S] Fd by physicochemical techniques. A high resolution (0.97 Å) three-dimensional structure of the CmFd D58N mutant molecule has been determined using the Fe-SAD phasing method to clarify the precise position of the Asn58 amide, as this substitution increases the electron-transfer ability relative to wild-type CmFd by a factor of 1.5. The crystal structure reveals an electro-positive surface surrounding Asn58 that may interact with ferredoxin NADP{sup +} reductase or cytochrome c.

  7. Sublethal concentrations of salicylic acid decrease the formation of reactive oxygen species but maintain an increased nitric oxide production in the root apex of the ethylene-insensitive Never ripe tomato mutants

    PubMed Central

    Poór, Péter; Gémes, Katalin

    2011-01-01

    The pattern of salicylic acid (SA)-induced production of reactive oxygen species (ROS) and nitric oxide (NO) were different in the apex of adventitious roots in wild-type and in the ethylene-insensitive Never ripe (Nr) mutants of tomato (Solanum lycopersicum L. cv Ailsa Craig). ROS were upregulated, while NO remained at the control level in apical root tissues of wildtype plants exposed to sublethal concentrations of SA. In contrast, Nr plants expressing a defective ethylene receptor displayed a reduced level of ROS and a higher NO content in the apical root cells. In wild-type plants NO production seems to be ROS(H2O2)-dependent at cell death-inducing concentrations of SA, indicating that ROS and NO may interact to trigger oxidative cell death. In the absence of significant ROS accumulation, the increased NO production caused moderate reduction in cell viability in root apex of Nr plants exposed to 10−3 M SA. This suggests that a functional ethylene signaling pathway is necessary for the control of ROS and NO production induced by SA. PMID:21847015

  8. Sublethal concentrations of salicylic acid decrease the formation of reactive oxygen species but maintain an increased nitric oxide production in the root apex of the ethylene-insensitive never ripe tomato mutants.

    PubMed

    Tari, Irma; Poór, Péter; Gémes, Katalin

    2011-09-01

    The pattern of salicylic acid (SA)-induced production of reactive oxygen species (ROS) and nitric oxide (NO) were different in the apex of adventitious roots in wild-type and in the ethylene-insensitive never ripe (Nr) mutants of tomato (Solanum lycopersicum L. cv Ailsa Craig). ROS were upregulated, while NO remained at the control level in apical root tissues of wildtype plants exposed to sublethal concentrations of SA. In contrast, Nr plants expressing a defective ethylene receptor displayed a reduced level of RO S and a higher NO content in the apical root cells. In wild-type plants NO production seems to be RO S(H2O2)-dependent at cell death-inducing concentrations of SA, indicating that ROS and NO may interact to trigger oxidative cell death. In the absence of significant RO S accumulation, the increased NO production caused moderate reduction in cell viability in root apex of Nr plants exposed to 10(-3) M SA. This suggests that a functional ethylene signaling pathway is necessary for the control of ROS and NO production induced by SA.

  9. Endonuclease IV (nfo) mutant of Escherichia coli.

    PubMed Central

    Cunningham, R P; Saporito, S M; Spitzer, S G; Weiss, B

    1986-01-01

    A cloned gene, designated nfo, caused overproduction of an EDTA-resistant endonuclease specific for apurinic-apyrimidinic sites in DNA. The sedimentation coefficient of the enzyme was similar to that of endonuclease IV. An insertion mutation was constructed in vitro and transferred from a plasmid to the Escherichia coli chromosome. nfo mutants had an increased sensitivity to the alkylating agents methyl methanesulfonate and mitomycin C and to the oxidants tert-butyl hydroperoxide and bleomycin. The nfo mutation enhanced the killing of xth (exonuclease III) mutants by methyl methanesulfonate, H2O2, tert-butyl hydroperoxide, and gamma rays, and it enhanced their mutability by methyl methanesulfonate. It also increased the temperature sensitivity of an xth dut (dUTPase) mutant that is defective in the repair of uracil-containing DNA. These results are consistent with earlier findings that endonuclease IV and exonuclease III both cleave DNA 5' to an apurinic-apyrimidinic site and that exonuclease III is more active. However, nfo mutants were more sensitive to tert-butyl hydroperoxide and to bleomycin than were xth mutants, suggesting that endonuclease IV might recognize some lesions that exonuclease III does not. The mutants displayed no marked increase in sensitivity to 254-nm UV radiation, and the addition of an nth (endonuclease III) mutation to nfo or nfo xth mutants did not significantly increase their sensitivity to any of the agents tested. Images PMID:2430946

  10. Protection against radiation-induced mutations at the hprt locus by spermine and N,N{double_prime}-(dithiodi-2,1-ethanediyl)bis-1,3-propanediamine (WR-33278)

    SciTech Connect

    Grdina, D.J.; Schwartz, J.L. |; Shigematsu, N.

    1993-06-01

    The polyamine spermine and the disulfide NN{double_prime}-(dithiodi-2,1-ethanediyl)bis-1,3-propanediamine (WR-33278) are structurally similar agents capable of binding to DNA. WR-33278 is the disulfide moiety of the clinically studied radioprotective agent (WR-2721). Because of their structural similarities, it was of interest to characterize and compare their radioprotective properties using the endpoints of cell survival and mutation induction at the hypoxanthine-guanine phosphoribosyl transferase (hprt) locus in Chinese hamster AA8 cells. In order to facilitate both the uptake of VM-33278 into cells and the direct comparison between the protective properties of WR-33278 and spermine, these agents were electroporated into cells. Electroporation alone reduced cell survival to 75% but had no effect on hprt mutation frequency. The electroporation of either spermine or WR-33278 at concentrations greater than 0.01 mM was extremely toxic. The exposure of cells to both electroporation and irradiation gave rise to enhanced cell killing and mutation induction. Cell survival values at a radiation dose of 750 cGy were enhanced by factors of 1.3 and 1.8 following electroporation of 0.01 mM of spermine and WR-33278, respectively, 30 min prior to irradiation. Neither agent was protective at a concentration of 0.001 mM. Protection against radiation-induced hprt mutations was observed for both spermine and WR-33278 under all experimental conditions tested.

  11. Hypoxanthine-guanine phosphoribosyl transferase regulates early developmental programming of dopamine neurons: implications for Lesch-Nyhan disease pathogenesis.

    PubMed

    Ceballos-Picot, Irene; Mockel, Lionel; Potier, Marie-Claude; Dauphinot, Luce; Shirley, Thomas L; Torero-Ibad, Raoul; Fuchs, Julia; Jinnah, H A

    2009-07-01

    Hypoxanthine-guanine phosphoribosyltransferase (HPRT) deficiency results in Lesch-Nyhan disease (LND), where affected individuals exhibit a characteristic neurobehavioral disorder that has been linked with dysfunction of dopaminergic pathways of the basal ganglia. Since the functions of HPRT, a housekeeping enzyme responsible for recycling purines, have no direct relationships with the dopaminergic pathways, the mechanisms whereby HPRT deficiency affect them remain unknown. The current studies demonstrate that HPRT deficiency influences early developmental processes controlling the dopaminergic phenotype, using several different cell models for HPRT deficiency. Microarray methods and quantitative PCR were applied to 10 different HPRT-deficient (HPRT(-)) sublines derived from the MN9D cell line. Despite the variation inherent in such mutant sublines, several consistent abnormalities were evident. Most notable were increases in the mRNAs for engrailed 1 and 2, transcription factors known to play a key role in the specification and survival of dopamine neurons. The increases in mRNAs were accompanied by increases in engrailed proteins, and restoration of HPRT reverted engrailed expression towards normal levels, demonstrating a functional relationship between HPRT and engrailed. The functional relevance of the abnormal developmental molecular signature of the HPRT(-) MN9D cells was evident in impoverished neurite outgrowth when the cells were forced to differentiate chemically. To verify that these abnormalities were not idiosyncratic to the MN9D line, HPRT(-) sublines from the SK-N-BE(2) M17 human neuroblastoma line were evaluated and an increased expression of engrailed mRNAs was also seen. Over-expression of engrailed occurred even in primary fibroblasts from patients with LND in a manner that suggested a correlation with disease severity. These results provide novel evidence that HPRT deficiency may affect dopaminergic neurons by influencing early developmental

  12. PDGFRA-mutant syndrome.

    PubMed

    Ricci, Riccardo; Martini, Maurizio; Cenci, Tonia; Carbone, Arnaldo; Lanza, Paola; Biondi, Alberto; Rindi, Guido; Cassano, Alessandra; Larghi, Alberto; Persiani, Roberto; Larocca, Luigi M

    2015-07-01

    Germline PDGFRA mutations cause multiple heterogeneous gastrointestinal mesenchymal tumors. In its familial form this disease, which was formerly termed intestinal neurofibromatosis/neurofibromatosis 3b (INF/NF3b), has been included among familial gastrointestinal stromal tumors (GISTs) because of its genotype, described when GIST was the only known PDGFRA-mutant gastrointestinal tumor. Shortly afterwards, however, inflammatory fibroid polyps also revealed PDGFRA mutations. Subsequently, gastrointestinal CD34+ 'fibrous tumors' of uncertain classification were described in a germline PDGFRA-mutant context. Our aim was to characterize the syndrome produced by germline PDGFRA mutations and establish diagnostic criteria and management strategies for this hitherto puzzling disease. We studied a kindred displaying multiple gastrointestinal mesenchymal tumors, comparing it with published families/individuals with possible analogous conditions. We identified a novel inherited PDGFRA mutation (P653L), constituting the third reported example of familial PDGFRA mutation. In adult mutants we detected inflammatory fibroid polyps, gastric GISTs and gastrointestinal fibrous tumors of uncertain nosology. We demonstrate that the syndrome formerly defined as INF/NF3b (exemplified by the family reported herein) is simplistically considered a form of familial GIST, because inflammatory fibroid polyps often prevail. Fibrous tumors appear variants of inflammatory fibroid polyps. 'INF/NF3b' and 'familial GIST' are misleading terms which we propose changing to 'PDGFRA-mutant syndrome'. In this condition, unlike KIT-dependent familial GIST syndromes, if present, GISTs are stomach-restricted and diffuse Cajal cell hyperplasia is not observed. This restriction of GISTs to the stomach in PDGFRA-mutant syndrome: (i) focuses oncological concern on gastric masses, as inflammatory fibroid polyps are benign; (ii) supports a selective role of gastric environment for PDGFRA mutations to elicit GISTs

  13. Control of carbohydrate processing: increased beta-1,6 branching in N-linked carbohydrates of Lec9 CHO mutants appears to arise from a defect in oligosaccharide-dolichol biosynthesis.

    PubMed Central

    Rosenwald, A G; Stanley, P; Krag, S S

    1989-01-01

    A correlation between increased beta-1,6 branching of N-linked carbohydrates and the ability of a cell to metastasize or to form a tumor has been observed in several experimental models. Lec9 Chinese hamster ovary (CHO) mutants exhibit a drastic reduction in tumorigenicity in nude mice, and this phenotype directly correlates with their ability to attach an increased proportion of beta-1,6-branched carbohydrates to the G glycoprotein of vesicular stomatitis virus (J. Ripka, S. Shin, and P. Stanley, Mol. Cell. Biol. 6:1268-1275, 1986). In this paper we provide evidence that cellular carbohydrates from Lec9 cells also contain an increased proportion of beta-1,6-branched carbohydrates, although they do not possess significantly increased activity of the beta-1,6 branching enzyme (GlcNAc-transferase V). Biosynthetic labeling experiments show that a substantial degree of underglycosylation occurs in Lec9 cells and that this affects several classes of glycoproteins. Lec9 cells synthesize ca. 40-fold less Glc3Man9GlcNAc2-P-P-lipid and ca. 2-fold less Man5GlcNAc2-P-P-lipid than parental cells do. In addition, Lec9 cells possess ca. fivefold less protein-bound oligosaccharide intermediates, and one major species is resistant to release by endo-beta-N-acetylglucosaminidase H (endo H). Membranes of Lec9 cells exhibit normal mannosylphosphoryldolichol synthase, glucosylphosphoryldolichol synthase, and N-acetylglucosaminylphosphate transferase activities in the presence of exogenous dolichyl phosphate. However, in the absence of exogenous dolichyl phosphate, mannosylphosphoryldolichol synthase and glucosylphosphoryldolichol synthase activities are reduced in membranes of Lec9 cells, indicating that membranes of Lec9 cells are deficient in lipid phosphate. This was confirmed by analysis of lipids labeled by [3H]mevalonate, which showed that Lec9 cells have less lipid phosphate than parental CHO cells. Mechanisms by which a defect in the synthesis of dolichol

  14. Brassinosteroid Mutants of Crops.

    PubMed

    Bishop, Gerard J.

    2003-12-01

    Plant steroid hormones, brassinosteroids (BRs), were originally isolated from extracts of pollen because of their growth-promoting properties and their potential use for enhancing crop production. Mutants in the biosynthesis, metabolism, and signaling of brassinolide (BL), the most bioactive BR, are important resources in helping to establish BRs' essential role in plant growth and development. The dark green and distinctive dwarf phenotype of BR-related mutants identified in pea, tomato, and rice highlights the importance of BRs in crops. These mutants are helping to elucidate both the conserved and the unique features of BR biosynthesis and signaling. Such insights are providing the key knowledge and understanding that will enable the development of strategies towards the production of crops with enhanced qualities.

  15. Characteristics of Agrobacterium tumefaciens auxotrophic mutant infectivity.

    PubMed

    Lippincott, B B; Lippincott, J A

    1966-10-01

    Lippincott, Barbara B. (Northwestern University, Evanston, Ill.), and James A. Lippincott. Characteristics of Agrobacterium tumefaciens auxotrophic mutant infectivity. J. Bacteriol. 92:937-945. 166.-Mutants of Agrobacterium tumefaciens auxotrophic for adenine, methionine, or asparagine are less infectious than the wild-type strain B6 from which they were derived and show increased infectivity on pinto bean leaves when the specific compounds required for growth of the mutants are added to the infected leaf. Reversion to a prototrophic form of nutrition is accompanied by increased infectivity. Tumors initiated by these auxotrophic mutants are shown to arise only at large wound sites where nutritional conditions may be less restricting. The data indicate that, after inoculation, the bacteria pass through a phase in which host-supplied nutrients are utilized for the production of one or more factors necessary for successful tumor initiation.

  16. Mutant p53: One, No One, and One Hundred Thousand.

    PubMed

    Walerych, Dawid; Lisek, Kamil; Del Sal, Giannino

    2015-01-01

    Encoded by the mutated variants of the TP53 tumor suppressor gene, mutant p53 proteins are getting an increased experimental support as active oncoproteins promoting tumor growth and metastasis. p53 missense mutant proteins are losing their wild-type tumor suppressor activity and acquire oncogenic potential, possessing diverse transforming abilities in cell and mouse models. Whether various mutant p53s differ in their oncogenic potential has been a matter of debate. Recent discoveries are starting to uncover the existence of mutant p53 downstream programs that are common to different mutant p53 variants. In this review, we discuss a number of studies on mutant p53, underlining the advantages and disadvantages of alternative experimental approaches that have been used to describe the numerous mutant p53 gain-of-function activities. Therapeutic possibilities are also discussed, taking into account targeting either individual or multiple mutant p53 proteins in human cancer.

  17. Increased expression of (pro)renin receptor does not cause hypertension or cardiac and renal fibrosis in mice.

    PubMed

    Rosendahl, Alva; Niemann, Gianina; Lange, Sascha; Ahadzadeh, Erfan; Krebs, Christian; Contrepas, Aurelie; van Goor, Harry; Wiech, Thorsten; Bader, Michael; Schwake, Michael; Peters, Judith; Stahl, Rolf; Nguyen, Geneviève; Wenzel, Ulrich O

    2014-08-01

    Binding of renin and prorenin to the (pro)renin receptor (PRR) increases their enzymatic activity and upregulates the expression of pro-fibrotic genes in vitro. Expression of PRR is increased in the heart and kidney of hypertensive and diabetic animals, but its causative role in organ damage is still unclear. To determine whether increased expression of PRR is sufficient to induce cardiac or renal injury, we generated a mouse that constitutively overexpresses PRR by knocking-in the Atp6ap2/PRR gene in the hprt locus under the control of a CMV immediate early enhancer/chicken beta-actin promoter. Mice were backcrossed in the C57Bl/6 and FVB/N strain and studied at the age of 12 months. In spite of a 25- to 80-fold renal and up to 400-fold cardiac increase in Atp6ap2/PRR expression, we found no differences in systolic blood pressure or albuminuria between wild-type and PRR overexpressing littermates. Histological examination did not show any renal or cardiac fibrosis in mutant mice. This was supported by real-time PCR analysis of inflammatory markers as well as of pro-fibrotic genes in the kidney and collagen in cardiac tissue. To determine whether the concomitant increase of renin would trigger fibrosis, we treated PRR overexpressing mice with the angiotensin receptor-1 blocker losartan over a period of 6 weeks. Renin expression increased eightfold in the kidney but no renal injury could be detected. In conclusion, our results suggest no major role for PRR in organ damage per se or related to its function as a receptor of renin.

  18. Sleep restores behavioral plasticity to Drosophila mutants.

    PubMed

    Dissel, Stephane; Angadi, Veena; Kirszenblat, Leonie; Suzuki, Yasuko; Donlea, Jeff; Klose, Markus; Koch, Zachary; English, Denis; Winsky-Sommerer, Raphaelle; van Swinderen, Bruno; Shaw, Paul J

    2015-05-18

    Given the role that sleep plays in modulating plasticity, we hypothesized that increasing sleep would restore memory to canonical memory mutants without specifically rescuing the causal molecular lesion. Sleep was increased using three independent strategies: activating the dorsal fan-shaped body, increasing the expression of Fatty acid binding protein (dFabp), or by administering the GABA-A agonist 4,5,6,7-tetrahydroisoxazolo-[5,4-c]pyridine-3-ol (THIP). Short-term memory (STM) or long-term memory (LTM) was evaluated in rutabaga (rut) and dunce (dnc) mutants using aversive phototaxic suppression and courtship conditioning. Each of the three independent strategies increased sleep and restored memory to rut and dnc mutants. Importantly, inducing sleep also reverses memory defects in a Drosophila model of Alzheimer's disease. Together, these data demonstrate that sleep plays a more fundamental role in modulating behavioral plasticity than previously appreciated and suggest that increasing sleep may benefit patients with certain neurological disorders.

  19. Targeting Oncogenic Mutant p53 for Cancer Therapy.

    PubMed

    Parrales, Alejandro; Iwakuma, Tomoo

    2015-01-01

    Among genetic alterations in human cancers, mutations in the tumor suppressor p53 gene are the most common, occurring in over 50% of human cancers. The majority of p53 mutations are missense mutations and result in the accumulation of dysfunctional p53 protein in tumors. These mutants frequently have oncogenic gain-of-function activities and exacerbate malignant properties of cancer cells, such as metastasis and drug resistance. Increasing evidence reveals that stabilization of mutant p53 in tumors is crucial for its oncogenic activities, while depletion of mutant p53 attenuates malignant properties of cancer cells. Thus, mutant p53 is an attractive druggable target for cancer therapy. Different approaches have been taken to develop small-molecule compounds that specifically target mutant p53. These include compounds that restore wild-type conformation and transcriptional activity of mutant p53, induce depletion of mutant p53, inhibit downstream pathways of oncogenic mutant p53, and induce synthetic lethality to mutant p53. In this review article, we comprehensively discuss the current strategies targeting oncogenic mutant p53 in cancers, with special focus on compounds that restore wild-type p53 transcriptional activity of mutant p53 and those reducing mutant p53 levels.

  20. Characterization of rag1 mutant zebrafish leukocytes

    PubMed Central

    Petrie-Hanson, Lora; Hohn, Claudia; Hanson, Larry

    2009-01-01

    Background Zebrafish may prove to be one of the best vertebrate models for innate immunology. These fish have sophisticated immune components, yet rely heavily on innate immune mechanisms. Thus, the development and characterization of mutant and/or knock out zebrafish are critical to help define immune cell and immune gene functions in the zebrafish model. The use of Severe Combined Immunodeficient (SCID) and recombination activation gene 1 and 2 mutant mice has allowed the investigation of the specific contribution of innate defenses in many infectious diseases. Similar zebrafish mutants are now being used in biomedical and fish immunology related research. This report describes the leukocyte populations in a unique model, recombination activation gene 1-/- mutant zebrafish (rag1 mutants). Results Differential counts of peripheral blood leukocytes (PBL) showed that rag1 mutants had significantly decreased lymphocyte-like cell populations (34.7%) compared to wild-types (70.5%), and significantly increased granulocyte populations (52.7%) compared to wild-types (17.6%). Monocyte/macrophage populations were similar between mutants and wild-types, 12.6% and 11.3%, respectively. Differential leukocyte counts of rag1 mutant kidney hematopoietic tissue showed a significantly reduced lymphocyte-like cell population (8%), a significantly increased myelomonocyte population (57%), 34.8% precursor cells, and 0.2% thrombocytes, while wild-type hematopoietic kidney tissue showed 29.4% lymphocytes/lymphocyte-like cells, 36.4% myelomonocytes, 33.8% precursors and 0.5% thrombocytes. Flow cytometric analyses of kidney hematopoietic tissue revealed three leukocyte populations. Population A was monocytes and granulocytes and comprised 34.7% of the gated cells in rag1 mutants and 17.6% in wild-types. Population B consisted of hematopoietic precursors, and comprised 50% of the gated cells for rag1 mutants and 53% for wild-types. Population C consisted of lymphocytes and lymphocyte

  1. Fluoroquinolone-resistant mutants of Burkholderia cepacia.

    PubMed

    Pope, C F; Gillespie, S H; Pratten, J R; McHugh, T D

    2008-03-01

    Fluoroquinolone-resistant Burkholderia cepacia mutants were selected on ciprofloxacin. The rate of mutation in gyrA was estimated to be 9.6 x 10(-11) mutations per division. Mutations in gyrA conferred 12- to 64-fold increases in MIC, and an additional parC mutation conferred a large increase in MIC (>256-fold). Growth rate, biofilm formation, and survival in water and during drying were not impaired in strains containing single gyrA mutations. Double mutants were impaired only in growth rate (0.85, relative to the susceptible parent).

  2. A Bystander Effect Observed in Boron Neutron Capture Therapy: A Study of the Induction of Mutations in the HPRT Locus

    SciTech Connect

    Kinashi, Yuko . E-mail: kinashi@rri.kyoto-u.ac.jp; Masunaga, Shinichiro; Nagata, Kenji; Suzuki, Minoru; Takahashi, Sentaro; Ono, Koji

    2007-06-01

    Purpose: To investigate bystander mutagenic effects induced by {alpha}-particles during boron neutron capture therapy, we mixed cells that were electroporated with borocaptate sodium (BSH), which led to the accumulation of {sup 10}B inside the cells, and cells that did not contain the boron compound. The BSH-containing cells were irradiated with {alpha}-particles produced by the {sup 10}B(n,{alpha}){sup 7}Li reaction, whereas cells without boron were affected only by the {sup 1}H(n,{gamma}){sup 2}H and {sup 14}N(n,{rho}){sup 14}C reactions. Methods and Materials: The lethality and mutagenicity measured by the frequency of mutations induced in the hypoxanthine-guanine phosphoribosyltransferase locus were examined in Chinese hamster ovary cells irradiated with neutrons (Kyoto University Research Reactor: 5 MW). Neutron irradiation of 1:1 mixtures of cells with and without BSH resulted in a survival fraction of 0.1, and the cells that did not contain BSH made up 99.4% of the resulting cell population. The molecular structures of the mutations were determined using multiplex polymerase chain reactions. Results: Because of the bystander effect, the frequency of mutations increased in the cells located nearby the BSH-containing cells compared with control cells. Molecular structural analysis indicated that most of the mutations induced by the bystander effect were point mutations and that the frequencies of total and partial deletions induced by the bystander effect were less than those induced by the original neutron irradiation. Conclusion: These results suggested that in boron neutron capture therapy, the mutations caused by the bystander effect and those caused by the original neutron irradiation are induced by different mechanisms.

  3. The zebrafish early arrest mutants.

    PubMed

    Kane, D A; Maischein, H M; Brand, M; van Eeden, F J; Furutani-Seiki, M; Granato, M; Haffter, P; Hammerschmidt, M; Heisenberg, C P; Jiang, Y J; Kelsh, R N; Mullins, M C; Odenthal, J; Warga, R M; Nüsslein-Volhard, C

    1996-12-01

    This report describes mutants of the zebrafish having phenotypes causing a general arrest in early morphogenesis. These mutants identify a group of loci making up about 20% of the loci identified by mutants with visible morphological phenotypes within the first day of development. There are 12 Class I mutants, which fall into 5 complementation groups and have cells that lyse before morphological defects are observed. Mutants at three loci, speed bump, ogre and zombie, display abnormal nuclei. The 8 Class II mutants, which fall into 6 complementation groups, arrest development before cell lysis is observed. These mutants seemingly stop development in the late segmentation stages, and maintain a body shape similar to a 20 hour embryo. Mutations in speed bump, ogre, zombie, specter, poltergeist and troll were tested for cell lethality by transplanting mutant cells into wild-type hosts. With poltergeist, transplanted mutant cells all survive. The remainder of the mutants tested were autonomously but conditionally lethal: mutant cells, most of which lyse, sometimes survive to become notochord, muscles, or, in rare cases, large neurons, all cell types which become postmitotic in the gastrula. Some of the genes of the early arrest group may be necessary for progression though the cell cycle; if so, the survival of early differentiating cells may be based on having their terminal mitosis before the zygotic requirement for these genes.

  4. Superior triacylglycerol (TAG) accumulation in starchless mutants of Scenedesmus obliquus: (I) mutant generation and characterization

    PubMed Central

    2014-01-01

    Background Microalgae are a promising platform for producing neutral lipids, to be used in the application for biofuels or commodities in the feed and food industry. A very promising candidate is the oleaginous green microalga Scenedesmus obliquus, because it accumulates up to 45% w/w triacylglycerol (TAG) under nitrogen starvation. Under these conditions, starch is accumulated as well. Starch can amount up to 38% w/w under nitrogen starvation, which is a substantial part of the total carbon captured. When aiming for optimized TAG production, blocking the formation of starch could potentially increase carbon allocation towards TAG. In an attempt to increase TAG content, productivity and yield, starchless mutants of this high potential strain were generated using UV mutagenesis. Previous studies in Chlamydomonas reinhardtii have shown that blocking the starch synthesis yields higher TAG contents, although these TAG contents do not surpass those of oleaginous microalgae yet. So far no starchless mutants in oleaginous green microalgae have been isolated that result in higher TAG productivities. Results Five starchless mutants have been isolated successfully from over 3,500 mutants. The effect of the mutation on biomass and total fatty acid (TFA) and TAG productivity under nitrogen-replete and nitrogen-depleted conditions was studied. All five starchless mutants showed a decreased or completely absent starch content. In parallel, an increased TAG accumulation rate was observed for the starchless mutants and no substantial decrease in biomass productivity was perceived. The most promising mutant showed an increase in TFA productivity of 41% at 4 days after nitrogen depletion, reached a TAG content of 49.4% (% of dry weight) and had no substantial change in biomass productivity compared to the wild type. Conclusions The improved S. obliquus TAG production strains are the first starchless mutants in an oleaginous green microalga that show enhanced TAG content under

  5. Neurobehavioral Mutants Identified in an ENU Mutagenesis Project

    SciTech Connect

    Cook, Melloni N.; Dunning, Jonathan P; Wiley, Ronald G; Chesler, Elissa J; Johnson, Dabney K; Goldowitz, Daniel

    2007-01-01

    We report on a behavioral screening test battery that successfully identified several neurobehavioral mutants among a large-scale ENU-mutagenized mouse population. Large numbers of ENU mutagenized mice were screened for abnormalities in central nervous system function based on abnormal performance in a series of behavior tasks. We developed and employed a high-throughput screen of behavioral tasks to detect behavioral outliers. Twelve mutant pedigrees, representing a broad range of behavioral phenotypes, have been identified. Specifically, we have identified two open field mutants (one displaying hyper-locomotion, the other hypo-locomotion), four tail suspension mutants (all displaying increased immobility), one nociception mutant (displaying abnormal responsiveness to thermal pain), two prepulse inhibition mutants (displaying poor inhibition of the startle response), one anxiety-related mutant (displaying decreased anxiety in the light/dark test), and one learning and memory mutant (displaying reduced response to the conditioned stimulus) These findings highlight the utility of a set of behavioral tasks used in a high throughput screen to identify neurobehavioral mutants. Further analysis (i.e., behavioral and genetic mapping studies) of mutants is in progress with the ultimate goal of identification of novel genes and mouse models relevant to human disorders as well as the identification of novel therapeutic targets.

  6. ECB deacylase mutants

    DOEpatents

    Arnold, Frances H.; Shao, Zhixin; Zhao, Huimin; Giver, Lorraine J.

    2002-01-01

    A method for in vitro mutagenesis and recombination of polynucleotide sequences based on polymerase-catalyzed extension of primer oligonucleotides is disclosed. The method involves priming template polynucleotide(s) with random-sequences or defined-sequence primers to generate a pool of short DNA fragments with a low level of point mutations. The DNA fragments are subjected to denaturization followed by annealing and further enzyme-catalyzed DNA polymerization. This procedure is repeated a sufficient number of times to produce full-length genes which comprise mutants of the original template polynucleotides. These genes can be further amplified by the polymerase chain reaction and cloned into a vector for expression of the encoded proteins.

  7. Phanerochaete mutants with enhanced ligninolytic activity

    SciTech Connect

    Kakar, S.N.; Perez, A.; Gonzales, J.

    1993-06-01

    In addition to lignin, the white rot fungus Phanerochaete chrysosporium has the ability to degrade a wide spectrum of recalcitrant organopollutants in soils and aqueous media. Although some of the organic compounds are degraded under nonligninolytic conditions, most are degraded under ligninolytic conditions with the involvement of the extracellular enzymes, lignin peroxidases, and manganese-dependent peroxidases, which are produced as secondary metabolites triggered by conditions of nutrient starvation (e.g., nitrogen limitation). The fungus and its enzymes can thus provide alternative technologies for bioremediation, biopulping, biobleaching, and other industrial applications. The efficiency and effectiveness of the fungus can be enhanced by increasing production and secretion of the important enzymes in large quantities and as primary metabolites under enriched conditions. One way this can be achieved is through isolation of mutants that are deregulated or are hyperproducers or supersecretors of key enzymes under enriched conditions. Through ultraviolet-light and gamma-rays mutagenesis we have isolated a variety of mutants, some of which produce key enzymes of the ligninolytic system under high-nitrogen growth conditions. One of the mutants produced 272 units (U) of lignin peroxidases enzyme activity per liter after nine days under high nitrogen. The mutant and the parent strains produced up to 54 U/L and 62 U/L, respectively, of the enzyme activity under low-nitrogen growth conditions during this period. In some experiments the mutant showed 281 U/L of enzyme activity under high nitrogen after 17 days.

  8. Isolation and characterization of transcription fidelity mutants.

    PubMed

    Strathern, Jeffrey N; Jin, Ding Jun; Court, Donald L; Kashlev, Mikhail

    2012-07-01

    Accurate transcription is an essential step in maintaining genetic information. Error-prone transcription has been proposed to contribute to cancer, aging, adaptive mutagenesis, and mutagenic evolution of retroviruses and retrotransposons. The mechanisms controlling transcription fidelity and the biological consequences of transcription errors are poorly understood. Because of the transient nature of mRNAs and the lack of reliable experimental systems, the identification and characterization of defects that increase transcription errors have been particularly challenging. In this review we describe novel genetic screens for the isolation of fidelity mutants in both Saccharomyces cerevisiae and Escherichia coli RNA polymerases. We obtained and characterized two distinct classes of mutants altering NTP misincorporation and transcription slippage both in vivo and in vitro. Our study not only validates the genetic schemes for the isolation of RNA polymerase mutants that alter fidelity, but also sheds light on the mechanism of transcription accuracy. This article is part of a Special Issue entitled: Chromatin in time and space.

  9. A series of N-terminal epitope tagged Hdh knock-in alleles expressing normal and mutant huntingtin: their application to understanding the effect of increasing the length of normal huntingtin’s polyglutamine stretch on CAG140 mouse model pathogenesis

    PubMed Central

    2012-01-01

    Background Huntington’s disease (HD) is an autosomal dominant neurodegenerative disease that is caused by the expansion of a polyglutamine (polyQ) stretch within Huntingtin (htt), the protein product of the HD gene. Although studies in vitro have suggested that the mutant htt can act in a potentially dominant negative fashion by sequestering wild-type htt into insoluble protein aggregates, the role of the length of the normal htt polyQ stretch, and the adjacent proline-rich region (PRR) in modulating HD mouse model pathogenesis is currently unknown. Results We describe the generation and characterization of a series of knock-in HD mouse models that express versions of the mouse HD gene (Hdh) encoding N-terminal hemaglutinin (HA) or 3xFlag epitope tagged full-length htt with different polyQ lengths (HA7Q-, 3xFlag7Q-, 3xFlag20Q-, and 3xFlag140Q-htt) and substitution of the adjacent mouse PRR with the human PRR (3xFlag20Q- and 3xFlag140Q-htt). Using co-immunoprecipitation and immunohistochemistry analyses, we detect no significant interaction between soluble full-length normal 7Q- htt and mutant (140Q) htt, but we do observe N-terminal fragments of epitope-tagged normal htt in mutant htt aggregates. When the sequences encoding normal mouse htt’s polyQ stretch and PRR are replaced with non-pathogenic human sequence in mice also expressing 140Q-htt, aggregation foci within the striatum, and the mean size of htt inclusions are increased, along with an increase in striatal lipofuscin and gliosis. Conclusion In mice, soluble full-length normal and mutant htt are predominantly monomeric. In heterozygous knock-in HD mouse models, substituting the normal mouse polyQ and PRR with normal human sequence can exacerbate some neuropathological phenotypes. PMID:22892315

  10. Characterization of Halobacterium halobium mutants defective in taxis.

    PubMed Central

    Sundberg, S A; Alam, M; Lebert, M; Spudich, J L; Oesterhelt, D; Hazelbauer, G L

    1990-01-01

    Mutant derivatives of Halobacterium halobium previously isolated by using a procedure that selected for defective phototactic response to white light were examined for an array of phenotypic characteristics related to phototaxis and chemotaxis. The properties tested were unstimulated swimming behavior, behaviorial responses to temporal gradients of light and spatial gradients of chemoattractants, content of photoreceptor pigments, methylation of methyl-accepting taxis proteins, and transient increases in rate of release of volatile methyl groups induced by tactic stimulation. Several distinct phenotypes were identified, corresponding to a mutant missing photoreceptors, a mutant defective in the methyltransferase, a mutant altered in control of the methylesterase, and mutants apparently defective in intracellular signaling. All except the photoreceptor mutant were defective in both chemotaxis and phototaxis. Images PMID:2332402

  11. Characterization of Halobacterium halobium mutants defective in taxis.

    PubMed

    Sundberg, S A; Alam, M; Lebert, M; Spudich, J L; Oesterhelt, D; Hazelbauer, G L

    1990-05-01

    Mutant derivatives of Halobacterium halobium previously isolated by using a procedure that selected for defective phototactic response to white light were examined for an array of phenotypic characteristics related to phototaxis and chemotaxis. The properties tested were unstimulated swimming behavior, behaviorial responses to temporal gradients of light and spatial gradients of chemoattractants, content of photoreceptor pigments, methylation of methyl-accepting taxis proteins, and transient increases in rate of release of volatile methyl groups induced by tactic stimulation. Several distinct phenotypes were identified, corresponding to a mutant missing photoreceptors, a mutant defective in the methyltransferase, a mutant altered in control of the methylesterase, and mutants apparently defective in intracellular signaling. All except the photoreceptor mutant were defective in both chemotaxis and phototaxis.

  12. The Outer Membrane of Brucella ovis Shows Increased Permeability to Hydrophobic Probes and Is More Susceptible to Cationic Peptides than Are the Outer Membranes of Mutant Rough Brucella abortus Strains

    PubMed Central

    Freer, Enrique; Pizarro-Cerdá, Javier; Weintraub, Andrej; Bengoechea, José-Antonio; Moriyón, Ignacio; Hultenby, Kjell; Gorvel, Jean-Pierre; Moreno, Edgardo

    1999-01-01

    The permeability of the outer membrane (OM) to hydrophobic probes and its susceptibility to bactericidal cationic peptides were investigated for natural rough Brucella ovis and for mutant rough Brucella abortus strains. The OM of B. ovis displayed an abrupt and faster kinetic profile than rough B. abortus during the uptake of the hydrophobic probe N-phenyl-naphthylamine. B. ovis was more sensitive than rough B. abortus to the action of cationic peptides. Bactenecins 5 and 7 induced morphological alterations on the OMs of both rough Brucella strains. B. ovis lipopolysaccharide (LPS) captured considerably more polymyxin B than LPSs from both rough and smooth B. abortus strains. Polymyxin B, poly-l-lysine, and poly-l-ornithine produced a thick coating on the surfaces of both strains, which was more evident in B. ovis than in rough B. abortus. The distinct functional properties of the OMs of these two rough strains correlate with some structural differences of their OMs and with their different biological behaviors in animals and culture cells. PMID:10531286

  13. Mutant maize variety containing the glt1-1 allele

    DOEpatents

    Nelson, O.E.; Pan, D.

    1994-07-19

    A maize plant has in its genome a non-mutable form of a mutant allele designated vitX-8132. The allele is located at a locus designated as glt which conditions kernels having an altered starch characteristic. Maize plants including such a mutant allele produce a starch that does not increase in viscosity on cooling, after heating. 2 figs.

  14. Mutant maize variety containing the glt1-1 allele

    DOEpatents

    Nelson, Oliver E.; Pan, David

    1994-01-01

    A maize plant has in its genome a non-mutable form of a mutant allele designated vitX-8132. The allele is located at a locus designated as glt which conditions kernels having an altered starch characteristic. Maize plants including such a mutant allele produce a starch that does not increase in viscosity on cooling, after heating.

  15. Amuvatinib has cytotoxic effects against NRAS-mutant melanoma but not BRAF-mutant melanoma.

    PubMed

    Fedorenko, Inna V; Fang, Bin; Koomen, John M; Gibney, Geoffrey T; Smalley, Keiran S M

    2014-10-01

    Effective targeted therapy strategies are still lacking for the 15-20% of melanoma patients whose melanomas are driven by oncogenic NRAS. Here, we report on the NRAS-specific behavior of amuvatinib, a kinase inhibitor with activity against c-KIT, Axl, PDGFRα, and Rad51. An analysis of BRAF-mutant and NRAS-mutant melanoma cell lines showed the NRAS-mutant cohort to be enriched for targets of amuvatinib, including Axl, c-KIT, and the Axl ligand Gas6. Increasing concentrations of amuvatinib selectively inhibited the growth of NRAS-mutant, but not BRAF-mutant melanoma cell lines, an effect associated with induction of S-phase and G2/M-phase cell cycle arrest and induction of apoptosis. Mechanistically, amuvatinib was noted to either inhibit Axl, AKT, and MAPK signaling or Axl and AKT signaling and to induce a DNA damage response. In three-dimensional cell culture experiments, amuvatinib was cytotoxic against NRAS-mutant melanoma cell lines. Thus, we show for the first time that amuvatinib has proapoptotic activity against melanoma cell lines, with selectivity observed for those harboring oncogenic NRAS.

  16. Native Mutant Huntingtin in Human Brain

    PubMed Central

    Sapp, Ellen; Valencia, Antonio; Li, Xueyi; Aronin, Neil; Kegel, Kimberly B.; Vonsattel, Jean-Paul; Young, Anne B.; Wexler, Nancy; DiFiglia, Marian

    2012-01-01

    Huntington disease (HD) is caused by polyglutamine expansion in the N terminus of huntingtin (htt). Analysis of human postmortem brain lysates by SDS-PAGE and Western blot reveals htt as full-length and fragmented. Here we used Blue Native PAGE (BNP) and Western blots to study native htt in human postmortem brain. Antisera against htt detected a single band broadly migrating at 575–850 kDa in control brain and at 650–885 kDa in heterozygous and Venezuelan homozygous HD brains. Anti-polyglutamine antisera detected full-length mutant htt in HD brain. There was little htt cleavage even if lysates were pretreated with trypsin, indicating a property of native htt to resist protease cleavage. A soluble mutant htt fragment of about 180 kDa was detected with anti-htt antibody Ab1 (htt-(1–17)) and increased when lysates were treated with denaturants (SDS, 8 m urea, DTT, or trypsin) before BNP. Wild-type htt was more resistant to denaturants. Based on migration of in vitro translated htt fragments, the 180-kDa segment terminated ≈htt 670–880 amino acids. If second dimension SDS-PAGE followed BNP, the 180-kDa mutant htt was absent, and 43–50 kDa htt fragments appeared. Brain lysates from two HD mouse models expressed native full-length htt; a mutant fragment formed if lysates were pretreated with 8 m urea + DTT. Native full-length mutant htt in embryonic HD140Q/140Q mouse primary neurons was intact during cell death and when cell lysates were exposed to denaturants before BNP. Thus, native mutant htt occurs in brain and primary neurons as a soluble full-length monomer. PMID:22375012

  17. The occurrence of new mutants in the X-linked recessive Lesch-Nyhan disease.

    PubMed Central

    Francke, U; Felsenstein, J; Gartler, S M; Migeon, B R; Dancis, J; Seegmiller, J E; Bakay, F; Nyhan, W L

    1976-01-01

    In a population at equilibrium for a sex-linked lethal, one-third of the genes for that lethal must arise anew each generation. Therefore, one-third of all cases of Lesch-Nyhan disease, a severe X-linked recessive lethal disorder, should be new mutants. To test this hypothesis, we have collected 47 families, 20 with a single proband and 27 with multiple affected males in which the patients' mothers and other female relatives had been studied for heterozygosity. Available carrier detection tests identify heterozygous for HPRT deficiency in hair roots and skin fibroblasts. Only four mothers were found not to be carriers. This result deviates significantly from expected (P less than .001). Statistical tests for ascertainment effects indicated absence of bias for multiple proband families but strong bias in favor of families with many heterozygous females. When the analysis was limited to single proband families, the deviation from expected was still significant (P less than .01). The incidence of new mutants among the heterozygous mothers, as determined by the ratio of +/+ to +/- maternal grandmothers, should be one-half (see Appendix). Of all 20 maternal grandmothers studied, five were +/+ and 15 were +/- (P less than .05). Considering only the single proband families, the ratio of 5 +/+ to 8 +/- was not significantly different from expected. In four of the five cases in which the heterozygous mother of an affected individual was a new mutation, the age of her parents was considerably higher than the mean parental age in the population. This raises the possibility of a paternal age effect on X-linked mutations. There appears to be a true deficiency of new mutatnts among males but not among females. Data on additional Lesch-Nyhan families are needed before conclusions regarding a possible higher mutation rate in males can be drawn. PMID:1266847

  18. Protection against radiation-induced mutations at the hprt locus by spermine and N,N{double_prime}-(dithiodi-2,1-ethanediyl)bis-1,3-propanediamine (WR-33278). WR-33278 and spermine protect against mutation induction

    SciTech Connect

    Grdina, D.J.; Shigematsu, N.; Schwartz, J.L.

    1994-08-01

    The polyamine spermine and the disulfide N,N{double_prime}-(dithiodi-2,1-ethanediyl)bis-1,3-propanediamine (WR-33278) are structurally similar agents capable of binding to DNA. WR-33278 is the disulfide moiety of the clinically studied radioprotective agent S-2-(3-aminopropylamino)ethylphosphorothioic acid (WR-2721). Because of their reported structural and functional similarities, it was of interest to characterize and compare their radioprotective properties using the endpoints of cell survival and mutation induction at the hypoxanthine-guanine phosphoribosyl transferase (hprt) locus in Chinese hamster AA8 cells. In order to facilitate both the uptake of WR-33278 into cells and the direct comparison between the protective properties of WR-33278 and spermine, these agents (at concentrations of 0.01 mM and 0.001 mM) were electroporated into cells. The exposure of cells to both electroporation and irradiation gave rise to enhanced cell killing and mutation induction, with the sequence of irradiation followed 3 h later by electroporation being the more toxic protocol. Enhanced cell survival was observed following electroporation of 0.01 mM of spermine and WR-33278 30 min prior to irradiation; protection factors (PF) of 1.3 and 1.8, respectively. Neither agent was protective at a concentration of 0.001 mM. Protection against radiation-induced hprt mutations was observed for both spermine and WR-33278 under all experimental conditions tested. These data suggest that the properties of radioprotection and chemoprevention exhibited by the phosphorothioate (WR-2721) and associated aminothiol (WR-1065) and disulfide (WR-33278) metabolites may be mediated via endogenous spermine-like polyamine processes. Such a mechanism would have important implications with respect to the design and development of new generation drugs for use in radioprotection and chemoprevention.

  19. Sleep restores behavioral plasticity to Drosophila mutants

    PubMed Central

    Dissel, Stephane; Angadi, Veena; Kirszenblat, Leonie; Suzuki, Yasuko; Donlea, Jeff; Klose, Markus; Koch, Zachary; English, Denis; Winsky-Sommerer, Raphaelle; van Swinderen, Bruno; Shaw, Paul J.

    2015-01-01

    SUMMARY Given the role that sleep plays in modulating plasticity, we hypothesized that increasing sleep would restore memory to canonical memory mutants without specifically rescuing the causal molecular-lesion. Sleep was increased using three independent strategies: activating the dorsal Fan Shaped Body (FB), increasing the expression of Fatty acid binding protein (dFabp) or by administering the GABA-A agonist 4,5,6,7-tetrahydroisoxazolo-[5,4-c]pyridine-3-ol (THIP). Short-term memory (STM) or Long-term memory (LTM) was evaluated in rutabaga (rut) and dunce (dnc) mutants using Aversive Phototaxic Suppression (APS) and courtship conditioning. Each of the three independent strategies increased sleep and restored memory to rut and dnc mutants. Importantly, inducing sleep also reverses memory defects in a Drosophila model of Alzheimer’s disease. Together these data demonstrate that sleep plays a more fundamental role in modulating behavioral plasticity than previously appreciated and suggests that increasing sleep may benefit patients with certain neurological disorders. PMID:25913403

  20. Yeast mutants overproducing iso-cytochromes c

    SciTech Connect

    Sherman, F.; Cardillo, T.S.; Errede, B.; Friedman, L.; McKnight, G.; Stiles, J.I.

    1980-01-01

    For over 15 years, the iso-cytochrome c system in the yeast Saccharomyces cerevisiae has been used to investigate a multitude of problems in genetics and molecular biology. More recently, attention has been focused on using mutants for examining translation and transcriptional processes and for probing regulatory regions governing gene expression. In an effort to explore regulatory mechanisms and to investigate mutational alterations that lead to increased levels of gene products, we have isolated and characterized mutants that overproduce cytochrome c. In this paper we have briefly summarized background information of some essential features of the iso-cytochrome c system and we have described the types of mutants that overproduce iso-1-cytochrome c or iso-2-cytochrome c. Genetic procedures and recombinant DNA procedures were used to demonstrate that abnormally high amounts of gene products occur in mutants as result of duplications of gene copies or of extended alteration of regulatory regions. The results summarized in this paper point out the requirements of gross mutational changes or rearrangements of chromosomal segments for augmenting gene products.

  1. Aminoglycoside-resistant mutants of Pseudomonas aeruginosa deficient in cytochrome d, nitrite reductase, and aerobic transport.

    PubMed Central

    Bryan, L E; Kwan, S

    1981-01-01

    Two gentamicin-resistant mutants of Pseudomonas aeruginosa PAO 503 were selected after ethyl methane sulfonate mutagenesis. Mutant PAO 2403 had significantly increased resistance to aminoglycoside but not to other antibiotics. Mutant PAO 2402 showed a similar spectrum of resistance but of lower magnitude. Both mutants showed no detectable cytochrome d and had a high frequency of reversion to a fully wild-type phenotype. PAO 2403 had a marked decrease and PAO 2402 had a moderate decrease in nitrite reductase activity. Both mutants had reduced uptake of gentamicin and dihydrostreptomycin. Mutant PAO 2403 showed a general decrease in transport rate of cationic compounds, whereas mutant PAO 2402 had only deficient glucose transport. Both mutants showed enhanced rates of glutamine transport and no change in glutamic acid transport. Other components of electron transport and oxidative phosphorylation were normal. These mutants involve ferrocytochrome C551 oxidoreductase formed only on anaerobic growth but illustrate transport defects in aerobically grown cells. PMID:6791588

  2. Comparative stability of dihydrofolate reductase mutants in vitro and in vivo.

    PubMed

    Leontiev, V V; Uversky, V N; Gudkov, A T

    1993-01-01

    Dihydrofolate reductase mutants with amino acid replacements in the active center (Thr35-->Asp mutant, Arg57-->His mutant and the mutant with triple replacement Thr35-->Asp, Asn37-->Ser, Arg57-->His) were obtained by site-directed mutagenesis. The stabilization effect of trimethoprim and NADP.H on the protein tertiary structure in vitro has been investigated. In the case of mutants with a 'weak' tertiary structure (Thr35-->Asp35 and the triple mutant) the separate addition of ligands does not affect their stability. The simultaneous addition of these ligands to Thr35-->Asp35 and the triple mutant leads to the large increase in their stability. A distinct correlation was found between the in vitro studied stability of the mutant proteins to the urea- or heat-induced denaturation and the level of proteolytic degradation of these mutants previously observed in vivo.

  3. Architectural phenotypes in the transparent testa mutants of Arabidopsis thaliana

    PubMed Central

    Buer, Charles S.; Djordjevic, Michael A.

    2009-01-01

    Flavonoids are low molecular weight secondary plant metabolites with a myriad of functions. As flavonoids affect auxin transport (an important growth-controlling hormone) and are biologically active in eukaryotes, flavonoid mutants were expected to have undescribed architectural phenotypes. The Arabidopsis thaliana transparent testa (tt) mutants are compromised in the enzymatic steps or transcriptional regulators affecting flavonoid synthesis. tt mutant seedlings were grown on hard-slanted agar (a stress condition), under varying light conditions, and in soil to examine the resulting growth patterns. These tt mutants revealed a wide variety of architectural phenotypes in root and aerial tissues. Mutants with increased inflorescences, siliques, and lateral root density or reduced stature are traits that could affect plant yield or performance under certain environmental conditions. The regulatory genes affected in architectural traits may provide useful molecular targets for examination in other plants. PMID:19129166

  4. Tetrahymena mutants with short telomeres.

    PubMed Central

    Ahmed, S; Sheng, H; Niu, L; Henderson, E

    1998-01-01

    Telomere length is dynamic in many organisms. Genetic screens that identify mutants with altered telomere lengths are essential if we are to understand how telomere length is regulated in vivo. In Tetrahymena thermophila, telomeres become long at 30 degrees, and growth rate slows. A slow-growing culture with long telomeres is often overgrown by a variant cell type with short telomeres and a rapid-doubling rate. Here we show that this variant cell type with short telomeres is in fact a mutant with a genetic defect in telomere length regulation. One of these telomere growth inhibited forever (tgi) mutants was heterozygous for a telomerase RNA mutation, and this mutant telomerase RNA caused telomere shortening when overexpressed in wild-type cells. Several other tgi mutants were also likely to be heterozygous at their mutant loci, since they reverted to wild type when selective pressure for short telomeres was removed. These results illustrate that telomere length can regulate growth rate in Tetrahymena and that this phenomenon can be exploited to identify genes involved in telomere length regulation. PMID:9755196

  5. Histological and Molecular Characterization of Grape Early Ripening Bud Mutant.

    PubMed

    Guo, Da-Long; Yu, Yi-He; Xi, Fei-Fei; Shi, Yan-Yan; Zhang, Guo-Hai

    2016-01-01

    An early ripening bud mutant was analyzed based on the histological, SSR, and methylation-sensitive amplified polymorphism (MSAP) analysis and a layer-specific approach was used to investigate the differentiation between the bud mutant and its parent. The results showed that the thickness of leaf spongy tissue of mutant (MT) is larger than that of wild type (WT) and the differences are significant. The mean size of cell layer L2 was increased in the mutant and the difference is significant. The genetic background of bud mutant revealed by SSR analysis is highly uniform to its parent; just the variations from VVS2 SSR marker were detected in MT. The total methylation ratio of MT is lower than that of the corresponding WT. The outside methylation ratio in MT is much less than that in WT; the average inner methylation ratio in MT is larger than that in WT. The early ripening bud mutant has certain proportion demethylation in cell layer L2. All the results suggested that cell layer L2 of the early ripening bud mutant has changed from the WT. This study provided the basis for a better understanding of the characteristic features of the early ripening bud mutant in grape.

  6. Histological and Molecular Characterization of Grape Early Ripening Bud Mutant

    PubMed Central

    Yu, Yi-He; Xi, Fei-Fei; Shi, Yan-Yan; Zhang, Guo-Hai

    2016-01-01

    An early ripening bud mutant was analyzed based on the histological, SSR, and methylation-sensitive amplified polymorphism (MSAP) analysis and a layer-specific approach was used to investigate the differentiation between the bud mutant and its parent. The results showed that the thickness of leaf spongy tissue of mutant (MT) is larger than that of wild type (WT) and the differences are significant. The mean size of cell layer L2 was increased in the mutant and the difference is significant. The genetic background of bud mutant revealed by SSR analysis is highly uniform to its parent; just the variations from VVS2 SSR marker were detected in MT. The total methylation ratio of MT is lower than that of the corresponding WT. The outside methylation ratio in MT is much less than that in WT; the average inner methylation ratio in MT is larger than that in WT. The early ripening bud mutant has certain proportion demethylation in cell layer L2. All the results suggested that cell layer L2 of the early ripening bud mutant has changed from the WT. This study provided the basis for a better understanding of the characteristic features of the early ripening bud mutant in grape. PMID:27610363

  7. Construction and physiological analysis of a Xanthomonas oryzae pv. oryzae recA mutant.

    PubMed

    Mongkolsuk, S; Rabibhadana, S; Sukchavalit, R; Vaughn, G

    1998-12-15

    A Xoo recA insertion inactivation mutant was constructed. The mutant, lacking RecA, showed increased sensitivity towards mutagen killing. This phenotype could be complemented by a cloned, functional recA. Unlike other bacteria, both the recA mutant and the parental strain had similar level of resistance to H2O2 killing and peroxide-induced mutagenesis.

  8. Acriflavine-Resistant Mutant of Streptococcus cremoris†

    PubMed Central

    Sinha, R.P.

    1977-01-01

    Selection for resistance to acriflavine in Streptococcus cremoris resulted in cross-resistance to the drugs neomycin, streptomycin, ethidium bromide, mitomycin C, and proflavine. Furthermore, the mutants showed resistance to lytic bacteriophages to which the parental strain was sensitive, and, unlike the parent, the mutants grew well at higher temperatures (40°C). Revertants selected independently either for temperature sensitivity or for acriflavine sensitivity lost resistance to all the drugs and dyes but retained the bacteriophage resistance phenotype. The acriflavine-resistant mutation resulted in an increase in resistance by the bacterial cells to sodium dodecyl sulfate, a potent solvent of lipopolysaccharide and lipoprotein. It is suggested that the acriflavine resistance mutation determines the synthesis of a membrane substance resistant to higher temperatures. PMID:907329

  9. Loss of BRCA1 leads to an increase in epidermal growth factor receptor expression in mammary epithelial cells, and epidermal growth factor receptor inhibition prevents estrogen receptor-negative cancers in BRCA1-mutant mice

    PubMed Central

    2011-01-01

    Introduction Women who carry a BRCA1 mutation typically develop "triple-negative" breast cancers (TNBC), defined by the absence of estrogen receptor (ER), progesterone receptor and Her2/neu. In contrast to ER-positive tumors, TNBCs frequently express high levels of epidermal growth factor receptor (EGFR). Previously, we found a disproportionate fraction of progenitor cells in BRCA1 mutation carriers with EGFR overexpression. Here we examine the role of EGFR in mammary epithelial cells (MECs) in the emergence of BRCA1-related tumors and as a potential target for the prevention of TNBC. Methods Cultures of MECs were used to examine EGFR protein levels and promoter activity in response to BRCA1 suppression with inhibitory RNA. EGFR was assessed by immunoblot and immunofluorescence analysis, real-time reverse transcriptase-polymerase chain reaction assay (RT-PCR) and flow cytometry. Binding of epidermal growth factor (EGF) to subpopulations of MECs was examined by Scatchard analysis. The responsiveness of MECs to the EGFR inhibitor erlotinib was assessed in vitro in three-dimensional cultures and in vivo. Mouse mammary tumor virus-Cre recombinase (MMTV-Cre) BRCA1flox/flox p53+/- mice were treated daily with erlotinib or vehicle control, and breast cancer-free survival was analyzed using the Kaplan-Meier method. Results Inhibition of BRCA1 in MECs led to upregulation of EGFR with an inverse correlation of BRCA1 with cellular EGFR protein levels (r2 = 0.87) and to an increase in cell surface-expressed EGFR. EGFR upregulation in response to BRCA1 suppression was mediated by transcriptional and posttranslational mechanisms. Aldehyde dehydrogenase 1 (ALDH1)-positive MECs expressed higher levels of EGFR than ALDH1-negative MECs and were expanded two- to threefold in the BRCA1-inhibited MEC population. All MECs were exquisitely sensitive to EGFR inhibition with erlotinib in vitro. EGFR inhibition in MMTV-Cre BRCA1flox/flox p53+/- female mice starting at age 3 months increased

  10. Mouse infection and pathogenesis by Trypanosoma brucei motility mutants.

    PubMed

    Kisalu, Neville K; Langousis, Gerasimos; Bentolila, Laurent A; Ralston, Katherine S; Hill, Kent L

    2014-06-01

    The flagellum of Trypanosoma brucei is an essential and multifunctional organelle that drives parasite motility and is receiving increased attention as a potential drug target. In the mammalian host, parasite motility is suspected to contribute to infection and disease pathogenesis. However, it has not been possible to test this hypothesis owing to lack of motility mutants that are viable in the bloodstream life cycle stage that infects the mammalian host. We recently identified a bloodstream-form motility mutant in 427-derived T. brucei in which point mutations in the LC1 dynein subunit disrupt propulsive motility but do not affect viability. These mutants have an actively beating flagellum, but cannot translocate. Here we demonstrate that the LC1 point mutant fails to show enhanced cell motility upon increasing viscosity of the surrounding medium, which is a hallmark of wild type T. brucei, thus indicating that motility of the mutant is fundamentally altered compared with wild type cells. We next used the LC1 point mutant to assess the influence of trypanosome motility on infection in mice. Wesurprisingly found that disrupting parasite motility has no discernible effect on T. brucei bloodstream infection. Infection time-course, maximum parasitaemia, number of waves of parasitaemia, clinical features and disease outcome are indistinguishable between motility mutant and control parasites. Our studies provide an important step toward understanding the contribution of parasite motility to infection and a foundation for future investigations of T. brucei interaction with the mammalian host.

  11. Isolation and characterization of gallium resistant Pseudomonas aeruginosa mutants.

    PubMed

    García-Contreras, Rodolfo; Lira-Silva, Elizabeth; Jasso-Chávez, Ricardo; Hernández-González, Ismael L; Maeda, Toshinari; Hashimoto, Takahiro; Boogerd, Fred C; Sheng, Lili; Wood, Thomas K; Moreno-Sánchez, Rafael

    2013-12-01

    Pseudomonas aeruginosa PA14 cells resistant to the novel antimicrobial gallium nitrate (Ga) were developed using transposon mutagenesis and by selecting spontaneous mutants. The mutants showing the highest growth in the presence of Ga were selected for further characterization. These mutants showed 4- to 12-fold higher Ga minimal inhibitory growth concentrations and a greater than 8-fold increase in the minimum biofilm eliminating Ga concentration. Both types of mutants produced Ga resistant biofilms whereas the formation of wild-type biofilms was strongly inhibited by Ga. The gene interrupted in the transposon mutant was hitA, which encodes a periplasmic iron binding protein that delivers Fe³⁺ to the HitB iron permease; complementation of the mutant with the hitA gene restored the Ga sensitivity. This hitA mutant showed a 14-fold decrease in Ga internalization versus the wild-type strain, indicating that the HitAB system is also involved in the Ga uptake. Ga uptake in the spontaneous mutant was also lower, although no mutations were found in the hitAB genes. Instead, this mutant harbored 64 non-silent mutations in several genes including those of the phenazine pyocyanin biosynthesis. The spontaneous mutant produced 2-fold higher pyocyanin basal levels than the wild-type; the addition of this phenazine to wild-type cultures protected them from the Ga bacteriostatic effect. The present data indicate that mutations affecting Ga transport and probably pyocyanin biosynthesis enable cells to develop resistance to Ga. Copyright © 2013 Elsevier GmbH. All rights reserved.

  12. Isolation and Characterization of mAMSA-hypersensitive Mutants

    PubMed Central

    Rogojina, Anna T.; Nitiss, John L.

    2008-01-01

    Topoisomerase II (Top2) is the primary target for active anti-cancer agents. We developed an efficient approach for identifying hypersensitive Top2 mutants and isolated a panel of mutants in yeast Top2 conferring hypersensitivity to the intercalator N-[4-(9-acridinylamino)-3-methoxyphenyl]methanesulphonanilide (mAMSA). Some mutants conferred hypersensitivity to etoposide as well as mAMSA, whereas other mutants exhibited hypersensitivity only to mAMSA. Two mutants in Top2, changing Pro473 to Leu and Gly737 to Val, conferred extraordinary hypersensitivity to mAMSA and were chosen for further characterization. The mutant proteins were purified, and their biochemical activities were assessed. Both mutants encode enzymes that are hypersensitive to inhibition by mAMSA and other intercalating agents and exhibited elevated levels of mAMSA-induced Top2:DNA covalent complexes. While Gly737 → Val Top2p generated elevated levels of Top2-mediated double strand breaks in vitro, the Pro473 → Leu mutant protein showed only a modest increase in Top2-mediated double strand breaks but much higher levels of Top2-mediated single strand breaks. In addition, the Pro473 → Leu mutant protein also generated high levels of mAMSA-stabilized covalent complexes in the absence of ATP. We tested the role of single strand cleavage in cell killing with alleles of Top2 that could generate single strand breaks, but not double strand breaks. Expression in yeast of a Pro473 → Leu mutant that could only generate single strand breaks conferred hypersensitivity to mAMSA. These results indicate that generation of single strand breaks by Top2-targeting agents can be an important component of cell killing by Top2-targeting drugs. PMID:18723844

  13. Identification of GRO1 as a critical determinant for mutant p53 gain of function.

    PubMed

    Yan, Wensheng; Chen, Xinbin

    2009-05-01

    Mutant p53 gain of function contributes to cancer progression, increased invasion and metastasis potentials, and resistance to anticancer therapy. The ability of mutant p53 to acquire its gain of function is shown to correlate with increased expression of progrowth genes, such as c-MYC, MDR1, and NF-kappaB2. However, most of the published studies to identify mutant p53 target genes were performed in a cell system that artificially overexpresses mutant p53. Thus, it remains unclear whether such mutant p53 targets can be regulated by endogenous physiological levels of mutant p53. Here, we utilized SW480 and MIA-PaCa-2 cells, in which endogenous mutant p53 can be inducibly knocked down, to identify mutant p53 target genes that potentially mediate mutant p53 gain of function. We found that knockdown of mutant p53 inhibits GRO1 expression, whereas ectopic expression of mutant R175H in p53-null HCT116 cells increases GRO1 expression. In addition, we found that endogenous mutant p53 is capable of binding to and activating the GRO1 promoter. Interestingly, ectopic expression of GRO1 can rescue the proliferative defect in SW480 and MIA-PaCa-2 cells induced by knockdown of mutant p53. Conversely, knockdown of endogenous GRO1 inhibits cell proliferation and thus abrogates mutant p53 gain of function in SW480 cells. Taken together, our findings define a novel mechanism by which mutant p53 acquires its gain of function via transactivating the GRO1 gene in cancer cells. Thus, targeting GRO1 for cancer therapy would be applicable to a large portion of human tumors with mutant p53, but the exploration of GRO1 as a potential target should take the mutation status of p53 into consideration.

  14. Combined Isothermal Titration and Differential Scanning Calorimetry Define Three-State Thermodynamics of fALS-Associated Mutant Apo SOD1 Dimers and an Increased Population of Folded Monomer.

    PubMed

    Broom, Helen R; Vassall, Kenrick A; Rumfeldt, Jessica A O; Doyle, Colleen M; Tong, Ming Sze; Bonner, Julia M; Meiering, Elizabeth M

    2016-01-26

    Many proteins are naturally homooligomers, homodimers most frequently. The overall stability of oligomeric proteins may be described in terms of the stability of the constituent monomers and the stability of their association; together, these stabilities determine the populations of different monomer and associated species, which generally have different roles in the function or dysfunction of the protein. Here we show how a new combined calorimetry approach, using isothermal titration calorimetry to define monomer association energetics together with differential scanning calorimetry to measure total energetics of oligomer unfolding, can be used to analyze homodimeric unmetalated (apo) superoxide dismutase (SOD1) and determine the effects on the stability of structurally diverse mutations associated with amyotrophic lateral sclerosis (ALS). Despite being located throughout the protein, all mutations studied weaken the dimer interface, while concomitantly either decreasing or increasing the marginal stability of the monomer. Analysis of the populations of dimer, monomer, and unfolded monomer under physiological conditions of temperature, pH, and protein concentration shows that all mutations promote the formation of folded monomers. These findings may help rationalize the key roles proposed for monomer forms of SOD1 in neurotoxic aggregation in ALS, as well as roles for other forms of SOD1. Thus, the results obtained here provide a valuable approach for the quantitative analysis of homooligomeric protein stabilities, which can be used to elucidate the natural and aberrant roles of different forms of these proteins and to improve methods for predicting protein stabilities.

  15. Ascorbate, added after irradiation, reduces the mutant yield and alters the spectrum of CD59- mutations in A(L) cells irradiated with high LET carbon ions

    NASA Technical Reports Server (NTRS)

    Ueno, Akiko; Vannais, Diane; Lenarczyk, Marek; Waldren, Charles A.; Chatterjee, A. (Principal Investigator)

    2002-01-01

    It has been reported that X-ray induced HPRT- mutation in cultured human cells is prevented by ascorbate added after irradiation. Mutation extinction is attributed to neutralization by ascorbate, of radiation-induced long-lived radicals (LLR) with half-lives of several hours. We here show that post-irradiation treatment with ascorbate (5 mM added 30 min after radiation) reduces, but does not eliminate, the induction of CD59- mutants in human-hamster hybrid A(L) cells exposed to high-LET carbon ions (LET of 100 KeV/microm). RibCys, [2(R,S)-D-ribo-1',2',3',4'-Tetrahydroxybutyl]-thiazolidene-4(R)-ca riboxylic acid] (4 mM) gave a similar but lesser effect. The lethality of the carbon ions was not altered by these chemicals. Preliminary data are presented that ascorbate also alters the spectrum of CD59- mutations induced by the carbon beam, mainly by reducing the incidence of small mutations and mutants displaying transmissible genomic instability (TGI), while large mutations are unaffected. Our results suggest that LLR are important in initiating TGI.

  16. Ascorbate, added after irradiation, reduces the mutant yield and alters the spectrum of CD59- mutations in A(L) cells irradiated with high LET carbon ions

    NASA Technical Reports Server (NTRS)

    Ueno, Akiko; Vannais, Diane; Lenarczyk, Marek; Waldren, Charles A.; Chatterjee, A. (Principal Investigator)

    2002-01-01

    It has been reported that X-ray induced HPRT- mutation in cultured human cells is prevented by ascorbate added after irradiation. Mutation extinction is attributed to neutralization by ascorbate, of radiation-induced long-lived radicals (LLR) with half-lives of several hours. We here show that post-irradiation treatment with ascorbate (5 mM added 30 min after radiation) reduces, but does not eliminate, the induction of CD59- mutants in human-hamster hybrid A(L) cells exposed to high-LET carbon ions (LET of 100 KeV/microm). RibCys, [2(R,S)-D-ribo-1',2',3',4'-Tetrahydroxybutyl]-thiazolidene-4(R)-ca riboxylic acid] (4 mM) gave a similar but lesser effect. The lethality of the carbon ions was not altered by these chemicals. Preliminary data are presented that ascorbate also alters the spectrum of CD59- mutations induced by the carbon beam, mainly by reducing the incidence of small mutations and mutants displaying transmissible genomic instability (TGI), while large mutations are unaffected. Our results suggest that LLR are important in initiating TGI.

  17. Resistant mechanism study of benzalkonium chloride selected Salmonella Typhimurium mutants.

    PubMed

    Guo, Wei; Cui, Shenghui; Xu, Xiao; Wang, Haoyan

    2014-02-01

    Benzalkonium chloride is one of the invaluable biocides that is extensively used in healthcare settings as well as in the food processing industry. After exposing wild-type Salmonella Typhimurium 14028s or its AcrAB inactivation mutant to gradually increasing levels of benzalkonium chloride, resistance mutants S-41, S-150, S-AB-23, S-AB-38, and S-AB-73 were selected and these mutants also showed a 2-64-fold stable minimum inhibitory concentration (MIC) increase to chloramphenicol, ciprofloxacin, nalidixic acid, and tetracycline. In S-41 and S-150, the expression of acrB was increased 2.7- and 7.6-fold, and ΔtolC or ΔacrAB mutants of S-41 and S-150 showed the same MICs to all tested antimicrobials as the equivalent Salmonella Typhimurium 14028s mutants. However, in S-AB-23, S-AB-38, and S-AB-73, the expression of acrF was increased 96-, 230-, and 267-fold, respectively, and ΔtolC or ΔacrEF mutants of S-AB-23, S-AB-38, and S-AB-73 showed the similar MICs to all tested antimicrobials as the ΔtolC mutant of Salmonella Typhimurium 14028s. Our data showed that constitutively over-expressed AcrAB working through TolC was the main resistance mechanism in ST14028s benzalkonium chloride resistance mutants. However, after AcrAB had been inactivated, benzalkonium chloride-resistant mutants could still be selected and constitutively over-expressed, AcrEF became the dominant efflux pump working through TolC and being responsible for the increasing antimicrobial resistance. These data indicated that different mechanisms existed for acrB and acrF constitutive over-expression. Since exposure to benzalkonium chloride may lead to Salmonella mutants with a decreased susceptibility to quinolones, which is currently one of the drugs of choice for the treatment of life-threatening salmonelosis, research into the pathogenesis and epidemiology of the benzalkonium chloride resistance mutants will be of increasing importance.

  18. Selective Chemosensitization of Rb Mutant Cells

    DTIC Science & Technology

    2000-07-01

    examined the ability of each E1A mutant to induce p53. Cells expressing full-length E1A dis - played a 20- to 30-fold increase in steady-state p53...retinoblastoma (Rb) protein, and inac- tivation of both is essential for viral transformation (Lane and Crawford 1979; Linzer and Levine 1979; De- Caprio et al...Western blotting. Despite the fact that wild-type MEFs expressing El A dis - played an -10-fold increase in p53 and Mdm2 levels as compared to their Ai?F

  19. Stability of Osaka Mutant and Wild-Type Fibril Models.

    PubMed

    Berhanu, Workalemahu M; Alred, Erik J; Hansmann, Ulrich H E

    2015-10-15

    Single amino acid mutations in amyloid-beta (Aβ) peptides can lead to early onset and increased severity of Alzheimer's disease. An example is the Osaka mutation (Aβ1-40E22D), which is more toxic than wild-type Aβ1-40. This mutant quickly forms early stage fibrils, one of the hallmarks of the disease, and these fibrils can even seed fibrilization of wild-type monomers. Using molecular dynamic simulations, we show that because of formation of various intra- and intermolecular salt bridges the Osaka mutant fibrils are more stable than wild-type fibrils. The mutant fibril also has a wider water channel with increased water flow than the wild type. These two observations can explain the higher toxicity and aggregation rate of the Osaka mutant over the wild type.

  20. Epilepsy-Related Slack Channel Mutants Lead to Channel Over-Activity by Two Different Mechanisms.

    PubMed

    Tang, Qiong-Yao; Zhang, Fei-Fei; Xu, Jie; Wang, Ran; Chen, Jian; Logothetis, Diomedes E; Zhang, Zhe

    2016-01-05

    Twelve sodium-activated potassium channel (KCNT1, Slack) genetic mutants have been identified from severe early-onset epilepsy patients. The changes in biophysical properties of these mutants and the underlying mechanisms causing disease remain elusive. Here, we report that seven of the 12 mutations increase, whereas one mutation decreases, the channel's sodium sensitivity. Two of the mutants exhibit channel over-activity only when the intracellular Na(+) ([Na(+)]i) concentration is ∼80 mM. In contrast, single-channel data reveal that all 12 mutants increase the maximal open probability (Po). We conclude that these mutant channels lead to channel over-activity predominantly by increasing the ability of sodium binding to activate the channel, which is indicated by its maximal Po. The sodium sensitivity of these epilepsy causing mutants probably determines the [Na(+)]i concentration at which these mutants exert their pathological effects.

  1. Escherichia coli mutants resistant to inactivation by high hydrostatic pressure.

    PubMed Central

    Hauben, K J; Bartlett, D H; Soontjens, C C; Cornelis, K; Wuytack, E Y; Michiels, C W

    1997-01-01

    Alternating cycles of exposure to high pressure and outgrowth of surviving populations were used to select for highly pressure-resistant mutants of Escherichia coli MG1655. Three barotolerant mutants (LMM1010, LMM1020, and LMM1030) were isolated independently by using outgrowth temperatures of 30, 37, and 42 degrees C, respectively. Survival of these mutants after pressure treatment for 15 min at ambient temperature was 40 to 85% at 220 MPa and 0.5 to 1.5% at 800 MPa, while survival of the parent strain, MG1655, decreased from 15% at 220 MPa to 2 x 10(-8)% at 700 MPa. Heat resistance of mutants LMM1020 and LMM1030 was also altered, as evident by higher D values at 58 and 60 degrees C and reduced z values compared to those for the parent strain. D and z values for mutant LMM1010 were not significantly different from those for the parent strain. Pressure sensitivity of the mutants increased from 10 to 50 degrees C, as opposed to the parent strain, which showed a minimum around 40 degrees C. The ability of the mutants to grow at moderately elevated pressure (50 MPa) was reduced at temperatures above 37 degrees C, indicating that resistance to pressure inactivation is unrelated to barotolerant growth. The development of high levels of barotolerance as demonstrated in this work should cause concern about the safety of high-pressure food processing. PMID:9055412

  2. Mechanisms of Azole Resistance in Petite Mutants of Candida glabrata

    PubMed Central

    Brun, Sophie; Bergès, Thierry; Poupard, Pascal; Vauzelle-Moreau, Carole; Renier, Gilles; Chabasse, Dominique; Bouchara, Jean-Philippe

    2004-01-01

    We previously showed that resistant colonies of Candida glabrata inside the azole inhibition zones had respiratory deficiency due to mutations in mitochondrial DNA. Here, we analyzed the mechanisms of azole resistance in petite mutants of C. glabrata obtained by exposure to fluconazole or induced by ethidium bromide. The respiratory deficiency of these mutants was confirmed by oxygraphy and flow cytometric analysis with rhodamine 123, and its mitochondrial origin was demonstrated by transmission electron microscopy and restriction endonuclease analysis of the mitochondrial DNA. Flow cytometry with rhodamine 6G suggested an increased drug efflux in mutant cells, which was further supported by Northern blot analysis of the expression of the C. glabrata CDR1 (CgCDR1) and CgCDR2 genes, encoding efflux pumps. Conversely, the expression of CgERG11, which encodes the azole target, was not affected by petite mutations, and no differences were seen in the sequence of this gene between parent isolates and mutants. Moreover, sterol analysis showed similar overall amount of sterols in parent and mutant cells, but quantitative modifications were observed in the mutants, with almost undetectable biosynthesis intermediates. Further analysis performed after separation of free sterols from steryl esters revealed a defect in sterol esterification in mutant cells, with free ergosterol representing 92% of the overall sterol content. Thus, resistance or decreased susceptibility to azoles in petite mutants of C. glabrata is associated with increased expression of CgCDR1 and, to a lesser extent, of CgCDR2. In addition, the marked increase in free ergosterol content would explain their increased susceptibility to polyenes. PMID:15105136

  3. Mechanisms of azole resistance in petite mutants of Candida glabrata.

    PubMed

    Brun, Sophie; Bergès, Thierry; Poupard, Pascal; Vauzelle-Moreau, Carole; Renier, Gilles; Chabasse, Dominique; Bouchara, Jean-Philippe

    2004-05-01

    We previously showed that resistant colonies of Candida glabrata inside the azole inhibition zones had respiratory deficiency due to mutations in mitochondrial DNA. Here, we analyzed the mechanisms of azole resistance in petite mutants of C. glabrata obtained by exposure to fluconazole or induced by ethidium bromide. The respiratory deficiency of these mutants was confirmed by oxygraphy and flow cytometric analysis with rhodamine 123, and its mitochondrial origin was demonstrated by transmission electron microscopy and restriction endonuclease analysis of the mitochondrial DNA. Flow cytometry with rhodamine 6G suggested an increased drug efflux in mutant cells, which was further supported by Northern blot analysis of the expression of the C. glabrata CDR1 (CgCDR1) and CgCDR2 genes, encoding efflux pumps. Conversely, the expression of CgERG11, which encodes the azole target, was not affected by petite mutations, and no differences were seen in the sequence of this gene between parent isolates and mutants. Moreover, sterol analysis showed similar overall amount of sterols in parent and mutant cells, but quantitative modifications were observed in the mutants, with almost undetectable biosynthesis intermediates. Further analysis performed after separation of free sterols from steryl esters revealed a defect in sterol esterification in mutant cells, with free ergosterol representing 92% of the overall sterol content. Thus, resistance or decreased susceptibility to azoles in petite mutants of C. glabrata is associated with increased expression of CgCDR1 and, to a lesser extent, of CgCDR2. In addition, the marked increase in free ergosterol content would explain their increased susceptibility to polyenes.

  4. Genetic analysis of sex chromosomal meiotic mutants in Drosophilia melanogaster.

    PubMed

    Baker, B S; Carpenter, A T

    1972-06-01

    A total of 209 ethyl methanesulfonate-treated X chromosomes were screened for meiotic mutants that either (1) increased sex or fourth chromosome nondisjunction at either meiotic division in males; (2) allowed recombination in such males; (3) increased nondisjunction of the X chromosome at either meiotic division in females; or (4) caused such females, when mated to males heterozygous for Segregation-Distorter (SD) and a sensitive homolog to alter the strength of meiotic drive in males.-Twenty male-specific meiotic mutants were found. Though the rates of nondisjunction differed, all twenty mutants were qualitatively similar in that (1) they alter the disjunction of the X chromosome from the Y chromosome; (2) among the recovered sex-chromosome exceptional progeny, there is a large excess of those derived from nullo-XY as compared to XY gametes; (3) there is a negative correlation between the frequency of sex-chromosome exceptional progeny and the frequency of males among the regular progeny. In their effects on meiosis these mutants are similar to In(1)sc(4L)sc(8R), which is deleted for the basal heterochromatin. These mutants, however, have normal phenotypes and viabilities when examined as X/0 males, and furthermore, a mapping of two of the mutants places them in the euchromatin of the X chromosome. It is suggested that these mutants are in genes whose products are involved in insuring the proper functioning of the basal pairing sites which are deleted in In(1)sc(4L)sc(8R), and in addition that there is a close connection, perhaps causal, between the disruption of normal X-Y pairing (and, therefore, disjunction) and the occurrence of meiotic drive in the male.-Eleven mutants were found which increased nondisjunction in females. These mutants were characterized as to (1) the division at which they acted; (2) their effect on recombination; (3) their dominance; (4) their effects on disjunction of all four chromosome pairs. Five female mutants caused a nonuniform

  5. Bacteriorhodopsin mutants of Halobacterium sp. GRB. II. Characterization of mutants.

    PubMed

    Soppa, J; Otomo, J; Straub, J; Tittor, J; Meessen, S; Oesterhelt, D

    1989-08-05

    The bacterioopsin genes of Halobacterium sp. GRB (Ebert, K., Goebel, W., and Pfeifer, F. (1984) Mol. & Gen. Genet. 194, 91-97) wild type and 10 independent mutants of different phenotypes have been cloned and sequenced. The wild type gene has two conservative changes compared to the gene of Halobacterium halobium, so that the proteins of the two species are identical. Six different mutations at five different codons have been found, leading to the following amino acid changes compared to the wild type: Trp10----Cys (three cases), Tyr57----Asn, Asp85----Glu, Asp06----Asn (three cases), Asp96----Gly, Trp138----Arg. A first characterization of the mutant proteins is given, and their implications for models of bacteriorhodopsin structure and function are discussed.

  6. Mutant power: using mutant allele collections for yeast functional genomics

    PubMed Central

    Norman, Kaitlyn L.

    2016-01-01

    The budding yeast has long served as a model eukaryote for the functional genomic analysis of highly conserved signaling pathways, cellular processes and mechanisms underlying human disease. The collection of reagents available for genomics in yeast is extensive, encompassing a growing diversity of mutant collections beyond gene deletion sets in the standard wild-type S288C genetic background. We review here three main types of mutant allele collections: transposon mutagen collections, essential gene collections and overexpression libraries. Each collection provides unique and identifiable alleles that can be utilized in genome-wide, high-throughput studies. These genomic reagents are particularly informative in identifying synthetic phenotypes and functions associated with essential genes, including those modeled most effectively in complex genetic backgrounds. Several examples of genomic studies in filamentous/pseudohyphal backgrounds are provided here to illustrate this point. Additionally, the limitations of each approach are examined. Collectively, these mutant allele collections in Saccharomyces cerevisiae and the related pathogenic yeast Candida albicans promise insights toward an advanced understanding of eukaryotic molecular and cellular biology. PMID:26453908

  7. Mutant power: using mutant allele collections for yeast functional genomics.

    PubMed

    Norman, Kaitlyn L; Kumar, Anuj

    2016-03-01

    The budding yeast has long served as a model eukaryote for the functional genomic analysis of highly conserved signaling pathways, cellular processes and mechanisms underlying human disease. The collection of reagents available for genomics in yeast is extensive, encompassing a growing diversity of mutant collections beyond gene deletion sets in the standard wild-type S288C genetic background. We review here three main types of mutant allele collections: transposon mutagen collections, essential gene collections and overexpression libraries. Each collection provides unique and identifiable alleles that can be utilized in genome-wide, high-throughput studies. These genomic reagents are particularly informative in identifying synthetic phenotypes and functions associated with essential genes, including those modeled most effectively in complex genetic backgrounds. Several examples of genomic studies in filamentous/pseudohyphal backgrounds are provided here to illustrate this point. Additionally, the limitations of each approach are examined. Collectively, these mutant allele collections in Saccharomyces cerevisiae and the related pathogenic yeast Candida albicans promise insights toward an advanced understanding of eukaryotic molecular and cellular biology. © The Author 2015. Published by Oxford University Press. All rights reserved. For permissions, please email: journals.permissions@oup.com.

  8. Induction of petite yeast mutants by membrane-active agents.

    PubMed

    Jiménez, J; Longo, E; Benítez, T

    1988-12-01

    Ethanol proved to be a strong mutagenic agent of Saccharomyces mitochondrial DNA. Other active membrane solvents, such as tert-butanol, isopropanol, and sodium dodecyl sulfate, also turned out to be powerful petite mutation [rho-] inducers. Mutants defective in ergosterol synthesis (erg mutants) showed an extremely high frequency of spontaneous petite cells, suggesting that mitochondrial membrane alterations that were caused either by changes in its composition, as in the erg mutants, or by the effects of organic solvents resulted in an increase in the proportion of petite mutants. Wine yeast strains were generally more tolerant to the mutagenic effects of alcohols on mitochondrial DNA and more sensitive to the effect of sodium dodecyl sulfate than laboratory strains. However, resistance to petite mutation formation in laboratory strains was increased by mitochondrial transfer from alcohol-tolerant wine yeasts. Hence, the stability of the [rho+] mitochondrial DNA in either the presence or absence of solvents depends in part on the nature of the mitochondrial DNA itself. The low frequency of petite mutants found in wine yeast-laboratory yeast hybrids and the fact that the high frequency of petite mutants of a particular wine spore segregated meiotically indicated that many nuclear genes also play an important role in the mitochondrial genome in both the presence and absence of membrane solvents.

  9. Induction of petite yeast mutants by membrane-active agents.

    PubMed Central

    Jiménez, J; Longo, E; Benítez, T

    1988-01-01

    Ethanol proved to be a strong mutagenic agent of Saccharomyces mitochondrial DNA. Other active membrane solvents, such as tert-butanol, isopropanol, and sodium dodecyl sulfate, also turned out to be powerful petite mutation [rho-] inducers. Mutants defective in ergosterol synthesis (erg mutants) showed an extremely high frequency of spontaneous petite cells, suggesting that mitochondrial membrane alterations that were caused either by changes in its composition, as in the erg mutants, or by the effects of organic solvents resulted in an increase in the proportion of petite mutants. Wine yeast strains were generally more tolerant to the mutagenic effects of alcohols on mitochondrial DNA and more sensitive to the effect of sodium dodecyl sulfate than laboratory strains. However, resistance to petite mutation formation in laboratory strains was increased by mitochondrial transfer from alcohol-tolerant wine yeasts. Hence, the stability of the [rho+] mitochondrial DNA in either the presence or absence of solvents depends in part on the nature of the mitochondrial DNA itself. The low frequency of petite mutants found in wine yeast-laboratory yeast hybrids and the fact that the high frequency of petite mutants of a particular wine spore segregated meiotically indicated that many nuclear genes also play an important role in the mitochondrial genome in both the presence and absence of membrane solvents. PMID:3066293

  10. Mutant models of prolonged life span.

    PubMed

    Mahler, J F

    2001-01-01

    Aging is an important biological process that affects all creatures. For humans, age-related diseases and the question of why we age and die also have tremendous social and philosophical impact. We can therefore expect that models to study mechanisms of the aging process will always attract much interest. Until recently, the mutant model approach to study molecular mechanisms of aging has been limited to lower animals such as yeast, worms, and flies. However, given the current power of genetic technology in mammals, we can expect that phenotypes of prolonged life span will increasingly be seen in mice and subject to evaluation by pathologists. A brief review of current models is presented.

  11. Leptin gene promoter DNA methylation in WNIN obese mutant rats.

    PubMed

    Kalashikam, Rajender Rao; Inagadapa, Padmavathi J N; Thomas, Anju Elizabeth; Jeyapal, Sugeetha; Giridharan, Nappan Veettil; Raghunath, Manchala

    2014-02-05

    Obesity has become an epidemic in worldwide population. Leptin gene defect could be one of the causes for obesity. Two mutant obese rats WNIN/Ob and WNIN/GROb, isolated at National Centre for Laboratory Animal Sciences (NCLAS), Hyderabad, India, were found to be leptin resistant. The present study aims to understand the regulatory mechanisms underlying the resistance by promoter DNA methylation of leptin gene in these mutant obese rats. Male obese mutant homozygous, carrier and heterozygous rats of WNIN/Ob and WNIN/GROb strain of 6 months old were studied to check the leptin gene expression (RT-PCR) and promoter DNA methylation (MassARRAY Compact system, SEQUENOM) of leptin gene by invivo and insilico approach. Homozygous WNIN/Ob and WNIN/GROb showed significantly higher leptin gene expression compared to carrier and lean counterparts. Leptin gene promoter DNA sequence region was analyzed ranging from transcription start site (TSS) to-550 bp length and found four CpGs in this sequence among them only three CpG loci (-309, -481, -502) were methylated in these WNIN mutant rat phenotypes. The increased percentage of methylation in WNIN mutant lean and carrier phenotypes is positively correlated with transcription levels. Thus genetic variation may have effect on methylation percentages and subsequently on the regulation of leptin gene expression which may lead to obesity in these obese mutant rat strains.

  12. Thymidine kinase mutants obtained by random sequence selection.

    PubMed

    Munir, K M; French, D C; Loeb, L A

    1993-05-01

    Knowledge of the catalytic properties and structural information regarding the amino acid residues that comprise the active site of an enzyme allows one, in principle, to use site-specific mutagenesis to construct genes that encode enzymes with altered functions. However, such information about most enzymes is not known and the effects of specific amino acid substitutions are not generally predictable. An alternative approach is to substitute random nucleotides for key codons in a gene and to use genetic selection to identify new and interesting enzyme variants. We describe here the construction, selection, and characterization of herpes simplex virus type 1 thymidine kinase mutants either with different catalytic properties or with enhanced thermostability. From a library containing 2 x 10(6) plasmid-encoded herpes thymidine kinase genes, each with a different nucleotide sequence at the putative nucleoside binding site, we obtained 1540 active mutants. Using this library and one previously constructed, we identified by secondary selection Escherichia coli harboring thymidine kinase mutant clones that were unable to grow in the presence of concentrations of 3'-azido-3'-deoxythymidine (AZT) that permits colony formation by E. coli harboring the wild-type plasmid. Two of the mutant enzymes exhibited a reduced Km for AZT, one of which displayed a higher catalytic efficiency for AZT over thymidine relative to that of the wild type. We also identified one mutant with enhanced thermostability. These mutants may have clinical potential as the promise of gene therapy is increasingly becoming a reality.

  13. Orc mutants arrest in metaphase with abnormally condensed chromosomes.

    PubMed

    Pflumm, M F; Botchan, M R

    2001-05-01

    The origin recognition complex (ORC) is a six subunit complex required for eukaryotic DNA replication initiation and for silencing of the heterochromatic mating type loci in Saccharomyces cerevisiae. Our discovery of the Drosophila ORC complex concentrated in the centric heterochromatin of mitotic cells in the early embryo and its interactions with heterochromatin protein 1 (HP-1) lead us to speculate that ORC may play some general role in chromosomal folding. To explore the role of ORC in chromosomal condensation, we have identified a mutant of subunit 5 of the Drosophila melanogaster origin recognition complex (Orc5) and have characterized the phenotypes of both the Orc5 and the previously identified Orc2 mutant, k43. Both Orc mutants died at late larval stages and surprisingly, despite a reduced number of S-phase cells, an increased fraction of cells were also detected in mitosis. For this latter population of cells, Orc mutants arrest in a defective metaphase with shorter and thicker chromosomes that fail to align at the metaphase plate within a poorly assembled mitotic spindle. In addition, sister chromatid cohesion was frequently lost. PCNA and MCM4 mutants had similar phenotypes to Orc mutants. We propose that DNA replication defects trigger the mitotic arrest, due to the fact that frequent fragmentation was observed. Thus, cells have a mitotic checkpoint that senses chromosome integrity. These studies also suggest that the density of functional replication origins and completion of S phase are requirements for proper chromosomal condensation.

  14. Mutant Kras copy number defines metabolic reprogramming and therapeutic susceptibilities.

    PubMed

    Kerr, Emma M; Gaude, Edoardo; Turrell, Frances K; Frezza, Christian; Martins, Carla P

    2016-03-03

    The RAS/MAPK (mitogen-activated protein kinase) signalling pathway is frequently deregulated in non-small-cell lung cancer, often through KRAS activating mutations. A single endogenous mutant Kras allele is sufficient to promote lung tumour formation in mice but malignant progression requires additional genetic alterations. We recently showed that advanced lung tumours from Kras(G12D/+);p53-null mice frequently exhibit Kras(G12D) allelic enrichment (Kras(G12D)/Kras(wild-type) > 1) (ref. 7), implying that mutant Kras copy gains are positively selected during progression. Here we show, through a comprehensive analysis of mutant Kras homozygous and heterozygous mouse embryonic fibroblasts and lung cancer cells, that these genotypes are phenotypically distinct. In particular, Kras(G12D/G12D) cells exhibit a glycolytic switch coupled to increased channelling of glucose-derived metabolites into the tricarboxylic acid cycle and glutathione biosynthesis, resulting in enhanced glutathione-mediated detoxification. This metabolic rewiring is recapitulated in mutant KRAS homozygous non-small-cell lung cancer cells and in vivo, in spontaneous advanced murine lung tumours (which display a high frequency of Kras(G12D) copy gain), but not in the corresponding early tumours (Kras(G12D) heterozygous). Finally, we demonstrate that mutant Kras copy gain creates unique metabolic dependences that can be exploited to selectively target these aggressive mutant Kras tumours. Our data demonstrate that mutant Kras lung tumours are not a single disease but rather a heterogeneous group comprising two classes of tumours with distinct metabolic profiles, prognosis and therapeutic susceptibility, which can be discriminated on the basis of their relative mutant allelic content. We also provide the first, to our knowledge, in vivo evidence of metabolic rewiring during lung cancer malignant progression.

  15. A genetic screen for zebrafish mutants with hepatic steatosis identifies a locus required for larval growth.

    PubMed

    Hugo, Sarah E; Schlegel, Amnon

    2017-03-01

    In a screen for zebrafish larval mutants with excessive liver lipid accumulation (hepatic steatosis), we identified harvest moon (hmn). Cytoplasmic lipid droplets, surrounded by multivesicular structures and mitochondria whose cristae appeared swollen, are seen in hmn mutant hepatocytes. Whole body triacylglycerol is increased in hmn mutant larvae. When we attempted to raise mutants, which were morphologically normal at the developmental stage that the screen was conducted, to adulthood, we observed that most hmn mutants do not survive to the juvenile period when raised. An arrest in growth occurs in the late larval period without obvious organ defects. Maternal zygotic mutants have no additional defects, suggesting that the mutation affects a late developmental process. The developmental window between embryogenesis and the metamorphosis remains under-studied, and hmn mutants might be useful for exploring the molecular and anatomic processes occurring during this transition period.

  16. Increased gene targeting in Ku70 and Xrcc4 transiently deficient human somatic cells.

    PubMed

    Bertolini, Luciana R; Bertolini, Marcelo; Maga, Elizabeth A; Madden, Knut R; Murray, James D

    2009-02-01

    The insertion of foreign DNA at a specific genomic locus directed by homologous DNA sequences, or gene targeting, is an inefficient process in mammalian somatic cells. Given the key role of non-homologous end joining (NHEJ) pathway in DNA double-strand break (DSB) repair in mammalian cells, we investigated the effects of decreasing NHEJ protein levels on gene targeting. Here we demonstrate that the transient knockdown of integral NHEJ proteins, Ku70 and Xrcc4, by RNAi in human HCT116 cells has a remarkable effect on gene targeting/random insertions ratios. A timely transfection of an HPRT-based targeting vector after RNAi treatment led to a 70% reduction in random integration events and a 33-fold increase in gene targeting at the HPRT locus. These findings bolster the role of NHEJ proteins in foreign DNA integration in vivo, and demonstrate that their transient depletion by RNAi is a viable approach to increase the frequency of gene targeting events. Understanding how foreign DNA integrates into a cell's genome is important to advance strategies for biotechnology and genetic medicine.

  17. Proposed Nomenclature for Mutants of Adenoviruses

    PubMed Central

    Ginsberg, Harold S.; Williams, James F.; Doerfler, Walter H.; Shimojo, Hiroto

    1973-01-01

    In accord with the nomenclature proposed for mutants of simian virus 40 the same rules, with minor modifications, are recommended for naming mutants of adenoviruses. It is further suggested that these rules, which pertain to a system of classification based primarily upon complementation analysis, also be applied to mutants of other DNA-containing animal viruses. PMID:4355864

  18. Problem-Solving Test: Tryptophan Operon Mutants

    ERIC Educational Resources Information Center

    Szeberenyi, Jozsef

    2010-01-01

    This paper presents a problem-solving test that deals with the regulation of the "trp" operon of "Escherichia coli." Two mutants of this operon are described: in mutant A, the operator region of the operon carries a point mutation so that it is unable to carry out its function; mutant B expresses a "trp" repressor protein unable to bind…

  19. Problem-Solving Test: Tryptophan Operon Mutants

    ERIC Educational Resources Information Center

    Szeberenyi, Jozsef

    2010-01-01

    This paper presents a problem-solving test that deals with the regulation of the "trp" operon of "Escherichia coli." Two mutants of this operon are described: in mutant A, the operator region of the operon carries a point mutation so that it is unable to carry out its function; mutant B expresses a "trp" repressor protein unable to bind…

  20. Ethylene Inhibitors Restore Nodulation to sym 5 Mutants of Pisum sativum L. cv Sparkle 12

    PubMed Central

    Fearn, Jeffrey C.; LaRue, Thomas A.

    1991-01-01

    The sym 5 mutants of pea, Pisum sativum L. cv Sparkle, do not differ in growth habit from their normal parent and nodulate poorly at a root temperature of 20°C. If inhibitors of ethylene formation or action (Co2+, aminoethoxyvinylglycine, or Ag+) are added to the substrate, nodulation of the sym 5 mutants is increased. Similar treatments of four other mutant sym lines do not restore nodulation. When Ag+ is added to the substrate from 4 days before to 4 days after inoculation with rhizobia, nodulation of sym 5 mutants is increased. The roots of the mutant need only be exposed to Ag+ for 4 hours to significantly increase nodule numbers. The content of free 1-aminocyclopropane-1-carboxylic acid and the production of ethylene in the lateral roots of sym 5 mutants do not differ from Sparkle. PMID:16668158

  1. C. elegans and mutants with chronic nicotine exposure as a novel model of cancer phenotype.

    PubMed

    Kanteti, Rajani; Dhanasingh, Immanuel; El-Hashani, Essam; Riehm, Jacob J; Stricker, Thomas; Nagy, Stanislav; Zaborin, Alexander; Zaborina, Olga; Biron, David; Alverdy, John C; Im, Hae Kyung; Siddiqui, Shahid; Padilla, Pamela A; Salgia, Ravi

    2016-01-01

    We previously investigated MET and its oncogenic mutants relevant to lung cancer in C. elegans. The inactive orthlogues of the receptor tyrosine kinase Eph and MET, namely vab-1 and RB2088 respectively, the temperature sensitive constitutively active form of KRAS, SD551 (let-60; GA89) and the inactive c-CBL equivalent mutants in sli-1 (PS2728, PS1258, and MT13032) when subjected to chronic exposure of nicotine resulted in a significant loss in egg-laying capacity and fertility. While the vab-1 mutant revealed increased circular motion in response to nicotine, the other mutant strains failed to show any effect. Overall locomotion speed increased with increasing nicotine concentration in all tested mutant strains except in the vab-1 mutants. Moreover, chronic nicotine exposure, in general, upregulated kinases and phosphatases. Taken together, these studies provide evidence in support of C. elegans as initial in vivo model to study nicotine and its effects on oncogenic mutations identified in humans.

  2. Isolation of prostrate turfgrass mutants via screening of dwarf phenotype and characterization of a perennial ryegrass prostrate mutant

    PubMed Central

    Chen, Junmei; Thammina, Chandra; Li, Wei; Yu, Hao; Yer, Huseyin; El-Tanbouly, Rania; Marron, Manon; Katin-Grazzini, Lorenzo; Chen, Yongqin; Inguagiato, John; McAvoy, Richard J.; Guillard, Karl; Zhang, Xian; Li, Yi

    2016-01-01

    Prostrate turf varieties are desirable because of their increased low mowing tolerance, heat resistance, traffic resistance and ground coverage compared with upright varieties. Mutation breeding may provide a powerful tool to create prostrate varieties, but there are no simple, straightforward methods to screen for such mutants. Elucidation of the molecular basis of the major ‘green revolution’ traits, dwarfism and semi-dwarfism, guided us to design a simple strategy for isolating dwarf mutants of perennial ryegrass (Lolium perenne L.). We have shown that gamma-ray-mediated dominant dwarf mutants can be easily screened for at the three-leaf stage. About 10% of dwarf mutant lines also displayed a prostrate phenotype at mature stages (>10 tillers). One prostrate line, Lowboy I, has been characterized in detail. Lowboy I had significantly shorter canopy, leaf blade and internode lengths compared with wild type. Lowboy I also exhibited greater tolerance to low mowing stress than wild type. Exogenous gibberellic acid (GA) restored Lowboy I to a wild-type phenotype, indicating that the dwarf and prostrate phenotypes were both due to GA deficiency. We further showed that phenotypes of Lowboy I were dominant and stably inherited through sexual reproduction. Prostrate turfgrass mutants are difficult to screen for because the phenotype is not observed at young seedling stages, therefore our method represents a simple strategy for easily isolating prostrate mutants. Furthermore, Lowboy I may provide an outstanding germplasm for breeding novel prostrate perennial ryegrass cultivars. PMID:26955481

  3. PIK3CA mutant tumors depend on oxoglutarate dehydrogenase.

    PubMed

    Ilic, Nina; Birsoy, Kıvanç; Aguirre, Andrew J; Kory, Nora; Pacold, Michael E; Singh, Shambhavi; Moody, Susan E; DeAngelo, Joseph D; Spardy, Nicole A; Freinkman, Elizaveta; Weir, Barbara A; Tsherniak, Aviad; Cowley, Glenn S; Root, David E; Asara, John M; Vazquez, Francisca; Widlund, Hans R; Sabatini, David M; Hahn, William C

    2017-04-25

    Oncogenic PIK3CA mutations are found in a significant fraction of human cancers, but therapeutic inhibition of PI3K has only shown limited success in clinical trials. To understand how mutant PIK3CA contributes to cancer cell proliferation, we used genome scale loss-of-function screening in a large number of genomically annotated cancer cell lines. As expected, we found that PIK3CA mutant cancer cells require PIK3CA but also require the expression of the TCA cycle enzyme 2-oxoglutarate dehydrogenase (OGDH). To understand the relationship between oncogenic PIK3CA and OGDH function, we interrogated metabolic requirements and found an increased reliance on glucose metabolism to sustain PIK3CA mutant cell proliferation. Functional metabolic studies revealed that OGDH suppression increased levels of the metabolite 2-oxoglutarate (2OG). We found that this increase in 2OG levels, either by OGDH suppression or exogenous 2OG treatment, resulted in aspartate depletion that was specifically manifested as auxotrophy within PIK3CA mutant cells. Reduced levels of aspartate deregulated the malate-aspartate shuttle, which is important for cytoplasmic NAD(+) regeneration that sustains rapid glucose breakdown through glycolysis. Consequently, because PIK3CA mutant cells exhibit a profound reliance on glucose metabolism, malate-aspartate shuttle deregulation leads to a specific proliferative block due to the inability to maintain NAD(+)/NADH homeostasis. Together these observations define a precise metabolic vulnerability imposed by a recurrently mutated oncogene.

  4. Nif- Hup- mutants of Rhizobium japonicum.

    PubMed Central

    Moshiri, F; Stults, L; Novak, P; Maier, R J

    1983-01-01

    Two H2 uptake-negative (Hup-) Rhizobium japonicum mutants were obtained that also lacked symbiotic N2 fixation (acetylene reduction) activity. One of the mutants formed green nodules and was deficient in heme. Hydrogen oxidation activity in this mutant could be restored by the addition of heme plus ATP to crude extracts. Bacteroid extracts from the other mutant strain lacked hydrogenase activity and activity for both of the nitrogenase component proteins. Hup+ revertants of the mutant strains regained both H2 uptake ability and nitrogenase activity. Images PMID:6874648

  5. Zebrafish Genomic Instability Mutants and Cancer Susceptibility

    PubMed Central

    Moore, Jessica L.; Rush, Lindsay M.; Breneman, Carol; Mohideen, Manzoor-Ali P. K.; Cheng, Keith C.

    2006-01-01

    Somatic loss of tumor suppressor gene function comprising the second hit of Knudson's two-hit hypothesis is important in human cancer. A genetic screen was performed in zebrafish (Danio rerio) to find mutations that cause genomic instability (gin), as scored by Streisinger's mosaic-eye assay that models this second hit. The assay, based on a visible test for loss of wild-type gene function at a single locus, golden, is representative of genomewide events. Twelve ENU-induced genomic instability (gin) mutations were isolated. Most mutations showed weak dominance in heterozygotes and all showed a stronger phenotype in homozygotes. Trans-heterozygosity for 7 of these mutations showed greatly enhanced instability. A variety of spontaneous tumors were found in heterozygous adults from all gin lines, consistent with the expectation that genomic instability (mutator) mutations can accelerate carcinogenesis. The incidence of spontaneous cancer at 30–34 months was increased 9.6-fold in heterozygotes for the mutant with the strongest phenotype, gin-10. Tumors were seen in skin, colon, kidney, liver, pancreas, ovary, testis, and neuronal tissues, with multiple tumors in some fish. The study of these mutants will add to our understanding of the mechanisms of somatic loss of gene function and how those mechanisms contribute to cancer susceptibility. PMID:16888336

  6. Method for rapid isolation of sensitive mutants

    DOEpatents

    Freyer, James P.

    1997-01-01

    Sensitive mammalian cell mutants are rapidly isolated using flow cytometry. A first population of clonal spheroids is established to contain both normal and mutant cells. The population may be naturally occurring or may arise from mutagenized cells. The first population is then flow sorted by size to obtain a second population of clonal spheroids of a first uniform size. The second population is then exposed to a DNA-damaging agent that is being investigated. The exposed second population is placed in a growth medium to form a third population of clonal spheroids comprising spheroids of increased size from the mammalian cells that are resistant to the DNA-damaging agent and spheroids of substantially the first uniform size formed from the mammalian cells that are sensitive to the DNA-damaging agent. The third population is not flow sorted to differentiate the spheroids formed from resistant mammalian cells from spheroids formed from sensitive mammalian cells. The spheroids formed from sensitive mammalian cells are now treated to recover viable sensitive cells from which a sensitive cell line can be cloned.

  7. Method for rapid isolation of sensitive mutants

    DOEpatents

    Freyer, J.P.

    1997-07-29

    Sensitive mammalian cell mutants are rapidly isolated using flow cytometry. A first population of clonal spheroids is established to contain both normal and mutant cells. The population may be naturally occurring or may arise from mutagenized cells. The first population is then flow sorted by size to obtain a second population of clonal spheroids of a first uniform size. The second population is then exposed to a DNA-damaging agent that is being investigated. The exposed second population is placed in a growth medium to form a third population of clonal spheroids comprising spheroids of increased size from the mammalian cells that are resistant to the DNA-damaging agent and spheroids of substantially the first uniform size formed from the mammalian cells that are sensitive to the DNA-damaging agent. The third population is not flow sorted to differentiate the spheroids formed from resistant mammalian cells from spheroids formed from sensitive mammalian cells. The spheroids formed from sensitive mammalian cells are now treated to recover viable sensitive cells from which a sensitive cell line can be cloned. 15 figs.

  8. Auxin physiology of the tomato mutant diageotropical

    SciTech Connect

    Daniel, S.G.; Rayle, D.L. ); Cleland, R.E. )

    1989-11-01

    The tomato (Lycopersicon esculentum, Mill.) mutant diageotropica (dgt) exhibits biochemical, physiological, and morphological abnormalities that suggest the mutation may have affected a primary site of auxin perception or action. We have compared two aspects of the auxin physiology of dgt and wild-type (VFN8) seedlings: auxin transport and cellular growth parameters. The rates of basipetal indole-3-acetic acid (IAA) polar transport are identical in hypocotyl sections of the two genotypes, but dgt sections have a slightly greater capacity for IAA transport. 2,3,5-Triiodobenzoic acid and ethylene reduce transport in both mutant and wild-type sections. The kinetics of auxin uptake into VFN8 and dgt sections are nearly identical. These results make it unlikely that an altered IAA efflux carrier or IAA uptake symport are responsible for the pleiotropic effects resulting from the dgt mutation. The lack of auxin-induced cell elongation in dgt plants is not due to insufficient turgor, as the osmotic potential of dgt cell sap is less (more negative) than that of VFN8. An auxin-induced increase in wall extensibility, as measured by the Instron technique, only occurs in the VFN8 plants. These data suggest dgt hypocotyls suffer a defect in the sequence of events culminating in auxin-induced cell wall loosening.

  9. Spontaneous Nif- mutants of Rhodopseudomonas capsulata.

    PubMed Central

    Wall, J D; Love, J; Quinn, S P

    1984-01-01

    Revertible, spontaneous Nif- mutants of Rhodopseudomonas capsulata have been shown to accumulate in cultures growing photosynthetically with an amino acid as the nitrogen source such that H2 is maximally produced. The majority of such strains carry mutations which are clustered in a short region of the chromosome, probably representing one or two genes. Because this cluster includes temperature-sensitive mutations, it is also likely that it identifies the structural gene of a polypeptide. The phenotypic characterization of these spontaneous mutants showed (i) an inability to grow with N2 as the nitrogen source, no measurable nitrogenase activity, a reduction or absence of the three polypeptides of the MoFe and Fe proteins of the nitrogenase complex, a faster growth rate on glutamate as the nitrogen source under saturating light, and frequently a small increase in glutamine synthetase activity relative to that of the wild type when grown with glutamate as the nitrogen source. Alterations in other ammonium-assimilatory enzyme activities were not observed. Taken together, these properties suggest that the mutations have affected a regulatory protein necessary for nitrogen fixation. Images PMID:6146598

  10. The LORE1 insertion mutant resource.

    PubMed

    Małolepszy, Anna; Mun, Terry; Sandal, Niels; Gupta, Vikas; Dubin, Manu; Urbański, Dorian; Shah, Niraj; Bachmann, Asger; Fukai, Eigo; Hirakawa, Hideki; Tabata, Satoshi; Nadzieja, Marcin; Markmann, Katharina; Su, Junyi; Umehara, Yosuke; Soyano, Takashi; Miyahara, Akira; Sato, Shusei; Hayashi, Makoto; Stougaard, Jens; Andersen, Stig U

    2016-10-01

    Long terminal repeat (LTR) retrotransposons are closely related to retroviruses, and their activities shape eukaryotic genomes. Here, we present a complete Lotus japonicus insertion mutant collection generated by identification of 640 653 new insertion events following de novo activation of the LTR element Lotus retrotransposon 1 (LORE1) (http://lotus.au.dk). Insertion preferences are critical for effective gene targeting, and we exploit our large dataset to analyse LTR element characteristics in this context. We infer the mechanism that generates the consensus palindromes typical of retroviral and LTR retrotransposon insertion sites, identify a short relaxed insertion site motif, and demonstrate selective integration into CHG-hypomethylated genes. These characteristics result in a steep increase in deleterious mutation rate following activation, and allow LORE1 active gene targeting to approach saturation within a population of 134 682 L. japonicus lines. We suggest that saturation mutagenesis using endogenous LTR retrotransposons with germinal activity can be used as a general and cost-efficient strategy for generation of non-transgenic mutant collections for unrestricted use in plant research.

  11. Auxin physiology of the tomato mutant diageotropica

    NASA Technical Reports Server (NTRS)

    Daniel, S. G.; Rayle, D. L.; Cleland, R. E.

    1989-01-01

    The tomato (Lycopersicon esculentum, Mill.) mutant diageotropica (dgt) exhibits biochemical, physiological, and morphological abnormalities that suggest the mutation may have affected a primary site of auxin perception or action. We have compared two aspects of the auxin physiology of dgt and wild-type (VFN8) seedlings: auxin transport and cellular growth parameters. The rates of basipetal indole-3-acetic acid (IAA) polar transport are identical in hypocotyl sections of the two genotypes, but dgt sections have a slightly greater capacity for IAA transport. 2,3,5-Triiodobenzoic acid and ethylene reduce transport in both mutant and wild-type sections. The kinetics of auxin uptake into VFN8 and dgt sections are nearly identical. These results make it unlikely that an altered IAA efflux carrier or IAA uptake symport are responsible for the pleiotropic effects resulting from the dgt mutation. The lack of auxin-induced cell elongation in dgt plants is not due to insufficient turgor, as the osmotic potential of dgt cell sap is less (more negative) than that of VFN8. An auxin-induced increase in wall extensibility, as measured by the Instron technique, only occurs in the VFN8 plants. These data suggest dgt hypocotyls suffer a defect in the sequence of events culminating in auxin-induced cell wall loosening.

  12. Auxin physiology of the tomato mutant diageotropica

    NASA Technical Reports Server (NTRS)

    Daniel, S. G.; Rayle, D. L.; Cleland, R. E.

    1989-01-01

    The tomato (Lycopersicon esculentum, Mill.) mutant diageotropica (dgt) exhibits biochemical, physiological, and morphological abnormalities that suggest the mutation may have affected a primary site of auxin perception or action. We have compared two aspects of the auxin physiology of dgt and wild-type (VFN8) seedlings: auxin transport and cellular growth parameters. The rates of basipetal indole-3-acetic acid (IAA) polar transport are identical in hypocotyl sections of the two genotypes, but dgt sections have a slightly greater capacity for IAA transport. 2,3,5-Triiodobenzoic acid and ethylene reduce transport in both mutant and wild-type sections. The kinetics of auxin uptake into VFN8 and dgt sections are nearly identical. These results make it unlikely that an altered IAA efflux carrier or IAA uptake symport are responsible for the pleiotropic effects resulting from the dgt mutation. The lack of auxin-induced cell elongation in dgt plants is not due to insufficient turgor, as the osmotic potential of dgt cell sap is less (more negative) than that of VFN8. An auxin-induced increase in wall extensibility, as measured by the Instron technique, only occurs in the VFN8 plants. These data suggest dgt hypocotyls suffer a defect in the sequence of events culminating in auxin-induced cell wall loosening.

  13. Thiostrepton-resistant mutants of Thermus thermophilus

    PubMed Central

    Cameron, Dale M.; Thompson, Jill; Gregory, Steven T.; March, Paul E.; Dahlberg, Albert E.

    2004-01-01

    Ribosomal protein L11 and its associated binding site on 23S rRNA together comprise one of the principle components that mediate interactions of translation factors with the ribosome. This site is also the target of the antibiotic thiostrepton, which has been proposed to act by preventing important structural transitions that occur in this region of the ribosome during protein synthesis. Here, we describe the isolation and characterization of spontaneous thiostrepton-resistant mutants of the extreme thermophile, Thermus thermophilus. All mutations were found at conserved positions in the flexible N-terminal domain of L11 or at conserved positions in the L11-binding site of 23S rRNA. A number of the mutant ribosomes were affected in in vitro EF-G-dependent GTP hydrolysis but all showed resistance to thiostrepton at levels ranging from high to moderate. Structure probing revealed that some of the mutations in L11 result in enhanced reactivity of adjacent rRNA bases to chemical probes, suggesting a more open conformation of this region. These data suggest that increased flexibility of the factor binding site results in resistance to thiostrepton by counteracting the conformation-stabilizing effect of the antibiotic. PMID:15199170

  14. Cytokinin production by plant growth promoting rhizobacteria and selected mutants.

    PubMed

    García de Salamone, I E; Hynes, R K; Nelson, L M

    2001-05-01

    One of the proposed mechanisms by which rhizobacteria enhance plant growth is through the production of plant growth regulators. Five plant growth promoting rhizobacterial (PGPR) strains produced the cytokinin dihydrozeatin riboside (DHZR) in pure culture. Cytokinin production by Pseudomonas fluorescens G20-18, a rifampicin-resistant mutant (RIF), and two TnphoA-derived mutants (CNT1, CNT2), with reduced capacity to synthesize cytokinins, was further characterized in pure culture using immunoassay and thin layer chromatography. G20-18 produced higher amounts of three cytokinins, isopentenyl adenosine (IPA), trans-zeatin ribose (ZR), and DHZR than the three mutants during stationary phase. IPA was the major metabolite produced, but the proportion of ZR and DHZR accumulated by CNT1 and CNT2 increased with time. No differences were observed between strain G20-18 and the mutants in the amounts of indole acetic acid synthesized, nor were gibberellins detected in supernatants of any of the strains. Addition of 10(-5) M adenine increased cytokinin production in 96- and 168-h cultures of strain G20-18 by approximately 67%. G20-18 and the mutants CNT1 and CNT2 may be useful for determination of the role of cytokinin production in plant growth promotion by PGPR.

  15. A combinatorial strategy for treating KRAS-mutant lung cancer.

    PubMed

    Manchado, Eusebio; Weissmueller, Susann; Morris, John P; Chen, Chi-Chao; Wullenkord, Ramona; Lujambio, Amaia; de Stanchina, Elisa; Poirier, John T; Gainor, Justin F; Corcoran, Ryan B; Engelman, Jeffrey A; Rudin, Charles M; Rosen, Neal; Lowe, Scott W

    2016-06-30

    Therapeutic targeting of KRAS-mutant lung adenocarcinoma represents a major goal of clinical oncology. KRAS itself has proved difficult to inhibit, and the effectiveness of agents that target key KRAS effectors has been thwarted by activation of compensatory or parallel pathways that limit their efficacy as single agents. Here we take a systematic approach towards identifying combination targets for trametinib, a MEK inhibitor approved by the US Food and Drug Administration, which acts downstream of KRAS to suppress signalling through the mitogen-activated protein kinase (MAPK) cascade. Informed by a short-hairpin RNA screen, we show that trametinib provokes a compensatory response involving the fibroblast growth factor receptor 1 (FGFR1) that leads to signalling rebound and adaptive drug resistance. As a consequence, genetic or pharmacological inhibition of FGFR1 in combination with trametinib enhances tumour cell death in vitro and in vivo. This compensatory response shows distinct specificities: it is dominated by FGFR1 in KRAS-mutant lung and pancreatic cancer cells, but is not activated or involves other mechanisms in KRAS wild-type lung and KRAS-mutant colon cancer cells. Importantly, KRAS-mutant lung cancer cells and patients’ tumours treated with trametinib show an increase in FRS2 phosphorylation, a biomarker of FGFR activation; this increase is abolished by FGFR1 inhibition and correlates with sensitivity to trametinib and FGFR inhibitor combinations. These results demonstrate that FGFR1 can mediate adaptive resistance to trametinib and validate a combinatorial approach for treating KRAS-mutant lung cancer.

  16. Mutant laboratory mice with abnormalities in pigmentation: annotated tables.

    PubMed

    Nakamura, Motonobu; Tobin, Desmond J; Richards-Smith, Beverly; Sundberg, John P; Paus, Ralf

    2002-01-01

    Mammalian pigment cell research has recently entered a phase of significantly increased activity due largely to the exploitation of the many mutant mouse stocks that are coming on stream. Numerous transgenic, targeted mutagenesis (so-called 'knockouts'), conditional (so-called 'gene switch') and spontaneous mutant mice develop abnormal coat color phenotypes. The number of mice that exhibit such abnormalities is increasing exponentially as genetic engineering methods become routine. Since defined abnormalities in such mutant mice provide important clues to the as yet often poorly understood functional roles of many gene products, this overview includes a corresponding, annotated table of mutant mice with pigmentation alterations. These range from early developmental defects via a large array of coat color abnormalities to a melanoma metastasis model. This overview should provide helpful pointers to investigators who are looking for mouse models to explore or to compare functional activities of genes of interest and for comparing coat color phenotypes of spontaneous or genetically engineered mouse mutants with novel ones. Secondly, this review includes a table of mouse models of specific human diseases with genetically defined pigmentation abnormalities. In summary, this annotated table should serve as a useful reference for anyone interested in the molecular controls of pigmentation.

  17. Respiratory-deficient Mutants in Saccharomyces lactis1

    PubMed Central

    Herman, Alberta I.; Griffin, Patricia S.

    1968-01-01

    Six independent ultraviolet-induced respiratory-deficient mutants (petites) of Saccharomyces lactis were isolated and characterized. Two possessed a normal cytochrome spectrum, another displayed an increased level of all the cytochromes, and three suffered from a partial or complete loss of one or more of the cytochromes a, b, c, and c1. All of the mutants were segregational petites; none was vegetative. Determination of linkage relationships between mutants was restricted because matings between mutants, homozygous or heterozygous, for loci affecting cytochrome content were blocked at various stages in the mating-sporulating sequence. At least three of the petites were genetically nonidentical. Three of the mutations appeared to occupy loci within the same linkage group; two of the three mutations that mapped within this region were cytochrome-deficient. Growth at high or low temperatures, under increased osmotic pressure or in media supplemented with various fatty acids or sterols, did not relieve the physiological defects in these mutants. Reasons for the differences in survival of segregational and vegetative petites within this species are examined. PMID:5674057

  18. Emergence of HA mutants during influenza virus pneumonia.

    PubMed

    Manríquez, Maria Eugenia Vázquez; Makino, Akiko; Tanaka, Motoko; Abe, Yasuhisa; Yoshida, Hiroyuki; Morioka, Ichiro; Arakawa, Soichi; Takeshima, Yasuhiro; Iwata, Kentaro; Takasaki, Jin; Manabe, Toshie; Nakaya, Takaaki; Nakamura, Shota; Iglesias, Anjarath Lorena Higuera; Rossales, Rosa Maria Rivera; Mirabal, Erika Pena; Ito, Tateki; Kitazawa, Toshio; Oka, Teruaki; Yamashita, Makoto; Kudo, Koichiro; Shinya, Kyoko

    2012-01-01

    During the influenza pandemic of 2009, the number of viral pneumonia cases showed a marked increase in comparison with seasonal influenza viruses. Mutations at amino acid 222 (D222G mutations) in the virus hemagglutinin (HA) molecule, known to alter the receptor-recognition properties of the virus, were detected in a number of the more severely-affected patients in the early phases of the pandemic. To understand the background for the emergence of the mutant amino acid D222G in human lungs, we conducted histological examinations on lung specimens of patients from Mexico who had succumbed in the pandemic. Prominent regenerative and hyperplastic changes in the alveolar type II pneumocytes, which express avian-type sialoglycan receptors in the respiratory tract of severely affected individuals, were observed in the Mexican patients. An infection model utilizing guinea pigs, which was chosen in order to best simulate the sialic acid distribution of severe pneumonia in human patients, demonstrated an increase of D222G mutants and a delay in the diminution of mutants in the lower respiratory tract in comparison to the upper respiratory tract. Our data suggests that the predominance of avian-type sialoglycan receptors in the pneumonic lungs may contribute to the emergence of viral HA mutants. This data comprehensively illustrates the mechanisms for the emergence of mutants in the clinical samples.

  19. A combinatorial strategy for treating KRAS mutant lung cancer

    PubMed Central

    Manchado, Eusebio; Weissmueller, Susann; Morris, John P.; Chen, Chi-Chao; Wullenkord, Ramona; Lujambio, Amaia; de Stanchina, Elisa; Poirier, John T.; Gainor, Justin F.; Corcoran, Ryan B.; Engelman, Jeffrey A.; Rudin, Charles M.; Rosen, Neal; Lowe, Scott W.

    2016-01-01

    Therapeutic targeting of KRAS-mutant lung adenocarcinoma represents a major goal of clinical oncology. KRAS itself has proven difficult to inhibit, and the effectiveness of agents that target key KRAS effectors has been thwarted by activation of compensatory or parallel pathways that limit their efficacy as single agents. Here we take a systematic approach towards identifying combination targets for trametinib, an FDA-approved MEK inhibitor that acts downstream of KRAS to suppress signaling through the mitogen-activated protein kinase (MAPK) cascade. Informed by a short-hairpin RNA (shRNA) screen, we show that trametinib provokes a compensatory response involving the fibroblast growth factor receptor 1 (FGFR1) that leads to signaling rebound and adaptive drug resistance. As a consequence, genetic or pharmacologic inhibition of FGFR1 in combination with trametinib enhances tumor cell death in vitro and in vivo. This compensatory response shows distinct specificities – it is dominated by FGFR1 in KRAS mutant lung and pancreatic cancer cells, but is not activated or involves other mechanisms in KRAS wild-type lung and KRAS-mutant colon cancer cells. Importantly, KRAS-mutant lung cancer cells and patient tumors treated with trametinib show an increase in FRS2 phosphorylation, a biomarker of FGFR activation; this increase is abolished by FGFR1 inhibition and correlates with sensitivity to trametinib and FGFR inhibitor combinations. These results demonstrate that FGFR1 can mediate adaptive resistance to trametinib and validate a combinatorial approach for treating KRAS-mutant lung cancer. PMID:27338794

  20. Auditory development in progressive motor neuronopathy mouse mutants.

    PubMed

    Volkenstein, Stefan; Brors, Dominik; Hansen, Stefan; Berend, Achim; Mlynski, Robert; Aletsee, Christoph; Dazert, Stefan

    2009-11-06

    The present study was performed to elucidate the hearing development in the progressive motor neuronopathy (pmn) mouse mutant. This mouse has been used as a model for human motoneuron disease. A missense mutation in the tubulin-specific chaperon E (Tbce) gene on mouse chromosome 13 was localized as the underlying genetic defect. The protein encoded by the Tbce gene is essential for the formation of primary tubulin complexes. Studies on motoneurons show disorganization in microtubules and disturbed axonal transport, followed by retrograde degeneration of the motoneurons. A similar pathomechanism is also possible for hearing disorders where disrupted microtubules could cause functional deficits in spiral ganglion neurons or in cochlear hair cells. Click auditory brainstem response (ABR) audiometry in homozygous pmn mutants showed a normal onset of hearing, but an increasing hearing threshold from postnatal day 26 (P26) on to death, compared to heterozygous mutants and wild-type mice. Histological sections of the cochlea at different ages showed a regular morphology. Additionally, spiral ganglion explants from mutant and wild-type mice were cultured. The neurite length from pmn mutants was shorter than in wild-type mice, and the neurite number/explant was significantly decreased in pmn mutants. We show that the pmn mouse mutant is a model for a progressive rapid hearing loss from P26 on, after initially normal hearing development. Heterozygous mice are not affected by this defect. With the knowledge of the well-known pathomechanism of this defect in motoneurons, a dysfunction of cellular mechanisms regulating tubulin assembling suggests that tubulin assembling plays an essential role in hearing function and maintenance.

  1. Selection of chemotaxis mutants of Dictyostelium discoideum

    PubMed Central

    1987-01-01

    A method has been developed for the efficient selection of chemotaxis mutants of Dictyostelium discoideum. Mutants defective in the chemotactic response to folate could be enriched up to 30-fold in one round of selection using a chamber in which a compartment that contained the chemoattractant was separated by a sandwich of four nitrocellulose filters from a compartment that contained buffer. Mutagenized cells were placed in the center of the filter layer and exposed to the attractant gradient built up between the compartments for a period of 3-4 h. While wild-type cells moved through the filters in a wave towards the compartment that contained attractant, mutant cells remained in the filter to which they were applied. After several repetitions of the selection procedure, mutants defective in chemotaxis made up 10% of the total cell population retained in that filter. Mutants exhibiting three types of alterations were collected: motility mutants with either reduced speed of movement, or altered rates of turning; a single mutant defective in production of the attractant- degrading enzyme, folate deaminase; and mutants with normal motility but reduced chemotactic responsiveness. One mutant showed drastically reduced sensitivity in folate-induced cGMP production. Morphogenetic alterations of mutants defective in folate chemotaxis are described. PMID:3793759

  2. Interaction of metronidazole with DNA repair mutants of Escherichia coli.

    PubMed Central

    Yeung, T C; Beaulieu, B B; McLafferty, M A; Goldman, P

    1984-01-01

    It has been proposed that one of metronidazole's partially reduced intermediates interacts either with DNA to exert a bactericidal effect or with water to form acetamide. To test this hypothesis we have examined the effect of metronidazole on several mutants of Escherichia coli that are defective in DNA repair. UV-susceptible RecA- and UvrB- point mutants have an increased susceptibility to metronidazole as manifested by both a decreased minimal inhibitory concentration and a greater bactericidal response to metronidazole in resting cultures. By these criteria, however, we find that UvrB- deletion mutants, which lack the ability to reduce nitrate and chlorate, are no more susceptible to metronidazole than is the wild type. We find, however, that these deletion mutants also lack the ability to reduce metronidazole and thus possibly to form its reactive species. When metronidazole's bactericidal effect is expressed in terms of the concurrent accumulation of acetamide derived from metronidazole, then all RecA- and UvrB- mutants are killed more efficiently than their wild types. The data are consistent, therefore, with metronidazole's lethal effect being mediated by a partially reduced intermediate on the metabolic pathway between metronidazole and acetamide. Defects in other aspects of the DNA repair system do not confer this increased susceptibility to the proposed intermediate. A Tag- mutant, for example, which is defective in 3-methyl-adenine-DNA glycosylase, does not have this increased susceptibility to the presumed precursor of acetamide. Thus, these results provide further support for the hypothesis that the bactericidal effect of metronidazole is mediated by a partially reduced intermediate in the metabolic conversion of metronidazole to acetamide and suggest that this intermediate interacts with DNA to produce a lesion similar to that caused by UV light. PMID:6367636

  3. Characterization of a mutant glucose isomerase from Thermoanaerobacterium saccharolyticum.

    PubMed

    Xu, Heng; Shen, Dong; Wu, Xue-Qiang; Liu, Zhi-Wei; Yang, Qi-He

    2014-10-01

    A series of site-directed mutant glucose isomerase at tryptophan 139 from Thermoanaerobacterium saccharolyticum strain B6A were purified to gel electrophoretic homogeneity, and the biochemical properties were determined. W139F mutation is the most efficient mutant derivative with a tenfold increase in its catalytic efficiency toward glucose compared with the native GI. With a maximal activity at 80 °C of 59.58 U/mg on glucose, this mutant derivative is the most active type ever reported. The enzyme activity was maximal at 90 °C and like other glucose isomerase, this mutant enzyme required Co(2+) or Mg(2+) for enzyme activity and thermal stability (stable for 20 h at 80 °C in the absence of substrate). Its optimum pH was around 7.0, and it had 86 % of its maximum activity at pH 6.0 incubated for 12 h at 60 °C. This enzyme was determined as thermostable and weak-acid stable. These findings indicated that the mutant GI W139F from T. saccharolyticum strain B6A is appropriate for use as a potential candidate for high-fructose corn syrup producing enzyme.

  4. 6-Aminonicotinamide-resistant mutants of Salmonella typhimurium.

    PubMed Central

    Hughes, K T; Cookson, B T; Ladika, D; Olivera, B M; Roth, J R

    1983-01-01

    Resistance to the nicotinamide analog 6-aminonicotinamide has been used to identify the following three new classes of mutants in pyridine nucleotide metabolism. (i) pncX mutants have Tn10 insertion mutations near the pncA locus which reduce but do not eliminate the pncA product, nicotinamide deamidase. (ii) nadB (6-aminonicotinamide-resistant) mutants have dominant alleles of the nadB gene, which we propose are altered in feedback inhibition of the nadB enzyme, L-aspartate oxidase. Many of these mutants also exhibit a temperature-sensitive nicotinamide requirement phenotype. (iii) nadD mutants have mutations that affect a new gene involved in pyridine nucleotide metabolism. Since a high proportion of nadD mutations are temperature-sensitive lethal mutations, this appears to be an essential gene for NAD and NADP biosynthesis. In vivo labeling experiments indicate that in all the above cases, resistance is gained by increasing the ratio of NAD to 6-aminonicotinamide adenine dinucleotide. 6-Aminonicotinamide adenine dinucleotide turns over significantly more slowly in vivo than does normal NAD. PMID:6222034

  5. Regulation of iron absorption in Hfe mutant mice.

    PubMed

    Ajioka, Richard S; Levy, Joanne E; Andrews, Nancy C; Kushner, James P

    2002-08-15

    Hereditary hemochromatosis is most commonly caused by homozygosity for a point mutation (C282Y) in the human hemochromatosis gene (HFE). The mechanism by which HFE regulates iron absorption is not known, but the C282Y mutation results in loss of cell surface expression of the human hemachromatosis protein (HFE) and hyperabsorption of iron by the duodenal enterocyte. Mice homozygous for a deletion in the mouse hemochromatosis gene (Hfe) or a mutation equivalent to that seen in human hereditary hemochromatosis (C282Y) were compared with wild-type animals for their ability to regulate iron absorption. Both mutant strains hyperabsorbed (59)Fe administered by gavage. Feeding a diet supplemented with carbonyl iron resulted in a more than 5-fold reduction of (59)Fe absorption in both wild-type and mutant mouse strains. Similarly, the iron loading associated with age in Hfe mutant mice resulted in nearly a 4-fold reduction in iron absorption. When mice were stimulated to absorb iron either by depleting iron stores or by inducing erythropoiesis, wild type and Hfe mutant strains increased absorption to similar levels, approximately 5-fold over control values. Our data indicate that Hfe mutant mice retain the ability to regulate iron absorption. Mouse hemachromatosis protein (Hfe) plays a minor role in down-regulation but does not influence the up-regulation of iron absorption.

  6. Repair effects of laser on mutants of filamentous fungi

    NASA Astrophysics Data System (ADS)

    Zhao, Yansheng; Xiao, Canpeng; Qian, Hailun; Su, Baoliang; Hu, Yujun; Deng, Jianhui

    1999-09-01

    The paper reports that penicillin-producing strains and lovastatin-producing strains were irradiated by UV and subsequently by laser (632.8 nm), and the reparation rate reached 297% and 264%. High-yield mutant was selected with improved potency of 24.5% and 30%, respectively; Gibberellin producing strains were treated with chemical agent LiCl, and then irradiated with 632.8 nm laser. One mutant with 189.6% increased potency was obtained. The experimental results indicated that using laser irradiation after UV or chemical agent mutation was a new useful method in breeding high-yield strains.

  7. On the quality of mutations in mammalian cells induced by high LET radiations

    NASA Astrophysics Data System (ADS)

    Baumstark-Khan, Christa; Rosendahl, Ilja M.; Rink, Hermann

    The deleterious effects of accelerated heavy ions as component of the space radiation environment on living cells are of increasing importance for long duration human space flight activities. The most important aspect of such densely ionizing particle radiation is attributed to the type and quality of biological damage induced by them. This issue is addressed by investigating cell inactivation and mutation induction at the Hprt locus (coding for hypoxanthine-guanine-phosphoribosyl-transferase) of cultured V79 Chinese hamster cells exposed to densely ionizing radiation (accelerated heavy ions with different LETs from oxygen to gold, specific energies ranging from 1.9 to 69.7 MeV/u, corresponding LET values range from 62 to 13,223 keV/μm) and to sparsely ionizing radiation (200 kV X-rays). 30 spontaneous, 40 X-ray induced and 196 heavy ion induced 6-thioguanine resistant Hprt mutant colonies were characterized by Southern technique using the restriction enzymes EcoRI, PstI and BglII and a full length Hprt cDNA probe isolated from the plasmid pHPT12. Restriction patterns of the spontaneous Hprt mutants were indistinguishable from the wild type pattern, as these mutants probably contain only small deletions or even point mutations in the Hprt locus. In contrast, the overall spectrum of heavy ion induced mutations revealed a majority of partial or total deletions of the Hprt gene. With constant particle fluence (3 × 10 6 particles/cm 2) the quality of heavy ion induced mutations in the Hprt locus depends on physical parameters of the beam (atomic number, specific energy, LET). This finding suggests a relationship between the type of DNA damage and track structure. The fraction of mutants with severe deletions in the Hprt locus after exposure to oxygen ions increases from 65% at 60 keV/μm up to a maximum (100%) at 300 keV/μm and declines with higher LET values to 75% at 750 keV/μm. With heavier ions (Ca- and Au-ions) and even higher LET-values this mutant fraction

  8. Mutant fatty acid desaturase and methods for directed mutagenesis

    DOEpatents

    Shanklin, John [Shoreham, NY; Whittle, Edward J [Greenport, NY

    2008-01-29

    The present invention relates to methods for producing fatty acid desaturase mutants having a substantially increased activity towards substrates with fewer than 18 carbon atom chains relative to an unmutagenized precursor desaturase having an 18 carbon chain length specificity, the sequences encoding the desaturases and to the desaturases that are produced by the methods. The present invention further relates to a method for altering a function of a protein, including a fatty acid desaturase, through directed mutagenesis involving identifying candidate amino acid residues, producing a library of mutants of the protein by simultaneously randomizing all amino acid candidates, and selecting for mutants which exhibit the desired alteration of function. Candidate amino acids are identified by a combination of methods. Enzymatic, binding, structural and other functions of proteins can be altered by the method.

  9. Changes in temperature preferences and energy homeostasis in dystroglycan mutants.

    PubMed

    Takeuchi, Ken-Ichi; Nakano, Yoshiro; Kato, Utako; Kaneda, Mizuho; Aizu, Masako; Awano, Wakae; Yonemura, Shigenobu; Kiyonaka, Shigeki; Mori, Yasuo; Yamamoto, Daisuke; Umeda, Masato

    2009-03-27

    Temperature affects the physiology, behavior, and evolution of organisms. We conducted mutagenesis and screens for mutants with altered temperature preference in Drosophila melanogaster and identified a cryophilic (cold-seeking) mutant, named atsugari (atu). Reduced expression of the Drosophila ortholog of dystroglycan (DmDG) induced tolerance to cold as well as preference for the low temperature. A sustained increase in mitochondrial oxidative metabolism caused by the reduced expression of DmDG accounted for the cryophilic phenotype of the atu mutant. Although most ectothermic animals do not use metabolically produced heat to regulate body temperature, our results indicate that their thermoregulatory behavior is closely linked to rates of mitochondrial oxidative metabolism and that a mutation in a single gene can induce a sustained change in energy homeostasis and the thermal responses.

  10. Ferritin Mutants of Escherichia coli Are Iron Deficient and Growth Impaired, and fur Mutants are Iron Deficient

    PubMed Central

    Abdul-Tehrani, Hossein; Hudson, Aaron J.; Chang, Yung-Sheng; Timms, Andrew R.; Hawkins, Chris; Williams, John M.; Harrison, Pauline M.; Guest, John R.; Andrews, Simon C.

    1999-01-01

    Escherichia coli contains at least two iron storage proteins, a ferritin (FtnA) and a bacterioferritin (Bfr). To investigate their specific functions, the corresponding genes (ftnA and bfr) were inactivated by replacing the chromosomal ftnA and bfr genes with disrupted derivatives containing antibiotic resistance cassettes in place of internal segments of the corresponding coding regions. Single mutants (ftnA::spc and bfr::kan) and a double mutant (ftnA::spc bfr::kan) were generated and confirmed by Western and Southern blot analyses. The iron contents of the parental strain (W3110) and the bfr mutant increased by 1.5- to 2-fold during the transition from logarithmic to stationary phase in iron-rich media, whereas the iron contents of the ftnA and ftnA bfr mutants remained unchanged. The ftnA and ftnA bfr mutants were growth impaired in iron-deficient media, but this was apparent only after the mutant and parental strains had been precultured in iron-rich media. Surprisingly, ferric iron uptake regulation (fur) mutants also had very low iron contents (2.5-fold less iron than Fur+ strains) despite constitutive expression of the iron acquisition systems. The iron deficiencies of the ftnA and fur mutants were confirmed by Mössbauer spectroscopy, which further showed that the low iron contents of ftnA mutants are due to a lack of magnetically ordered ferric iron clusters likely to correspond to FtnA iron cores. In combination with the fur mutation, ftnA and bfr mutations produced an enhanced sensitivity to hydroperoxides, presumably due to an increase in production of “reactive ferrous iron.” It is concluded that FtnA acts as an iron store accommodating up to 50% of the cellular iron during postexponential growth in iron-rich media and providing a source of iron that partially compensates for iron deficiency during iron-restricted growth. In addition to repressing the iron acquisition systems, Fur appears to regulate the demand for iron, probably by controlling

  11. [Synergism between aggregation mutants of Dictyostelium discoideum].

    PubMed

    Barra, J

    1977-02-21

    The cells of an aggregateless mutant of Dictyostelium discoïdeum, agip 235, can cooperate with other aggregateless or wild strains to form differentiated aggregates. A soluble mediator liberated by the coaggregating cells seems responsible for the development of agip 235. In most cases, the development of mutant agip 235 stops at the aggregation stage; however, its coaggregation with the mutant 518 results in cosporulation, with the production of viable spores of each genotype, effecting a phenotypic suppression of both mutations.

  12. The TOC159 mutant of Arabidopsis thaliana accumulates altered levels of saturated and polyunsaturated fatty acids.

    PubMed

    Afitlhile, Meshack; Fry, Morgan; Workman, Samantha

    2015-02-01

    We evaluated whether the TOC159 mutant of Arabidopsis called plastid protein import 2-2 (ppi2-2) accumulates normal levels of fatty acids, and transcripts of fatty acid desaturases and galactolipid synthesis enzymes. The ppi2-2 mutant accumulates decreased pigments and total fatty acid content. The MGD1 gene was downregulated and the mutant accumulates decreased levels of monogalactosyldiacylglycerol (MGDG) and 16:3, which suggests that the prokaryotic pathway was impaired in the mutant. The HY5 gene, which encodes long hypocotyl5 transcription factor, was upregulated in the mutant. The DGD1 gene, an HY5 target was marginally increased and the mutant accumulates digalactosyldiacylglycerol at the control level. The mutant had increased expression of 3-ketoacyl-ACP synthase II gene, which encodes a plastid enzyme that elongates 16:0 to 18:0. Interestingly, glycerolipids in the mutant accumulate increased levels of 18:0. A gene that encodes stearoyl-ACP desaturase (SAD) was expressed at the control level and 18:1 was increased, which suggest that SAD may be strongly regulated at the posttranscriptional level. The molar ratio of MGDG to bilayer forming plastid lipids was decreased in the cold-acclimated wild type but not in the ppi2-2 mutant. This indicates that the mutant was unresponsive to cold-stress, and is consistent with increased levels of 18:0, and decreased 16:3 and 18:3 in the ppi2-2 mutant. Overall, these data indicate that a defective Toc159 receptor impaired the synthesis of MGDG, and affected desaturation of 16 and 18-carbon fatty acids. We conclude that expression of the MGD1 gene and synthesis of MGDG are tightly linked to plastid biogenesis.

  13. In vivo fitness and virulence of a drug-resistant herpes simplex virus 1 mutant.

    PubMed

    Pesola, Jean M; Coen, Donald M

    2007-05-01

    Two important issues regarding a virus mutant that is resistant to an antiviral drug are its ability to replicate in animal hosts (in vivo fitness) relative to other genetic variants, including wild type, and its ability to cause disease. These issues have been investigated for a herpes simplex virus 1 mutant that is resistant to thiourea compounds, which inhibit encapsidation of viral DNA. Following corneal inoculation of mice, the mutant virus replicated very similarly to its wild-type parent in the eye, trigeminal ganglion and brain. The mutant virus was as lethal to mice as its wild-type parent following this route of inoculation. Indeed, it exhibited increased virulence. Thus, unlike most drug-resistant virus mutants, this mutant retained in vivo fitness and virulence.

  14. Identifying representative drug resistant mutants of HIV

    PubMed Central

    2015-01-01

    Background Drug resistance is one of the most important causes for failure of anti-AIDS treatment. During therapy, multiple mutations accumulate in the HIV genome, eventually rendering the drugs ineffective in blocking replication of the mutant virus. The huge number of possible mutants precludes experimental analysis to explore the molecular mechanisms of resistance and develop improved antiviral drugs. Results In order to solve this problem, we have developed a new algorithm to reveal the most representative mutants from the whole drug resistant mutant database based on our newly proposed unified protein sequence and 3D structure encoding method. Mean shift clustering and multiple regression analysis were applied on genotype-resistance data for mutants of HIV protease and reverse transcriptase. This approach successfully chooses less than 100 mutants with the highest resistance to each drug out of about 10K in the whole database. When considering high level resistance to multiple drugs, the numbers reduce to one or two representative mutants. Conclusion This approach for predicting the most representative mutants for each drug has major importance for experimental verification since the results provide a small number of representative sequences, which will be amenable for in vitro testing and characterization of the expressed mutant proteins. PMID:26678327

  15. Electrophysiological study of Drosophila rhodopsin mutants

    PubMed Central

    1986-01-01

    Electrophysiological investigations were carried out on several independently isolated mutants of the ninaE gene, which encodes opsin in R1-6 photoreceptors, and a mutant of the ninaD gene, which is probably important in the formation of the rhodopsin chromophore. In these mutants, the rhodopsin content in R1-6 photoreceptors is reduced by 10(2)-10(6)-fold. Light-induced bumps recorded from even the most severely affected mutants are physiologically normal. Moreover, a detailed noise analysis shows that photoreceptor responses of both a ninaE mutant and a ninaD mutant follow the adapting bump model. Since any extensive rhodopsin-rhodopsin interactions are not likely in these mutants, the above results suggest that such interactions are not needed for the generation and adaptation of light-induced bumps. Mutant bumps are strikingly larger in amplitude than wild-type bumps. This difference is observed both in ninaD and ninaE mutants, which suggests that it is due to severe depletion of rhodopsin content, rather than to any specific alterations in the opsin protein. Lowering or buffering the intracellular calcium concentration by EGTA injection mimics the effects of the mutations on the bump amplitude, but, unlike the mutations, it also affects the latency and kinetics of light responses. PMID:3097245

  16. Cell surface properties of organic solvent-tolerant mutants of Escherichia coli K-12.

    PubMed Central

    Aono, R; Kobayashi, H

    1997-01-01

    In this study, we examined cell surface properties of mutants of Escherichia coli for which organic solvent tolerance levels were elevated. The cell surface of each mutant was less hydrophobic than that of the parent, probably due to an increase in lipopolysaccharide content. OmpF synthesis was repressed in the mutants. Organic solvent bound readily to viable E. coli cells in response to the polarity of the solvent. The mutants were bound less abundantly with the organic solvent than was the parent. PMID:9293016

  17. Susceptibility of lipopolysaccharide-defective mutants of Pseudomonas aeruginosa strain PAO to dyes, detergents, and antibiotics.

    PubMed Central

    Kropinski, A M; Chan, L; Milazzo, F H

    1978-01-01

    Lipopolysaccharide-defective mutants of Pseudomonas aeruginosa strain PAO have been isolated on the basis of their resistance to lipopolysaccharide-specific bacteriophages. These mutants have been differentiated by their agglutination in NaCl and acriflavine, phage sensitivity, and chemical analysis of the lipopolysaccharides. The susceptibility of the wild-type strain and four mutants to a series of twenty-six agents, including dyes, detergents, antibiotics, and lysozyme, was examined. The roughest mutant (AK-43) exhibited increased susceptibility to sodium deoxycholate, hexadecylpyridinium chloride, benzalkonium chloride, ampicillin, penicillin G, erythromycin, colymycin, and polymyxin B. The role of cell envelope fractions in antibiotic resistance in P. aeruginosa is discussed. PMID:122525

  18. Membrane function in lipid mutants of Arabidopsis. First year progress report

    SciTech Connect

    Browse, J.A.

    1993-06-01

    Progress on the biochemical characterization of the fad3 mutants deficient in 18:3 fatty acid synthesis and the fab2 mutant that accumulates increased amounts of 18:0 is described. Studies of the cell biology and physiology of the fab2 and fad2 mutants have provided evidence for some of the critical roles played by unsaturated fatty acids as components of plant membranes. Finally, the fab2 mutant has allowed us to carry out the first isolation and characterization of intergenic suppressor mutations in a higher plant.

  19. Cytochrome abnormalities and cyanide-resistant respiration in extranuclear mutants of Aspergillus nidulans.

    PubMed Central

    Turner, G; Rowlands, R T

    1976-01-01

    The cytochrome spectra of two extranuclear mutants of Aspergillus nidulans and the double-mutant recombinant formed from them have been examined both at room temperature and at the temperature of liquid N2 and compared with those of the wild-type strain. The oligomycin-resistant, slow growing mutant contained an increased amount of cytochrome c without any loss of cytochromes b and a,a3. The cold-sensitive mutant, apparently normal when grown at 37 C, showed an increased amount of cytochrome c and a partial loss of cytochromes b and a,a3 when grown at 20 C. A combination of these effects was observed in the double-mutant recombinant. Cyanide-resistant respiration was present in both mutant strains and in the recombinant at much higher levels than in the wild-type strain. In the oligomycin-resistant mutant, this was usually present together with cyanide-sensitive respiration, whereas in the cold-sensitive mutant and recombinant grown at 20 C cyanide-resistant approached 100%. Inhibitor and growth yield studies indicated that the cyanide-resistant pathway was not used by the cold-sensitive mutant during growth at 20 C. PMID:1107321

  20. 'Green revolution' genes encode mutant gibberellin response modulators.

    PubMed

    Peng, J; Richards, D E; Hartley, N M; Murphy, G P; Devos, K M; Flintham, J E; Beales, J; Fish, L J; Worland, A J; Pelica, F; Sudhakar, D; Christou, P; Snape, J W; Gale, M D; Harberd, N P

    1999-07-15

    World wheat grain yields increased substantially in the 1960s and 1970s because farmers rapidly adopted the new varieties and cultivation methods of the so-called 'green revolution'. The new varieties are shorter, increase grain yield at the expense of straw biomass, and are more resistant to damage by wind and rain. These wheats are short because they respond abnormally to the plant growth hormone gibberellin. This reduced response to gibberellin is conferred by mutant dwarfing alleles at one of two Reduced height-1 (Rht-B1 and Rht-D1) loci. Here we show that Rht-B1/Rht-D1 and maize dwarf-8 (d8) are orthologues of the Arabidopsis Gibberellin Insensitive (GAI) gene. These genes encode proteins that resemble nuclear transcription factors and contain an SH2-like domain, indicating that phosphotyrosine may participate in gibberellin signalling. Six different orthologous dwarfing mutant alleles encode proteins that are altered in a conserved amino-terminal gibberellin signalling domain. Transgenic rice plants containing a mutant GAI allele give reduced responses to gibberellin and are dwarfed, indicating that mutant GAI orthologues could be used to increase yield in a wide range of crop species.

  1. Mutant calreticulin requires both its mutant C-terminus and the thrombopoietin receptor for oncogenic transformation

    PubMed Central

    Elf, Shannon; Abdelfattah, Nouran S.; Chen, Edwin; Perales-Patón, Javier; Rosen, Emily A.; Ko, Amy; Peisker, Fabian; Florescu, Natalie; Giannini, Silvia; Wolach, Ofir; Morgan, Elizabeth A.; Tothova, Zuzana; Losman, Julie-Aurore; Schneider, Rebekka K.; Al-Shahrour, Fatima; Mullally, Ann

    2016-01-01

    Somatic mutations in calreticulin (CALR) are present in approximately 40% of patients with myeloproliferative neoplasms (MPN) but the mechanism by which mutant CALR is oncogenic remains unclear. Here, we demonstrate that expression of mutant CALR alone is sufficient to engender MPN in mice and recapitulates the disease phenotype of CALR-mutant MPN patients. We further show that the thrombopoietin receptor, MPL is required for mutant CALR-driven transformation through JAK-STAT pathway activation, thus rendering mutant CALR-transformed hematopoietic cells sensitive to JAK2 inhibition. Finally, we demonstrate that the oncogenicity of mutant CALR is dependent on the positive electrostatic charge of the C-terminus of the mutant protein, which is necessary for physical interaction between mutant CALR and MPL. Together, our findings elucidate a novel paradigm of cancer pathogenesis and reveal how CALR mutations induce MPN. PMID:26951227

  2. Erythritol production with minimum by-product using Candida magnoliae mutant.

    PubMed

    Ghezelbash, G R; Nahvi, I; Malekpour, A

    2014-01-01

    In order to enhance erythritol production, mutants of Candida magnoliae DSM70638 were generated by ultraviolet and chemical mutagenesis. Erythritol productivity of samples was analyzed by TLC and HPLC with the refractive index detector. One of the mutants named mutant 12-2 gave a 2.4-fold increase in erythritol (20.32 g/L) and a 5.5-fold decrease in glycerol production compared to the wild strain. A sequence-based map of erythrose reductase gene in this mutant showed a replacement of the A321 by G321 that did not cause any amino acid exchange in protein structure. Therefore, the reason of higher erythritol production in C. magnoliae mutant 12-2 is probably the increase in expression of the open reading frame gene. This study revealed that a mutation or minor change in the sequence of genes involved in a production pathway can lead to a significant increase in protein translation.

  3. The phenotype alterations showed by the res tomato mutant disappear when the plants are grown under semi-arid conditions: Is the res mutant tolerant to multiple stresses?

    PubMed

    Garcia-Abellan, José O; Albaladejo, Irene; Egea, Isabel; Flores, Francisco B; Capel, Carmen; Capel, Juan; Angosto, Trinidad; Lozano, Rafael; Bolarin, Maria C

    2016-02-23

    The res (restored cell structure by salinity) mutant, recently identified as the first tomato mutant accumulating jasmonate (JA) without stress, exhibited important morphological alterations when plants were grown under control conditions but these disappeared under salt stress. Since the defense responses against stresses are activated in the res mutant as a consequence of the increased expression of genes from the JA biosynthetic and signaling pathways, the mutant may display a tolerance response not only to salt stress but also to multiple stresses. Here, we show that when res mutant plants are grown under the summer natural conditions of the Mediterranean area, with high temperatures and low relative humidity, the characteristic leaf chlorosis exhibited by the mutant disappears and leaves become dark green over time, with a similar aspect to WT leaves. Moreover, the mutant plants are able to achieve chlorophyll and fluorescence levels similar to those of WT. These results hint that research on res tomato mutant may allow very significant advances in the knowledge of defense responses activated by JA against multiple stresses.

  4. Analyses of Tomato Fruit Brightness Mutants Uncover Both Cutin-Deficient and Cutin-Abundant Mutants and a New Hypomorphic Allele of GDSL Lipase[C][W][OPEN

    PubMed Central

    Petit, Johann; Bres, Cécile; Just, Daniel; Garcia, Virginie; Mauxion, Jean-Philippe; Marion, Didier; Bakan, Bénédicte; Joubès, Jérôme; Domergue, Frédéric; Rothan, Christophe

    2014-01-01

    The cuticle is a protective layer synthesized by epidermal cells of the plants and consisting of cutin covered and filled by waxes. In tomato (Solanum lycopersicum) fruit, the thick cuticle embedding epidermal cells has crucial roles in the control of pathogens, water loss, cracking, postharvest shelf-life, and brightness. To identify tomato mutants with modified cuticle composition and architecture and to further decipher the relationships between fruit brightness and cuticle in tomato, we screened an ethyl methanesulfonate mutant collection in the miniature tomato cultivar Micro-Tom for mutants with altered fruit brightness. Our screen resulted in the isolation of 16 glossy and 8 dull mutants displaying changes in the amount and/or composition of wax and cutin, cuticle thickness, and surface aspect of the fruit as characterized by optical and environmental scanning electron microscopy. The main conclusions on the relationships between fruit brightness and cuticle features were as follows: (1) screening for fruit brightness is an effective way to identify tomato cuticle mutants; (2) fruit brightness is independent from wax load variations; (3) glossy mutants show either reduced or increased cutin load; and (4) dull mutants display alterations in epidermal cell number and shape. Cuticle composition analyses further allowed the identification of groups of mutants displaying remarkable cuticle changes, such as mutants with increased dicarboxylic acids in cutin. Using genetic mapping of a strong cutin-deficient mutation, we discovered a novel hypomorphic allele of GDSL lipase carrying a splice junction mutation, thus highlighting the potential of tomato brightness mutants for advancing our understanding of cuticle formation in plants. PMID:24357602

  5. Analyses of tomato fruit brightness mutants uncover both cutin-deficient and cutin-abundant mutants and a new hypomorphic allele of GDSL lipase.

    PubMed

    Petit, Johann; Bres, Cécile; Just, Daniel; Garcia, Virginie; Mauxion, Jean-Philippe; Marion, Didier; Bakan, Bénédicte; Joubès, Jérôme; Domergue, Frédéric; Rothan, Christophe

    2014-02-01

    The cuticle is a protective layer synthesized by epidermal cells of the plants and consisting of cutin covered and filled by waxes. In tomato (Solanum lycopersicum) fruit, the thick cuticle embedding epidermal cells has crucial roles in the control of pathogens, water loss, cracking, postharvest shelf-life, and brightness. To identify tomato mutants with modified cuticle composition and architecture and to further decipher the relationships between fruit brightness and cuticle in tomato, we screened an ethyl methanesulfonate mutant collection in the miniature tomato cultivar Micro-Tom for mutants with altered fruit brightness. Our screen resulted in the isolation of 16 glossy and 8 dull mutants displaying changes in the amount and/or composition of wax and cutin, cuticle thickness, and surface aspect of the fruit as characterized by optical and environmental scanning electron microscopy. The main conclusions on the relationships between fruit brightness and cuticle features were as follows: (1) screening for fruit brightness is an effective way to identify tomato cuticle mutants; (2) fruit brightness is independent from wax load variations; (3) glossy mutants show either reduced or increased cutin load; and (4) dull mutants display alterations in epidermal cell number and shape. Cuticle composition analyses further allowed the identification of groups of mutants displaying remarkable cuticle changes, such as mutants with increased dicarboxylic acids in cutin. Using genetic mapping of a strong cutin-deficient mutation, we discovered a novel hypomorphic allele of GDSL lipase carrying a splice junction mutation, thus highlighting the potential of tomato brightness mutants for advancing our understanding of cuticle formation in plants.

  6. Genetic interactions among homologous recombination mutants in Candida albicans.

    PubMed

    Bellido, Alberto; Andaluz, Encarnación; Gómez-Raja, Jonathan; Álvarez-Barrientos, Alberto; Larriba, Germán

    2015-01-01

    rad52-ΔΔ and, to a lesser extent, rad51-ΔΔ deletants of Candidaalbicans displayed slow growth and aberrant filamentous morphology whereas rad59-ΔΔ mutants, both by growth rate and morphology resembled wild type. In this study, we have constructed pair-wise double deletants to analyze genetic interactions among these homologous recombination (HR) proteins that affect growth and morphology traits. When grown in liquid YPD medium, double mutant rad51-ΔΔ rad59-ΔΔ exhibited growth rates, cell and colony morphologies, and plating efficiencies that were not significantly different from those observed for rad51-ΔΔ. The same was true for rad52-ΔΔ rad59-ΔΔ compared to rad52-ΔΔ. Slow growth and decreased plating efficiency were caused, at least in part, by a decreased viability, as deduced from FUN1 staining. Flow cytometry and microscopic studies of filamentous mutant populations revealed major changes in cell ploidy, size and morphology, whereas DAPI staining identified complex nuclear rearrangements in yeast and filamentous cells. These phenotypes were not observed in the rad59-ΔΔ mutant populations. Our results show that abolishing Rad51 functions induces the appearance of a subpopulation of aberrant yeast and filamentous forms with increased cell size and ploidy. The size of this complex subpopulation was exacerbated in rad52-ΔΔ mutants. The combination of filamentous cell morphology and viability phenotypes was reflected on the colony morphology of the respective mutants. We conclude that the rad52 mutation is epistatic to rad51 for all the morphological traits analyzed. We discuss these results in the light of the several functions of these recombination genes. Copyright © 2014 Elsevier Inc. All rights reserved.

  7. Metabolic flexibility of a butyrate pathway mutant of Clostridium acetobutylicum.

    PubMed

    Yoo, Minyeong; Croux, Christian; Meynial-Salles, Isabelle; Soucaille, Philippe

    2017-01-31

    Clostridium acetobutylicum possesses two homologous buk genes, buk (or buk1) and buk2, which encode butyrate kinases involved in the last step of butyrate formation. To investigate the contribution of buk in detail, an in-frame deletion mutant was constructed. However, in all the Δbuk mutants obtained, partial deletions of the upstream ptb gene were observed, and low phosphotransbutyrylase and butyrate kinase activities were measured. This demonstrates that i) buk (CA_C3075) is the key butyrate kinase-encoding gene and that buk2 (CA_C1660) that is poorly transcribed only plays a minor role; and ii) strongly suggests that a Δbuk mutant is not viable if the ptb gene is not also inactivated, probably due to the accumulation of butyryl-phosphate, which might be toxic for the cell. One of the ΔbukΔptb mutants was subjected to quantitative transcriptomic (mRNA molecules/cell) and fluxomic analyses in acidogenic, solventogenic and alcohologenic chemostat cultures. In addition to the low butyrate production, drastic changes in metabolic fluxes were also observed for the mutant: i) under acidogenic conditions, the primary metabolite was butanol and a new metabolite, 2-hydroxy-valerate, was produced ii) under solventogenesis, 58% increased butanol production was obtained compared to the control strain under the same conditions, and a very high yield of butanol formation (0.3gg(-1)) was reached; and iii) under alcohologenesis, the major product was lactate. Furthermore, at the transcriptional level, adhE2, which encodes an aldehyde/alcohol dehydrogenase and is known to be a gene specifically expressed in alcohologenesis, was surprisingly highly expressed in all metabolic states in the mutant. The results presented here not only support the key roles of buk and ptb in butyrate formation but also highlight the metabolic flexibility of C. acetobutylicum in response to genetic alteration of its primary metabolism.

  8. Differential thermosensitivity in mixed syndrome cardiac sodium channel mutants.

    PubMed

    Abdelsayed, Mena; Peters, Colin H; Ruben, Peter C

    2015-09-15

    Cardiac arrhythmias are often associated with mutations in SCN5A the gene that encodes the cardiac paralogue of the voltage-gated sodium channel, NaV 1.5. The NaV 1.5 mutants R1193Q and E1784K give rise to both long QT and Brugada syndromes. Various environmental factors, including temperature, may unmask arrhythmia. We sought to determine whether temperature might be an arrhythmogenic trigger in these two mixed syndrome mutants. Whole-cell patch clamp was used to measure the biophysical properties of NaV 1.5 WT, E1784K and R1193Q mutants. Recordings were performed using Chinese hamster ovary (CHOk1) cells transiently transfected with the NaV 1.5 α subunit (WT, E1784K, or R1193Q), β1 subunit, and eGFP. The channels' voltage-dependent and kinetic properties were measured at three different temperatures: 10ºC, 22ºC, and 34ºC. The E1784K mutant is more thermosensitive than either WT or R1193Q channels. When temperature is elevated from 22°C to 34°C, there is a greater increase in late INa and use-dependent inactivation in E1784K than in WT or R1193Q. However, when temperature is lowered to 10°C, the two mutants show a decrease in channel availability. Action potential modelling using Q10 fit values, extrapolated to physiological and febrile temperatures, show a larger transmural voltage gradient in E1784K compared to R1193Q and WT with hyperthermia. The E1784K mutant is more thermosensitive than WT or R1193Q channels. This enhanced thermosensitivity may be a mechanism for arrhythmogenesis in patients with E1784K sodium channels.

  9. Enhancers of Conidiation Mutants in Aspergillus Nidulans

    PubMed Central

    Gems, D. H.; Clutterbuck, A. J.

    1994-01-01

    Mutants at a number of loci, designated sthenyo, have been isolated as enhancers of the oligoconidial mutations at the medA locus. Two loci have been mapped: sthA on linkage group I, and sthB on linkage group V. Two probable alleles have been identified at each locus but two further mutants were unlinked to either sthA or sthB. Neither sthA nor sthB mutants have conspicuous effects on morphology on their own, nor could the sthA1 sthB2 double mutant be distinguished from wild type. Mutants at both loci also interact with the temperature-sensitive brlA42 mutant at the permissive temperature to give a phenotype described as ``Abacoid.'' sthA1 also induces a slight modification of the phenotype of an abaA mutant. We conclude that sthenyo genes act mainly at the phialide stage of conidiation. We also describe the isolation of new medA mutants arising spontaneously as outgrowths on brlA42 colonies. PMID:8056325

  10. Regulation of Mutant p53 Protein Expression.

    PubMed

    Vijayakumaran, Reshma; Tan, Kah Hin; Miranda, Panimaya Jeffreena; Haupt, Sue; Haupt, Ygal

    2015-01-01

    For several decades, p53 has been detected in cancer biopsies by virtue of its high protein expression level which is considered indicative of mutation. Surprisingly, however, mouse genetic studies revealed that mutant p53 is inherently labile, similar to its wild type (wt) counterpart. Consistently, in response to stress conditions, both wt and mutant p53 accumulate in cells. While wt p53 returns to basal level following recovery from stress, mutant p53 remains stable. In part, this can be explained in mutant p53-expressing cells by the lack of an auto-regulatory loop with Mdm2 and other negative regulators, which are pivotal for wt p53 regulation. Further, additional protective mechanisms are acquired by mutant p53, largely mediated by the co-chaperones and their paralogs, the stress-induced heat shock proteins. Consequently, mutant p53 is accumulated in cancer cells in response to chronic stress and this accumulation is critical for its oncogenic gain of functions (GOF). Building on the extensive knowledge regarding wt p53, the regulation of mutant p53 is unraveling. In this review, we describe the current understanding on the major levels at which mutant p53 is regulated. These include the regulation of p53 protein levels by microRNA and by enzymes controlling p53 proteasomal degradation.

  11. Eyespot-assembly mutants in Chlamydomonas reinhardtii.

    PubMed Central

    Lamb, M R; Dutcher, S K; Worley, C K; Dieckmann, C L

    1999-01-01

    Chlamydomonas reinhardtii is a single-celled green alga that phototaxes toward light by means of a light-sensitive organelle, the eyespot. The eyespot is composed of photoreceptor and Ca(++)-channel signal transduction components in the plasma membrane of the cell and reflective carotenoid pigment layers in an underlying region of the large chloroplast. To identify components important for the positioning and assembly of a functional eyespot, a large collection of nonphototactic mutants was screened for those with aberrant pigment spots. Four loci were identified. eye2 and eye3 mutants have no pigmented eyespots. min1 mutants have smaller than wild-type eyespots. mlt1(ptx4) mutants have multiple eyespots. The MIN1, MLT1(PTX4), and EYE2 loci are closely linked to each other; EYE3 is unlinked to the other three loci. The eye2 and eye3 mutants are epistatic to min1 and mlt1 mutations; all double mutants are eyeless. min1 mlt1 double mutants have a synthetic phenotype; they are eyeless or have very small, misplaced eyespots. Ultrastructural studies revealed that the min1 mutants are defective in the physical connection between the plasma membrane and the chloroplast envelope membranes in the region of the pigment granules. Characterization of these four loci will provide a beginning for the understanding of eyespot assembly and localization in the cell. PMID:10511552

  12. A halotolerant mutant of Saccharomyces cerevisiae.

    PubMed Central

    Gaxiola, R; Corona, M; Zinker, S

    1996-01-01

    FRD, a nuclear and dominant spontaneous mutant of Saccharomyces cerevisiae capable of growing in up to 2 M NaCl, was isolated. Compared with parental cells, the mutant cells have a lower intracellular Na+/K+ ratio, shorter generation times in the presence of 1 M NaCl, and alterations in gene expression. PMID:8631691

  13. Saint Louis Encephalitis Temperature-Sensitive Mutants.

    DTIC Science & Technology

    1979-09-01

    Several mutants have been used in pairwise crosses to determine complementation Accessionl For VTIS G!RA& DTIC T %B 3 fl!, -r,. AD) Av ’Di [ Annual...Ghendon (1973) indicate that a large number of polio virus ts mutants producing a pathologic change in infected monkeys were assayed for virus produccion

  14. Comparable clinical outcomes in patients with HER2-mutant and EGFR-mutant lung adenocarcinomas.

    PubMed

    Gow, Chien-Hung; Chang, Hou-Tai; Lim, Chor-Kuan; Liu, Chao-Yu; Chen, Jin-Shing; Shih, Jin-Yuan

    2017-05-01

    HER2 is a major proliferative driver in lung cancer. HER2 gene aberrations impact the prognosis of lung adenocarcinoma (ADC). A one-step reverse transcription-polymerase chain reaction was performed using RNA samples from 888 Asian lung cancer patients to detect HER2, EGFR, KRAS, ALK, and ROS1 mutations. The demographic data and treatment outcomes of HER2 mutation-positive lung ADC patients were analyzed and compared to those with HER2 mutation-negative tumors. HER2 mutation was identified in 40 (4.5%) lung ADC patients. HER2 mutations tended to occur in male patients with advanced-stage disease and never-smokers. A775_G776insYVMA (n = 22, 55%) was the most prevalent HER2 mutation, followed by P780_Y781insGSP (n = 4, 10%). For patients diagnosed with stage-IIIB/IV disease, HER2-mutant patients showed clinical outcomes comparable to EGFR-mutant patients (P = 0.721, log-rank test) and a better overall survival (OS) compared to patients lacking driver mutations in the investigated genes (P = 0.033, Breslow test). Specifically, lung ADC patients with stage-IV HER2-mutant tumors treated with chemotherapy or targeted agents, even without afatinib or anti-HER2 targeted therapy, showed similar clinical outcomes to lung ADC patients harboring EGFR exon 19 deletion or L858R mutations (P = 0.870). In addition, multivariate analysis indicated that HER2 mutation status was not a major risk factor for diminished OS in stage-IV lung cancer. In conclusion, lung ADC harboring HER2 mutations showed distinct characteristics from other driver mutations, including increased chemosensitivity with in advanced stage disease.

  15. Radiation-sensitive mutants of Arabidopsis thaliana

    SciTech Connect

    Jenkins, M.E.; Harlow, G.R.; Liu, Z.

    1995-06-01

    Five Arabidopsis mutants have been isolated on the basis of hypersensitivity of leaf tissue to UV light. For each mutant, the UV-hypersensitive phenotype (uvh) was inherited as a single recessive Mendelian trait. In addition, each uvh mutant represented a separate complementation group. Three of the mutations producing the UV hypersensitive phenotype have been mapped relative to either genetic markers or physical microsatellite polymorphisms. Locus UVH1 is linked to nga76 on chromosome 5, UVH3 to GL1 on chromosome three, and UVH6 to nga59 on chromosome 1. Each uvh mutant has a characteristic pattern of sensitivity based on UV sensitivity of leaf tissue, UV sensitivity of root tissue, and ionizing radiation sensitivity of seeds. On the basis of these patterns, possible molecular defects in these mutants are discussed. 30 refs., 3 figs., 5 tabs.

  16. Suppression of inhibitor of differentiation 2, a target of mutant p53, is required for gain-of-function mutations.

    PubMed

    Yan, Wensheng; Liu, Gang; Scoumanne, Ariane; Chen, Xinbin

    2008-08-15

    Overexpression of mutant p53 is a common theme in human tumors, suggesting a tumor-promoting gain-of-function for mutant p53. To elucidate whether and how mutant p53 acquires its gain-of-function, mutant p53 is inducibly knocked down in the SW480 colon cancer cell line, which contains mutant p53(R273H/P309S), and the MIA PaCa-2 pancreatic cancer cell line, which contains mutant p53(R248W). We found that knockdown of mutant p53 markedly inhibits cell proliferation. In addition, knockdown of mutant p53 sensitizes tumor cells to growth suppression by various chemotherapeutic drugs. To determine whether a gene involved in cell growth and survival is regulated by mutant p53, gene expression profiling analysis was performed and showed that the expression level of Id2, a member of the inhibitor of differentiation (Id) family, was markedly increased upon knockdown of mutant p53. To confirm this, Northern blot analysis was performed and showed that the expression level of Id2 was regulated by various mutant p53s in multiple cell lines. In addition, we found that the Id2 promoter is responsive to mutant but not wild-type p53, and mutant p53 binds to the Id2 promoter. Consistent with these observations, expression of endogenous Id2 was found to be inhibited by exogenous mutant p53 in p53-null HCT116 cells. Finally, we showed that knockdown of Id2 can restore the proliferative potential of tumor cells inhibited by withdrawal of mutant p53. Together, these findings suggest that one mechanism by which mutant p53 acquires its gain-of-function is through the inhibition of Id2 expression.

  17. [Characteristics of mutants of Streptomyces peucetius subsp. caesius ATCC 27952 resistant to anthracycline antibiotics].

    PubMed

    Dubyts'ka, L P; Fedorenko, V O

    2001-01-01

    Daunorubicine (DNR) and doxorubicine (DXR) resistance to anthracycline antibiotics and antibiotical activity of the strain Streptomyces peucetius subsp. caesius ATCC 27952-2 and its daunorubicine- and doxorubicine-resistant (DNRr and DXRr) mutants have been studied. It has been found that strain S. peucetius subsp. caesius ATCC 27952-2 is much more resistant to DXR than to DNR. It has been shown that frequency of appearance of spontaneous mutants of the strain S. peucetius subsp. caesius ATCC 27952-2, resistant to DNR (10-30 micrograms/ml) makes 1.1 x 10(-6)-1.0 x 10(-8) and DXR (10-30 micrograms/ml) 7.7 x 10(-4)-3.0 x 10(-4). Treatment of spores of the initial strain by N-methyl-N'-nitro-N-nitrosoguanidin resulted in the 60-700-fold increase of arising frequency of DNRr-mutants while frequency of arising of DXRr-mutants did not change essentially. The majority of DNRr-mutants (76.9%) were characterised by high resistance to DXR, while there are such mutants among DXRr-mutants which possess the cross-resistance to DNR (below 30%). The mean value of antibiotic activity was the highest among DXRr-mutants induced by NG and sampled in the medium with 30 micrograms/ml of DXR. The majority of the tested DNRr- and DXRr-mutants (13 of 15) synthesized 1.8-10.9 times more of anthracycline compounds as compared with the initial strain. All the mutants synthesized 2.6-28.3 and 1.6-20.7 times more of DNR and DXR, respectively, than the initial strain. The results obtained prove that it is expedient to search for the strains with high production of DXR among DNR1-mutants, while DXRr-mutants are promising for choosing the producers of the both antibiotics.

  18. Sequential evaluation of CALR mutant burden in patients with myeloproliferative neoplasms.

    PubMed

    Cavalloni, Chiara; Rumi, Elisa; Ferretti, Virginia V; Pietra, Daniela; Roncoroni, Elisa; Bellini, Marta; Ciboddo, Michele; Casetti, Ilaria C; Landini, Benedetta; Fugazza, Elena; Troletti, Daniela; Astori, Cesare; Cazzola, Mario

    2017-05-16

    We investigated the variation of CALR-mutant burden during follow-up in 105 CALR-mutant MPN and compared it to the variation of JAK2-mutant burden in 226 JAK2-mutant MPN.The median allele burden at last evaluation was significantly higher than at first evaluation in essential thrombocythemia (ET) (49.5% vs 45%, P < .001) but not in primary myelofibrosis (PMF) (52% vs 51%, P 0.398). Median values of slope were positive both in ET (0.071) and in PMF (0.032). In CALR-mutant ET there was a difference between natural and therapy-related slope (P 0.006).In the JAK2-mutated cohort, the median allele burden at last evaluation was not different respect to that at first evaluation, neither in ET (22.9% vs 23.2%, P = 0.216) nor in PMF (50.5% vs 45.0%, P = 0.809), despite a positive slope. Multivariate analysis to evaluate the effect of mutation (CALR vs JAK2) on the slope of mutant burden in not treated pts with a positive slope adjusting for diagnosis (ET vs PMF) showed a trend toward a higher increase of mutant burden in CALR vs JAK2 (β = 0.19, P = 0.061) with no difference between diagnosis (P = 0.419). The findings of this study suggest that clonal expansion in CALR-mutant MPN is faster than that observed in JAK2-mutant MPN.

  19. Sequential evaluation of CALR mutant burden in patients with myeloproliferative neoplasms

    PubMed Central

    Ferretti, Virginia V.; Pietra, Daniela; Roncoroni, Elisa; Bellini, Marta; Ciboddo, Michele; Casetti, Ilaria C.; Landini, Benedetta; Fugazza, Elena; Troletti, Daniela; Astori, Cesare; Cazzola, Mario

    2017-01-01

    We investigated the variation of CALR-mutant burden during follow-up in 105 CALR-mutant MPN and compared it to the variation of JAK2-mutant burden in 226 JAK2-mutant MPN. The median allele burden at last evaluation was significantly higher than at first evaluation in essential thrombocythemia (ET) (49.5% vs 45%, P < .001) but not in primary myelofibrosis (PMF) (52% vs 51%, P 0.398). Median values of slope were positive both in ET (0.071) and in PMF (0.032). In CALR-mutant ET there was a difference between natural and therapy-related slope (P 0.006). In the JAK2-mutated cohort, the median allele burden at last evaluation was not different respect to that at first evaluation, neither in ET (22.9% vs 23.2%, P = 0.216) nor in PMF (50.5% vs 45.0%, P = 0.809), despite a positive slope. Multivariate analysis to evaluate the effect of mutation (CALR vs JAK2) on the slope of mutant burden in not treated pts with a positive slope adjusting for diagnosis (ET vs PMF) showed a trend toward a higher increase of mutant burden in CALR vs JAK2 (β = 0.19, P = 0.061) with no difference between diagnosis (P = 0.419). The findings of this study suggest that clonal expansion in CALR-mutant MPN is faster than that observed in JAK2-mutant MPN. PMID:28422716

  20. Metabolism of Proline, Glutamate, and Ornithine in Proline Mutant Root Tips of Zea mays (L.)

    PubMed Central

    Dierks-Ventling, Christa; Tonelli, Chiara

    1982-01-01

    In excised pro1-1 mutant and corresponding normal type roots of Zea mays L. the uptake and interconversion of [14C]proline, [14C]glutamic acid, [14C]glutamine, and [14C]ornithine and their utilization for protein synthesis was measured with the intention of finding an explanation for the proline requirement of the mutant. Uptake of these four amino acids, with the exception of proline, was the same in mutant and normal roots, but utilization differed. Higher than normal utilization rates for proline and glutamic acid were noted in mutant roots leading to increased CO2 production, free amino acid interconversion, and protein synthesis. Proline was synthesized from either glutamic acid (or glutamine) or ornithine in both mutant and normal roots; it did not accumulate but rather was used for protein synthesis. Ornithine was not a good precursor for proline in either system, but was preferentially converted to arginine and glutamine, particularly in mutant roots. The pro1-1 mutant was thus not deficient in its ability to make proline. Based on these findings, and on the fact that ornithine, arginine, glutamic acid and aspartic acid are elevated as free amino acids in mutant roots, it is suggested that in the pro1-1 mutant proline catabolism prevails over proline synthesis. PMID:16662144

  1. Altered desferrioxamine-mediated iron utilization is a common trait of bald mutants of Streptomyces coelicolor.

    PubMed

    Lambert, Stéphany; Traxler, Matthew F; Craig, Matthias; Maciejewska, Marta; Ongena, Marc; van Wezel, Gilles P; Kolter, Roberto; Rigali, Sébastien

    2014-08-01

    Streptomyces coelicolor is an important model organism for developmental studies of filamentous GC-rich actinobacteria. The genetic characterization of mutants of S. coelicolor blocked at the vegetative mycelium stage, the so-called bald (bld) mutants that are unable to erect spore-forming aerial hyphae, has opened the way to discovering the molecular basis of development in actinomycetes. Desferrioxamine (DFO) production and import of ferrioxamines (FO; iron-complexed DFO) are key to triggering morphogenesis of S. coelicolor and we show here that growth of S. coelicolor on the reference medium for Streptomyces developmental studies is fully dependent on DFO biosynthesis. UPLC-ESI-MS analysis revealed that all bld mutants tested are affected in DFO biosynthesis, with bldA, bldJ, and ptsH mutants severely impaired in DFO production, while bldF, bldK, crr and ptsI mutants overproduce DFO. Morphogenesis of bldK and bldJ mutants was recovered by supplying exogenous iron. Transcript analysis showed that the bldJ mutant is impaired in expression of genes involved in the uptake of FO, whereas transcription of genes involved in both DFO biosynthesis and FO uptake is increased in bldK mutants. Our study allows proposing altered DFO production and/or FO uptake as a novel phenotypic marker of many S. coelicolor bld mutants, and strengthe