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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. PMID:12539752

  2. 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.

  3. Hprt mutants in a transplantable murine tumour arise more frequently in vivo than in vitro.

    PubMed Central

    Wilkinson, D.; Sandhu, J. K.; Breneman, J. W.; Tucker, J. D.; Birnboim, H. C.

    1995-01-01

    A model system was developed to allow investigation of the frequency at which clastogenic and/or mutagenic events occur in situ in a transplantable murine fibrosarcoma tumour (MC1A-C1) compared with in vitro culture. The marker selected for detecting these events was the X-linked hprt (hypoxanthine-guanine phosphoribosyltransferase) gene. We found that the hprt gene in MC1A-C1 was not suitable for this purpose, most likely because multiple active copies were present. To circumvent the problem, HPRT- [6-thioguanine (6-TG)-resistant] clones were isolated by inactivating all hprt genes with methylnitrosourea. Spontaneous revertants to hypoxanthine/aminopterin/thymidine resistance (HATR) were isolated and found to be approximately 1000 times more sensitive than the parental tumour to induction of 6-TGR mutants by cobalt-60 gamma-rays. This sensitivity is expected for a heterozygous marker, these revertants may therefore possess only one functional hprt locus but two or more active X chromosomes. A clone with a stable hprt gene was identified and a neo gene was introduced. The resulting cell line (MN-11) could be grown as a subcutaneous tumour in syngeneic C57BL/6 animals. The frequency of mutations arising in vivo in the marker hprt gene could be estimated by culturing explanted tumour cells in the presence of 6-TG, using G418 selection to distinguish tumour from host cells. The frequency of mutants in MN-11 cells grown as tumours was found to be 3.4-fold higher than in tissue culture for an equivalent period of time. These data provide the first direct evidence for the existence of mutagenic factors in a tumour environment that might contribute to tumour progression. Images Figure 1 PMID:7577474

  4. 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

  5. Longitudinal study of the in vivo hprt mutant frequency in human T-lymphocytes as determined by a cell cloning assay

    SciTech Connect

    O'Neill, J.P.; Sullivan, L.M.; Booker, J.K.; Pornelos, B.S.; Falta, M.T.; Greene, C.J.; Albertini, R.J. )

    1989-01-01

    The in vivo frequency of mutants resulting from mutation at the hprt locus in human T-lymphocytes can be determined by a cloning assay. This assay quantifies the frequency of 6-thioguanine-resistant (TG{sup r}) T-cells through growth of colonies in 96-well microtiter dishes. The reproducibility of the TG{sup r} mutant frequency values has now been assessed in a longitudinal study of six individuals employing 4-5 blood samples over a 26-37 week time period. Cloning assays were performed with both fresh and cryopreserved cell samples. No significant differences were found among the mutant frequency values for multiple samples from each individual with both fresh and cryopreserved cell samples. These results demonstrate the reproducibility of this cloning assay for in vivo mutant frequency determinations in human T-lymphocytes.

  6. 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. PMID:25914368

  7. Use of the clonal assay for the measurement of frequencies of HPRT mutants in T-lymphocytes from five control populations.

    PubMed

    Tates, A D; van Dam, F J; van Mossel, H; Schoemaker, H; Thijssen, J C; Woldring, V M; Zwinderman, A H; Natarajan, A T

    1991-10-01

    The clonal assay was used to measure frequencies of 6-thioguanine-resistant (HPRT) T-lymphocytes in 111 donors from the following 5 control populations: 55 adult healthy volunteers; 20 untreated cancer patients; 8 healthy hospital workers serving as controls for 9 hospital workers sterilizing equipment with ethylene oxide; 15 factory workers serving as controls for 15 workers occupationally exposed to high doses of ethylene oxide; 13 pretreatment samples from donors undergoing a diagnostic test with Technetium-99m for an analysis of heart function. With respect to mutant frequency (MF), cloning efficiency (CE) and age distribution, the first 4 populations were identical. The Technetium group had significantly higher MFs and lower CEs but this can be attributed to the higher mean age of this group. Using the total data base, we calculated the following relationships between MF, CE, age and smoking: (1) ln MF = 4.23-0.63 x ln CE indicating that a doubling of the CE has the effect of decreasing the MF by 37%, (2) ln MF = 0.71 + 0.03 x age meaning that the MF increases by 3% from one year to the next, (3) ln CE = 4.87-0.04 X age indicating that the CE decreases by 0.98% from one year to the next, (4) ln MF = 3.25-0.52 x ln CE + 0.02 X age being the equation quantifying the interrelationship between MF, CE and age, (5) ln MF = 3.32-0.56 x ln CE + 0.01 x age + 0.31 s (where s = 1 for smokers and s = 0 for nonsmokers). Using the latter equation, which allows for effects of CE and age on the MF, a statistically significant effect of smoking could be established. For any combination of CE and age smoking has the effect of increasing the MF by 36%. The above conclusions and calculations remain essentially the same when donors with cloning efficiencies lower than 10 or 20% are excluded from the data base. PMID:1922146

  8. 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

  9. Increased frequency of in vivo hprt gene-mutated T cells in the peripheral blood of patients with systemic sclerosis.

    PubMed Central

    Sfikakis, P P; Tesar, J; Theocharis, S; Klipple, G L; Tsokos, G C

    1994-01-01

    OBJECTIVE--Activated T lymphocytes are involved in the pathogenesis of scleroderma (systemic sclerosis, SSc); such cells rapidly divide in vivo and are thus theoretically subject to random mutation more frequently than resting cells. To study whether SSc is associated with rapidly expanding T cell clones the frequency was determined of in vivo mutated T cells (MF) at the hypoxanthine guanine phosphoribosyl transferase (hprt) gene in the peripheral blood from patients with SSc. Specific clinical or serological associations were also investigated. METHODS--Peripheral blood lymphocytes from 16 healthy individuals and 20 patients with SSc were cultured using an hprt clonal assay; mutated and wild T cell clones were established to assess individual values of T cell MF. T cell clones were further expanded in vitro and their phenotype was determined by standard immunofluorescence technique. Enzyme-linked immunosorbent assays were used for simultaneous measurements of plasma levels of soluble Interleukin-2 receptors (s-IL-2R) and Intercellular adhesion molecule-1 (s-ICAM-1). RESULT--Mean (SD) value of T cell MF in patients with SSc was 2.5-fold higher than the normal mean (SD) value [10.6 (6.6) x 10(-6) v [4.4 (2.8) x 10(-6), p = 0.0007]. Eleven of 20 patients with SSc (55%) had T cell MF values greater than two SD above the normal mean value. The majority (84%) of mutated T cells had a helper/inducer, memory phenotype while 12% were cytotoxic/suppressor T cells. There was no association between T cell MF and the extent of skin involvement or the duration of Raynaud's phenomenon. High individual T cell MF values were not related to a possible concurrent immune overactivity as assessed by plasma levels of s-IL-2R and s-ICAM-1. Patients with long standing skin disease, however, had almost double T cell MF values than patients with early skin disease [(13.6 (7.4)) x 10(-6) v (7.5 (4.3)) x 10(-6), p = 0.03], suggesting that increased T cell MF in SSc may reflect an ongoing

  10. HPRT Mutations in Lymphocytes from 1,3-Butadiene-Exposed Workers in China

    PubMed Central

    Liu, Shengxue; Ao, Lin; Du, Bing; Zhou, Yanhong; Yuan, Jian; Bai, Yang; Zhou, Ziyuan; Cao, Jia

    2008-01-01

    Background 1,3-Butadiene (BD) is an important industrial chemical and an environmental and occupational pollutant. The carcinogenicity of BD in rodents has been proved, but its carcinogenic and mutagenic molecular mechanism(s) are not fully elucidated in humans. Objectives In the present study, we compared the mutation frequencies and exon deletions of BD-exposed workers with that of control subjects in China to identify the characteristic mutations associated with BD exposure in the human HPRT (hypoxanthine–guanine–phosphoribosyltransferase) gene. Methods Seventy-four workers exposed to BD via inhalation and 157 matched controls were evaluated in Nanjing, China. Molecular analysis of HPRT mutant T lymphocytes from BD-exposed workers and nonexposed control subjects was conducted to identify changes in the structure of the HPRT gene. A total of 783 HPRT mutants were analyzed by multiplex polymerase chain reaction, in which 368 HPRT mutants were isolated from BD-exposed workers and 415 mutants from control subjects. Results The BD-exposed workers showed a higher mutation frequency (18.2 ± 9.4 × 10−6) than the control subjects (12.7 ± 7.3 × 10−6), but the difference was not significant (p > 0.05). The frequency of exon deletions in BD-exposed workers (27.4%) was significantly higher than that in control subjects (12.5%) (p < 0.05), which mainly included multiplex exon deletions (2–8 exons). Conclusions The results of the present study suggest that BD should increase the frequency of large deletions of HPRT gene in human lymphocytes This change confirms and supports the previous findings in BD-exposed workers. PMID:18288319

  11. LARGE DELETIONS ARE TOLERATED AT THE HPRT LOCUS OF IN-VIVO DERIVED HUMAN T-LYMPHOCYTES

    EPA Science Inventory

    A cloning assay was used to recover hprt T-lymphocytes from adult human males. nalysis of crude cellular extracts by polymerase chain reactions (PCRs) demonstrated that 8% (18/218) of the hprt mutations were due to total deletion of the hprt gene. ourteen of the 18 mutants were e...

  12. 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

  13. NAD metabolism in HPRT-deficient mice

    PubMed Central

    Jacomelli, Gabriella; Di Marcello, Federica; Notarantonio, Laura; Sestini, Silvia; Cerboni, Barbara; Bertelli, Matteo; Pompucci, Giuseppe; Jinnah, Hyder A.

    2016-01-01

    The activity of hypoxanthine-guanine phosphoribosyltransferase (HPRT) is virtually absent in Lesch-Nyhan disease (LND), an X-linked genetic disorder characterized by uric acid accumulation and neurodevelopmental dysfunction. The biochemical basis for the neurological and behavioral abnormalities have not yet been completely explained. Prior studies of cells from affected patients have shown abnormalities of NAD metabolism. In the current studies, NAD metabolism was evaluated in HPRT gene knock-out mice. NAD content and the activities of the enzymes required for synthesis and breakdown of this coenzyme were investigated in blood, brain and liver of HPRT− and control mice. NAD concentration and enzyme activities were found to be significantly increased in liver, but not in brain or blood of the HPRT− mice. These results demonstrate that changes in NAD metabolism occur in response to HPRT deficiency depending on both species and tissue type. PMID:19319672

  14. 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

  15. Aflatoxin B1-induced Hprt mutations in splenic lymphocytes of Fischer 344 rats. Results of an intermittent feeding trial.

    PubMed

    Morris, S M; Aidoo, A; Chen, J J; Chou, M W; Casciano, D A

    1999-01-25

    In a previous study, we found an increase in the mutant frequency at the Hypoxanthine phosphoribosyl transferase (Hprt) locus in the splenic lymphocytes of Fischer 344 rats acutely exposed to aflatoxin B1 (AFB1). Because an acute exposure may not reflect the exposure pattern of individuals whose diet may contain AFB1-contaminated foodstuffs, we sought to determine if the feeding regimen affected the induction of Hprt mutations in the rat splenic lymphocyte. Thus, Fischer 344 rats were fed either (A) a control diet, (B) various doses of AFB1 for three four-week periods interspersed with two four-week periods of the control diet, or (C) continuously fed 1.6 ppm of AFB1. Not only was a significant increase in the mutant frequency detected in the lymphocytes of rats fed a dose as low as 0. 01 ppm of AFB1, but the increase in the mutant frequency at the end of the 20-week experimental period was consistent with an accumulation of damage induced by AFB1. These results indicate that the rat lymphocyte/Hprt assay is useful for detecting chronic low level exposures. Further, these data suggest that an intermittent, low-level exposure to AFB1 may present a human health risk. PMID:10029671

  16. 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.

  17. PRTFDC1 Is a Genetic Modifier of HPRT-Deficiency in the Mouse

    PubMed Central

    Gaval-Cruz, Meriem; Freeman, Kimberly G.; Edwards, Gaylen L.; Weinshenker, David; Thomas, James W.

    2011-01-01

    Lesch-Nyhan disease (LND) is a severe X-linked neurological disorder caused by a deficiency of hypoxanthine phosphoribosyltransferase (HPRT). In contrast, HPRT-deficiency in the mouse does not result in the profound phenotypes such as self-injurious behavior observed in humans, and the genetic basis for this phenotypic disparity between HPRT-deficient humans and mice is unknown. To test the hypothesis that HPRT deficiency is modified by the presence/absence of phosphoribosyltransferase domain containing 1 (PRTFDC1), a paralog of HPRT that is a functional gene in humans but an inactivated pseudogene in mice, we created transgenic mice that express human PRTFDC1 in wild-type and HPRT-deficient backgrounds. Male mice expressing PRTFDC1 on either genetic background were viable and fertile. However, the presence of PRTFDC1 in the HPRT-deficient, but not wild-type mice, increased aggression as well as sensitivity to a specific amphetamine-induced stereotypy, both of which are reminiscent of the increased aggressive and self-injurious behavior exhibited by patients with LND. These results demonstrate that PRTFDC1 is a genetic modifier of HPRT-deficiency in the mouse and could therefore have important implications for unraveling the molecular etiology of LND. PMID:21818316

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

    PubMed Central

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

    1988-01-01

    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. Images PMID:3267219

  19. Increase in NRAS mutant allele percentage during metastatic melanoma progression.

    PubMed

    Funck-Brentano, Elisa; Hélias-Rodzewicz, Zofia; Longvert, Christine; Mokhtari, Karima; Saiag, Philippe; Emile, Jean-François

    2016-06-01

    One-fifth of cutaneous melanomas have dominant gain-of-function mutations of the NRAS oncogene. We report the first two cases of increasing NRAS mutant allele frequency in melanoma metastases and show that the chromosomal mechanism of this homozygosity is an increased polysomy of chromosome 1. We observed an increase in NRAS mutant allele percentage (NRAS-MA%) in the metastatic melanoma progression from 2 patients with melanomas harbouring a NRAS mutation (p.Q61K in case 1 and p.Q61R in case 2). In case 1, we observed a NRAS-MA% increase from 18% within the first metastatic node to 81%, 92% and 85% respectively in the three subsequent metastases: lymph node, brain and subcutaneous metastases biopsied 1, 6 and 17 months, respectively, after the initial lymph node biopsy. In case 2, we observed an increase in NRAS-MA% from 40% within the primary melanoma to 63% within the metastatic lymph node. FISH analysis showed the same results in both cases: a frequent polysomy of chromosome 1 in metastasis samples with NRAS mutant allele percentage >60%, while most cells were disomic in the samples with well-balanced heterozygous mutations. The percentage of NRAS mutant allele may increase during metastatic progression and may be associated with chromosomal instability. Further studies are needed to evaluate the prognostic impact of the NRAS homozygous status and/or polyploidy in metastatic cutaneous melanomas. PMID:26990546

  20. 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.

  1. 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.

  2. 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.

  3. 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.

  4. Mapping the end points of large deletions affecting the hprt locus in human peripheral blood cells and cell lines

    SciTech Connect

    Nelson, S.L.; Grosovsky, A.J.; Jones, I.M.; Burkhart-Schultz, K.; Fuscoe, J.C.

    1995-01-01

    We have examined the extent of of HPRT{sup {minus}} total gene deletions in three mutant collections: spontaneous and X-ray-induced deletions in TK6 human B lymphoblasts, and HPRT{sup {minus}} deletions arising in vivo in T cells. A set of 13 Xq26 STS markers surrounding hprt and spanning approximately 3.3 Mb was used. Each marker used was observed to be missing in at least one of the hprt deletion mutants analyzed. The largest deletion observed encompassed at least 3 Mb. Nine deletions extended outside of the mapped region in the centromeric direction (>1.7 Mb). In contrast, only two telomeric deletions extended to marker 342R (1.26 Mb), and both exhibited slowed or limited cell growth. These data suggest the existence of a gene, within the vicinity of 342R, which establishes the telomeric limit of recoverable deletions. Most (25/41) X-ray-induced total gene deletion mutants exhibited marker loss, but only 1/8 of the spontaneous deletions encompassed any Xq26 markers (P = 0.0187). Furthermore, nearly half (3/8) of the spontaneous 3{prime} total deletion breakpoints were within 14 kb of the hprt coding sequence. In contrast, 40/41 X-ray-induced HPRT{sup {minus}} total deletions extended beyond this point (P = 0.011). Although the overall representation of total gene deletions in the in vivo spectrum is low, 4/5 encompass Xq26 markers flanking hprt. This pattern differs significantly from spontaneous HPRT{sup {minus}} large deletions occurring in vitro (P = 0.032) but resembles the spectrum of X-ray-induced deletions. 24 refs., 6 figs., 1 tab.

  5. Evaluation of an Hprt-Luciferase Reporter Gene on a Mammalian Artificial Chromosome in Response to Cytotoxicity

    PubMed Central

    Endo, Takeshi; Noda, Natsumi; Kuromi, Yasushi; Kokura, Kenji; Kazuki, Yasuhiro; Oshimura, Mitsuo; Ohbayashi, Tetsuya

    2016-01-01

    Background Hypoxanthine guanine phosphoribosyltransferase (Hprt) is known as a house-keeping gene, and has been used as an internal control for real-time quantitative RT-PCR and various other methods of gene expression analysis. To evaluate the Hprt mRNA levels as a reference standard, we engineered a luciferase reporter driven by a long Hprt promoter and measured its response to cytotoxicity. Methods We constructed a reporter vector that harbored a phiC31 integrase recognition site and a mouse Hprt promoter fused with green-emitting luciferase (SLG) coding sequence. The Hprt-SLG vector was loaded onto a mouse artificial chromosome containing a multi-integrase platform using phiC31 integrase in mouse A9 cells. We established three independent clones. Results The established cell lines had similar levels of expression of the Hprt-SLG reporter gene. Hprt-SLG activity increased proportionately under growth conditions and decreased under cytotoxic conditions after blasticidin or cisplatin administration. Similar increases and decreases in the SLG luminescent were observed under growth and cytotoxic conditions, respectively, to those in the fluorescent obtained using the commercially available reagent, alamarBlue. Conclusion By employing a reliable and stable expression system in a mammalian artificial chromosome, the activity of an Hprt-SLG reporter can reflect cell numbers under cell growth condition and cell viability in the evaluation of cytotoxic conditions. PMID:27493490

  6. Analysis of in vivo mutation in the Hprt and Tk genes of mouse lymphocytes.

    PubMed

    Dobrovolsky, Vasily N; Shaddock, Joseph G; Heflich, Robert H

    2014-01-01

    Assays measuring mutant frequencies in endogenous reporter genes are used for identifying potentially genotoxic environmental agents and discovering phenotypes prone to genomic instability and diseases, such as cancer. Here, we describe methods for identifying mouse spleen lymphocytes with mutations in the endogenous X-linked hypoxanthine guanine phosphoribosyl transferase (Hprt) gene and the endogenous autosomal thymidine kinase (Tk) gene. The selective clonal expansion of mutant lymphocytes is based upon the phenotypic properties of HPRT- and TK-deficient cells. The same procedure can be utilized for quantifying Hprt mutations in most strains of mice (and, with minor changes, in other mammalian species), while mutations in the Tk gene can be determined only in transgenic mice that are heterozygous for inactivation of this gene. Expanded mutant clones can be further analyzed to classify the types of mutations in the Tk gene (small intragenic mutations vs. large chromosomal mutations) and to determine the nature of intragenic mutation in both the Hprt and Tk genes. PMID:24623234

  7. HPRT gene alterations in umbilical cord blood T-lymphocytes in newborns of mothers exposed to tobacco smoke during pregnancy.

    PubMed

    Keohavong, Phouthone; Xi, Liqiang; Day, Richard D; Zhang, Lifang; Grant, Stephen G; Day, Billy W; Ness, Roberta B; Bigbee, William L

    2005-05-01

    Prenatal exposure to tobacco smoke has been associated with an increased risk of pediatric malignancies, yet the transplacental induction of genetic alterations by tobacco smoke carcinogens and their implication to childhood diseases remain poorly understood. We characterized mutations in the HPRT gene in umbilical cord blood T-lymphocytes of self-reported 103 never-smoking mothers and 104 smoking mothers (54 mothers smoked throughout and 50 mothers quit smoking during pregnancy). The results showed the illegitimate V(D)J recombinase-mediated deletion of HPRT exons 2-3 was the most prominent alteration occurring in 48.2% (26/54) of mutants from neonates of the smoking mothers who smoked during pregnancy, compared with 28.0% (14/50) from those of smoking mothers who quit smoking during pregnancy (p=0.035, Fisher's exact test), 34.9% (36/103) from never-smoking mothers (p=0.08), or 32.7% (50/153) of those of neonates born from the latter two groups of mothers combined (p=0.043). There was no significant difference in the frequency of this deletion between neonates of the never-smoking mothers and the smoking mothers who quit smoking during pregnancy (34.9% versus 28.0%, respectively, p=0.39). The results show an increase in illegitimate V(D)J recombinase-mediated deletion of HPRT exons 2-3 in cord blood T-lymphocytes of newborns of mothers who smoked during pregnancy, compared with the group of mothers who did not smoke during pregnancy, implying an increase in illegitimate V(D)J recombinase-mediated alteration, a genetic recombination event associated with childhood malignancies, may be induced in utero during pregnancy by maternal exposure to tobacco smoke-derived genotoxicants. PMID:15790499

  8. Mutagenicity monitoring following battlefield exposures: Molecular analysis of HPRT mutations in Gulf War I veterans exposed to depleted uranium.

    PubMed

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

    2015-08-01

    Molecular studies that involved cDNA and genomic DNA sequencing as well as multiplex PCR of the HPRT gene were performed to determine the molecular mutational spectrum for 1,377 HPRT mutant isolates obtained from 61 Veterans of the 1991 Gulf War, most of whom were exposed to depleted uranium (DU). Mutant colonies were isolated from one to four times from each Veteran (in 2003, 2005, 2007, and/or 2009). The relative frequencies of the various types of mutations (point mutations, deletions, insertions, etc.) were compared between high versus low DU exposed groups, (based on their urine U concentration levels), with HPRT mutant frequency (as determined in the companion paper) and with a database of historic controls. The mutational spectrum includes all classes of gene mutations with no significant differences observed in Veterans related to their DU exposures. PMID:25914382

  9. 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. PMID:24623237

  10. 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}.

  11. 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

  12. 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

    2016-01-01

    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

  13. 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

  14. Molecular analysis of mutations affecting hprt mRNA splicing in human T-lymphocytes in vivo

    SciTech Connect

    Rossi, A.M. Pisa Univ. ); Tates, A.D.; van Zeeland, A.A.; Vrieling, H. )

    1992-01-01

    Molecular analysis of hypoxanthine-guanine phosphoribosyltransferase (hprt) cDNA from 6-thioguanine-resistant T-lymphocytes cloned from smoking and non-smoking adult donors showed that 35% of these mutants were defective in splicing of hprt mRNA. Among a set of 42 hprt splice mutants, the authors observed (1) complete loss of one or more exons, (2) partial loss of one exon, or (3) inclusion of part of an intron sequence between adjacent exons. Loss of exon 4 was significantly more frequent than of the other exons, suggesting that the sequences that regulate splicing of this exon are either larger than those of the other exons or especially prone to mutation. In order to identify the molecular nature of DNA alterations causing aberrant splicing of hprt mRNA, 17 splice mutants were analyzed in more detail by sequencing the genomic regions flanking the mis-spliced exon. Base pair substitutions or small deletions causing defective splicing were either detected in exon sequences or in splice site consensus sequences of introns.

  15. 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

  16. 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.

  17. Fine structure mapping of the hypoxanthine-guanine phosphoribosyltransferase (HPRT) gene region of the human X chromosome (Xq26).

    PubMed Central

    Nicklas, J A; Hunter, T C; O'Neill, J P; Albertini, R J

    1991-01-01

    The Xq26-q27 region of the X chromosome is interesting, as an unusually large number of genes and anonymous RFLP probes have been mapped in this area. A number of studies have used classical linkage analysis in families to map this region. Here, we use mutant human T-lymphocyte clones known to be deleted for all or part of the hypoxanthine-guanine phosphoribosyltransferase (hprt) gene, to order anonymous probes known to map to Xq26. Fifty-seven T-cell clones were studied, including 44 derived from in vivo mutation and 13 from in vitro irradiated T-lymphocyte cultures. Twenty anonymous probes (DXS10, DXS11, DXS19, DXS37, DXS42, DXS51, DXS53, DXS59, DXS79, DXS86, DXS92, DXS99, DXS100d, DXS102, DXS107, DXS144, DXS172, DXS174, DXS177, and DNF1) were tested for codeletion with the hprt gene by Southern blotting methods. Five of these probes (DXS10, DXS53, DXS79, DXS86 and DXS177) showed codeletion with hprt in some mutants. The mutants established the following unambiguous ordering of the probes relative to the hprt gene: DXS53-DXS79-5'hprt3'-DXS86-DXS10-DXS177 . The centromere appears to map proximal to DXS53. These mappings order several closely linked but previously unordered probes. In addition, these studies indicate that rather large deletions of the functionally haploid X chromosome can occur while still retaining T-cell viability. Images Figure 1 PMID:1678246

  18. The Electrogenic Bacterium Shewanella Oneidensis MR-1 and its Mutants with Increased Reducing Capacity

    NASA Astrophysics Data System (ADS)

    Voeikova, T. A.; Emelyanova, L. K.; Novikova, L. M.; Mordkovich, N. N.; Shakulov, R. S.; Debabov, V. G.

    2013-02-01

    Mutants of Shewanella oneidensis MR-1 resistant to fosfomycin, a toxic analogue of phosphoenolpyruvate, were obtained. The mutants exhibited an increased reducing activity and a higher rate of lactate utilization. A correlation was shown between the rates of metabolism of oxidized substrates and the rate of reduction of methylene blue, a mediator of electron transport. The mutants of S.oneidensis MR-1 will be used in microbial fuel cells (MFC) to enhance energy production from organic compounds. The strain S. oneidensis MR-1 and its mutants with an increased electron production will be used as a good source of bioelectricity in MFC in the experiments on the International Space Station.

  19. 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.

  20. 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. PMID:19716523

  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 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

  3. 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. PMID:23299430

  4. 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. PMID:23456361

  5. 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.

  6. 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.

  7. Identification of an arsenic tolerant double mutant with a thiol-mediated component and increased arsenic tolerance in phyA mutants.

    PubMed

    Sung, Dong-Yul; Lee, David; Harris, Hugh; Raab, Andrea; Feldmann, Jörg; Meharg, Andrew; Kumabe, Bryan; Komives, Elizabeth A; Schroeder, Julian I

    2007-03-01

    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. PMID:17335514

  8. Characterizing mutagenesis in the hprt gene of rat alveolar epithelial cells

    SciTech Connect

    Driscoll, K.E.; Deyo, L.C.; Howard, B.W.

    1995-12-31

    A clonal selection assay was developed for mutation in the hypoxanthine-guanine phosphoribosyl transferase (hprt) gene of rat alveolar epithelial cells. Studies were conducted to establish methods for isolation and long-term culture of rat alveolar epithelial cells. When isolated by pronase digestion purified on a Nycodenz gradient and cultured in media containing 7.5% fetal bovine serum (FBS), pituitary extract, EGF, insulin, and IGF-1, rat alveolar epithelial cells could be maintained in culture for several weeks with cell doubling times of 2-4 days. The rat alveolar epithelial cell cultures were exposed in vitro to the mutagens ethylnitrosourea (ENU) and H{sub 2}O{sub 2}, and mutation in the hprt gene was selected for by culture in the presence of the toxic purine analog, 6-thioguanine (6TG). In vitro exposure to ENU or H{sub 2}O produced a dose-dependent increase in hprt mutation frequency in the alveolar epithelial cells. To determine if the assay system could be used to evaluate mutagenesis in alveolar type II cells after in vivo mutagen or carcinogen exposure, cells were isolated from rats treated previously with ENU or {alpha}-quartz. A significant increase in hprt mutation frequency was detected in alveolar epithelial cells obtained from rats exposed to ENU or {alpha}-quartz; the latter observation is the first demonstration that crystalline silica exposure is mutagenic in vivo. In summary, these studies show that rat alveolar epithelial cells isolated by pronase digestion and Nycodenz separation techniques and cultured in a defined media can be used in a clonal selection assay for mutation in the hprt gene. This assay demonstrates that ENU and H{sub 2}O{sub 2} in vitro and ENU and {alpha}-quartz in vivo are mutagenic for rat alveolar epithelial cells. This model should be useful for investigating the genotoxic effects of chemical and physical agents on an important lung cell target for neoplastic transformation. 41 refs., 4 figs., 3 tabs.

  9. 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. PMID:26869603

  10. In vitro mutational spectrum of cyclopenta[cd]pyrene in the human HPRT gene.

    PubMed

    Keohavong, P; Melacrinos, A; Shukla, R

    1995-04-01

    Cyclopenta[cd]pyrene (CPP) is a widely distributed polycyclic aromatic hydrocarbon with potent mutagenic and carcinogenic activity. In order to acquire an understanding of the mutagenic pathways of CPP, we studied mutations induced by this chemical in human cells. Four independent cultures of a human cell line expressing cytochrome P450 CYP1A1 (cell line MCL-5) were treated with CPP, and mutants at the hypoxanthine phosphoribosyltransferase (HPRT) locus were selected en masse by 6-thioguanine (6TG) resistance. The kinds and positions of the mutations were analyzed using the combination of high-fidelity polymerase chain reaction (hifi-PCR) and denaturing gradient gel electrophoresis (DGGE). The third exon of the HPRT gene was amplified from the 6TG-resistant cells using the hifi-PCR and the amplified fragment was subsequently analyzed by DGGE to separate mutant sequences from the wild-type sequence. Mutant bands were excised from the gel, amplified using PCR and sequenced. Sixteen different mutations were identified and consisted mostly of the G to T and A to T transversions. Other mutations identified included G to A and A to G transitions, a G to C transversion, and a single G deletion. Of these mutations, six occurred within a run of six guanines. The predominance of transversions involving a guanine or an adenine observed with CPP is similar to the data previously reported for the racemic mixtures of benzo[a]pyrene (B[a]P), suggesting that the mechanisms of mutation induced by CPP may be similar to those induced by B[a]P. PMID:7728967

  11. 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. PMID:26453761

  12. Partial HPRT Deficiency with a Novel Mutation of the HPRT Gene in Combination with Four Previously Reported Variants Associated with Hyperuricemia.

    PubMed

    Kurajoh, Masafumi; Koyama, Hidenori; Hatayama, Miki; Okazaki, Hirokazu; Shoji, Takuhito; Moriwaki, Yuji; Yamamoto, Tetsuya; Nakayama, Tomitaka; Namba, Mitsuyoshi

    2015-01-01

    A 15-year-old boy was referred to our department due to gout. The laboratory findings showed hyperuricemia with a decreased erythrocyte hypoxanthine phosphoribosyl transferase (HPRT) activity. The HPRT cDNA sequence was revealed to be 206A>T, which has not been previously reported. In addition, direct sequencing of genomic DNA showed the patient to possess four variants reported to be associated with hyperuricemia. This is the first case report of partial HPRT deficiency due to a novel HPRT mutation accompanied by variants associated with hyperuricemia. Combination treatment consisting of benzbromarone and febuxostat had a significant effect in reducing the urate level in our patient. PMID:26073243

  13. Positive selection of Caenorhabditis elegans mutants with increased stress resistance and longevity.

    PubMed

    Muñoz, Manuel J; Riddle, Donald L

    2003-01-01

    We developed selective conditions for long-lived mutants of the nematode Caenorhabditis elegans by subjecting the first larval stage (L1) to thermal stress at 30 degrees for 7 days. The surviving larvae developed to fertile adults after the temperature was shifted to 15 degrees. A total of one million F(2) progeny and a half million F(3) progeny of ethyl-methanesulfonate-mutagenized animals were treated in three separate experiments. Among the 81 putative mutants that recovered and matured to the reproductive adult, 63 retested as thermotolerant and 49 (80%) exhibited a >15% increase in mean life span. All the known classes of dauer formation (Daf) mutant that affect longevity were found, including six new alleles of daf-2, and a unique temperature-sensitive, dauer-constitutive allele of age-1. Alleles of dyf-2 and unc-13 were isolated, and mutants of unc-18, a gene that interacts with unc-13, were also found to be long lived. Thirteen additional mutations define at least four new genes. PMID:12586705

  14. Implementation of a large-scale ENU mutagenesis program: towards increasing the mouse mutant resource.

    PubMed

    Nolan, P M; Peters, J; Vizor, L; Strivens, M; Washbourne, R; Hough, T; Wells, C; Glenister, P; Thornton, C; Martin, J; Fisher, E; Rogers, D; Hagan, J; Reavill, C; Gray, I; Wood, J; Spurr, N; Browne, M; Rastan, S; Hunter, J; Brown, S D

    2000-07-01

    Systematic approaches to mouse mutagenesis will be vital for future studies of gene function. We have begun a major ENU mutagenesis program incorporating a large genome-wide screen for dominant mutations. Progeny of ENU-mutagenized mice are screened for visible defects at birth and weaning, and at 5 weeks of age by using a systematic and semi-quantitative screening protocol-SHIRPA. Following this, mice are screened for abnormal locomotor activity and for deficits in prepulse inhibition of the acoustic startle response. Moreover, in the primary screen, blood is collected from mice and subjected to a comprehensive clinical biochemical analysis. Subsequently, secondary and tertiary screens of increasing complexity can be used on animals demonstrating deficits in the primary screen. Frozen sperm is archived from all the male mice passing through the screen. In addition, tail tips are stored for DNA. Overall, the program will provide an extensive new resource of mutant and phenotype data to the mouse and human genetics communities at large. The challenge now is to employ the expanding mouse mutant resource to improve the mutant map of the mouse. An improved mutant map of the mouse will be an important asset in exploiting the growing gene map of the mouse and assisting with the identification of genes underlying novel mutations-with consequent benefits for the analysis of gene function and the identification of novel pathways. PMID:10886012

  15. 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.

  16. Increased Rrm2 gene dosage reduces fragile site breakage and prolongs survival of ATR mutant mice

    PubMed Central

    Specks, Julia; Barlow, Jacqueline H.; Ambrogio, Chiara; Desler, Claus; Vikingsson, Svante; Rodrigo-Perez, Sara; Green, Henrik; Rasmussen, Lene Juel; Murga, Matilde; Nussenzweig, André

    2015-01-01

    In Saccharomyces cerevisiae, absence of the checkpoint kinase Mec1 (ATR) is viable upon mutations that increase the activity of the ribonucleotide reductase (RNR) complex. Whether this pathway is conserved in mammals remains unknown. Here we show that cells from mice carrying extra alleles of the RNR regulatory subunit RRM2 (Rrm2TG) present supraphysiological RNR activity and reduced chromosomal breakage at fragile sites. Moreover, increased Rrm2 gene dosage significantly extends the life span of ATR mutant mice. Our study reveals the first genetic condition in mammals that reduces fragile site expression and alleviates the severity of a progeroid disease by increasing RNR activity. PMID:25838540

  17. 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. PMID:17179647

  18. p53 increases caspase-6 expression and activation in muscle tissue expressing mutant huntingtin.

    PubMed

    Ehrnhoefer, Dagmar E; Skotte, Niels H; Ladha, Safia; Nguyen, Yen T N; Qiu, Xiaofan; Deng, Yu; Huynh, Khuong T; Engemann, Sabine; Nielsen, Signe M; Becanovic, Kristina; Leavitt, Blair R; Hasholt, Lis; Hayden, Michael R

    2014-02-01

    Activation of caspase-6 in the striatum of both presymptomatic and affected persons with Huntington's disease (HD) is an early event in the disease pathogenesis. However, little is known about the role of caspase-6 outside the central nervous system (CNS) and whether caspase activation might play a role in the peripheral phenotypes, such as muscle wasting observed in HD. We assessed skeletal muscle tissue from HD patients and well-characterized mouse models of HD. Cleavage of the caspase-6 specific substrate lamin A is significantly increased in skeletal muscle obtained from HD patients as well as in muscle tissues from two different HD mouse models. p53, a transcriptional activator of caspase-6, is upregulated in neuronal cells and tissues expressing mutant huntingtin. Activation of p53 leads to a dramatic increase in levels of caspase-6 mRNA, caspase-6 activity and cleavage of lamin A. Using mouse embryonic fibroblasts (MEFs) from YAC128 mice, we show that this increase in caspase-6 activity can be mitigated by pifithrin-α (pifα), an inhibitor of p53 transcriptional activity, but not through the inhibition of p53's mitochondrial pro-apoptotic function. Remarkably, the p53-mediated increase in caspase-6 expression and activation is exacerbated in cells and tissues of both neuronal and peripheral origin expressing mutant huntingtin (Htt). These findings suggest that the presence of the mutant Htt protein enhances p53 activity and lowers the apoptotic threshold, which activates caspase-6. Furthermore, these results suggest that this pathway is activated both within and outside the CNS in HD and may contribute to both loss of CNS neurons and muscle atrophy. PMID:24070868

  19. Hajdu Cheney Mouse Mutants Exhibit Osteopenia, Increased Osteoclastogenesis, and Bone Resorption.

    PubMed

    Canalis, Ernesto; Schilling, Lauren; Yee, Siu-Pok; Lee, Sun-Kyeong; Zanotti, Stefano

    2016-01-22

    Notch receptors are determinants of cell fate and function and play a central role in skeletal development and bone remodeling. Hajdu Cheney syndrome, a disease characterized by osteoporosis and fractures, is associated with NOTCH2 mutations resulting in a truncated stable protein and gain-of-function. We created a mouse model reproducing the Hajdu Cheney syndrome by introducing a 6955C→T mutation in the Notch2 locus leading to a Q2319X change at the amino acid level. Notch2(Q2319X) heterozygous mutants were smaller and had shorter femurs than controls; and at 1 month of age they exhibited cancellous and cortical bone osteopenia. As the mice matured, cancellous bone volume was restored partially in male but not female mice, whereas cortical osteopenia persisted in both sexes. Cancellous bone histomorphometry revealed an increased number of osteoclasts and bone resorption, without a decrease in osteoblast number or bone formation. Osteoblast differentiation and function were not affected in Notch2(Q2319X) cells. The pre-osteoclast cell pool, osteoclast differentiation, and bone resorption in response to receptor activator of nuclear factor κB ligand in vitro were increased in Notch2(Q2319X) mutants. These effects were suppressed by the γ-secretase inhibitor LY450139. In conclusion, Notch2(Q2319X) mice exhibit cancellous and cortical bone osteopenia, enhanced osteoclastogenesis, and increased bone resorption. PMID:26627824

  20. Loss of Cell Adhesion Increases Tumorigenic Potential of Polarity Deficient Scribble Mutant Cells

    PubMed Central

    Waghmare, Indrayani

    2016-01-01

    Epithelial polarity genes are important for maintaining tissue architecture, and regulating growth. The Drosophila neoplastic tumor suppressor gene scribble (scrib) belongs to the basolateral polarity complex. Loss of scrib results in disruption of its growth regulatory functions, and downregulation or mislocalization of Scrib is correlated to tumor growth. Somatic scribble mutant cells (scrib-) surrounded by wild-type cells undergo apoptosis, which can be prevented by introduction of secondary mutations that provide a growth advantage. Using genetic tools in Drosophila, we analyzed the phenotypic effects of loss of scrib in different growth promoting backgrounds. We investigated if a central mechanism that regulates cell adhesion governs the growth and invasive potential of scrib mutant cells. Here we show that increased proliferation, and survival abilities of scrib- cells in different genetic backgrounds affect their differentiation, and intercellular adhesion. Further, loss of scrib is sufficient to cause reduced cell survival, activation of the JNK pathway and a mild reduction of cell adhesion. Our data show that for scrib cells to induce aggressive tumor growth characterized by loss of differentiation, cell adhesion, increased proliferation and invasion, cooperative interactions that derail signaling pathways play an essential role in the mechanisms leading to tumorigenesis. Thus, our study provides new insights on the effects of loss of scrib and the modification of these effects via cooperative interactions that enhance the overall tumorigenic potential of scrib deficient cells. PMID:27327956

  1. Utilization of stiff culm trait of rice smos1 mutant for increased lodging resistance.

    PubMed

    Hirano, Ko; Okuno, Ayako; Hobo, Tokunori; Ordonio, Reynante; Shinozaki, Yusuke; Asano, Kenji; Kitano, Hidemi; Matsuoka, Makoto

    2014-01-01

    Although the introduction of semi-dwarf trait into rice has led to improved lodging resistance making it capable of supporting high grain yield, lodging still remains a concern when attempting to further increase the grain yield of rice. However, improving the lodging resistance in rice by depending on the semi-dwarf trait alone is possible only up to a certain limit, beyond which other traits may be needed for reinforcement. To search for alternative traits relating to high lodging resistance, we identified 9 rice mutant lines possessing improved culm strength. To evaluate whether such lines can be useful for breeding lodging resistant rice, small organ size1 (smos1) mutant having increased lodging resistance but low tiller number and low grain yield, was chosen as a representative for a breeding trial. smos1 was crossed with ST-4 (from the Stock rice collection of Nagoya University Togo field #4), a cultivar with high tiller number and high grain yield, and from their progeny, LRC1 (lodging resistance candidate-1) was selected. Although the low tiller number trait of smos1 was not fully reversed in LRC1, this was compensated by an increase in grain weight per panicle, thereby resulting in high grain yield per plant. This important attribute of LRC1 was further enhanced by the improved lodging resistance trait inherited from smos1. Such improved lodging resistance in LRC1 and smos1 was revealed to be mainly due to increased culm diameter and culm thickness, which led to a high section modulus (SM) value, a parameter defining the physical strength of the culm. Since smos1 possesses high breaking-type lodging resistance which is different from semi-dwarf plants with high bending-type lodging resistance, an alternative approach of using thick culm lines for the creation of rice with increased lodging resistance is hereby proposed. PMID:24987959

  2. Increased persistent Na+ current contributes to seizure in the slamdance bang-sensitive Drosophila mutant

    PubMed Central

    Marley, Richard

    2011-01-01

    There is clinical need to extend the understanding of epilepsy and to find novel approaches to treat this condition. Bang-sensitive (bs) Drosophila mutants, which exhibit reduced thresholds for seizure, offer an attractive possibility to combine tractable genetics, electrophysiology, and high-throughput screening. However, despite these advantages, the precise electrophysiological aberrations that contribute to seizure have not been identified in any bs mutant. Because of this, the applicability of Drosophila as a preclinical model has not yet been established. In this study, we show that electroshock of bs slamdance (sda) larvae was sufficient to induce extended seizure-like episodes. Whole cell voltage-clamp recordings from identified motoneurons (termed aCC and RP2) showed synaptic currents that were greatly increased in both amplitude and duration. Current-clamp recordings indicated that these inputs produced longer-lived plateau depolarizations and increased action potential firing in these cells. An analysis of voltage-gated currents in these motoneurons, in both first and third instar larvae, revealed a consistently increased persistent Na+ current (INap) and a reduced Ca2+ current in first instar larvae, which appeared normal in older third instar larvae. That increased INap may contribute to seizure-like activity is indicated by the observation that feeding sda larvae the antiepileptic drug phenytoin, which was sufficient to reduce INap, rescued both seizure-like episode duration and synaptic excitation of motoneurons. In contrast, feeding of either anemone toxin, a drug that preferentially increases INap, or phenytoin to wild-type larvae was sufficient to induce a bs behavioral phenotype. Finally, we show that feeding of phenytoin to gravid sda females was sufficient to both reduce INap and synaptic currents and rescue the bs phenotype in their larval progeny, indicating that a heightened predisposition to seizure may arise as a consequence of abnormal

  3. Lack of increased genetic damage in 1,3-butadiene-exposed Chinese workers studied in relation to EPHX1 and GST genotypes

    PubMed Central

    Zhang, Luoping; Hayes, Richard B.; Guo, Weihong; McHale, Cliona M.; Yin, Songnian; Wiencke, John K.; O’Neill, J. Patrick; Rothman, Nathaniel; Li, Gui-Lan; Smith, Martyn T.

    2005-01-01

    1,3-Butadiene (BD) is an important industrial chemical and pollutant. Its ability to induce genetic damage and cause hematological malignancies in humans is controversial. We have examined chromosome damage by fluorescence in situ hybridization (FISH) and mutations in the HPRT gene in the blood of Chinese workers exposed to BD. Peripheral blood samples were collected and cultured from 39 workers exposed to BD (median level 2 ppm, 6 h time-weighted average) and 38 matched controls in Yanshan, China. No difference in the level of aneuploidy or structural changes in chromosomes 1, 7, 8, and 12 was detected in metaphase cells from exposed subjects in comparison with matched controls, nor was there an increase in the frequency of HPRT mutations in the BD-exposed workers. Because genetic polymorphisms in glutathione S-transferase (GST) enzymes and microsomal epoxide hydrolase (EPHX1) may affect the genotoxic effects of BD and its metabolites, we also related chromosome alterations and gene mutations to GSTT1, GSTM1 and EPHX1 genotypes. Overall, there was no effect of variants in these genotypes on numerical or structural changes in chromosomes 1, 7, 8 and 12 or on HPRT mutant frequency in relation to BD exposure, but the GST genotypes did influence background levels of both hyperdiploidy and HPRT mutant frequency. In conclusion, our data show no increase in chromosomal aberrations or HPRT mutations among workers exposed to BD, even in potentially susceptible genetic subgroups. The study is, however, quite small and the levels of BD exposure are not extremely high, but our findings in China do support those from a similar study conducted in the Czech Republic. Together, these studies suggest that low levels of occupational BD exposure do not pose a significant risk of genetic damage. PMID:15036120

  4. 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. PMID:24450179

  5. 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.

  6. 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

  7. Benomyl-resistant mutant strain of Trichoderma sp. with increased mycoparasitic activity.

    PubMed

    Olejníková, P; Ondrusová, Z; Krystofová, S; Hudecová, D

    2010-01-01

    Application of UV radiation to the strain Trichoderma sp. T-bt (isolated from lignite) resulted in the T-brm mutant which was resistant to the systemic fungicide benomyl. The tub2 gene sequence in the T-brm mutant differed from the parent as well as the collection strain (replacing tyrosine with histidine in the TUB2 protein). Under in vitro conditions this mutant exhibited a higher mycoparasitic activity toward phytopathogenic fungi. PMID:20336512

  8. 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. PMID:15858385

  9. V(D)J RECOMBINASE-MEDIATED DELETION OF THE HPRT GENE IN T-LYMPHOCYTES FROM ADULT HUMANS

    EPA Science Inventory

    The hprt T-cell cloning assay allows the detection of mutations occurring in vivo in the hypoxanthine guanine phosphoribosyltransferase (hprt) gene of T-lymphocytes. e have shown previously that the illegitimate activity of V(D)J recombinase accounts for about 40% of the hprt mut...

  10. MULTIPLEX PCR ANALYSIS OF IN VIVO-ARISING DELETION MUTATIONS IN THE HPRT GENE OF HUMAN T-LYMPHOCYTES

    EPA Science Inventory

    A multiplex polymerase chain reaction (PCR) procedure was adapted for the rapid and efficient evaluation of the hypoxanthine guanine phosphoribosyltransferase (hprt) gene in human T-lymphocytes for deletions. he hprt clonal assay was used to isolate in-vivo-arising hprt-deficient...

  11. Mutant SOD1 Increases APP Expression and Phosphorylation in Cellular and Animal Models of ALS

    PubMed Central

    Rabinovich-Toidman, Polina; Rabinovich-Nikitin, Inna; Ezra, Assaf; Barbiro, Beka; Fogel, Hilla; Slutsky, Inna; Solomon, Beka

    2015-01-01

    Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease and it is the most common adult onset neurodegenerative disorder affecting motor neurons. There is currently no effective treatment for ALS and our understanding of the pathological mechanism is still far away from prevention and/or treatment of this devastating disease. Amyloid precursor protein (APP) is a transmembrane protein that undergoes processing either by β-secretase or α-secretase, followed by γ-secretase. In the present study, we show that APP levels, and aberrant phosphorylation, which is associated with enhanced β-secretase cleavage, are increased in SOD1G93A ALS mouse model. Fluorescence resonance energy transfer (FRET) analysis suggests a close interaction between SOD1 and APP at hippocampal synapses. Notably, SOD1G93A mutation induces APP-SOD1 conformational changes, indicating a crosstalk between these two signaling proteins. Inhibition of APP processing via monoclonal antibody called BBS that blocks APP β-secretase cleavage site, resulted in reduction of mutant SOD1G93A levels in animal and cellular models of ALS, significantly prolonged life span of SOD1G93A mice and diminished inflammation. Beyond its effect on toxic mutant SOD1G93A, BBS treatment resulted in a reduction in the levels of APP, its processing product soluble APPβ and pro-apoptotic p53. This study demonstrates that APP and its processing products contribute to ALS pathology through several different pathways; thus BBS antibody could be a promising neuroprotective strategy for treatment of this disease. PMID:26600047

  12. 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. PMID:8293973

  13. 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

  14. Lycium barbarum polysaccharide attenuates the cytotoxicity of mutant huntingtin and increases the activity of AKT.

    PubMed

    Fang, Fang; Peng, Ting; Yang, Shiming; Wang, Weixi; Zhang, Yinong; Li, He

    2016-08-01

    Huntington's disease (HD) is an inherited neurodegenerative disease that is caused by the abnormal expansion of CAG repeats in the gene encoding huntingtin (Htt). Reduced AKT phosphorylation and inhibited AKT activity have been shown to be involved in mutant Htt (mHtt)-induced cell death. Lycium barbarum polysaccharide (LBP), the main bioactive component of Lycium barbarum, reportedly has neuroprotective roles in neural injuries, including neurodegenerative diseases. Here, we report that treatment with LBP can increased the viability of HEK293 cells that stably expressed mHtt containing 160 glutamine repeats and significantly improved motor behavior and life span in HD-transgenic mice. Furthermore, we found that in LBP-treated HEK293 cells expressing mHtt, mHtt levels were reduced and the phosphorylation of AKT at Ser473 (p-AKT-Ser473) was significantly increased. We also found that treatment with LBP increased p-AKT-Ser473 and decreased mHtt in the cortex, hippocampus and striatum in HD-transgenic mice. The level of phosphorylation of p-GSK3β-Ser9 remained unchanged in both cultured cells and HD-transgenic mice. Our findings suggest that LBP alleviates the cytotoxicity of mHtt by activating AKT and reducing mHtt levels, indicating that LBP may be potentially useful for treating HD. PMID:27196502

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

    PubMed Central

    Rogers, Ryan P.; Rogina, Blanka

    2014-01-01

    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 PMID:24827528

  16. 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

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

    PubMed Central

    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

  18. Increasing prevalence of a novel triple-mutant dihydropteroate synthase genotype in Plasmodium falciparum in western Kenya.

    PubMed

    Lucchi, Naomi W; Okoth, Sheila Akinyi; Komino, Franklin; Onyona, Philip; Goldman, Ira F; Ljolje, Dragan; Shi, Ya Ping; Barnwell, John W; Udhayakumar, Venkatachalam; Kariuki, Simon

    2015-07-01

    The molecular basis of sulfadoxine-pyrimethamine (SP) resistance lies in a combination of single-nucleotide polymorphisms (SNPs) in two genes coding for Plasmodium falciparum dihydrofolate reductase (Pfdhfr) and P. falciparum dihydropteroate synthase (Pfdhps), targeted by pyrimethamine and sulfadoxine, respectively. The continued use of SP for intermittent preventive treatment in pregnant women in many African countries, despite SP's discontinuation as a first-line antimalarial treatment option due to high levels of drug resistance, may further increase the prevalence of SP-resistant parasites and/or lead to the selection of new mutations. An antimalarial drug resistance surveillance study was conducted in western Kenya between 2010 and 2013. A total of 203 clinical samples from children with uncomplicated malaria were genotyped for SNPs associated with SP resistance. The prevalence of the triple-mutant Pfdhfr C50 I51R59N108: I164 genotype and the double-mutant Pfdhps S436 G437E540: A581A613 genotype was high. Two triple-mutant Pfdhps genotypes, S436 G437E540G581: A613 and H436G437E540: A581A613, were found, with the latter thus far being uniquely found in western Kenya. The prevalence of the S436 G437E540G581: A613 genotype was low. However, a steady increase in the prevalence of the Pfdhps triple-mutant H436G437E540: A581A613 genotype has been observed since its appearance in early 2000. Isolates with these genotypes shared substantial microsatellite haplotypes with the most common double-mutant allele, suggesting that this triple-mutant allele may have evolved locally. Overall, these findings show that the prevalence of the H436G437E540: A581A613 triple mutant may be increasing in this population and could compromise the efficacy of SP for intermittent preventive treatment in pregnant women if it increases the resistance threshold further. PMID:25896703

  19. Increased transmitter release and aberrant synapse morphology in a Drosophila calmodulin mutant.

    PubMed Central

    Arredondo, L; Nelson, H B; Beckingham, K; Stern, M

    1998-01-01

    The ubiquitous calcium-binding protein calmodulin (CaM) has been implicated in the development and function of the nervous system in a variety of eukaryotic organisms. We have generated mutations in the single Drosophila Calmodulin (Cam) gene and examined the effects of these mutations on behavior, synaptic transmission at the larval neuromuscular junction, and structure of the larval motor nerve terminal. Flies hemizygous for Cam3c1, a mutation in the first Ca2+-binding site, exhibit behavioral, neurophysiological, and neuroanatomical abnormalities. In particular, adults exhibit defects in locomotion, coordination, and flight. Larvae exhibit increased neurotransmitter release from the motor nerve terminal at low [Ca2+] in the presence of the K+ channel-blocking drug quinidine. In addition, synaptic bouton structure at motor nerve terminals is altered. These effects are distinct from those produced by altering the activity of the CaM target enzymes CaM-activated kinase II (CaMKII) and CaM-activated adenylyl cyclase (CaMAC). Furthermore, previous in vitro studies of mutant Cam3c1 demonstrated that although its Ca2+ affinity is decreased, Cam3c1 protein can activate CaMKII, CaMAC, and CaM-activated phosphatase calcineurin in a manner similar to wild-type CaM. Thus, the Cam3c1 mutation might affect Ca2+ buffering or interfere with the activation or inhibition of a CaM target distinct from CaMKII or CaMAC. PMID:9725845

  20. Tipping the mutation-selection balance: Limited migration increases the frequency of deleterious mutants.

    PubMed

    Cooper, Jacob D; Neuhauser, Claudia; Dean, Antony M; Kerr, Benjamin

    2015-09-01

    Typical mutation-selection models assume well-mixed populations, but dispersal and migration within many natural populations is spatially limited. Such limitations can lead to enhanced variation among locations as different types become clustered in different places. Such clustering weakens competition between unlike types relative to competition between like types; thus, the rate by which a fitter type displaces an inferior competitor can be affected by the spatial scale of movement. In this paper, we use a birth-death model to show that limited migration can affect asexual populations by creating competitive refugia. We use a moment closure approach to show that as population structure is introduced by limiting migration, the equilibrial frequency of deleterious mutants increases. We support and extend the model through stochastic simulation, and we use a spatially explicit cellular automaton approach to corroborate the results. We discuss the implications of these results for standing variation in structured populations and adaptive valley crossing in Wright's "shifting balance" process. PMID:25983046

  1. 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. PMID:26721420

  2. 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. PMID:23074187

  3. Pseudogene-free amplification of HPRT1 in quantitative reverse transcriptase polymerase chain reaction.

    PubMed

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

    2015-09-15

    Quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) provides a powerful tool for precise gene expression analysis. The accuracy of the results highly depends on careful selection of a reference gene for data normalization. HPRT1 (hypoxanthine phosphoribosyl transferase 1) is a frequently used housekeeping gene for normalizing relative expression values. However, the existence of processed pseudogenes for HPRT1 might interfere with reliable results obtained in qRT-PCR due to amplification of unintended products. Here, we designed a primer pair for pseudogene-free amplification of HPRT1 in qRT-PCR. We demonstrate that this primer pair specifically amplified HPRT1 messenger RNA (mRNA) sequence while avoiding coamplification of the pseudogenes. PMID:26050630

  4. Biophysical characterization of mutants of Bacillus subtilis lipase evolved for thermostability: Factors contributing to increased activity retention

    PubMed Central

    Augustyniak, Wojciech; Brzezinska, Agnieszka A; Pijning, Tjaard; Wienk, Hans; Boelens, Rolf; Dijkstra, Bauke W; Reetz, Manfred T

    2012-01-01

    Previously, Lipase A from Bacillus subtilis was subjected to in vitro directed evolution using iterative saturation mutagenesis, with randomization sites chosen on the basis of the highest B-factors available from the crystal structure of the wild-type (WT) enzyme. This provided mutants that, unlike WT enzyme, retained a large part of their activity after heating above 65°C and cooling down. Here, we subjected the three best mutants along with the WT enzyme to biophysical and biochemical characterization. Combining thermal inactivation profiles, circular dichroism, X-ray structure analyses and NMR experiments revealed that mutations of surface amino acid residues counteract the tendency of Lipase A to undergo precipitation under thermal stress. Reduced precipitation of the unfolding intermediates rather than increased conformational stability of the evolved mutants seems to be responsible for the activity retention. PMID:22267088

  5. ISOLATED MEDICAGO TRUNCATULA MUTANTS WITH INCREASED CALCIUM OXALATE CRYSTAL ACCUMULATION HAVE DECREASED ASCORBIC ACID LEVELS

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The mechanisms controlling oxalate biosynthesis and calcium oxalate formation in plants remains largely unknown. As an initial step toward gaining insight into these regulatory mechanisms we initiated a mutant screen to identify plants that over-accumulate crystals of calcium oxalate. Four new mut...

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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...

  7. 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.

  8. In Vitro selection of Neisseria gonorrhoeae mutants with elevated MIC values and increased resistance to cephalosporins.

    PubMed

    Johnson, Steven R; Grad, Yonatan; Ganakammal, Satishkumar Ranganathan; Burroughs, Mark; Frace, Mike; Lipsitch, Marc; Weil, Ryan; Trees, David

    2014-11-01

    Strains of Neisseria gonorrhoeae with mosaic penA genes bearing novel point mutations in penA have been isolated from ceftriaxone treatment failures. Such isolates exhibit significantly higher MIC values to third-generation cephalosporins. Here we report the in vitro isolation of two mutants with elevated MICs to cephalosporins. The first possesses a point mutation in the transpeptidase region of the mosaic penA gene, and the second contains an insertion mutation in pilQ. PMID:25199775

  9. Increased outer membrane resistance to ethylenediaminetetraacetate and cations in novel lipid A mutants.

    PubMed Central

    Vaara, M

    1981-01-01

    Polymyxin-resistant pmrA mutants of Salmonella typhimurium differed from their parents in that they were resistant to tris(hydroxymethyl)aminomethane-ethylenediaminetetraacetate-lysozyme, tris(hydroxymethyl)aminomethane-ethylenediaminetetraacetate-deoxycholate, and tris(hydroxymethyl)aminomethane-ethylenediaminetetraacetate-bacitracin. Tris(hydroxymethyl)aminomethane-ethylenediaminetetraacetate released about 50% less lipopolysaccharide from the pmrA strains than from the parental strains when the bacteria were grown in L-broth containing 2 mM Ca2+. Protamine, polylysine, octapeptin, benzalkonium chloride, cold NaCl, cold MgCl2, or cold tris(hydroxymethyl)aminomethane hydrochloride (pH 7.2) caused no leakage or markedly less leakage of periplasmic beta-lactamase from a pmrA mutant than from its parent strain. pmrA mutants were more resistant than their parent strains to protamine and polylysine but not to octapeptin or benzalkonium chloride, as measured by the ability of these agents to kill the bacteria or to sensitize them to deoxycholate-induced lysis. The pmrA strains did not differ from their parent strains in sensitivity to several antibiotics, in porin function (as measured by cephaloridine diffusion across the outer membrane), or in outer membrane-associated phospholipase A activity. PMID:6795177

  10. Inducible expression of mutant alpha-synuclein decreases proteasome activity and increases sensitivity to mitochondria-dependent apoptosis.

    PubMed

    Tanaka, Y; Engelender, S; Igarashi, S; Rao, R K; Wanner, T; Tanzi, R E; Sawa, A; L Dawson, V; Dawson, T M; Ross, C A

    2001-04-15

    Parkinson's disease (PD) is a common progressive neurodegenerative disorder caused by the loss of dopaminergic neurons in the substantia nigra. Although mutations in alpha-synuclein have been identified in autosomal dominant PD, the mechanism by which dopaminergic neural cell death occurs remains unknown. Proteins encoded by two other genes in which mutations cause familial PD, parkin and UCH-L1, are involved in regulation of the ubiquitin-proteasome pathway, suggesting that dysregulation of the ubiquitin-proteasome pathway is involved in the mechanism by which these mutations cause PD. We established inducible PC12 cell lines in which wild-type or mutant alpha-synuclein can be de-repressed by removing doxycycline. Differentiated PC12 cell lines expressing mutant alpha-synuclein showed decreased activity of proteasomes without direct toxicity. Cells expressing mutant alpha-synuclein showed increased sensitivity to apoptotic cell death when treated with sub-toxic concentrations of an exogenous proteasome inhibitor. Apoptosis was accompanied by mitochondrial depolarization and elevation of caspase-3 and -9, and was blocked by cyclosporin A. These data suggest that expression of mutant alpha-synuclein results in sensitivity to impairment of proteasome activity, leading to mitochondrial abnormalities and neuronal cell death. PMID:11309365

  11. Spirogyra varians mutant generated by high dose gamma-irradiation shows increased antioxidant properties

    NASA Astrophysics Data System (ADS)

    Lee, Hak-Jyung; Yoon, Minchul; Sung, Nak-Yun; Choi, Jong-il

    2012-08-01

    The aim of this study was to evaluate the antioxidant properties of a Spirogyra varians mutant (Mut) produced by gamma irradiation. Methanol extracts were prepared from Spirogyra varians wild-type and Mut plants, and their antioxidant activities and total phenolic content (TPC) were determined. Antioxidant parameters, including the 2-diphenyl-1-picrylhydrazyl radical-scavenging activity and ferric-reducing/antioxidant power, were higher in the Mut extract. Moreover, the TPC level was higher (P<0.05) in the Mut methanol extract. Therefore, these results suggest that gamma irradiation-induced S. varians Mut has superior antioxidant properties.

  12. 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

    2016-02-01

    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

  13. 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

  14. Multi-walled carbon nanotubes (NM401) induce ROS-mediated HPRT mutations in Chinese hamster lung fibroblasts.

    PubMed

    Rubio, Laura; El Yamani, Naouale; Kazimirova, Alena; Dusinska, Maria; Marcos, Ricard

    2016-04-01

    Although there is an important set of data showing potential genotoxic effects of nanomaterials (NMs) at the DNA (comet assay) and chromosome (micronucleus test) levels, few studies have been conducted to analyze their potential mutagenic effects at gene level. We have determined the ability of multi-walled carbon nanotubes (MWCNT, NM401), to induce mutations in the HPRT gene in Chinese hamster lung (V79) fibroblasts. NM401, characterized in the EU NanoGenotox project, were further studied within the EU Framework Programme Seven (FP7) project NANoREG. From the proliferation assay data we selected a dose-range of 0.12 to 12µg/cm(2) At these range we have been able to observe significant cellular uptake of MWCNT by using transmission electron microscopy (TEM), as well as a concentration-dependent induction of intracellular reactive oxygen species. In addition, a clear concentration-dependent increase in the induction of HPRT mutations was also observed. Data support a potential genotoxic/ carcinogenic risk associated with MWCNT exposure. PMID:26774957

  15. Expression of colSR Genes Increased in the rpf Mutants of Xanthomonas oryzae pv. oryzae KACC10859

    PubMed Central

    Noh, Young-Hee; Kim, Sun-Young; Han, Jong-Woo; Seo, Young-Su; Cha, Jae-Soon

    2014-01-01

    The rpf genes and colSXOO1207/colRXOO1208 were known to require for virulence of Xanthomonas oryzae pv. oryzae (Xoo). In Xoo KACC10331 genome, two more colS/colR genes, colSXOO3534 (raxH)/colRXOO3535 (raxR) and colSXOO3762/colRXOO3763 were annotated. The colSXOO3534/colRXOO3535 were known to control AvrXa21 activity and functions of colSXOO3762/colRXOO3763 were unknown in Xoo. To characterize the relationship between rpf and colS/colR genes, expression of colS/colR genes in Rpf mutants of Xoo were analyzed with quantitative reverse transcription PCR (qRT-PCR). Expressions of all three colS/colR genes increased in the rpfF mutant in which DSF synthesis is defective. Expression of colSXOO1207/colRXOO1208, colSXOO3534/colRXOO3535 and colSXOO3762/colRXOO3763 increased 2, 2–7, 3–13 folds respectively. Expression of colSXOO3534 and colSXOO3762 also increased 2–4 folds in the rpfG mutant in which the signal from DSF is no longer transferred to down-stream. Expression of the other colS/colR genes was not significantly changed in the rpfG mutant compared to the wild type. Since RpfF and RpfG are responsible for DSF synthesis and signal transfer from DSF to down-stream to regulate virulence gene expression, these results suggest that the DSF and DSF-mediated signal regulate negatively three colS/colR genes in Xoo. PMID:25289017

  16. Charge separation in Rhodobacter sphaeroides mutant reaction centers with increased midpoint potential of the primary electron donor.

    PubMed

    Khmelnitskiy, A Yu; Khatypov, R A; Khristin, A M; Leonova, M M; Vasilieva, L G; Shuvalov, V A

    2013-01-01

    Primary charge separation dynamics in four mutant reaction centers (RCs) of the purple bacterium Rhodobacter sphaeroides with increased midpoint potential of the primary electron donor P (M160LH, L131LH, M197FH, and M160LH + L131LH + M197FH) have been studied by femtosecond transient absorption spectroscopy at room temperature. The decay of the excited singlet state in the wild-type and mutant RCs is complex and has two main exponential components, which indicates heterogeneity of electron transfer rates or the presence of reverse electron transfer reactions. The radical anion band of monomeric bacteriochlorophyll B(A) at 1020 nm was first observed in transient absorbance difference spectra of single mutants. This band remains visible, although with somewhat reduced amplitude, even at delays up to tens of picoseconds when stimulated emission is absent and the reaction centers are in the P(+)H(A)(-) state. The presence of this band in this time period indicates the existence of thermodynamic equilibrium between the P(+)B(A)(-)H(A) and P(+)B(A)H(A)(-) states. The data give grounds for assuming that the value of the energy difference between the states P*, P(+)B(A)(-)H(A), and P(+)B(A)H(A)(-) at early times is of the same order of magnitude as the energy kT at room temperature. Besides, monomeric bacteriochlorophyll B(A) is found to be an immediate electron acceptor in the single mutant RCs, where electron transfer is hampered due to increased energy of the P(+)B(A)(-) state with respect to P*. PMID:23379560

  17. Mutations induced by 1,3-butadiene metabolites, butadiene diolepoxide, and 1,2,3,4-diepoxybutane at the Hprt locus in CHO-K1 cells.

    PubMed

    Lee, Dong-Hyun; Kim, Tae-Ho; Lee, Sun-Young; Kim, Hyun-Jo; Rhee, Seung Keun; Yoon, ByoungSu; Pfeifer, Gerd P; Lee, Chong-Soon

    2002-12-31

    Butadiene (BD) is an important industrial chemical that is classified as a probable human carcinogen. Butadiene diolepoxide (BDE) and 1,2,3,4-diepoxybutane (DEB) are metabolites of carcinogenic BD and contain the DNA-reactive one and two epoxides, respectively. In this study, the mutation frequencies and mutation spectra that are induced by BDE and DEB have been investigated at the hprt locus in CHO-K1 cells. The BDE- and DEB-treated CHO-K1 cells were allowed to grow for several days, then seeded in a medium that contained 6-thioguanine in order to select the hprt mutants. BDE exhibited the mutagenic activity at concentrations that were approximately 100-times higher than DEB. The mutation spectra for BDE and DEB were determined by a reverse transcription-polymerase chain reaction of hprt mRNA, which was followed by automatic DNA sequencing of the PCR products. The mutational spectrum for BDE was exon deletions (16/41), G x C --> A x T transitions (11/41), and A x T --> G x C transitions (5/41). The mutational spectrum for DEB was exon deletions (15/39), G x C --> A x T transitions (11/39), and A x T --> T x A transversions (5/39). The most common base substitution that was induced by both BDE and DEB was G x C --> A x T transitions. The sites of the single base substitutions that were induced by BDE and DEB were guanine and adenine, which was consistent with the DNA adduct profiles. The high frequencies of the exon deletions by each metabolite occurred in the regions of exons 2, 3, or 4. These data indicate that BDE and DEB are mutagenic carcinogens by forming DNA adducts at the site of adenine and guanine, and inducing large exon deletions and single base substitutions. PMID:12521305

  18. Evidence that the LRRK2 ROC domain Parkinson's disease-associated mutants A1442P and R1441C exhibit increased intracellular degradation.

    PubMed

    Greene, Izabella D; Mastaglia, Francis; Meloni, Bruno P; West, Kristin A; Chieng, Joanne; Mitchell, Chris J; Gai, Wei-Ping; Boulos, Sherif

    2014-04-01

    Mutations in the leucine-rich repeat kinase 2 (lrrk2) gene are the leading genetic cause of Parkinson's disease (PD). In characterizing the novel ROC domain mutant A1442P, we compared its steady-state protein levels, propensity to aggregate, and toxicity with the pathogenic R1441C mutant and wild-type (WT) LRRK2. Mutant (R1441C and A1442P) and WT LRRK2 fused to green fluorescent protein (GFP) and FLAG were transiently expressed in HEK293 cells using plasmid constructs. Western analysis and fluorescence microscopy consistently demonstrated lower mutant LRRK2 protein levels compared with WT. A time-course expression study using flow cytometry showed that WT LRRK2 expression increased initially but then plateaued by 72 hr. Conversely, R1441C and A1442P mutant expression attained 85% and 74% of WT levels at 24 hr but fell to 68% and 55% of WT levels by 72 hr, respectively. We found that proteasome inhibition markedly increased mutant LRRK2 to levels approaching those of WT. Taken together, our findings reveal increased intracellular degradation for both mutants. Furthermore, the impact of mutant and WT LRRK2 expression on HEK293 cell viability was assessed under normative and oxidative (hydrogen peroxide) conditions and found not to differ. Expression of WT and mutant LRRK2 protein gave rise to intracellular aggregates of similar appearance and cellular localization. In summary, we provide evidence that the novel A1442P mutant and the previously investigated R1441C pathogenic mutant exhibit increased intracellular degradation, a property reportedly demonstrated for the pathogenic LRRK2 kinase domain mutant I2020T. PMID:24375786

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

    PubMed

    Nguyen, Khue Vu; Nyhan, William L

    2016-08-01

    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 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. PMID:27379977

  20. 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

  1. 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

  2. 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.

  3. Enriching CRISPR-Cas9 targeted cells by co-targeting the HPRT gene.

    PubMed

    Liao, Shuren; Tammaro, Margaret; Yan, Hong

    2015-11-16

    The CRISPR-Cas9 system uses guide RNAs to direct the Cas9 endonuclease to cleave target sequences. It can, in theory, target essentially any sequence in a genome, but the efficiency of the predicted guide RNAs varies dramatically. If no targeted cells are obtained, it is also difficult to know why the experiment fails. We have developed a transient transfection based method to enrich successfully targeted cells by co-targeting the hypoxanthine phosphoribosyltransferase (HPRT) gene. Cells are transfected with two guide RNAs that target respectively HPRT and the gene of interest. HPRT targeted cells are selected by resistance to 6-thioguanine (6-TG) and then examined for potential alterations to the gene targeted by the co-transfected guide RNA. Alterations of many genes, such as AAVS1, Exo1 and Trex1, are highly enriched in the 6-TG resistant cells. This method works in both HCT116 cells and U2OS cells and can easily be scaled up to process multiple guide RNAs. When co-targeting fails, it is straightforward to determine whether the target gene is essential or the guide RNA is ineffective. HPRT co-targeting thus provides a simple, efficient and scalable way to enrich gene targeting events and to identify the cause of failure. PMID:26130722

  4. Neurotransmitter and their metabolite concentrations in different areas of the HPRT knockout mouse brain.

    PubMed

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

    2016-06-15

    Lesch-Nyhan syndrome (LNS) is characterized by uric acid overproduction and severe neurobehavioral symptoms, such as recurrent self-mutilative behavior. To learn more about the pathophysiology of the disease, we quantified neurotransmitters and their metabolites in the cerebral hemisphere, cerebellum and the medulla oblongata of HPRT knockout mice, an animal model for LNS, in comparison to the corresponding wild-type. Our analyses included l-glutamate, 4-aminobutanoic acid (GABA), acetylcholine, serotonin, 5-hydroxyindoleacetic acid (5-HIAA), norepinephrine, l-normetanephrine, epinephrine and l-metanephrine and were conducted via high performance liquid chromatography (HPLC) coupled to tandem mass spectrometry (MS/MS). Among these neurotransmitter systems, we did not find any abnormalities in the HPRT knockout mouse brains. On one side, this might indicate that HPRT deficiency most severely affects dopamine signaling, while brain functioning based on other neurotransmitters is more or less spared. On the other hand, our findings may reflect a compensating mechanism for impaired purine salvage that protects the brain in HPRT-deficient mice but not in LNS patients. PMID:27206901

  5. Increases of efficacy as vaccine against Brucella abortus infection in mice by simultaneous inoculation with avirulent smooth bvrS/bvrR and rough wbkA mutants.

    PubMed

    Grilló, María Jesús; Manterola, Lorea; de Miguel, María Jesús; Muñoz, Pilar María; Blasco, José María; Moriyón, Ignacio; López-Goñi, Ignacio

    2006-04-01

    The Brucella abortus S19 and RB51 strains are the most widely used live vaccines against bovine brucellosis. However, both can induce abortion and milk excretion, S19 vaccination interferes in serological tests, and RB51 is less effective. We have shown previously that a rough wbkAB. abortus mutant is attenuated and a better vaccine than RB51 in BALB/c mice, and that mutants in the two-component regulatory system bvrS/bvrR are markedly attenuated while keeping a smooth lipopolysaccharide (S-LPS). In this work, we tested whether simultaneous inoculation with live bvrS increases wbkA vaccine efficacy in mice. Even at high doses, the bvrS mutant was cleared much faster from spleens than the wbkA mutant. The splenic persistence of the wbkA mutant increased when inoculated along with the bvrS mutant, but also with inactivated bvrS cells or with purified B. abortus S-LPS, strongly suggesting that S-LPS in the bvrS mutant played a determinant role in the wbkA persistence. When inoculated alone, both mutants protected against virulent B. abortus but less than when inoculated simultaneously, and the protection afforded by the combination was better than that obtained with B. abortus S19. Increased protection was also obtained after simultaneous inoculation of the wbkA mutant and inactivated bvrS cells or purified S-LPS, showing again the role played by the S-LPS in the bvrS cells. In mice, the bvrS-wbkA combination induced an antibody response reduced with respect to B. abortus S19 vaccination. Thus, the simultaneous use of live bvrS and wbkA B. abortus mutants seems a promising approach to overcome the problems of the S19 andRB51 vaccines. PMID:16439039

  6. Fasting increases survival to cold in FOXO, DIF, autophagy mutants and in other genotypes of Drosophila melanogaster.

    PubMed

    Le Bourg, Éric; Massou, Isabelle

    2015-08-01

    Fasting increases survival to a severe cold stress in young and middle-aged wild-type flies, this effect being lowered or absent at old age. As an attempt to determine the mechanisms of this effect, genes involved in metabolism (dFOXO), autophagy (Atg7), innate immunity (Dif (1) ), and resistance to cold (Frost) were studied. The 12 mutant, RNAi and control lines tested in this study displayed an increased survival to cold after fasting. This shows that fasting has a robust effect on survival to cold in many genotypes, but the mechanism of this effect remains unknown. This mechanism does not seem to be linked to metabolic pathways often considered to play a critical role in ageing and longevity determinations (insulin/insulin-like growth factor-1 pathway and autophagy). PMID:25663303

  7. Isolation and Characterization of Arbuscules from Roots of an Increased-arbuscule-forming Mutant of Lotus japonicus

    PubMed Central

    Senoo, Keishi; Solaiman, Zakaria; Tanaka, Satoki; Kawaguchi, Masayoshi; Imaizumi-Anraku, Haruko; Akao, Shoichiro; Tanaka, Akiyoshi; Obata, Hitoshi

    2007-01-01

    Background and Aims Previous methods for isolation of arbuscules from mycorrhizal roots are time-consuming, complex and expensive. Therefore, a simple, rapid and inexpensive method for the isolation of metabolically active arbuscules from plant root of an increased-arbuscule-forming mutant of Lotus japonicus (Ljsym78-2) is described. Method Roots of the L. japonicus mutant plants Ljsym78-2 colonized by Glomus sp. were separated from soil, washed with water, immersed in CaSO4 before being cut into 5-mm pieces and homogenized with a Waring blender at 6000 rpm for 30 s. The arbuscules were purified by separation from plant tissues with a 50-μm nylon mesh, finally collecting on a 30-μm nylon mesh. Enzyme histochemical staining showed that the collected arbuscules had succinate dehydrogenase, alkaline phosphatase and acid phosphatase activities. Key Results and Conclusions The enzymic activity of the arbuscules was not affected after the isolation process. The establishment of this simple, rapid and inexpensive method for the isolation of metabolically active arbuscules will be useful to clarify the biochemical processes occurring in nutrient exchange at the arbuscular interface. PMID:17921523

  8. Construction of Mutant Glucose Oxidases with Increased Dye-Mediated Dehydrogenase Activity

    PubMed Central

    Horaguchi, Yohei; Saito, Shoko; Kojima, Katsuhiro; Tsugawa, Wakako; Ferri, Stefano; Sode, Koji

    2012-01-01

    Mutagenesis studies on glucose oxidases (GOxs) were conducted to construct GOxs with reduced oxidase activity and increased dehydrogenase activity. We focused on two representative GOxs, of which crystal structures have already been reported—Penicillium amagasakiense GOx (PDB ID; 1gpe) and Aspergillus niger GOx (PDB ID; 1cf3). We constructed oxygen-interacting structural models for GOxs, and predicted the residues responsible for oxidative half reaction with oxygen on the basis of the crystal structure of cholesterol oxidase as well as on the fact that both enzymes are members of the glucose/methanol/choline (GMC) oxidoreductase family. Rational amino acid substitution resulted in the construction of an engineered GOx with drastically decreased oxidase activity and increased dehydrogenase activity, which was higher than that of the wild-type enzyme. As a result, the dehydrogenase/oxidase ratio of the engineered enzyme was more than 11-fold greater than that of the wild-type enzyme. These results indicate that alteration of the dehydrogenase/oxidase activity ratio of GOxs is possible by introducing a mutation into the putative functional residues responsible for oxidative half reaction with oxygen of these enzymes, resulting in a further increased dehydrogenase activity. This is the first study reporting the alteration of GOx electron acceptor preference from oxygen to an artificial electron acceptor. PMID:23203056

  9. 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

  10. JAK2 exon 12 mutant mice display isolated erythrocytosis and changes in iron metabolism favoring increased erythropoiesis.

    PubMed

    Grisouard, Jean; Li, Sai; Kubovcakova, Lucia; Rao, Tata Nageswara; Meyer, Sara C; Lundberg, Pontus; Hao-Shen, Hui; Romanet, Vincent; Murakami, Masato; Radimerski, Thomas; Dirnhofer, Stephan; Skoda, Radek C

    2016-08-11

    Mutations in JAK2 exon 12 are frequently found in patients with polycythemia vera (PV) that do not carry a JAK2-V617F mutation. The majority of these patients display isolated erythrocytosis. We generated a mouse model that expresses JAK2-N542-E543del, the most frequent JAK2 exon 12 mutation found in PV patients. Mice expressing the human JAK2-N542-E543del (Ex12) showed a strong increase in red blood cell parameters but normal neutrophil and platelet counts, and reduced overall survival. Erythropoiesis was increased in the bone marrow and spleen, with normal megakaryopoiesis and absence of myelofibrosis in histopathology. Erythroid progenitors and precursors were increased in hematopoietic tissues, but the numbers of megakaryocytic precursors were unchanged. Phosphorylation Stat3 and Erk1/2 proteins were increased, and a trend toward increased phospho-Stat5 and phospho-Stat1 was noted. However, Stat1 knock out in Ex12 mice induced no changes in platelet or red cell parameters, indicating that Stat1 does not play a central role in mediating the effects of Ex12 signaling on megakaryopoiesis or erythropoiesis. Ex12 mice showed decreased expression of hepcidin and increased expression of transferrin receptor-1 and erythroferrone, suggesting that the strong erythroid phenotype in Ex12 mutant mice is favored by changes in iron metabolism that optimize iron availability to allow maximal production of red cells. PMID:27288519

  11. Increased dopaminergic innervation in the brain of conditional mutant mice overexpressing Otx2: effects on locomotor behavior and seizure susceptibility.

    PubMed

    Tripathi, P P; Di Giovannantonio, L G; Sanguinetti, E; Acampora, D; Allegra, M; Caleo, M; Wurst, W; Simeone, A; Bozzi, Y

    2014-03-01

    The homeobox-containing transcription factor Otx2 controls the identity, fate and proliferation of mesencephalic dopaminergic (mesDA) neurons. Transgenic mice, in which Otx2 was conditionally overexpressed by a Cre recombinase expressed under the transcriptional control of the Engrailed1 gene (En1(Cre/+); tOtx2(ov/+)), show an increased number of mesDA neurons during development. In adult mice, Otx2 is expressed in a subset of neurons in the ventral tegmental area (VTA) and its overexpression renders mesDA more resistant to 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-HCl (MPTP) neurotoxin. Here we further investigated the neurological consequences of the increased number of mesDA neurons in En1(Cre/+); tOtx2(ov/+) adult mice. Immunohistochemistry for the active, glycosylated form of the dopamine transporter (glyco-Dat) showed that En1(Cre/+); tOtx2(ov/+) adult mice display an increased density of mesocortical DAergic fibers, as compared to control animals. Increased glyco-Dat staining was accompanied by a marked hypolocomotion in En1(Cre/+); tOtx2(ov/+) mice, as detected in the open field test. Since conditional knockout mice lacking Otx2 in mesDA precursors (En1(Cre/+); Otx2(floxv/flox) mice) show a marked resistance to kainic acid (KA)-induced seizures, we investigated the behavioral response to KA in En1(Cre/+); tOtx2(ov/+) and control mice. No difference was observed between mutant and control mice, but En1(Cre/+); tOtx2(ov/+) mice showed a markedly different c-fos mRNA induction profile in the cerebral cortex and hippocampus after KA seizures, as compared to controls. Accordingly, an increased density of parvalbumin (PV)-positive inhibitory interneurons was detected in the deep layers of the frontal cortex of naïve En1(Cre/+); tOtx2(ov/+) mice, as compared to controls. These data indicate that Otx2 overexpression results in increased DAergic innervation and PV cell density in the fronto-parietal cortex, with important consequences on spontaneous locomotor

  12. 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

  13. The Housekeeping Gene Hypoxanthine Guanine Phosphoribosyltransferase (HPRT) Regulates Multiple Developmental and Metabolic Pathways of Murine Embryonic Stem Cell Neuronal Differentiation

    PubMed Central

    Bader, Joel S.; Friedmann, Theodore

    2013-01-01

    The mechanisms by which mutations of the purinergic housekeeping gene hypoxanthine guanine phosphoribosyltransferase (HPRT) cause the severe neurodevelopmental Lesch Nyhan Disease (LND) are poorly understood. The best recognized neural consequences of HPRT deficiency are defective basal ganglia expression of the neurotransmitter dopamine (DA) and aberrant DA neuronal function. We have reported that HPRT deficiency leads to dysregulated expression of multiple DA-related developmental functions and cellular signaling defects in a variety of HPRT-deficient cells, including human induced pluripotent stem (iPS) cells. We now describe results of gene expression studies during neuronal differentiation of HPRT-deficient murine ESD3 embryonic stem cells and report that HPRT knockdown causes a marked switch from neuronal to glial gene expression and dysregulates expression of Sox2 and its regulator, genes vital for stem cell pluripotency and for the neuronal/glial cell fate decision. In addition, HPRT deficiency dysregulates many cellular functions controlling cell cycle and proliferation mechanisms, RNA metabolism, DNA replication and repair, replication stress, lysosome function, membrane trafficking, signaling pathway for platelet activation (SPPA) multiple neurotransmission systems and sphingolipid, sulfur and glycan metabolism. We propose that the neural aberrations of HPRT deficiency result from combinatorial effects of these multi-system metabolic errors. Since some of these aberrations are also found in forms of Alzheimer's and Huntington's disease, we predict that some of these systems defects play similar neuropathogenic roles in diverse neurodevelopmental and neurodegenerative diseases in common and may therefore provide new experimental opportunities for clarifying pathogenesis and for devising new potential therapeutic targets in developmental and genetic disease. PMID:24130677

  14. Metabolic analysis of the increased adventitious rooting mutant of Artemisia annua reveals a role for the plant monoterpene borneol in adventitious root formation.

    PubMed

    Tian, Na; Liu, Shuoqian; Li, Juan; Xu, Wenwen; Yuan, Lin; Huang, Jianan; Liu, Zhonghua

    2014-08-01

    Adventitious root (AR) formation is a critical process for plant clonal propagation. The role of plant secondary metabolites in AR formation is still poorly understood. Chemical and physical mutagenesis in combination with somatic variation were performed on Artemisia annua in order to obtain a mutant with changes in adventitious rooting and composition of plant secondary metabolites. Metabolic and morphological analyses of the iar (increased adventitious rooting) mutant coupled with in vitro assays were used to elucidate the relationship between plant secondary metabolites and AR formation. The only detected differences between the iar mutant and wild-type were rooting capacity and borneol/camphor content. Consistent with this, treatment with borneol in vitro promoted adventitious rooting in wild-type. The enhanced rooting did not continue upon removal of borneol. The iar mutant displayed no significant differences in AR formation upon treatment with camphor. Together, our results suggest that borneol promotes adventitious rooting whereas camphor has no effect on AR formation. PMID:24329606

  15. 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

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

    PubMed

    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

  17. Regulatory elements in the introns of the human HPRT gene are necessary for its expression in embryonic stem cells.

    PubMed Central

    Reid, L H; Gregg, R G; Smithies, O; Koller, B H

    1990-01-01

    We have examined the expression of transfected human hypoxanthine phosphoribosyltransferase minigenes (HPRT) in mouse embryonic stem (ES) cells. cDNA constructs of this gene that have been successfully used in somatic cell lines failed to confer hypoxanthine/aminopterin/thymidine (HAT) resistance in ES cells. In contrast, constructs containing introns 1 and 2 from the HPRT gene produced a high frequency of HAT-resistant colonies. This observation allowed us to identify two sequences in these introns that influence expression of the HPRT gene in ES cells. One element, located in intron 2, is required for effective HPRT expression in these cells; the other element, located in intron 1, acts as an enhancer of HPRT expression. Using this information, we have constructed an HPRT minigene that can be used for either positive or negative selection in ES cell experiments. This dual capability allows the design of "in-out" procedures to create subtle changes in target genes by homologous recombination with the aid of this selectable minigene. PMID:2349238

  18. TRAIL causes deletions at the HPRT and TK1 loci of clonogenically competent cells.

    PubMed

    Miles, Mark A; Shekhar, Tanmay M; Hall, Nathan E; Hawkins, Christine J

    2016-05-01

    When chemotherapy and radiotherapy are effective, they function by inducing DNA damage in cancerous cells, which respond by undergoing apoptosis. Some adverse effects can result from collateral destruction of non-cancerous cells, via the same mechanism. Therapy-related cancers, a particularly serious adverse effect of anti-cancer treatments, develop due to oncogenic mutations created in non-cancerous cells by the DNA damaging therapies used to eliminate the original cancer. Physiologically achievable concentrations of direct apoptosis inducing anti-cancer drugs that target Bcl-2 and IAP proteins possess negligible mutagenic activity, however death receptor agonists like TRAIL/Apo2L can provoke mutations in surviving cells, probably via caspase-mediated activation of the nuclease CAD. In this study we compared the types of mutations sustained in the HPRT and TK1 loci of clonogenically competent cells following treatment with TRAIL or the alkylating agent ethyl methanesulfonate (EMS). As expected, the loss-of-function mutations in the HPRT or TK1 loci triggered by exposure to EMS were almost all transitions. In contrast, only a minority of the mutations identified in TRAIL-treated clones lacking HPRT or TK1 activity were substitutions. Almost three quarters of the TRAIL-induced mutations were partial or complete deletions of the HPRT or TK1 genes, consistent with sub-lethal TRAIL treatment provoking double strand breaks, which may be mis-repaired by non-homologous end joining (NHEJ). Mis-repair of double-strand breaks following exposure to chemotherapy drugs has been implicated in the pathogenesis of therapy-related cancers. These data suggest that TRAIL too may provoke oncogenic damage to the genomes of surviving cells. PMID:26943263

  19. 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. PMID:25636690

  20. Use of the HPRT gene to study nuclease-induced DNA double-strand break repair

    PubMed Central

    Gravells, Polly; Ahrabi, Sara; Vangala, Rajani K.; Tomita, Kazunori; Brash, James T.; Brustle, Lena A.; Chung, Christopher; Hong, Julia M.; Kaloudi, Aikaterini; Humphrey, Timothy C.; Porter, Andrew C.G.

    2015-01-01

    Understanding the mechanisms of chromosomal double-strand break repair (DSBR) provides insight into genome instability, oncogenesis and genome engineering, including disease gene correction. Research into DSBR exploits rare-cutting endonucleases to cleave exogenous reporter constructs integrated into the genome. Multiple reporter constructs have been developed to detect various DSBR pathways. Here, using a single endogenous reporter gene, the X-chromosomal disease gene encoding hypoxanthine phosphoribosyltransferase (HPRT), we monitor the relative utilization of three DSBR pathways following cleavage by I-SceI or CRISPR/Cas9 nucleases. For I-SceI, our estimated frequencies of accurate or mutagenic non-homologous end-joining and gene correction by homologous recombination are 4.1, 1.5 and 0.16%, respectively. Unexpectedly, I-SceI and Cas9 induced markedly different DSBR profiles. Also, using an I-SceI-sensitive HPRT minigene, we show that gene correction is more efficient when using long double-stranded DNA than single- or double-stranded oligonucleotides. Finally, using both endogenous HPRT and exogenous reporters, we validate novel cell cycle phase-specific I-SceI derivatives for investigating cell cycle variations in DSBR. The results obtained using these novel approaches provide new insights into template design for gene correction and the relationships between multiple DSBR pathways at a single endogenous disease gene. PMID:26423459

  1. Optimization of biomass production of a mutant of Yarrowia lipolytica with an increased lipase activity using raw glycerol.

    PubMed

    Galvagno, Miguel A; Iannone, Leopoldo J; Bianchi, Jorgelina; Kronberg, Florencia; Rost, Enrique; Carstens, Maria R; Cerrutti, Patricia

    2011-01-01

    The yeast Yarrowia lipolytica accumulates oils and is able to produce extracellular lipases when growing in different carbon sources including glycerol, the principal by-product of the biodiesel industry. In this study, biomass production of a novel mutant strain of Y. lipolytica was statistically optimized by Response Surface Methodology in media containing biodiesel-derived glycerol as main carbon source. This strain exhibited distinctive morphological and fatty acid profile characteristics, and showed an increased extracellular lipase activity. An organic source of nitrogen and the addition of 1.0 g/l olive oil were necessary for significant lipase production. Plackett-Burman and Central Composite Statistical Designs were employed for screening and optimization of fermentation in shaken flasks cultures, and the maximum values obtained were 16.1 g/l for biomass and 12.2 Units/ml for lipase, respectively. Optimized batch bioprocess was thereafter scaled in aerated bioreactors and the values reached for lipase specific activity after 95 % of the glycerol had been consumed, were three-fold higher than those obtained in shaken flasks cultures. A sustainable bioprocess to obtain biomass and extracellular lipase activity was attained by maximizing the use of the by-products of biodiesel industry. PMID:22430997

  2. 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. PMID:23333947

  3. 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

  4. Uranyl acetate induces hprt mutations and uranium-DNA adducts in Chinese hamster ovary EM9 cells.

    PubMed

    Stearns, Diane M; Yazzie, Monica; Bradley, Andrew S; Coryell, Virginia H; Shelley, Jake T; Ashby, Adam; Asplund, Craig S; Lantz, R Clark

    2005-11-01

    Questions about possible adverse health effects from exposures to uranium have arisen as a result of uranium mining, residual mine tailings and use of depleted uranium in the military. The purpose of the current study was to measure the toxicity of depleted uranium as uranyl acetate (UA) in mammalian cells. The activity of UA in the parental CHO AA8 line was compared with that in the XRCC1-deficient CHO EM9 line. Cytotoxicity was measured by clonogenic survival. A dose of 200 microM UA over 24 h produced 3.1-fold greater cell death in the CHO EM9 than the CHO AA8 line, and a dose of 300 microM was 1.7-fold more cytotoxic. Mutagenicity at the hypoxanthine (guanine) phosphoribosyltransferase (hprt) locus was measured by selection with 6-thioguanine. A dose of 200 microM UA produced approximately 5-fold higher averaged induced mutant frequency in the CHO EM9 line relative to the CHO AA8 line. The generation of DNA strand breaks was measured by the alkaline comet assay at 40 min and 24 h exposures. DNA strand breaks were detected in both lines; however a dose response may have been masked by U-DNA adducts or crosslinks. Uranium-DNA adducts were measured by inductively coupled plasma optical emission spectroscopy (ICP-OES) at 24 and 48 h exposures. A maximum adduct level of 8 U atoms/10(3) DNA-P for the 300 microM dose was found in the EM9 line after 48 h. This is the first report of the formation of uranium-DNA adducts and mutations in mammalian cells after direct exposure to a depleted uranium compound. Data suggest that uranium could be chemically genotoxic and mutagenic through the formation of strand breaks and covalent U-DNA adducts. Thus the health risks for uranium exposure could go beyond those for radiation exposure. PMID:16195314

  5. 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

  6. Disease causing mutants of TDP-43 nucleic acid binding domains are resistant to aggregation and have increased stability and half-life

    PubMed Central

    Austin, James A.; Wright, Gareth S. A.; Watanabe, Seiji; Grossmann, J. Günter; Antonyuk, Svetlana V.; Yamanaka, Koji; Hasnain, S. Samar

    2014-01-01

    Over the last two decades many secrets of the age-related human neural proteinopathies have been revealed. A common feature of these diseases is abnormal, and possibly pathogenic, aggregation of specific proteins in the effected tissue often resulting from inherent or decreased structural stability. An archetype example of this is superoxide dismutase-1, the first genetic factor to be linked with amyotrophic lateral sclerosis (ALS). Mutant or posttranslationally modified TAR DNA binding protein-32 (TDP-43) is also strongly associated with ALS and an increasingly large number of other neurodegenerative diseases, including frontotemporal lobar degeneration (FTLD). Cytoplasmic mislocalization and elevated half-life is a characteristic of mutant TDP-43. Furthermore, patient age at the onset of disease symptoms shows a good inverse correlation with mutant TDP-43 half-life. Here we show that ALS and FTLD-associated TDP-43 mutations in the central nucleic acid binding domains lead to elevated half-life and this is commensurate with increased thermal stability and inhibition of aggregation. It is achieved without impact on secondary, tertiary, or quaternary structure. We propose that tighter structural cohesion contributes to reduced protein turnover, increasingly abnormal proteostasis and, ultimately, faster onset of disease symptoms. These results contrast our perception of neurodegenerative diseases as misfolded proteinopathies and delineate a novel path from the molecular characteristics of mutant TDP-43 to aberrant cellular effects and patient phenotype. PMID:24591609

  7. A Mutant Form of the Neisseria gonorrhoeae Pilus Secretin Protein PilQ Allows Increased Entry of Heme and Antimicrobial Compounds†

    PubMed Central

    Chen, Ching-ju; Tobiason, Deborah M.; Thomas, Christopher E.; Shafer, William M.; Seifert, H. Steven; Sparling, P. Frederick

    2004-01-01

    A spontaneous point mutation in pilQ (pilQ1) resulted in phenotypic suppression of a hemoglobin (Hb) receptor mutant (hpuAB mutant), allowing gonococci to grow on Hb as the sole source of iron. PilQ, formerly designated OMP-MC, is a member of the secretin family of proteins located in the outer membrane and is required for pilus biogenesis. The pilQ1 mutant also showed decreased piliation and transformation efficiency. Insertional inactivation of pilQ1 resulted in the loss of the Hb utilization phenotype and decreased entry of free heme. Despite the ability of the pilQ1 mutant to use Hb for iron acquisition and porphyrin, there was no demonstrable binding of Hb to the cell surface. The pilQ1 mutant was more sensitive to the toxic effect of free heme in growth medium and hypersensitive to the detergent Triton X-100 and multiple antibiotics. Double mutation in pilQ1 and tonB had no effect on these phenotypes, but a double pilQ1 pilT mutant showed a reduction in Hb-dependent growth and decreased sensitivity to heme and various antimicrobial agents. Insertional inactivation of wild-type pilQ also resulted in reduced entry of heme, Triton X-100, and some antibiotics. These results show that PilQ forms a channel that allows entry of heme and certain antimicrobial compounds and that a gain-of function point mutation in pilQ results in TonB-independent, PilT-dependent increase of entry. PMID:14729699

  8. Increased dopaminergic neuron sensitivity to 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) in transgenic mice expressing mutant A53T alpha-synuclein.

    PubMed

    Yu, Wai Haung; Matsuoka, Yasuji; Sziráki, István; Hashim, Audrey; Lafrancois, John; Sershen, Henry; Duff, Karen E

    2008-05-01

    Familial Parkinson's disease (PD) has been linked to point mutations and duplication of the alpha-synuclein gene and mutant alpha-synuclein expression increases the vulnerability of neurons to exogenous insults. In this study, we analyzed the levels of dopamine and its metabolites in the olfactory bulb (OB), and nigrostriatal regions of transgenic mice expressing human, mutant A53T alpha-synuclein (alpha-syn tg) and their non-transgenic (ntg) littermates using a sub-toxic, moderate dose of MPTP to determine if mutant human alpha-synuclein sensitizes the central dopaminergic systems to oxidative stress. We observed that after a single, sub-lethal MPTP injection, dopamine levels were reduced in striatum and SN in both the alpha-syn tg and ntg mice. In the olfactory bulb, a region usually resistant to MPTP toxicity, levels were reduced only in the alpha-syn tg mice. In addition, we identified a significant increase in dopamine metabolism in the alpha-syn transgenic, but not ntg mice. Finally, MPTP treatment of alpha-syn tg mice was associated with a marked elevation in the oxidative product, 3-nitrotyrosine that co-migrated with alpha-synuclein. Cumulatively, the data support the hypothesis that mutant alpha-synuclein sensitizes dopaminergic neurons to neurotoxic insults and is associated with greater oxidative stress. The alpha-syn tg line is therefore useful to study the genetic and environmental inter-relationship in PD. PMID:17999181

  9. 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

    2015-01-01

    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. PMID:26132002

  10. 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. PMID:25965333

  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. Production of 1,3-PDO and butanol by a mutant strain of Clostridium pasteurianum with increased tolerance towards crude glycerol

    PubMed Central

    2012-01-01

    The production of biodiesel results in a concomitant production of crude glycerol (10% w/w). Clostridium pasteurianum can utilize glycerol as sole carbon source and converts it into 1,3-propanediol, ethanol, butanol, and CO2. Reduced growth and productivities on crude glycerol as compared to technical grade glycerol have previously been observed. In this study, we applied random mutagenesis mediated by ethane methyl sulfonate (EMS) to develop a mutant strain of C. pasteurianum tolerating high concentrations of crude glycerol. At an initial crude glycerol concentration of 25 g/l the amount of dry cell mass produced by the mutant strain was six times higher than the amount produced by the wild type. Growth of the mutant strain was even detected at an initial crude glycerol concentration of 105 g/l. A pH controlled reactor with in situ removal of butanol by gas-stripping was used to evaluate the performance of the mutant strain. Utilizing stored crude glycerol, the mutant strain showed significantly increased rates compared to the wild type. A maximum glycerol utilization rate of 7.59 g/l/h was observed along with productivities of 1.80 g/l/h and 1.21 g/l/h of butanol and 1,3-PDO, respectively. These rates are higher than what previously has been published for C. pasteurianum growing on technical grade glycerol in fed batch reactors. In addition, high yields of the main products (butanol and 1,3-PDO) were detected and these two products were efficiently separated in two steams using gas-stripping. PMID:22901717

  13. Increased Expression of Clumping Factor and Fibronectin-Binding Proteins by hemB Mutants of Staphylococcus aureus Expressing Small Colony Variant Phenotypes

    PubMed Central

    Vaudaux, Pierre; Francois, Patrice; Bisognano, Carmelo; Kelley, William L.; Lew, Daniel P.; Schrenzel, Jacques; Proctor, Richard A.; McNamara, Peter J.; Peters, G.; Von Eiff, Christof

    2002-01-01

    Small colony variants (SCVs) of Staphylococcus aureus are slow-growing subpopulations that cause persistent and relapsing infections. The altered phenotype of SCV can arise from defects in menadione or hemin biosynthesis, which disrupt the electron transport chain and decrease ATP concentrations. With SCVs, virulence is altered by a decrease in exotoxin production and susceptibility to various antibiotics, allowing their intracellular survival. The expression of bacterial adhesins by SCVs is poorly documented. We tested fibrinogen- and fibronectin-mediated adhesion of a hemB mutant of S. aureus 8325-4 that is defective for hemin biosynthesis and exhibits a complete SCV phenotype. In this strain, adhesion to fibrinogen and fibronectin was significantly higher than that of its isogenic, normally growing parent and correlated with the increased surface display of these adhesins as assessed by flow cytometry. Real-time quantitative reverse transcription-PCR demonstrated increased expression of clfA and fnb genes by the hemB mutant compared to its isogenic parent. The influence of the hemB mutation on altered adhesin expression was confirmed by showing complete restoration of the wild-type adhesive phenotype in the hemB mutant, either by complementing with intact hemB or by supplementing the growth medium with hemin. Increased surface display of fibrinogen and fibronectin adhesins by the hemB mutation occurred independently from agr, a major regulatory locus of virulence factors in S. aureus. Both agr-positive and agr-lacking hemB mutants were also more efficiently internalized by human embryonic kidney cells than were their isogenic controls, presumably because of increased surface display of their fibronectin adhesins. PMID:12228267

  14. 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. PMID:26094596

  15. A mutation in the dynein heavy chain gene compensates for energy deficit of mutant SOD1 mice and increases potentially neuroprotective IGF-1

    PubMed Central

    2011-01-01

    Background Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by a progressive loss of motor neurons. ALS patients, as well as animal models such as mice overexpressing mutant SOD1s, are characterized by increased energy expenditure. In mice, this hypermetabolism leads to energy deficit and precipitates motor neuron degeneration. Recent studies have shown that mutations in the gene encoding the dynein heavy chain protein are able to extend lifespan of mutant SOD1 mice. It remains unknown whether the protection offered by these dynein mutations relies on a compensation of energy metabolism defects. Results SOD1(G93A) mice were crossbred with mice harboring the dynein mutant Cramping allele (Cra/+ mice). Dynein mutation increased adipose stores in compound transgenic mice through increasing carbohydrate oxidation and sparing lipids. Metabolic changes that occurred in double transgenic mice were accompanied by the normalization of the expression of key mRNAs in the white adipose tissue and liver. Furthermore, Dynein Cra mutation rescued decreased post-prandial plasma triglycerides and decreased non esterified fatty acids upon fasting. In SOD1(G93A) mice, the dynein Cra mutation led to increased expression of IGF-1 in the liver, increased systemic IGF-1 and, most importantly, to increased spinal IGF-1 levels that are potentially neuroprotective. Conclusions These findings suggest that the protection against SOD1(G93A) offered by the Cramping mutation in the dynein gene is, at least partially, mediated by a reversal in energy deficit and increased IGF-1 availability to motor neurons. PMID:21521523

  16. 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.

  17. Mutations that alter RNA splicing of the human HPRT gene: a review of the spectrum.

    PubMed

    O'Neill, J P; Rogan, P K; Cariello, N; Nicklas, J A

    1998-11-01

    The human HPRT gene contains spans approximately 42,000 base pairs in genomic DNA, has a mRNA of approximately 900 bases and a protein coding sequence of 657 bases (initiation codon AUG to termination codon UAA). This coding sequence is distributed into 9 exons ranging from 18 (exon 5) to 184 (exon 3) base pairs. Intron sizes range from 170 (intron 7) to 13,075 (intron 1) base pairs. In a database of human HPRT mutations, 277 of 2224 (12.5%) mutations result in alterations in splicing of the mRNA as analyzed by both reverse transcriptase mediated production of a cDNA followed by PCR amplification and cDNA sequencing and by genomic DNA PCR amplification and sequencing. Mutations have been found in all eight 5' (donor) and 3' (acceptor) splice sequences. Mutations in the 5' splice sequences of introns 1 and 5 result in intron inclusion in the cDNA due to the use of cryptic donor splice sequences within the introns; mutations in the other six 5' sites result in simple exon exclusion. Mutations in the 3' splice sequences of introns 1, 3, 7 and 8 result in partial exon exclusion due to the use of cryptic acceptor splice sequences within the exons; mutations in the other four 3' sites result in simple exon exclusion. A base substitution in exon 3 (209G-->T) creates a new 5' (donor) splice site which results in the exclusion of 110 bases of exon 3 from the cDNA. Two base substitutions in intron 8 (IVS8-16G-->A and IVS8-3T-->G) result in the inclusion of intron 8 sequences in the cDNA due to the creation of new 3' (acceptor) splice sites. Base substitution within exons 1, 3, 4, 6 and 8 also result in splice alterations in cDNA. Those in exons 1 and 6 are at the 3' end of the exon and may directly affect splicing. Those within exons 3 and 4 may be the result of the creation of nonsense codons, while those in exon 8 cannot be explained by this mechanism. Lastly, many mutations that affect splicing of the HPRT mRNA have pleiotropic effects in that multiple cDNA products are

  18. 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. PMID:25492221

  19. 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

  20. Mutant Alleles of lptD Increase the Permeability of Pseudomonas aeruginosa and Define Determinants of Intrinsic Resistance to Antibiotics

    PubMed Central

    Grabowicz, Marcin

    2015-01-01

    Gram-negative bacteria provide a particular challenge to antibacterial drug discovery due to their cell envelope structure. Compound entry is impeded by the lipopolysaccharide (LPS) of the outer membrane (OM), and those molecules that overcome this barrier are often expelled by multidrug efflux pumps. Understanding how efflux and permeability affect the ability of a compound to reach its target is paramount to translating in vitro biochemical potency to cellular bioactivity. Herein, a suite of Pseudomonas aeruginosa strains were constructed in either a wild-type or efflux-null background in which mutations were engineered in LptD, the final protein involved in LPS transport to the OM. These mutants were demonstrated to be defective in LPS transport, resulting in compromised barrier function. Using isogenic strain sets harboring these newly created alleles, we were able to define the contributions of permeability and efflux to the intrinsic resistance of P. aeruginosa to a variety of antibiotics. These strains will be useful in the design and optimization of future antibiotics against Gram-negative pathogens. PMID:26596941

  1. Novel angiogenin mutants with increased cytotoxicity enhance the depletion of pro-inflammatory macrophages and leukemia cells ex vivo.

    PubMed

    Cremer, Christian; Braun, Hanna; Mladenov, Radoslav; Schenke, Lea; Cong, Xiaojing; Jost, Edgar; Brümmendorf, Tim H; Fischer, Rainer; Carloni, Paolo; Barth, Stefan; Nachreiner, Thomas

    2015-12-01

    Immunotoxins are fusion proteins that combine a targeting component such as an antibody fragment or ligand with a cytotoxic effector component that induces apoptosis in specific cell populations displaying the corresponding antigen or receptor. Human cytolytic fusion proteins (hCFPs) are less immunogenic than conventional immunotoxins because they contain human pro-apoptotic enzymes as effectors. However, one drawback of hCFPs is that target cells can protect themselves by expressing endogenous inhibitor proteins. Inhibitor-resistant enzyme mutants that maintain their cytotoxic activity are therefore promising effector domain candidates. We recently developed potent variants of the human ribonuclease angiogenin (Ang) that were either more active than the wild-type enzyme or less susceptible to inhibition because of their lower affinity for the ribonuclease inhibitor RNH1. However, combining the mutations was unsuccessful because although the enzyme retained its higher activity, its susceptibility to RNH1 reverted to wild-type levels. We therefore used molecular dynamic simulations to determine, at the atomic level, why the affinity for RNH1 reverted, and we developed strategies based on the introduction of further mutations to once again reduce the affinity of Ang for RNH1 while retaining its enhanced activity. We were able to generate a novel Ang variant with remarkable in vitro cytotoxicity against HL-60 cells and pro-inflammatory macrophages. We also demonstrated the pro-apoptotic potential of Ang-based hCFPs on cells freshly isolated from leukemia patients. PMID:26472728

  2. 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.

  3. A novel tomato mutant, Solanum lycopersicum elongated fruit1 (Slelf1), exhibits an elongated fruit shape caused by increased cell layers in the proximal region of the ovary.

    PubMed

    Chusreeaeom, Katarut; Ariizumi, Tohru; Asamizu, Erika; Okabe, Yoshihiro; Shirasawa, Kenta; Ezura, Hiroshi

    2014-06-01

    Genes controlling fruit morphology offer important insights into patterns and mechanisms determining organ shape and size. In cultivated tomato (Solanum lycopersicum L.), a variety of fruit shapes are displayed, including round-, bell pepper-, pear-, and elongate-shaped forms. In this study, we characterized a tomato mutant possessing elongated fruit morphology by histologically analyzing its fruit structure and genetically analyzing and mapping the genetic locus. The mutant line, Solanum lycopersicum elongated fruit 1 (Slelf1), was selected in a previous study from an ethylmethane sulfonate-mutagenized population generated in the background of Micro-Tom, a dwarf and rapid-growth variety. Histological analysis of the Slelf1 mutant revealed dramatically increased elongation of ovary and fruit. Until 6 days before flowering, ovaries were round and they began to elongate afterward. We also determined pericarp thickness and the number of cell layers in three designated fruit regions. We found that mesocarp thickness, as well as the number of cell layers, was increased in the proximal region of immature green fruits, making this the key sector of fruit elongation. Using 262 F2 individuals derived from a cross between Slelf1 and the cultivar Ailsa Craig, we constructed a genetic map, simple sequence repeat (SSR), cleaved amplified polymorphism sequence (CAPS), and derived CAPS (dCAPS) markers and mapped to the 12 tomato chromosomes. Genetic mapping placed the candidate gene locus within a 0.2 Mbp interval on the long arm of chromosome 8 and was likely different from previously known loci affecting fruit shape. PMID:24519535

  4. 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. PMID:26542441

  5. 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

  6. 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

  7. Overexpression of Sis2, Which Contains an Extremely Acidic Region, Increases the Expression of Swi4, Cln1 and Cln2 in Sit4 Mutants

    PubMed Central

    Di-Como, C. J.; Bose, R.; Arndt, K. T.

    1995-01-01

    The Saccharomyces cerevisiae SIS2 gene was identified by its ability, when present on a high copy number plasmid, to increase dramatically the growth rate of sit4 mutants. SIT4 encodes a type 2A-related protein phosphatase that is required in late G1 for normal G1 cyclin expression and for bud initiation. Overexpression of SIS2, which contains an extremely acidic carboxyl terminal region, stimulated the rate of CLN1, CLN2, SWI4 and CLB5 expression in sit4 mutants. Also, overexpression of SIS2 in a CLN1 cln2 cln3 strain stimulated the growth rate and the rate of CLN1 and CLB5 RNA accumulation during late G1. The SIS2 protein fractionated with nuclei and was released from the nuclear fraction by treatment with either DNase I or micrococcal nuclease, but not by RNase A. This result, combined with the finding that overexpression of SIS2 is extremely toxic to a strain containing lower than normal levels of histones H2A and H2B, suggests that SIS2 might function to stimulate transcription via an interaction with chromatin. PMID:7705654

  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 14day 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 14days post-weaning (38days 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. PMID:26850554

  9. Microsecond Molecular Dynamics Simulations of Influenza Neuraminidase Suggest a Mechanism for the Increased Virulence of Stalk-Deletion Mutants

    PubMed Central

    2016-01-01

    Deletions in the stalk of the influenza neuraminidase (NA) surface protein are associated with increased virulence, but the mechanisms responsible for this enhanced virulence are unclear. Here we use microsecond molecular dynamics simulations to explore the effect of stalk deletion on enzymatic activity, contrasting NA proteins from the A/swine/Shandong/N1/2009 strain both with and without a stalk deletion. By modeling and simulating neuraminidase apo glycoproteins embedded in complex-mixture lipid bilayers, we show that the geometry and dynamics of the neuraminidase enzymatic pocket may differ depending on stalk length, with possible repercussions on the binding of the endogenous sialylated-oligosaccharide receptors. We also use these simulations to predict previously unrecognized druggable “hotspots” on the neuraminidase surface that may prove useful for future efforts aimed at structure-based drug design. PMID:27141956

  10. Microsecond Molecular Dynamics Simulations of Influenza Neuraminidase Suggest a Mechanism for the Increased Virulence of Stalk-Deletion Mutants.

    PubMed

    Durrant, Jacob D; Bush, Robin M; Amaro, Rommie E

    2016-08-25

    Deletions in the stalk of the influenza neuraminidase (NA) surface protein are associated with increased virulence, but the mechanisms responsible for this enhanced virulence are unclear. Here we use microsecond molecular dynamics simulations to explore the effect of stalk deletion on enzymatic activity, contrasting NA proteins from the A/swine/Shandong/N1/2009 strain both with and without a stalk deletion. By modeling and simulating neuraminidase apo glycoproteins embedded in complex-mixture lipid bilayers, we show that the geometry and dynamics of the neuraminidase enzymatic pocket may differ depending on stalk length, with possible repercussions on the binding of the endogenous sialylated-oligosaccharide receptors. We also use these simulations to predict previously unrecognized druggable "hotspots" on the neuraminidase surface that may prove useful for future efforts aimed at structure-based drug design. PMID:27141956

  11. 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. PMID:8691031

  12. The Reaumuria trigyna leucoanthocyanidin dioxygenase (RtLDOX) gene complements anthocyanidin synthesis and increases the salt tolerance potential of a transgenic Arabidopsis LDOX mutant.

    PubMed

    Zhang, Huirong; Du, Chao; Wang, Yan; Wang, Jia; Zheng, Linlin; Wang, Yingchun

    2016-09-01

    Reaumuria trigyna is a typical, native desert halophyte that grows under extreme conditions in Inner Mongolia. In a previous transcriptomic profiling analysis, flavonoid pathway-related genes in R. trigyna showed significant differences in transcript abundance under salt stress. Leucoanthocyanidin dioxygenase (LDOX, EC 1.14.11.19) is one of three dioxygenases in the flavonoid pathway that catalyzes the formation of anthocyanidins from leucoanthocyanidins. In this study, we cloned the full-length cDNA of R. trigyna LDOX (RtLDOX), and found RtLDOX recombinant protein was able to replace flavanone-3-hydroxylase (F3H, EC 1.14.11.9), another dioxygenase in the flavonoid pathway, to convert naringenin to dihydrokaempferol in vitro. R. trigyna LDOX can complement the Arabidopsis LDOX mutant transparent testa11 (tt11-11), which has reduced proanthocyanin (PA) and anthocyanin levels in seeds, to accumulate these two compounds. Thus, RtLDOX acts as a multifunctional dioxygenase to effect the synthesis of PA and anthocyanins and can perform F3H dioxygenase activities in the flavonoid biosynthesis pathway. The RtLDOX promoter harbored many cis-acting elements that might be recognized and bound by transcription factors related to stress response. RtLDOX expression was strongly increased under salt stress, and RtLDOX transgenic Arabidopsis mutant under NaCl stress accumulated the content of flavonoids leading to an increased antioxidant activities and plant biomass. These results suggest that RtLDOX as a multifunctional dioxygenase in flavonoid biosynthesis involves in enhancing plant response to NaCl stress. PMID:27219053

  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. Target binding to S100B reduces dynamic properties and increases Ca2+-binding affinity for wild type and EF-hand mutant proteins

    PubMed Central

    Liriano, Melissa A.; Varney, Kristen M.; Wright, Nathan T.; Hoffman, Cassandra L.; Toth, Eric A.; Ishima, Rieko; Weber, David J.

    2012-01-01

    Mutations in the second EF-hand (D61N, D63N, D65N, E72A) of S100B were used to study its Ca2+-binding and dynamic properties in the absence and presence of abound target, TRTK-12. With D63NS100B as an exception (D63NKD = 50 ± 9 µM), Ca2+-binding to EF2-hand mutants were reduced by more than 8-fold in the absence of TRTK-12 (D61NKD = 412 ± 67 µM; D65NKD = 968 ± 171 µM; E72AKD = 471 ± 133 µM), when compared to wild-type protein (WTKD = 56 ± 9 µM). For the TRTK-12 complexes, the Ca2+-binding affinity to wild type (WT+TRTKKD = 12 ± 10 µM) and the EF2 mutants were increased by 5- to 19-fold versus in the absence of target (D61N+TRTKKD = 29 ± 1.2 µM; D63N+TRTKKD = 10 ± 2.2 µM; D65N+TRTKKD = 73 ± 4.4 µM; E72A+TRTKKD = 18 ± 3.7 µM). In addition, Rex, as measured using relaxation dispersion for side chain 15N resonances of Asn63 (D63NS100B) was reduced upon TRTK-12 binding when measured by nuclear magnetic resonance (NMR). Likewise, backbone motions on multiple time scales (ps-ms) throughout wild type, D61NS100B D63NS100B, and D65NS100B were lowered upon binding TRTK-12. However, the X-ray structures of Ca2+-bound (2.0 Å) and TRTK-bound (1.2 Å) D63NS100B showed no change in Ca2+ coordination, so these and analogous structural data for the wild-type protein could not be used to explain how target binding increased Ca2+-binding affinity in solution. Thus, a model for how S100B-TRTK12 complex formation increases Ca2+ binding is discussed, which considers changes in protein dynamics upon binding the target TRTK-12. PMID:22824086

  15. 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. PMID:17603848

  16. 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

  17. Co-culturing of Lactobacillus paracasei subsp. paracasei with a Lactobacillus delbrueckii subsp. delbrueckii mutant to make high cell density for increased lactate productivity from cassava bagasse hydrolysate.

    PubMed

    John, Rojan Pappy; Nampoothiri, K Madhavan

    2011-03-01

    To increase the productivity of lactic acid, a co-culture of lactobacilli was made by mixing 1:1 ratio of Lactobacillus paracasei subsp. paracasei and a fast growing L. delbrueckii subsp. delbrueckii mutant. The culture was embedded on to polyurethane foam (PUF) cubes as a biofilm and used for fermentation. In order to prevent the cell leakage, the PUF cubes were further entrapped in calcium cross-linked alginate. The maximum lactic acid production using a high cell density free culture was >38 g l(-1) from ~40 g l(-1) of reducing sugar within 12 h of fermentation. Using PUF biofilms, the same yield of lactic acid attained after 24 h. When the cubes were further coated with alginate it took 36 h for the maximum yield. Even though, the productivity is slightly lesser with the alginate coating, cell leakage was decreased and cubes were reused without much decrease in production in repeated batches. Using a conventional control inoculum (3%, w/v), it took 120 h to yield same amount of lactic acid. PMID:20972788

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

    PubMed Central

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

    2015-01-01

    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 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 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. PMID:25645379

  19. 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

  20. 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

  1. Isolation and characterization of mutants of Arabidopsis thaliana with increased resistance to growth inhibition by indoleacetic acid-amino acid conjugates.

    PubMed Central

    Campanella, J J; Ludwig-Mueller, J; Town, C D

    1996-01-01

    Two mutants of Arabidopsis thaliana that are resistant to growth inhibition by indole-3-acetic acid (IAA)-phenylalanine have been isolated. Both mutants were 2- to 3-fold more resistant than wild type to inhibition by IAA-phenylalanine, IAA-alanine, and IAA-glycine in root growth assays. The mutant icr1 (but not icr2) also shows some resistance to IAA-aspartate. Studies using 3H-labeled IAA-phenylalanine showed that the uptake of conjugate from the medium by icr1 was the same as wild type and was reduced by about 25% in icr2. No differences in hydrolysis of the exogenous conjugate were detected between the mutants and their wild-type parents. There was no significant metabolism of the IAA released from the [3H]IAA-phenylalanine, whereas exogenous [3H]IAA was rapidly metabolized to two unidentified products considerably more polar than IAA. Analysis of a cross between icr1 and icr2 indicated that these mutations were at distinct loci and that their effects were additive, and preliminary mapping data indicated that icr1 and icr2 were located at the top and bottom of chromosome V, respectively. PMID:8883385

  2. Increased leaf photosynthesis caused by elevated stomatal conductance in a rice mutant deficient in SLAC1, a guard cell anion channel protein

    PubMed Central

    Kusumi, Kensuke

    2012-01-01

    In rice (Oryza sativa L.), leaf photosynthesis is known to be highly correlated with stomatal conductance; however, it remains unclear whether stomatal conductance dominantly limits the photosynthetic rate. SLAC1 is a stomatal anion channel protein controlling stomatal closure in response to environmental [CO2]. In order to examine stomatal limitations to photosynthesis, a SLAC1-deficient mutant of rice was isolated and characterized. A TILLING screen of N-methyl-N-nitrosourea-derived mutant lines was conducted for the rice SLAC1 orthologue gene Os04g0674700, and four mutant lines containing mutations within the open reading frame were obtained. A second screen using an infrared thermography camera revealed that one of the mutants, named slac1, had a constitutive low leaf temperature phenotype. Measurement of leaf gas exchange showed that slac1 plants grown in the greenhouse had significantly higher stomatal conductance (g s), rates of photosynthesis (A), and ratios of internal [CO2] to ambient [CO2] (C i/C a) compared with wild-type plants, whereas there was no significant difference in the response of photosynthesis to internal [CO2] (A/C i curves). These observations demonstrate that in well-watered conditions, stomatal conductance is a major determinant of photosynthetic rate in rice. PMID:22915747

  3. 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...

  4. 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.

  5. In vivo footprint analysis and genomic sequencing of the human hypoxanthine-phosphoribosyl transferase (HPRT) 5 prime region on the active and inactive X chromosome

    SciTech Connect

    Hornstra, I.K.; Yang, T.P. )

    1991-03-11

    In female placental mammals, one of the two X chromosome in each somatic cell is randomly inactivated during female embryogenesis as a mechanism for dosage compensation. Once a given X chromosome is inactivated, all mitotic progeny maintain the same X chromosome in the inactive state. DNA-protein interactions and DNA methylation are hypothesized to maintain this allele-specific system of differential gene expression. Ligation-mediated polymerase chain reaction (LMPCR) in vivo footprinting and genomic sequencing were used to study DNA-protein interactions and DNA-methylation within the 5{prime} region of the X-linked human HPRT gene on the active and inactive X chromosomes. In vivo footprint analysis reveals at least one DNA-protein interaction specific to the active HPRT allele in human male fibroblast cells and hamster-human hybrid cells containing only the active human X chromosome. In the region examined, all CpG dinucleotides are methylated on the inactive HPRT allele and unmethylated on the active X allele in hamster-human hybrid cells carrying either the inactive or active human X chromosome, respectively. Thus, DNA-methylation may be mediating the differential binding of sequence-specific DNA-binding proteins to the active or inactive HPRT alleles.

  6. Oral treatment with Cu(II)(atsm) increases mutant SOD1 in vivo but protects motor neurons and improves the phenotype of a transgenic mouse model of amyotrophic lateral sclerosis.

    PubMed

    Roberts, Blaine R; Lim, Nastasia K H; McAllum, Erin J; Donnelly, Paul S; Hare, Dominic J; Doble, Philip A; Turner, Bradley J; Price, Katherine A; Lim, Sin Chun; Paterson, Brett M; Hickey, James L; Rhoads, Timothy W; Williams, Jared R; Kanninen, Katja M; Hung, Lin W; Liddell, Jeffrey R; Grubman, Alexandra; Monty, Jean-Francois; Llanos, Roxana M; Kramer, David R; Mercer, Julian F B; Bush, Ashley I; Masters, Colin L; Duce, James A; Li, Qiao-Xin; Beckman, Joseph S; Barnham, Kevin J; White, Anthony R; Crouch, Peter J

    2014-06-01

    Mutations in the metallo-protein Cu/Zn-superoxide dismutase (SOD1) cause amyotrophic lateral sclerosis (ALS) in humans and an expression level-dependent phenotype in transgenic rodents. We show that oral treatment with the therapeutic agent diacetyl-bis(4-methylthiosemicarbazonato)copper(II) [Cu(II)(atsm)] increased the concentration of mutant SOD1 (SOD1G37R) in ALS model mice, but paradoxically improved locomotor function and survival of the mice. To determine why the mice with increased levels of mutant SOD1 had an improved phenotype, we analyzed tissues by mass spectrometry. These analyses revealed most SOD1 in the spinal cord tissue of the SOD1G37R mice was Cu deficient. Treating with Cu(II)(atsm) decreased the pool of Cu-deficient SOD1 and increased the pool of fully metallated (holo) SOD1. Tracking isotopically enriched (65)Cu(II)(atsm) confirmed the increase in holo-SOD1 involved transfer of Cu from Cu(II)(atsm) to SOD1, suggesting the improved locomotor function and survival of the Cu(II)(atsm)-treated SOD1G37R mice involved, at least in part, the ability of the compound to improve the Cu content of the mutant SOD1. This was supported by improved survival of SOD1G37R mice that expressed the human gene for the Cu uptake protein CTR1. Improving the metal content of mutant SOD1 in vivo with Cu(II)(atsm) did not decrease levels of misfolded SOD1. These outcomes indicate the metal content of SOD1 may be a greater determinant of the toxicity of the protein in mutant SOD1-associated forms of ALS than the mutations themselves. Improving the metal content of SOD1 therefore represents a valid therapeutic strategy for treating ALS caused by SOD1. PMID:24899723

  7. 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.

  8. Importance of β2-β3 Loop Motion for the Increased Binding and Decreased Selectivity of the ΔLL Mutant of the Human Papillomavirus Type 6 E2 Protein.

    PubMed

    Gray, Geoffrey M; van der Vaart, Arjan

    2015-08-11

    The binding affinity of the human papillomavirus type 6 E2 protein is strongly mediated by the sequence of the DNA linker region, with high affinity for the AATT linker and low affinity for the CCGG linker. When two terminal leucine residues are removed from the protein, the level of binding to both strands increases, but unequally, resulting in a significant decrease in selectivity for the AATT linker strand. To rationalize this behavior, we performed molecular dynamics simulations of the wild-type and mutant protein in the apo state and bound to DNA with high-affinity AATT and low-affinity CCGG linker strands. While no stable contacts were made between the β2-β3 loop and DNA in the wild type, this loop was repositioned in the mutant complexes and formed electrostatic contacts with the DNA backbone. More contacts were formed when the mutant was bound to the CCGG linker strand than to the AATT linker strand, resulting in a more favorable change in interaction energy for the CCGG strand. In addition, significant differences in correlated motions were found, which further explained the differences in binding. The simulations suggest that β2-β3 loop motions are responsible for the increased affinity and decreased selectivity of the mutant protein. PMID:26169609

  9. 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

  10. 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...

  11. Molecular and clonal analysis of in vivo hprt (hypoxanthine-guanine phosphoribosyl-transferase) mutations in human cells

    SciTech Connect

    Albertini, R.J.; O'Neill, J.P.; Nicklas, J.A.; Allegretta, M. . Genetics Lab.); Recio, L.; Skopek, T.R. )

    1989-08-08

    There is no longer doubt that gene mutations occur in vivo in human somatic cells, and that methods can be developed to detect, quantify and study them. Four assays are now available for such purpose; two detecting mutations that arise in bone marrow erythroid stem cells and two defining mutations that occur in T-lymphocytes. The red cell assays measure changes in mature red blood cells that involve either the blood group glycophorin-A locus or the hemoglobin loci; the lymphocyte assays score for genetic events at either the X-chromosomal hypoxanthine-guanine phosphoribosyl-transferase (hprt) locus. We describe here our attempts in studying in vivo gene mutations in human T-lymphocytes. 35 refs., 3 figs., 3 tabs.

  12. Mosaic mice with teratocarcinoma-derived mutant cells deficient in hypoxanthine phosphoribosyltransferase.

    PubMed

    Dewey, M J; Martin, D W; Martin, G R; Mintz, B

    1977-12-01

    Mutagenized stem cells of a cultured mouse teratocarcinoma cell line were selected for resistance to the purine base analog 6-thioguanine. Cells of a resistant clone were completely deficient in activity of the enzyme hypoxanthine phosphoribosyltransferase (HPRT, IMP:pyrophosphate phosphoribosyltransferase, EC 2.4.2.8), the same X-linked lesion as occurs in human Lesch-Nyhan disease. After microinjection into blastocysts of another genetic strain, the previously malignant cells successfully participated in normal embryogenesis and tumor-free, viable mosaic mice were obtained. Cells of tumor lineage were identified by strain markers in virtually all tissues of some individuals. Mature function of those cells was evident from their tissue-specific products (e.g., melanins, liver proteins). These mutagenized teratocarcinoma cells are therefore developmentally totipotent. Retention of the severe HPRT deficiency in the differentiated state was documented in extracts of mosaic tissues by depressed specific activity of the enzyme, and also by presence of unlabeled clones in autoradiographs of explanted cells incubated in [(3)H]hypoxanthine. Some mosaic individuals had mutant-strain cells in only one or a few tissues. Such animals may provide unique opportunities to identify the tissue sources of particular aspects of the complex disease syndrome. The tissue distribution of HPRT-deficient cells suggests that selection against them is particularly strong in blood of the mosaic mice, as is already known to be the case in human heterozygotes. This phenotypic parallelism supports the expectation that afflicted F(1) male mice that might be obtained from mutant germ cells can serve as a model of the human disease. PMID:271982

  13. 2-O-methylation of fucosyl residues of a rhizobial lipopolysaccharide is increased in response to host exudate and is eliminated in a symbiotically defective mutant.

    PubMed

    Noel, K Dale; Box, Jodie M; Bonne, Valerie J

    2004-03-01

    When Rhizobium etli CE3 was grown in the presence of Phaseolus vulgaris seed extracts containing anthocyanins, its lipopolysaccharide (LPS) sugar composition was changed in two ways: greatly decreased content of what is normally the terminal residue of the LPS, di-O-methylfucose, and a doubling of the 2-O-methylation of other fucose residues in the LPS O antigen. R. etli strain CE395 was isolated after Tn5 mutagenesis of strain CE3 by screening for mutant colonies that did not change antigenically in the presence of seed extract. The LPS of this strain completely lacked 2-O-methylfucose, regardless of whether anthocyanins were present during growth. The mutant gave only pseudonodules in association with P. vulgaris. Interpretation of this phenotype was complicated by a second LPS defect exhibited by the mutant: its LPS population had only about 50% of the normal amount of O-antigen-containing LPS (LPS I). The latter defect could be suppressed genetically such that the resulting strain (CE395 alpha 395) synthesized the normal amount of an LPS I that still lacked 2-O-methylfucose residues. Strain CE395 alpha 395 did not elicit pseudonodules but resulted in significantly slower nodule development, fewer nodules, and less nitrogenase activity than lps(+) strains. The relative symbiotic deficiency was more severe when seeds were planted and inoculated with bacteria before they germinated. These results support previous conclusions that the relative amount of LPS I on the bacterial surface is crucial in symbiosis, but LPS structural features, such as 2-O-methylation of fucose, also may facilitate symbiotic interactions. PMID:15006776

  14. 2-O-Methylation of Fucosyl Residues of a Rhizobial Lipopolysaccharide Is Increased in Response to Host Exudate and Is Eliminated in a Symbiotically Defective Mutant

    PubMed Central

    Noel, K. Dale; Box, Jodie M.; Bonne, Valerie J.

    2004-01-01

    When Rhizobium etli CE3 was grown in the presence of Phaseolus vulgaris seed extracts containing anthocyanins, its lipopolysaccharide (LPS) sugar composition was changed in two ways: greatly decreased content of what is normally the terminal residue of the LPS, di-O-methylfucose, and a doubling of the 2-O-methylation of other fucose residues in the LPS O antigen. R. etli strain CE395 was isolated after Tn5 mutagenesis of strain CE3 by screening for mutant colonies that did not change antigenically in the presence of seed extract. The LPS of this strain completely lacked 2-O-methylfucose, regardless of whether anthocyanins were present during growth. The mutant gave only pseudonodules in association with P. vulgaris. Interpretation of this phenotype was complicated by a second LPS defect exhibited by the mutant: its LPS population had only about 50% of the normal amount of O-antigen-containing LPS (LPS I). The latter defect could be suppressed genetically such that the resulting strain (CE395α395) synthesized the normal amount of an LPS I that still lacked 2-O-methylfucose residues. Strain CE395α395 did not elicit pseudonodules but resulted in significantly slower nodule development, fewer nodules, and less nitrogenase activity than lps+ strains. The relative symbiotic deficiency was more severe when seeds were planted and inoculated with bacteria before they germinated. These results support previous conclusions that the relative amount of LPS I on the bacterial surface is crucial in symbiosis, but LPS structural features, such as 2-O-methylation of fucose, also may facilitate symbiotic interactions. PMID:15006776

  15. Infection with Cytotoxic T-Lymphocyte Escape Mutants Results in Increased Mortality and Growth Retardation in Mice Infected with a Neurotropic Coronavirus

    PubMed Central

    Pewe, Lecia; Xue, Shurong; Perlman, Stanley

    1998-01-01

    C57BL/6 mice infected with mouse hepatitis virus strain JHM (MHV-JHM) develop a chronic demyelinating encephalomyelitis several weeks after inoculation. Previously, we showed that mutations in the immunodominant CD8 T-cell epitope (S-510-518) could be detected in nearly all samples of RNA and virus isolated from these mice. These mutations abrogated recognition by T cells harvested from the central nervous systems of infected mice in direct ex vivo cytotoxicity assays. These results suggested that cytotoxic T-lymphocyte (CTL) escape mutants contributed to virus amplification and the development of clinical disease in mice infected with wild-type virus. In the present study, the importance of these mutations was further evaluated by infecting naive mice with MHV-JHM variants isolated from infected mice and in which epitope S-510-518 was mutated. Compared to mice infected with wild-type virus, variant virus-infected animals showed higher mortality and morbidity manifested by decreased weight gain and neurological signs. Although a delay in the kinetics of virus clearance has been demonstrated in previous studies of CTL escape mutants, this is the first illustration of significant changes in clinical disease resulting from infection with viruses able to evade the CD8 T-cell immune response. PMID:9621053

  16. 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.

  17. 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

  18. 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. PMID:25870145

  19. 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

  20. Mouse model for somatic mutation at the HPRT (hypoxanthine phosphoribosyl-transferase) gene: Molecular and cellular analyses

    SciTech Connect

    Burkhart-Schultz, K.; Strout, C.L.; Jones, I.M.

    1989-07-11

    Our goal is to use the mouse to model the organismal, cellular and molecular factors that affect somatic mutagenesis in vivo. A fundamental tenet of genetic toxicology is that the principles of mutagenesis identified in one system can be used to predict the principles of mutagenesis in another system. The validity of this tenet depends upon the comparability of the systems involved. To begin to achieve an understanding of somatic mutagenesis in vivo, we have been studying mutations that occur in the hypoxanthine phosphoribosyl-transferase (HPRT) gene of lymphocytes of mice. Our in vivo model for somatic mutation allows us to analyse factors that affect somatic mutation. Having chosen the mouse, we are working with cells in which the karyotype is normal, and metabolic and DNA repair capacity are defined by the mouse strain chosen. At the organismal level, we can vary sex, age, the exposure history, and the tissue source of cells analysed. (All studies reported here have, however, used male mice.) At the cellular level, T lymphocytes and their precursors are the targets and reporters of mutation. 26 refs., 1 fig., 1 tab.

  1. 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.

  2. 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. PMID:26069184

  3. 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

  4. Changes in Expression of Virulence Mechanisms in Three Related Salmonella Typhimurium Mutants with Increasing Multi-Drug Resistance Properties, as Determined by Microarray Analysis

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Salmonella Typhimurium is a common cause of Salmonellosis and has been associated with multi-drug resistance. Previously, the wild-type strain (Salmonella Typhimurium ATCC 14028) was exposed to increasing concentrations of nalidixic acid to derive naturally occurring drug resistant isolates. Three d...

  5. Enzymatic Dysfunction of Mitochondrial Complex I of the Candida albicans goa1 Mutant Is Associated with Increased Reactive Oxidants and Cell Death ▿

    PubMed Central

    Li, Dongmei; Chen, Hui; Florentino, Abigail; Alex, Deepu; Sikorski, Patricia; Fonzi, William A.; Calderone, Richard

    2011-01-01

    We have previously shown that deletion of GOA1 (growth and oxidant adaptation) of Candida albicans results in a loss of mitochondrial membrane potential, ATP synthesis, increased sensitivity to oxidants and killing by human neutrophils, and avirulence in a systemic model of candidiasis. We established that translocation of Goa1p to mitochondria occurred during peroxide stress. In this report, we show that the goa1Δ (GOA31), compared to the wild type (WT) and a gene-reconstituted (GOA32) strain, exhibits sensitivity to inhibitors of the classical respiratory chain (CRC), including especially rotenone (complex I [CI]) and salicylhydroxamic acid (SHAM), an inhibitor of the alternative oxidase pathway (AOX), while potassium cyanide (KCN; CIV) causes a partial inhibition of respiration. In the presence of SHAM, however, GOA31 has an enhanced respiration, which we attribute to the parallel respiratory (PAR) pathway and alternative NADH dehydrogenases. Interestingly, deletion of GOA1 also results in a decrease in transcription of the alternative oxidase gene AOX1 in untreated cells as well as negligible AOX1 and AOX2 transcription in peroxide-treated cells. To explain the rotenone sensitivity, we measured enzyme activities of complexes I to IV (CI to CIV) and observed a major loss of CI activity in GOA31 but not in control strains. Enzymatic data of CI were supported by blue native polyacrylamide gel electrophoresis (BN-PAGE) experiments which demonstrated less CI protein and reduced enzyme activity. The consequence of a defective CI in GOA31 is an increase in reactive oxidant species (ROS), loss of chronological aging, and programmed cell death ([PCD] apoptosis) in vitro compared to control strains. The increase in PCD was indicated by an increase in caspase activity and DNA fragmentation in GOA31. Thus, GOA1 is required for a functional CI and partially for the AOX pathway; loss of GOA1 compromises cell survival. Further, the loss of chronological aging is new to

  6. Maternal diet supplementation with methyl donors and increased parity affect the incidence of craniofacial defects in the offspring of twisted gastrulation mutant mice.

    PubMed

    Billington, Charles J; Schmidt, Brian; Zhang, Lei; Hodges, James S; Georgieff, Michael K; Schotta, Gunnar; Gopalakrishnan, Rajaram; Petryk, Anna

    2013-03-01

    Diets rich in methyl-donating compounds, including folate, can provide protection against neural tube defects, but their role in preventing craniofacial defects is less clear. Mice deficient in Twisted gastrulation (TWSG1), an extracellular modulator of bone morphogenetic protein signaling, manifest both midline facial defects and jaw defects, allowing study of the effects of methyl donors on various craniofacial defects in an experimentally tractable animal model. The goal of this study was to examine the effects of maternal dietary supplementation with methyl donors on the incidence and type of craniofacial defects among Twsg1(-/-) offspring. Nulliparous and primiparous female mice were fed an NIH31 standard diet (control) or a methyl donor supplemented (MDS) diet (folate, vitamin B-12, betaine, and choline). Observed defects in the pups were divided into those derived mostly from the first branchial arch (BA1) (micrognathia, agnathia, cleft palate) and midline facial defects in the holoprosencephaly spectrum (cyclopia, proboscis, and anterior truncation). In the first pregnancy, offspring of mice fed the MDS diet had lower incidence of BA1-derived defects (12.8% in MDS vs. 32.5% in control; P = 0.02) but similar incidence of midline facial defects (6.4% in MDS vs. 5.2% in control; P = 1.0). Increased maternal parity was independently associated with increased incidence of craniofacial defects after adjusting for diet (from 37.7 to 59.5% in control, P = 0.04 and from 19.1 to 45.3% in MDS, P = 0.045). In conclusion, methyl donor supplementation shows protective effects against jaw defects, but not midline facial defects, and increased parity can be a risk factor for some craniofacial defects. PMID:23343680

  7. 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

    Ngo, Ha Thi; Hetland, Ragna Bogen; Nygaard, Unni Cecilie; Steffensen, Inger-Lise

    2015-01-01

    We have studied how spontaneous or carcinogen-induced intestinal tumorigenesis was affected by genetic or diet-induced obesity in C57BL/6J-ApcMin/+ X C57BL/6J-Lepob/+ 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 ApcMin/+ 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

  8. Increased Ca2+ sensitivity of the ryanodine receptor mutant RyR2R4496C underlies catecholaminergic polymorphic ventricular tachycardia.

    PubMed

    Fernández-Velasco, María; Rueda, Angélica; Rizzi, Nicoletta; Benitah, Jean-Pierre; Colombi, Barbara; Napolitano, Carlo; Priori, Silvia G; Richard, Sylvain; Gómez, Ana María

    2009-01-30

    Cardiac ryanodine receptor (RyR2) mutations are associated with autosomal dominant catecholaminergic polymorphic ventricular tachycardia, suggesting that alterations in Ca(2+) handling underlie this disease. Here we analyze the underlying Ca(2+) release defect that leads to arrhythmia in cardiomyocytes isolated from heterozygous knock-in mice carrying the RyR2(R4496C) mutation. RyR2(R4496C-/-) littermates (wild type) were used as controls. [Ca(2+)](i) transients were obtained by field stimulation in fluo-3-loaded cardiomyocytes and viewed using confocal microscopy. In our basal recording conditions (2-Hz stimulation rate), [Ca(2+)](i) transients and sarcoplasmic reticulum Ca(2+) load were similar in wild-type and RyR2(R4496C) cells. However, paced RyR2(R4496C) ventricular myocytes presented abnormal Ca(2+) release during the diastolic period, viewed as Ca(2+) waves, consistent with the occurrence of delayed afterdepolarizations. The occurrence of this abnormal Ca(2+) release was enhanced at faster stimulation rates and by beta-adrenergic stimulation, which also induced triggered activity. Spontaneous Ca(2+) sparks were more frequent in RyR2(R4496C) myocytes, indicating increased RyR2(R4496C) activity. When permeabilized cells were exposed to different cytosolic [Ca(2+)](i), RyR2(R4496C) showed a dramatic increase in Ca(2+) sensitivity. Isoproterenol increased [Ca(2+)](i) transient amplitude and Ca(2+) spark frequency to the same extent in wild-type and RyR2(R4496C) cells, indicating that the beta-adrenergic sensitivity of RyR2(R4496C) cells remained unaltered. This effect was independent of protein expression variations because no difference was found in the total or phosphorylated RyR2 expression levels. In conclusion, the arrhythmogenic potential of the RyR2(R4496C) mutation is attributable to the increased Ca(2+) sensitivity of RyR2(R4496C), which induces diastolic Ca(2+) release and lowers the threshold for triggered activity. PMID:19096022

  9. 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

    2014-10-01

    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. PMID:25017139

  10. 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. PMID:27154328

  11. The gain-of-function enhancement of IP3-receptor channel gating by familial Alzheimer's disease-linked presenilin mutants increases the open probability of mitochondrial permeability transition pore.

    PubMed

    Toglia, Patrick; Ullah, Ghanim

    2016-07-01

    Mutants in presenilins (PS1 or PS2) are the major cause of familial Alzheimer's disease (FAD). They affect intracellular Ca(2+) homeostasis by increasing the open probability (Po) of inositol 1,4,5-trisposphate (IP3) receptor (IP3R) Ca(2+) release channel located on the endoplasmic reticulum (ER) leading to exaggerated Ca(2+) release into a cytoplasmic microdomain formed by neighboring cluster of a few IP3R channels and mitochondrial Ca(2+) uniporter (MCU). Ca(2+) concentration in the microdomain ( [Formula: see text] ) depends on the distance between the cluster and MCU (r); the number of IP3R in the cluster releasing Ca(2+) to the cytoplasm ( [Formula: see text] ), and Po of IP3R. Using experimental whole-cell IP3R-mediated cytosolic Ca(2+) data, in conjunction with a computational model of cell bioenergetics, a data-driven Markov chain model for IP3R gating, and a model for the dynamics of the mitochondrial permeability transition pore (PTP), we explore differences in mitochondrial Ca(2+) uptake in cells expressing wild type (PS1-WT) and FAD-causing mutant (PS1-M146L) PS. We find that increased mitochondrial [Formula: see text] due to the gain-of-function enhancement of IP3R channels in the cells expressing PS1-M146L leads to the opening of PTP in high conductance state (PTPh), where the latency of opening is inversely correlated with r and proportional to [Formula: see text] . Furthermore, we observe diminished inner mitochondrial membrane potential (ΔΨm), [NADH], [Formula: see text] , and [ATP] when PTP opens. Additionally, we explore how parameters such as the pH gradient, inorganic phosphate concentration, and the rate of the Na(+)/Ca(2+)-exchanger affect the latency of PTP to open in PTPh. PMID:27184076

  12. Synaptic gain-of-function effects of mutant Cav2.1 channels in a mouse model of familial hemiplegic migraine are due to increased basal [Ca2+]i.

    PubMed

    Di Guilmi, Mariano N; Wang, Tiantian; Inchauspe, Carlota Gonzalez; Forsythe, Ian D; Ferrari, Michel D; van den Maagdenberg, Arn M J M; Borst, J Gerard G; Uchitel, Osvaldo D

    2014-05-21

    Specific missense mutations in the CACNA1A gene, which encodes a subunit of voltage-gated CaV2.1 channels, are associated with familial hemiplegic migraine type 1 (FHM1), a rare monogenic subtype of common migraine with aura. We used transgenic knock-in (KI) mice harboring the human pathogenic FHM1 mutation S218L to study presynaptic Ca(2+) currents, EPSCs, and in vivo activity at the calyx of Held synapse. Whole-cell patch-clamp recordings of presynaptic terminals from S218L KI mice showed a strong shift of the calcium current I-V curve to more negative potentials, leading to an increase in basal [Ca(2+)]i, increased levels of spontaneous transmitter release, faster recovery from synaptic depression, and enhanced synaptic strength despite smaller action-potential-elicited Ca(2+) currents. The gain-of-function of transmitter release of the S218L mutant was reproduced in vivo, including evidence for an increased release probability, demonstrating its relevance for glutamatergic transmission. This synaptic phenotype may explain the misbalance between excitation and inhibition in neuronal circuits resulting in a persistent hyperexcitability state and other migraine-relevant mechanisms such as an increased susceptibility to cortical spreading depression. PMID:24849341

  13. Allele Specific p53 Mutant Reactivation

    PubMed Central

    Yu, Xin; Vazquez, Alexei; Levine, Arnold J.; Carpizo, Darren R.

    2012-01-01

    Summary Rescuing the function of mutant p53 protein is an attractive cancer therapeutic strategy. Using the NCI anticancer drug screen data, we identified two compounds from the thiosemicarbazone family that manifest increased growth inhibitory activity in mutant p53 cells, particularly for the p53R175 mutant. Mechanistic studies reveal that NSC319726 restores WT structure and function to the p53R175 mutant. This compound kills p53R172H knock-in mice with extensive apoptosis and inhibits xenograft tumor growth in a 175-allele specific mutant p53 dependent manner. This activity depends upon the zinc ion chelating properties of the compound as well as redox changes. These data identify NSC319726 as a p53R175 mutant reactivator and as a lead compound for p53 targeted drug development. PMID:22624712

  14. Abnormal lignin in a loblolly pine mutant

    SciTech Connect

    Ralph, J.; MacKay, J.J.; Hatfield, R.D.

    1997-07-11

    Novel lignin is formed in a mutant loblolly pine (Pinus taeda L.) severely depleted in cinnamyl alcohol dehydrogenase (E.C. 1.1.1.195), which converts coniferaldehyde to coniferyl alcohol, the primary lignin precursor in pines. Dihydroconiferyl alcohol, a monomer not normally associated with the lignin biosynthetic pathway, is the major component of the mutant`s lignin, accounting for {approximately}30 percent (versus {approximately}3 percent in normal pine) of the units. The level of aldehydes, including new 2-methoxybenzaldehydes, is also increased. The mutant pines grew normally indicating that, even within a species, extensive variations in lignin composition need not disrupt the essential functions of lignin.

  15. 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.

  16. Mutant p53: One, No One, and One Hundred Thousand

    PubMed Central

    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. PMID:26734571

  17. Arabidopsis mutants impaired in cosuppression.

    PubMed Central

    Elmayan, T; Balzergue, S; Béon, F; Bourdon, V; Daubremet, J; Guénet, Y; Mourrain, P; Palauqui, J C; Vernhettes, S; Vialle, T; Wostrikoff, K; Vaucheret, H

    1998-01-01

    Post-transcriptional gene silencing (cosuppression) results in the degradation of RNA after transcription. A transgenic Arabidopsis line showing post-transcriptional silencing of a 35S-uidA transgene and uidA-specific methylation was mutagenized using ethyl methanesulfonate. Six independent plants were isolated in which uidA mRNA accumulation and beta-glucuronidase activity were increased up to 3500-fold, whereas the transcription rate of the 35S-uidA transgene was increased only up to threefold. These plants each carried a recessive monogenic mutation that is responsible for the release of silencing. These mutations defined two genetic loci, called sgs1 and sgs2 (for suppressor of gene silencing). Transgene methylation was distinctly modified in sgs1 and sgs2 mutants. However, methylation of centromeric repeats was not affected, indicating that sgs mutants differ from ddm (for decrease in DNA methylation) and som (for somniferous) mutants. Indeed, unlike ddm and som mutations, sgs mutations were not able to release transcriptional silencing of a 35S-hpt transgene. Conversely, both sgs1 and sgs2 mutations were able to release cosuppression of host Nia genes and 35S-Nia2 transgenes. These results therefore indicate that sgs mutations act in trans to impede specifically transgene-induced post-transcriptional gene silencing. PMID:9761800

  18. Decrease in Leaf Sucrose Synthesis Leads to Increased Leaf Starch Turnover and Decreased RuBP-limited Photosynthesis But Not Rubisco-limited Photosynthesis in Arabidopsis Null Mutants of SPSA1

    Technology Transfer Automated Retrieval System (TEKTRAN)

    SPS (Sucrose phosphate synthase) isoforms from dicots cluster into families A, B and C. In this study, we investigated the individual effect of null mutations of each of the four SPS genes in Arabidopsis (spsa1, spsa2, spsb and spsc) on photosynthesis and carbon partitioning. Null mutants spsa1 and ...

  19. A link between increased transforming activity of lymphoma-derived MYC mutant alleles, their defective regulation by p107, and altered phosphorylation of the c-Myc transactivation domain.

    PubMed Central

    Hoang, A T; Lutterbach, B; Lewis, B C; Yano, T; Chou, T Y; Barrett, J F; Raffeld, M; Hann, S R; Dang, C V

    1995-01-01

    The c-Myc protein is a transcription factor with an N-terminal transcriptional regulatory domain and C-terminal oligomerization and DNA-binding motifs. Previous studies have demonstrated that p107, a protein related to the retinoblastoma protein, binds to the c-Myc transcriptional activation domain and suppresses its activity. We sought to characterize the transforming activity and transcriptional properties of lymphoma-derived mutant MYC alleles. Alleles encoding c-Myc proteins with missense mutations in the transcriptional regulatory domain were more potent than wild-type c-Myc in transforming rodent fibroblasts. Although the mutant c-Myc proteins retained their binding to p107 in in vitro and in vivo assays, p107 failed to suppress their transcriptional activation activities. Many of the lymphoma-derived MYC alleles contain missense mutations that result in substitution for the threonine at codon 58 or affect sequences flanking this amino acid. We observed that in vivo phosphorylation of Thr-58 was absent in a lymphoma cell line with a mutant MYC allele containing a missense mutation flanking codon 58. Our in vitro studies suggest that phosphorylation of Thr-58 in wild-type c-Myc was dependent on cyclin A and required prior phosphorylation of Ser-62 by a p107-cyclin A-CDK complex. In contrast, Thr-58 remained unphosphorylated in two representative mutant c-Myc transactivation domains in vitro. Our studies suggest that missense mutations in MYC may be selected for during lymphomagenesis, because the mutant MYC proteins have altered functional interactions with p107 protein complexes and fail to be phosphorylated at Thr-58. PMID:7623799

  20. 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.

  1. Targeting Oncogenic Mutant p53 for Cancer Therapy

    PubMed Central

    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. PMID:26732534

  2. Escherichia coli mutants deficient in deoxyuridine triphosphatase.

    PubMed Central

    Hochhauser, S J; Weiss, B

    1978-01-01

    Mutants deficient in deoxyuridine triphosphatase (dUTPase) were identified by enzyme assays of randomly chosen heavily mutagenized clones. Five mutants of independent origin were obtained. One mutant produced a thermolabile enzyme, and it was presumed to have a mutation in the structural gene for dUTPase, designated dut. The most deficient mutant had the following associated phenotypes: less than 1% of parental dUTPase activity, prolonged generation time, increased sensitivity to 5'-fluorodeoxyuridine, increased rate of spontaneous mutation, increased rate of recombination (hyper-Rec), an inhibition of growth in the presence of 2 mM uracil, and a decreased ability to support the growth of phage P1 (but not T4 or lambda). This mutation also appeared to be incompatible with pyrE mutations. A revertant selected by its faster growth had regained dUTPase activity and lost its hyper-Rec phenotype. Many of the properties of the dut mutants are compatible with their presumed increased incorporation of uracil into DNA and the subsequent transient breakage of the DNA by excision repair. PMID:148458

  3. Arabidopsis mutants with a reduced seed dormancy.

    PubMed Central

    Léon-Kloosterziel, K M; van de Bunt, G A; Zeevaart, J A; Koornneef, M

    1996-01-01

    The development of seed dormancy is an aspect of seed maturation, the last stage of seed development. To isolate mutants of Arabidopsis thaliana that are affected in this process, we selected directly for the absence of dormancy among freshly harvested M2 seeds. The screen yielded two mutants exhibiting a reduced dormancy, rdo1 and rdo2, that are specifically affected in dormancy determined by the embryo. The rdo1 and rdo2 mutants show normal levels of abscisic acid and the same sensitivity to abscisic acid, ethylene, auxin, and cytokinin as the wild type. The rdo2 mutant but not the rdo1 mutant has a reduced sensitivity to the gibberellin biosynthesis inhibitor tetcyclacis. Double-mutant analysis suggested that the RDO1 and RDO2 genes are involved in separate pathways leading to the development of dormancy. We assume that the RDO2 gene controls a step in the induction of dormancy that is most likely induced by abscisic acid and is expressed as an increase of the gibberellin requirement for germination. PMID:8587986

  4. Increased Prevalence of Mutant Allele Pfdhps 437G and Pfdhfr Triple Mutation in Plasmodium falciparum Isolates from a Rural Area of Gabon, Three Years after the Change of Malaria Treatment Policy

    PubMed Central

    Ndong Ngomo, Jacques-Mari; Mawili-Mboumba, Denise Patricia; M'Bondoukwe, Noé Patrick; Nikiéma Ndong Ella, Rosalie; Bouyou Akotet, Marielle Karine

    2016-01-01

    In Gabon, sulfadoxine-pyrimethamine (SP) is recommended for intermittent preventive treatment during pregnancy (IPTp-SP) and for uncomplicated malaria treatment through ACTs drug. P. falciparum strains resistant to SP are frequent in areas where this drug is highly used and is associated with the occurrence of mutations on Plasmodium falciparum dihydrofolate reductase (Pfdhfr) and dihydropteroate synthetase (Pfdhps) genes. The aim of the study was to compare the proportion of mutations on Pfdhfr and Pfdhps genes in isolates collected at Oyem in northern Gabon, in 2005 at the time of IPTp-SP introduction and three years later. Point mutations were analyzed by nested PCR-RFLP method. Among 91 isolates, more than 90% carried Pfdhfr 108N and Pfdhfr 59R alleles. Frequencies of Pfdhfr 51I (98%) and Pfdhps 437G (67.7%) mutant alleles were higher in 2008. Mutations at codons 164, 540, and 581 were not detected. The proportion of the triple Pfdhfr mutation and quadruple mutation including A437G was high: 91.9% in 2008 and 64.8% in 2008, respectively. The present study highlights an elevated frequency of Pfdhfr and Pfdhps mutant alleles, although quintuple mutations were not found in north Gabon. These data suggest the need of a continuous monitoring of SP resistance in Gabon. PMID:27190671

  5. Increased Prevalence of Mutant Allele Pfdhps 437G and Pfdhfr Triple Mutation in Plasmodium falciparum Isolates from a Rural Area of Gabon, Three Years after the Change of Malaria Treatment Policy.

    PubMed

    Ndong Ngomo, Jacques-Mari; Mawili-Mboumba, Denise Patricia; M'Bondoukwe, Noé Patrick; Nikiéma Ndong Ella, Rosalie; Bouyou Akotet, Marielle Karine

    2016-01-01

    In Gabon, sulfadoxine-pyrimethamine (SP) is recommended for intermittent preventive treatment during pregnancy (IPTp-SP) and for uncomplicated malaria treatment through ACTs drug. P. falciparum strains resistant to SP are frequent in areas where this drug is highly used and is associated with the occurrence of mutations on Plasmodium falciparum dihydrofolate reductase (Pfdhfr) and dihydropteroate synthetase (Pfdhps) genes. The aim of the study was to compare the proportion of mutations on Pfdhfr and Pfdhps genes in isolates collected at Oyem in northern Gabon, in 2005 at the time of IPTp-SP introduction and three years later. Point mutations were analyzed by nested PCR-RFLP method. Among 91 isolates, more than 90% carried Pfdhfr 108N and Pfdhfr 59R alleles. Frequencies of Pfdhfr 51I (98%) and Pfdhps 437G (67.7%) mutant alleles were higher in 2008. Mutations at codons 164, 540, and 581 were not detected. The proportion of the triple Pfdhfr mutation and quadruple mutation including A437G was high: 91.9% in 2008 and 64.8% in 2008, respectively. The present study highlights an elevated frequency of Pfdhfr and Pfdhps mutant alleles, although quintuple mutations were not found in north Gabon. These data suggest the need of a continuous monitoring of SP resistance in Gabon. PMID:27190671

  6. Connexin Mutants and Cataracts

    PubMed Central

    Beyer, Eric C.; Ebihara, Lisa; Berthoud, Viviana M.

    2013-01-01

    The lens is a multicellular, but avascular tissue that must stay transparent to allow normal transmission of light and focusing of it on the retina. Damage to lens cells and/or proteins can cause cataracts, opacities that disrupt these processes. The normal survival of the lens is facilitated by an extensive network of gap junctions formed predominantly of connexin46 and connexin50. Mutations of the genes that encode these connexins (GJA3 and GJA8) have been identified and linked to inheritance of cataracts in human families and mouse lines. In vitro expression studies of several of these mutants have shown that they exhibit abnormalities that may lead to disease. Many of the mutants reduce or modify intercellular communication due to channel alterations (including loss of function or altered gating) or due to impaired cellular trafficking which reduces the number of gap junction channels within the plasma membrane. However, the abnormalities detected in studies of other mutants suggest that they cause cataracts through other mechanisms including gain of hemichannel function (leading to cell injury and death) and formation of cytoplasmic accumulations (that may act as light scattering particles). These observations and the anticipated results of ongoing studies should elucidate the mechanisms of cataract development due to mutations of lens connexins and abnormalities of other lens proteins. They may also contribute to our understanding of the mechanisms of disease due to connexin mutations in other tissues. PMID:23596416

  7. An ethA-ethR-Deficient Mycobacterium bovis BCG Mutant Displays Increased Adherence to Mammalian Cells and Greater Persistence In Vivo, Which Correlate with Altered Mycolic Acid Composition

    PubMed Central

    Ang, Michelle Lay Teng; Siti, Zarina Zainul Rahim; Shui, Guanghou; Dianišková, Petronela; Madacki, Jan; Lin, Wenwei; Koh, Vanessa Hui Qi; Martinez Gomez, Julia Maria; Sudarkodi, Sukumar; Bendt, Anne; Wenk, Markus; Mikušová, Katarína; Korduláková, Jana; Pethe, Kevin

    2014-01-01

    Tuberculosis remains a major worldwide epidemic because of its sole etiological agent, Mycobacterium tuberculosis. Ethionamide (ETH) is one of the major antitubercular drugs used to treat infections with multidrug-resistant M. tuberculosis strains. ETH is a prodrug that requires activation within the mycobacterial cell; its bioactivation involves the ethA-ethR locus, which encodes the monooxygenase EthA, while EthR is a transcriptional regulator that binds to the intergenic promoter region of the ethA-ethR locus. While most studies have focused on the role of EthA-EthR in ETH bioactivation, its physiological role in mycobacteria has remained elusive, although a role in bacterial cell detoxification has been proposed. Moreover, the importance of EthA-EthR in vivo has never been reported on. Here we constructed and characterized an EthA-EthR-deficient mutant of Mycobacterium bovis BCG. Our results indicate that absence of the ethA-ethR locus led to greater persistence of M. bovis BCG in the mouse model of mycobacterial infection, which correlated with greater adherence to mammalian cells. Furthermore, analysis of cell wall lipid composition by thin-layer chromatography and mass spectrometry revealed differences between the ethA-ethR KO mutant and the parental strain in the relative amounts of α- and keto-mycolates. Therefore, we propose here that M. bovis BCG ethA-ethR is involved in the cell wall-bound mycolate profile, which impacts mycobacterial adherence properties and in vivo persistence. This study thus provides some experimental clues to the possible physiological role of ethA-ethR and proposes that this locus is a novel factor involved in the modulation of mycobacterial virulence. PMID:24566628

  8. 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. PMID:9007229

  9. Germinal HPRT splice donor site mutation results in multiple RNA splicing products in T-lymphocyte cultures

    SciTech Connect

    Hunter, T.C.; Albertini, R.J.; O`Neill, J.P.

    1996-03-01

    Fanconi anemia (FA) is an autosomal recessive disease characterized by birth defects, progressive bone marrow failure and increased risk for leukemia. FA cells display chromosome breakage and increased cell killing in response to DNA crosslinking agents. At least 5 genes have been defined by cell complementation studies, but only one of these, FAC has been cloned to date. Efforts to map and isolate new FA genes by functional complementation have been hampered by the lack of immortalized FA fibroblast cell lines. Here we report the use of a novel immortalization strategy to create 4 new immortalized FA fibroblast lines, including one from the rare complementation group D. 16 refs., 3 tabs.

  10. 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.

  11. 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.

  12. 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.

  13. 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.

  14. Sorghum Brown Midrib Mutants, Tools to Improve Biomass for Biofuels

    Technology Transfer Automated Retrieval System (TEKTRAN)

    To improve sorghum for cellulosic bioenergy uses, brown midrib mutants are being investigated for their ability to increase the conversion efficiency of biomass. brown midrib 6 and 12 (bmr6 and 12) mutants affect monolignol biosynthesis resulting in reduced lignin content and altered lignin composi...

  15. 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.

  16. 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.

  17. Escherichia coli mutants deficient in exonuclease VII.

    PubMed Central

    Chase, J W; Richardson, C C

    1977-01-01

    Mutants of Escherichia coli having reduced levels of exonuclease VII activity have been isolated by a mass screening procedure. Nine mutants, five of which are known to be of independent origin, were obtained and designated xse. The defects in these strains lie at two or more loci. One of these loci, xseA, lies in the interval between purG and purC; it is 93 to 97% co-transducible with guaA. The order of the genes in this region is purG-xseA guaA,B-purC. The available data do not allow xseA to be ordered with respect to guaA,B. Exonuclease VII purified from E. coli KLC3 xseA3 is more heat labile than exonuclease VII purified from the parent, E. coli PA610 xse+. Therefore, xseA is the structural gene for exonuclease VII. Mutants with defects in the xseA gene show increased sensitivity to nalidixic acid and have an abnormally high frequency of recombination (hyper-Rec phenotype) as measured by the procedure of Konrad and Lehlman (1974). The hyper-Rec character of xseA strains is approximately one-half that of the polAex1 mutant defective in the 5' leads to 3' hydrolytic activity of deoxyribonucleic acid polymerase I. The double mutant, polAex1 xseA7, is twice as hyper-Rec as the polAex1 mutant alone. The xseA- strains are slightly more sensitive to ultraviolet irradiation than the parent strain. Bacteriophages T7, fd, and lambdared grow normally in xseA- strains. Images PMID:320198

  18. 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

  19. 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.

  20. 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

  1. 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

  2. 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

  3. 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. PMID:27610363

  4. Nonchemotactic Mutants of Escherichia coli

    PubMed Central

    Armstrong, John B.; Adler, Julius; Dahl, Margaret M.

    1967-01-01

    We have isolated 40 mutants of Escherichia coli which are nonchemotactic as judged by their failure to swarm on semisolid tryptone plates or to make bands in capillary tubes containing tryptone broth. All the mutants have normal flagella, a fact shown by their shape and reaction with antiflagella serum. All are fully motile under the microscope and all are sensitive to the phage chi. Unlike its parent, one of the mutants, studied in greater detail, failed to show chemotaxis toward oxygen, glucose, serine, threonine, or aspartic acid. The failure to exhibit chemotaxis does not result from a failure to use the chemicals. The swimming of this mutant was shown to be random. The growth rate was normal under several conditions, and the growth requirements were unchanged. Images PMID:5335897

  5. Motility mutants of Dictyostelium discoideum

    PubMed Central

    1982-01-01

    We describe six motility mutants of Dictyostelium discoideum in this report. They were identified among a group of temperature-sensitive growth (Tsg) mutants that had been previously isolated using an enrichment for phagocytosis-defective cells. The Tsg mutants were screened for their ability to produce tracks on gold-coated cover slips, and several strains were found that were temperature-sensitive for migration in this assay. Analysis of spontaneous Tsg+ revertants of 10 migration-defective strains identified six strains that co-reverted the Tsg and track formation phenotypes. Characterization of these six strains indicated that they were defective at restrictive temperature in track formation, phagocytosis of bacteria, and pseudopodial and filopodial activity, while retaining normal rates of oxygen consumption and viability. Because they had lost this group of motile capabilities, these strains were designated motility mutants. The Tsg+ revertants of these mutants, which coordinately recovered all of the motile activities, were found at frequencies consistent with single genetic events. Analysis of the motility mutants and their revertants suggests a relationship between the motility mutations in some of these strains and genes affecting axenic growth. PMID:7118999

  6. 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

  7. 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.

  8. 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

    2016-01-01

    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. PMID:26725113

  9. 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. PMID:23987991

  10. New Salmonella typhimurium mutants with altered outer membrane permeability.

    PubMed Central

    Sukupolvi, S; Vaara, M; Helander, I M; Viljanen, P; Mäkelä, P H

    1984-01-01

    We describe three new classes of Salmonella typhimurium mutants with increased sensitivity to hydrophobic agents. In contrast to many previously described mutants, the phage sensitivity pattern of these mutants did not give any indication of defective lipopolysaccharide. Furthermore, they had no detectable changes in their phospholipid or outer membrane protein composition, and their growth rate and cell morphology were normal. Class B mutants were nearly as sensitive to novobiocin, fusidic acid, erythromycin, rifampin, and clindamycin as are deep rough (heptoseless) mutants; in addition they were sensitive to methicillin, penicillin (to which heptoseless mutants are resistant), gentian violet, and anionic and cationic detergents. Class A and C mutants had less sensitive, but characteristic phenotypes. None of the three classes were sensitive to serum bactericidal action. The class B mutation mapped between map positions 7 and 11 on the S. typhimurium chromosome, and the class C mutation mapped between positions 5 and 7. The map position for the class A mutation remained undefined, but it was separate from the class B and C mutations and, like those, did not correspond to any gene loci known to participate in the synthesis of major outer membrane constituents. Images PMID:6378889

  11. Enhanced Symbiotic Performance by Rhizobium tropici Glycogen Synthase Mutants

    PubMed Central

    Marroquí, Silvia; Zorreguieta, Angeles; Santamaría, Carmen; Temprano, Francisco; Soberón, Mario; Megías, Manuel; Downie, J. Allan

    2001-01-01

    We isolated a Tn5-induced Rhizobium tropici mutant that has enhanced capacity to oxidize N,N-dimethyl-p-phenylendiamine (DMPD) and therefore has enhanced respiration via cytochrome oxidase. The mutant had increased levels of the cytochromes c1 and CycM and a small increase in the amount of cytochrome aa3. In plant tests, the mutant increased the dry weight of Phaseolus vulgaris plants by 20 to 38% compared with the control strain, thus showing significantly enhanced symbiotic performance. The predicted product of the mutated gene is homologous to glycogen synthases from several bacteria, and the mutant lacked glycogen. The DNA sequence of the adjacent gene region revealed six genes predicted to encode products homologous to the following gene products from Escherichia coli: glycogen phosphorylase (glgP), glycogen branching enzyme (glgB), ADP glucose pyrophosphorylase (glgC), glycogen synthase (glgA), phosphoglucomutase (pgm), and glycogen debranching enzyme (glgX). All six genes are transcribed in the same direction, and analysis with lacZ gene fusions suggests that the first five genes are organized in one operon, although pgm appears to have an additional promoter; glgX is transcribed independently. Surprisingly, the glgA mutant had decreased levels of high-molecular-weight exopolysaccharide after growth on glucose, but levels were normal after growth on galactose. A deletion mutant was constructed in order to generate a nonpolar mutation in glgA. This mutant had a phenotype similar to that of the Tn5 mutant, indicating that the enhanced respiration and symbiotic nitrogen fixation and decreased exopolysaccharide were due to mutation of glgA and not to a polar effect on a downstream gene. PMID:11208782

  12. 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.

  13. An Arabidopsis mutant defective in the general phenylpropanoid pathway.

    PubMed Central

    Chapple, C C; Vogt, T; Ellis, B E; Somerville, C R

    1992-01-01

    Mutants of Arabidopsis deficient in a major leaf phenylpropanoid ester, 2-O-sinapoyl-L-malate, were identified by thin-layer chromatographic screening of methanolic leaf extracts from several thousand mutagenized plants. Mutations at a locus designated SIN1 also eliminate accumulation of the sinapic acid esters characteristic of seed tissues. Because of increased transparency to UV light, the sin1 mutants exhibit a characteristic red fluorescence under UV light, whereas wild-type plants have a blue-green appearance due to the fluorescence of sinapoyl malate in the upper epidermis. As determined by in vivo radiotracer feeding experiments, precursor supplementation studies, and enzymatic assays, the defect in the sin1 mutants appears to block the conversion of ferulate to 5-hydroxyferulate in the general phenylpropanoid pathway. As a result, the lignin of the mutant lacks the sinapic acid-derived components typical of wild-type lignin. PMID:1477555

  14. PATHOGENICITY AND IMMUNOGENICITY OF STREPTOMYCIN-DEPENDENT MUTANTS OF BRUCELLA

    PubMed Central

    Simon, Ellen M.; Berman, David T.

    1962-01-01

    Simon, Ellen M. (University of Wisconsin, Madison) and David T. Berman. Pathogenicity and immunogenicity of streptomycin-dependent mutants of Brucella. J. Bacteriol. 83:1347–1355. 1962.—Streptomycin-dependent (Sd) mutants of Brucella suis and B. abortus were avirulent for guinea pigs whether selected in the presence of streptomycin only or streptomycin and normal or immune serum. Administration of large quantities of streptomycin to guinea pigs increased the numbers of organisms which could be recovered, but did not cause the development of progressive infections. Vaccination with Sd mutants of B. abortus diminished the pathological response of guinea pigs infected with a large challenge dose of virulent B. abortus, but equal numbers of organisms were recovered from vaccinated animals and unvaccinated controls. Vaccination with Sd mutants of B. suis protected some guinea pigs from small challenge doses. Immunization by multiple injections or by one injection plus streptomycin was superior to a single inoculation of organisms. PMID:13913089

  15. Proteomic analysis of the flooding tolerance mechanism in mutant soybean.

    PubMed

    Komatsu, Setsuko; Nanjo, Yohei; Nishimura, Minoru

    2013-02-21

    Flooding stress of soybean is a serious problem because it reduces growth; however, flooding-tolerant cultivars have not been identified. To analyze the flooding tolerance mechanism of soybean, the flooding-tolerant mutant was isolated and analyzed using a proteomic technique. Flooding-tolerance tests were repeated five times using gamma-ray irradiated soybeans, whose root growth (M6 stage) was not suppressed even under flooding stress. Two-day-old wild-type and mutant plants were subjected to flooding stress for 2days, and proteins were identified using a gel-based proteomic technique. In wild-type under flooding stress, levels of proteins related to development, protein synthesis/degradation, secondary metabolism, and the cell wall changed; however, these proteins did not markedly differ in the mutant. In contrast, an increased number of fermentation-related proteins were identified in the mutant under flooding stress. The root tips of mutant plants were not affected by flooding stress, even though the wild-type plants had damaged root. Alcohol dehydrogenase activity in the mutant increased at an early stage of flooding stress compared with that of the wild-type. Taken together, these results suggest that activation of the fermentation system in the early stages of flooding may be an important factor for the acquisition of flooding tolerance in soybean. PMID:23313221

  16. HDAC6 Regulates Mutant SOD1 Aggregation through Two SMIR Motifs and Tubulin Acetylation*

    PubMed Central

    Gal, Jozsef; Chen, Jing; Barnett, Kelly R.; Yang, Liuqing; Brumley, Erin; Zhu, Haining

    2013-01-01

    Histone deacetylase 6 (HDAC6) is a tubulin deacetylase that regulates protein aggregation and turnover. Mutations in Cu/Zn superoxide dismutase (SOD1) linked to familial amyotrophic lateral sclerosis (ALS) make the mutant protein prone to aggregation. However, the role of HDAC6 in mutant SOD1 aggregation and the ALS etiology is unclear. Here we report that HDAC6 knockdown increased mutant SOD1 aggregation in cultured cells. Different from its known role in mediating the degradation of poly-ubiquitinated proteins, HDAC6 selectively interacted with mutant SOD1 via two motifs similar to the SOD1 mutant interaction region (SMIR) that we identified previously in p62/sequestosome 1. Expression of the aggregation-prone mutant SOD1 increased α-tubulin acetylation, and the acetylation-mimicking K40Q α-tubulin mutant promoted mutant SOD1 aggregation. Our results suggest that ALS-linked mutant SOD1 can modulate HDAC6 activity and increase tubulin acetylation, which, in turn, facilitates the microtubule- and retrograde transport-dependent mutant SOD1 aggregation. HDAC6 impairment might be a common feature in various subtypes of ALS. PMID:23580651

  17. Mutant Kras copy number defines metabolic reprogramming and therapeutic susceptibilities.

    PubMed

    Kerr, Emma M; Gaude, Edoardo; Turrell, Frances K; Frezza, Christian; Martins, Carla P

    2016-03-01

    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. PMID:26909577

  18. Mutagenic effects of alpha particles in normal human skin fibroblasts

    SciTech Connect

    Chen, D.J.; Carpenter, S.; Hanks, T.

    1992-12-31

    Alpha-irradiation to the bronchial airways from inhaled radon progeny increases the risk of developing lung cancer. The molecular mechanism of radon-induced lung cancer is not clear, but one of the most important genetic effects of ionizing radiation is the induction of gene mutation. Mutations, especially those associated with visible chromosome abnormalities in humans, have been associated with cancer. Therefore, our objective is to use a well-defined model system to determine the mutagenic potential of alpha particles in normal human skin cells and to define this action at the molecular level. Normal human skin fibroblasts were irradiated with alpha particles (3.59 MeV, LET 115 keV {mu}m{sup {minus}1}) emitted from the decay of {sup 238}Pu. Mutagenicity was determined at the X-linked hypoxanthine guanine phosphoribosyl transferase (HPRT) locus. Results from this study indicate that beta particles were more efficient in mutation induction than gamma rays. Based on the initial slopes of the dose-response curves, the RBE for mutation is about 8 for alpha particles. HPRT-deficient mutants which are resistant to 6-thioguanine have been isolated and analyzed by the Southern blot technique. To date, we have characterized 69 gamma-ray-induced and 195 alpha-particle-induced HPRT-deficient mutants. Our data indicate that more than 50% of all gamma-ray-induced mutants have band patterns identical to that observed for the normal structural HPRT gene, whereas the remaining mutants (45%) contain either a rearrangement, partial deletion, or total deletion of the HPRT gene. In contrast, only 30% of alpha-particle-induced human HPRT mutants contain a normal Southern blot pattern, and about 50% indicate total deletion of the HPRT gene. Our results support the notion that high-LET radiation produces more unrepaired or misrepaired DNA damage than do gamma rays.

  19. Isolation of prostrate turfgrass mutants via screening of dwarf phenotype and characterization of a perennial ryegrass prostrate mutant.

    PubMed

    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

  20. 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

  1. 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…

  2. Mutational effects of retrovirus insertion on the genome of V79 cells by an attenuated retrovirus vector: implications for gene therapy.

    PubMed

    Themis, M; May, D; Coutelle, C; Newbold, R F

    2003-09-01

    Attenuated retroviruses are currently the most widely used vectors in clinical gene therapy because of their potential to effect stable and permanent gene transfer. Since gene delivery is accompanied by random insertion of foreign genetic material into the recipient chromosomal DNA, the potential for insertional mutagenesis exists. In this study, we used a defective retrovirus vector containing a selectable marker, the hygromycin phosphotransferase gene, to investigate the mutagenic effects of vector integration on the mammalian genome. V79 Chinese hamster cells were infected with virus supernatants or by coculture with virus producer cells, and provirus insertion events occurred at low and high frequencies, respectively. The frequency of hprt mutagenesis was increased by a factor of 2.3 over the spontaneous hprt mutation frequency only following multiple provirus insertions/cell genome. Multiple provirus insertions (>3/genome) resulted in instability at the hprt locus in 63% of the virally induced hprt mutants, as indicated by rearrangements at the molecular level, whereas no rearrangements were found when the provirus copy number was 1-2/genome. To demonstrate direct proviral involvement in mutagenesis, the defective MLV vector was retrieved along with flanking genomic hprt sequences from one mutant, and localized within intron 5 of the hprt gene. These data suggest that provirus copy number is a key factor when considering the potential hazards of using retrovirus vectors for gene therapy. PMID:12923569

  3. Altered lipid composition in Streptococcus pneumoniae cpoA mutants

    PubMed Central

    2014-01-01

    Background Penicillin-resistance in Streptococcus pneumoniae is mainly due to alterations in genes encoding the target enzymes for beta-lactams, the penicillin-binding proteins (PBPs). However, non-PBP genes are altered in beta-lactam-resistant laboratory mutants and confer decreased susceptibility to beta-lactam antibiotics. Two piperacillin resistant laboratory mutants of Streptococcus pneumoniae R6 contain mutations in the putative glycosyltransferase gene cpoA. The CpoA gene is part of an operon including another putative glycosyltransferase gene spr0982, both of which being homologous to glycolipid synthases present in other Gram-positive bacteria. Results We now show that the cpoA mutants as well as a cpoA deletion mutant are defective in the synthesis of galactosyl-glucosyl-diacylglycerol (GalGlcDAG) in vivo consistent with the in vitro function of CpoA as α-GalGlcDAG synthase as shown previously. In addition, the proportion of phosphatidylglycerol increased relative to cardiolipin in cpoA mutants. Moreover, cpoA mutants are more susceptible to acidic stress, have an increased requirement for Mg2+ at low pH, reveal a higher resistance to lysis inducing conditions and are hypersensitive to bacitracin. Conclusions The data show that deficiency of the major glycolipid GalGlcDAG causes a pleitotropic phenotype of cpoA mutant cells consistent with severe membrane alterations. We suggest that the cpoA mutations selected with piperacillin are directed against the lytic response induced by the beta-lactam antibiotic. PMID:24443834

  4. 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. PMID:11400730

  5. 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.

  6. 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.

  7. 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.

  8. 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.

  9. Auditory development in progressive motor neuronopathy mouse mutants.

    PubMed

    Volkenstein, Stefan; Brors, Dominik; Hansen, Stefan; Berend, Achim; Mlynski, Robert; Aletsee, Christoph; Dazert, Stefan

    2009-11-01

    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. PMID:19735697

  10. Neurospora Mutant Exhibiting Hyperproduction of Amylase and Invertase

    PubMed Central

    Gratzner, Howard; Sheehan, D. N.

    1969-01-01

    A mutant strain of Neurospora crassa has been isolated which is derepressed for amylase and β-fructofuranosidase (invertase). Large amounts of the two enzymes were secreted into the culture medium upon depletion of exogenous carbon source. The resulting increases of the two extracellular enzymes were prevented by actinomycin D, cycloheximide, and glycerol. The starving cells of the mutant strain produced amylase and invertase de novo, as evidenced by incorporation of radioactive amino acids into the enzymes. Preliminary genetic studies indicate that these elevated enzyme levels described are due to a single gene mutation. PMID:5773010