Glyceraldehyde 3 Phosphate Dehydrogenase Mediates Anoxia

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1174 A R Mendenhall B LaRue and P A Padilla, Wild type C elegans exposed to anoxia 0 001 kPa O2 daf 2 e1370 high temperature anoxia survival pheno. in laboratory culture and temperature 20 conditions type we used RNA interference RNAi to decrease the. enters a reversible state of suspended animation In the expression of specific genes upregulated in the daf. anoxic environment embryonic and postembryonic de 2 e1370 strain We determined that gpd 2 and gpd 3 two. velopment arrests Furthermore the postembryonic nearly identical genes in an operon that encode the. animal stops feeding becomes immobile and does not glycolytic enzyme glyceraldehyde 3 phosphate dehydro. lay eggs After reexposure to a normoxic environment genase GAPDH are required for response to anoxia. the nematodes proceed with development C elegans at all Here we suggest that gpd 2 and gpd 3 are involved with. stages of development survive at least 1 day of anoxia with cell and tissue protection during anoxia exposure The. a viability of 90 Van Voorhies and Ward 2000 reduction of expression of other glycolytic genes by. Padilla et al 2002 Hajeri et al 2005 The dauer larvae RNAi does not result in phenotypes similar to that of daf. a developmentally arrested larval stage survive several 2 e1370 gpd 2 3 RNAi suggesting that it may not be. days of anoxia making this stage the most resistant to the mere flux through glycolysis that is required for. anoxia Anderson 1978 Padilla et al 2002 The long anoxia survival. term anoxia survival phenotype of dauer larvae is con. sistent with its ability to survive stress Yet further studies. are needed to understand the molecular mechanism that MATERIALS AND METHODS. the dauer larvae use to survive anoxia, Given that the dauer larvae have an enhanced anoxia Strains and growth conditions The wild type Bristol strain. N2 and mutant strains were cultured using NGM plates. survival phenotype it is of interest to investigate the rela seeded with Escherichia coli OP50 and raised at 20 as. tionship of anoxia survival and the genetic pathways described Sulston and Hodgkin 1988 Synchronized pop. involved with dauer formation One of the dauer reg ulations of animals were obtained by allowing embryos. ulatory pathways includes the daf 2 and daf 16 genes obtained from hypochlorite treated adults to grow to specific. which encode the insulin IGF 1 receptor like protein developmental stages Anatomical markers such as gonad. morphology or the hours after L4 molt were used to determine. and a fork head transcription factor respectively the developmental stage of the animal Wild type or daf. Larsen et al 1995 Kimura et al 1997 Riddle and 2 e1370 adult animals were collected at 21 25 or 17 21 hr. Albert 1997 Gems et al 1998 In the daf 2 e1370 ani after the L4 to adulthood molt respectively The following. mal which has a mutation in the insulin receptor kinase genetic strains were obtained from either the Caenorhabditis. domain the DAF 16 protein is translocated to the nu elegans Genetics Center or colleagues daf 2 e1370 III daf. 16 m26 I daf 2 e1370 III daf 16 mu86 I daf 16 m26 I and. cleus Phenotypes observed in the daf 2 e1370 animal PD4792 mIs11 IV. include constitutive dauer formation at increased tem Oxygen deprivation experiments For all experiments the. perature alteration in gene expression increased long nematodes were exposed to an anoxic environment using the. evity resistance to a variety of stresses and changes in BioBag type A environmental chamber as previously described. metabolism Kenyon et al 1993 Vanfleteren and De Becton and Dickinson Cockeysville MD Padilla et al. 2002 The anoxia exposure time 1 5 days and the temper. Vreese 1996 Kimura et al 1997 Ogg et al 1997 Gems ature conditions 20 or 28 are noted for each experiment. et al 1998 Lee et al 2003 McElwee et al 2003 2004 For the purpose of these studies we refer to long term anoxia. Munoz 2003 Murphy et al 2003 Van Voorhies 2003 as 3 days of 0 001 kPa O2 at 20 and high temperature. Burnell et al 2005 DNA microarray studies deter anoxia as 1 day of 0 001 kPa O2 at 28. mined that genes involved with stress response and Anoxia survival assays Nematodes either at the L2 larval. stage or at the young adult hermaphrodite stage were assayed. metabolism are upregulated in the daf 2 mutant How for anoxia survival The L2 larvae were collected 24 hr after. ever it is not known how gene expression and metabolic newly hatched L1 were placed on seeded plates Due to dif. changes are involved with the mechanism by which the ferent developmental progression rates between the wild type. daf 2 mutant animal survives stress and daf 2 e1370 animals the actual time to reach adulthood. A study in which researchers screened for genetic varied between the two strains Therefore we collected adult. wild type and daf 2 e1370 animals 16 5 25 hr after the L4 to. mutants that survive hypoxia at a high temperature adulthood molt The adult hermaphrodites assayed were at a. 20 hr of 0 3 O2 28 identified a mutation in the daf 2 developmental stage where their offspring consisted of em. gene thus providing the first direct genetic evidence bryos and newly hatched L1 larvae Nematodes were exposed. that the daf 2 daf 16 insulin like signaling pathway is to anoxia as described above and 24 hr postanoxic animals. involved with oxygen deprivation survival in C elegans were examined for survival by visual inspection for movement. or reaction to a platinum wire To determine if anoxia exposed. Scott et al 2002 In this report we compared the animals had an uncoordinated phenotype the animals were. phenotypes of the daf 2 e1370 and wild type animals inspected for abnormal movement or inability to move along. exposed to long term anoxia defined here as 3 days the agar surface The data collected for nematode survival and. of 0 001 kPa O2 20 as well as to high temperature phenotype analysis included at least three independent ex. anoxia defined here as 1 day of 0 001 kPa O2 at 28 periments consisting of 50 150 worms experiment. RNA interference assays We used RNAi to inhibit the ex. We determined that unlike wild type animals the daf pression of several genes including genes upregulated by. 2 e1370 animals survive long term anoxia and high DAF 16 and genes predicted to encode a glycolytic enzyme. temperature anoxia To identify genes involved with the Lee et al 2003 Murphy et al 2003 Briefly the daf 2 e1370. Anoxia Responses in C elegans 1175, or N2 synchronized L1 larvae were collected and grown to Identification of putative glycolytic genes in C elegans We. adulthood on NGM 200 mg ml ampicillin 12 5 mg ml tetra identified C elegans genes homologous to mammalian glyco. cycline and 0 5 mg ml IPTG seeded with the E coli strain for lytic genes using BLASTP analysis and Wormbase Briefly the. RNAi of a specified gene The E coli strains were developed by primary sequences of human glycolytic genes were obtained. the J Ahringer laboratory and obtained from the MRC using the NCBI protein database and the C elegans ortholog s. Geneservice Cambridge UK Timmons et al 2001 Kamath was identified using the NCBI and Wormbase BLASTP. et al 2003 The bacterial clones used for RNAi of gpd 2 and program. gpd 3 were K10B3 8 or K10B3 7 respectively Throughout most Real time quantitative PCR assays Real time quantitative. of these experiments we used the K10B3 8 clone and refer to PCR assay RT qPCR was used to assay RNAi efficiency as well. the RNAi experiment as gpd 2 3 RNAi The long term anoxia as to compare the gpd 2 3 transcript level in daf 2 and wild type. survival phenotype observed for daf 2 e1370 gpd 2 3 RNAi animals Briefly animals were placed in pureZOL Bio Rad. was more pronounced when the bacteria were grown as fol Hercules CA frozen in liquid nitrogen and then thawed at. lows Bacteria were plated on LB plates supplemented with 200 37 This freeze thaw process was repeated three times before. mg ml ampicillin amp and 12 5 mg ml tetracycline tet RNA was extracted using the Aurum Fatty and Fibrous Tissue. From this plate a single colony was grown in 1 ml of LB amp RNA extraction kit Bio Rad RNA quality and quantity were. tet 200 mg ml ampicillin 12 5 mg ml tetracycline for 6 8 hr determined using the Experion system Bio Rad RT qPCR. and 15 ml of this culture was used to inoculate 5 ml of LB amp was conducted using the iScript One step RT PCR kit Bio. tet liquid which was grown for 4 hr before seeding onto NGM Rad of which 10 ng of total RNA was loaded into each reac. IPTG agar plates These NGM IPTG plates were placed at 37 tion Reactions were normalized against a nonvariable control. for 24 hr and then transferred to 15 or 20 for 24 48 hr before gene F23B2 13 Link et al 2003 The DCT with a reference. L1 larvae were placed onto the plates L1 larvae were grown on gene method was used to calculate fold decrease or increase in. the RNAi food to adulthood The adult nematodes were ex transcript Real time PCR Applications Guide Bio Rad All. posed to anoxia and assayed for viability as described above experiments were from at least four independent experiments. The F01F1 12 RNAi and F25H5 3 RNAi results in embryo and each experiment was performed in triplicate. lethality or larvae arrest respectively Therefore for these. RNAi experiments the daf 2 e1370 animals were grown to the. L4 stage on control food for 72 hr and transferred to the ap. propriate RNAi food for 48 hr and then these adults were RESULTS. assayed for high temperature anoxia survival As a control we. grew the E coli strain which has no insert in the plasmid on The daf 2 e1370 animal survives long term anoxia. identical NGM IPTG plates to account for any differences and high temperature anoxia Wild type adult hermaph. attributable to the vector rodites survive 1 day of anoxia at 20 yet the survival rate. Motility assays of nematodes exposed to anoxia The wild dramatically decreases when the animals are exposed to. type gpd 2 3 RNAi daf 2 e1370 daf 2 e1370 gpd 2 3 RNAi. daf 16 m26 I and daf 16 mu86 I adult hermaphrodites were. long term anoxia 3 days 20 Van Voorhies and, used to compare and analyze nematode motion in anoxia Ward 2000 Padilla et al 2002 When considering the. Nematode strains were placed on separate NGM plates in the developmental stage of C elegans the dauer larvae have. same anoxia BioBag chamber to ensure that the samples were the highest long term anoxia 3 days survival rate. treated equally To assay animal motility during anoxia ex Anderson 1978 Padilla et al 2002 Unlike wild type. posure nematodes were observed through the anoxia Bio. Bag over a 4 5 min period at 30 sec intervals every hour for. animals the daf 2 e1370 animals raised to adulthood at. the time indicated in each experiment Motility was assayed 20 and then transferred to high temperature hypoxia. and images were obtained using a Zeiss M2Bio stereoscope 0 3 O2 28 have a high survival rate Scott et al. and the Openlab software 3 17 imaging software Improvision 2002 To determine if daf 2 e1370 and wild type ani. Lexington MA Nematodes were maintained at 20 and were mals have different anoxia survival rates both strains. at only room temperature during image collection which took. were exposed to anoxia at 20 Wild type and daf, 20 min for each sampling For the wild type gpd 2 3 RNAi.
daf 2 e1370 and daf 2 e1370 gpd 2 3 RNAi comparisons a 2 e1370 animals were collected as L2 larvae or gravid. worm was scored positive for movement if the nematode adults and exposed to 1 3 4 or 5 days of anoxia at 20. displayed forward or reverse motion along the agar surface Figure 1 shows that wild type L2 larvae and adult ani. Data include at least three independent experiments For fur mals had a high survival rate when exposed to 1 day of. ther analysis of worm motility during anoxia exposure nema. anoxia however the survival rate decreased when ex. todes were observed as described above except the images. were collected at 5 sec intervals over a 4 5 min period The posed to 3 days of anoxia and the animals did not survive. collected images were imported into QuickTime 7 Pro Apple 4 or 5 days of anoxia In comparison the daf 2 e1370. Computer for visualization larvae and adult animals survived long term anoxia ex. Nomarski microscopy analysis Nematodes were exposed to posures at a significantly higher rate in comparison to. normoxic or anoxic conditions and allowed to recover in air wild type animals The daf 2 e1370 adults survived. for the time indicated for each experiment The animals were. placed on a 2 5 agar pad containing 10 mm sodium azide and 5 days of anoxia better than the L2 larvae P 0 001. covered with a coverslip Animals were also analyzed without Given that the DAF 16 transcription factor is translo. the use of sodium azide to verify that this chemical did not cated to the nucleus in the daf 2 e1370 animal we. contribute factors to the analysis of the phenotype For each wanted to determine if the daf 2 e1370 long term. experiment at least three independent evaluations were done anoxia phenotype was mediated by DAF 16 The daf. Microscopy was conducted using a motorized Zeiss Axioscope. with a 3100 objective lens and imaging Openlab software 3 17 16 m26 daf 2 e1370 adults and larvae animals exposed. Images were prepared using Adobe Photoshop CS Adobe to 5 days of anoxia at 20 had an average viability of. Systems 1 65 6 2 71 n 353 and 3 32 6 5 04 n 458,1176 A R Mendenhall B LaRue and P A Padilla. Figure 1 The survival rate of daf 2 e1370 and wild type. animals exposed to long term anoxia at 20 Wild type L2 lar Figure 2 The survival rate of daf 2 e1370 wild type and. Glyceraldehyde 3 Phosphate Dehydrogenase Mediates Anoxia Response and Survival in Caenorhabditis elegans Alexander R Mendenhall Bobby LaRue and Pamela A Padilla1 Department of Biological Sciences University of North Texas Denton Texas 76203 Manuscript received June 17 2006 Accepted for publication August 22 2006 ABSTRACT Oxygen deprivation has a role in the pathology of many human

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