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1.
Intra- and interspecific cross combinations between the tetraploid treefrog Hyla versicolor, and between H. versicolor and the diploid treefrog Hyla chrysoscelis were performed. Progeny phenotypes resulting from these crosses were examined electrophoretically using a polymorphic glutamate oxaloacetic transminase (GOT-1) locus, to determine the mechanism of chromosome segregation in H. versicolor, and to test theoretical expectations for isozyme expression in interspecific (2n x 4n or 4n x 2n) hybrids. In some intraspecific tetraploid crosses progeny phenotypes fit a disomic mode of segregation, whereas in other crosses a tetrasomic mode of segregation was the most probable. Additional crosses produced phenotypic ratios that conformed to either a disomic or tetrasomic mode of segregation. These results suggest that a polymorphism, with respect to segregation of gametes, exists in H. versicolor, resulting from differences in chromosome pairings during meiosis I. This polymorphism in gametic segregation occurred in both sexes. Certain crosses, however, produced phenotypic ratios that did not conform to any chromosome segregation model. Progeny phenotypes observed from most interspecific crosses conformed to expected interspecific isozyme staining intensity models. Symmetrical heterozygotes, representing either a single dose for both alternate alleles or double doses for both alternate alleles, were also observed. Such phenotypes are unexpected in triploid progeny. A null allele was postulated to account for the aberrant segregation ratios and phenotypes observed in certain intra- and interspecific crosses.  相似文献   

2.
Electrophoretic survey of malate dehydrogenase (EC 1.1.1.37) in Opuntia basilaris showed intraspecific polymorphism. Further experiments with microbody malate dehydrogenase-specific antiserum suggest that the polymorphism occurs in microbody malate dehydrogenase independent of the soluble and mitochondrial forms. The pattern of polymorphism is one expected from a two-allele Mendelian system.  相似文献   

3.
Marsden JE  Schwager SJ  May B 《Genetics》1987,116(2):299-311
The recently evolved autotetraploid frog, Hyla versicolor , was examined electrophoretically for evidence of genomic restructuring leading to diploidization. Loci were tested against the progeny ratios expected if inheritance was disomic vs. tetrasomic. Two loci (Mpi and Sod-2) appeared to be inherited tetrasomically, one (Mdh-2) appeared to be inherited disomically, and one (Tpi) appeared to be inherited disomically in one family and tetrasomically in another family, when tested conventionally against 1:2:1 and 1:4:1 segregation ratios. The minimum number of progeny required for this type of analysis for codominant alleles is shown to be 92. Progeny resulting from double reduction were observed, and the occurrence of a null allele class at Mpi was noted. A reexamination of expected progeny ratios in tetraploid organisms reveals that tetrasomic inheritance patterns cannot be predicted without adequate knowledge of the amount of crossing-over, the proportion of tetravalents vs. random bivalents that are formed, and the ratio and types of centromere segregation (alternate and adjacent) that occur from tetravalents in the species being studied. However, disomic inheritance can be unambiguously confirmed only by the production of all heteroallelic gametes from homobivalent, symmetrically heterozygous individuals. In addition, a method is described for estimating genecentromere distances using the ratio of progeny genotypes in certain crosses in tetraploid species.  相似文献   

4.
The kinetics of malate dehydrogenase (MDH) catalyzed oxidation/reduction of L-malate/oxaloacetate is pH-dependent due to the proton generated/taken up during the reaction. Previous kinetic studies on the mitochondrial MDH did not yield a consensus kinetic model that explains both substrate and pH dependency of the initial velocity. In this study, we propose, to our knowledge, a new kinetic mechanism to explain kinetic data acquired over a range of pH and substrate concentrations. Progress curves in the forward and reverse reaction directions were obtained under a variety of reactant concentrations to identify associated kinetic parameters. Experiments were conducted at physiologically relevant ionic strength of 0.17 M, pH ranging between 6.5 and 9.0, and at 25°C. The developed model was built on the prior observation of proton uptake upon binding of NADH to MDH, and that the MDH-catalyzed oxidation of NADH may follow an ordered bi-bi mechanism with NADH/NAD binding to the enzyme first, followed by the binding of oxaloacetate/L-malate. This basic mechanism was expanded to account for additional ionic states to explain the pH dependency of the kinetic behavior, resulting in what we believe to be the first kinetic model explaining both substrate and pH dependency of the reaction velocity.  相似文献   

5.
6.
Over a range of concentrations from less than 0.1 mm to more than 70 mm, sweet potato root mitochondria display a bimodal substrate saturation isotherm for malate. The high affinity portion of the isotherm has an apparent Km for malate of 0.85 mm and fits a rectangular hyperbolic function. The low affinity portion of the isotherm is sigmoid in character and gives an apparent S(0.5) of 40.6 mm and a Hill number of 3.7.Extracts of sweet potato mitochondria contain both malate dehydrogenase and NAD malic enzyme. The malate dehydrogenase, assayed in the forward direction at pH 7.2, shows typical Michaelis-Menten kinetics with a Km for malate of 0.38 mm. The NAD malic enzyme shows pronounced sigmoidicity in response to malate with a Hill number of 3.5 and an S(0.5) of 41.6 mm.On the basis of the normal kinetics, the Km, and the fact that oxaloacetate production from malate by mitochondria appears most active at low malate concentrations, the high affinity portion of the malate isotherm with mitochondria is attributed to malate dehydrogenase. The low affinity portion of the malate isotherm with mitochondria is thought, on the basis of the similarity of S(0.5) values, the Hill numbers, and the greater production of pyruvate from malate at high malate concentrations, to represent the activity of the NAD malic enzyme.  相似文献   

7.
8.
9.
From a collection of electrophoretic variants of XDH obtained from laboratory strains and natural populations, a stock was isolated that was associated with much greater than normal levels of XDH activity. Preliminary recombination experiments demonstrated that this character maps to the rosy locus. While a series of observations failed to relate this phenotype to alteration in the structure of the XDH polypeptide, kinetic and immunological experiments did succeed in associating this character with variation in number of molecules of XDH/fly. Large scale fine structure recombination experiments locate the genetic basis for this variation in number of molecules of XDH/fly to a site very close to, but definitely outside of, the genetic boundaries of the XDH structural information. Observations are described which eliminate the possibility that we are dealing with a tandem duplication of the XDH structural element. Turning to a regulatory role for this genetic element located adjacent to the XDH structural information, a simple experiment is described which demonstrates that it functions as a "cis-acting" regulator of the XDH structural element.  相似文献   

10.
The influence of starvation on malate dehydrogenase (MDH) in rat liver was investigated. Native electrophoresis revealed two MDH isoforms in non-starved rats and three isoenzymes in starved rats. After sucrose density gradient centrifugation of cell organelles from liver, MDH activity was detected in the mitochondrial and cytosolic fractions from non-starved rats. However, additional activity was found in the peroxisomal fraction from starved rats. The latter was identified as the electrophoretically new isoform in starved animals. The three isoforms of malate dehydrogenase from hepatocytes were separated and partially purified by chromatography on DEAE-Toyopearl. Several kinetic and regulatory properties of the three isoforms were rather similar. It is suggested that the newly expressed isoform of MDH operates in the glyoxylate cycle of liver peroxisomes of food-starved animals.  相似文献   

11.
Malate dehydrogenases (MDHs) play crucial roles in the physiological processes of plant growth and development. In this study, 13 and 25 MDH genes were identified from Gossypium raimondii and Gossypium hirsutum, respectively. Using these and 13 previously reported Gossypium arboretum MDH genes, a comparative molecular analysis between identified MDH genes from G. raimondii, G. hirsutum, and G. arboretum was performed. Based on multiple sequence alignments, cotton MDHs were divided into five subgroups: mitochondrial MDH, peroxisomal MDH, plastidial MDH, chloroplastic MDH and cytoplasmic MDH. Almost all of the MDHs within the same subgroup shared similar gene structure, amino acid sequence, and conserved motifs in their functional domains. An analysis of chromosomal localization suggested that segmental duplication played a major role in the expansion of cotton MDH gene families. Additionally, a selective pressure analysis indicated that purifying selection acted as a vital force in the evolution of MDH gene families in cotton. Meanwhile, an expression analysis showed the distinct expression profiles of GhMDHs in different vegetative tissues and at different fiber developmental stages, suggesting the functional diversification of these genes in cotton growth and fiber development. Finally, a promoter analysis indicated redundant but typical cis-regulatory elements for the potential functions and stress activity of many MDH genes. This study provides fundamental information for a better understanding of cotton MDH gene families and aids in functional analyses of the MDH genes in cotton fiber development.  相似文献   

12.
关洪英  唐志权  李辉 《遗传学报》2006,33(6):501-506
苹果酸脱氢酶(Malate Dehydrogenase,MD)是一种氧化还原性酶,参与体内多种能量代谢反应.它可以催化苹果酸氧化脱羧生成丙酮酸和CO2,并使NADP+还原成NADPH,NADPH是脂肪酸合成所必需的载体,棕榈酸可以利用生成的NADPH来合成长链脂肪酸,MD的活性与脂肪酸合成效率之间存在密切的相关,MD也参与体内骨骼肌、心肌的能量代谢,并对肌纤维的生长有一定的调节作用.根据鸡MD基因的5侧翼区序列设计一对引物,用直接测序的方法在侧翼区检测多态性位点,在235bp(GenBank登录号U49693)处发现一个SNP位点,此位点是一个限制性内切酶(SphⅠ酶)发生变化的位点.以东北农业大学高低脂双向选择系的第8世代肉鸡和东农F2资源群体为实验材料,用PCR-RFLP的方法进行基因型分析,建立适合的统计模型,进行基因型与生长和体组成性状的相关分析.结果表明在高低脂系第8世代肉鸡中AA基因型个体的腹脂重和腹脂率显著高于BB基因型个体(P<0.05);BB基因型个体的大胸肌重和大胸肌率显著高于AA基因型个体(P<0.05).在东农F2资源家系中BB基因型个体的大胸肌重和大胸肌率显著高于AA和AB基因型个体(P<0.05);AA基因型个体的肝脏重和肝脏率显著高于BB基因型个体(P<0.05).综上所述,MD基因可能是影响鸡生长和体组成性状的主效基因或与控制生长和体组成性状的主效基因相连锁.  相似文献   

13.
Evidence is presented for the existence of a second homoserine dehydrogenase in Salmonella typhimurium. The formation, but not the activity, of this enzyme is controlled by methionine. Two distinct homoserine dehydrogenases were separated from wild-type cells by diethylaminoethyl (cellulose) column chromatography. Sucrose gradient ultracentrifugation gave molecular weight estimates for the threonine-regulated enzyme (HSD I) of 220,000 to 240,000 and for the methionine controlled enzyme (HSD II) of 130,000 to 140,000. Approximately 12% of the total HSD activity in wild-type cells was accounted for by HSD II. A threonine-requiring strain of S. typhimurium was found to lack HSD I but not HSD II. Under certain conditions, this mutant grew rapidly in minimal medium. Rapid growth in minimal medium was correlated with the appearance of an enzyme with similar characteristics to HSD I. The possible origins of this HSD I-like enzyme are presented.  相似文献   

14.
Crouau-Roy B  Clayton J 《Genetica》2002,114(1):17-23
Two possible mechanisms for the development and maintenance of gametic association have been subject to test using family studies (HLA typed) and direct analysis of two linked markers in individual sperm. The mechanisms are: (1) haplotypes frozen against recombination and (2) balanced segregation distortion. In the study of 1320 families, 86 individuals were identified in the offspring of whom recombination was confirmed or suspected. These individuals displayed allele frequencies of HLA similar to the others in the study, but had significantly different haplotype frequencies. In general, they displayed reduced (or absent) frequencies for the more common haplotypes. Similarly, in the study of meiotic products, recombination was observed in the individual whose HLA phenotypes suggested rare haplotypes, but not in the other individual. Both results were of marginal significance. When the family data were used to test for segregation distortion at HLA-DR, only a marginally significant result was found. However, when the data were classified according to the sex of the parent, a highly significant result was found in females, but not in males. Male segregation distortion was found in the sperm analysis, but was only of marginal statistical significance. The concordant results of two different experimental systems suggest that both mechanisms remain feasible explanations of the gametic associations in this area of the genome, although of the two, segregation distortion is preferable on theoretical grounds.  相似文献   

15.
A chemically defined medium was developed for the production of intracellular malate dehydrogenases by Streptomyces aureofaciens NRRL-B 1286. The composition of the medium (per liter) was as follows: 50 g of starch, 4 g of ammonium sulfate, 7.32 g of l-aspartic acid, 13.8 g of MgSO(4) . 7H(2)O, 1.7 g of K(2)HPO(4), 0.01 g of ZnSO(4) . 7H(2)O, 0.01 g of FeSO(4) . 7H(2)O, 0.01 g of MnSO(4) . H(2)O, and 0.005 g of CoSO(4) . 7H(2)O. The pH of the medium was adjusted to 6.7 to 7.0 after sterilization. The activity of the intracellular malate dehydrogenases of the crude cell extract was greatest after 40 h of mycelium growth in a rotary shaker at 30 degrees C. The best temperature for the enzyme reactions was approximately 35 degrees C for NAD activity at pH 9.7 and 40 degrees C for NADP -linked enzyme at pH 9.0. The NAD activity required Mg, and both activities were sensitive to SH-group reagents. The NADP -dependent activity remained completely stable, and the NAD -dependent activity decreased to a very low residual level after 30 min at 60 degrees C.  相似文献   

16.
Maize mitochondrial malate dehydrogenase is coded by four genetic loci, Mdh1, Mdh2, Mdh3 and Mdh4. Two of the four loci have been located on the long arm of chromosome 6, using trisomic analysis and B-A translocations.  相似文献   

17.
18.
Malate dehydrogenase (MD) is a key enzyme that plays an important role in energy metabolism. It catalyzes the oxidative decarboxylation of L-malate to yield CO2 and pyruvate, while simultaneously generating NADPH from NADP+. The NADPH generated can be utilized in de novo synthesis of palmitate, which is the precursor molecule for the formation of other long-chain fatty acids. And high levels of MD will also activate muscle development. The current study was designed to investigate the effects of MD gene on growth and body-composition traits in chicken. The eighth generation population of Northeast Agricultural University broiler lines divergently selected for its abdominal fat and Northeast Agricultural University F2 resource population were used in the research. Polymorphisms were detected by DNA sequencing and PCR-RFLP method was then developed to screen the population. A single mutation at the position of the 235 bp (Accession No. U49693) of MD 5′-flanking region was found. The correlation analysis between the polymorphism of the MD gene and growth and body composition traits was carried out using the appropriate statistic model. Least-square analysis showed that the BB genotype birds had much higher pectoralis major weight and percentage of pectoralis major than AA genotype birds (P<0.05). The abdominal fat weight, percentage of abdominal fat, the liver weight and percentage of liver weight of the AA genotype birds were much higher than those of BB genotype birds (P<0.05). These results indicate that MD gene is the major gene or is linked to the major gene that affects the growth and body composition traits in chicken.  相似文献   

19.
Malate: A Possible Source of Error in the NAD Glutamate Dehydrogenase Assay   总被引:2,自引:0,他引:2  
The effects of externally induced metabolic perturbations areoften studied through changes of the enzyme activity patternsin crude plant extracts. From glutamate dehydrogenase (GDH)it is reported that environmental changes not only influencethe amount of the enzymatic activity, but also the ratio ofthe aminating to the deaminating activities (NADH/NAD+ ratio).Using crude cell extracts of suspension cultures of wheat (Triticumaestivum L. cv. Heines Koga II) we find evidence that the pretreatmentof the homogenate directly influences this ratio. Dialysis ofthese crude cell extracts resulted in a 70% loss of the NAD+activity, while the NADH activity remained unchanged. The deaminatingactivity in the dialysed extract could be completely restoredupon addition of a dialysable factor which was identified tobe malate. The interference of malate with the glutamate dehydrogenasereaction is caused through the action of malate dehydrogenaseand glutamate oxaloacetate transaminase which are both presentin high activities in the extracts. Only in exhaustively dialysedcell extracts can the proper deaminating GDH activity be determined.The results are discussed in the light of the controversialreports on the variable ratio of the NADH/NAD+ activity of GDH. Key words: Glutamate dehydrogenase, malate, NADH/NAD+, activity, Triticum aestivum  相似文献   

20.
In illuminated chloroplasts, one mechanism involved in reduction-oxidation (redox) homeostasis is the malate-oxaloacetate (OAA) shuttle. Excess electrons from photosynthetic electron transport in the form of nicotinamide adenine dinucleotide phosphate, reduced are used by NADP-dependent malate dehydrogenase (MDH) to reduce OAA to malate, thus regenerating the electron acceptor NADP. NADP-MDH is a strictly redox-regulated, light-activated enzyme that is inactive in the dark. In the dark or in nonphotosynthetic tissues, the malate-OAA shuttle was proposed to be mediated by the constitutively active plastidial NAD-specific MDH isoform (pdNAD-MDH), but evidence is scarce. Here, we reveal the critical role of pdNAD-MDH in Arabidopsis (Arabidopsis thaliana) plants. A pdnad-mdh null mutation is embryo lethal. Plants with reduced pdNAD-MDH levels by means of artificial microRNA (miR-mdh-1) are viable, but dark metabolism is altered as reflected by increased nighttime malate, starch, and glutathione levels and a reduced respiration rate. In addition, miR-mdh-1 plants exhibit strong pleiotropic effects, including dwarfism, reductions in chlorophyll levels, photosynthetic rate, and daytime carbohydrate levels, and disordered chloroplast ultrastructure, particularly in developing leaves, compared with the wild type. pdNAD-MDH deficiency in miR-mdh-1 can be functionally complemented by expression of a microRNA-insensitive pdNAD-MDH but not NADP-MDH, confirming distinct roles for NAD- and NADP-linked redox homeostasis.Reduction-oxidation (redox) reactions play pivotal roles for most metabolic processes and occur in all cellular compartments. The origin of all reducing power in plants is the chloroplast thylakoid membrane system, where light-driven photosynthetic electron transport leads to the coupled formation of ATP and the reducing equivalent NADPH (Dietz and Pfannschmidt, 2011). Sudden changes in light intensity and withdrawal of ATP and NADPH for biosynthetic processes in varying amounts can potentially disturb the ATP:NADPH ratio. Maintaining this ratio within certain limits, however, is crucial for plant metabolism, because it avoids the accumulation of excess electrons and the production of cytotoxic reactive oxygen species and allows for the continued production of ATP (Apel and Hirt, 2004; Logan, 2006; Scheibe and Dietz, 2012). Accordingly, plants have several mechanisms to dissipate excess electrons, avoid damage to cellular components, and maintain redox homeostasis. These mechanisms include nonphotochemical energy quenching, chlororespiration, cyclic electron transport, and the Mehler reaction (Scheibe et al., 2005).Reducing equivalents in the form of dedicated electron carriers or reduced cofactors (e.g. ferredoxin and NADH) are not generally transported directly across membranes; however, they can be shuttled indirectly as malate in exchange for oxaloacetic acid (OAA). This redox-poising mechanism is known as the malate valve in illuminated plastids or more generally, the malate-OAA shuttle (Heber, 1974; Scheibe, 2004; Taniguchi and Miyake, 2012). The key enzyme of the malate-OAA shuttle is malate dehydrogenase (MDH), which catalyses the reversible interconversion of malate and OAA. Isoforms of MDH are present in various cell compartments (Gietl, 1992), and each isoform is specific to either cosubstrate NAD (NAD-MDH; EC 1.1.1.37) or NADP (NADP-MDH; EC 1.1.1.82). The Arabidopsis genome encodes eight putative NAD-MDH isoforms: two isoforms are peroxisomal MDH (PMDH; PMDH1 and PMDH2; Pracharoenwattana et al., 2007; Eubel et al., 2008), two isoforms are mitochondrial MDH (MMDH; MMDH1 and MMDH2; Millar et al., 2001; Lee et al., 2008; Tomaz et al., 2010), and one isoform is plastidial MDH (plastid-localized NAD-dependent MDH [pdNAD-MDH]; Berkemeyer et al., 1998). The remaining three isoforms have no detectable target sequence and are thought to be cytosolic MDH (CMDH; CMDH1, CMDH2, and CMDH3). The Arabidopsis genome also encodes an additional NADP-dependent isoform of MDH, which is localized to the plastid (Hebbelmann et al., 2012).The physiological role of the different isoforms depends on the subcellular localization and the different metabolic pathways occurring there. For instance, MMDH was reported to be involved in two processes that are at least partly mitochondrial: leaf respiration and photorespiration (Tomaz et al., 2010). An MMDH null mutant (mmdh1 mmdh2) was slow growing and showed elevated leaf respiration in the dark and the light, although photosynthetic capacity was not affected. Tomaz et al. (2010) proposed that MMDH uses NADH to reduce OAA to malate, which is then shuttled to the cytosol, rather than generate NADH to fuel mitochondrial respiration (Tomaz et al., 2010). PMDH might serve at least two different functions. First, during fatty acid β-oxidation, which generates NADH, PMDH is proposed to regenerate the electron acceptor NAD by reducing OAA to malate, which is then shuttled to the cytosol in exchange for OAA (Pracharoenwattana et al., 2007). Second, PMDH is thought to generate NADH during photorespiration by oxidation of malate imported from the cytosol (Reumann and Weber, 2006; Cousins et al., 2008). Arabidopsis mutants lacking PMDH (pmdh1 pmdh2) are severely impaired in β-oxidation, and seedling establishment is strongly impaired and dependent on the supply of exogenous sugar (Pracharoenwattana et al., 2007), a phenotype characteristic of β-oxidation mutants (Pinfield-Wells et al., 2005; Baker et al., 2006). However, after transfer of established pmdh1 pmdh2 seedlings to compost, they grew only slightly slower than wild-type plants (Pracharoenwattana et al., 2007).Until recently, genetic evidence for the roles of the plastidial MDH isoforms was scarce. In most C4 plants, NADP-MDH is directly involved in CO2 fixation, catalyzing the formation of the stable CO2 carrier malate from the primary CO2 fixation product OAA (Scheibe, 1987). However, in C3 plants, NADP-MDH has long been proposed to have its major function in the malate valve, leading to shuttling of reducing power (as malate) from the chloroplast to the cytosol during the day and thereby regenerating the electron acceptor NADP inside the chloroplasts (Heber, 1974; Lance and Rustin, 1984; Scheibe, 1987). NADP-MDH is redox activated by thioredoxins in the light and essentially inactive in the dark (Scheibe, 1987; Buchanan and Balmer, 2005). The widely accepted belief that chloroplasts only possess this one strictly light-/redox-activated NADP-MDH temporarily led to the conclusion that the malate valve only works in illuminated chloroplasts (Berkemeyer et al., 1998; Scheibe, 2004). However, a recent study showed that Arabidopsis plants lacking NADP-MDH (nadp-mdh) were indistinguishable from wild-type plants, even under conditions that are supposed to provoke the accumulation of excess electrons and the production of cytotoxic reactive oxygen species (high light and short days; Hebbelmann et al., 2012). This finding indicates that NADP-MDH is not crucial for providing electron acceptors in chloroplasts, but it rather suggests that other mechanisms can counteract or prevent overreduction of the chloroplast.The existence of a second MDH isoform in plastids, which uses NAD as cofactor, has been questioned, because it could not be ruled out that NAD-MDH activity detected in isolated chloroplasts was caused by contamination from other organelles (Siebke et al., 1991; Backhausen et al., 1998). In 1998, Berkemeyer et al. (1998) reported the cloning, heterologous expression, and in vitro characterization of a pdNAD-MDH from Arabidopsis (At3g47520). In contrast to NADP-MDH, pdNAD-MDH is active under both light and dark conditions in isolated chloroplasts, and the activities of both enzymes are within the same range in the light (Backhausen et al., 1998; Berkemeyer et al., 1998). However, up to now, genetic evidence for the in vivo function of pdNAD-MDH is missing, and experimental data are scarce. Backhausen et al. (1998) showed that chloroplasts and heterotrophic chromoplasts isolated from different sources followed by incubation in the dark concomitantly produced 3-phosphoglycerate and malate on addition of dihydroxyacetone phosphate and OAA to the medium. It was proposed that 3-phosphoglycerate production was in a glycolytic step involving glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and that pdNAD-MDH regenerates the electron acceptor NAD required by GAPDH through reduction of OAA to malate, thus operating the malate-OAA shuttle in the dark and in nongreen tissues (Scheibe, 2004; Taniguchi and Miyake, 2012).Here, we aimed to evaluate the function of this MDH isoform in plastid metabolism by analyzing Arabidopsis plants with a transposon insertion in the pdNAD-MDH gene and Arabidopsis plants with reduced pdNAD-MDH by means of artificial microRNA silencing.  相似文献   

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