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1.
Genetically engineered kanamycin-resistant Escherichia coli HB101 containing the mutant chimeric plasmid pAW6194-T17 specifying biphenyl dioxygenase and dihydrodiol dehydrogenase and lacking the ability to produce active 3-phenylcatechol dioxygenase was used to produce 1,2-dihydroxy-4'-chlorobiphenyl (DHCB) from 4-chlorobiphenyl. Resting-cell suspensions of genetically engineered E. coli in mineral salts medium (pH 7.0) containing 880 microM 4-chlorobiphenyl produced 110 microM DHCB. The Km for 4-chlorobiphenyl was 3.3 mM. Biotransformation of DHCB from 4-chlorobiphenyl was maximum when cells (2.5 mg of protein per ml) were incubated with shaking (150 rpm) at pH 7.0 and 30 degrees C for 6 h. The enzymatically produced DHCB was a suitable substrate for assaying 3-phenylcatechol dioxygenase activity. Biologically produced DHCB showed UV and mass spectra similar to those of chemically synthesized DHCB. The bioconversion rate of ortho-substituted chlorobiphenyl was slower than that of the para- or meta-substituted chlorobiphenyl.  相似文献   

2.
Genes capable of 4-chlorobiphenyl (4-CBP) degradation were cloned from 4-CBP-degrading Pseudomonas putida OU83 by using a genomic library which was constructed in the broad-host-range cosmid vector pCP13. P. putida AC812 containing chimeric cosmid-expressing enzymes involved in the 4-CBP degradation pathway were identified by detecting 3-phenylcatechol dioxygenase activity (3-PDA). Chimeric cosmid clones pOH83, pOH84, pOH85, pOH87, and pOH88 positive for 3-PDA grew in synthetic basal medium containing 4-CBP (5 mM) as a carbon source. Restriction digestion analysis of recombinant cosmids showed DNA inserts ranging from 6 to 30 kilobase pairs. Southern hybridization data revealed that the cloned DNA inserts originated from strain OU83. Gas chromatography-mass spectrometry analysis of the metabolites of P. putida AC812(pOH88) incubated with 4-CBP and 4'-chloro-3-phenylcatechol showed the formation of 4-chlorobenzoic acid and benzoic acid. These results demonstrate that the cloned DNA fragments contain genes encoding for chlorobiphenyl dioxygenase (cbpA), dihydrodiol dehydrogenase (cbpB), 4'-chloro-3-phenylcatechol dioxygenase (cbpC), a meta-cleavage compound (a chloro derivative of 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoate) hydrolase (cbpD), and a new dechlorinating activity (dcpE). The location of the cbpC gene specifying 3-PDA was determined by subcloning an EcoRI DNA fragment (9.8 kilobase pairs) of pOH88 in plasmid vector pUC19. The cloned gene encoding 3-PDA was expressed in Escherichia coli HB101 and had substrate specificity only for 3-phenylcatechol and 4'-chloro-3-phenylcatechol.  相似文献   

3.
Genes capable of 4-chlorobiphenyl (4-CBP) degradation were cloned from 4-CBP-degrading Pseudomonas putida OU83 by using a genomic library which was constructed in the broad-host-range cosmid vector pCP13. P. putida AC812 containing chimeric cosmid-expressing enzymes involved in the 4-CBP degradation pathway were identified by detecting 3-phenylcatechol dioxygenase activity (3-PDA). Chimeric cosmid clones pOH83, pOH84, pOH85, pOH87, and pOH88 positive for 3-PDA grew in synthetic basal medium containing 4-CBP (5 mM) as a carbon source. Restriction digestion analysis of recombinant cosmids showed DNA inserts ranging from 6 to 30 kilobase pairs. Southern hybridization data revealed that the cloned DNA inserts originated from strain OU83. Gas chromatography-mass spectrometry analysis of the metabolites of P. putida AC812(pOH88) incubated with 4-CBP and 4'-chloro-3-phenylcatechol showed the formation of 4-chlorobenzoic acid and benzoic acid. These results demonstrate that the cloned DNA fragments contain genes encoding for chlorobiphenyl dioxygenase (cbpA), dihydrodiol dehydrogenase (cbpB), 4'-chloro-3-phenylcatechol dioxygenase (cbpC), a meta-cleavage compound (a chloro derivative of 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoate) hydrolase (cbpD), and a new dechlorinating activity (dcpE). The location of the cbpC gene specifying 3-PDA was determined by subcloning an EcoRI DNA fragment (9.8 kilobase pairs) of pOH88 in plasmid vector pUC19. The cloned gene encoding 3-PDA was expressed in Escherichia coli HB101 and had substrate specificity only for 3-phenylcatechol and 4'-chloro-3-phenylcatechol.  相似文献   

4.
Genes of Pseudomonas putida strains that are capable of degrading polychlorinated biphenyls were cloned in the plasmid vector pUC19. The resultant hybrid plasmid, pAW6194, contained cbpABCD genes on a 9.0-kb DNA fragment that was necessary for the catabolism of polychlorinated biphenyls. These genes were further subcloned on an 8.0-kb HindIII fragment of pAW540. Degradation of 3-chlorobiphenyl, 2,4-dichlorobiphenyl, and 2,4,5-trichlorobiphenyl into a chloro derivative of benzoic acid was found in Escherichia coli harboring chimeric plasmid pAW540. Expression of cbpA (biphenyl dioxygenase, 6.2 U/mg of protein) and cbpC (3-phenylcatechol dioxygenase, 611.00 U/mg of protein) genes was also found in E. coli containing the hybrid plasmid pAW540. These enzyme activities were up to 10-fold higher than those found in P. putida OU83. These results led us to conclude that cbpABCD genes of P. putida OU83 were encoded on cloned DNA and expressed in E. coli. Whether the expression of cbpABCD genes of P. putida OU83 was driven by its own promoters located on the cloned DNA or by the lacZ promoter of pUC19 was examined by subcloning a 8.0-kb DNA fragment encoding the cbpABCD genes, in both orientations, in the HindIII site of the promoter probe vector pKK232-8. The resulting recombinant plasmids, pAW560 and pAW561, expressed cbpABCD genes and conferred chloramphenicol resistance only in E. coli harboring pAW560, indicating that the expression of chloramphenicol acetyltransferase is independent of cbpABCD gene expression.(ABSTRACT TRUNCATED AT 250 WORDS)  相似文献   

5.
Genes of Pseudomonas putida strains that are capable of degrading polychlorinated biphenyls were cloned in the plasmid vector pUC19. The resultant hybrid plasmid, pAW6194, contained cbpABCD genes on a 9.0-kb DNA fragment that was necessary for the catabolism of polychlorinated biphenyls. These genes were further subcloned on an 8.0-kb HindIII fragment of pAW540. Degradation of 3-chlorobiphenyl, 2,4-dichlorobiphenyl, and 2,4,5-trichlorobiphenyl into a chloro derivative of benzoic acid was found in Escherichia coli harboring chimeric plasmid pAW540. Expression of cbpA (biphenyl dioxygenase, 6.2 U/mg of protein) and cbpC (3-phenylcatechol dioxygenase, 611.00 U/mg of protein) genes was also found in E. coli containing the hybrid plasmid pAW540. These enzyme activities were up to 10-fold higher than those found in P. putida OU83. These results led us to conclude that cbpABCD genes of P. putida OU83 were encoded on cloned DNA and expressed in E. coli. Whether the expression of cbpABCD genes of P. putida OU83 was driven by its own promoters located on the cloned DNA or by the lacZ promoter of pUC19 was examined by subcloning a 8.0-kb DNA fragment encoding the cbpABCD genes, in both orientations, in the HindIII site of the promoter probe vector pKK232-8. The resulting recombinant plasmids, pAW560 and pAW561, expressed cbpABCD genes and conferred chloramphenicol resistance only in E. coli harboring pAW560, indicating that the expression of chloramphenicol acetyltransferase is independent of cbpABCD gene expression.(ABSTRACT TRUNCATED AT 250 WORDS)  相似文献   

6.
In this work, we examined the profile of metabolites produced from the doubly para-substituted biphenyl analogs 4,4′-dihydroxybiphenyl, 4-hydroxy-4′-chlorobiphenyl, 3-hydroxy-4,4′-dichlorobiphenyl, and 3,3′-dihydroxy-4,4′-chlorobiphenyl by biphenyl-induced Pandoraea pnomenusa B356 and by its biphenyl dioxygenase (BPDO). 4-Hydroxy-4′-chlorobiphenyl was hydroxylated principally through a 2,3-dioxygenation of the hydroxylated ring to generate 2,3-dihydro-2,3,4-trihydroxy-4′-chlorobiphenyl and 3,4-dihydroxy-4′-chlorobiphenyl after the removal of water. The former was further oxidized by the biphenyl dioxygenase to produce ultimately 3,4,5-trihydroxy-4′-chlorobiphenyl, a dead-end metabolite. 3-Hydroxy-4,4′-dichlorobiphenyl was oxygenated on both rings. Hydroxylation of the nonhydroxylated ring generated 2,3,3′-trihydroxy-4′-chlorobiphenyl with concomitant dechlorination, and 2,3,3′-trihydroxy-4′-chlorobiphenyl was ultimately metabolized to 2-hydroxy-4-chlorobenzoate, but hydroxylation of the hydroxylated ring generated dead-end metabolites. 3,3′-Dihydroxy-4,4′-dichlorobiphenyl was principally metabolized through a 2,3-dioxygenation to generate 2,3-dihydro-2,3,3′-trihydroxy-4,4′-dichlorobiphenyl, which was ultimately converted to 3-hydroxy-4-chlorobenzoate. Similar metabolites were produced when the biphenyl dioxygenase of Burkholderia xenovorans LB400 was used to catalyze the reactions, except that for the three substrates used, the BPDO of LB400 was less efficient than that of B356, and unlike that of B356, it was unable to further oxidize the initial reaction products. Together the data show that BPDO oxidation of doubly para-substituted hydroxychlorobiphenyls may generate nonnegligible amounts of dead-end metabolites. Therefore, biphenyl dioxygenase could produce metabolites other than those expected, corresponding to dihydrodihydroxy metabolites from initial doubly para-substituted substrates. This finding shows that a clear picture of the fate of polychlorinated biphenyls in contaminated sites will require more insights into the bacterial metabolism of hydroxychlorobiphenyls and the chemistry of the dihydrodihydroxylated metabolites derived from them.  相似文献   

7.
Optimized plant-microbe bioremediation processes in which the plant initiates the metabolism of xenobiotics and releases the metabolites in the rhizosphere to be further degraded by the rhizobacteria is a promising alternative to restore contaminated sites in situ. However, such processes require that plants produce the metabolites that bacteria can readily oxidize. The biphenyl dioxygenase is the first enzyme of the bacterial catabolic pathway involved in the degradation of polychlorinated biphenyls. This enzyme consists of three components: the two sub-unit oxygenase (BphAE) containing a Rieske-type iron-sulfur cluster and a mononuclear iron center, the Rieske-type ferredoxin (BphF), and the FAD-containing ferredoxin reductase (BphG). In this work, based on analyses with Nicotiana benthamiana plants transiently expressing the biphenyl dioxygenase genes from Burkholderia xenovorans LB400 and transgenic Nicotiana tabacum plants transformed with each of these four genes, we have shown that each of the three biphenyl dioxygenase components can be produced individually as active protein in tobacco plants. Therefore, when BphAE, BphF, and BphG purified from plant were used to catalyze the oxygenation of 4-chlorobiphenyl, detectable amounts of 2,3-dihydro-2, 3-dihydroxy-4'-chlorobiphenyl were produced. This suggests that creating transgenic plants expressing simultaneously all four genes required to produce active biphenyl dioxygenase is feasible.  相似文献   

8.
Axenic methanotrophic culture strain CSC1, which was derived from a mixed-culture aquifer sample, oxidized 2-chlorobiphenyl to a hydroxylated chlorobiphenyl intermediate identified as 2-hydroxy-3-chlorobiphenyl. Minor amounts of another hydroxylated chlorobiphenyl isomer, assumed to be the nonmigrated product 3-hydroxy-2-chlorobiphenyl, were found in axenic culture incubation extracts. The occurrence of chlorine migration during oxidation indicates that the metabolite is formed via a concerted oxidation mechanism involving an arene oxide, which rearranges spontaneously to the hydroxylated intermediate via an NIH shift mechanism.  相似文献   

9.
Summary A bioconversion process was demonstrated by manipulation of catabolic genes. Catabolic intermediates of various biphenyl compounds could be efficiently produced by Pseudomonas aeruginosa carrying recombinant plasmids containing a set of cloned bph genes. A dihydrodiol compound was produced by the strain carrying plasmid pMFB4 containing bphA (encoding biphenyl dioxygenase) gene. A dihydroxy compound was produced from 4-chlorobiphenyl by the strain carrying plasmid pMFB6 containing bphA and bphB (encoding dihydrodiol dehydrogenase) genes. Tetrahydroxybiphenyl was accumulated as the final product via dihydroxybiphenyl from biphenyl by the same pMFB6 carrying strain. Meta-cleavage yellow compounds were produced from biphenyl and its derivatives substituted with methyl, chloro, bromo, or nitro group on one of the biphenyl rings by the strain carrying plasmid pMFB2 containing bphA, bphB and bphC (encoding dihydroxybiphenyl dioxygenase) genes.  相似文献   

10.
The role of cell shape in chondrogenesis was studied by using rat mesenchymal cells cultured with cartilage-inducing factor (CIF). Here we report that enhanced expression of chondroblastic markers by induced cells was attained by culturing cells in monolayer in the presence of dihydrocytochalasin B (DHCB). This effect was optimal at 3 microM DHCB and was apparent after 3 days in culture. Mesenchymal cells cultured with DHCB alone exhibited no detectable increase in cartilage proteoglycan synthesis, whereas cells cultured with 3 microM DHCB and 0.1 nM CIF showed a 4-5 fold increase in proteoglycan synthesis. When cells were cultured with CIF alone on plastic, only small increases in proteoglycan synthesis were observed. Cells cultured with CIF in monolayer and then transferred to a permissive environment (either agarose or cultured with DHCB) showed enhanced synthesis of chondroblastic proteins. These results suggest that expression, but not induction, of a chondroblastic phenotype by CIF is inhibited by growth in monolayer. The altering of cell shape with DHCB releases that inhibition.  相似文献   

11.
C Wyndham  S Safe 《Biochemistry》1978,17(2):208-215
The in vitro metabolism, mechanism of metabolism, and macromolecular binding of a monochlorobiphenyl component of commercial polychlorinated biphenyls (PCB) have been investigated. 4-Chlorobiphenyl was metabolized by rat liver microsomes in the presence of NADPH to yield a major metabolite, 4'-chloro-4-biphenylol, and a number of minor metabolites. The metabolism of deuterium-labeled 4-chlorobiphenyl proceeded with the NIH shift of the isotope and no observed isotope effect thus indicating the intermediacy of an arene oxide. Noninduced rat liver microsomes mediated the covalent binding between the 4-chlorobiphenyl and 4'-chloro-4-biphenylol substrates and endogenous microsomal protein. Prior in vivo administration of a commericial PCB preparation, Aroclor 1248 (Monsanto Chemical Co., containing 48 percent by weight of chlorine), resulted in an induced microsomal preparation which significantly increased the substrate-protein binding. The effect of various inhibitors on protein binding was investigated. Aroclor 1248 induced microsomes mediated binding of 4-chlorobiphenyl to endogenous and exogenous nucleic acids, indicating a possible mechanism for the previously reported mutagenic action of this chlorobiphenyl. The spectral properties of Aroclor 1248 induced cytochrome P-450 were investigated and compared with the pentobarbital-induced cytochrome fraction.  相似文献   

12.
Growth of five strains of sulfur-oxidizing bacteria Acidithiobacillus thiooxidans, including strain NB1-3, was inhibited completely by 50 microM of sodium tungstate (Na(2)WO(4)). When the cells of NB1-3 were incubated in 0.1 M beta-alanine-SO(4)(2-) buffer (pH 3.0) with 100 microM Na(2)WO(4) for 1 h, the amount of tungsten bound to the cells was 33 microg/mg protein. Approximately 10 times more tungsten was bound to the cells at pH 3.0 than at pH 7.0. The tungsten binding to NB1-3 cells was inhibited by oxyanions such as sodium molybdenum and ammonium vanadate. The activities of enzymes involved in elemental sulfur oxidation of NB1-3 cells such as sulfur oxidase, sulfur dioxygenase, and sulfite oxidase were strongly inhibited by Na(2)WO(4). These results indicate that tungsten binds to NB1-3 cells and inhibits the sulfur oxidation enzyme system of the cells, and as a result, inhibits cell growth. When portland cement bars supplemented with 0.075% metal nickel and with 0.075% metal nickel and 0.075% calcium tungstate were exposed to the atmosphere of a sewage treatment plant containing 28 ppm of H(2)S for 2 years, the weight loss of the portland cement bar with metal nickel and calcium tungstate was much lower than the cement bar containing 0.075% metal nickel.  相似文献   

13.
李朔  许楹  周宁一 《微生物学通报》2017,44(7):1513-1524
【目的】研究Sphingomonas sp.YL-JM2C菌株的生长特性,确定以三氯卡班作为碳源的生长情况。挖掘菌株YL-JM2C潜在的邻苯二酚1,2-双加氧酶及邻苯二酚2,3-双加氧酶基因,在大肠杆菌(Escherichia coli)中异源表达邻苯二酚双加氧酶基因并研究其酶学性质。【方法】优化S.sp.YL-JM2C菌株以三氯卡班作为碳源时的培养条件,并利用全自动生长曲线测定仪测定菌株生长情况,绘制生长曲线。通过生物信息学方法挖掘潜在的邻苯二酚双加氧酶基因,并分别在Escherichia coli BL21(DE3)中进行异源表达,通过AKTA快速纯化系统纯化蛋白,分别以邻苯二酚、3-和4-氯邻苯二酚为底物检测重组蛋白的酶学特性。【结果】菌株在pH为7.0-7.5时生长最优。在以浓度为4-8 mg/L的三氯卡班做为底物时,菌株适宜生长。当R2A培养基仅含有0.01%酵母提取物和无机盐时,加入终浓度为4 mg/L的三氯卡班可促进菌株生长。挖掘到6个潜在的邻苯二酚双加氧酶基因stcA1、stcA2、stcA3、stcE1、stcE2和stcE3,表达并通过粗酶液分析证明其中5个基因stcA1、stcA2、stcA3、stcE1和stcE2编码的酶均具有邻苯二酚双加氧酶和氯邻苯二酚双加氧酶的活性;纯化酶的底物范围研究揭示了StcA1、StcA2和StcA3均属于Ⅱ型邻苯二酚1,2-双加氧酶,StcE1和StcE2为两个新型邻苯二酚2,3-双加氧酶;它们酶动力学分析研究证明了5个酶对邻苯二酚的亲和力和催化效率最高,4-氯邻苯二酚次之。【结论】在同一菌株中发现了5个具有功能的邻苯二酚双加氧酶基因,stcA1、stcA2和stcA3编码的酶均属于Ⅱ型邻苯二酚1,2-双加氧酶,stcE1和stcE2为两个新型邻苯二酚2,3-双加氧酶编码基因。5个酶均具有催化邻苯二酚和氯邻苯二酚开环反应的功能,这为更好地理解微生物基因组内代谢邻苯二酚及其衍生物氯代邻苯二酚基因的多样性奠定了基础。  相似文献   

14.
In this work we have characterized the galA gene product from Pseudomonas putida KT2440, a ring-cleavage dioxygenase that acts specifically on gallate to produce 4-oxalomesaconate. The protein is a trimer composed by three identical subunits of 47.6 kDa (419 amino acids) that uses Fe2+ as the main cofactor. The gallate dioxygenase showed maximum activity at pH 7.0, and the Km and Vmax values for gallate were 144 microM and 53.2 micromol/min/mg of protein, respectively. A phylogenetic study suggests that the gallate dioxygenase from P. putida KT2440 is the prototype of a new subgroup of type II extradiol dioxygenases that share a common ancestor with protocatechuate 4,5-dioxygenases and whose two-domain architecture might have evolved from the fusion of the large and small subunits of the latter. A three-dimensional model for the N-terminal domain (residues 1-281) and C-terminal domain (residues 294-420) of the gallate dioxygenase from P. putida KT2440 was generated by comparison with the crystal structures of the large (LigB) and small (LigA) subunits of the protocatechuate 4,5-dioxygenase from Sphingomonas paucimobilis SYK-6. The expression of the galA gene was specifically induced when P. putida KT2440 cells grew in the presence of gallate. A P. putida KT2440 galA mutant strain was unable to use gallate as the sole carbon source and it did not show gallate dioxygenase activity, suggesting that the GalA protein is the only dioxygenase involved in gallate cleavage in this bacterium. This work points to the existence of a new pathway that is devoted to the catabolism of gallic acid and that remained unknown in the paradigmatic P. putida KT2440 strain.  相似文献   

15.
The bphC and bphD genes of Pseudomonas putida involved in the catabolism of polychlorinated biphenyls or biphenyl were identified, localized, and studied for expression in Escherichia coli. This was achieved by cloning a 2.4-kilobase (kb) DNA fragment of recombinant cosmid pOH101 into HindIII site of pUC plasmids downstream of a lacZ promoter and measuring the enzyme activities of 3-phenylcatechol dioxygenase (3-PDase; a product of bphC) and the meta-cleavage product 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoate hydrolase (a product of bphD). The amount of 3-PDase produced in E. coli was about 20 times higher than that of the enzyme produced by the parent, P. putida. Determination of expression of the bphC and bphD genes through their own promoter sequences or by using the lacZ promoter of pUC plasmids was done by cloning the DNA that encodes bphC and bphD genes in a HindIII site of a promoter selection vector (pKK232-8) upstream of the gene for chloramphenicol acetyltransferase (CAT). The recombinant plasmid (pAW787) constructed by inserting the 2.4-kb DNA in pKK232-8 expressed both 3-PDase and CAT activities. Another hybrid construct (pAW786) in which the DNA insert was cloned in the opposite orientation lacked CAT activity but produced normal amounts of 3-PDase activity. On the basis of these results, we suggest that the bphC and bphD genes were expressed by using promoter sequences that are independent of the promoter that expresses CAT activity in E. coli. The locations of the bphC and bphD genes were determined by insertional inactivation of the open reading frames of structural genes bphC and bphD by Tn5 mutagenesis.(ABSTRACT TRUNCATED AT 250 WORDS)  相似文献   

16.
A cDNA clone encoding amorpha-4,11-diene synthase from Artemisia annua was subcloned into a bacterial expression vector in frame with a His6-tag. Recombinant amorpha-4,11-diene synthase was produced in Escherichia coli and purified to apparent homogeneity. The enzyme showed pH optimum at pH 6.5, and a minimum at pH 7.5. Substantial activity was observed in the presence of Mg2+, Mn2+ or Co2+ as cofactor. The enzyme exhibits a low activity in the presence of Ni2+ and essentially no activity with Cu2+ or Zn2+. The sesquiterpenoids produced from farnesyl diphosphate in the presence of Mg2+ were analyzed by GC-MS. In addition to amorpha-4,11-diene, 15 sesquiterpenoids were produced. Only small quantitative differences in product pattern were observed at pH 6.5, 7.5, or 9.5. Amorpha-4,11-diene synthase showed significant increased product selectivity in the presence of Mn2+ or Co2+. Km for farnesyl diphosphate was 3.3, 8.0, and 0.7 microM in the presence of Mg2+, Mn2+ or Co2+, respectively. The corresponding kcat-values were 6.8, 15.0, and 1.3 x 10(-3) s(-1), respectively. Km and kcat for geranyl diphosphate were 16.9 microM and 7.0 x 10(-4) s(-1), respectively, at pH 6.5, in the presence of Mn2+.  相似文献   

17.
Phenotypic expression of chondrocytes can be modulated in vitro by changing the culture technique and by agents such vitamins and growth factors. We studied the effects of ascorbic acid, retinoic acid (0.5 and 10 microM), and dihydrocytochalasin B (3, 10, 20 microM DHCB), separately or in combination (ascorbic acid + retinoic acid or ascorbic acid + DHCB), on the induction of maturation of fetal bovine epiphyseal chondrocytes grown for up to 4 weeks at high density in medium containing 10% fetal calf serum and the various agents. In the absence of any agent or with retinoic acid or DHCB alone, the metabolic activity of the cells remained very low after day 6, with no induction of type I or X collagen synthesis nor increase in alkaline phosphatase activity. Chondrocytes treated with fresh ascorbic acid showed active protein synthesis associated with expression of types I and X after 6 and 13 days, respectively. This maturation was not accompanied by obvious hypertrophy of the cells or high alkaline phosphatase activity. Addition of retinoic acid to the ascorbic acid-treated cultures decreased the level of type II collagen synthesis and delayed the induction of types I and X collagen, which were present only after 30 days. A striking increase in alkaline phosphatase activity (15-20-fold) was observed in the presence of both ascorbic acid and the highest dose of retinoic acid (10 microM). DHCB was also a potent inhibitor of the maturation induced by treatment with ascorbic acid, as the chondrocytes maintained their rounded shape and synthesized type II collagen without induction of type I or X collagen. The pattern of protein secretion was compared under all culture conditions by two-dimensional gel electrophoresis. The different regulations of chondrocyte differentiation by ascorbic acid, retinoic acid, and DHCB were confirmed by the important qualitative and quantitative changes in the pattern of secreted proteins observed by two-dimensional gel electrophoresis along the study.  相似文献   

18.
The bphC and bphD genes of Pseudomonas putida involved in the catabolism of polychlorinated biphenyls or biphenyl were identified, localized, and studied for expression in Escherichia coli. This was achieved by cloning a 2.4-kilobase (kb) DNA fragment of recombinant cosmid pOH101 into HindIII site of pUC plasmids downstream of a lacZ promoter and measuring the enzyme activities of 3-phenylcatechol dioxygenase (3-PDase; a product of bphC) and the meta-cleavage product 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoate hydrolase (a product of bphD). The amount of 3-PDase produced in E. coli was about 20 times higher than that of the enzyme produced by the parent, P. putida. Determination of expression of the bphC and bphD genes through their own promoter sequences or by using the lacZ promoter of pUC plasmids was done by cloning the DNA that encodes bphC and bphD genes in a HindIII site of a promoter selection vector (pKK232-8) upstream of the gene for chloramphenicol acetyltransferase (CAT). The recombinant plasmid (pAW787) constructed by inserting the 2.4-kb DNA in pKK232-8 expressed both 3-PDase and CAT activities. Another hybrid construct (pAW786) in which the DNA insert was cloned in the opposite orientation lacked CAT activity but produced normal amounts of 3-PDase activity. On the basis of these results, we suggest that the bphC and bphD genes were expressed by using promoter sequences that are independent of the promoter that expresses CAT activity in E. coli. The locations of the bphC and bphD genes were determined by insertional inactivation of the open reading frames of structural genes bphC and bphD by Tn5 mutagenesis.(ABSTRACT TRUNCATED AT 250 WORDS)  相似文献   

19.
Abstract In this report we describe the development and construction of a DNA module which encodes bph genes for the metabolism of PCBs and which is capable of stable integration into the chromosome of Gram negative bacteria. Introduction of the bph -module into Pseudomonas putida KT2442, Pseudomonas sp. strain B13 and its genetically engineered derivative B13FR1 expanded the biodegradative ability of these strains to include biphenyl and 4-chlorobiphenyl. The bph operon was stably inherited under laboratory conditions. Behavior of the genetically engineered strains was evaluated under simulated natural habitat conditions in lake sediment microcosms with respect to survival and removal of 4-chlorobiphenyl. The genetically engineered strains persisted under these conditions and were effective in degrading 4-chlorobiphenyl over a five day incubation period.  相似文献   

20.
The reaction of superoxide radical (O2-) with the heme protein indoleamine 2,3-dioxygenase has been investigated by the use of pulse radiolysis. In the absence of the substrate tryptophan (Trp), the ferric enzyme reacted quantitatively with O2- to form the oxygenated enzyme. The rate constant for the reaction (8.0 x 10(6) M-1 s-1 at pH 7.0) increased with a decrease in pH. In the presence of low concentrations of L-Trp (approximately 50 microM), under which the catalytic site of the ferric enzyme is greater than 99% Trp-free at pH 7.0, the only spectral species observed upon O2- binding was L-Trp-bound oxygenated enzyme, the ternary complex. This suggests that under the conditions employed O2- binds first to the ferric enzyme to form the oxygenated enzyme and is followed by rapid binding of L-Trp. It was also found that absorbance changes (delta A) for the enzyme after the pulse were significantly decreased when an increased L-Trp concentration was employed. A 50% decrease in delta A was caused with approximately 50 microM L-Trp at pH 7.0. Similar results were also observed with other indole derivatives with decreasing delta A values in the order of indole, 3-indoleethanol, alpha-methyl-DL-Trp, and D-Trp. These results suggest that there exists a binding site for these compounds in the dioxygenase different from the catalytic site for Trp and, most significantly, that binding of Trp to the effector binding site of the ferric enzyme markedly inhibits its reaction with O2-.  相似文献   

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