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1.
J Clin Invest ; 129(2): 863-874, 2019 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-30667376

RESUMO

Inherited retinal degenerations are a common cause of untreatable blindness worldwide, with retinitis pigmentosa and cone dystrophy affecting approximately 1 in 3500 and 1 in 10,000 individuals, respectively. A major limitation to the development of effective therapies is the lack of availability of animal models that fully replicate the human condition. Particularly for cone disorders, rodent, canine, and feline models with no true macula have substantive limitations. By contrast, the cone-rich macula of a nonhuman primate (NHP) closely mirrors that of the human retina. Consequently, well-defined NHP models of heritable retinal diseases, particularly cone disorders that are predictive of human conditions, are necessary to more efficiently advance new therapies for patients. We have identified 4 related NHPs at the California National Primate Research Center with visual impairment and findings from clinical ophthalmic examination, advanced retinal imaging, and electrophysiology consistent with achromatopsia. Genetic sequencing confirmed a homozygous R565Q missense mutation in the catalytic domain of PDE6C, a cone-specific phototransduction enzyme associated with achromatopsia in humans. Biochemical studies demonstrate that the mutant mRNA is translated into a stable protein that displays normal cellular localization but is unable to hydrolyze cyclic GMP (cGMP). This NHP model of a cone disorder will not only serve as a therapeutic testing ground for achromatopsia gene replacement, but also for optimization of gene editing in the macula and of cone cell replacement in general.


Assuntos
Distrofia de Cones , Nucleotídeo Cíclico Fosfodiesterase do Tipo 6 , Modelos Animais de Doenças , Proteínas do Olho , Mutação de Sentido Incorreto , Retinose Pigmentar , Substituição de Aminoácidos , Animais , Defeitos da Visão Cromática/enzimologia , Defeitos da Visão Cromática/genética , Defeitos da Visão Cromática/patologia , Distrofia de Cones/enzimologia , Distrofia de Cones/genética , Distrofia de Cones/patologia , Nucleotídeo Cíclico Fosfodiesterase do Tipo 6/genética , Nucleotídeo Cíclico Fosfodiesterase do Tipo 6/metabolismo , Proteínas do Olho/genética , Proteínas do Olho/metabolismo , Feminino , Células HEK293 , Humanos , Macaca mulatta , Masculino , Retinose Pigmentar/enzimologia , Retinose Pigmentar/genética , Retinose Pigmentar/patologia
2.
Cell Signal ; 37: 74-80, 2017 09.
Artigo em Inglês | MEDLINE | ID: mdl-28583373

RESUMO

Mutations in PDE6 genes encoding the effector enzymes in rods and cones underlie severe retinal diseases including retinitis pigmentosa (RP), autosomal dominant congenital stationary night blindness (adCSNB), and achromatopsia (ACHM). Here we examined a spectrum of pathogenic missense mutations in PDE6 using the system based on co-expression of cone PDE6C with its specialized chaperone AIPL1 and the regulatory Pγ subunit as a potent co-chaperone. We uncovered two mechanisms of PDE6C mutations underlying ACHM: (a) folding defects leading to expression of catalytically inactive proteins and (b) markedly diminished ability of Pγ to co-chaperone mutant PDE6C proteins thereby dramatically reducing the levels of functional enzyme. The mechanism of the Rambusch adCSNB associated with the H258N substitution in PDE6B was probed through the analysis of the model mutant PDE6C-H262N. We identified two interrelated deficits of PDE6C-H262N: disruption of the inhibitory interaction of Pγ with mutant PDE6C that markedly reduced the ability of Pγ to augment the enzyme folding. Thus, we conclude that the Rambusch adCSNB is triggered by low levels of the constitutively active PDE6. Finally, we examined PDE6C-L858V, which models PDE6B-L854V, an RP-linked mutation that alters the protein isoprenyl modification. This analysis suggests that the type of prenyl modifications does not impact the folding of PDE6, but it modulates the enzyme affinity for its trafficking partner PDE6D. Hence, the pathogenicity of PDE6B-L854V likely arises from its trafficking deficiency. Taken together, our results demonstrate the effectiveness of the PDE6C expression system to evaluate pathogenicity and elucidate the mechanisms of PDE6 mutations in retinal diseases.


Assuntos
Nucleotídeo Cíclico Fosfodiesterase do Tipo 6/genética , Proteínas do Olho/genética , Mutação de Sentido Incorreto , Doenças Retinianas/genética , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Defeitos da Visão Cromática/genética , Defeitos da Visão Cromática/metabolismo , Nucleotídeo Cíclico Fosfodiesterase do Tipo 6/análise , Nucleotídeo Cíclico Fosfodiesterase do Tipo 6/metabolismo , Oftalmopatias Hereditárias/genética , Oftalmopatias Hereditárias/metabolismo , Proteínas do Olho/análise , Proteínas do Olho/metabolismo , Expressão Gênica , Doenças Genéticas Ligadas ao Cromossomo X/genética , Doenças Genéticas Ligadas ao Cromossomo X/metabolismo , Células HEK293 , Humanos , Camundongos , Modelos Moleculares , Miopia/genética , Miopia/metabolismo , Cegueira Noturna/genética , Cegueira Noturna/metabolismo , Dobramento de Proteína , Prenilação de Proteína , Doenças Retinianas/metabolismo
3.
J Biol Chem ; 291(31): 16282-91, 2016 07 29.
Artigo em Inglês | MEDLINE | ID: mdl-27268253

RESUMO

Phosphodiesterase 6 (PDE6) is the effector enzyme in the phototransduction cascade and is critical for the health of both rod and cone photoreceptors. Its dysfunction, caused by mutations in either the enzyme itself or AIPL1 (aryl hydrocarbon receptor-interacting protein-like 1), leads to retinal diseases culminating in blindness. Progress in research on PDE6 and AIPL1 has been severely hampered by failure to express functional PDE6 in a heterologous expression system. Here, we demonstrated that AIPL1 is an obligate chaperone of PDE6 and that it enables low yield functional folding of cone PDE6C in cultured cells. We further show that the AIPL1-mediated production of folded PDE6C is markedly elevated in the presence of the inhibitory Pγ-subunit of PDE6. As illustrated in this study, a simple and sensitive system in which AIPL1 and Pγ are co-expressed with PDE6 represents an effective tool for probing structure-function relationships of AIPL1 and reliably establishing the pathogenicity of its variants.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Nucleotídeo Cíclico Fosfodiesterase do Tipo 6/metabolismo , Chaperonas Moleculares/metabolismo , Doenças Retinianas/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Células COS , Chlorocebus aethiops , Nucleotídeo Cíclico Fosfodiesterase do Tipo 6/genética , Células HEK293 , Humanos , Camundongos , Chaperonas Moleculares/genética , Doenças Retinianas/genética
4.
FEBS J ; 282(3): 550-61, 2015 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-25425538

RESUMO

Uncoordinated 119 protein (UNC119) is a partner of transducin-α subunit (Gαt ) that is essential for transducin trafficking in rod photoreceptors. The interaction is known to involve binding of the acylated N terminus of Gαt to the hydrophobic pocket of UNC119. To gain insights into the mechanism of transducin trafficking, we isolated a highly pure protein complex between myristoylated chimeric Gαt (Gαt *) and UNC11950₋240, and examined the solution structure by small angle X-ray scattering and chemical crosslinking. The solution structure of the Gαt -UNC11950₋240 complex was derived with rigid body/ab initio modeling against the small angle X-ray scattering data by use of known atomic structures of Gαt and UNC119, and a distance constraint based on the protein crosslinking with p-phenyldimaleimide. The model of the Gαt -UNC11950₋240 complex indicates rotation and bending of the N-terminal α-helix of Gαt from its position in the structure of the heterotrimeric G-protein transducin (Gt ). This allows a considerably more compact complex conformation, which also suggests a novel interface involving the switch II/α3-ß5 surface of Gαt . Supporting a novel interface, UNC119 was found to bind full-length Gαt * more strongly than the Gαt N-terminal peptide. Furthermore, UNC119 competed with the effector molecule phosphodiesterase-6 γ-subunit, which is known to bind to the same surface of Gαt . The solution structure of the Gαt -UNC119 complex suggests that the ability of UNC119 to dissociate Gt subunits and release Gαt from the membrane is attributable to disruption and sterical occlusion of the Gß1γ1-binding sites on Gαt .


Assuntos
Transducina/química , Transducina/metabolismo , Sítios de Ligação , Modelos Moleculares , Estrutura Secundária de Proteína , Células Fotorreceptoras Retinianas Bastonetes/metabolismo
5.
J Biol Chem ; 288(29): 21320-21328, 2013 Jul 19.
Artigo em Inglês | MEDLINE | ID: mdl-23737531

RESUMO

Aryl hydrocarbon receptor-interacting protein-like 1 (AIPL1) is a photoreceptor specific chaperone of the visual effector enzyme phosphodiesterase-6 (PDE6). AIPL1 has been shown to bind the farnesylated PDE6A subunit. Mutations in AIPL1 are thought to destabilize PDE6 and thereby cause Leber congenital amaurosis type 4 (LCA4), a severe form of childhood blindness. Here, we examined the solution structure of AIPL1 by small angle x-ray scattering. A structural model of AIPL1 with the best fit to the scattering data features two independent FK506-binding protein (FKBP)-like and tetratricopeptide repeat domains. Guided by the model, we tested the hypothesis that AIPL1 directly binds the farnesyl moiety. Our studies revealed high affinity binding of the farnesylated-Cys probe to the FKBP-like domain of AIPL1, thus uncovering a novel function of this domain. Mutational analysis of the potential farnesyl-binding sites on AIPL1 identified two critical residues, Cys-89 and Leu-147, located in close proximity in the structure model. The L147A mutation and the LCA-linked C89R mutation prevented the binding of the farnesyl-Cys probe to AIPL1. Furthermore, Cys-89 and Leu-147 flank the unique insert region of AIPL1, deletion of which also abolished the farnesyl interaction. Our results suggest that the binding of PDE6A farnesyl is essential to normal function of AIPL1 and its disruption is one of the mechanisms underlying LCA.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Prenilação , Proteínas Adaptadoras de Transdução de Sinal/química , Animais , Sítios de Ligação , Cisteína/metabolismo , Humanos , Amaurose Congênita de Leber/genética , Camundongos , Modelos Moleculares , Proteínas Mutantes/metabolismo , Mutação/genética , Ligação Proteica , Estrutura Terciária de Proteína , Espalhamento a Baixo Ângulo , Difração de Raios X
6.
Biochemistry ; 51(8): 1617-24, 2012 Feb 28.
Artigo em Inglês | MEDLINE | ID: mdl-22324825

RESUMO

The molecular nature of transducin-α subunits (Gα(t)) may contribute to the distinct physiology of cone and rod photoreceptors. Biochemical properties of mammalian cone Gα(t2) subunits and their differences with rod Gα(t1) are largely unknown. Here, we examined properties of chimeric Gα(t2) in comparison with its rod counterpart. The key biochemical difference between the rod- and cone-like Gα(t) was ~10-fold higher intrinsic nucleotide exchange on the chimeric Gα(t2). Presented mutational analysis suggests that weaker interdomain interactions between the GTPase (Ras-like) domain and the helical domain in Gα(t2) are in part responsible for its increased spontaneous nucleotide exchange. However, the rates of R*-dependent nucleotide exchange of chimeric Gα(t2) and Gα(t1) were equivalent. Furthermore, chimeric Gα(t2) and Gα(t1) exhibited similar rates of intrinsic GTPase activity as well as similar acceleration of GTP hydrolysis by the RGS domain of RGS9. Our results suggest that the activation and inactivation properties of cone and rod Gα(t) subunits in an in vitro reconstituted system are comparable.


Assuntos
Transducina/química , Animais , Bovinos , Células Cultivadas , GTP Fosfo-Hidrolases/química , GTP Fosfo-Hidrolases/metabolismo , Estrutura Secundária de Proteína , Células Fotorreceptoras Retinianas Cones/metabolismo , Células Fotorreceptoras Retinianas Bastonetes/metabolismo , Transducina/metabolismo
7.
J Biol Chem ; 286(33): 28954-28962, 2011 Aug 19.
Artigo em Inglês | MEDLINE | ID: mdl-21712387

RESUMO

The key visual G protein, transducin undergoes bi-directional translocations between the outer segment (OS) and inner compartments of rod photoreceptors in a light-dependent manner thereby contributing to adaptation and neuroprotection of rods. A mammalian uncoordinated 119 protein (UNC119), also known as Retina Gene 4 protein (RG4), has been recently implicated in transducin transport to the OS in the dark through its interaction with the N-acylated GTP-bound transducin-α subunit (Gα(t1)). Here, we demonstrate that the interaction of human UNC119 (HRG4) with transducin is dependent on the N-acylation, but does not require the GTP-bound form of Gα(t1). The lipid specificity of UNC119 is unique: UNC119 bound the myristoylated N terminus of Gα(t1) with much higher affinity than a prenylated substrate, whereas the homologous prenyl-binding protein PrBP/δ did not interact with the myristoylated peptide. UNC119 was capable of interacting with Gα(t1)GDP as well as with heterotrimeric transducin (G(t)). This interaction of UNC119 with G(t) led to displacement of Gß(1)γ(1) from the heterotrimer. Furthermore, UNC119 facilitated solubilization of G(t) from dark-adapted rod OS membranes. Consistent with these observations, UNC119 inhibited rhodopsin-dependent activation of G(t), but had no effect on the GTP-hydrolysis by Gα(t1). A model for the role of UNC119 in the IS→OS translocation of G(t) is proposed based on the UNC119 ability to dissociate G(t) subunits from each other and the membrane. We also found that UNC119 inhibited activation of G(o) by D2 dopamine receptor in cultured cells. Thus, UNC119 may play conserved inhibitory role in regulation of GPCR-G protein signaling in non-visual tissues.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Membrana Celular/metabolismo , Modelos Biológicos , Células Fotorreceptoras Retinianas Bastonetes/metabolismo , Transducina/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Bovinos , Membrana Celular/genética , Proteínas Heterotriméricas de Ligação ao GTP/genética , Proteínas Heterotriméricas de Ligação ao GTP/metabolismo , Humanos , Transporte Proteico/fisiologia , Receptores Dopaminérgicos/genética , Receptores Dopaminérgicos/metabolismo , Células Fotorreceptoras Retinianas Bastonetes/citologia , Transdução de Sinais/fisiologia , Transducina/genética
8.
J Med Virol ; 82(2): 239-48, 2010 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-20029802

RESUMO

Hepatitis C is an oncogenic virus although the mechanisms responsible for this behavior are not clear. We studied the effects of hepatitis C virus (HCV) core protein expression on Telomerase, an enzyme closely associated with cellular immortalization and neoplasia. The aim of this study was to investigate the effects of HCV core protein on the regulation of Telomerase activity in human hepatoma cells. Regulation and expression of human Telomerase reverse transcriptase (TERT) was compared in Huh7 cells stably transfected with HCV core protein or cells expressing vector alone. Telomerase activity was measured using Quantitative Telomerase Detection (QTD) and telomere length was measured by fluorescence in situ hybridization (FISH). Transient transfection and luciferase assay were used to evaluate TERT promoter activity. Telomerase activity was increased twofold in Huh7 cells expressing HCV core protein compared to controls (P < 0.01). This was accompanied by a 1.4-fold increase of TERT mRNA and 1.9-fold increase in TERT protein (P < 0.01 in either case). Cellular fractionation and immunocytochemical studies showed increased localization of TERT in the nucleus of core-expressing cells as compared to controls. FISH assay confirmed that telomeres of HCV core-expressing Huh7 cells were relatively longer than those of control cells (0.22 + 0.05 vs. 0.12 + 0.03, P < 0.01). TERT promoter activity was enhanced about 30% in HCV core-expressing Huh7 cells compared to control cells (P < 0.02). HCV core protein is associated with increased Telomerase activity in hepatoma cells. These findings suggest that enhancement of Telomerase activity by HCV core protein may contribute to the oncogenicity of HCV.


Assuntos
Hepacivirus/patogenicidade , Hepatócitos/virologia , Interações Hospedeiro-Patógeno , Telomerase/biossíntese , Proteínas do Core Viral/metabolismo , Fusão Gênica Artificial , Linhagem Celular Tumoral , Núcleo Celular/química , Perfilação da Expressão Gênica , Genes Reporter , Humanos , Hibridização in Situ Fluorescente/métodos , Luciferases/biossíntese , Luciferases/genética , Telômero/genética , Regulação para Cima
9.
Protein Expr Purif ; 50(2): 196-202, 2006 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-16829135

RESUMO

Recent studies have indicated that the loop harboring the S1 specificity site (residues 185-189 in chymotrypsin numbering) of coagulation proteases has several charged residues with important structural and functional roles for the catalytic activity of these proteases. This loop is allosterically linked to the Na(+)-binding site in both factor Xa and thrombin. There are three candidate residues (His-185, Glu-186, and Arg-188) on this loop of factor IXa (fIXa) whose side chains can influence the Na(+) binding and the catalytic function of the protease in the intrinsic Xase complex. In this study, we developed a novel expression/purification vector system, substituted all three residues of factor IX individually with Ala, and expressed the mutant zymogens in mammalian cells. Following activation, all three fIXa mutants exhibited normal activity towards a fIXa-specific chromogenic substrate in the presence of Ca(2+) with no obvious requirement for Na(+) in the reaction. Furthermore, all three mutants interacted with factor VIIIa with near normal affinity and catalyzed the activation of factor X in the intrinsic Xase complex with a normal catalytic efficiency. These results suggest that, unlike thrombin and factor Xa, the charged residues of this loop do not play a functional role in modulating the catalytic function of fIXa in the intrinsic Xase complex.


Assuntos
Fator IXa/isolamento & purificação , Proteínas Recombinantes/isolamento & purificação , Alanina/genética , Alanina/metabolismo , Cálcio/metabolismo , Catálise , Cisteína Endopeptidases/metabolismo , Ativação Enzimática , Fator IXa/biossíntese , Fator IXa/genética , Fator VIIIa/metabolismo , Fator X/metabolismo , Vetores Genéticos , Humanos , Modelos Moleculares , Mutação , Proteínas de Neoplasias/metabolismo , Plasmídeos , Ligação Proteica , Conformação Proteica , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética , Sódio/metabolismo
10.
Thromb Haemost ; 95(6): 936-41, 2006 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-16732371

RESUMO

Sodium plays an important role in modulating both the amidolytic and proteolytic activities of thrombin. By contrast, while the optimal amidolytic activity of factor Xa requires Na(+), the proteolytic activity of factor Xa in the prothrombinase complex is minimally affected by the monovalent cation. In this study, we analyzed the effect of Na(+) on the amidolytic and proteolytic activity of factor IXa in the absence and presence of factor VIIIa. Factor IXa exhibited normal activity towards a fIXa-specific chromogenic substrate and antithrombin in the presence of physiological concentrations of Ca(2+) with no obvious requirement for Na(+) in either reaction. Further studies revealed that factor IXa binds to its cofactor factor VIIIa with a normal affinity in the absence of Na(+) and that the catalytic function in the intrinsic Xase complex is also independent of Na(+) in the presence of physiological concentrations of Ca(2+). These results suggest that unlike the important role that Na(+) plays in modulating the macromolecular substrate specificity of thrombin, the monovalent cation is not required for the physiological function of factor Ixa in the intrinsic Xase complex.


Assuntos
Fatores de Coagulação Sanguínea/química , Fator IXa/química , Sódio/química , Regulação Alostérica , Antitrombinas/química , Antitrombinas/metabolismo , Sítios de Ligação , Cálcio/química , Cálcio/metabolismo , Compostos Cromogênicos , Cisteína Endopeptidases/química , Cisteína Endopeptidases/metabolismo , Fator IXa/metabolismo , Fator VIIIa/química , Fator VIIIa/metabolismo , Fator X/química , Fator X/metabolismo , Humanos , Cinética , Proteínas de Neoplasias/química , Proteínas de Neoplasias/metabolismo , Oligopeptídeos , Ligação Proteica , Sódio/metabolismo
11.
Biochemistry ; 43(24): 7725-35, 2004 Jun 22.
Artigo em Inglês | MEDLINE | ID: mdl-15196015

RESUMO

A recently discovered enzyme in the mandelate pathway of Pseudomonas putida, mandelamide hydrolase (MAH), catalyzes the hydrolysis of mandelamide to mandelic acid and ammonia. Sequence analysis suggests that MAH is a member of the amidase signature family, which is widespread in nature and contains a novel Ser-cis-Ser-Lys catalytic triad. Here we report the expression in Escherichia coli, purification, and characterization of both wild-type and His(6)-tagged MAH. The recombinant enzyme was stable, exhibited a pH optimum of 7.8, and was able to hydrolyze both enantiomers of mandelamide with little enantiospecificity. The His-tagged variant showed no significant change in kinetic constants. Phenylacetamide was found to be the best substrate, with changes in chain length or replacement of the phenyl group producing greatly decreased values of k(cat)/K(m). As with another member of this family, fatty acid amide hydrolase, MAH has the uncommon ability to hydrolyze esters and amides at similar rates. MAH is even more unusual in that it will only hydrolyze esters and amides with little steric bulk. Ethyl and larger esters and N-ethyl and larger amides are not substrates, suggesting that the MAH active site is very sterically hindered. Mutation of each residue in the putative catalytic triad to alanine resulted in total loss of activity for S204A and K100A, while S180A exhibited a 1500-fold decrease in k(cat) and significant increases in K(m) values. Overall, the MAH data are similar to those of fatty acid amide hydrolase and support the suggestion that there are two distinct subgroups within the amidase signature family.


Assuntos
Amidoidrolases/química , Pseudomonas putida/enzimologia , Amidoidrolases/genética , Amidoidrolases/isolamento & purificação , Amidoidrolases/metabolismo , Sequência de Aminoácidos , Sequência de Bases , Dicroísmo Circular , Primers do DNA , Espectroscopia de Ressonância Magnética , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Ressonância Magnética Nuclear Biomolecular , Homologia de Sequência de Aminoácidos
12.
J Bacteriol ; 185(8): 2451-6, 2003 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-12670968

RESUMO

The enzymes of the mandelate metabolic pathway permit Pseudomonas putida ATCC 12633 to utilize either or both enantiomers of mandelate as the sole carbon source. The genes encoding the mandelate pathway were found to lie on a single 10.5-kb restriction fragment. Part of that fragment was shown to contain the genes coding for mandelate racemase, mandelate dehydrogenase, and benzoylformate decarboxylase arranged in an operon. Here we report the sequencing of the remainder of the restriction fragment, which revealed three further open reading frames, denoted mdlX, mdlY, and mdlD. All were transcribed in the opposite direction from the genes of the mdlABC operon. Sequence alignments suggested that the open reading frames encoded a regulatory protein (mdlX), a member of the amidase signature family (mdlY), and an NAD(P)(+)-dependent dehydrogenase (mdlD). The mdlY and mdlD genes were isolated and expressed in Escherichia coli, and the purified gene products were characterized as a mandelamide hydrolase and an NAD(P)(+)-dependent benzaldehyde dehydrogenase, respectively.


Assuntos
Aldeído Oxirredutases/metabolismo , Amidoidrolases/metabolismo , Ácidos Mandélicos/metabolismo , Pseudomonas putida/enzimologia , Aldeído Oxirredutases/genética , Aldeído Oxirredutases/isolamento & purificação , Amidoidrolases/genética , Amidoidrolases/isolamento & purificação , Sequência de Aminoácidos , Benzaldeído Desidrogenase (NADP+) , Clonagem Molecular , Escherichia coli/metabolismo , Dados de Sequência Molecular , Fases de Leitura Aberta , Óperon , Pseudomonas putida/genética , Mapeamento por Restrição , Alinhamento de Sequência , Transcrição Gênica
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