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1.
Opt Express ; 25(16): 18853-18862, 2017 Aug 07.
Artigo em Inglês | MEDLINE | ID: mdl-29041077

RESUMO

Fully integrated monolithic, multi-channel InP-based coherent receiver PICs and transceiver modules with extended C-band tunability are described. These PICs operate at 33 and 44 Gbaud per channel under dual polarization (DP) 16-QAM modulation. Fourteen-channel monolithic InP receiver PICs show integration and data rate scaling capability to operate at 44 Gbaud under DP 16-QAM modulation for combined 4.9 Tb/s total capacity. Six channel simultaneous operation of a commercial transceiver module at 33 Gbaud is demonstrated for a variety of modulation formats including DP 16-QAM for >1.2Tbit/s aggregate data capacity.

2.
Opt Express ; 19(26): B154-8, 2011 Dec 12.
Artigo em Inglês | MEDLINE | ID: mdl-22274012

RESUMO

In this work, a 10-wavelength, polarization-multiplexed, monolithically integrated InP coherent QPSK transmitter PIC is demonstrated to operate at 112 Gb/sec per wavelength and total chip superchannel bandwidth of 1.12 Tb/s. This demonstration suggests that increasing data capacity to multi-Tb/s per chip is possible and likely in the future.

3.
Opt Express ; 15(9): 5388-93, 2007 Apr 30.
Artigo em Inglês | MEDLINE | ID: mdl-19532793

RESUMO

We describe modulation responses and relative intensity noise (RIN) spectra of an InAs/GaAs quantum dot laser operating near 1300 nm. A very large nonlinear gain compression coefficient yields a highly damped modulation response with a maximum 3 dB bandwidth of ~6.5 GHz and flat RIN spectra which reach as low a level as -158/-160 dB/Hz at frequencies up to 10 GHz.

4.
J Microsc ; 212(Pt 3): 280-91, 2003 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-14629554

RESUMO

Atomic force microscopy (AFM) in aqueous solution was used to investigate native nacre of the marine snail Haliotis laevigata on the microscopic scale and the interaction of purified nacre proteins with calcium carbonate crystals on the nanoscopic scale. These investigations were controlled by scanning electron microscopy (SEM), light microscopy (LM) and biochemical methods. For investigations with AFM and SEM, nacre was cleaved parallel to the aragonite tablets in this biogenic polymer/mineral composite. Multilamellar organic sheets consisting of a core of chitin with layers of proteins attached on both sides lay between the aragonite layers consisting of confluent aragonite tablets. Cleavage appeared to occur between the aragonite tablet layer and the protein layer. AFM images revealed a honeycomb-like structure to the organic material with a diameter of the 'honeycombs' equalling that of the aragonite tablets. The walls of the structures consisted of filaments, which were suggested to be collagen. The flat regions of the honeycomb-like structures exhibited a hole with a diameter of more than 100 nm. When incubated in saturated calcium carbonate solution, aragonite needles with perfect vertical orientation grew on the proteinacous surface. After treatment with proteinase K, no growth of orientated aragonite needles was detected. Direct AFM measurements on dissolving and growing calcite crystals revealed a surface structure with straight steps the number of which decreased with crystal growth. When the purified nacre protein perlucin was added to the growth solution (a super-saturated calcium carbonate solution) new layers were nucleated and the number of steps increased. Anion exchange chromatography of the water-soluble proteins revealed a mixture of about 10 different proteins. When this mixture was dialysed against saturated calcium carbonate solution and sodium chloride, calcium carbonate crystals precipitated together with perlucin leaving the other proteins in the supernatant. Thus perlucin was shown to be a protein able to nucleate calcium carbonate layers on calcite surfaces, and in the presence of sodium chloride, is incorporated as an intracrystalline protein into calcium carbonate crystals.


Assuntos
Carbonato de Cálcio/química , Lectinas/química , Cristalização , Microscopia de Força Atômica , Microscopia Eletrônica de Varredura
5.
J Exp Bot ; 52(360): 1545-54, 2001 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-11457915

RESUMO

Plastid lipid-associated proteins, also termed fibrillin/CDSP34 proteins, are known to accumulate in fibrillar-type chromoplasts such as those of ripening pepper fruit, and in leaf chloroplasts from Solanaceae plants under abiotic stress conditions. It is shown here that treatments generating active oxygen species (high light combined with low temperature, gamma irradiation or methyl viologen treatment) result in potato CDSP34 gene induction and protein accumulation in leaves. Using transgenic tomato plants containing the pepper fibrillin promoter, a significant increase in promoter activity in leaves subjected to biotic stress, namely bacterial infections, was observed. In WT, a higher level of the endogenous fibrillin/CDSP34 protein is also observed after infection by E. chrysanthemi strain 3739. In addition to stress-related induction, a progressive increase in the fibrillin promoter activity is noticed during ageing in various tomato photosynthetic tissues and this increase correlates with a higher abundance of the endogenous protein in WT leaves. It is proposed that a mechanism related to oxidative events plays an essential role in the regulation of fibrillin/CDSP34 genes during stress and also during development. Using a biolistic transient expression assay, the pepper fibrillin promoter is found to be active in various dicot species, but not in monocots. Further, substantially increased levels of fibrillin/ CDSP34 proteins are shown in various dicotyledonous and monocotyledonous plants in response to water deficit.


Assuntos
Regulação da Expressão Gênica no Desenvolvimento , Regulação da Expressão Gênica de Plantas , Proteínas dos Microfilamentos/metabolismo , Proteínas de Plantas/metabolismo , Plastídeos/metabolismo , Solanaceae/fisiologia , Adaptação Fisiológica , Fibrilinas , Luz , Estresse Oxidativo , Folhas de Planta , Plantas Geneticamente Modificadas , Regiões Promotoras Genéticas , RNA de Plantas/isolamento & purificação , Especificidade da Espécie , Fatores de Tempo , Ativação Transcricional , Água/metabolismo
6.
Trends Plant Sci ; 6(1): 31-6, 2001 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-11164375

RESUMO

Inactivation of a plastid located quinone-oxygen oxidoreductase gene in the immutans Arabidopsis mutant leads to a photobleached phenotype because of a lack of photoprotective carotenoids. Inactivation of the corresponding gene in the ghost tomato mutant leads to a similar phenotype in leaves and to carotenoid deficiency in petals and ripe fruits. This plastid terminal oxidase (the first to be cloned and biochemically characterized) resembles the mitochondrial cyanide-insensitive alternative oxidase. Here, we propose a model integrating this novel oxidase as a component of an electron transport chain associated to carotenoid desaturation, as well as to a respiratory activity within plastids.


Assuntos
Carotenoides/biossíntese , Oxirredutases/metabolismo , Oxigênio/metabolismo , Plastídeos/enzimologia , Arabidopsis/enzimologia , Oxirredutases/genética
7.
Philos Trans R Soc Lond B Biol Sci ; 355(1402): 1447-54, 2000 Oct 29.
Artigo em Inglês | MEDLINE | ID: mdl-11127998

RESUMO

Besides electron transfer reactions involved in the 'Z' scheme of photosynthesis, alternative electron transfer pathways have been characterized in chloroplasts. These include cyclic electron flow around photosystem I (PS I) or a respiratory chain called chlororespiration. Recent work has supplied new information concerning the molecular nature of the electron carriers involved in the non-photochemical reduction of the plastoquinone (PQ) pool. However, until now little is known concerning the nature of the electron carriers involved in PQ oxidation. By using mass spectrometric measurement of oxygen exchange performed in the presence of 18O-enriched O2 and Chlamydomonas mutants deficient in PS I, we show that electrons can be directed to a quinol oxidase sensitive to propyl gallate but insensitive to salicyl hydroxamic acid. This oxidase has immunological and pharmacological similarities with a plastid protein involved in carotenoid biosynthesis.


Assuntos
Cloroplastos/enzimologia , Complexos de Proteínas Captadores de Luz , Oxirredutases/metabolismo , Fotossíntese/fisiologia , Complexo de Proteínas do Centro de Reação Fotossintética/metabolismo , Complexo de Proteína do Fotossistema I , Tilacoides/metabolismo , Animais , Respiração Celular , Chlamydomonas reinhardtii/metabolismo , Cloroplastos/metabolismo , Grupo dos Citocromos b/metabolismo , Complexo Citocromos b6f , Transporte de Elétrons , Proteínas de Membrana/genética , Proteínas de Membrana/fisiologia , Oxigênio/metabolismo , Complexo de Proteínas do Centro de Reação Fotossintética/genética , Proteínas de Plantas/genética , Proteínas de Plantas/fisiologia
8.
J Agric Food Chem ; 48(10): 4676-80, 2000 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-11052718

RESUMO

Pepper leaves treated with the herbicide J852 show an accumulation of phytoene and zeta-carotene, whereas treatment with norflurazon led to an accumulation of only phytoene. The effects of these herbicides were examined in vitro after the expression of carotenoid desaturases in Escherichia coli. Whereas norflurazon is a potent inhibitor of phytoene desaturase (PDS) (I(50) = 0.12 microM) but not of zeta-carotene desaturase (ZDS) (I(50) = 144 microM), J852 inhibits both PDS (I(50) = 23 microM) and ZDS (I(50) = 49 microM). The influence of PDS/ZDS inhibition on gene expression was examined by comparative RT-PCR. None of the examined genes, namely, encoding phytoene synthase, PDS, ZDS, or the terminal oxidase associated with phytoene desaturation, were induced upon herbicide treatment in pepper leaves or seedlings. This was unexpected because inhibition of carotene desaturation led to an up-regulation of the carotenoid biosynthetic capacity (higher amounts of accumulating precursors plus remaining colored carotenoids are present in treated tissues versus control).


Assuntos
Capsicum/metabolismo , Carotenoides/biossíntese , Herbicidas/farmacologia , Plantas Medicinais , Capsicum/química , Capsicum/efeitos dos fármacos , Depressão Química , Oxirredutases/metabolismo , RNA Mensageiro/biossíntese , Reação em Cadeia da Polimerase Via Transcriptase Reversa
9.
Plant Physiol ; 123(4): 1427-36, 2000 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-10938359

RESUMO

The Arabidopsis IMMUTANS gene encodes a plastid homolog of the mitochondrial alternative oxidase, which is associated with phytoene desaturation. Upon expression in Escherichia coli, this protein confers a detectable cyanide-resistant electron transport to isolated membranes. In this assay this activity is sensitive to n-propyl-gallate, an inhibitor of the alternative oxidase. This protein appears to be a plastid terminal oxidase (PTOX) that is functionally equivalent to a quinol:oxygen oxidoreductase. This protein was immunodetected in achlorophyllous pepper (Capsicum annuum) chromoplast membranes, and a corresponding cDNA was cloned from pepper and tomato (Lycopersicum esculentum) fruits. Genomic analysis suggests the presence of a single gene in these organisms, the expression of which parallels phytoene desaturase and zeta-carotene desaturase gene expression during fruit ripening. Furthermore, this PTOX gene is impaired in the tomato ghost mutant, which accumulates phytoene in leaves and fruits. These data show that PTOX also participates in carotenoid desaturation in chromoplasts in addition to its role during early chloroplast development.


Assuntos
Proteínas de Arabidopsis , Carotenoides/biossíntese , Proteínas Nucleares/genética , Plantas/genética , Plastídeos/metabolismo , Sequência de Aminoácidos , Arabidopsis/genética , Arabidopsis/metabolismo , Sequência de Bases , Southern Blotting , Capsicum/genética , Capsicum/metabolismo , Carotenoides/metabolismo , Cloroplastos/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Immunoblotting , Solanum lycopersicum/genética , Solanum lycopersicum/metabolismo , Dados de Sequência Molecular , Mutação , Proteínas Nucleares/metabolismo , Plantas/metabolismo , Plantas Medicinais , RNA Mensageiro/análise , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Homologia de Sequência de Aminoácidos
10.
Plant J ; 21(5): 483-94, 2000 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-10758499

RESUMO

Proteins homologous to fibrillin, a pepper plastid lipid-associated protein involved in carotenoid storage in fruit chromoplasts, have been recently identified in leaf chloroplasts from several species and shown to be induced upon environmental stress. To further investigate the role of the protein, transgenic Nicotiana tabacum plants over-expressing fibrillin using a constitutive promoter were generated. Transgenics grown under standard light intensities (300 micromol photons m-2 sec-1) were found to contain substantial amounts of fibrillin in flowers and leaves. In leaves, the protein was immunolocalized within chloroplasts in both stromal and thylakoid subfractions. No change was noticed in thylakoid structures from transgenics, but chloroplasts contained an increased number of plastoglobules organized in clusters. In petals, leucoplasts were also found to contain more agglutinated plastoglobules. The effects of environmental factors on fibrillin gene expression and protein localization were studied in tobacco leaves. Less fibrillin was present in plants grown under low light intensities, which can be explained by the involvement of a light-dependent splicing step in the control of fibrillin gene expression in leaves. Analysis of protein subfractions from plants subjected to drought or high light showed that both stresses resulted in fibrillin association with thylakoids. Whereas no growth difference between wild-type (WT) and transgenic plants was noticed under low light conditions, transgenics exhibit a longer main stem, enhanced development of lateral stems and accelerated floral development under higher light intensities. These data suggest that fibrillin-related proteins fulfil an important function in plant development in relation to environmental constraints.


Assuntos
Capsicum/genética , Expressão Gênica , Proteínas dos Microfilamentos/genética , Nicotiana/genética , Proteínas de Plantas/genética , Plantas Medicinais , Plantas Tóxicas , Plastídeos/ultraestrutura , Capsicum/crescimento & desenvolvimento , Capsicum/ultraestrutura , Fibrilinas , Microscopia Eletrônica , Água
11.
J Biol Chem ; 275(23): 17256-62, 2000 Jun 09.
Artigo em Inglês | MEDLINE | ID: mdl-10748104

RESUMO

In Chlamydomonas reinhardtii mutants deficient in photosystem I because of inactivation of the chloroplast genes psaA or psaB, oxygen evolution from photosystem II occurs at significant rates and is coupled to a stimulation of oxygen uptake. Both activities can be simultaneously monitored by continuous mass spectrometry in the presence of (18)O(2). The light-driven O(2) exchange was shown to involve the plastoquinone pool as an electron carrier, but not cytochrome b(6)f. Photosystem II-dependent O(2) production and O(2) uptake were observed in isolated chloroplast fractions. Photosystem II-dependent oxygen exchange was insensitive to a variety of inhibitors (azide, carbon monoxide, cyanide, antimycin A, and salicylhydroxamic acid) and radical scavengers. It was, however, sensitive to propyl gallate. From inhibitors effects and electronic requirements of the O(2) uptake process, we conclude that an oxidase catalyzing oxidation of plastoquinol and reduction of oxygen to water is present in thylakoid membranes. From the sensitivity of flash-induced O(2) exchange to propyl gallate, we conclude that this oxidase is involved in chlororespiration. Clues to the identity of the protein implied in this process are given by pharmacological and immunological similarities with a protein (IMMUTANS) identified in Arabidopsis chloroplasts.


Assuntos
Chlamydomonas reinhardtii/genética , Chlamydomonas reinhardtii/metabolismo , Cloroplastos/metabolismo , Oxirredutases/metabolismo , Consumo de Oxigênio , Oxigênio/metabolismo , Complexo de Proteínas do Centro de Reação Fotossintética/genética , Complexo de Proteínas do Centro de Reação Fotossintética/metabolismo , Animais , Antimicina A/farmacologia , Azidas/farmacologia , Monóxido de Carbono/farmacologia , Clorofila/metabolismo , Transporte de Elétrons , Sequestradores de Radicais Livres/farmacologia , Cinética , Complexos de Proteínas Captadores de Luz , Fotossíntese/efeitos dos fármacos , Complexo de Proteína do Fotossistema I , Complexo de Proteína do Fotossistema II , Salicilamidas/farmacologia
12.
J Steroid Biochem Mol Biol ; 74(4): 169-78, 2000 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-11162922

RESUMO

The expression of high levels of full-length human estrogen receptor alpha (hERalpha) in Escherichia coli has proven difficult. We found that expression of the ER DNA binding domain is highly toxic to E. coli, resulting in rapid loss of the expression plasmid. Using a tightly regulated arabinose expression system and the antibiotic Timentin, we were able to overcome ER toxicity and express substantial levels of ER. The expressed ER exhibited protease cleavage at a single site near the N-terminus of the hinge region. Of the many measures we tested to eliminate ER cleavage, only addition of carbonyl cyanide m-chlorophenyl-hydrazone (CCCP), an uncoupler of oxidative phosphorylation, completely blocked intracellular proteolysis of the ER. Using CCCP and our expression methods, full-length FLAG epitope-tagged hERalpha (fER) was expressed in E. coli at approximately 1 mg/l. The fER was purified to homogeneity in a single step by immunoaffinity chromatography with anti-FLAG monoclonal antibody. Purified full-length bacterial fER binds 17beta-estradiol with the same affinity as hER expressed in human cells (K(D) approximately 0.5 nM). At high concentrations of fER (20 nM), a bell-shaped estrogen binding curve with a Hill coefficient of 1.7 was seen. Bacterially-expressed fER exhibits a reduced affinity for the estrogen response element (ERE). Anti-FLAG antibody restores high affinity binding of the fER to the ERE, suggesting that impaired dimerization may be responsible for the reduced affinity of bacterially-expressed fER for the ERE. The use of Timentin and CCCP may provide a general method for high level bacterial expression of steroid/nuclear receptors and other proteins important in hormone action.


Assuntos
Carbonil Cianeto m-Clorofenil Hidrazona/farmacologia , Escherichia coli/genética , Engenharia de Proteínas/métodos , Receptores de Estrogênio/genética , Receptores de Estrogênio/metabolismo , Desacopladores/farmacologia , Sequência de Bases , Sítios de Ligação , DNA/metabolismo , Escherichia coli/efeitos dos fármacos , Escherichia coli/metabolismo , Estradiol/metabolismo , Dados de Sequência Molecular , Oxirredução , Fosforilação , Receptores de Estrogênio/efeitos dos fármacos , Proteínas Recombinantes/efeitos dos fármacos , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Elementos de Resposta/fisiologia
13.
Plant Physiol Biochem ; 37(11): 859-868, 1999 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-10580286

RESUMO

Fibrillin was originally identified as a chromoplast protein involved in the assembly of carotenoid-containing fibrils and was also found to accumulate in chloroplasts of wounded or water-stressed leaves. We now show that the promoter from the pepper fibrillin (nuclear) gene can be induced in leaves of stable tomato transformants by various stresses, namely wounding, drought, cold and salt stress, in light but not in darkness, as well as by high light intensities. Various herbicides causing reactive oxygen (superoxide, singlet oxygen) production in chloroplasts also induce the promoter. Higher expression levels are observed in transgenic tobacco plants which are apparently more sensitive to photo-oxidative stress than tomato. Similarly, wounding which causes strong induction of the promoter in tobacco, produces only weak induction in tomato. Hydrogen peroxide produced in plastids or added exogenously causes the induction of this nuclear gene. Our data suggest that the ascorbate/glutathione pathway (which eliminates hydrogen peroxide) can influence indirectly the induction of the fibrillin promoter. We propose a generalized model which links stresses of external origin to nuclear gene induction, via the plastid compartment which is subjected to photo-oxidative stress.

14.
Eur J Biochem ; 265(1): 376-83, 1999 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-10491195

RESUMO

The zeta-carotene desaturase from Capsicum annuum (EC 1.14.99.-) was expressed in Escherichia coli, purified and characterized biochemically. The enzyme acts as a monomer with lipophilic quinones as cofactors. Km values for the substrate zeta-carotene or the intermediate neurosporene in the two-step desaturation reaction are almost identical. Product analysis showed that different lycopene isomers are formed, including substantial amounts of the all-trans form, together with 7,7',9,9'-tetracis prolycopene via the corresponding neurosporene isomers. The application of different geometric isomers as substrates revealed that the zeta-carotene desaturase has no preference for certain isomers and that the nature of the isomers formed during catalysis depends strictly on the isomeric composition of the substrate.


Assuntos
Capsicum/enzimologia , Carotenoides/metabolismo , Oxirredutases/metabolismo , Plantas Medicinais , Capsicum/genética , Escherichia coli/genética , Isomerismo , Cinética , Licopeno , Oxirredutases/efeitos dos fármacos , Oxirredutases/genética , Oxirredutases/isolamento & purificação , Plastoquinona/análogos & derivados , Plastoquinona/farmacologia , Proteínas Recombinantes/efeitos dos fármacos , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Ubiquinona/análogos & derivados , Ubiquinona/farmacologia
15.
Plant Cell ; 11(1): 57-68, 1999 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-9878632

RESUMO

The immutans (im) mutant of Arabidopsis shows a variegated phenotype comprising albino and green somatic sectors. We have cloned the IM gene by transposon tagging and show that even stable null alleles give rise to a variegated phenotype. The gene product has amino acid similarity to the mitochondrial alternative oxidase. We show that the IM protein is synthesized as a precursor polypeptide that is imported into chloroplasts and inserted into the thylakoid membrane. The albino sectors of im plants contain reduced levels of carotenoids and increased levels of the carotenoid precursor phytoene. The data presented here are consistent with a role for the IM protein as a cofactor for carotenoid desaturation. The suggested terminal oxidase function of IM appears to be essential to prevent photooxidative damage during early steps of chloroplast formation. We propose a model in which IM function is linked to phytoene desaturation and, possibly, to the respiratory activity of the chloroplast.


Assuntos
Proteínas de Arabidopsis , Arabidopsis/genética , Carotenoides/metabolismo , Cloroplastos/genética , Proteínas Nucleares/genética , Oxirredutases/metabolismo , Pigmentação/genética , Sequência de Aminoácidos , Arabidopsis/fisiologia , Sequência de Bases , Carotenoides/biossíntese , Cloroplastos/enzimologia , Elementos de DNA Transponíveis/genética , Dados de Sequência Molecular , Mutagênese Insercional , Proteínas Nucleares/metabolismo , Fenótipo , Alinhamento de Sequência
16.
Plant Mol Biol ; 36(5): 785-9, 1998 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-9526511

RESUMO

The red colour of pepper fruits is determined by the y+ dominant allele and the yellow colour by the y recessive allele. The capsanthin-capsorubin synthase (CCS) gene is activated specifically during the final stages of pepper fruit ripening. RFLP and specific-PCR polymorphisms derived from the CCS gene were analysed in a F2 progeny of a red by yellow-fruited cross. They cosegregated completely with fruit colour. Our results support the hypothesis that the yellow phenotype might result from a deletion of the CCS gene. These specific markers were integrated into the genetic map and will be useful for marker assisted plant breeding.


Assuntos
Genes de Plantas , Oxirredutases/genética , Proteínas de Plantas , Verduras/enzimologia , Verduras/genética , Alelos , Sequência de Bases , Mapeamento Cromossômico , Primers do DNA/genética , Deleção de Genes , Regulação da Expressão Gênica no Desenvolvimento , Regulação Enzimológica da Expressão Gênica , Regulação da Expressão Gênica de Plantas , Marcadores Genéticos , Fenótipo , Pigmentação/genética , Reação em Cadeia da Polimerase , Polimorfismo Genético , Polimorfismo de Fragmento de Restrição , Seleção Genética , Verduras/crescimento & desenvolvimento
17.
J Biol Chem ; 272(44): 27949-56, 1997 Oct 31.
Artigo em Inglês | MEDLINE | ID: mdl-9346945

RESUMO

In this work, we provide a rationale for the finding that the estrogen receptor (ER) binds to its DNA response element as a homodimer in vivo. Binding of the monomer estrogen receptor DNA binding domain (ER DBD) to a palindromic, consensus estrogen response element (ERE) is increased 5-6-fold when the ER DBD is dimerized either by a monoclonal antibody that recognizes an attached epitope tag or by expressing the ER DBD as a single molecule in which the two monomers are joined by a peptide linker. Most of the increase in binding is due to stabilization of the ER DBD.ERE complex. We observed only an approximately 2.5-fold reduction in binding when a consensus ERE was replaced with widely spaced ERE half-sites, suggesting that the interaction between ER DBDs on the ERE is relatively weak, and that in full-length ER the DBDs can move independently of each other. To test binding to an imperfect palindrome, typical of the imperfect EREs found in almost all natural estrogen receptor responsive genes, we used the pS2 ERE. Even at high concentrations of ER DBD, specific binding of the ER DBD to the imperfect pS2 ERE was undetectable. Both of the dimerized ER DBDs exhibited efficient binding to the imperfect pS2 ERE, with an affinity at least 25-fold greater than monomer ER DBD. These data support the view that steroid receptor dimerization provides an important mechanism facilitating the recognition of naturally occurring, imperfect hormone response elements.


Assuntos
Proteínas de Ligação a DNA/metabolismo , Estrogênios/metabolismo , Receptores de Estrogênio/metabolismo , Anticorpos/química , Sequência de Bases , Primers do DNA , Dimerização , Humanos , Dados de Sequência Molecular , Ligação Proteica , Receptores de Estrogênio/química
18.
Plant J ; 8(3): 417-24, 1995 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-7550379

RESUMO

The biosynthetic pathway of cyclic carotenoid is known to be quantitatively and qualitatively different in the non-green plastids of Capsicum annuum fruits compared with chloroplasts. Here, the cloning is described of a novel cDNA from this organism, which encodes an enzyme catalyzing the cyclization of lycopene to beta-carotene when expressed in Escherichia coli. The corresponding gene is constitutively expressed during fruit development. Significant amino acid sequence identity was observed between this enzyme and capsanthin/capsorubin synthase which is involved in the synthesis of the species-specific red carotenoids of C. annuum fruits. The latter enzyme was found also to possess a lycopene beta-cyclase activity when expressed in E. coli. A model is proposed for the origin of the capsanthin/capsorubin synthase gene and the role of this enzyme, together with the newly cloned lycopene cyclase, in the specific re-channeling of linear carotenoids into beta-cyclic carotenoids in C. annuum ripening fruits.


Assuntos
Capsicum/metabolismo , Carotenoides/metabolismo , Cloroplastos/metabolismo , Liases Intramoleculares , Isomerases/metabolismo , Plantas Medicinais , Sequência de Aminoácidos , Sequência de Bases , Capsicum/enzimologia , Capsicum/genética , Clonagem Molecular , Sequência Consenso , DNA Complementar , Escherichia coli , Isomerases/biossíntese , Isomerases/química , Licopeno , Dados de Sequência Molecular , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Homologia de Sequência de Aminoácidos , beta Caroteno
19.
FEBS Lett ; 372(2-3): 199-202, 1995 Sep 25.
Artigo em Inglês | MEDLINE | ID: mdl-7556669

RESUMO

We have cloned a cDNA from the plant Capsicum annuum which encodes a novel enzyme mediating the dehydrogenation of zeta-carotene and neurosporene to lycopene when expressed in E. coli cells accumulating zeta-carotene or neurosporene. This enzyme is unable to dehydrogenate either phytoene or lycopene. The deduced amino acid sequence suggests that this cDNA encodes a polypeptide whose mature size is ca. 59 kDa and which is synthesized as a precursor with a NH2-terminal extension resembling transit peptides for plastid targeting. Sequence comparison reveals 33-35% similarity with previously cloned plant or cyanobacterial phytoene desaturases. In contrast, only limited sequence similarity is found with a zeta-carotene desaturase from the cyanobacterium Anabaena.


Assuntos
Carotenoides/metabolismo , Enzimas/isolamento & purificação , Proteínas de Plantas/isolamento & purificação , Plantas/enzimologia , Sequência de Aminoácidos , Sequência de Bases , Clonagem Molecular , Enzimas/genética , Escherichia coli/enzimologia , Escherichia coli/genética , Dados de Sequência Molecular , Proteínas de Plantas/genética , Alinhamento de Sequência
20.
Proc Natl Acad Sci U S A ; 92(12): 5630-4, 1995 Jun 06.
Artigo em Inglês | MEDLINE | ID: mdl-7777561

RESUMO

Structural evidence has accumulated suggesting that fusion and/or translocation factors are involved in plastid membrane biogenesis. To test this hypothesis, we have developed an in vitro system in which the extent of fusion and/or translocation is monitored by the conversion of the xanthophyll epoxide (antheraxanthin) into the red ketocarotenoid (capsanthin). Only chromoplast membrane vesicles from red pepper fruits (Capsicum annuum) contain the required enzyme. Vesicles prepared from the mutant yellow cultivar are devoid of this enzyme and accumulate antheraxanthin. The fusion and/or translocation activity is characterized by complementation due to the synthesis of capsanthin and the parallel decrease of antheraxanthin when the two types of vesicles are incubated together in the presence of plastid stroma. We show that the extent of conversion is dependent upon an ATP-requiring protein that is sensitive to N-ethylmaleimide. Further purification and immunological analysis have revealed that the active factor, designated plastid fusion and/or translocation factor (Pftf), resides in a protein of 72 kDa. cDNA cloning revealed that mature Pftf has significant homology to yeast and animal (NSF) or bacterial (Ftsh) proteins involved in vesicle fusion or membrane protein translocation.


Assuntos
Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Plastídeos/metabolismo , Sequência de Aminoácidos , Sequência de Bases , Transporte Biológico , Capsicum , Clonagem Molecular , DNA Complementar , Membranas Intracelulares/metabolismo , Proteínas de Membrana/química , Proteínas de Membrana/fisiologia , Microscopia Eletrônica , Dados de Sequência Molecular , Proteínas de Plantas/química , Proteínas de Plantas/fisiologia , Plantas Medicinais , Plastídeos/ultraestrutura , Homologia de Sequência de Aminoácidos
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