RESUMEN
Multiple myeloma (MM) is a malignant neoplasm of plasma cells. Although new molecular targeting agents against MM have been developed based on the better understanding of the underlying pathogenesis, MM still remains an incurable disease. We previously demonstrated that ß-catenin, a downstream effector in the Wnt pathway, is a potential target in MM using RNA interference in an in vivo experimental mouse model. In this study, we have screened a library of more than 100 000 small-molecule chemical compounds for novel Wnt/ß-catenin signaling inhibitors using a high-throughput transcriptional screening technology. We identified AV-65, which diminished ß-catenin protein levels and T-cell factor transcriptional activity. AV-65 then decreased c-myc, cyclin D1 and survivin expression, resulting in the inhibition of MM cell proliferation through the apoptotic pathway. AV-65 treatment prolonged the survival of MM-bearing mice. These findings indicate that this compound represents a novel and attractive therapeutic agent against MM. This study also illustrates the potential of high-throughput transcriptional screening to identify candidates for anticancer drug discovery.
RESUMEN
Abl tyrosine kinase inhibitors (TKIs) such as imatinib and dasatinib are ineffective against Bcr-Abl(+) leukemic stem cells. Thus, the identification of novel agents that are effective in eradicating quiescent Bcr-Abl(+) stem cells is needed to cure leukemias caused by Bcr-Abl(+) cells. Human Bcr-Abl(+) cells engrafted in the bone marrow of immunodeficient mice survive under severe hypoxia. We generated two hypoxia-adapted (HA)-Bcr-Abl(+) sublines by selection in long-term hypoxic cultures (1.0% O(2)). Interestingly, HA-Bcr-Abl(+) cells exhibited stem cell-like characteristics, including more cells in a dormant, increase of side population fraction, higher beta-catenin expression, resistance to Abl TKIs, and a higher transplantation efficiency. Compared with the respective parental cells, HA-Bcr-Abl(+) cells had higher levels of protein and higher enzyme activity of glyoxalase-I (Glo-I), an enzyme that detoxifies methylglyoxal, a cytotoxic by-product of glycolysis. In contrast to Abl TKIs, Glo-I inhibitors were much more effective in killing HA-Bcr-Abl(+) cells both in vitro and in vivo. These findings indicate that Glo-I is a novel molecular target for treatment of Bcr-Abl(+) leukemias, and, in particular, Abl TKI-resistant quiescent Bcr-Abl(+) leukemic cells that have acquired stem-like characteristics in the process of adapting to a hypoxic environment.
Asunto(s)
Lactoilglutatión Liasa/antagonistas & inhibidores , Leucemia Mielógena Crónica BCR-ABL Positiva/tratamiento farmacológico , Células Madre Neoplásicas/efectos de los fármacos , Animales , Benzamidas , Hipoxia de la Célula , Línea Celular Tumoral , Dasatinib , Humanos , Mesilato de Imatinib , Lactoilglutatión Liasa/metabolismo , Leucemia Mielógena Crónica BCR-ABL Positiva/enzimología , Ratones , Piperazinas/farmacología , Inhibidores de Proteínas Quinasas/farmacología , Pirimidinas/farmacología , Tiazoles/farmacología , Trasplante Heterólogo , beta Catenina/metabolismoAsunto(s)
Anafilaxia/etiología , Antígenos de Plantas/inmunología , Aspirina/efectos adversos , Asma Inducida por Ejercicio/inmunología , Ejercicio Físico , Hipersensibilidad a los Alimentos/diagnóstico , Prunus/inmunología , Anafilaxia/fisiopatología , Anafilaxia/terapia , Aspirina/administración & dosificación , Asma Inducida por Ejercicio/complicaciones , Asma Inducida por Ejercicio/diagnóstico , Asma Inducida por Ejercicio/fisiopatología , Niño , Reacciones Cruzadas , Disnea , Epinefrina/uso terapéutico , Eritema , Hipersensibilidad a los Alimentos/complicaciones , Hipersensibilidad a los Alimentos/inmunología , Hipersensibilidad a los Alimentos/fisiopatología , Humanos , Masculino , PruritoRESUMEN
Both biological treatment processes including conventional activated sludge (CAS) and biological nutrient removal (BNR) processes, and physico-chemical treatment processes including ozonation process and Title 22 process consisting of coagulation, sedimentation and filtration followed by UV or chlorination disinfection after the above biological processes, were compared from the viewpoint of removal efficiency. 66 pharmaceuticals including antibiotics, analgesics, psychoneurotic agents were measured with SPE-LC/MS/MS. 26 compounds out of 66 were detected in the influent ranging ng/L to microg/L order. Particularly, disopyramide, sulpiride, and dipyridamole that have been rarely detected before in the WWTP, occurred at concentration levels of more than 100 ng/L. The total concentration of the individual pharmaceuticals in the influent was efficiently removed by 80% during the biological treatment. But removal efficiencies of carbamazepine and crotamiton were less than 30%. The total concentration of the individual pharmaceuticals in the effluent from CAS process was 1.5 times higher than that from BNR process. Further, the total concentration of the individual pharmaceuticals in the discharge from WWTPs applying ozonation following activated sludge process was reduced to less than 20%. Physico-chemical treatment train called Title 22 treatment after CAS could not efficiently remove the pharmaceuticals. However, ozonation process followed by biological activated carbon process could efficiently reduce all the residual pharmaceuticals below their quantification limits.
Asunto(s)
Preparaciones Farmacéuticas/aislamiento & purificación , Eliminación de Residuos Líquidos/métodos , Contaminantes Químicos del Agua/aislamiento & purificación , Purificación del Agua/métodos , Japón , Preparaciones Farmacéuticas/químicaRESUMEN
A new member of the GT-2 family of transcription factors, GmGT-2, was isolated from soybean while screening a cDNA library with a protein binding site (D1) in the promoter of Aux28, a member of the Aux/IAA family of auxin-responsive genes. GmGT-2 possesses various primary amino acid sequence characteristics common to all GT-2 factors thus far isolated, including sequence identity in the twin trihelix DNA-binding domains. Recombinant GmGT-2 expressed in Escherichia coli binds oligotetramers of both D1 and various GT-boxes. However, unlike other known members of the GT-2 family, GmGT-2 message levels are down-regulated by light in a phytochrome-dependent manner. Evidence is presented that the expression levels of Aux28 mRNA are also down-regulated by phytochrome. These results and other referenced data implicate the possible convergence of phytochrome and auxin signaling pathways.
Asunto(s)
Glycine max/genética , Luz , Fitocromo/fisiología , Factores de Transcripción/genética , Secuencia de Aminoácidos , ADN Complementario/química , ADN Complementario/genética , ADN Complementario/aislamiento & purificación , ADN de Plantas/química , ADN de Plantas/genética , ADN de Plantas/metabolismo , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Regulación hacia Abajo , Regulación del Desarrollo de la Expresión Génica/efectos de la radiación , Regulación de la Expresión Génica de las Plantas/efectos de la radiación , Hipocótilo/genética , Hipocótilo/crecimiento & desarrollo , Hipocótilo/metabolismo , Ácidos Indolacéticos/farmacología , Datos de Secuencia Molecular , Proteínas de Plantas/efectos de los fármacos , Proteínas de Plantas/genética , Regiones Promotoras Genéticas/efectos de los fármacos , Regiones Promotoras Genéticas/genética , Unión Proteica/efectos de la radiación , ARN Mensajero/genética , ARN Mensajero/metabolismo , Alineación de Secuencia , Análisis de Secuencia de ADN , Homología de Secuencia de Aminoácido , Glycine max/crecimiento & desarrollo , Transcripción GenéticaRESUMEN
To evaluate the effect of antihypertensive therapy on platelet activation in essential hypertension, the plasma levels of beta-thromboglobulin (beta-TG) were examined in 45 patients with essential hypertension and 20 age-matched normotensive control subjects. Hypertensive patients were assigned to monotherapy with one of five different antihypertensive drugs for 6 months, and the change of plasma levels of beta-TG was reexamined after the completion of the monotherapy. The plasma beta-TG increased in hypertensive patients compared with levels in normotensive control subjects. Monotherapy with each drug resulted in sufficient blood pressure control in all hypertensive patients. The plasma beta-TG decreased significantly after monotherapy with an alpha-blocker or an angiotensin-converting enzyme inhibitor (ACEI). The plasma beta-TG increased with the use of a diuretic but did not change with the use of a beta-blocker or calcium antagonist. The platelet activation observed in patients with essential hypertension is reversed by monotherapy with an alpha-blocker or an ACEI. It is possible that these drugs reduce the development of hypertensive vascular complications due to suppression of platelet activation in patients with essential hypertension.
Asunto(s)
Antihipertensivos/uso terapéutico , Plaquetas/efectos de los fármacos , Plaquetas/fisiología , Hipertensión/sangre , Hipertensión/tratamiento farmacológico , Prazosina/análogos & derivados , Antagonistas Adrenérgicos alfa/uso terapéutico , Inhibidores de la Enzima Convertidora de Angiotensina/uso terapéutico , Presión Sanguínea/efectos de los fármacos , Captopril/uso terapéutico , Diuréticos/uso terapéutico , Femenino , Humanos , Hipertensión/fisiopatología , Masculino , Persona de Mediana Edad , Prazosina/uso terapéutico , Valores de Referencia , Triclormetiazida/uso terapéutico , beta-Tromboglobulina/análisisRESUMEN
Two separate nuclear binding activities (B1 and B2) in the soybean apical hypocotyl have been identified that interact with a palindromic C-box sequence (TGACGTCA) and which are developmentally regulated in an inverse manner. The bZIP factors responsible for these two binding activities, B1 and B2, were isolated from a cDNA library and designated STGA1 and STFs (STF1 and STF2), respectively. Sequence analysis shows that the STFs contain both a zinc-finger domain and a bZIP domain. The two zinc finger sequences of Cys4-Cys4 are most related to the RING zinc-finger motif carrying a Cys3-His-Cys4. In addition the bZIP domain of STFs is highly homologous to the HY5 protein of Arabidopsis. DNA binding studies revealed that STF1 binding to the TGACGT sequence requires distinct flanking sequences. Furthermore, STF1 binds to the Hex sequence as a heterodimer with G-box binding factors (GBFs), a feature not observed with STGA1. Since STF1 expression is most prevalent in apical and elongating hypocotyls, it is proposed that STF1 may be a transcription factor involved in the process of hypocotyl elongation.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Glycine max/metabolismo , Proteínas de Soja , Factores de Transcripción/metabolismo , Secuencia de Aminoácidos , Secuencia de Bases , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico , Sitios de Unión , ADN Complementario , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/genética , Dimerización , Factores de Unión a la G-Box , Biblioteca de Genes , Leucina Zippers , Datos de Secuencia Molecular , Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Factores de Transcripción/química , Dedos de ZincRESUMEN
To overcome the disadvantages of conventional three-dimensional time-of-flight MR angiography (3D-TOF), such as saturation or intravoxel dephasing, black blood MR angiography (BB-MRA) using an interleaved multi-slab 3D fast spin echo sequence was developed and evaluated clinically. In major branches, the contrast-to-noise ratio of the flow was not as good as 3D-TOF in BB-MRA. However, in-plane slow flow and large aneurysm were visualized better on BB-MRA than on 3D-TOF. Furthermore, BB-MRA could provide wider coverage than 3D-TOF. BB-MRA using interleaved multi-slab 3D fast spin echo is now feasible and complementary to 3D-TOF.
Asunto(s)
Circulación Cerebrovascular , Angiografía por Resonancia Magnética/métodos , Trastornos Cerebrovasculares/diagnóstico , Trastornos Cerebrovasculares/fisiopatología , Humanos , Procesamiento de Imagen Asistido por ComputadorRESUMEN
The effect of the proline analog azetidine-2-carboxylic acid (Aze) on the induction and the regulation of heat-shock (HS) mRNA accumulation and heat-shock protein (HSP) synthesis in soybean (Glycine max) seedlings was studied. Treatment with Aze elicited an HS-like response at the normal growth temperature, 28[deg]C, with seven of nine HS cDNA clones tested. Two cDNA clones, Gm-Hsp22.5 and pFS2033, share 78% identity; however, transcripts hybridizing to GmHsp22.5 but not pFS2033 accumulated with Aze treatment at 28[deg]C. Substantial incorporation of radioactive amino acid into high molecular weight HSPs but not low molecular weight HSPs was observed in vivo during Aze treatment at 28[deg]C. Low molecular weight HSPs were detected using antibodies raised against an abundant member of low molecular weight class I HSPs, indicating that low molecular weight HSPs were synthesized at normal growth temperatures during Aze treatment despite a lack of substantial in vivo radioactive amino acid incorporation. In summary, Aze treatment induced accumulation of most but not all HS mRNAs and HSPs in soybean seedlings; the observations presented here suggest differential regulation among various HS genes at the transcriptional and posttranscriptional levels.
RESUMEN
Three cDNA clones (GmHSP23.9, GmHSP22.3, and GmHSP22.5) representing three different members of the low-molecular-weight (LMW) heat shock protein (HSP) gene superfamily were isolated and characterized. A fourth cDNA clone, pFS2033, was partially characterized previously as a full-length genomic clone GmHSP22.0. The deduced amino acid sequences of all four cDNA clones have the conserved carboxyl-terminal LMW HSP domain. Sequence and hydropathy analyses of GmHSP22, GmHSP22.3, and GmHSP22.5, representing HSPs in the 20 to 24 kDa range, indicate they contain amino-terminal signal peptides. The mRNAs from GmHSP22, GmHSP22.3, and GmHSP22.5 were preferentially associated in vivo with endoplasmic reticulum (ER)-bound polysomes. GmHSP22 and GmHSP22.5 encode strikingly similar proteins; they are 78% identical and 90% conserved at the amino acid sequence level, and both possess the C-terminal tetrapeptide KDEL which is similar to the consensus ER retention motif KDEL; the encoded polypeptides can be clearly resolved from each other by two-dimensional gel analysis of their hybrid-arrest translation products. GmHSP22.3 is less closely related to GmHSP22 (48% identical and 70% conserved) and GmHSP22.5 (47% identical and 65% conserved). The fourth cDNA clone, GmHSP23.9, encodes a HSP of ca. 24 kDa with an amino terminus that has characteristics of some mitochondrial transit sequences, and in contrast to GmHSP22, GmHSP22.3, and GmHSP22.5, the corresponding mRNA is preferentially associated in vivo with free polysomes. It is proposed that the LMW HSP gene superfamily be expanded to at least six classes to include a mitochondrial class and an additional endomembrane class of LMW HSPs.
Asunto(s)
Glycine max/genética , Secuencia de Aminoácidos , Secuencia de Bases , ADN Complementario/genética , Biblioteca de Genes , Proteínas de Choque Térmico/química , Proteínas de Choque Térmico/clasificación , Membranas/química , Datos de Secuencia Molecular , Polirribosomas/química , Conformación Proteica , ARN de Planta/análisis , Análisis de Secuencia de ADN , Homología de Secuencia de AminoácidoRESUMEN
G-box binding factors (GBFs) constitute a family of plant DNA-binding proteins that bind to the G-box motif, a regulatory cis element present in many plant genes with a palindromic DNA motif of CACGTG. Previously TCCACGTGTC, a G-box motif, from an auxin responsive gene GmAux28 has been identified as a sequence-specific protein-binding site. Here the isolation of two soybean cDNA clones, referred to as SGBF-1 and SGBF-2, encoding proteins which bind to the G-box motif is reported. The primary structure of SGBF-1 and SGBF-2 predicts that these proteins contain a basic leucine zipper (bZIP) DNA-binding domain and an N-terminal proline-rich domain. A dramatic difference in the pattern of protein-DNA complex formation was observed when recombinant SGBF-1 and SGBF-2 proteins were analyzed by electrophoretic mobility shift assays (EMSAs). The SGBF-1 binding pattern obtained with the G-box probe resulted in three major retarded bands while the SGBF-2 formed a single complex. This shows that the characteristically diffuse banding pattern of plant nuclear proteins interacting with the G-box is also observed in a binding assay using only one recombinant GBF. EMSAs were performed with a few selected binding sequences to study the effect of flanking nucleotides to the hexanucleotide G-box core motif. The binding specificity of the SGBF proteins resembles that described for type A cauliflower nuclear G-box binding proteins which bind class I G-box elements [(G/T)(C/A)CACGTG(G/T)(A/C)]. Phylogenetic analysis of 13 GBF-like proteins from various plant species reveals that the SGBF-1 and SGBF-2 proteins belong to different lineages, suggesting that they may have distinct functions in activating transcription.
Asunto(s)
ADN de Plantas/metabolismo , Proteínas de Unión al ADN/metabolismo , Regulación de la Expresión Génica de las Plantas , Glycine max/genética , Ácidos Indolacéticos/metabolismo , Secuencias Reguladoras de Ácidos Nucleicos/genética , Factores de Transcripción , Secuencia de Aminoácidos , Secuencia de Bases , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico , Northern Blotting , Southern Blotting , Clonación Molecular , ADN Complementario/genética , ADN de Plantas/genética , Proteínas de Unión al ADN/genética , Factores de Unión a la G-Box , Genes de Plantas/genética , Datos de Secuencia Molecular , Familia de Multigenes/genética , Unión Proteica , Conformación Proteica , Análisis de Secuencia de ADNRESUMEN
A cDNA clone encoding a 101-kD heat shock protein (HSP101) of soybean was isolated and sequenced. Genomic DNA gel blot analysis indicated that the corresponding gene is a member of a multigene family. The mRNA for HSP101 was not detected in 2-day-old etiolated soybean seedlings grown at 28 degrees C but was induced by elevated temperatures. DNA sequence comparison has shown that the corresponding gene belongs to the Clp (caseinolytic protease) (or Hsp100) gene family, which is evolutionarily conserved and found in both prokaryotes and eukaryotes. On the basis of the spacer length between the two conserved ATP binding regions, this gene has been identified as a member of the ClpB subfamily. Unlike other Clp genes previously isolated from higher plants, the expression of this soybean Hsp101 gene is heat inducible, and it does not have an N-terminal signal peptide for targeting to chloroplasts. Transformation of the soybean Hsp101 gene into a yeast HSP104 deletion mutant complemented restoration of acquired thermotolerance, a process in which cells survive an otherwise lethal heat stress after they are given a permissive heat treatment.
Asunto(s)
Adaptación Fisiológica/genética , Prueba de Complementación Genética , Glycine max/genética , Proteínas de Choque Térmico/genética , Proteínas de Plantas/genética , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Secuencia de Aminoácidos , Secuencia de Bases , ADN Complementario , ADN de Plantas , Proteínas de Choque Térmico/fisiología , Calor , Datos de Secuencia Molecular , Mutación , Eliminación de SecuenciaRESUMEN
In this study, we report on two patients diagnosed with active pulmonary tuberculosis who later developed complications of lung cancer. In both instances, lung cancer was not detected until after cessation of tuberculostatic drugs. Both patients were initially considered to be experiencing exacerbation of pulmonary tuberculosis. Patient 1 was a 77-year-old female. A roentgenogram of her chest revealed a cavitary lesion with infiltration into the right lung field. Her sputum tested positive for acid-fast bacilli. Although she was treated with isoniazid (INH), rifampicin (RFP) and streptomycin sulfate (SM), the RFP and INH treatments had to be discontinued due to liver dysfunction. Her general condition was deteriorated, and pleural effusion appeared on a subsequent chest roentgenogram. Primary squamous-cell lung cancer was confirmed by conducting a transbronchial biopsy. Patient 2 was a 59-year-old male. A roentgenogram of his chest revealed multiple cavitary lesions with infiltration into the bilateral lung field. His sputum also tested positive for acid-fast bacilli. Although he was treated with INH, RFP and SM, INH and RFP treatment had to be discontinued due to liver dysfunction and high fever. The shadow infiltrating the left lung field subsided, but a massive shadow appeared in the right lung field. Primary small-cell lung cancer was confirmed after conducting a sputum cytology. The patients was then administered cisplatin and etoposide. Patient 1 was diagnosed with lung cancer five months after being admitted to the hospital, and Patient 2 ten months after admission. Both patients succumbed due to lung cancer at seven and 26 months, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)
Asunto(s)
Neoplasias Pulmonares/complicaciones , Tuberculosis Pulmonar/complicaciones , Anciano , Carcinoma de Células Pequeñas/complicaciones , Carcinoma de Células Escamosas/complicaciones , Femenino , Humanos , Masculino , Persona de Mediana EdadRESUMEN
We have characterized three different soybean (Glycine max) mRNAs that encode apoproteins of extensins, a family of cell wall hydroxyproline-rich glycoproteins (HRGPs). These transcripts encoded distinctive Tyr-rich proteins containing characteristic Ser-Pro4 sequences organized in higher-order repetitive units. The first transcript encoded an extensin SbHRGP-1 containing the 16-amino acid repeat Ser-Pro4-Ser-Pro-Ser-Pro4-Tyr-Val-Tyr-Lys, with Val occasionally replaced by Ile or Tyr. The second transcript encoded the SbHRGP-2 protein containing the 16-amino acid repeat Ser-Pro4-Ser-Pro-Ser-Pro4-Tyr-Tyr-Tyr-Lys/His. The third transcript encoded the SbHRGP-3 protein containing a variant of 9- or 10-amino acid canonical repeats: Ser-Pro4-Tyr-Lys-Tyr-Pro, Ser-Pro5-Tyr-Lys-Tyr-Pro, and Ser-Pro4-Val-Tyr-Lys-Tyr-Lys, respectively. The dramatic amino acid substitutions in the Tyr-rich blocks (Tyr-X-Tyr-Lys) among these HRGPs indicate that each SbHRGP may have a different function in cell wall architecture.
Asunto(s)
Glycine max/genética , Glicoproteínas/genética , Proteínas de Plantas/genética , Secuencia de Aminoácidos , Secuencia de Bases , Northern Blotting , Clonación Molecular , ADN Complementario/genética , ADN Complementario/aislamiento & purificación , Datos de Secuencia Molecular , Homología de Secuencia de Aminoácido , Transcripción GenéticaRESUMEN
Five constructions containing deletions of the promoter from an auxin-inducible gene of Arabidopsis thaliana, AtAux2-11, were fused to the coding region of the reporter gene LacZ, which encodes beta-galactosidase, and a polyadenylation 3'-untranslated nopaline synthase sequence from Agrobacterium. These chimeric genes were introduced into Arabidopsis by Agrobacterium tumefaciens-mediated transformation, and expression of the gene was examined by spectrophotometric and histochemical analyses. A 600 bp fragment from the AtAux2-11 promoter conferred histochemical patterns of staining similar to the longest 5' promoter tested, a 3.0 kb fragment. Localization of AtAux2-11/LacZ activity in the transgenic plants revealed spatial and temporal expression patterns that correlated with tissues and cells undergoing physiological processes modulated by auxin. LacZ activity was expressed in the elongating region of roots, etiolated hypocotyls, and anther filaments. Expression was detected in the vascular cylinder of the root and the vascular tissue, epidermis, and cortex of the hypocotyl, and filament. The AtAux2-11/LacZ gene was preferentially expressed in cells on the elongating side of hypocotyls undergoing gravitropic curvature. Expression of the chimeric gene in the hypocotyls of light-grown seedlings was less than that in etiolated seedling hypocotyls. The AtAux2-11/LacZ gene was active in the root cap, and expression in the root stele increased at sites of lateral root initiation. Staining was evident in cell types that develop lignified cell walls, e.g. trichomes, anther endothecial cells, and especially developing xylem. The chimeric gene was not expressed in primary meristems. While the magnitude of expression increased after application of exogenous auxin (2,4-D), the histochemical localization of AtAux2-11/LacZ remained unchanged. Transgenic plants with a 600 bp promoter construct (-0.6 kb AtAux2-11/LacZ) had higher levels of basal and auxin-inducible expression than plants with a 3.0 kb promoter construct. Transgenic plants with a -500 bp promoter had levels of expression similar to the -3.0 kb construct. The -0.6 kb AtAux2-11/LacZ gene responded maximally to a concentration of 5 x 10(-6) to 5 x 10(-5) M 2,4-D and was responsive to as little as 5 x 10(-8) M. The evidence presented here suggests that this gene may play a role in several auxin-mediated developmental and physiological processes.
Asunto(s)
Arabidopsis/genética , Genes de Plantas , Ácidos Indolacéticos/fisiología , Aminoácido Oxidorreductasas/biosíntesis , Aminoácido Oxidorreductasas/genética , Arabidopsis/anatomía & histología , Arabidopsis/crecimiento & desarrollo , Secuencia de Bases , ADN , Regulación de la Expresión Génica , Operón Lac , Datos de Secuencia Molecular , Plantas Modificadas Genéticamente , Regiones Promotoras Genéticas , Rhizobium/enzimología , Rhizobium/genética , Semillas/metabolismo , Transformación Genética , beta-Galactosidasa/biosíntesis , beta-Galactosidasa/genéticaRESUMEN
The effects of expressing a chimeric gene consisting of a soybean heat shock gene promoter and a sequence that encodes an enzyme catalyzing the synthesis of a potent phytohormone, the cytokinin iPMP, have been analyzed in transgenic tobacco plants. The production of cytokinin endogenously produced several effects previously undocumented. The differentiation of shoots independent of exogenous cytokinin from heat-treated transgenic plant leaf explants demonstrates that long-term heat treatments do not interfere with complex developmental processes. This extends the potential usefulness of heat shock gene promoters to conditionally express genes during windows of development that span several weeks.
Asunto(s)
Transferasas Alquil y Aril , Citocininas/biosíntesis , Regulación de la Expresión Génica , Glycine max/genética , Nicotiana/metabolismo , Plantas Tóxicas , Adenosina/análogos & derivados , Adenosina/análisis , Citocininas/farmacología , Inducción Enzimática , Genes de Plantas/genética , Proteínas de Choque Térmico/genética , Calor , Isopenteniladenosina/análogos & derivados , Isopenteniladenosina/análisis , Morfogénesis/efectos de los fármacos , Plantas Modificadas Genéticamente , Regiones Promotoras Genéticas/genética , Nicotiana/efectos de los fármacos , Nicotiana/genética , Transferasas/genética , Transferasas/metabolismoRESUMEN
Five cDNA clones (ADR6, ADR11-1, ADR11-2, ADR12-1 and ADR12-2), representing three families of auxin down-regulated (ADR) genes were isolated and characterized. These were isolated by screening a lambda Zap cDNA library with the partial cDNA clones p6, p11 and p12, isolated earlier (Baulcombe and Key, J Biol Chem 255: 8907-8913, 1980). Hybrid-select translation of ADR6, ADR11-2 and ADR12-2 clones produced polypeptides of 33 kDa 22.5 kDa and a 6 and 7 kDa respectively, when analyzed by SDS-PAGE. ADR6 and ADR12-2 gave one and two spots, respectively, on an IEF-SDS 2D gel. ADR11-2 probably encodes a basic protein as it was only resolved on non-equilibrium pH gradient gel electrophoresis (NEPHGE). Genomic Southern blot analysis of ADR6, ADR11 and ADR12 suggests that each represents a small multigene family. The RNA levels corresponding to ADR6, ADR11 and ADR12 decrease in response to applied auxin by 100-, 15- and 10-fold, respectively (Baulcombe and Key, 1980). Runoff transcription, done in the presence and absence of auxin, showed that the rate of transcription of the genes corresponding to ADR6, ADR11-2 and ADR12-2 was reduced in the presence of auxin, but the decrease was small relative to the decrease in the cytoplasmic levels of these mRNAs, in response to auxin. A comparative analysis of the influence of auxin on in vitro transcription and steady state RNA levels corresponding to these ADR cDNAs suggests that the decrease in rate of transcription due to auxin is not enough to account for the auxin-induced decrease in the steady state levels. Northern analysis showed developmental and organ/tissue-specific response of these ADR genes. Furthermore, the expression of the genes corresponding to ADR6 and ADR12-1 appears to be up-regulated by light, whereas the gene corresponding to ADR11 appears to be down-regulated by light.
Asunto(s)
Genes de Plantas/genética , Glycine max/genética , Ácidos Indolacéticos/fisiología , Secuencia de Aminoácidos , Secuencia de Bases , Clonación Molecular , Regulación hacia Abajo/genética , Luz , Datos de Secuencia Molecular , Familia de Multigenes , Especificidad de Órganos , Poli A/metabolismo , ARN Mensajero/metabolismo , Transcripción Genética/fisiología , Regulación hacia Arriba/genéticaRESUMEN
The promoter region of a soybean auxin-responsive gene, GmAux28, was analyzed to identify protein-binding DNA sequences that may be involved in regulation of expression. Using DNase I footprinting and gel mobility shift assays, multiple regions of interaction, including eight major protein-binding sites, were observed in the GmAux28 gene. Two sequence motifs, TGACGACA and TCCACGTGTC, related to as-1/Hex and G-box elements, respectively, found in several plant promoters, were identified. Four distinct A/T-rich domains were identified; such A/T-rich domains appear to modulate, but not to specify, the expression of many genes. Two new sequence motifs, delta-1 (D1) and delta-4 (D4) were also identified. D1 and D4 share a very similar core sequence, TAGTxxCTGT and TAGTxCTGT, respectively. In gel mobility shift analyses, D1 and D4 elements exhibit a complex interaction of binding proteins. The GmAux22 promoter also contains D1-related elements which compete with the GmAux28 elements. Sequence comparisons have identified D1/D4-like sequences in several other auxin-responsive genes suggesting the possible importance of D1/D4 and the respective binding proteins in the regulation of expression of these genes.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Regulación de la Expresión Génica , Genes de Plantas , Glycine max/genética , Ácidos Indolacéticos/fisiología , Regiones Promotoras Genéticas , Secuencia de Bases , Sitios de Unión , ADN/metabolismo , Datos de Secuencia Molecular , Proteínas Nucleares/metabolismoRESUMEN
Three related gene families of low-molecular-weight (LMW) heat shock proteins (HSPs) have been characterized in plants. We describe a fourth LMW HSP family, represented by PsHSP22.7 from Pisum sativum and GmHSP22.0 from Glycine max, and demonstrate that this family of proteins is endomembrane localized. PsHSP22.7 and GmHSP22.0 are 76.7% identical at the amino acid level. Both proteins have amino-terminal signal peptides and carboxyl-terminal sequences characteristic of endoplasmic reticulum (ER) retention signals. The two proteins closely resemble class I cytoplasmic LMW HSPs, suggesting that they evolved from the cytoplasmic proteins through the addition of the signal peptide and ER retention motif. The endomembrane localization of these proteins was confirmed by cell fractionation. The polypeptide product of PsHSP22.7 mRNA was processed to a smaller-M(r) form by canine pancreatic microsomes; in vivo, GmHSP22.0 polysomal mRNA was found to be predominantly membrane bound. In vitro-processed PsHSP22.7 corresponded in mass and pI to one of two proteins detected in ER fractions from heat-stressed plants by using anti-PsHSP22.7 antibodies. Like other LMW HSPs, PsHSP22.7 was observed in higher-molecular-weight structures with apparent masses of between 80 and 240 kDa. The results reported here indicate that members of this new class of LMW HSPs are most likely resident ER proteins and may be similar in function to related LMW HSPs in the cytoplasm. Along with the HSP90 and HSP70 classes of HSPs, this is the third category of HSPs localized to the ER.