RESUMEN
The expression, regulation and cellular localization of ZmHyPRP, a gene marker of embryo differentiation whose expression declines after ABA induction, was studied. ZmHyPRP is a proline-rich protein with a C-terminal domain having eight cysteines in a CM8 pattern. Transient expression in onion epidermal cells, transformed with a 2x35S::ZmHyPRP-GFP construction, indicated the protein is present in vesicles lining the membrane of the cell. The ZmHyPRP gene expression is under the control of classic promoter seed-specific regulatory elements such as Sph/RY and G-boxes, suggesting regulation by B3 and b-ZIP transcription factors. Promoter deletion analysis, by particle-bombardment transient transformation of maize immature embryos with serial deletions of the promoter fused to GUS, showed the presence of two negative regulatory elements, NE1 (-2070 to -1280) and NE2 (-232 to -178), in the ZmHyPRP promoter. By selective deletion or mutation of ZmHyPRP regulatory promoter elements we conclude that the promoter expression is attenuated by the NE2 element as well as by the G-box2 and the Sph1-2 box together with the G-box2.
Asunto(s)
Regulación de la Expresión Génica de las Plantas/genética , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Regiones Promotoras Genéticas/genética , Zea mays/genética , Zea mays/metabolismo , Ácido Abscísico/farmacología , Secuencia de Bases , ADN de Plantas/genética , Regulación de la Expresión Génica de las Plantas/efectos de los fármacos , Datos de Secuencia Molecular , Mutación , Cebollas/genética , Cebollas/metabolismo , Especificidad de Órganos , Plantas Modificadas Genéticamente/genética , Plantas Modificadas Genéticamente/metabolismo , Estructura Terciaria de Proteína , Transporte de Proteínas , Proteínas Recombinantes de Fusión , Semillas/genética , Semillas/metabolismo , Análisis de Secuencia de ADN , Eliminación de Secuencia , Zea mays/efectos de los fármacos , Zea mays/embriologíaRESUMEN
The expression of a cytosolic glutamine synthetase (GS1; EC 6.3.1.2) gene was examined in cotyledons of Scots pine seedlings. Light strongly stimulated GS1 mRNA accumulation during development. Similarly, steady-state levels of GS1 transcripts increased in dark-grown seedlings transferred to light and decreased in dark-adapted seedlings. Light/dark adaptation affected rbcS and lhcb2 mRNA levels and chlorophyll contents in the same manner. Light-grown seedlings in the presence of the herbicide norflurazon showed a drastic decrease in mRNA for GS and photosynthetic proteins, whereas the effect of the herbicide on mitochondrial beta-ATP synthase mRNA was limited. These results indicate that factors associated with developing chloroplasts could be required for maximal GS1 gene expression during seedling development. The level of GS polypeptide, determined by immunoblot, was up-regulated during seedling development in the light or dark. However, the levels of the polypeptide detected were unaltered by the light/dark adaptation treatments. The analysis of GS1 mRNA association with polysomes indicated that the discrepancies between GS protein and mRNA levels are not a result of a differential translational rate of the transcript in darkness relative to light. Two GS isoproteins with different isoelectric point were resolved by two-dimensional PAGE in light- and dark-germinated plants. The relative abundance of one of them was markedly affected by light and correlated with the observed changes in GS mRNA, suggesting that the other form is not a product derived from the detected transcript. In situ hybridization of cotyledon sections showed the presence of GS1 mRNAs in mesophyll and phloem cells confirming gene expression in photosynthetic tissues. High levels of transcript were detected also in meristematic cells of apical primordia. These data suggest a dual role for the GS1 gene associated with chloroplast development/activity and glutamine biosynthesis for nitrogen mobilization during early growth of Scots pine.
Asunto(s)
Cotiledón/genética , Glutamato-Amoníaco Ligasa/genética , Plantas/genética , Árboles/genética , Cloroplastos/fisiología , Cotiledón/enzimología , Citosol/enzimología , Regulación del Desarrollo de la Expresión Génica/efectos de la radiación , Regulación Enzimológica de la Expresión Génica/efectos de la radiación , Regulación de la Expresión Génica de las Plantas/efectos de la radiación , Glutamato-Amoníaco Ligasa/metabolismo , Hibridación in Situ , Luz , Plantas/enzimología , ARN Mensajero/genética , ARN Mensajero/metabolismo , ARN Mensajero/efectos de la radiación , Distribución Tisular , Transcripción Genética , Árboles/enzimología , Árboles/crecimiento & desarrolloRESUMEN
A clone encoding a proline-rich protein (ZmPRP) has been obtained from maize root by differential screening of a maturing elongation root cDNA library. The amino acid sequence deduced from the full-length cDNA contains a putative signal peptide and a highly repetitive sequence containing the PEPK motif, indicating that the ZmPRP mRNA may code for a cell wall protein. The PEPK repeat is also found in a previously reported wheat sequence but differs from the repeated sequences found in hydroxyproline-rich glycoproteins (HRGP) and in dicot proline-rich proteins (PRP). In the maize genome, the ZmPRP protein is encoded by a single gene that is expressed in maturing regions of the root, in the hypocotyl and in the pericarp. In these organs, the ZmPRP mRNA accumulates in the xylem and surrounding cells, and in the epidermis. No ZmPRP mRNA was found in the phloem. The pattern of mRNA accumulation is very similar to the one observed for genes coding for proteins involved in lignin biosynthesis and, like most cell wall proteins, ZmPRP synthesis is also induced by wounding. These data support the hypothesis that ZmPRP is a member of a new class of fibrous proteins involved in the secondary cell wall formation in monocot species.
Asunto(s)
Proteínas de Plantas/metabolismo , ARN Mensajero/metabolismo , Zea mays/genética , Secuencia de Aminoácidos , Pared Celular/fisiología , Clonación Molecular , ADN Complementario/química , ADN Complementario/genética , Dosificación de Gen , Regulación de la Expresión Génica de las Plantas , Hipocótilo/citología , Hipocótilo/metabolismo , Hibridación in Situ , Datos de Secuencia Molecular , Proteínas de Plantas/genética , Raíces de Plantas/citología , Raíces de Plantas/metabolismo , ARN Mensajero/genética , Distribución Tisular , Zea mays/químicaRESUMEN
The pattern of expression of two genes coding for proline-rich proteins, zmHyPRP and zmHRGP, in Zea mays is modified when the embryogenesis programme is altered by placing the embryos in conditions which promote either precocious germination or callogenesis. zmHyPRP gene expression is rapidly arrested when the embryogenesis programme is altered. zmHRGP mRNA is highly induced in scutellum within a few hours of callogenesis or precocious germination.
Asunto(s)
Regulación del Desarrollo de la Expresión Génica , Glicoproteínas/genética , Péptidos/genética , Proteínas de Plantas/genética , Prolina , Zea mays/genética , Germinación , Glicoproteínas/química , Proteínas de Plantas/química , Dominios Proteicos Ricos en Prolina , Zea mays/embriologíaRESUMEN
The pattern of expression of two genes coding for proteins rich in proline, HyPRP (hybrid proline-rich protein) and HRGP (hydroxyproline-rich glycoprotein), has been studied in maize (Zea mays) embryos by RNA analysis and in situ hybridization. mRNA accumulation is high during the first 20 d after pollination, and disappears in the maturation stages of embryogenesis. The two genes are also expressed during the development of the pistillate spikelet and during the first stages of embryo development in adjacent but different tissues. HyPRP mRNA accumulates mainly in the scutellum and HRGP mRNA mainly in the embryo axis and the suspensor. The two genes appear to be under the control of different regulatory pathways during embryogenesis. We show that HyPRP is repressed by abscisic acid and stress treatments, with the exception of cold treatment. In contrast, HRGP is affected positively by specific stress treatments.
RESUMEN
A gene from maize that encodes a hybrid proline-rich protein (HyPRP) formed by two well-defined domains, proline-rich and hydrophobic, respectively, has been characterized at the level of its structure and expression. The proline-rich domain is composed of elements PPYV and PPTPRPS, similar to those found in PRP proteins from soybean. The hydrophobic domain is rich in cysteine and is similar to seed proteins, mainly to a soybean hydrophobic seed protein. In maize, HyPRP is encoded by a single gene, and its mRNA accumulates in immature maize zygotic embryos, with a maximum accumulation between 12 and 18 days after pollination. The HyPRP mRNA can also be detected in ovary prior to pollination. In situ hybridization experiments on embryo sections show an expression of the gene in scutellum and in nonvascular cells from the embryo axis. Functional hypotheses related to HyPRP are discussed.
Asunto(s)
Proteínas de Plantas/genética , Zea mays/genética , Secuencia de Aminoácidos , Secuencia de Bases , Clonación Molecular , ADN , Datos de Secuencia Molecular , Hibridación de Ácido Nucleico , Prolina/genética , ARN Mensajero/genética , Agua , Zea mays/embriologíaRESUMEN
Rat liver nuclear extracts were tested for the presence of factors which might be common to the transcriptional regulation of both the albumin and alpha-foetoprotein genes. Gel shift assay showed the formation of three complexes (I, II and III) with the albumin probe. Two of them (I and III) could be displaced by the alpha-foetoprotein promoter. Analysis of nuclear extracts from liver, kidney, spleen and brain and competition experiments using several oligonucleotides covering regions from the albumin and alpha-foetoprotein promoters showed that complex III results from the binding of the ubiquitous nuclear factor 1, while complex II involves a CCAAT-box-binding protein also detected in brain and spleen extracts. Complex I is formed upon binding of a liver-specific factor to a proximal element of the rat albumin promoter. This factor also binds to a similar sequence in the alpha-foetoprotein promoter and is closely related to the hepatocyte nuclear factor 1, as shown by competition experiments using an oligonucleotide covering its target sequence on the beta-fibrinogen promoter. Transfection competition experiments indicated that, in vivo, this factor acts as a positive trans-acting element in the expression of both the rat albumin and alpha-foetoprotein genes.
Asunto(s)
Albúminas/genética , Hígado/metabolismo , Factores de Transcripción/aislamiento & purificación , alfa-Fetoproteínas/genética , Albúminas/metabolismo , Animales , Secuencia de Bases , Sitios de Unión , Unión Competitiva , Química Encefálica , Núcleo Celular/metabolismo , Sondas de ADN , Regulación de la Expresión Génica , Genes Homeobox , Técnicas In Vitro , Riñón/análisis , Hígado/análisis , Plásmidos , Regiones Promotoras Genéticas , Ratas , Ratas Endogámicas , Bazo/análisis , Factores de Transcripción/fisiología , Transcripción Genética , Transfección , alfa-Fetoproteínas/metabolismoRESUMEN
Proteins putatively involved in the transcriptional control of rat (alpha-fetoprotein (AFP) gene expression in the liver were identified by analyzing the in vitro binding of proteins from nuclear extracts of fetal and adult rat livers to the cloned rat AFP promoter region (-197 to +48) using a combination of gel shift and DNase I footprinting assays. Three stable and specific high affinity complexes (I, II, and III) were detected by gel shift analysis of fetal rat liver extracts. Complex II was specific to extracts from fetal liver while the two others (I and III) were also formed with extracts from adult liver. Complex I was highly liver-specific since it was not detected with extracts of kidney, spleen, and brain. DNase I and gel shift competition experiments using a synthetic oligonucleotide indicated that it is formed upon binding of a liver-specific factor to region -65 to -46 of the rat AFP promoter. This region, perfectly conserved in the rat, mouse, and man, had been previously shown to be crucial for liver-specific expression of the mouse AFP gene. The binding of this liver-specific factor to this DNA region may thus represent a key step in the specificity of expression of AFP gene in liver. Gel shift and DNase I footprinting competition experiments showed that complex III was formed by binding of the widely distributed nuclear factor I to the rat AFP promoter in the region -125 to -100. It is potentially significant that, in extracts from fetal liver, nuclear factor is also involved with another binding factor in the formation of the stage-specific complex II.
Asunto(s)
Proteínas Potenciadoras de Unión a CCAAT , Proteínas de Unión al ADN/metabolismo , Genes , Hígado/metabolismo , Proteínas Nucleares/metabolismo , Regiones Promotoras Genéticas , Factores de Transcripción , Transcripción Genética , alfa-Fetoproteínas/genética , Animales , Secuencia de Bases , Regulación de la Expresión Génica , Genes Reguladores , Hígado/embriología , Datos de Secuencia Molecular , Factores de Transcripción NFI , Especificidad de Órganos , Ratas , Proteína 1 de Unión a la Caja YRESUMEN
Functional and structural approaches were used to characterize the transcription units of the rat alpha-feto-protein (AFP) and albumin genes. A cell-free nuclear transcription assay and several genomic clones were used to show that: 1) the rate of transcription of these genes is closely related to the levels of corresponding mRNAs in the yolk sac and during rat liver development, indicating that the expression of the albumin and AFP genes is mainly regulated at the transcriptional level in the rat, and 2) the in vivo 5' end boundaries of the rat AFP and albumin transcription domains were mapped near the respective first exons. Due to the presence of repeated sequences, the 3' end boundary of both genes could not be accurately defined in the same manner. 3) No transcription could be detected until 7 kilobases upstream from the cap site of these genes. In addition, the organization of the rat AFP gene was analyzed by restriction endonuclease mapping, S1 nuclease mapping, and nucleotide sequencing. Our results indicate that: 1) the rat AFP gene is 20 kilobase pairs long and is split into 15 exons by 14 intervening sequences; 2) the transcription initiation site of the rat AFP gene is heterogenous; 3) the 5'-flanking region upstream from the rat AFP gene exhibits 60-90% similarity with the mouse and human AFP genes while no major nucleotide identity is found with the rat albumin gene; 4) a 90-base pair sequence present as one copy upstream from the rat and mouse AFP genes is present as two copies in the human genome; 5) several inverted repeats are mapped in the 5'-flanking region indicating potential stem-loop structures. One highly conserved structure encompasses an enhancer-like core sequence and the sequence recognized by the TGGCA-binding protein.