RESUMEN
Nodulin genes are specifically expressed in the nitrogen-fixing root nodules. We have identified a novel type of DNA-binding protein (CPP1) interacting with the promoter of the soybean leghemoglobin gene Gmlbc3. The DNA-binding domain of CPP1 contains two similar Cys-rich domains with 9 and 10 Cys, respectively. Genes encoding similar domains have been identified in Arabidopsis thaliana, Caenorhabditis elegans, the mouse, and human. The domains also have some homology to a Cys-rich region present in some polycomb proteins. The cpp1 gene is induced late in nodule development and the expression is confined to the distal part of the central infected tissue of the nodule. A constitutively expressed cpp1 gene reduces the expression of a Gmlbc3 promoter-gusA reporter construct in Vicia hirsuta roots. These data therefore suggest that CPP1 might be involved in the regulation of the leghemoglobin genes in the symbiotic root nodule.
Asunto(s)
Proteínas de Unión al ADN/genética , Genes de Plantas , Glycine max/genética , Leghemoglobina/genética , Raíces de Plantas/microbiología , Secuencia de Aminoácidos , Sitios de Unión , Compartimento Celular , Núcleo Celular/química , Cisteína , Regulación de la Expresión Génica de las Plantas , Leghemoglobina/biosíntesis , Datos de Secuencia Molecular , Proteínas de Plantas/genética , Unión Proteica , Secuencias Repetitivas de Aminoácido , Rhizobiaceae , Homología de Secuencia de Aminoácido , Simbiosis , Distribución TisularRESUMEN
A DNA-binding protein, VsENBP1, previously isolated from Vicia sativa was shown to bind in a sequence-specific manner to the early nodulin ENOD12 gene promoter from Pisum sativum. Here, the functional importance of the VsENBP1 binding sites on the PsENOD12B promoter has been studied in vivo. A promoter-gusA fusion in which a mutation was introduced at the putative target sequence, AATAA, was inactive in nodules of transgenic Vicia hirsuta roots. Gel retardation assays showed that VsENBP1 does not bind to the mutated promoter segment, suggesting that VsENBP1 activates the PsENOD12B expression in nodules through its interaction with its target sequence. In the presence of the 35S enhancer, an ENOD12 promoter-GUS construct gave expression in root vascular tissue in addition to the root nodules. Overexpression of Vsenbp1 in transgenic V. hirsuta roots reduced the leaky expression in root vascular tissue in contrast to nodules in which a small increase in GUS expression was observed. The results indicate that VsENBP1 acts as a repressor of ENOD12 expression in root tissue.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Factores de Transcripción/metabolismo , Secuencia de Bases , ADN de Plantas/genética , ADN de Plantas/metabolismo , Proteínas de Unión al ADN/genética , Elementos de Facilitación Genéticos , Fabaceae/genética , Fabaceae/metabolismo , Regulación de la Expresión Génica de las Plantas , Genes de Plantas , Genes Reporteros , Datos de Secuencia Molecular , Mutación , Pisum sativum/genética , Plantas Modificadas Genéticamente , Plantas Medicinales , Regiones Promotoras Genéticas , Factores de Transcripción/genéticaRESUMEN
A cDNA containing a homeobox sequence was isolated from a soybean nodule-specific expression library. This homeobox cDNA, Ndx (nodulin homeobox), represents a small gene family with at least two members in soybean (Glycine max) and three in Lotus japonicus. One complete 3304 bp Ndx cDNA from L. japonicus encodes a protein, NDX, of 958 amino acids. An unusual type of homeodomain that differs in two of the most conserved amino acid positions in the consensus sequence is located close to the C-terminal and appears to be the only DNA-binding domain. Weak Ndx gene expression in the root increases very shortly after infection with Rhizobium and remains throughout nodule development. In situ hybridizations show cell-specific expression patterns that suggest developmentally separate regions in maturing determinate nodules. Thus in the maturing nodule Ndx and leghemoglobin genes are expressed in a mutually exclusive fashion. The Ndx transcript is also detectable in the young nodule primordium. Ndx expression is not confined to the root nodule since Ndx is also expressed in shoot and root meristems, indicating that the Ndx gene products might also be involved in developmental processes in other plant tissues.
Asunto(s)
Genes Homeobox , Genes de Plantas , Proteínas de Homeodominio/genética , Magnoliopsida/genética , Proteínas de la Membrana , Raíces de Plantas/genética , Simbiosis/genética , Secuencia de Aminoácidos , Secuencia Conservada , ADN Complementario/genética , Proteínas de Homeodominio/clasificación , Proteínas de Homeodominio/aislamiento & purificación , Hibridación in Situ , Leghemoglobina/genética , Familia de Multigenes , Proteínas de Plantas/biosíntesis , Estructura Secundaria de Proteína , ARN Mensajero/aislamiento & purificación , ARN de Planta/aislamiento & purificación , Homología de Secuencia de Aminoácido , Glycine max/genética , Distribución TisularRESUMEN
The pea genes PsENOD12A and PsENOD12B are expressed in the root hairs shortly after infection with the nitrogen-fixing bacterium Rhizobium leguminosarum bv. viciae or after application of purified Nod factors. A 199 bp promoter fragment of the PsENOD12B gene contains sufficient information for Nod factor-induced tissue-specific expression. We have isolated a Vicia sativa cDNA encoding a 1641 amino acid protein, ENBP1, that interacts with the 199 bp ENOD12 promoter. Two different DNA-binding domains were identified in ENBP1. A domain containing six AT-hooks interacts specifically with an AT-rich sequence located between positions -95 and -77 in the PsENOD12B promoter. A second domain in ENBP1 is a cysteine-rich region that binds to the ENOD12 promoter in a sequence non-specific but metal-dependent way. ENBP1 is expressed in the same cell types as ENOD12. However, additional expression is observed in the nodule parenchyma and meristem. The presence of three small overlapping ORFs in the 5'-untranslated region of the ENBP1 cDNA indicates that ENBP1 expression might be regulated at the translational level. The interaction of ENBP1 with a conserved AT-rich element within the ENOD12 promoter and the presence of the ENBP1 transcript in cells expressing ENOD12 strongly suggest that ENBP1 is a transcription factor involved in the regulation of ENOD12. Finally, the C-terminal region of ENBP1 shows strong homology to a protein from rat that is specifically expressed in testis tissue.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Proteínas de la Membrana , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Regiones Promotoras Genéticas , Factores de Transcripción/metabolismo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Sitios de Unión , ADN Complementario , ADN de Plantas/metabolismo , Proteínas de Unión al ADN/genética , Fabaceae , Expresión Génica , Datos de Secuencia Molecular , Plantas Medicinales , Ratas , Homología de Secuencia de Aminoácido , Factores de Transcripción/genéticaRESUMEN
We have isolated a new hemoglobin gene from soybean. It is expressed in cotyledons, stems of seedlings, roots, young leaves, and in some cells in the nodules that are associated with the nitrogen-fixing Bradyrhizobium symbiont. This contrasts with the expression of the leghemoglobins, which are active only in the infected cells of the nodules. The deduced protein sequence of the new gene shows only 58% similarity to one of the soybean leghemoglobins, but 85-87% similarity to hemoglobins from the nonlegumes Parasponia, Casuarina, and barley. The pattern of expression and the gene sequence indicate that this new gene is a nonsymbiotic legume hemoglobin. The finding of this gene in legumes and similar genes in other species strengthens our previous suggestion that genomes of all plants contain hemoglobin genes. The specialized leghemoglobin gene family may have arisen from a preexisting nonsymbiotic hemoglobin by gene duplication.
Asunto(s)
Glycine max/genética , Hemoglobinas/genética , Proteínas de la Membrana , Secuencia de Aminoácidos , Secuencia de Bases , Secuencia de Consenso , ADN de Plantas , Expresión Génica , Hemoglobinas/fisiología , Datos de Secuencia Molecular , Hibridación de Ácido Nucleico , Proteínas de Plantas/genética , Regiones Promotoras Genéticas , Homología de Secuencia de Aminoácido , Homología de Secuencia de Ácido NucleicoRESUMEN
Casuarina glauca has a gene encoding hemoglobin (cashb-nonsym). This gene is expressed in a number of plant tissues. Casuarina also has a second family of hemoglobin genes (cashb-sym) expressed at a high level in the nodules that Casuarina forms in a nitrogen-fixing symbiosis with the actinomycete Frankia. Both the nonsymbiotic and symbiotic genes retained their specific patterns of expression when introduced into the legume Lotus corniculatus. We interpret this finding to mean that the controls of expression of the symbiotic gene in Casuarina must be similar to the controls of expression of the leghemoglobin genes that operate in nodules formed during the interaction between rhizobia and legumes. Deletion analyses of the promoters of the Casuarina symbiotic genes delineated a region that contains nodulin motifs identified in legumes; this region is critical for the controlled expression of the Casuarina gene. The finding that the nonsymbiotic Casuarina gene is also correctly expressed in L. corniculatus suggests to us that a comparable non-symbiotic hemoglobin gene will be found in legume species.
Asunto(s)
Genes de Plantas/genética , Hemoglobinas/genética , Plantas/genética , Actinomycetales/metabolismo , Secuencia de Aminoácidos , Secuencia de Bases , Evolución Biológica , Datos de Secuencia Molecular , Fijación del Nitrógeno , Alineación de Secuencia , Simbiosis/genéticaRESUMEN
A nodule nuclear factor, NAT2, interacts with two AT-rich binding sites (NAT2 BS1 and NAT2 BS2) in the soybean leghemoglobin (lb) c3 promoter. In transgenic Lotus corniculatus nodules, an oligonucleotide containing NAT2 BS1 activated an inactive -159 lbc3 promoter when placed immediately upstream of the promoter. The activation was independent of the orientation of NAT2 BS1 but was dependent on its position in the promoter. The abilities of different mutated binding sites to activate expression in vivo were correlated to their respective in vitro affinities for binding NAT2. This suggested that the interaction between NAT2 and NAT2 BS1 is responsible for the observed reactivation. Further activation experiments with the lbc3 and the leaf-specific Nicotiana plumbaginifolia ribulose bisphosphate carboxylase/oxygenase small subunit (rbcS-8B) promoter suggested that another specific cis element(s) is required for the function of NAT2 BS1. Thus, the -102 lbc3 promoter lacking the organ-specific element (-139 to -102) was not reactivated by the presence of the binding site, and the rbcS-8B promoter required sequences between -312 and -257 to be activated by NAT2 BS1. This implies that NAT2 has to work in combination with other trans-acting factor(s) to increase expression. The finding of NAT2-like binding activities in different plant organs and the specific expression of the hybrid NAT2 BS1/-312 rbcS-8B promoter in leaves suggest that NAT2 is a general activator of transcription.
Asunto(s)
Arilamina N-Acetiltransferasa/metabolismo , Proteínas de Unión al ADN/metabolismo , ADN/metabolismo , Expresión Génica , Glycine max/genética , Leghemoglobina/genética , Regiones Promotoras Genéticas , Composición de Base , Secuencia de Bases , Sitios de Unión , Cloranfenicol O-Acetiltransferasa/biosíntesis , Cloranfenicol O-Acetiltransferasa/genética , ADN/química , Datos de Secuencia Molecular , Mutagénesis Insercional , Oligodesoxirribonucleótidos , Rhizobium/genética , Especificidad por Sustrato , Transcripción Genética , Transformación GenéticaRESUMEN
All patients contacts at the four psychiatric emergency rooms in Copenhagen and Frederiksberg were registered every tenth day during 1985 (a total of 1969 patient contacts). Based on this material, we have compared patients who were given the diagnosis schizophrenia with other patients attending the psychiatric emergency rooms as regards demographic data, attendance patterns and treatment. There were 387 contacts from schizophrenic patients, of which 69.8% were men and 30.2% women. The schizophrenic patients were significantly younger than the other patients, and significantly more of them were unmarried, living alone and on pensions. A quarter of the schizophrenic patient contacts ended in hospital admission, either directly or after spending the night in the emergency room, 10% spent the night only, and 65% left the emergency room the same day with or without further appointments. There were signs that many of the schizophrenic patients, especially the men, used the emergency rooms as a means of human contact and a "shelter", compensating for the lack of a more personal social network. This is seen in connection with the fact that the male patients were to a greater degree without a family network. On the other hand, significantly more female schizophrenic patients were admitted to hospital or were offered overnight stays in the emergency room. Schizophrenic patients are regarded as large-scale users of psychiatric emergency rooms, which is seen in relation to recent years' limited capacity for hospital admission and lack of relevant options for housing, treatment and rehabilitation. The plans for community psychiatry in Copenhagen are expected to consider the special problems which these patients face.
Asunto(s)
Servicios Comunitarios de Salud Mental/estadística & datos numéricos , Servicios de Urgencia Psiquiátrica/estadística & datos numéricos , Admisión del Paciente/estadística & datos numéricos , Esquizofrenia , Adulto , Dinamarca/epidemiología , Femenino , Humanos , Masculino , Sistema de Registros , Esquizofrenia/epidemiología , Esquizofrenia/terapia , Psicología del Esquizofrénico , Factores SocioeconómicosRESUMEN
On the basis of a prospective random sample investigation of 611 alcohol-related visits to the four psychiatric emergency units of the City of Copenhagen, demographic variables, referral sources and dispositions of treatment are described. On every 10th day throughout 1985 all visits were registered. The distribution of all variables except age and sex deviate significantly from those of non-alcohol-related visits. Thus fewer alcoholics cohabit and more are divorced. 25% of the alcohol-related visits resulted in an overnight stay in the unit, while 10% resulted in admission to the psychiatric ward. For non-alcohol-related visits the proportions were the reverse.
Asunto(s)
Intoxicación Alcohólica/psicología , Alcoholismo/psicología , Servicio de Urgencia en Hospital/estadística & datos numéricos , Servicios de Urgencia Psiquiátrica/estadística & datos numéricos , Adulto , Delirio por Abstinencia Alcohólica/psicología , Delirio por Abstinencia Alcohólica/terapia , Intoxicación Alcohólica/terapia , Alcoholismo/terapia , Dinamarca/epidemiología , Femenino , Humanos , Masculino , Persona de Mediana Edad , Estudios Prospectivos , Servicio de Psiquiatría en Hospital/estadística & datos numéricos , Psicosis Alcohólicas/psicología , Psicosis Alcohólicas/terapia , Factores SocioeconómicosRESUMEN
Three different nuclear factors recognizing short AT-rich DNA sequences were identified in different organs of soybean. One factor (NAT2) was found to be present in mature nodules, another factor (NAT1) was detected in roots and nodules, and a third one (LAT1) was only observed in leaves. All three factors recognized several DNA sequences in the promoter region of the soybean nodulin N23 gene. Footprinting, deletion, and point mutation analyses revealed different binding properties for all three factors and further showed that even single base pair substitutions had a dramatic effect on binding affinity. The LAT1 and NAT1 factors were released from chromatin by extraction with a low-salt buffer and were soluble in 2% trichloroacetic acid, implying a relationship to high-mobility group (HMG) proteins. DNA binding studies further indicated a functional relationship of these factors to the human HMG I protein. Purification of the LAT1 factor from leaf nuclei revealed the presence of two polypeptides with molecular masses of 21 kilodaltons and 23 kilodaltons, respectively, binding the same DNA sequence with equal affinity.
Asunto(s)
Glycine max/genética , Proteínas del Grupo de Alta Movilidad/metabolismo , Proteínas de la Membrana/genética , Proteínas de Plantas/genética , Regiones Promotoras Genéticas/genética , Secuencia de Bases , Sitios de Unión/genética , Núcleo Celular/metabolismo , ADN/metabolismo , Análisis Mutacional de ADN , Proteínas del Grupo de Alta Movilidad/aislamiento & purificación , Datos de Secuencia Molecular , Glycine max/metabolismoRESUMEN
The primary structure of a leghemoglobin (lb) gene from the stem-nodulated, tropical legume Sesbania rostrata and two lb gene promoter regions was analysed. The S. rostrata lb gene structure and Lb amino acid composition were found to be highly conserved with previously described lb genes and Lb proteins. Distinct DNA elements were identified in the S. rostrata lb promoter regions, which share a high degree of homology with cis-active regulatory elements found in the soybean (Glycine max) lbc3 promoter. One conserved DNA element was found to interact specifically with an apparently universal, trans-acting factor present in nuclear extracts of nodules. These results suggest a conserved mechanism for nodule specific induction of lb genes in leguminous plants.
Asunto(s)
Genes , Hemoproteínas/genética , Leghemoglobina/genética , Plantas/genética , Regiones Promotoras Genéticas , Secuencia de Aminoácidos , Secuencia de Bases , Southern Blotting , Fabaceae/genética , Intrones , Datos de Secuencia Molecular , Hibridación de Ácido Nucleico , Plantas Medicinales , Homología de Secuencia de Ácido NucleicoRESUMEN
Nuclear extracts from soybean nodules, leaves and roots were used to investigate protein-DNA interactions in the 5' upstream (promoter) region of the soybean leghaemoglobin lbc(3) gene. Two distinct regions were identified which strongly bind a nodule specific factor. A Bal31 deletion analysis delimited the DNA elements responsible for the binding of this factor, which map at nucleotides -223 to -246 (element 1) and -161 to -176 (element 2), relative to the start point of transcription. Competition experiments strongly suggest that both elements bind to the same nodule specific factor, but with different affinities. Elements 1 and 2 share a common motif, although their AT-rich DNA sequences differ. Element 2 is highly conserved at an analogous position in other soybean lb gene 5' upstream regions.
RESUMEN
Poly(A)+ RNA isolated from root nodules of yellow lupin (Lupinus luteus, var. Ventus) has been used as a template for the construction of a cDNA library. The ds cDNA was synthesized and inserted into the Hind III site of plasmid pBR 322 using synthetic Hind III linkers. Clones containing sequences specific for nodules were selected by differential colony hybridization using 32P-labeled cDNA synthesized either from nodule poly(A)+ RNA or from poly(A)+ RNA of uninfected root as probes. Among the recombinant plasmids, the cDNA gene for leghemoglobin was identified. The protein structure derived from its nucleotide sequence was consistent with known amino acid sequence of lupin Lb II. The cloned lupin Lb cDNA hybridized to poly(A)+ RNA from nodules only, which is in accordance with the general concept, that leghemoglobin is expressed exclusively in nodules.
Asunto(s)
Clonación Molecular , ADN/genética , Hemoproteínas/genética , Leghemoglobina/genética , Plantas/genética , Secuencia de Aminoácidos , Secuencia de Bases , ADN Recombinante , Fabaceae , Hibridación de Ácido Nucleico , Plantas Medicinales , Plásmidos , Poli A/genética , ARN/genética , ARN MensajeroRESUMEN
The TM1 yeast mutant was transformed with a 2 micron-derived plasmid (YEp24) which carries a chimaeric gene containing the Escherichia coli chloramphenicol acetyl transferase (CAT) gene fused to the 5'- and 3'-flanking regions of the soybean leghemoglobin (Lb) c3 gene. Expression of the chimaeric CAT gene is controlled specifically by heme at a post-transcriptional level, most likely by regulating the efficiencies of translation. Expression of another chimaeric gene consisting of the neomycin phosphotransferase (NPTII) gene fused to only the 5'-flanking region of the Lbc3 gene is regulated by heme in a similar way. Thus, in yeast, heme modulates the translation of the chimaeric mRNAs through interactions with the 5' Lbc3 non-coding region.
Asunto(s)
Quimera , Genes , Hemo/farmacología , Hemoproteínas/genética , Leghemoglobina/genética , Plantas/genética , Saccharomyces cerevisiae/genética , Acetiltransferasas/genética , Secuencia de Bases , Cloranfenicol O-Acetiltransferasa , Enzimas de Restricción del ADN , Escherichia coli/enzimología , Escherichia coli/genética , Genes/efectos de los fármacos , Genes Bacterianos , Vectores Genéticos , Mutación , Plásmidos , Procesamiento Postranscripcional del ARN , Saccharomyces cerevisiae/efectos de los fármacos , Glycine max , Transcripción GenéticaRESUMEN
During the early stages of soybean nodule development the leghemoglobin (Lb) genes are activated sequentially in the opposite order to which they are arranged in the soybean genome. At a specific stage after the initial activation of all the Lb genes, a large increment occurs in the transcription of the Lb(c1), Lb(c3) and Lb(a) genes while the transcription of the Lb(c2) gene is not amplified to a similar extent. All the Lb genes retain significant activity for a long period during the lifetime of a nodule. Consequently the soybean Lb genes are not regulated by a developmental gene switching mechanism as is the case for vertebrate globin genes. Concomitantly with the increase in Lb gene transcription some of the other nodule specific plant genes are activated. These specific changes in the activities of the Lb and nodulin genes precede the activation of the bacterial nitrogenase gene. Thus the alteration in bacterial metabolism due to nitrogen fixation is not responsible for the observed changes in the transcriptional activities of the Lb and nodule-specific genes.
RESUMEN
A clone containing an unusual leghemoglobin (Lb) gene was isolated from a soybean DNA library present in Charon 4A phage. DNA sequence analysis revealed that the isolated Lb gene has three intervening sequences (IVS-1, IVS-2 and IVS-3) located in the same positions as those found in other Lb genes. Due to a large increase of IVS-2 and IVS-3, the isolated Lb gene is about twice the size of a normal Lb gene. The coding sequence derived from the DNA sequence corresponds to no known soybean Lb and attempts to find a corresponding mRNA failed. In addition, the 5'-flanking sequence of the Lb gene is mutated in two regions which seem to be important for transcription. It is, therefore, tentatively suggested that the isolated Lb gene is non-functional, and consequently is an Lb pseudogene.
Asunto(s)
Genes de Plantas , Glycine max/genética , Leghemoglobina/genética , Secuencia de Aminoácidos , Secuencia de Bases , Mapeo Cromosómico , ADN de Plantas/análisis , Datos de Secuencia Molecular , Recombinación Genética , Homología de Secuencia de Aminoácido , Homología de Secuencia de Ácido NucleicoRESUMEN
Clones containing six leghemoglobin (Lb) genes have been isolated from two genomic libraries of soybean. They encompass two independent DNA regions: a 40-kb region containing four genes in the order 5' Lba-Lbc(1)-[unk]Lb-Lbc(3) 3' with the same transcriptional polarity, and another 40-kb region containing two genes in the order 5' Lbc(4)-Lbc(2) 3' with the same polarity. The order in which the Lb genes are arranged in the soybean genome imply that they are activated in the opposite order to which they are arranged on the chromosome. There is a close similarity between corresponding DNA regions outside the Lb genes in the two clusters. Thus, a moderately repetitive DNA element is present in corresponding positions in each cluster. In addition, at least two different non-Lb genes are linked to each Lb gene cluster in corresponding positions. These genes are apparently regulated in a way which differs from that of the Lb genes. The existence of two very similar Lb gene clusters in soybean suggest that soybean may have evolved from an ancestral form by genome duplication.
RESUMEN
We present the complete nucleotide sequences of two leghemoglobin genes isolated from soybean DNA. Both genes contain three intervening sequences in identical positions. Comparison of the coding sequences with known amino-acid sequences of soybean leghemoglobins suggest that the two genes correspond to leghemoglobin C2 and leghemoglobin C3, respectively.
Asunto(s)
Genes , Hemoproteínas/genética , Leghemoglobina/genética , Plantas/genética , Secuencia de Bases , Clonación Molecular , Enzimas de Restricción del ADN , Glycine maxRESUMEN
We present the complete nucleotide sequences of two leghemoglobin genes isolated from soybean DNA. Both genes contain three intervening sequences which interrupt the two coding sequences in identical positions. The 5' and 3' flanking sequences in both genes contain conserved sequences similar to those found in corresponding positions in other eukaryotic genes. Thus, the general DNA sequence organization of these plant genes is similar to that of other eukaryotic genes.