RESUMEN
In Asian cultivated rice (Oryza sativa L.), aroma is one of the most valuable traits in grain quality and 2-ACP is the main volatile compound contributing to the characteristic popcorn-like odour of aromatic rices. Although the major locus for grain fragrance (frg gene) has been described recently in Basmati rice, this gene has not been characterised in true japonica varieties and molecular information available on the genetic diversity and evolutionary origin of this gene among the different varieties is still limited. Here we report on characterisation of the frg gene in the Azucena variety, one of the few aromatic japonica cultivars. We used a RIL population from a cross between Azucena and IR64, a non-aromatic indica, the reference genomic sequence of Nipponbare (japonica) and 93-11 (indica) as well as an Azucena BAC library, to identify the major fragance gene in Azucena. We thus identified a betaine aldehyde dehydrogenase gene, badh2, as the candidate locus responsible for aroma, which presented exactly the same mutation as that identified in Basmati and Jasmine-like rices. Comparative genomic analyses showed very high sequence conservation between Azucena and Nipponbare BADH2, and a MITE was identified in the promotor region of the BADH2 allele in 93-11. The badh2 mutation and MITE were surveyed in a representative rice collection, including traditional aromatic and non-aromatic rice varieties, and strongly suggested a monophylogenetic origin of this badh2 mutation in Asian cultivated rices. Altogether these new data are discussed here in the light of current hypotheses on the origin of rice genetic diversity.
Asunto(s)
Genes de Plantas , Variación Genética , Oryza/genética , Alelos , Asia , Secuencia de Bases , Cromatografía de Gases , Mapeo Cromosómico , Genotipo , Datos de Secuencia Molecular , Fenotipo , Regiones Promotoras Genéticas/genética , Análisis de Secuencia de ADN , Homología de Secuencia de Ácido NucleicoRESUMEN
Single nucleotide polymorphisms (SNP) are the most abundant type of DNA polymorphism found in animal and plant genomes. They provide an important new source of molecular markers that are useful in genetic mapping, map-based positional cloning, quantitative trait locus mapping and the assessment of genetic distances between individuals. Very little is known on the frequency of SNPs in cassava. We have exploited the recently-developed collection of cassava expressed sequence tags (ESTs) to detect SNPs in the five cultivars of cassava used to generate the sequences. The frequency of intra-cultivar and inter-cultivar SNPs after analysis of 111 contigs was one polymorphism per 905 and one per 1,032 bp, respectively; totaling 1 each 509 bp. We have obtained further information on the frequency of SNPs in six cassava cultivars by analysis of 33 amplicons obtained from 3' EST and BAC end sequences. Overall, about 11 kb of DNA sequence was obtained for each cultivar. A total of 186 SNPs (136 and 50 from ESTs and BAC ends, respectively) were identified. Among these, 146 were intra-cultivar polymorphisms, while 80 were inter-cultivar polymorphisms. Thus the total frequency of SNPs was one per 62 bp. This information will help to develop new strategies for EST mapping as well as their association with phenotypic characteristics.
Asunto(s)
Etiquetas de Secuencia Expresada , Manihot/genética , Polimorfismo de Nucleótido Simple , Biología Computacional , Análisis de Secuencia de ADN , Especificidad de la EspecieRESUMEN
The Cdc25 protein phosphatase is a key enzyme involved in the regulation of the G(2)/M transition in metazoans and yeast. However, no Cdc25 ortholog has so far been identified in plants, although functional studies have shown that an activating dephosphorylation of the CDK-cyclin complex regulates the G(2)/M transition. In this paper, the first green lineage Cdc25 ortholog is described in the unicellular alga Ostreococcus tauri. It encodes a protein which is able to rescue the yeast S. pombe cdc25-22 conditional mutant. Furthermore, microinjection of GST-tagged O. tauri Cdc25 specifically activates prophase-arrested starfish oocytes. In vitro histone H1 kinase assays and anti-phosphotyrosine Western Blotting confirmed the in vivo activating dephosphorylation of starfish CDK1-cyclinB by recombinant O. tauri Cdc25. We propose that there has been coevolution of the regulatory proteins involved in the control of M-phase entry in the metazoan, yeast and green lineages.
Asunto(s)
Fosfatasas cdc25/química , Secuencia de Aminoácidos , Animales , Western Blotting , Ciclo Celular , Proteínas de Ciclo Celular/metabolismo , Linaje de la Célula , Clonación Molecular , Relación Dosis-Respuesta a Droga , Fase G2 , Glutatión Transferasa/metabolismo , Mitosis , Datos de Secuencia Molecular , Mutación , Oocitos/metabolismo , Monoéster Fosfórico Hidrolasas/metabolismo , Fosfotirosina/metabolismo , Filogenia , Proteínas Quinasas/metabolismo , Proteínas Recombinantes/química , Schizosaccharomyces/metabolismo , Homología de Secuencia de Aminoácido , Estrellas de Mar , Temperatura , Factores de Tiempo , Fosfatasas cdc25/metabolismoRESUMEN
Several cDNA libraries were constructed using mRNA isolated from roots, panicles, cell suspensions and leaves of non-stressed Oryza sativa indica (IR64) and japonica (Azucena) plants, from wounded leaves, and from leaves of both cultivars inoculated with Rice Yellow Mottle Virus (RYMV). A total of 5549 cleaned expressed sequence tags (ESTs) were generated from these libraries. They were classified into functional categories on the basis of homology, and analyzed for redundancy within each library. The expression profiles represented by each library revealed great differences between indica and japonica backgrounds. EST frequencies during the early stages of RYMV infection indicated that changes in the expression of genes involved in energy metabolism and photosynthesis are differentially accentuated in susceptible and partially resistant cultivars. Mapping of these ESTs revealed that several co-localize with previously described resistance gene analogs and QTLs (quantitative trait loci).
Asunto(s)
Regulación de la Expresión Génica de las Plantas/genética , Oryza/genética , Oryza/virología , Virus de Plantas/genética , Transcripción Genética , Mapeo Cromosómico , ADN Complementario/genética , Etiquetas de Secuencia Expresada , Biblioteca de Genes , Marcadores Genéticos , Oryza/clasificación , Enfermedades de las Plantas/virología , Hojas de la Planta/virología , Sitios de Carácter Cuantitativo , Especificidad de la EspecieRESUMEN
Plant disease resistance genes (R genes) show significant similarity amongst themselves in terms of both their DNA sequences and structural motifs present in their protein products. Oligonucleotide primers designed from NBS (Nucleotide Binding Site) domains encoded by several R-genes have been used to amplify NBS sequences from the genomic DNA of various plant species, which have been called Resistance Gene Analogues (RGAs) or Resistance Gene Candidates (RGCs). Using specific primers from the NBS and TIR (Toll/Interleukin-1 Receptor) regions, we identified twelve classes of RGCs in cassava (Manihot esculenta Crantz). Two classes were obtained from the PCR-amplification of the TIR domain. The other 10 classes correspond to the NBS sequences and were grouped into two subfamilies. Classes RCa1 to RCa5 are part of the first subfamily and were linked to a TIR domain in the N terminus. Classes RCa6 to RCa10 corresponded to non-TIR NBS-LRR encoding sequences. BAC library screening with the 12 RGC classes as probes allowed the identification of 42 BAC clones that were assembled into 10 contigs and 19 singletons. Members of the two TIR and non-TIR NBS-LRR subfamilies occurred together within individual BAC clones. The BAC screening and Southern hybridization analyses showed that all RGCs were single copy sequences except RCa6 that represented a large and diverse gene family. One BAC contained five NBS sequences and sequence analysis allowed the identification of two complete RGCs encoding two highly similar proteins. This BAC was located on linkage group J with three other RGC-containing BACs. At least one of these genes, RGC2, is expressed constitutively in cassava tissues.
Asunto(s)
Genes de Plantas , Inmunidad Innata/genética , Manihot/genética , Secuencia de Aminoácidos , Secuencia de Bases , Clonación Molecular , Proteínas de Unión al ADN , Datos de Secuencia Molecular , Filogenia , Enfermedades de las Plantas , Receptores de Interleucina-1 , Alineación de SecuenciaRESUMEN
We investigated the potential of an improved Agrobacterium tumefaciens-mediated transformation procedure of japonica rice ( Oryza sativa L.) for generating large numbers of T-DNA plants that are required for functional analysis of this model genome. Using a T-DNA construct bearing the hygromycin resistance ( hpt), green fluorescent protein ( gfp) and beta-glucuronidase ( gusA) genes, each individually driven by a CaMV 35S promoter, we established a highly efficient seed-embryo callus transformation procedure that results both in a high frequency (75-95%) of co-cultured calli yielding resistant cell lines and the generation of multiple (10 to more than 20) resistant cell lines per co-cultured callus. Efficiencies ranged from four to ten independent transformants per co-cultivated callus in various japonica cultivars. We further analysed the T-DNA integration patterns within a population of more than 200 transgenic plants. In the three cultivars studied, 30-40% of the T(0) plants were found to have integrated a single T-DNA copy. Analyses of segregation for hygromycin resistance in T(1) progenies showed that 30-50% of the lines harbouring multiple T-DNA insertions exhibited hpt gene silencing, whereas only 10% of lines harbouring a single T-DNA insertion was prone to silencing. Most of the lines silenced for hpt also exhibited apparent silencing of the gus and gfp genes borne by the T-DNA. The genomic regions flanking the left border of T-DNA insertion points were recovered in 477 plants and sequenced. Adapter-ligation Polymerase chain reaction analysis proved to be an efficient and reliable method to identify these sequences. By homology search, 77 T-DNA insertion sites were localized on BAC/PAC rice Nipponbare sequences. The influence of the organization of T-DNA integration on subsequent identification of T-DNA insertion sites and gene expression detection systems is discussed.
Asunto(s)
ADN Bacteriano/genética , Genoma de Planta , Genómica , Oryza/genética , Agrobacterium tumefaciens/fisiología , Secuencia de Bases , Caulimovirus/genética , Cartilla de ADN , Glucuronidasa/genética , Proteínas Fluorescentes Verdes , Proteínas Luminiscentes/genética , Regiones Promotoras Genéticas , Transformación GenéticaRESUMEN
Expressed sequence tags (ESTs) from the Arabidopsis thaliana sequencing project were used to construct a genetic RFLP map for Brassica oleracea. Of the 110 A. thaliana ESTs tested, 95 were found to be informative RFLP probes in map construction. In total, 212 new loci corresponding to the 95 ESTs were added to the existing genetic map of B. oleracea. The enriched map covers all nine basic linkage groups and confirms that the chromosomes of B. oleracea and A. thaliana are similar in linear organization. However, varying levels of sequence conservation between the chromosomes of B. oleracea and A. thaliana were detected in different regions of the genomes. Long conserved regions encompassing entire chromosome arms in both genomes were identified; these are probably shared by descent. On the other hand, extensive rearrangements were observed in numerous chromosome regions, producing a mosaic of A. thaliana-like segments in the genome of Brassica. The presence of extensive chromosome duplication in A. thaliana was taken into consideration in the construction of the comparative maps of B. oleracea and A. thaliana.
Asunto(s)
Arabidopsis/genética , Brassica/genética , Cromosomas de las Plantas/genética , Evolución Biológica , Mapeo Cromosómico , Etiquetas de Secuencia Expresada , Eliminación de Gen , Duplicación de Gen , Reordenamiento Génico , Ligamiento Genético , Genoma de Planta , Polimorfismo de Longitud del Fragmento de Restricción , Poliploidía , Especificidad de la EspecieRESUMEN
The Arabidopsis genome contains at least 18 genes encoding members of the 70-kilodalton heat shock protein (Hsp70) family, 14 in the DnaK subfamily and 4 in the Hsp110/SSE subfamily. While the Hsp70s are highly conserved, a phylogenetic analysis including all members of this family in Arabidopsis and in yeast indicates the homology of Hsp70s in the subgroups, such as those predicted to localize in the same subcellular compartment and those similar to the mammalian Hsp110 and Grp170. Gene structure and genome organization suggest duplication in the origin of some genes. The Arabidopsis hsp70s exhibit distinct expression profiles; representative genes of the subgroups are expressed at relatively high levels during specific developmental stages and under thermal stress.
Asunto(s)
Proteínas de Arabidopsis/genética , Arabidopsis/genética , Proteínas de Escherichia coli , Proteínas HSP70 de Choque Térmico/genética , Arabidopsis/fisiología , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/metabolismo , Mapeo Cromosómico , Regulación de la Expresión Génica de las Plantas/fisiología , Genes de Plantas , Genoma de Planta , Proteínas HSP70 de Choque Térmico/química , Proteínas HSP70 de Choque Térmico/clasificación , Proteínas HSP70 de Choque Térmico/metabolismo , Filogenia , Estructura Terciaria de ProteínaRESUMEN
Eukaryotic ribosomes are made of two components, four ribosomal RNAs, and approximately 80 ribosomal proteins (r-proteins). The exact number of r-proteins and r-protein genes in higher plants is not known. The strong conservation in eukaryotic r-protein primary sequence allowed us to use the well-characterized rat (Rattus norvegicus) r-protein set to identify orthologues on the five haploid chromosomes of Arabidopsis. By use of the numerous expressed sequence tag (EST) accessions and the complete genomic sequence of this species, we identified 249 genes (including some pseudogenes) corresponding to 80 (32 small subunit and 48 large subunit) cytoplasmic r-protein types. None of the r-protein genes are single copy and most are encoded by three or four expressed genes, indicative of the internal duplication of the Arabidopsis genome. The r-proteins are distributed throughout the genome. Inspection of genes in the vicinity of r-protein gene family members confirms extensive duplications of large chromosome fragments and sheds light on the evolutionary history of the Arabidopsis genome. Examination of large duplicated regions indicated that a significant fraction of the r-protein genes have been either lost from one of the duplicated fragments or inserted after the initial duplication event. Only 52 r-protein genes lack a matching EST accession, and 19 of these contain incomplete open reading frames, confirming that most genes are expressed. Assessment of cognate EST numbers suggests that r-protein gene family members are differentially expressed.
Asunto(s)
Arabidopsis/genética , Genoma de Planta , Proteínas Ribosómicas/genética , Animales , Arabidopsis/metabolismo , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Mapeo Cromosómico , Citoplasma/genética , Citoplasma/metabolismo , Etiquetas de Secuencia Expresada , Regulación de la Expresión Génica de las Plantas , Familia de Multigenes , ARN de Planta , ARN Ribosómico , Ratas , Proteínas Ribosómicas/química , Proteínas Ribosómicas/metabolismo , Ribosomas/genética , Ribosomas/metabolismo , Especificidad de la EspecieRESUMEN
The EMB 506 gene has been characterised as essential for embryo development. To provide insights into the role of EMB 506, which is hidden by the embryo defective phenotype, the ABI3 promoter was fused to the EMB 506 cDNA. The expression of such a transgene should provide sufficient protein during embryogenesis to ensure normal embryo development in homozygous emb 506 seeds. We show that homozygous emb 506 seedlings, partially complemented with the ABI3::EMB 506 transgene, can be obtained. Most of the rescued emb 506 plants are able to flower and to set normal seeds, but show mild to severe depigmentation of rosette leaves and/or inflorescences. This effect on chloroplast development indicated a putative chloroplast localisation of the EMB 506 protein, which was demonstrated by GFP-protein fusion. However, EMB 506 cannot be considered as a chloroplast housekeeping protein only, since EMB 506 is not present in all photosynthetic tissues. This study demonstrates the power of this simple strategy, which could be widely applied to other emb mutants and which may reveal similar or additional roles for EMB genes at vegetative stages of the life cycle.
Asunto(s)
Arabidopsis/genética , Prueba de Complementación Genética , Mutación , Proteínas de Plantas/genética , Secuencia de Bases , Cartilla de ADN , ADN Complementario , Genotipo , FenotipoRESUMEN
The promoter of the Arabidopsis thaliana L. AtEm1 gene encoding a late embryogenesis abundant protein was fused to the beta-glucuronidase reporter gene and introduced into Brassica napus. The promoter is highly active in the vascular tissues of embryo and pollen grains and also active in petals, sepals, caulinar leaves, and carpels.
Asunto(s)
Arabidopsis/genética , Brassica/genética , Regulación de la Expresión Génica de las Plantas , Proteínas de Plantas/genética , Arabidopsis/embriología , Fusión Artificial Génica , Transporte Biológico/genética , Genes de Plantas , Genes Reporteros , Glucuronidasa/genética , Estructuras de las Plantas/genética , Plantas Modificadas Genéticamente , Polen/genética , Regiones Promotoras Genéticas , Semillas/genéticaRESUMEN
Low erucic acid rapeseed (LEAR) is characterised by a near absence of very long chain fatty acids in the seed oil which has been correlated with a lack of acyl-CoA elongation activity. Here we show that the absence of acyl-CoA and ATP-dependent elongation activities in microsomes isolated from LEAR embryos is associated with an absence of beta-ketoacyl-CoA synthase activity encoded by the Bn-fatty acid elongation 1 (FAE1) genes. Size exclusion chromatography of solubilised microsomes revealed the presence of a high molecular mass acyl-CoA elongase complex in high erucic acid rapeseed which was absent in microsomes isolated from LEAR seeds. Although transcripts for the Bn-FAE1 genes were detected in LEAR embryos, immunoblots using antisera raised against the beta-ketoacyl-CoA synthase indicated an absence of this protein. Comparison of the deduced amino acid sequences of immature embryo cDNAs reveals that LEAR alleles of Bn-FAE1 encode variant beta-ketoacyl-CoA synthase proteins.
Asunto(s)
3-Oxoacil-(Proteína Transportadora de Acil) Sintasa/metabolismo , Acetiltransferasas/metabolismo , Brassica/enzimología , Ácidos Erucicos/metabolismo , Mutación , Acetiltransferasas/genética , Secuencia de Aminoácidos , Brassica/metabolismo , Cromatografía en Gel , ADN Complementario/análisis , Elongasas de Ácidos Grasos , Variación Genética , Inmunoensayo , Datos de Secuencia Molecular , Homología de Secuencia de Aminoácido , SolubilidadRESUMEN
During the last ten years, Arabidopsis thaliana has become the most favoured plant system for the study of many aspects of development and adaptation to adverse conditions and diseases. The sequencing of the Arabidopsis thaliana genome is nearly completed with more than 90% of the sequence being released in public databases. This is the first plant genome to be analysed and it has revealed a tremendous amount of information about the nature of the genes it contains and its largely duplicated organisation. French groups have been involved in Arabidopsis genomics at several steps: EST (expressed sequence tags) sequencing, construction and ordering (physical mapping of chromosomes) of a YAC (yeast artificial chromosomes) library, genomic sequencing. In parallel an extensive programme of functional genomics is being undertaken through the systematic analysis of insertional mutants. This information provides a support for analysing other more economically important plant genomes such as the rice genome and constitutes the beginning of a systematic investigation on plant gene functions and will promote new strategies for plant improvement.
Asunto(s)
Arabidopsis/genética , Genómica , Oryza/genética , Cromosomas Artificiales de Levadura/genética , Francia , Genoma de PlantaRESUMEN
Arabidopsis thaliana is an important model system for plant biologists. In 1996 an international collaboration (the Arabidopsis Genome Initiative) was formed to sequence the whole genome of Arabidopsis and in 1999 the sequence of the first two chromosomes was reported. The sequence of the last three chromosomes and an analysis of the whole genome are reported in this issue. Here we present the sequence of chromosome 3, organized into four sequence segments (contigs). The two largest (13.5 and 9.2 Mb) correspond to the top (long) and the bottom (short) arms of chromosome 3, and the two small contigs are located in the genetically defined centromere. This chromosome encodes 5,220 of the roughly 25,500 predicted protein-coding genes in the genome. About 20% of the predicted proteins have significant homology to proteins in eukaryotic genomes for which the complete sequence is available, pointing to important conserved cellular functions among eukaryotes.
Asunto(s)
Arabidopsis/genética , Genoma de Planta , Mapeo Cromosómico , ADN de Plantas , Duplicación de Gen , Humanos , Proteínas de Plantas/genética , Análisis de Secuencia de ADNRESUMEN
P67, a new protein binding to a specific RNA probe, was purified from radish seedlings [Echeverria, M. and Lahmy, S. (1995) Nucleic Acids Res. 23, 4963-4970]. Amino acid sequence information obtained from P67 microsequencing allowed the isolation of genes encoding P67 in radish and Airabidopsis thaliana. Immunolocalisation experiments in transfected protoplasts demonstrated that this protein is addressed to the chloroplast. The RNA-binding activity of recombinant P67 was found to be similar to that of the native protein. A significant similarity with the maize protein CRP1 [Fisk, D.G., Walker, M.B. and Barkan, A. (1999) EMBO J. 18, 2621-2630] suggests that P67 belongs to the PPR family and could be involved in chloroplast RNA processing.
Asunto(s)
Cloroplastos/metabolismo , Proteínas de Plantas/metabolismo , Proteínas de Unión al ARN/metabolismo , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Arabidopsis , Secuencia de Bases , Brassica , Clonación Molecular , ADN de Plantas , Expresión Génica , Genes de Plantas , Datos de Secuencia Molecular , Proteínas de Plantas/clasificación , Proteínas de Plantas/genética , ARN Mensajero , Proteínas de Unión al ARN/clasificación , Proteínas de Unión al ARN/genética , Proteínas Recombinantes de Fusión/clasificación , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Fracciones SubcelularesRESUMEN
Late embryogenesis abundant (lea) genes are a large and diverse group of genes highly expressed during late stages of seed development. Five major groups of LEA proteins have been described. Two Em genes (group I lea genes) are present in the genome of Arabidopsis thaliana L., AtEm1 and AtEm6. Both genes encode for very similar proteins which differ basically in the number of repetitions of a highly hydrophilic amino acid motif. The spatial patterns of expression of the two Arabidopsis Em genes have been studied using in situ hybridization and transgenic plants transformed with the promoters of the genes fused to the beta-glucuronidase reporter gene (uidA). In the embryo, AtEm1 is preferentially expressed in the pro-vascular tissues and in meristems. In contrast, AtEm6 is expressed throughout the embryo. The activity of both promoters disappears rapidly after germination, but is ABA-inducible in roots of young seedlings, although in different cells: the AtEm1 promoter is active in the internal tissues (vasculature and pericycle) whereas the AtEm6 promoter is active in the external tissues (cortex, epidermis and root hairs). The AtEm1 promoter, but not AtEm6, is also active in mature pollen grains and collapsed nectaries of young siliques. These data indicate that the two Em proteins could carry out at least slightly different functions and that the expression of AtEm1 and AtEm6 is controlled at, at least, three different levels: temporal, spatial and hormonal (ABA).
Asunto(s)
Arabidopsis/genética , Regulación de la Expresión Génica de las Plantas , Proteínas de Plantas/metabolismo , Estructuras de las Plantas/metabolismo , Secuencias de Aminoácidos , Arabidopsis/metabolismo , Fusión Artificial Génica , Genes Reporteros , Germinación , Glucuronidasa/genética , Hibridación in Situ , Proteínas de Plantas/genética , Estructuras de las Plantas/genética , Plantas Modificadas Genéticamente , Regiones Promotoras GenéticasRESUMEN
Arabidopsis thaliana L. leafy cotyledon1 (lec1) and fusca3 (fus3) mutants show multiple phenotypic defects during seed development. In this report the effects of these mutations are examined at the molecular level. The patterns of protein accumulation in lec1 and fus3 seeds are severly altered. In lec1 seeds the steady-state mRNA levels of several late embryogenesis genes were reduced. Different patterns of expression were observed, indicating the occurrence of several regulatory pathways. The effect of lec1 mutations on the expression of the late-embryogenesis abundant AtEm1 gene was examined in detail. In lec1-1 seeds, the AtEm1 gene was expressed at a higher level than in the wild type and earlier in development. The activity of an AtEm1 promoter/beta-glucuronidase reporter gene construct in transgenic A. thaliana plants was studied. Changes in promoter activity in lec1-1 with respect to wild-type seeds were correlated with changes in corresponding mRNA steady-state levels. fus3-2 mutation produced similar changes in AtEm1 promoter activity as lec1-1, which is consistent with the hypothesis that LEC1 and FUS3 might act in the same regulatory pathway. Transgenic analysis using 5'-promoter deletions demonstrated that at least two regions of AtEm1 gene promoter interact with the LEC1-dependent transcriptional regulatory pathway. In spite of expression of the AtEm1 promoter and accumulation of AtEm1 mRNA, the corresponding Em1 protein does not accumulate in lec1-1 seeds. The ABA inducibility of the AtEm1 promoter was not affected by the lec1 mutation.
Asunto(s)
Proteínas de Arabidopsis , Arabidopsis/genética , Proteínas Potenciadoras de Unión a CCAAT/genética , Semillas/genética , Factores de Transcripción/genética , Arabidopsis/crecimiento & desarrollo , Arabidopsis/metabolismo , Proteínas Potenciadoras de Unión a CCAAT/metabolismo , Regulación de la Expresión Génica de las Plantas , Genes Reporteros , Glucuronidasa/genética , Glucuronidasa/metabolismo , Mutación , Proteínas de Plantas/análisis , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Plantas Modificadas Genéticamente , Regiones Promotoras Genéticas , ARN Mensajero/análisis , ARN de Planta/análisis , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Semillas/crecimiento & desarrollo , Semillas/metabolismo , Eliminación de Secuencia , Factores de Transcripción/metabolismoRESUMEN
Previously, we described two cDNA clones corresponding to a new family of cold regulated genes, pBnC24a and pBnC24b homologous to the human BBC1 (Breast Basic Conserved) gene. In order to further analyze the function and regulation of these two genes we have prepared antibodies against a recombinant fusion protein, MBP-BnC24A and used them to study expression at the protein level. In contrast to the increased mRNA accumulation induced by cold, immuno-blot analysis showed that the quantity of the BnC24 protein was not correlated with the accumulation of BnC24 transcripts and is identical in both control and low temperature treated (4 degrees C) plants, suggesting a translational or post-translational regulation. This was confirmed by overexpressing BnC24 in transgenic Arabidopsis thaliana plants. Despite a substantial increase in mRNA, the BnC24 protein level is unchanged. In addition, we demonstrated by subcellular fractionation and immunodetection that a significant fraction of BnC24 protein is located in the nucleus. Using a GUS fusion construction and biolistic experiments it was found that a portion of the amino terminal region is sufficient to target this protein to the nucleus. These results are consistent with the recent finding that BBC1 and its homologues code for the ribosomal large subunit protein L13. In addition they illustrate the difficulty of correlating accumulation of a given mRNA in response to a given stimulus with a biologically significant role in the adaptation to a new environment.
RESUMEN
Systematic analysis of the Arabidopsis genome provides a basis for detailed studies of genome structure and evolution. Members of multigene families were mapped, and random sequence alignment was used to identify regions of extended similarity in the Arabidopsis genome. Detailed analysis showed that the number, order, and orientation of genes were conserved over large regions of the genome, revealing extensive duplication covering the majority of the known genomic sequence. Fine mapping analysis showed much rearrangement, resulting in a patchwork of duplicated regions that indicated deletion, insertion, tandem duplication, inversion, and reciprocal translocation. The implications of these observations for evolution of the Arabidopsis genome as well as their usefulness for analysis and annotation of the genomic sequence and in comparative genomics are discussed.