Browsing by Author "Gómez, I"

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    Nuclear SDH2-1 and SDH2-2 genes, encoding the iron-sulfur subunit of mitochondrial complex II in arabidopsis, have distinct cell-specific expression patterns and promoter activities
    (2004) Elorza, A; León, G; Gómez, I; Mouras, A; Holuigue, L; Araya, A; Jordana, X
    Three different nuclear genes encode the essential iron-sulfur subunit of mitochondrial complex 11 (succinate dehydrogenase) in Arabidopsis (Arabidopsis thaliana), raising interesting questions about their origin and function. To find clues about their role, we have undertaken a detailed analysis of their expression. Two genes (SDH2-1 and SDH2-2) that likely arose via a relatively recent duplication event are expressed in all organs from adult plants, whereas transcripts from the third gene (SDH2-3) were not detected. The tissue- and cell-specific expression of SDH2-1 and SDH2-2 was investigated by in situ hybridization. In flowers, both genes are regulated in a similar way. Enhanced expression was observed in floral meristems and sex organ primordia at early stages of development. As flowers develop, SDH2-1 and SDH2-2 transcripts accumulate in anthers, particularly in the tapetum, pollen mother cells, and microspores, in agreement with an essential role of mitochondria during anther development. Interestingly, in contrast to the situation in flowers, only SDH2-2 appears to be expressed at a significant level in root tips. Strong labeling was observed in all cell layers of the root meristematic zone, and a cell-specific pattern of expression was found with increasing distance from the root tip, as cells attain their differentiated state. Analysis of transgenic Arabidopsis plants carrying SDH2-1 and SDH2-2 promoters fused to the beta-glucuronidase reporter gene indicate that both promoters have similar activities in flowers, driving enhanced expression in anthers and/or pollen, and that only the SDH2-2 promoter is active in root tips. These beta-glucuronidase staining patterns parallel those obtained by in situ hybridization, suggesting transcriptional regulation of these genes. Progressive deletions of the promoters identified regions important for SDH2-1 expression in anthers and/or pollen and for SDH2-2 expression in anthers and/or pollen and root tips. Interestingly, regions driving enhanced expression in anthers are differently located in the two promoters.
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    The amino acidic substitution of cysteine 167 by serine (C167S) in BstVI restriction endonuclease of Bacillus stearothermophilus V affects its conformation and thermostability
    (1999) Loyola, C; Saavedra, C; Gómez, I; Vásquez, C
    The restriction endonuclease BstVI from Bacillus stearothermophilus V contains three cysteine residues at positions 134, 167 and 180. Titration of Cys residues with DTNB showed that none of them are involved in disulphide bond formation. Cysteine triplets 134 and 167 were modified by recombinant PCR to introduce a serine residue in each case. The mutated genes were cloned into pGEM-T vector and transformed into E. coli JM109. Even though pGEM-T is not designed for expression, the mutant proteins were efficiently expressed in E. coli. The endonuclease carrying the mutation (CS)-S-134 was purified to homogeneity but appeared to be very unstable. In contrast, the (CS)-S-167 mutant enzyme was stable when pure and was studied biochemically. This mutant enzyme was as stable and resistant to protein-denaturing agents as the wild type enzyme. The activity of both enzymes was not affected by preincubations of 2 h at 80 degrees C. A short preincubation at 95 degrees C caused a complete inactivation of the mutant enzyme while the wild type endonuclease retained 30% of its activity. Moreover, the (CS)-S-167 BstVI was more susceptible to be hydrolyzed by proteinase K and trypsine compared to the wild type endonuclease. These results show that the substitution Cys --> Ser at position 167 affects the configuration and thermostability of BstVI restriction endonuclease. (C) Societe francaise de biochimie et biologie moleculaire / Elsevier, Paris.
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    Transfer of RPS14 and RPL5 from the mitochondrion to the nucleus in grasses
    (2004) Sandoval, P; León, G; Gómez, I; Carmona, R; Figueroa, P; Holuigue, L; Araya, A; Jordana, X
    Gene transfer from the mitochondrion to the nucleus, a process of outstanding importance to the evolution of the eukaryotic cell, is an ongoing phenomenon in higher plants. After transfer, the mitochondrial gene has to be adapted to the nuclear context by acquiring a new promoter and targeting information to direct the protein back to the organelle. To better understand the strategies developed by higher plants to transfer organellar genes during evolution, we investigated the fate of the mitochondrial PPL5-RPS14 locus in grasses. While maize mitochondrial genome does not contain RPS14 and RPL5 genes, wheat mitochondrial DNA contains an intact RPL5 gene and a nonfunctional RPS14 pseudogene. RPL5 and psiRPS14 are co-transcribed and their transcripts are edited. In wheat, the functional RPS14 gene is located in the nucleus, within the intron of the respiratory complex II iron-sulfur subunit gene (SDH2). Its organization and expression mechanisms are similar to those previously described in maize and rice, allowing us to conclude that RPS14 transfer and nuclear activation occurred before divergence of these grasses. Unexpectedly, we found evidence for a more recent RPL5 transfer to the nucleus in wheat. This nuclear wheat RPL5 acquired its targeting information by duplication of an existing targeting presequence for another mitochondrial protein, ribosomal protein L4. Thus, mitochondrial and nuclear functional RPL5 genes appear to be maintained in wheat, supporting the hypothesis that in an intermediate stage of the transfer process, both nuclear and mitochondrial functional genes coexist. Finally, we show that RPL5 has been independently transferred to the nucleus in the maize lineage and has acquired regulatory elements for its expression and a mitochondrial targeting peptide from an unknown source. (C) 2003 Elsevier B.V. All rights reserved.
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    Transfer of rps14 from the mitochondrion to the nucleus in maize implied integration within a gene encoding the iran-sulphur subunit of succinate dehydrogenase and expression by alternative splicing
    (1999) Figueroa, P; Gómez, I; Holuigue, L; Araya, A; Jordana, X
    The maize mitochondrial genome does not contain a gene coding for ribosomal protein S14. In this paper we show that the functional rps14 gene was translocated to the nucleus and acquired the signals conferring expression and product targeting to the mitochondrion in a way not previously described. Transferred rps14 was found integrated between both exons of a gene encoding the iron-sulphur subunit of the respiratory complex II (sdh2). Sdh2 exon 1 and rps14 were separated by a typical plant nuclear intron that was spliced to give a mature poly(A)+ mRNA of 1.4 kb. This processed mRNA encoded a chimeric SDH2 (truncated)-RPS14 polypeptide, and we show that this chimeric polypeptide is targeted into isolated plant mitochondria, where it is proteolytically processed in a complex way. An alternative splicing event utilizing the same 5' splice site and a different downstream 3' splice site generated a second mature poly(A)+ mRNA of 1.3 kb that contained both sdh2 exons. This sdh2 transcript encoded an SDH2 polypeptide highly conserved compared with its homologues in other organisms, and it contained the three cysteine-rich clusters that made up the three nonheme iron-sulphur centres responsible for electron transport. To our knowledge, these results constitute the first evidence of alternative splicing playing a role in the expression and targeting of two mitochondrial proteins with different functions from the same gene.
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    Transport through a quantum wire with a side quantum-dot array -: art. no. 085321
    (2003) Orellana, PA; Domínguez-Adame, F; Gómez, I; de Guevara, MLL
    A noninteracting quantum-dot array side coupled to a quantum wire is studied. Transport through the quantum wire is investigated by using a noninteracting Anderson tunneling Hamiltonian. The conductance at zero temperature develops an oscillating band with resonances and antiresonances due to constructive and destructive interference in the ballistic channel, respectively. Moreover, we have found an odd-even parity in the system, whose conductance vanishes for an odd number of quantum dots while it becomes 2e(2)/h for an even number. We established an explicit relation between this odd-even parity and the positions of the resonances and antiresonances of the conductivity with the spectrum of the isolated quantum-dot array.