cis Recognition elements in plant mitochondrion RNA editing.

RNA editing in higher plant mitochondria modifies mRNA sequences by means of C-to-U conversions at highly specific sites. To determine the cis elements involved in recognition of an editing site in plant mitochondria, deletion and site-directed mutation constructs containing the cognate cox II mitochondrial gene were introduced into purified mitochondria by electroporation. The RNA editing status was analyzed for precursor and spliced transcripts from the test construct. We found that only a restricted number of nucleotides in the vicinity of the target C residue were necessary for recognition by the editing machinery and that the nearest neighbor 3' residues were crucial for the editing process. We provide evidence that two functionally distinguishable sequences can be defined: the 16-nucleotide 5' region, which can be replaced with the same region from another editing site, and a 6-nucleotide 3' region specific to the editing site. The latter region may play a role in positioning the actual editing residue.

Transcription initiation sites for the potato mitochondrial gene coding for subunit 9 of ATP synthase (atp9).

The potato mitochondrial atp9 gene has a simple expression pattern. To determine where transcription initiates, primary mitochondrial RNAs were labeled by in vitro capping and hybridized to the 5' flanking sequences of the atp9 gene. A single transcription initiation region was identified. Primer extension and nuclease S1 protection analyses were used to precisely map the transcript 5' termini in this region. These results indicate that transcription initiates at 121-128 bp upstream of the atp9 open reading frame, in a sequence which does not present any homology with proposed consensus sequences for plant mitochondrial promoters. Nuclease S1 protection were also used to map 3' termini 67-71 nucleotides downstream of a putative single-stem loop structure.

Isolation and nucleotide sequence of the Thiobacillus ferrooxidans genes for the small and large subunits of ribulose 1,5-bisphosphate carboxylase/oxygenase.

The genes encoding for the large (rbcL) and small (rbcS) subunits of ribulose-1,5-bisphosphate carboxylase (RuBisCO) were cloned from the obligate autotroph Thiobacillus ferrooxidans, a bacterium involved in the bioleaching of minerals. Nucleotide sequence analysis of the cloned DNA showed that the two coding regions are separated by a 30-bp intergenic region, the smallest described for the RuBisCO genes. The rbcL and rbcS genes encode polypeptides of 473 and 118 amino acids, respectively. Comparison of the nucleotide and amino acid sequences with those of the genes for rbcL and rbcS found in other species demonstrated that the T. ferrooxidans genes have the closest degree of identity with those of Chromatium vinosum and of Alvinoconcha hessleri endosymbiont. Both T. ferrooxidans enzyme subunits contain all the conserved amino acids that are known to participate in the catalytic process or in holoenzyme assembly.

Control of gene expression by base deamination: the case of RNA editing in wheat mitochondria.

The term 'RNA editing' was used for the first time in 1986 to describe the process of uridylate insertion into trypanosomal mitochondrial transcripts. Since then, the term has been used more generally to describe a large variety of processes involving base insertions, deletions and conversions that generate RNAs with a primary sequence different to those encoded by the gene. RNA editing has been observed in the mitochondrial fraction of trypanosomes, plants and other organisms, in the animal nuclear fraction in the case of the apolipoprotein B and glutamate brain receptors mRNAs as well as in viruses like paramyxovirus, hepatitis delta and probably HIV. The role of cytidine and adenine deamination leading to C to U and A to I transitions has became pivotal to explain this process by base conversion. In this review we will focus mainly on the work performed in our group on plant mitochondria and more specifically on the mechanism and the functional significance of RNA editing in wheat organelles. The original contributions of our laboratory in this field are: i) showing that RNA editing is reflected at the protein level; ii) settling three in vitro systems to assay C to U conversion using a wheat mitochondrial lysate as source of enzymes and factors, and unedited mRNA from the same source, as substrate; iii) determination by double labelling of the unedited substrate that RNA editing in wheat mitochondria occurs via a deamination step; and iv) that introducing unedited proteins in the mitochondria of transgenic plants leads to the emergence of cytoplasmic male sterility supporting the idea that the role of this process is to produce functional proteins. Using the antisense approach in transgenic plants we were able to obtain a significant male fertility restoration.

Inhibition by phenothiazine derivatives of the adenylate cyclase of amphibian oocytes.

The adenylate cyclase activity of membranes of Xenopus laevis oocytes and follicle cells was affected by the presence of 2-chloro-10-(3-aminopropyl)phenothiazine (CAPP) and two other antipsychotic drugs, fluphenazine and penfluridol. CAPP, at concentrations of 10 and 100 microM, had opposite effects on the activation of the oocyte adenylate cyclase by effectors that act through the G/F regulatory subunit. Under these conditions, the drug stimulated the activation by fluoride and drastically inhibited the activation by guanyl-5'-yl-imidodiphosphate [Gpp(NH)p] and by cholera toxin and GTP. The activity of the catalytic subunit measured in the presence of either Mn2+ or forskolin was not affected by 100 microM CAPP. however, concentrations of this drug above 100 microM inhibited the adenylate cyclase activated by fluoride or by forskolin and also inhibited the activity of a calmodulin-independent cyclic nucleotide phosphodiesterase present in the same oocyte membrane preparation. Oocyte adenylate cyclase has been shown previously to be inhibited by the hormone progesterone. The inhibitory effect of CAPP is additive to that measured with the hormone, indicating that these compounds act through different mechanisms. CAPP did not modify the concentration of Gpp(NH)p required to yield half-maximal activation and, although the drug inhibited more strongly at lower concentrations of Gpp(NH)p, saturating amounts of the guanine nucleotide did not reverse completely the inhibition caused by CAPP. The effects of these antipsychotic drugs on oocyte adenylate cyclase did not require the presence of free Ca2+ and were not altered by the addition of exogenous calmodulin and calcium.

La antropología forense al servicio de la justicia y la historia: las fosas de la Guerra Civil / Forensic anthropology serving justice and history: the Spanish Civil War mass graves

La antropología forense tiene por objeto tanto la identificación del individuo, como la determinación de la causa y circunstancias de la muerte. En este sentido, la antropología forense es esencial para la recuperación de los restos de personas desaparecidas y que fueron enterrados en fosas comunes durante la Guerra Civil y la dictadura franquista, para su posterior retorno a los familiares. En este trabajo se presentan los resultados de la intervención llevada a cabo en la fosa de Gurb, en la que fueron enterrados cuatro soldados republicanos vecinos de Gavà y cuyos familiares habían solicitado su exhumación. El trabajo multidisciplinar llevado a cabo permitió: 1) recuperar toda la información ante mortem disponible en relación a la fosa y los desaparecidos, 2) recuperar mediante metodología arqueológica y directrices antropológico-forenses los restos de los 13 individuos enterrados en la fosa, y 3) analizar los restos en el laboratorio a través de técnicas antropológicas, forenses, moleculares y de superposición craneofacial. Los resultados permitieron tanto la identificación de las cuatro personas buscadas, como la determinación de las causas y circunstancias de su muerte, relacionadas todas ellas con heridas por arma de fuego, acaecidas en un contexto de batalla (AU)

Forensic Anthropology seeks both to identify the individual, such as determining the cause and circumstances of death. In this sense, forensic anthropology is essential for recovery remains of missing persons who were buried in mass graves during the Spanish Civil War and Franco's dictatorship, for subsequent return to their families. This paper presents the results of work done at the grave of Gurb, where Republican soldiers were buried; four families of Gavà had applied for exhumation of their relatives buried in it. The multidisciplinary work carried out allowed us: 1) to retrieve all available ante mortem information regarding the pit and the missing persons, 2) to recover the remains of 13 individuals buried in the grave by archaeological methods and forensic-anthropological guidelines, and 3) to analyze their remains in the laboratory through anthropological, forensics, molecular and craniofacial superimposition techniques. The results allowed both the identification of the four wanted persons, such as determining the causes and circumstances of his death, in all cases due to battle related trauma, specifically firearm injuries (AU)

A reverse transcriptase activity in potato mitochondria.

A reverse transcriptase activity has been detected in potato mitochondria using special RNAs as templates: a bacterial RNA coding for neomycin phosphotransferase (neo pa RNA) and a Neurospora crassa mitochondrial RNA (184 nt RNA). Surprisingly, no exogenous primer addition was required. These RNA templates share a primary and secondary structure similar to the T psi CG loop of tRNAs that could constitute the recognition site for the enzyme. Reverse transcriptase activity was inhibited by ddTTP, ethidium bromide and aphidicolin, while potato mitochondrial DNA polymerase was not inhibited by aphidicolin indicating that these activities correspond to distinct enzymes. A conserved sequence of reverse transcriptases was detected in potato mitochondrial DNA suggesting that this enzyme could be mitochondrially encoded.

The nuclear-encoded SDH2-RPS14 precursor is proteolytically processed between SDH2 and RPS14 to generate maize mitochondrial RPS14.

In maize, the functional gene encoding mitochondrial ribosomal protein S14 (rps14) has been translocated to the nucleus where it became integrated between both exons of a gene encoding the iron-sulfur subunit of succinate dehydrogenase (sdh2). Two transcripts are generated from this locus by alternative splicing. One transcript encodes a precursor for a functional SDH2 protein, while the second transcript encodes a chimeric SDH2(t)-RPS14 precursor protein. In this paper we show that the same mitochondrial targeting presequence is able to direct the import of both precursors into isolated mitochondria and is removed during import. This processing event generates a 28 kDa protein from the SDH2 precursor, which corresponds to the iron-sulfur subunit of respiratory complex II present in maize mitochondria. In addition to cleavage of the presequence, the chimeric precursor undergoes proteolytical processing between SDH2 and RPS14. This processing generates RPS14, which is found assembled into mitochondrial ribosomes, and a truncated SDH2 protein which is degraded. Therefore, our results support a role of the SDH2 domain in the chimeric precursor only in providing a mitochondrial targeting function for RPS14.

Studies on the mechanism of inhibition of amphibian oocyte adenylate cyclase by progesterone.

Progesterone treatment induces the meiotic maturation of Xenopus laevis oocytes. Previous evidence indicates that this hormonal effect may be due to inhibition of oocyte adenylate cyclase. The present work studies several aspects of the mechanism of adenylate cyclase inhibition by this hormone. Forskolin greatly stimulates oocyte adenylate cyclase in the absence of guanine nucleotides and this activity is not sensitive to progesterone inhibition. In addition the forskolin-activated enzyme is not inhibited by a wide range of guanine nucleotide, in the presence or absence of hormone. The time course of cAMP synthesis catalyzed by oocyte adenylate cyclase in the presence of guanyl-5'l-imidodiphosphate (Gpp(NH)p) shows an initial lag period that does not depend on the concentration of Gpp(NH)p. Progesterone causes a very significant increase in the hysteresis of the reaction, at least doubling the half-time of enzyme activation. The hormonal effect on the lag cannot be reversed by saturating concentrations of Gpp(NH)p. Progesterone also decreases the steady-state rates of the reaction. This effect, however, depends on the concentration of Gpp(NH)p. High concentrations of Gpp(NH)p almost completely reverse the inhibition of the steady-state rates. Progesterone does not inhibit if it is added to the reaction after the initial lag period. Guanosine-5'-O-(2-thiodiphosphate) (GDP-beta-S) is an efficient competitive inhibitor of Gpp(NH)p activation of adenylate cyclase. Progesterone inhibition is observed at all concentrations of GDP-beta-S and is potentiated at high ratios of GDP-beta-S to Gpp(NH)p. These data indicate that progesterone inhibits by interfering with the activation of the Ns subunit of the enzyme by guanine nucleotides, rather than through a mechanism involving a separate Ni subunit.

Cloning and characterization of the cDNA coding for the catalytic alpha subunit of CK2 from tobacco.

We have previously reported the participation of the protein kinase CK2 in the mechanism by which salicylic acid activates transcription of genes, such as those coding for glutathion S-transferases, in tobacco. With the purpose of further studying the participation of CK2 in this signal transduction pathway, we isolated and sequenced the cDNA from the NtCK2A gene, coding for the catalytic alpha subunit of CK2 from tobacco. The NtCK2A cDNA was isolated by screening of a tobacco cDNA library with a heterologous probe from Arabidopsis thaliana, followed by 3' RACE to obtain the 3' region. Sequence analysis of the NtCK2A cDNA showed a high level of identity between this CK2alpha protein sequence and the corresponding sequences of other plant species such as Arabidopsis and maize (92-95% identity), or those of animal species such as human and Xenopus laevis (75% identity). The expression of the NtCK2A gene in different tissues from tobacco plants was analyzed by Northern blot. High levels of expression of this gene were observed in proliferating tissues such as shoot and root apical meristems. A recombinant CK2alpha protein was obtained after expression of the NtCK2A cDNA in Escherichia coli. The ability of this recombinant CK2alpha subunit to phosphorylate casein was inhibited by heparin and stimulated by the CK2beta subunit from Xenopus laevis.

Three different genes encode the iron-sulfur subunit of succinate dehydrogenase in Arabidopsis thaliana.

The iron-sulfur protein is an essential component of mitochondrial complex II (succinate dehydrogenase, SDH), which is a functional enzyme of both the citric acid cycle and the respiratory electron transport chain. This protein is encoded by a single-copy nuclear gene in mammals and fungi and by a mitochondrial gene in Rhodophyta and the protist Reclinomonas americana. In Arabidopsis thaliana, the homologous protein is now found to be encoded by three nuclear genes. Two genes (sdh2-1 and sdh2-2) likely arose from a relatively recent duplication event since they have similar structures, encode nearly identical proteins and show similar expression patterns. Both genes are interrupted by a single intron located at a conserved position. Expression was detected in all tissues analysed, with the highest steady-state mRNA levels found in flowers and inflorescences. In contrast, the third gene (sdh2-3) is interrupted by 4 introns, is expressed at a low level, and encodes a SDH2-3 protein which is only 67% similar to SDH2-1 and SDH2-2 and has a different N-terminal presequence. Interestingly, the proteins encoded by these three genes are probably functional because they are highly conserved compared with their homologues in other organisms. These proteins contain the cysteine motifs involved in binding the three iron-sulfur clusters essential for electron transport. Furthermore, the three polypeptides are found to be imported into isolated plant mitochondria.

Phosphorylation of nuclear proteins directs binding to salicylic acid-responsive elements.

The cis-located DNA sequence as-1 (Activation Sequence-1) from CaMV 35S promoter has been previously identified as an element that can confer inducibility by salicylic acid (SA) with immediate early kinetics. This sequence specifically binds to ASF-1 (Activation Sequence Factor-1), previously characterized in tobacco nuclear extracts. To assess whether modulation of ASF-1 binding activity can explain the activation of the as-1 sequence observed in vivo, we performed electrophoretic mobility shift assays using nuclear extracts from SA-treated and water-treated tobacco plants. Our results indicate that treatment of plants with SA increases ASF-1 binding to as-1 and to ocs, an as-1-like element from the Agrobacterium octopine synthase gene. In contrast, SA treatment has no effect on the binding of GT-1 factor to its target light-inducible box II element. Furthermore, treatment of nuclear extracts from SA-treated plants with alkaline phosphatase decreases ASF-1 binding to the as-1 element. This can be reversed by pretreatment with 10 mM NaF. Accordingly, pretreatment of nuclear extracts from control water-treated plants with ATP produces an increase in ASF-1 binding activity similar to that observed with SA. This effect of ATP is reversed by treatment with alkaline phosphatase and prevented by quercetin, a casein kinase II inhibitor. These results support the hypothesis that a nuclear protein kinase is involved in the immediate early events of transcriptional activation triggered by SA.

Bifid mandibular condyle: archaeological case report of a rare anomaly.

In this paper, an archaeological case of unilateral bifid mandibular condyle is presented. This uncommon anomaly is characterized by a division of the mandibular condylar head. In this case, the left condyle was divided into two articulating surfaces oriented mediolaterally; two articular facets on the anterior wall of the glenoid fossa for the double condyle were observed. The morphological and radiological analysis do not show any evidence of injuries or degenerative pathology. Taking into account the two main causes of bifid condyle suggested in the literature (traumatic and developmental), an embryopathy by teratogenic agents is proposed as a possible aetiology of the bifid condyle reported here.

Splicing and editing of rps10 transcripts in potato mitochondria.

The structure and expression of the potato mitochondrial gene rps10, encoding ribosomal protein S10, has been characterized. The RPS10 polypeptide of 129 amino acids is encoded by two exons of 307 bp and 80 bp respectively, which are separated by a 774-bp class-II intron. Editing of the complete rps10 coding region was studied by sequence analysis of spliced cDNAs. Four C residues are edited into U, resulting in the creation of a putative translational initiation codon, a new stop codon which eliminated ten carboxy-terminal residues, and two additional amino-acid alterations. All these changes increase the similarity between the potato and liverwort polypeptides. One additional C-to-U RNA editing event, observed in the intron sequence of unspliced cDNAs, improves the stability of the secondary structure in stem I (i) of domain I and may thus be required for the splicing reaction. All spliced cDNAs, and most unspliced cDNAs, were completely edited, suggesting that editing is an early step of rps10 mRNA processing and precedes splicing. Earlier work on potato rps10 (Zanlungo et al. 1994) is now known to comprise only a partial analysis of the gene, since the short downstream exon was not identified.

A ribosomal protein S10 gene is found in the mitochondrial genome in Solanum tuberosum.

The S10 ribosomal protein gene (rps10), which has not been previously reported in any angiosperm mitochondrial genome, was identified by sequence analysis in the potato mitochondrial DNA. This gene is found downstream of a truncated non-functional apocytochrome b (cob) pseudogene, and is expressed as multiple transcripts ranging in size from 0.8 to 5.0 kb. Southern hybridization analysis indicates that rps10-homologous sequences are not present in the wheat mitochondrial genome. Sequence analysis of a single-copy region of the pea mitochondrial genome located upstream of cox1 [11] shows that a non-functional rps10 pseudogene is present in this species. These results suggest that the functional genes coding for wheat and pea mitochondrial RPS10 polypeptides have been translocated to the nucleus.

A nuclear casein kinase 2 activity is involved in early events of transcriptional activation induced by salicylic acid in tobacco.

Salicylic acid (SA) activates immediate early transcription of genes controlled by a family of DNA promoter elements named as-1-like elements. These elements are functional in the promoter of glutathione S-transferase genes. We have previously shown that SA increases the binding of tobacco (Nicotiana tabacum cv Xanthi nc) nuclear factors to the as-1 sequence in a process mediated by protein phosphorylation. In this study we give evidence for the participation of a nuclear protein kinase CK2 (casein kinase 2) in the pathway activated by SA in tobacco. The first line of evidence comes from the evaluation of the CK2 activity in nuclear extracts prepared from tobacco plants treated with SA or water as a control. Results from these experiments indicate that SA increases the nuclear CK2 activity. The second line of evidence derives from the evaluation of the in vivo effect of 5,6-dichloro-1-(beta-D-ribofuranosyl) benzimidazole (DRB), a cell-permeable CK2 inhibitor, on the responsiveness of the as-1 sequence to SA. Results from these experiments indicate that DRB impairs the activating effect of SA on the transcription of both, the GUS reporter gene controlled by a tetramer of the as-1 element, and the endogenous gnt35 gene encoding a glutathione S-transferase, in transgenic tobacco plants. DRB also impaired the increasing effect of SA on the binding of nuclear factors to the as-1 element. Furthermore, transcription of the as-1/GUS reporter gene activated by the synthetic auxin 2,4-dichlorophenoxyacetic acid and by methyl jasmonate was also inhibited by DRB. To our knowledge, this is the first report in which activation of a CK2 enzyme by a plant hormone is reported.

The rpl5-rps14-cob gene arrangement in Solanum tuberosum: rps14 is a transcribed and unedited pseudogene.

The L5 ribosomal protein gene (rpl5) and a S14 ribosomal protein pseudogene were identified by sequence analysis in the potato mitochondrial genome. The two genes are separated by one nucleotide and are found upstream of the apocytochrome b gene (cob), an arrangement conserved also in Arabidopsis and Brassica. The rpl5 gene has an intact open reading frame while the rps14 locus is disrupted by a five nucleotide duplication that introduces a frameshift in the reading frame. Editing of rpl5 and pseudorps14 cotranscripts has been studied by cDNA sequence analysis. Eight C residues are edited into U in the rpl5 coding region, resulting in eight amino acid changes that increase the homology between potato and other RPL5 polypeptides. Interestingly, the rps14 pseudogene sequence is not edited at any nucleotide position.

Transfer of the mitochondrial rps10 gene to the nucleus in rice: acquisition of the 5' untranslated region followed by gene duplication.

Mitochondrial ribosomal protein S10 (rps10) is encoded by the mitochondrial genome in potato and pea. Here we show that the rps10 gene is absent from the mitochondrial genome of rice and has been transferred to the nucleus. Cloning and transcriptional analysis show that there are two rps10 genes in the rice nuclear genome and that their transcripts differ in abundance. Western analysis detected the RPS10 protein in the soluble fraction of rice mitochondria, although neither RPS10 has any obvious N-terminal presequence for targeting to mitochondria. This result suggests that targeting information is present in the internal region of rice RPS10. Genomic sequence analysis indicated that each rps10 gene has an intron in the 5' untranslated region (5' UTR) and that these intron sequences are homologous to each other. This result strongly suggests that a duplication event occurred after transfer of the rps10 gene to the nucleus. The duplicated rps10 genes have since been translocated to different chromosomes, because the two rps10 genes were mapped on chromosomes 6 and 12 by RFLP analysis. Interestingly, the 5' UTR and the intron of the rice rps10 genes are homologous to sequences found in several rice genes with various functions, such as osk4, EF-1beta2 and RAG1, suggesting a common origin and a functional role for the 5' UTR. Acquisition of the 5' flanking region might have accelerated the activation of the mitochondrial rps10 gene which was transferred to the nuclear genome.

RNA editing of apocytochrome b (cob) transcripts in mitochondria from two genera of plants.

Editing of the complete coding region of cob transcripts from two genera of plants has been studied by cDNA sequence analysis. Eighteen and nine C residues are edited into U in the mitochondrial transcripts from wheat and potato respectively. Both systems share eight common editing sites; ten codons edited in wheat are "pre-edited" at the genomic level in potato, and one codon edited in potato is "pre-edited" in wheat. Most amino-acid modifications lead to hydrophobic residues and increase the homology between the COB polypeptides and the corresponding protein of other species. In two out of the nine potato cDNA clones, an additional C-to-T modification, which also leads to a change in the encoded amino acid, was identified. Heterogeneity observed at the carboxy-terminus of the COB open reading frame in Triticum aestivum and Triticum timopheevi is not corrected by editing.