RESUMO
The rejection of composite veneerings in fixed partial dentures is primarily caused by the inadequacy of the bonds between the metal framework and the composite veneering. The development of improved veneering composites necessitates an investigation of their clinical suitability compared with ceramic materials. Nineteen patients with at least two suitable, adjacent natural teeth for crowning were treated with 64 high noble alloy crowns. The adjacent crowns were veneered with ceramic (IPS inline) and composite materials (SR Adoro). Seven follow-up examinations were carried out over a period of 10 years. The crowns were investigated for mechanical defects, periodontal parameters, and discolorations. The survival rates of the ceramic veneered metal crowns (CeMCs) and composite veneered metal crowns (CoMCs) at the 10-year follow-up were 87.1 and 87.9%, respectively. The success rates of the crowns after 10 years were 83.9% for CeMCs and 51.5% for CoMCs (log-rank test, p = 0.009). No significant differences between the groups were found in the periodontal parameters (Kruskal-Wallis one-way analysis of variance, ANOVA p ⟩ 0.05). After 10 years, discoloration patterns of the two materials differed significantly (Mann-Whitney-U-test, p = 0.017). Thus, despite the improvements associated with CoMCs, CeMCs remain the gold standard for veneered metal crowns.
Assuntos
Coroas , Porcelana Dentária , Cerâmica , Prótese Parcial Fixa , HumanosRESUMO
INTRODUCTION: Computer-aided design/computer-aided manufacturing (CAD/CAM) of fixed prosthetic restorations has gained popularity in the last decade. However, this field of dentistry has not been integrated in the dental curriculum at most universities. MATERIAL AND METHODS: According to the method of Kern, a curriculum was designed and established on a voluntary basis in the prosthetic education of a German dental school. The success of the implementation was measured by evaluation carried out by the participants on a visual analogue scale. Furthermore, the clinical performance of the fabricated restorations was evaluated. RESULTS: Ninety-four percent of all students participated in the CAD/CAM curriculum indicating considerable interest. Nearly half of all students used the acquired knowledge to design crowns for their patients. All restorations fabricated by participants of the new CAD/CAM programme showed good clinical performance. DISCUSSION: By phasing-in the CAD/CAM training programme, independent CAD/CAM-based fabrication of all-ceramic crowns increased student's self-confidence in tooth preparation. A tendency was found that students using CAD/CAM technology prepared more teeth than their fellow students who did not use CAD/CAM technology. Further studies are required to investigate the influence of independent CAD/CAM-based single-crown fabrication on the quality of the preparation.
Assuntos
Desenho Assistido por Computador , Currículo , Prótese Dentária , Prostodontia/educação , Materiais Dentários , Planejamento de Prótese Dentária/métodos , Europa (Continente) , Humanos , Faculdades de OdontologiaRESUMO
BACKGROUND: After radiation treatment of head-and-neck cancer, the impairment of patient's quality of life still remains an issue. After completion of the treatment course, a substantial number of patients develop so-called radiation caries. In addition, almost 50% of all cases of infectious osteoradionecrosis (iORN) of the jaws are directly associated with radiation caries. This review addresses our current knowledge on the etiology and pathogenesis of radiation caries including possible preventive strategies. MATERIALS AND METHODS: A PubMed search using the terms "radiation caries" ("radiation related caries", "radiation related damage to dentition") and "radiogenic caries" ("postradiation caries", "dental complications and radiotherapy") was performed. The analysis of its content focused on the etiology, the pathogenesis, and the available knowledge on prophylaxis as well as treatment of radiation caries. RESULTS: For this review, 60 publications were selected. As main causal factors for radiogenic caries, either indirect impairment, resulting from alterations in the oral environment (e.g., radiation-induced xerostomia) or direct radiation-induced damage in teeth hard tissues are discussed. Radiation caries remains a lifelong threat and, therefore, requires permanent prevention programs. CONCLUSION: To enable optimal medical care of the patients during the time course of radiotherapy as well as afterwards, close interdisciplinary cooperation between radiotherapists, oral surgeons, otorhinolaryngologists, and dentists is absolutely essential.
Assuntos
Cárie Dentária/etiologia , Neoplasias Otorrinolaringológicas/radioterapia , Lesões por Radiação/etiologia , Dente/efeitos da radiação , Adulto , Cárie Dentária/prevenção & controle , Relação Dose-Resposta à Radiação , Humanos , Lactente , Odontogênese/efeitos da radiação , Lesões por Radiação/prevenção & controle , Planejamento da Radioterapia Assistida por Computador , Radioterapia de Intensidade Modulada , Fatores de Risco , Anormalidades Dentárias/etiologia , Anormalidades Dentárias/prevenção & controle , Xerostomia/complicações , Xerostomia/etiologiaRESUMO
The self-splicing bl1 intron lariat from mitochondria of Saccharomyces cerevisiae catalyzed the insertion of nucleotidyl monomers derived from the 3' end of a donor RNA into an acceptor RNA in a 3' to 5' direction in vitro. In this catalyzed reaction, the site specificity provided by intermolecular base pair interactions, the formation of chimeric intermediates, the polarity of the nucleotidyl insertion, and its reversibility all resemble such properties in previously proposed models of RNA editing in kinetoplastid mitochondria. These results suggest that RNA editing occurs by way of a concerted, two-step transesterification mechanism and that RNA splicing and RNA editing might be prebiotically related mechanisms; possibly, both evolved from a primordial demand for self-replication.
Assuntos
Adenosina/metabolismo , Íntrons , Edição de RNA , RNA Catalítico/metabolismo , RNA Fúngico/metabolismo , RNA Guia de Cinetoplastídeos/metabolismo , Sequência de Bases , Esterificação , Mitocôndrias/metabolismo , Dados de Sequência Molecular , Splicing de RNA , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismoRESUMO
In order to reach the inner membrane of the mitochondrion, multispanning carrier proteins must cross the aqueous intermembrane space. Two essential proteins of that space, Tim10p and Tim12p, were shown to mediate import of multispanning carriers into the inner membrane. Both proteins formed a complex with the inner membrane protein Tim22p. Tim10p readily dissociated from the complex and was required to transport carrier precursors across the outer membrane; Tim12p was firmly bound to Tim22p and mediated the insertion of carriers into the inner membrane. Neither protein was required for protein import into the other mitochondrial compartments. Both proteins may function as intermembrane space chaperones for the highly insoluble carrier proteins.
Assuntos
Proteínas de Transporte/metabolismo , Proteínas Fúngicas/metabolismo , Membranas Intracelulares/metabolismo , Proteínas de Membrana/metabolismo , Proteínas de Membrana Transportadoras , Mitocôndrias/metabolismo , Proteínas de Saccharomyces cerevisiae , Transporte Biológico , Clonagem Molecular , Proteínas Fúngicas/genética , Genes Fúngicos , Temperatura Alta , Potenciais da Membrana , Proteínas de Membrana/genética , Translocases Mitocondriais de ADP e ATP/metabolismo , Proteínas de Transporte da Membrana Mitocondrial , Proteínas do Complexo de Importação de Proteína Precursora Mitocondrial , Modelos Biológicos , Chaperonas Moleculares/metabolismo , Mutagênese , Proteínas de Ligação a Fosfato , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , SolubilidadeRESUMO
Loss of the MDM38 gene product in yeast mitochondria results in a variety of phenotypic effects including reduced content of respiratory chain complexes, altered mitochondrial morphology and loss of mitochondrial K(+)/H(+) exchange activity resulting in osmotic swelling. By use of doxycycline-regulated shut-off of MDM38 gene expression, we show here that loss of K(+)/H(+) exchange activity and mitochondrial swelling are early events, associated with a reduction in membrane potential and fragmentation of the mitochondrial reticulum. Changes in the pattern of mitochondrially encoded proteins are likely to be secondary to the loss of K(+)/H(+) exchange activity. The use of a novel fluorescent biosensor directed to the mitochondrial matrix revealed that the loss of K(+)/H(+) exchange activity was immediately followed by morphological changes of mitochondria and vacuoles, the close association of these organelles and finally uptake of mitochondrial material by vacuoles. Nigericin, a K(+)/H(+) ionophore, fully prevented these effects of Mdm38p depletion. We conclude that osmotic swelling of mitochondria triggers selective mitochondrial autophagy or mitophagy.
Assuntos
Autofagia , Proteínas de Membrana/metabolismo , Mitocôndrias/metabolismo , Dilatação Mitocondrial , Antiportadores de Potássio-Hidrogênio/metabolismo , Potássio/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/fisiologia , Expressão Gênica , Hidrogênio/metabolismo , Proteínas de Membrana/genética , Mitocôndrias/ultraestrutura , Proteínas Mitocondriais , Mutação , Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/ultraestrutura , Proteínas de Saccharomyces cerevisiae/genéticaRESUMO
The Rab escort protein (REP) is an essential component of the heterotrimeric enzyme Rab geranylgeranyl transferase that modifies the carboxy-terminal cysteines of the Ras-like small G proteins belonging to the Rab/Ypt family. Deletions in the human CHM locus, encoding one of the two REPs known in humans, result in a retinal degenerative syndrome called choroideremia. The only known yeast homologue of the choroideremia gene product is encoded by an essential gene called MRS6. Besides three structurally conserved regions (SCRs) previously detected in the amino-terminal half of REPs and RabGDIs, three other regions in the carboxy-terminal domain (RCR 1-3) are here identified as being characteristic of REPs alone. We have performed the first mutational analysis of a REP protein to experimentally define the regions functionally important for Rab/Ypt protein binding, making use of the genetic system of the yeast Saccharomyces cerevisiae. This analysis has shown that the SCRs are necessary but not sufficient for Ypt1p binding by the yeast REP, the carboxy-terminal region also being required.
Assuntos
Alquil e Aril Transferases , Proteínas de Transporte/química , Proteínas Fúngicas/química , Proteínas de Ligação ao GTP/metabolismo , Inibidores de Dissociação do Nucleotídeo Guanina , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/metabolismo , Transferases/metabolismo , Proteínas rab de Ligação ao GTP , Proteínas Adaptadoras de Transdução de Sinal , Sequência de Aminoácidos , Animais , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Bovinos , Coroideremia/genética , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , GTP Fosfo-Hidrolases/metabolismo , Proteínas de Ligação ao GTP/química , Proteínas de Ligação ao GTP/genética , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Prenilação de Proteína , Processamento de Proteína Pós-Traducional , Deleção de Sequência , Relação Estrutura-Atividade , Transferases/química , Transferases/genéticaRESUMO
The RNA-catalysed self-splicing reaction of group II intron RNA is assumed to proceed by two consecutive transesterification steps, accompanied by lariat formation. This is effectively analogous to the small nuclear ribonucleoprotein (snRNP)-mediated nuclear pre-mRNA splicing process. Upon excision from pre-RNA, a group II lariat intervening sequence (IVS) has the capacity to re-integrate into its cognate exons, reconstituting the original pre-RNA. The process of reverse self-splicing is presumed to be a true reversion of both transesterification steps used in forward splicing. To investigate the fate of the esterified phosphate groups in splicing we assayed various exon substrates (5'E-*p3'E) containing a unique 32P-labelled phosphodiester at the ligation junction. In combined studies of alternating reverse and forward splicing we have demonstrated that the labelled phosphorus atom is displaced in conjunction with the 3' exon from the ligation junction to the 3' splice site and vice versa. Neither the nature of the 3' exon sequence nor its sequence composition acts as a prominent determinant for both substrate specificity and site-specific transesterification reactions catalysed by bI1 IVS. A cytosine ribonucleotide (pCp; pCOH) or even deoxyoligonucleotides could function as an efficient substitute for the authentic 3' exon in reverse and in forward splicing. Furthermore, the 3' exon can be single monophosphate group. Upon incubation of 3' phosphorylated 5' exon substrate (5'E-*p) with lariat IVS the 3'-terminal phosphate group is transferred in reverse and forward splicing like an authentic 3' exon, but with lower efficiency. In the absence of 3' exon nucleotides, it appears that substrate specificity is provided predominantly by the base-pairing interactions of the intronic exon binding site (EBS) sequences with the intron binding site (IBS) sequences in the 5' exon. These studies substantiate the predicted transesterification pathway in forward and reverse splicing and extend the catalytic repertoire of group II IVS in that they can act as a potential and sequence-specific transferase in vitro.
Assuntos
Precursores de Ácido Nucleico/metabolismo , Fosfatos/química , Splicing de RNA , Sequência de Bases , Catálise , DNA/química , Ésteres , Éxons , Íntrons , Dados de Sequência Molecular , RNA/química , Relação Estrutura-AtividadeRESUMO
The recent report on RNA-mediated group II intron (IVS, intervening sequence) transposition in mitochondria (mt) of Saccharomyces cerevisiae and Podospora anserina and the demonstration of reverse transcriptase (RT) activity encoded by the mobile S. cerevisiae intron cox1-aI1 suggests that group II introns constitute a new class of site-specific retro-like (retroid) elements. This is supported by the finding that the mitochondrial cob1-bI1 intron from the fission yeast Schizosaccharomyces pombe, encoding an RT-like open reading frame, is transposed in mtDNA populations. In agreement with the involvement of an RNA-intermediate in IVS transposition: First, the insertion sites were preceded by at least an IBS1-like (intron binding site) motif, which corresponds to the upstream exon and suffices to form the IBS1/EBS1 (EBS: exon binding site) base-pairing interactions. Second, intron transposition was conservative with respect to sequences flanking the insertion sites. We formulated the hypothesis that transient IVS insertion at non-allelic sites followed by recombination can be viewed as a general molecular mechanism, applicable equally well to site-specific genomic instabilities involving splice-site borders of group II introns and to the formation of extra-genomic IVS plasmid DNAs (plDNAs). We used polymerase chain reaction (PCR) techniques to detect infrequent rearrangements in mtDNA and report here on duplicative IVS transposition, twintron formation (e.g. bI1 insertion into another bI1 intron), and IVS insertions at canonical 5' exon-intron borders in S. pombe (cob1-bI1) and in S. cerevisiae (cox1-aI1). These data substantiate the concept that group II intron homing, IVS transposition and circular IVS plDNA formation involve a common RNA-mediated mechanism. Finally, the findings suggest that extra-genomic group II IVS copies are not restricted to senescence mycelia of P. anserina, but constitute natural components of group II IVS-containing genomes.
Assuntos
Elementos de DNA Transponíveis/genética , DNA Fúngico/genética , Íntrons/genética , Schizosaccharomyces/genética , Apoproteínas/genética , Sequência de Bases , Grupo dos Citocromos b/genética , Citocromos b , DNA Mitocondrial/genética , Rearranjo Gênico , Genoma Fúngico , Dados de Sequência Molecular , RNA Fúngico/genética , Saccharomyces cerevisiae/genéticaRESUMO
In the realms of RNA, transposable elements created by self-inserting introns recombine novel combinations of exon sequences in the background of replicating molecules. Although intermolecular RNA recombination is a wide-spread phenomenon reported for a variety of RNA-containing viruses, direct evidence to support the theory that modern splicing systems, together with the exon-intron structure, have evolved from the ability of RNA to recombine, is lacking. Here, we used an in vitro deletion-complementation assay to demonstrate trans-activation of forward and reverse self-splicing of a fragmented derivative of the group II intron bI1 from yeast mitochondria. We provide direct evidence for the functional interchangeability of analogous but non-identical domain 1 RNA molecules of group II introns that result in trans-activation of intron transposition and RNA-based exon shuffling. The data extend theories on intron evolution and raise the intriguing possibility that naturally fragmented group III and spliceosomal introns themselves can create transposons, permitting rapid evolution of protein-coding sequences by splicing reactions.
Assuntos
Éxons/genética , Íntrons/genética , Splicing de RNA/genética , RNA Catalítico/genética , RNA Catalítico/metabolismo , Recombinação Genética/genética , Sequência de Bases , Sítios de Ligação , Catálise , Evolução Molecular , Teste de Complementação Genética , Cinética , Modelos Genéticos , Peso Molecular , Conformação de Ácido Nucleico , RNA/química , RNA/genética , RNA/metabolismo , RNA Catalítico/química , RNA Mitocondrial , Deleção de Sequência/genética , Homologia de Sequência do Ácido Nucleico , Leveduras/enzimologia , Leveduras/genéticaRESUMO
When present in high copy number plasmids, the nuclear genes MRS3 and MRS4 from Saccharomyces cerevisiae can suppress the mitochondrial RNA splicing defects of several mit- intron mutations. Both genes code for closely related proteins of about Mr 32,000; they are 73% identical. Sequence comparisons indicate that MRS3 and MRS4 may be related to the family of mitochondrial carrier proteins. Support for this notion comes from a structural analysis of these proteins. Like the ADP/ATP carrier protein (AAC), the mitochondrial phosphate carrier protein (PiC) and the uncoupling protein (UCP), the two MRS proteins have a tripartite structure; each of the three repeats consists of two hydrophobic domains that are flanked by specific amino acid residues. The spacing of these specific residues is identical in all domains of all proteins of the family, whereas spacing between the hydrophobic domains is variable. Like the AAC protein, the MRS3 and MRS4 proteins are imported into mitochondria in vitro and without proteolytic cleavage of a presequence and they are located in the inner mitochondrial membrane. In vivo studies support this mitochondrial localization of the MRS proteins. Overexpression of the MRS3 and MRS4 proteins causes a temperature-dependent petite phenotype; this is consistent with a mitochondrial function of these proteins. Disruption of these genes affected neither mitochondrial functions nor cellular viability. Their products thus have no essential function for mitochondrial biogenesis or for whole yeast cells that could not be taken over by other gene products. The findings are discussed in relation to possible functions of the MRS proteins in mitochondrial solute translocation and RNA splicing.
Assuntos
Proteínas de Transporte/genética , Proteínas de Transporte de Cátions , Proteínas Fúngicas/genética , Genes Supressores , Mitocôndrias/metabolismo , Splicing de RNA , RNA/genética , Proteínas Repressoras , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Sequência de Aminoácidos , Sequência de Bases , Northern Blotting , Southern Blotting , Proteínas de Transporte/metabolismo , Mapeamento Cromossômico , Cromossomos Fúngicos , Proteínas Fúngicas/metabolismo , Regulação Fúngica da Expressão Gênica , Genes Fúngicos , Proteínas Mitocondriais , Dados de Sequência Molecular , Fenótipo , Plasmídeos , RNA/metabolismo , RNA Fúngico/genética , RNA Fúngico/metabolismo , RNA Mitocondrial , Mapeamento por Restrição , Homologia de Sequência do Ácido Nucleico , TemperaturaRESUMO
We report here a novel human gene, hMRS3/4, encoding a putative mitochondrial transporter structurally and functionally homologous to the yeast mitochondrial RNA splicing proteins 3 and 4. These proteins belong to the family of mitochondrial carrier proteins (MCF) and are likely to function as solute carriers. hMRS3/4 spans approximately 10 kb of genomic DNA on chromosome 10q24 and consists of four exons that encode a 364-aa protein with six transmembrane domains. A putative splice variant, encoding a 177-aa protein with three transmembrane domains, was also identified. hMRS3/4 has a well-conserved signature sequence of MCF and is targeted into the mitochondria. When expressed in yeast, hMRS3/4 efficiently restores the mitochondrial functions in mrs3(o)mrs4(o) knock-out mutants. Ubiquitous expression in human tissues and a well-conserved structure and function suggest an important role for hMRS3/4 in human cells.
Assuntos
Processamento Alternativo , Proteínas de Transporte/genética , Proteínas de Transporte de Cátions , Cromossomos Humanos Par 10 , Proteínas de Membrana Transportadoras , Mitocôndrias/metabolismo , Proteínas Repressoras , Proteínas de Saccharomyces cerevisiae , Sequência de Aminoácidos , Sequência de Bases , Proteínas de Transporte/metabolismo , Mapeamento Cromossômico , DNA Complementar , Proteínas Fúngicas/genética , Perfilação da Expressão Gênica , Humanos , Proteínas Mitocondriais , Dados de Sequência Molecular , Mutagênese , Oftalmoplegia Externa Progressiva Crônica/genética , Splicing de RNA , Saccharomyces cerevisiae/genética , Ataxias Espinocerebelares/genética , Distribuição Tecidual , TransfecçãoAssuntos
Proteínas de Transporte/química , Proteínas Fúngicas/química , Proteínas de Membrana/química , Microcorpos/química , Mitocôndrias/metabolismo , Sequência de Aminoácidos , Aminoácidos/metabolismo , Animais , Candida , Proteínas de Transporte/metabolismo , Cátions/metabolismo , Proteínas Fúngicas/metabolismo , Proteínas de Membrana/metabolismo , Microcorpos/metabolismo , Dados de Sequência Molecular , Ratos , Saccharomyces cerevisiae , Homologia de Sequência de AminoácidosRESUMO
Group II intron bl1 from yeast mitochondria can undergo self-splicing in vitro. Exons become correctly ligated, and the excised intron has a lariat structure similar to that of introns from nuclear mRNA. The branch point of the bl1 lariat is located eight or nine nucleotides upstream of the 3' end of the intron and is part of a hairpin structure that is well conserved among group II introns. Several mutations next to the branch point and in other parts of the core structure of group II introns are shown to affect lariat formation. One of them, carried by strain M4873, abolishes splicing in vivo and in vitro, apparently by changing the architecture of the hairpin structure containing the branch point. Similarities between group II introns and nuclear pre-mRNA introns are discussed in terms of evolutionary relatedness.
Assuntos
Grupo dos Citocromos b/genética , DNA Mitocondrial/genética , Splicing de RNA , Sequência de Bases , Mapeamento Cromossômico , Clonagem Molecular , Guanosina/fisiologia , Mutação , Conformação de Ácido Nucleico , Precursores de Ácido Nucleico/genética , Processamento Pós-Transcricional do RNA , Saccharomyces cerevisiae/genética , Espermidina/fisiologiaRESUMO
The inheritance of an extrakaryotic mutation conferring temperature-sensitive growth on non-fermentable substrates and a high frequency of mutation to rho- has been studied. Multifactorial crosses (rho+ X rho+) involving this mutation TS8 and mitochondrial mutations conferring resistance to chloramphenicol, erythromycin, oligomycin or paromomycin revealed: a) Mutation TS8 is localized on the mitDNA, referring to a new gene locus TSM1. b) Locus TSM1 appears to be weakly linked to the locus PAR1 and to the loci RIB1 and RIB3 but unlinked to the locus OLI1. c) The position of TSM1 is between PAR1 and the two closely linked loci RIB1 and RIB3, OLI1 is outside and not linked to the segment PAR-TSM-RIB. d) Mutation TS8 does not significantly influence the process of mitochondrial recombination and its control by the mitochondrial locus omega.
Assuntos
Mitocôndrias/fisiologia , Mutação , Saccharomyces cerevisiae/fisiologia , Mapeamento Cromossômico , Cruzamentos Genéticos , Genótipo , Fenótipo , Recombinação Genética , Especificidade da Espécie , TemperaturaRESUMO
We have investigated the processing of transcripts of the split gene COB in yeast mitochondrial DNA from cells whose mitochondrial translation was blocked by chloramphenicol for several generations of cell growth. First analysis of transcripts by electrophoresis and RNA/DNA-hybridization clearly showed that cell growth in the presence of CAP leads to an inhibition of processing yielding an increasing amount of splicing intermediates of the COB transcript and decreasing amounts of the 18S mRNA coding for apocytochrome b. This observation is in accordance with the now widely favoured idea that mitochondrial proteins are involved in splicing of COB transcripts and that their reduction should hamper processing and - therefore - lead to an accumulation of pre-mRNAs. However, further information obtained by pulse-labeling of pre-mRNA in vivo in the presence of CAP for various times shows that even 30 minutes after addition of CAP a reduction of the processing rate is obtained. Based on these findings we conclude that maturation of mtRNAs is not only dependent on mitochondrial proteins, but also on a more direct interaction of the translation machinery and RNA processing whose nature is so far unknown.
Assuntos
DNA Fúngico/genética , DNA Mitocondrial/genética , Splicing de RNA , Ribossomos/metabolismo , Saccharomyces cerevisiae/genética , Transcrição Gênica/efeitos dos fármacos , Cloranfenicol/farmacologia , Enzimas de Restrição do DNA , Hibridização de Ácido Nucleico , Ribossomos/efeitos dos fármacos , Saccharomyces cerevisiae/efeitos dos fármacosRESUMO
Recognition of 5' splice points by group I and group II self-splicing introns involves the interaction of exon sequences--directly preceding the 5' splice site--with intronic sequence elements. We show here that the exon binding sequences (EBS) of group II intron aI5c can accept various substitutes of the authentic intron binding sites (IBS) provided in cis or in trans. The efficiency of cleavages at these cryptic 5' splice sites was enhanced by deletion of the authentic IBS2 element. All cryptic 5' cleavage sites studied here were preceded by an IBS1 like sequence; indicating that the IBS1/EBS1 pairing alone is sufficient for proper 5' splice site selection by the intronic EBS element. The results are discussed in terms of minimal requirements for 5' cleavages and position effects of IBS sites relative to the intron.
Assuntos
Íntrons , Splicing de RNA , RNA Mensageiro/genética , Éxons , Precursores de Ácido Nucleico/metabolismo , Plasmídeos , Sequências Reguladoras de Ácido Nucleico , Relação Estrutura-AtividadeRESUMO
GC clusters constitute the major repetitive elements in the mitochondrial (mt) genome of the yeast Saccharomyces cerevisiae. Many of these clusters are optional and thus contribute much to the polymorphism of yeast mtDNAs. We have made a systematic search for polymorphic sites by comparing mtDNA sequences of various yeast strains. Most of the 26 di- or polymorphic sites found differ by the presence or absence of a GC cluster of the majority class, here referred to as the M class, which terminate with an AGGAG motif. Comparison of sequences with and without the GC clusters reveal that elements of the subclasses M1 and M2 are inserted 3' to a TAG, flanked by A + T rich sequences. M3 elements, in contrast, only occur in tandem arrays of two to four GC clusters; they are consistently inserted 3' to the AGGAG terminal sequence of a preexisting cluster. The TAG or the terminal AGGAG, therefore, are regarded as being part of the target sites for M1 and M2 or M3 elements, respectively. The dinucleotide AG is in common to both target sites; it also occurs at the 3' terminus (AGGAG). This suggests its duplication during GC cluster insertion. This notion is supported by the observation that GC clusters of the minor classes G and V similarily repeat at their 3' terminus a GT or an AA dinucleotide, respectively, from their putative target sites.
Assuntos
Citidina/genética , DNA Fúngico/genética , DNA Mitocondrial/genética , Guanosina/genética , Saccharomyces cerevisiae/genética , Sequência de Bases , Mapeamento Cromossômico , Clonagem Molecular , DNA Fúngico/isolamento & purificação , DNA Mitocondrial/isolamento & purificação , Dados de Sequência Molecular , Polimorfismo Genético , Sequências Repetitivas de Ácido NucleicoRESUMO
We have previously shown that two CRE elements situated on a 31 bp region of the cauliflower mosaic virus (CaMV) 35S promoter activate gene expression in the yeast Saccharomyces cerevisiae and are regulated by cAMP. Studies with the yeast transcription factors GCN4, SKO1 and YAP1, which bind CRE-like sequences, showed no influence on expression of the 35S promoter indicating that a yet unknown factor is involved in activation. Band shift experiments with the 31 bp promoter region revealed binding of similar factors in yeast and plant protein extracts. In a previous study this promoter region was shown to confer tissue-specific expression in plants and to interact with the transcription factor TGA1. To test whether expression of TGA1 in yeast also stimulates transcription of the 35S promoter, we co-transformed yeast cells with a cDNA clone of this transcription factor and a 35S promoter/reporter gene construct. Promoter activity studies revealed that TGA1 confers enhanced expression of a reporter gene under the control of the 35S promoter in yeast cells. Yeast cells that were transformed with a 35S promoter construct that containing a mutated TGA1-binding site showed that both TGA1 and the intact binding site are necessary for this activation. These results suggest that stimulation of the 35S promoter by TGA1 is mediated by competition with an endogenous down-regulating yeast factor that is modulated by the nutritional state of the cells.