RESUMEN
Excision by transposons is associated with chromosome breaks; generally, host-cell proteins repair this damage, often introducing mutations. Many transposons also use host proteins in the transposition mechanism or in regulation. Transposition in systems lacking host factors that influence the behaviour of these transpositions is useful in determining what those factors are and how they work. In addition, features of transposition and regulation intrinsic to the element itself can be determined. Maize Activator/Dissociation (Ac/Ds) elements transpose in a wide variety of heterologous plants, but their characteristics in these other systems differ from those in maize, including their response to increasing genetic dosage and the types of repair products recovered following excision. Two Arabidopsis thaliana mutants (iae1 and iae2) show increased Ac transposition frequencies. These mutants, and the differences mentioned above, suggest the involvement of host proteins in Ac/Ds activity and potential differences between these proteins among plant species. Here we report that Ac/Ds elements, members of the hAT (hobo, Ac, Tam3) superfamily, transpose in the yeast Saccharomyces cerevisiae, an organism lacking class II ('cut and paste') transposons. This demonstrates that plant-specific proteins are not essential for Ac/Ds transposition. The yeast system is valuable for dissecting the Ac/Ds transposition mechanism and identifying host factors that can influence transposition and the repair of DNA damage induced by Ac/Ds. Mutations caused by Ds excision in yeast suggest formation of a DNA-hairpin intermediate, and reinsertions occur throughout the genome with a frequency similar to that in plants. The high proportion of Ac/Ds reinsertions also makes this system an in vivo mutagenesis and reverse genetics tool in yeast and, presumably, other eukaryotic systems.
Asunto(s)
Elementos Transponibles de ADN , Saccharomyces cerevisiae/genética , Zea mays/genética , Secuencia de Bases , Reparación del ADN , Datos de Secuencia Molecular , Mutagénesis Insercional , Conformación de Ácido Nucleico , Oligodesoxirribonucleótidos , Plásmidos , Saccharomyces cerevisiae/enzimología , Zea mays/enzimologíaRESUMEN
Patterns of transposable element activity often provide useful information about how and when organisms regulate gene expression. The maize lowered Ac/Ds germinal reversion 1 (LAG1)-O mutation causes unusually low rates of germinal reversion by Ac/Ds-induced alleles even though these same alleles revert frequently and early in somatic development. LAG1-O suppresses Ds transposition at multiple, unlinked loci, and does not affect Spm elements, indicating that the mutation acts in trans and may be specific to Ac/Ds elements. Our data suggest that LAG1-O suppression gradually reduces Ac/Ds activity in the meristem and newly formed leaves until, by the floral transition, transposition is undetectable even with PCR-based assays. This suppression persists during tassel development and does not appear to be released until some point after meiosis. Competitive RT-PCR results show no difference in Ac transposase mRNA levels between LAG1-O and lag1(+) tassels, suggesting that suppression is post-transcriptional. The pattern of LAG1-O expression is consistent with a model in which at least some gene expression specific to those meristem cells that will ultimately give rise to floral tissue and therefore gametes begins very early in plant development, and then persists throughout development.
Asunto(s)
Elementos Transponibles de ADN/genética , Zea mays/genética , Linaje de la Célula , Expresión Génica , Genes de Plantas , Marcadores Genéticos , Germinación/genética , Mutación , Fenotipo , Transposasas/biosíntesisRESUMEN
Interest in the use of low-copy nuclear genes for phylogenetic analyses of plants has grown rapidly, because highly repetitive genes such as those commonly used are limited in number. Furthermore, because low-copy genes are subject to different evolutionary processes than are plastid genes or highly repetitive nuclear markers, they provide a valuable source of independent phylogenetic evidence. The gene for granule-bound starch synthase (GBSSI or waxy) exists in a single copy in nearly all plants examined so far. Our study of GBSSI had three parts: (1) Amino acid sequences were compared across a broad taxonomic range, including grasses, four dicotyledons, and the microbial homologs of GBSSI. Inferred structural information was used to aid in the alignment of these very divergent sequences. The informed alignments highlight amino acids that are conserved across all sequences, and demonstrate that structural motifs can be highly conserved in spite of marked divergence in amino acid sequence. (2) Maximum-likelihood (ML) analyses were used to examine exon sequence evolution throughout grasses. Differences in probabilities among substitution types and marked among-site rate variation contributed to the observed pattern of variation. Of the parameters examined in our set of likelihood models, the inclusion of among-site rate variation following a gamma distribution caused the greatest improvement in likelihood score. (3) We performed cladistic parsimony analyses of GBSSI sequences throughout grasses, within tribes, and within genera to examine the phylogenetic utility of the gene. Introns provide useful information among very closely related species, but quickly become difficult to align among more divergent taxa. Exons are variable enough to provide extensive resolution within the family, but with low bootstrap support. The combined results of amino acid sequence comparisons, maximum-likelihood analyses, and phylogenetic studies underscore factors that might affect phylogenetic reconstruction. In this case, accommodation of the variable rate of evolution among sites might be the first step in maximizing the phylogenetic utility of GBSSI.
Asunto(s)
Evolución Molecular , Filogenia , Poaceae/enzimología , Poaceae/genética , Estructura Secundaria de Proteína , Almidón Sintasa/química , Almidón Sintasa/genética , Secuencia de Aminoácidos , Animales , Bacterias/enzimología , Bacterias/genética , Secuencia de Bases , Gránulos Citoplasmáticos/enzimología , ADN de Plantas/química , ADN de Plantas/genética , Intrones , Mamíferos/genética , Modelos Moleculares , Datos de Secuencia Molecular , Fosforilasas/química , Fosforilasas/genética , Poaceae/clasificación , Reacción en Cadena de la Polimerasa , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Homología de Secuencia de Ácido NucleicoRESUMEN
Genetic fine structure analysis of the maize wx locus has determined that the ratio of genetic to physical distance within wx was one to two orders of magnitude higher than the average for the maize genome. Similar results have been found at other maize loci. In this study, we examined several mechanisms that could account for this pattern. First, crossovers in two other maize genes resolve preferentially at specific sites. By mapping exchanges between wx-B1 and wx-I relative to a polymorphic SstI site, we found no evidence for such a hotspot at wx. Second, deletion of promoter sequences from wx alleles had little effect on recombination frequencies, in contrast to results in yeast where promoter sequences are important for initiating recombination in some genes. Third, high levels of insertion polymorphism may suppress intergenic recombination. However, the presence of a 2-kb Ds element 470 bp upstream of the wx transcription start site did not further suppress recombination between Ds insertions in nearby wx sequences. Thus, none of these mechanisms is sufficient to explain the difference between intergenic and intragenic recombination rates at wx.
Asunto(s)
Proteínas de Plantas/genética , Recombinación Genética , Almidón Sintasa/genética , Zea mays/genética , Intercambio Genético , Mutagénesis Insercional , Polimorfismo Genético , Regiones Promotoras Genéticas , Eliminación de SecuenciaRESUMEN
Ac/Ds transposable elements often leave short DNA rearrangements, or 'footprints,' at the sites where they excise. Previous studies at the maize waxy (wx) gene suggest that the DNA repair that forms transposon footprints is not random. Each excision site consistently displays a different, predominant repair product suggesting flanking DNA may influence footprint formation. We have expanded these studies to show that predominant end-joining products also form in association with Ac/Ds excision in Arabidopsis and that chromosomal location of the Ac-containing construct does not appear to influence this repair. The predominant repair product is identical in both maize and Arabidopsis for Ac elements with the same adjacent DNA sequences. However, a broader range of minor footprint types is observed in Arabidopsis, including footprints that are rare in maize, suggesting potential differences in the host proteins involved in either transposition, repair or both. The data also suggest that the sequences influencing footprint formation are within 39 bp 5' and 18 bp 3' of the transposon. These studies demonstrate that transgenic Ac/Ds-containing plants will be useful tools in dissecting plant DNA repair processes.
Asunto(s)
Arabidopsis/genética , Reparación del ADN , Elementos Transponibles de ADN , Zea mays/genética , Secuencia de Bases , Mapeo Cromosómico , Huella de ADN , ADN de Plantas/química , ADN de Plantas/genética , Conformación de Ácido NucleicoRESUMEN
Plants form their gametes late in somatic development and, as a result, often pass somatic mutations on to their progeny. Classic examples of this process are the germinal revertants of unstable, Ac/Ds transposon-induced kernel mutations in maize: frequent and early reversion events during somatic development are generally correlated with a high frequency of revertant gametes. We have characterized a Ds allele of the maize waxy (wx) gene, wx-m5:CS7, for which the correlation between somatic and germinal reversion frequencies no longer holds. The ability of wx-m5:CS7 (CS7) to produce revertant gametes is suppressed approximately 100-fold in comparison with a second Ds allele, wx-m5:CS8 (CS8), which has an identical insertion at Wx and the same frequent and early somatic reversion pattern in endosperm. The excision of Ds from wx is not reduced 100-fold in the somatic tissues of CS7 plants as compared with CS8 plants. Suppressed formation of CS7 revertant gametes is independent of the Ac transposase source and is heritably passed to the embryos of progeny kernels; however, frequent and early somatic reversion is observed again in endosperms of these progeny kernels. This suppression appears to be caused by a dominant mutation in a trans-acting product that can suppress the germinal reversion of other Ds-induced alleles as well; the mutation is tightly linked to Wx but is not in the CS7 Ds itself. Taken together, the data suggest a novel mode of developmental control of Ac/Ds elements by the host plant, suppressing element excision in the shoot meristem.
Asunto(s)
Proteínas de Plantas/genética , Almidón Sintasa/genética , Zea mays/genética , Alelos , Clonación Molecular , Elementos Transponibles de ADN , ADN de Plantas/análisis , ADN de Plantas/genética , Regulación de la Expresión Génica de las Plantas , Biblioteca de Genes , Genes de Plantas , Células Germinativas/crecimiento & desarrollo , Mutagénesis Insercional , Reacción en Cadena de la Polimerasa , Eliminación de Secuencia , Transposasas/metabolismo , Zea mays/crecimiento & desarrolloRESUMEN
Mobile elements transposing via DNA intermediates often leave small rearrangements, or "transposon footprints," at sites where they excise. Each excision event leaves its own footprint and, at any given site, these vary in size and sequence. Footprint formation involves DNA repair of sequences flanking the element. We have analyzed the footprints formed by a 2-kb Ds element excising from six different sites in exons of the maize waxy (Wx) gene. We find that groups of footprints left at individual sites are surprisingly nonrandom; different excision products predominate consistently at each site. Less frequent footprints left by each insertion appear related to the predominant type. The data suggest that flanking sequences affect the DNA repair processes associated with element excision. Two models have been proposed to explain footprint formation, one featuring a 5' exonuclease and the other featuring hairpin loop formation and an endonuclease. Our data have interesting implications for both these models. Evidence is also presented to support the presence of a separate excision mechanism that can remove Ac/Ds elements without leaving any footprint and that operates in parallel with the footprint-forming mechanism.
Asunto(s)
Reparación del ADN/genética , Elementos Transponibles de ADN , Zea mays/genética , Alelos , Secuencia de Bases , Clonación Molecular , Huella de ADN , Cartilla de ADN/genética , ADN de Plantas/genética , Exonucleasas/metabolismo , Genes de Plantas , Modelos Genéticos , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa , Zea mays/metabolismoRESUMEN
The transposable Dissociation (Ds) element of maize was first discovered as a site of high-frequency chromosome breakage. Because both Ds-mediated breakage and transposition require the presence of the Activator (Ac) element, it has been suggested that chromosome breakage may be the outcome of an aberrant transposition event. This idea is consistent with the finding that only complex structures containing multiple Ds or Ac and Ds elements have been correlated with chromosome breakage. In this report, we describe two chromosome-breaking maize alleles that contain pairs of closely linked but separate Ds elements inserted at the Waxy locus. A polymerase chain reaction assay was utilized to isolate intermediates in the breakage process. The DNA sequence of these intermediates reveals deletions and base pair changes consistent with transposon footprints that may represent the junctions between fused sister chromatids. These results provide direct molecular evidence that chromosome breakage is the result of aberrant transposition events.
Asunto(s)
Cromosomas , Elementos Transponibles de ADN , Zea mays/genética , Alelos , Secuencia de Bases , Clonación Molecular , ADN , Modelos Genéticos , Datos de Secuencia Molecular , Reacción en Cadena de la PolimerasaRESUMEN
The molecular basis for the unusual phenotype conditioned by the waxy(Wx)-m5 Ds allele has been elucidated. Unlike most Ds alleles, Wx-m5 is phenotypically wild-type in the absence of Ac. We find that the Wx-m5 gene contains a 2-kb Ds element at -470 relative to the start of Wx transcription, representing the most 5' insertion of any transposable element allele characterized to date in plants. Despite its wild type phenotype, Wx-m5 has reduced levels of Wx enzymatic activity indicating that Ds insertion influences Wx gene expression. In the presence of Ac, Wx-m5 kernels have sectors of null expression on a wild-type background and give rise to stable wx and unstable wx-m germinal derivatives. Seventeen of 20 derivatives examined are wx-m alleles and at least 15 of these appear to result from intragenic transposition of Ds from -470 to new sites within the Wx gene. Three wx-m alleles contain two Ds elements, one at -470 and a second in Wx coding sequences. Surprisingly, only 3 out of 20 derivatives are stable wx mutants and these have sustained gross rearrangements of Wx and flanking sequences. For most other maize transposable element alleles somatic sectors and germinal derivatives usually arise following element excision or deletions of element sequences. In contrast, element insertion following intragenic transposition is apparently responsible for most of the somatic sectors and germinal derivatives of Wx-m5.
Asunto(s)
Elementos Transponibles de ADN/genética , Proteínas de Plantas/genética , Zea mays/genética , Alelos , Secuencia de Bases , Southern Blotting , Regulación de la Expresión Génica/genética , Datos de Secuencia Molecular , FenotipoRESUMEN
The DNA sequences encoding component C of methyl coenzyme M reductase (mcr genes) in Methanothermus fervidus, Methanobacterium thermoautotrophicum, Methanococcus vannielii, and Methanosarcina barkeri have been published. Comparisons of transcription initiation and termination sites and of the amino acid sequences of the mcr gene products are presented. Structural features conserved within the amino acid sequences are identified and a comparison of methyl reductase with other disulfide bond synthesizing enzymes is presented.
Asunto(s)
Euryarchaeota/genética , Genes Bacterianos , Oxidorreductasas/genética , Secuencia de Aminoácidos , Secuencia de Bases , ADN Bacteriano/genética , Euryarchaeota/enzimología , Datos de Secuencia Molecular , Especificidad de la Especie , Transcripción GenéticaRESUMEN
A 6-kilobase-pair (kbp) region of the genome of the extremely thermophilic arachaebacterium Methanothermus fervidus which encodes the alpha, beta, and gamma subunit polypeptides of component C of methyl coenzyme M reductase was cloned and sequenced. Genes encoding the beta (mcrB) and gamma (mcrG) subunits were separated by two open reading frames (designated mcrC and mcrD) which encode unknown gene products. The M. fervidus genes were preceded by ribosome-binding sites, separated by short A + T-rich intergenic regions, contained unexpectedly few NNC codons, and exhibited inflexible codon usage at some locations. Sites of transcription initiation and termination flanking the mcrBDCGA cluster of genes in M. fervidus were identified. The sequences of the genes, the encoded polypeptides, and transcription regulatory signals in M. fervidus were compared with the functionally equivalent sequences from two mesophilic methanogens (Methanococcus vannielii and Methanosarcina barkeri) and from a moderate thermophile (Methanobacterium thermoautotrophicum Marburg). The amino acid sequences of the polypeptides encoded by the mcrBCGA genes in the two thermophiles were approximately 80% identical, whereas all other pairs of these gene products contained between 50 and 60% identical amino acid residues. The mcrD gene products have diverged more than the products of the other mcr genes. Identification of highly conserved regions within mcrA and mcrB suggested oligonucleotide sequences which might be developed as hybridization probes which could be used for identifying and quantifying all methanogens.
Asunto(s)
Euryarchaeota/genética , Oxidorreductasas/genética , Secuencia de Aminoácidos , Secuencia de Bases , Clonación Molecular , Codón , Genes Bacterianos , Calor , Datos de Secuencia Molecular , Mapeo Restrictivo , Regiones Terminadoras Genéticas , Transcripción GenéticaRESUMEN
A restriction fragment of Methanococcus thermolithotrophicus genomic DNA was cloned into pUC8 to produce plasmid pET9301, which complements mutations in the hisA gene of Escherichia coli. Sequencing the DNA (2,155 base pairs) cloned from this thermophilic methanogen demonstrated that the M. thermolithotrophicus hisA gene is located within a cluster of open reading frames (ORFs) and is 68 and 69% homologous at the nucleotide level to the hisA genes of the mesophilic methanococci M. voltae and M. vannielii, respectively. The ORF (ORF 206) immediately 5' to the hisA gene of M. thermolithotrophicus is partially deleted in the genomes of the two mesophilic species, whereas ORF 114, which is 5' to ORF 206, is conserved in all three species.
Asunto(s)
ADN Bacteriano/análisis , Euryarchaeota/genética , Genes Bacterianos , Secuencia de Aminoácidos , Archaea/genética , Secuencia de Bases , Clonación Molecular , Escherichia coli/genética , Histidina/genética , Datos de Secuencia Molecular , Mutación , Plásmidos , Secuencias Repetitivas de Ácidos Nucleicos , Homología de Secuencia de Ácido Nucleico , Transformación BacterianaRESUMEN
We have complemented the riboB2 mutation of Aspergillus nidulans by transformation with a plasmid library of wild-type (wt) sequences. We have isolated, by marker rescue from a riboB+ transformant, a plasmid that complements riboB2 efficiently. From this plasmid we have subcloned an A. nidulans sequence that complements riboB2 efficiently and that integrates by homologous recombination at a site closely linked to the riboB locus. We conclude that this sequence contains the wt riboB+ allele.
Asunto(s)
Aspergillus nidulans/genética , Riboflavina/metabolismo , Mapeo Cromosómico , Clonación Molecular , Enzimas de Restricción del ADN , Genes , Genes Fúngicos , Prueba de Complementación Genética , Mutación , Recombinación GenéticaRESUMEN
We identified four mutations in two previously undescribed loci involved in microtubule function in Aspergillus nidulans as extragenic suppressors of benA33, a heat-sensitive beta-tubulin mutation. Three of the four mutations map to a locus closely linked to riboB on linkage group VIII; we designated this locus mipA (for microtubule-interacting protein). We were not able to map the remaining suppressor because of chromosomal rearrangements. However, since it recombines with riboB at a significantly higher frequency than the mipA alleles, it is unlikely to be in mipA; thus, we designated it mipB1. The mip mutations are not allelic to the previously identified loci that encode alpha- and beta-tubulin, and it is likely that mipA and mipB encode previously unidentified nontubulin proteins involved in microtubule function. Each of the mip mutations suppresses the heat sensitivity conferred by benA33 and suppresses the blockage of nuclear division and movement conferred by this mutation at high temperatures. Interactions between mipA and benA are allele specific. All of the mipA mutations are cryptic in a wild-type benA background but cause cold sensitivity in combination with benA33. These mutations also confer cold sensitivity in combination with benA31 and benA32 and reduce the resistance conferred by these mutations to the antimicrotubule agent benomyl but do not suppress the heat sensitivity conferred by these alleles. Finally, the mipA alleles suppress the heat sensitivity conferred by benA11, benA17, and benA21 but do not confer cold sensitivity in combination with these alleles.