RESUMEN
The Ty5 retrotransposons of Saccharomyces cerevisiae integrate preferentially into regions of silent chromatin at the telomeres and silent mating loci (HMR and HML). We define a Ty5-encoded targeting domain that spans 6 amino acid residues near the C terminus of integrase (LXSSXP). The targeting domain establishes silent chromatin when it is tethered to a weakened HMR-E silencer, and it disrupts telomeric silencing when it is overexpressed. As determined by both yeast two-hybrid and in vitro binding assays, the targeting domain interacts with the C terminus of Sir4p, a structural component of silent chromatin. This interaction is abrogated by mutations in the targeting domain that disrupt integration into silent chromatin, suggesting that recognition of Sir4p by the targeting domain is the primary determinant in Ty5 target specificity.
Asunto(s)
Cromatina/genética , Proteínas Fúngicas/metabolismo , Silenciador del Gen , Integrasas/metabolismo , Retroelementos , Saccharomyces cerevisiae/genética , Proteínas Reguladoras de Información Silente de Saccharomyces cerevisiae , Secuencias de Aminoácidos , Sitios de Unión , Proteínas Fúngicas/genética , Genes Fúngicos , Integrasas/química , Mutagénesis Sitio-Dirigida , Telómero/genética , Transformación Genética , Técnicas del Sistema de Dos HíbridosRESUMEN
Retroelements (retrotransposons and retroviruses) have two genes in common: gag, which specifies structural proteins that form a virus or virus-like particle, and pol, which specifies catalytic proteins required for replication. For many retroelements, gag and pol are present on separate reading frames. Their expression is highly regulated, and the ratio of Gag to Pol is critical for retroelement replication. The Saccharomyces retrotransposon Ty5 contains a single open reading frame, and we characterized Gag and Pol expression by generating transpositionally active Ty5 elements with epitope tags at the N terminus or C terminus or within the integrase coding region. Immunoblot analysis identified two Gag species (Gag-p27 and Gag-p37), reverse transcriptase (Pol-p59), and integrase (Pol-p80), all of which are largely insoluble in the absence of urea or ionic detergent. These proteins result from proteolytic processing of a polyprotein, because elements with mutations in the presumed active site of Ty5 protease express a single tagged protein (Gag-Pol-p182). Protease mutants are also transpositionally inactive. In a time course experiment, we monitored protein expression, proteolytic processing, and transposition of a Ty5 element with identical epitope tags at its N and C termini. Both transposition and the abundance of Gag-p27 increased over time. In contrast, the levels of Gag-p37 and reverse transcriptase peaked after approximately 14 h of induction and then gradually decreased. This may be due to differences in stability of Gag-p27 relative to Gag-p37 and reverse transcriptase. The ratio of Ty5 Gag to Pol averaged 5:1 throughout the time course experiment, suggesting that differential protein stability regulates the amounts of these proteins.
Asunto(s)
Retroelementos/fisiología , Saccharomyces/genética , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Proteínas de Fusión gag-pol/genética , Proteínas de Fusión gag-pol/metabolismo , Immunoblotting , Procesamiento Proteico-Postraduccional , ARN Mensajero/metabolismo , Retroelementos/genética , Saccharomyces/metabolismoRESUMEN
Variegated plants have green- and white-sectored leaves. Cells in the green sectors contain morphologically normal chloroplasts, whereas cells in the white sectors contain non-pigmented plastids that lack organized lamellar structures. Many variegations are caused by mutations in nuclear genes that affect plastid function, yet in only a few cases have the responsible genes been cloned. We show that mutations in the nuclear VAR2 locus of Arabidopsis cause variegation due to loss of a chloroplast thylakoid membrane protein that bears similarity to the FtsH family of AAA proteins (ATPases associated with diverse cellular activities). Escherichia coli FtsH is a chaperone metalloprotease that functions in a number of diverse membrane-associated events. Although FtsH homologs have been identified in multicellular organisms, their functions and activities are largely unknown; we provide genetic in vivo evidence that VAR2 functions in thylakoid membrane biogenesis. We have isolated four var2 alleles and they have allowed us to define domains of the protein that are required for activity. These include two putative ATP-binding sites. VAR2 protein amounts generally correlate with the severity of the var2 mutant phenotype. One allele lacks detectable VAR2 protein, suggesting that the mechanism of var2 variegation involves the action of a redundant activity in the green sectors. We conclude that redundant activities may be a general mechanism to explain nuclear gene-induced plant variegations.
Asunto(s)
Arabidopsis/genética , Cloroplastos/enzimología , Proteínas de la Membrana/metabolismo , Mutación , Proteasas ATP-Dependientes , Alelos , Secuencia de Aminoácidos , Arabidopsis/enzimología , Arabidopsis/ultraestructura , Proteínas de Arabidopsis , Clonación Molecular , Datos de Secuencia Molecular , Hojas de la Planta/enzimología , Hojas de la Planta/ultraestructura , Homología de Secuencia de AminoácidoRESUMEN
Retrotransposon and retroviral insertions are not randomly distributed on chromosomes, suggesting that retroelements actively select integration sites. This is the case for the yeast Ty5 retrotransposons, which preferentially integrate into domains of silent chromatin at the HM loci and telomeres. Here we demonstrate that loss of Sir3p or Sir4p-components of silent chromatin-causes a greater than ninefold decrease in Ty5 targeting to the HM loci and largely randomizes chromosomal integration patterns. Strains with a deletion of SIR4 also display an approximately 10-fold increase in cDNA recombination, which is due both to the expression a- and alpha-mating-type information and the loss of Sir4p. It is known that in old yeast cells or in strains carrying the sir4-42 allele, the Sir complex relocalizes to the rDNA. About 26% of Ty5 insertions occur within the rDNA in sir4-42 strains compared with 3% in wild type. Ty5, therefore, is sensitive to changes in chromatin, indicating that retrotransposons may be useful for dissecting chromatin dynamics that occur during developmental programs such as aging.
Asunto(s)
Cromatina/genética , Retroelementos/genética , Saccharomyces cerevisiae/genética , Proteínas Reguladoras de Información Silente de Saccharomyces cerevisiae , Alelos , Secuencia de Bases , Mapeo Cromosómico , Cromosomas Fúngicos/genética , Cartilla de ADN/genética , ADN Complementario/genética , ADN de Hongos/genética , ADN Ribosómico/genética , Proteínas Fúngicas/genética , Eliminación de Gen , Genes Fúngicos , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa , Recombinación Genética , Transactivadores/genéticaRESUMEN
Retrotransposons and retroviruses replicate by reverse transcription of an mRNA intermediate. Most retroelements initiate reverse transcription from a host-encoded tRNA primer. DNA synthesis typically extends from the 3'-OH of the acceptor stem, which is complementary to sequences on the retroelement mRNA (the primer binding site, PBS). However, for some retrotransposons, including the yeast Ty5 elements, sequences in the anticodon stem-loop of the initiator methionine tRNA (IMT) are complementary to the PBS. We took advantage of the genetic tractability of the yeast system to investigate the mechanism of Ty5 priming. We found that transposition frequencies decreased at least 800-fold for mutations in the Ty5 PBS that disrupt complementarity with the IMT. Similarly, transposition was reduced at least 200-fold for IMT mutations in the anticodon stem-loop. Base pairing between the Ty5 PBS and IMT is essential for transposition, as compensatory changes that restored base pairing between the two mutant RNAs restored transposition significantly. An analysis of 12 imt mutants with base changes outside of the region of complementarity failed to identify other tRNA residues important for transposition. In addition, assays carried out with heterologous IMTs from Schizosaccharomyces pombe and Arabidopsis thaliana indicated that residues outside of the anticodon stem-loop have at most a fivefold effect on transposition. Our genetic system should make it possible to further define the components required for priming and to understand the mechanism by which Ty5's novel primer is generated.
Asunto(s)
Anticodón , ARN de Transferencia de Metionina/genética , Retroelementos , Saccharomyces cerevisiae/genética , Transcripción Genética , Secuencia de Aminoácidos , Secuencia de Bases , Prueba de Complementación Genética , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Mutación , Conformación de Ácido Nucleico , Plásmidos , ARN , ARN de Transferencia de Metionina/química , Schizosaccharomyces/genética , Homología de Secuencia de Ácido NucleicoRESUMEN
Nuclear gene-induced variegation mutants provide a powerful system to dissect interactions between the genetic systems of the nucleus-cytoplasm, the chloroplast, and the mitochondrion. The immutans (im) variegation mutation of Arabidopsis is nuclear and recessive and results in the production of green- and white-sectored leaves. The green sectors contain cells with normal chloroplasts, whereas the white sectors are heteroplastidic and contain cells with abnormal, pigment-deficient plastids as well as some normal chloroplasts. White sector formation can be promoted by enhanced light intensities, but sectoring becomes irreversible early in leaf development. The white sectors accumulate the carotenoid precursor phytoene. We have positionally cloned IM and found that the gene encodes a 40.5-kD protein with sequence motifs characteristic of alternative oxidase, a mitochondrial protein that functions as a terminal oxidase in the respiratory chains of all plants. However, phylogenetic analyses revealed that the IM protein is only distantly related to these other alternative oxidases, suggesting that IM is a novel member of this protein class. We sequenced three alleles of im, and all are predicted to be null. Our data suggest a model of variegation in which the IM protein functions early in chloroplast biogenesis as a component of a redox chain responsible for phytoene desaturation but that a redundant electron transfer function is capable of compensating for IM activity in some plastids and cells.
Asunto(s)
Proteínas de Arabidopsis , Arabidopsis/genética , Cloroplastos/genética , Proteínas Nucleares/genética , Oxidorreductasas/genética , Pigmentación/genética , Secuencia de Aminoácidos , Secuencia de Bases , Clonación Molecular , Mitocondrias/enzimología , Mitocondrias/genética , Datos de Secuencia Molecular , Mutación , Proteínas Nucleares/metabolismo , Filogenia , Hojas de la Planta/genética , Homología de Secuencia de Ácido NucleicoRESUMEN
The yeast retrotransposon Ty5 preferentially integrates into regions of silent chromatin. Ty5 cDNA also recombines with homologous sequences, generating tandem elements or elements that have exchanged markers between cDNA and substrate. In this study, we demonstrate that Ty5 integration depends upon the conserved DD(35)E domain of integrase and cis-acting sequences at the end of the long terminal repeat (LTR) implicated in integrase binding. cDNA recombination requires Rad52p, which is responsible for homologous recombination. Interestingly, Ty5 cDNA recombines at least three times more frequently with substrates in silent chromatin than with a control substrate at an internal chromosomal locus. This preference depends upon the Ty5 targeting domain that is responsible for integration specificity, suggesting that localization of cDNA to silent chromatin results in the enhanced recombination. Recombination with a telomeric substrate occasionally generates highly reiterated Ty5 arrays, and mechanisms for tandem element formation were explored by using a plasmid-based recombination assay. Point mutations were introduced into plasmid targets, and recombination products were characterized to determine recombination initiation sites. Despite our previous observation of the importance of the LTR in forming tandem elements, recombination cannot simply be explained by crossover events between the LTRs of substrate and cDNA. We propose an alternative model based on single-strand annealing, where single-stranded cDNA initiates tandem element formation and the LTR is required for strand displacement to form a looped intermediate. Retrotransposons are increasingly found associated with chromosome ends, and amplification of Ty5 by both integration and recombination exemplifies how retroelements can contribute to telomere dynamics.
Asunto(s)
Cromatina , Genes Fúngicos , Recombinación Genética , Retroelementos , Saccharomyces cerevisiae/genética , Secuencia de Aminoácidos , Intercambio Genético , ADN Complementario , Proteínas de Unión al ADN/metabolismo , Integrasas/metabolismo , Datos de Secuencia Molecular , Mutación , Proteína Recombinante y Reparadora de ADN Rad52 , Proteínas de Saccharomyces cerevisiae , Telómero , Secuencias Repetidas TerminalesRESUMEN
Many retrotransposons and retroviruses are thought to select integration sites through interactions with specific chromosomal proteins. In yeast, the Ty5 retrotransposon integrates preferentially with regions bound by silent chromatin, namely the telomeres and the HMR and HML mating loci. A Ty5 mutant (M3) was identified with an approximately 20-fold decrease in targeted integration as measured by a plasmid-based targeting assay. Often chromosomal insertions generated by M3, none were located at the telomeres or silent mating loci. A single amino acid change at the boundary of integrase and reverse transcriptase is responsible for the mutant phenotype. We predict that this mutation lies within a targeting domain that mediates Ty5 target choice by interacting with a component of silent chromatin.
Asunto(s)
Cromatina/genética , Cromosomas Fúngicos/genética , Retroelementos/genética , Saccharomyces cerevisiae/genética , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Elementos Transponibles de ADN/genética , Integrasas/genética , Datos de Secuencia Molecular , Mutagénesis Insercional/métodos , Mutación Puntual/genética , ADN Polimerasa Dirigida por ARN/genética , Recombinación Genética , Homología de Secuencia de AminoácidoRESUMEN
Tat1 was originally identified as an insertion near the Arabidopsis thaliana SAM1 gene. We provide evidence that Tat1 is a retrotransposon and that previously described insertions are solo long terminal repeats (LTRs) left behind after the deletion of coding regions of full-length elements. Three Tat1 insertions were characterized that have retrotransposon features, including a primer binding site complementary to an A. thaliana asparagine tRNA and an open reading frame (ORF) with approximately 44% amino acid sequence similarity to the gag protein of the Zea mays retrotransposon Zeon-1. Tat1 elements have large, polymorphic 3' noncoding regions that may contain transduced DNA sequences; a 477-base insertion in the 3' noncoding region of the Tat1-3 element contains part of a related retrotransposon and sequences similar to the nontranslated leader sequence of AT-P5C1, a gene for pyrroline-5-carboxylate reductase. Analysis of DNA sequences generated by the A. thaliana genome project identified 10 families of Ty3/gypsy retrotransposons, which share up to 51 and 62% amino-acid similarity to the ORFs of Tat1 and the A. thaliana Athila element, respectively. Phylogenetic analyses resolved the plant Ty3/gypsy elements into two lineages, one of which includes homologs of Tat1 and Athila. Four families of A. thaliana elements within the Tat/Athila lineage encode a conserved ORF after integrase at a position occupied by the envelope gene in retroviruses and in some insect Ty3/gypsy retrotransposons. Like retroviral envelope genes, this ORF encodes a transmembrane domain and, in some insertions, a putative secretory signal sequence. This suggests that Tat/Athila retrotransposons may produce enveloped virions and may be infectious.
Asunto(s)
Arabidopsis/genética , Genes de Plantas/genética , Proteínas de Plantas/genética , Retroelementos/genética , Retroviridae/genética , Homología de Secuencia de Aminoácido , Proteínas del Envoltorio Viral/genética , Secuencia de Aminoácidos , Secuencia de Bases , Sitios de Unión/genética , Secuencia Conservada , Cartilla de ADN/metabolismo , Dosificación de Gen , Genoma de Planta , Datos de Secuencia Molecular , Filogenia , Proteínas de Plantas/química , Secuencias Repetitivas de Ácidos Nucleicos , Homología de Secuencia de Ácido Nucleico , Proteínas del Envoltorio Viral/químicaRESUMEN
We conducted a genome-wide survey of Saccharomyces cerevisiae retrotransposons and identified a total of 331 insertions, including 217 Ty1, 34 Ty2, 41 Ty3, 32 Ty4, and 7 Ty5 elements. Eighty-five percent of insertions were solo long terminal repeats (LTRs) or LTR fragments. Overall, retrotransposon sequences constitute >377 kb or 3.1% of the genome. Independent evolution of retrotransposon sequences was evidenced by the identification of a single-base pair insertion/deletion that distinguishes the highly similar Ty1 and Ty2 LTRs and the identification of a distinct Ty1 subfamily (Ty1'). Whereas Ty1, Ty2, and Ty5 LTRs displayed a broad range of sequence diversity (typically ranging from 70%-99% identity), Ty3 and Ty4 LTRs were highly similar within each element family (most sharing >96% nucleotide identity). Therefore, Ty3 and Ty4 may be more recent additions to the S. cerevisiae genome and perhaps entered through horizontal transfer or past polyploidization events. Distribution of Ty elements is distinctly nonrandom: 90% of Ty1, 82% of Ty2, 95% of Ty3, and 88% of Ty4 insertions were found within 750 bases of tRNA genes or other genes transcribed by RNA polymerase III. tRNA genes are the principle determinant of retrotransposon distribution, and there is, on average, 1.2 insertions per tRNA gene. Evidence for recombination was found near many Ty elements, particularly those not associated with tRNA gene targets. For these insertions, 5'- and 3'-flanking sequences were often duplicated and rearranged among multiple chromosomes, indicating that recombination between retrotransposons can influence genome organization. S. cerevisiae offers the first opportunity to view organizational and evolutionary trends among retrotransposons at the genome level, and we hope our compiled data will serve as a starting point for further investigation and for comparison to other, more complex genomes.
Asunto(s)
Genoma Fúngico , Retroelementos/genética , Saccharomyces cerevisiae/genética , Secuencia de Bases , Mapeo Cromosómico , Cromosomas Fúngicos/genética , Secuencia de Consenso/genética , ADN de Hongos/análisis , Reordenamiento Génico , Familia de Multigenes , Sistemas de Lectura/genética , Análisis de Secuencia de ADNRESUMEN
The Saccharomyces retrotransposon Ty5 integrates preferentially into transcriptionally inactive regions (silent chromatin) at the HM loci and telomeres. We found that silent chromatin represses basal Ty5 transcription, indicating that these elements are encompassed by silent chromatin in their native genomic context. Because transcription is a requirement for transposition, integration into silent chromatin would appear to prevent subsequent rounds of replication. Using plasmid-borne Ty5-lacZ constructs, we found that Ty5 expression is haploid specific and is repressed 10-fold in diploid strains. Ty5 transcription is also regulated by the pheromone response pathway and is induced approximately 20-fold upon pheromone treatment. Deletion analysis of the Ty5 LTR promoter revealed that a 33 bp region with three perfect matches to the pheromone response element is responsible for both mating pheromone and cell-type regulation. Transcriptional repression of Ty5 by silent chromatin can be reversed by pheromone treatment, which leads to transcription and transposition. Ty5 replication, therefore, is normally repressed by silent chromatin and appears to be induced during mating. This is the first example of transcriptional activation of a gene that naturally resides within silent chromatin.
Asunto(s)
Cromatina/genética , Regulación Fúngica de la Expresión Génica , Feromonas/farmacología , Retroelementos/genética , Saccharomyces cerevisiae/genética , Secuencia de Bases , Diploidia , Haploidia , Factor de Apareamiento , Modelos Genéticos , Datos de Secuencia Molecular , Péptidos/farmacología , Secuencias Reguladoras de Ácidos Nucleicos , Transducción de Señal , Transcripción GenéticaRESUMEN
Retroelement cDNA can integrate into the genome using the element-encoded integrase or it can recombine with preexisting elements using the recombination system of the host. Recombination is a particularly important pathway for the yeast retrotransposon Ty5 and accounts for approximately 30% of the putative transposition events when a homologous substrate is carried on a plasmid and approximately 7% when the substrate is located at the chromosomal URA3 locus. Characterization of recombinants revealed that they are either simple replacements of the marker gene tandem elements. Using an assay system in which the donor element and recombination substrates are separated, we found that the long terminal repeats (LTRs) are critical for tandem element formation. LTR-containing substrates generate tandem elements at frequencies more than 10-fold higher than similarly sized internal Ty5 sequences. Internal sequences, however, facilitate tandem element formation when associated with an LTR, and there is a linear relationship between frequencies of tandem element formation and the length of LTR-containing substrates. We propose that recombination is initiated between the LTRs of the cDNA and substrate and that internal sequences promote tandem element formation by facilitating sequence alignment. Because of its location in subtelomeric regions, recombinational amplification of Ty5 may contribute to the organizations of chromosome ends.
Asunto(s)
Recombinación Genética , Secuencias Repetitivas de Ácidos Nucleicos , Retroelementos , Saccharomyces cerevisiae/genética , Cromosomas Fúngicos , ADN ComplementarioRESUMEN
We have developed a genetic means to recover sequences from YAC-ends near the yeast selectable marker URA3. This strategy is based on the ability of URA3 to complement mutations in pyrF, an Escherichia coli gene required for pyrimidine biosynthesis. We have developed an E.coli strain with a non-reverting allele of pyrF that is also suitable for cloning (recA-, hsdR-). We demonstrate the utility of this complementation strategy to obtain right-end clones from three YACs containing Arabidopsis thaliana DNA.
Asunto(s)
Cromosomas Artificiales de Levadura/química , Escherichia coli/genética , Proteínas Fúngicas/genética , Mutación , Clonación Molecular , Técnicas GenéticasRESUMEN
The HML and HMR mating loci of Saccharomyces cerevisiae are bound in silent chromatin, which is assembled at the flanking E and I transcriptional silencers. The retrotransposon Ty5 preferentially integrates into regions of silent chromatin, and Ty5 insertions near the HMR-E silencer account for approximately 2% of total transposition events. Most Ty5 insertions occur within 800 bp on either side of the autonomously replicating consensus sequence within HMR-E. Ty5 target preference is determined by silent chromatin, because integration near HMR-E is abolished in strains with silencer mutations that alleviate transcriptional repression. The recognition of specific DNA sequences per se does not direct integration, rather, it is the protein complex assembled at the silencers. As demonstrated here for Ty5, recognition of specific chromatin domains may be a general mechanism by which retrotransposons and retroviruses determine integration sites.
Asunto(s)
Cromatina/genética , Cromosomas Fúngicos/genética , Retroelementos/genética , Saccharomyces cerevisiae/genética , Mutación , Reacción en Cadena de la PolimerasaRESUMEN
Retrotransposons are ubiquitous components of eukaryotic genomes suggesting that they have played a significant role in genome organization. In Saccharomyces cerevisiae, eight of 10 endogenous insertions of the Ty5 retrotransposon family are located within 15 kb of chromosome ends, and two are located near the subtelomeric HMR locus. This genomic organization is the consequence of targeted transposition, as 14 of 15 newly transposed Ty5 elements map to telomeric regions on 10 different chromosomes. Nine of these insertions are within 0.8 kb and three are within 1.5 kb of the autonomously replicating consensus sequence in the subtelomeric X repeat. This suggests that the X repeat plays an important role in directing Ty5 integration. Analysis of endogenous insertions from S.cerevisiae and its close relative S.paradoxus revealed that only one of 12 insertions has target site duplications, indicating that recombination occurs between elements. This is further supported by the observation that Ty5 insertions mark boundaries of sequence duplications and rearrangements in these species. These data suggest that transposable elements like Ty5 can shape the organization of chromosome ends through both transposition and recombination.
Asunto(s)
Cromosomas Fúngicos/ultraestructura , Retroelementos/genética , Saccharomyces cerevisiae/genética , Secuencia de Bases , Mapeo Cromosómico , Secuencia de Consenso , Elementos Transponibles de ADN/genética , ADN de Hongos/química , ADN de Hongos/genética , Datos de Secuencia Molecular , Recombinación Genética , Secuencias Repetitivas de Ácidos Nucleicos , TelómeroRESUMEN
The nonrandom integration of retrotransposons and retroviruses suggests that chromatin influences target choice. Targeted integration, in turn, likely affects genome organization. In Saccharomyces, native Ty5 retrotransposons are located near telomeres and the silent mating locus HMR. To determine whether this distribution is a consequence of targeted integration, we isolated a transposition-competent Ty5 element from S. paradoxus, a species closely related to S. cerevisiae. This Ty5 element was used to develop a transposition assay in S. cerevisiae to investigate target preference of de novo transposition events. Of 87 independent Ty5 insertions, approximately 30% were located on chromosome III, indicating this small chromosome (approximately 1/40 of the yeast genome) is a highly preferred target. Mapping of the exact location of 19 chromosome III insertions showed that 18 were within or adjacent to transcriptional silencers flanking HML and HMR or the type X subtelomeric repeat. We predict Ty5 target preference is attributable to interactions between transposition intermediates and constituents of silent chromatin assembled at these sites. Ty5 target preference extends the link between telomere structure and reverse transcription as carried out by telomerase and Drosophila retrotransposons.
Asunto(s)
Cromatina/genética , Péptidos/genética , Retroelementos/genética , Saccharomyces/genética , Telómero/genética , Secuencia de Aminoácidos , Secuencia de Bases , Northern Blotting , Mapeo Cromosómico , Cromosomas Fúngicos , Regulación Fúngica de la Expresión Génica , Factor de Apareamiento , Modelos Genéticos , Datos de Secuencia Molecular , ARN de Hongos/análisis , ARN Mensajero/análisis , Secuencias Repetitivas de Ácidos Nucleicos , Saccharomyces cerevisiae/genética , Análisis de Secuencia de ADN , Homología de Secuencia de Aminoácido , Especificidad de la Especie , Transcripción GenéticaRESUMEN
DNA sequence analysis near the Arabidopsis thaliana ABI3 gene revealed the presence of a non-LTR retrotransposon insertion that we have designated Ta11-1. This insertion is 6.2 kb in length and encodes two overlapping reading frames with similarity to non-LTR retrotransposon proteins, including reverse transcriptase. A polymerase chain reaction assay was developed based on conserved amino acid sequences shared between the Ta11-1 reverse transcriptase and those of non-LTR retrotransposons from other species. Seventeen additional A. thaliana reverse transcriptases were identified that range in nucleotide similarity from 48-88% (Ta12-Ta28). Phylogenetic analyses indicated that the A. thaliana sequences are more closely related to each other than to elements from other organisms, consistent with the vertical evolution of these sequences over most of their evolutionary history. One sequence, Ta17, is located in the mitochondrial genome. The remaining are nuclear and of low copy number among 17 diverse A. thaliana ecotypes tested, suggesting that they are not highly active in transposition. The paucity of retrotransposons and the small genome size of A. thaliana support the hypothesis that most repetitive sequences have been lost from the genome and that mechanisms may exist to prevent amplification of extant element families.