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At least 433 records · Page 24Linked to original sources

PubMed1996

Enhancement of frame-shift mutation by the overproduction of msDNA in Escherichia coli.

A minor population of wild Escherichia coli strains contain retroelements called retrons, which produce a peculiar satellite DNA, multicopy single-stranded DNA (msDNA). It has been reported that mismatched base pairs in the secondary structure formed in msDNA are mutagenic in E. coli[Maas et al.(1994) Mol.Microbiol. 14,437-441; Maas et al. (1996) Mol. Microbiol, 19, 505-509]. We reexamined this proposal by converting mismatched base pairs to matched base pairs using a single msDNA species, msDNA-Ec86, or by deleting mismatched regions using msDNA-Ec73. We also examined the effect of reverse transcriptases (RT) without msDNA production on mutagenesis. All the constructs are under the lpp/lac promoter-operator control so that their mutagenic effects can be tested in the absence and the presence of a lac inducer. It was found that when the production of msDNA-Ec86 or Ec73 was induced, reversion frequencies from Lac- to Lac+ significantly increased in the case of a Lac- mutation caused by a frame-shift mutation, but much less by a substitution mutation. The removal of mismatched base pairs eliminated the high mutation frequencies, and the inducible expression of RT alone was not mutagenic. These results are consistent with the hypothesis of Maas and his associates that mismatched base pairs in msDNA sequester a cellular mismatch repair system, resulting in the increase of frame-shift mutations.

Base Composition↗

PubMed1999

The distribution of rates of spontaneous mutation over viruses, prokaryotes, and eukaryotes.

Although mutation has chaotic aspects, spontaneous mutation rates assume certain characteristic values when expressed per genome per genome duplication. The rate among lytic RNA viruses is roughly 1, while the rate among retroelements is roughly 0.2. The rate among viral and cellular microbes with DNA chromosomes is close to 0.0034. Mutation rates among higher eukaryotes, estimated from specific-locus studies, vary greatly. Most of this variation can be suppressed if the rates are expressed per cell division instead of per sexual generation, and if the genome size is taken to be only a little larger than the sum of the protein-encoding sequences; then, the mutation rate is roughly 0.01. The reasons for different characteristic mutation rates among different organism groups remain mysterious and pose a substantial challenge to students of evolution.

Animals↗

PubMed1996

UVB irradiation upregulation of the Drosophila 1731 retrotransposon LTR requires the same short sequence of U3 region in a human epithelial cell line as in Drosophila cells.

Phylogenetic analysis of the retrotransposon and retrovirus suggests an evolutionary relationship between them and indicates that transactivation of the long terminal repeat (LTR)-containing retroelements could be ubiquitous. Using constructs expressing a reporter gene under the control of the entire or deleted LTR of 1731, which is a retrotransposable element of Drosophila melanogaster, we were able to show that the UVB-irradiation activation of the 1731-LTR requires the same short sequence of U3 region in a human epithelial cell line as in Schneider's Drosophila cell line (S2). This sequence is similar to the binding sequence of the members of the nuclear factor-kappa B (NF-kappa B)/rel family. In addition, human colonic carcinoma cells (HT29), in response to UVB-irradiation, produce some extracellular factor(s) that activates the 1731-LTR in nonirradiated cells.

Animals↗

PubMed2003

ARGONAUTE4 control of locus-specific siRNA accumulation and DNA and histone methylation.

Proteins of the ARGONAUTE family are important in diverse posttranscriptional RNA-mediated gene-silencing systems as well as in transcriptional gene silencing in Drosophila and fission yeast and in programmed DNA elimination in Tetrahymena. We cloned ARGONAUTE4 (AGO4) from a screen for mutants that suppress silencing of the Arabidopsis SUPERMAN (SUP) gene. The ago4-1 mutant reactivated silent SUP alleles and decreased CpNpG and asymmetric DNA methylation as well as histone H3 lysine-9 methylation. In addition, ago4-1 blocked histone and DNA methylation and the accumulation of 25-nucleotide small interfering RNAs (siRNAs) that correspond to the retroelement AtSN1. These results suggest that AGO4 and long siRNAs direct chromatin modifications, including histone methylation and non-CpG DNA methylation.

Alleles↗

PubMed2004

RNA branching and debranching in the yeast retrovirus-like element Ty1.

Ty elements of Saccharomyces cerevisiae are long terminal repeat (LTR) retroelements related to retroviruses. Normal levels of Ty1 transposition require Dbr1p, a cellular enzyme that cleaves 2'-5' RNA bonds. We show that Ty1 RNAs lacking identifiable 5' ends accumulate in virus-like particles (VLPs) in dbr1 mutants. Debranching this RNA in vitro with Dbr1p creates an uncapped version of the normal Ty1 RNA 5' end. We show that the 5' nucleotide (nt) of Ty1 RNA forms a 2'-5' bond with a nt near the 3' end of the same RNA, creating a lariat. The properties of the lariat suggest it forms by a novel mechanism and that branching and debranching may play roles in Ty1 reverse transcription at the minus-strand transfer step.

Base Pairing↗

PubMed2005

Comparative genomics of trypanosomatid parasitic protozoa.

A comparison of gene content and genome architecture of Trypanosoma brucei, Trypanosoma cruzi, and Leishmania major, three related pathogens with different life cycles and disease pathology, revealed a conserved core proteome of about 6200 genes in large syntenic polycistronic gene clusters. Many species-specific genes, especially large surface antigen families, occur at nonsyntenic chromosome-internal and subtelomeric regions. Retroelements, structural RNAs, and gene family expansion are often associated with syntenic discontinuities that-along with gene divergence, acquisition and loss, and rearrangement within the syntenic regions-have shaped the genomes of each parasite. Contrary to recent reports, our analyses reveal no evidence that these species are descended from an ancestor that contained a photosynthetic endosymbiont.

Animals↗

PubMed1996

Nested retrotransposons in the intergenic regions of the maize genome.

The relative organization of genes and repetitive DNAs in complex eukaryotic genomes is not well understood. Diagnostic sequencing indicated that a 280-kilobase region containing the maize Adh1-F and u22 genes is composed primarily of retrotransposons inserted within each other. Ten retroelement families were discovered, with reiteration frequencies ranging from 10 to 30,000 copies per haploid genome. These retrotransposons accounted for more than 60 percent of the Adh1-F region and at least 50 percent of the nuclear DNA of maize. These elements were largely intact and are dispersed throughout the gene-containing regions of the maize genome.

Chromosomes, Artificial, Yeast↗

PubMed1995

Requirement of RNA polymerase III transcription factors for in vitro position-specific integration of a retroviruslike element.

The yeast retroviruslike element Ty3 inserts at the transcription initiation sites of genes transcribed by RNA polymerase III (Pol III). An in vitro integration assay was developed with the use of Ty3 viruslike particles and a modified SUP2 tyrosine transfer RNA (tRNA(Tyr)) gene target. Integration was position-specific and required Ty3 integrase, Pol III transcription factor (TF) IIIB-, TFIIIC-, and Pol III-containing fractions showed that TFIIIB and TFIIIC, together, were sufficient for position-specific Ty3 integration, but not for transcription. This report demonstrates that in vitro integration of a retroelement can be targeted by cellular proteins.

DNA Nucleotidyltransferases↗

PubMed2006

Cooperation between reverse transcriptase and integrase during reverse transcription and formation of the preintegrative complex of Ty1.

Reverse transcriptase (RT) and integrase (IN) play a central role in the replication and transposition of retroelements. Increasing evidence suggests that the interaction between these two enzymes is functional and plays an important role in replication. In the yeast Saccharomyces cerevisiae retrotransposon Ty1, the interaction of IN with RT is critical for the formation of an active conformation of RT. We show here that the RT associated with VLPs is active only if it is in close interaction with IN. To probe the IN-RT cis-trans relationship, we have used a complementation assay based on coexpressing two transposons. We show that IN acts in cis to activate RT and that a functional integrase provided in trans is not able to complement replication and transposition defects of IN deletion or IN active-site mutant elements. Our data support a model in which IN not only interacts closely with RT during reverse transcription but also remains associated with RT during the formation of the preintegrative complex.

Binding Sites↗

PubMed1998

Proteolytic processing and assembly of gag and gag-pol proteins of TED, a baculovirus-associated retrotransposon of the gypsy family.

TED (transposable element D) is an env-containing member of the gypsy family of retrotransposons that represents a possible retrovirus of invertebrates. This lepidopteran (moth) retroelement contains gag and pol genes that encode proteins capable of forming viruslike particles (VLP) with reverse transcriptase. Since VLP are likely intermediates in TED transposition, we investigated the roles of gag and pol in TED capsid assembly and maturation. By using constructed baculovirus vectors and TED Gag-specific antiserum, we show that the principal translation product of gag (Pr55(gag)) is cleaved to produce a single VLP structural protein, p37(gag). Replacement of Asp436 within the retrovirus-like active site of the pol-encoded protease (PR) abolished Pr55(gag) cleavage and demonstrated the requirement for PR in capsid processing. As shown by expression of an in-frame fusion of TED gag and pol, PR is derived from the Gag-Pol polyprotein Pr195(gag-pol). The PR cleavage site within Pr55(gag) was mapped to a position near the junction of a basic, nucleocapsid-like domain and a C-terminal acidic domain. Once released by cleavage, the C-terminal fragment was not detected. This acidic fragment was dispensable for VLP assembly, as demonstrated by the formation of VLP by C-terminal Pr55(gag) truncation proteins and replacement of the acidic domain with a heterologous protein. In contrast, C-terminal deletions that extended into the adjacent nucleocapsid-like domain of Pr55(gag) abolished VLP recovery and demonstrated that this central region contributes to VLP assembly or stability, or both. Collectively, these data suggest that the single TED protein p37(gag) provides both capsid and nucleocapsid functions. TED may therefore use a simple processing strategy for VLP assembly and genome packaging.

Amino Acid Sequence↗

PubMed2000

Novel mouse type D endogenous proviruses and ETn elements share long terminal repeat and internal sequences.

The repetitive ETn (early transposon) family of sequences represents an active "mobile mutagen" in the mouse genome. The presence of long terminal repeats (LTRs) and other diagnostic features indicate that ETns are retrotransposons but they contain no long open reading frames or documented similarity to the genes of known retroviruses or other retroelements. Thus, the mechanisms responsible for the mobility of this family have been unknown. In this study, we used computer searches to detect a small region of previously unrecognized type D retroviral pol homology within ETn elements. This small region was used to isolate two mouse endogenous proviral elements with gag, pro, and pol genes similar to simian type D viruses. This new family of mouse endogenous proviruses, termed MusD, is present in several hundred copies in the genome. Interestingly, the MusD LTRs, 3' internal region, and the 5' region expected to contain the packaging signal are very closely related to members of the ETn subfamily that have recently transposed. Analysis of different mouse strains indicates that MusD elements predate the existence of the mobile subfamily of ETns. These findings indicate that the ETn family was likely created via recombination events resulting in a near complete substitution of MusD coding sequences with unrelated DNA. Furthermore, these results suggest that ETn transcripts retrotranspose using proteins provided by MusD proviruses.

Amino Acid Sequence↗

PubMed2002

A nucleocapsid functionality contained within the amino terminus of the Ty1 protease that is distinct and separable from proteolytic activity.

Ty1 is the most successful of the five endogenous yeast retrotransposons. The life cycle of Ty1 dictates that a number of nucleocapsid (NC)-facilitated events occur although the protein(s) responsible for these events has not been identified. The positioning of the NC peptide is conserved at the carboxy terminus of the Gag protein among most long terminal repeat (LTR)-containing retroelements. An analogous region of Ty1 that simultaneously encodes part of Gag, protease (PR), and the C-terminal p4 peptide was mutagenized. Some of these mutations result in smaller-than-normal virus-like particles (VLPs). The mutants were also found to impair an NC-like functionality contained within the amino terminus of the protease that is distinct and separable from its proteolytic activity. Remarkably, these mutants have distinct defects in reverse transcription.

Endopeptidases↗

PubMed1996

The retrotransposon Tf1 assembles virus-like particles that contain excess Gag relative to integrase because of a regulated degradation process.

The retrotransposon Tf1, isolated from Schizosaccharomyces pombe, contains a single open reading frame with sequences encoding Gag, protease, reverse transcriptase, and integrase (IN). Tf1 has previously been shown to possess significant transposition activity. Although Tf1 proteins do assemble into virus-like particles, the assembly does not require readthrough of a translational reading frame shift or stop codon, common mechanisms used by retroelements to express Gag in molar excess of the polymerase proteins. This study was designed to determine if Tf1 particles contain equal amounts of Gag and polymerase proteins or whether they contain the typical molar excess of Gag. After using two separate methods to calibrate the strength of our antibodies, we found that both S. pombe extracts and partially purified Tf1 particles contained a 26-fold molar excess of Gag relative to IN. Knowing that Gag and IN are derived from the same Tf1 primary translation product, we concluded that the excess Gag most likely resulted from specific degradation of IN. We obtained evidence of regulated IN degradation in comparisons of Tf1 protein extracted from log-phase cells and that extracted from stationary-phase cells. The log-phase cells contained equal molar amounts of Gag and IN, whereas cells approaching stationary phase rapidly degraded IN, leaving an excess of Gag. Analysis of the reverse transcripts indicated that the bulk of reverse transcription occurred within the particles that possess a molar excess of Gag.

Base Sequence↗

PubMed1998

Yeast Ty1 retrotransposition is stimulated by a synergistic interaction between mutations in chromatin assembly factor I and histone regulatory proteins.

A screen for host mutations which increase the rate of transposition of Ty1 and Ty2 into a chromosomal target was used to identify factors influencing retroelement transposition. The fortuitous presence of a mutation in the CAC3 gene in the strain in which this screen was undertaken enabled us to discover that double mutaions of cac3 and hir3, but neither of the two single mutations, caused a dramatic increase in the rate of retrotransposition. We further showed that this effect was not due to an increase in the overall level of Ty1 mRNA. Two subtle cac3 phenotypes, slight methyl methanesulfonate (MMS) sensitivity and reduction of telomeric silencing, were significantly enhanced in the cac3 hir3 double mutant. In addition, the growth rate of the double mutant was reduced. HIR3 belongs to a class of HIR genes that regulate the transcription of histones, while Cac3p, together with Cac1p and Cac2p, forms chromatin assembly factor I. Other combinations of mutations in cac and hir genes (cac3 hir1, cac3 hir2, and cac2 hir3) also increase Ty transposition and MMS sensitivity and reduce the growth rate. A model explaining the synergistic interaction between cac and hir mutations in terms of alterations in chromatin structure is proposed.

Alleles↗

PubMed1999

cDNA of the yeast retrotransposon Ty5 preferentially recombines with substrates in silent chromatin.

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.

Amino Acid Sequence↗

PubMed1999

Involvement of DNA end-binding protein Ku in Ty element retrotransposition.

Saccharomyces cerevisiae Ty elements are retrotransposons whose life cycles are strikingly similar to those of retroviruses. They transpose via an RNA intermediate that is converted to linear double-stranded cDNA and then inserted into the host genome. Although Ty integration is mediated by the element-encoded integrase, it has been proposed that host factors are involved in this process. Here, we show that the DNA end-binding protein Ku, which functions in DNA double-strand break repair, potentiates retrotransposition. Specifically, by using a galactose-inducible Ty1 system, we found that in vivo, Ty1 retrotransposition rates were substantially reduced in the absence of Ku. In contrast, this phenotype was not observed with yeast strains containing mutations in other genes that are involved in DNA repair. We present evidence that Ku associates with Ty1 viruslike particles both in vitro and in vivo. These results provide an additional role for Ku and suggest that it might function in the life cycles of retroelements in other systems.

Antigens, Nuclear↗

PubMed2000

Multiple homing pathways used by yeast mitochondrial group II introns.

The yeast mitochondrial DNA group II introns aI1 and aI2 are retroelements that insert site specifically into intronless alleles by a process called homing. Here, we used patterns of flanking marker coconversion in crosses with wild-type and mutant aI2 introns to distinguish three coexisting homing pathways: two that were reverse transcriptase (RT) dependent (retrohoming) and one that was RT independent. All three pathways are initiated by cleavage of the recipient DNA target site by the intron-encoded endonuclease, with the sense strand cleaved by partial or complete reverse splicing, and the antisense strand cleaved by the intron-encoded protein. The major retrohoming pathway in standard crosses leads to insertion of the intron with unidirectional coconversion of upstream exon sequences. This pattern of coconversion suggests that the major retrohoming pathway is initiated by target DNA-primed reverse transcription of the reverse-spliced intron RNA and completed by double-strand break repair (DSBR) recombination with the donor allele. The RT-independent pathway leads to insertion of the intron with bidirectional coconversion and presumably occurs by a conventional DSBR recombination mechanism initiated by cleavage of the recipient DNA target site by the intron-encoded endonuclease, as for group I intron homing. Finally, some mutant DNA target sites shift up to 43% of retrohoming to another pathway not previously detected for aI2 in which there is no coconversion of flanking exon sequences. This new pathway presumably involves synthesis of a full-length cDNA copy of the inserted intron RNA, with completion by a repair process independent of homologous recombination, as found for the Lactococcus lactis Ll.LtrB intron. Our results show that group II intron mobility can occur by multiple pathways, the ratios of which depend on the characteristics of both the intron and the DNA target site. This remarkable flexibility enables group II introns to use different recombination and repair enzymes in different host cells.

Base Sequence↗

PubMed2004

Targeted nuclear import of open reading frame 1 protein is required for in vivo retrotransposition of a telomere-specific non-long terminal repeat retrotransposon, SART1.

Non-long terminal repeat (non-LTR) retrotransposons, most of which carry two open reading frames (ORFs), are abundant mobile elements that are distributed widely among eukaryotes. ORF2 encodes enzymatic domains, such as reverse transcriptase, that are conserved in all retroelements, but the functional roles of ORF1 in vivo are little understood. We show with green fluorescent protein-ORF1 fusion proteins that the ORF1 proteins of SART1, a telomeric repeat-specific non-LTR retrotransposon in Bombyx mori, are transported into the nucleus to produce a dotted localization pattern. Nuclear localization signals N1 (RRKR) and N2 (PSKRGRG) at the N terminus and a highly basic region in the center of SART1 ORF1 are involved in nuclear import and the dotted localization pattern in the nucleus, respectively. An in vivo retrotransposition assay clarified that at least three ORF1 domains, N1/N2, the central basic domain, and CCHC zinc fingers are required for SART1 retrotransposition. The nuclear import activity of SART1 ORF1 makes it clear that the ORF1 proteins of non-LTR retrotransposons work mainly in the nucleus, in contrast to the cytoplasmic action of Gag proteins of LTR elements. The functional domains found here in SART1 ORF1 will be useful for developing a more efficient and target-specific LINE-based gene delivery vector.

Amino Acid Sequence↗