Retroshuffling the genomic deck.
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Animals↗
Biomedical subjects
J D Boeke
Publications and source records attributed to J D Boeke.
The yeast retrotransposon Ty5 uses the anticodon stem-loop of the initiator methionine tRNA as a primer for reverse transcription.
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.
Amino Acid Sequence↗
The Schizosaccharomyces pombe hst4(+) gene is a SIR2 homologue with silencing and centromeric functions.
Although silencing is a significant form of transcriptional regulation, the functional and mechanistic limits of its conservation have not yet been established. We have identified the Schizosaccharomyces pombe hst4(+) gene as a member of the SIR2/HST silencing gene family that is defined in organisms ranging from bacteria to humans. hst4Delta mutants grow more slowly than wild-type cells and have abnormal morphology and fragmented DNA. Mutant strains show decreased silencing of reporter genes at both telomeres and centromeres. hst4(+) appears to be important for centromere function as well because mutants have elevated chromosome-loss rates and are sensitive to a microtubule-destabilizing drug. Consistent with a role in chromatin structure, Hst4p localizes to the nucleus and appears concentrated in the nucleolus. hst4Delta mutant phenotypes, including growth and silencing phenotypes, are similar to those of the Saccharomyces cerevisiae HSTs, and at a molecular level, hst4(+) is most similar to HST4. Furthermore, hst4(+) is a functional homologue of S. cerevisiae HST3 and HST4 in that overexpression of hst4(+) rescues the temperature-sensitivity and telomeric silencing defects of an hst3Delta hst4Delta double mutant. These results together demonstrate that a SIR-like silencing mechanism is conserved in the distantly related yeasts and is likely to be found in other organisms from prokaryotes to mammals.
Centromere↗
The conserved core of a human SIR2 homologue functions in yeast silencing.
Silencing is a universal form of transcriptional regulation in which regions of the genome are reversibly inactivated by changes in chromatin structure. Sir2 (Silent Information Regulator) protein is unique among the silencing factors in Saccharomyces cerevisiae because it silences the rDNA as well as the silent mating-type loci and telomeres. Discovery of a gene family of Homologues of Sir Two (HSTs) in organisms from bacteria to humans suggests that SIR2's silencing mechanism might be conserved. The Sir2 and Hst proteins share a core domain, which includes two diagnostic sequence motifs of unknown function as well as four cysteines of a putative zinc finger. We demonstrate by mutational analyses that the conserved core and each of its motifs are essential for Sir2p silencing. Chimeras between Sir2p and a human Sir2 homologue (hSir2Ap) indicate that this human protein's core can substitute for that of Sir2p, implicating the core as a silencing domain. Immunofluorescence studies reveal partially disrupted localization, accounting for the yeast-human chimeras' ability to function at only a subset of Sir2p's target loci. Together, these results support a model for the involvement of distinct Sir2p-containing complexes in HM/telomeric and rDNA silencing and that HST family members, including the widely expressed hSir2A, may perform evolutionarily conserved functions.
Amino Acid Sequence↗
A genetic screen for ribosomal DNA silencing defects identifies multiple DNA replication and chromatin-modulating factors.
Transcriptional silencing in Saccharomyces cerevisiae occurs at several genetic loci, including the ribosomal DNA (rDNA). Silencing at telomeres (telomere position effect [TPE]) and the cryptic mating-type loci (HML and HMR) depends on the silent information regulator genes, SIR1, SIR2, SIR3, and SIR4. However, silencing of polymerase II-transcribed reporter genes integrated within the rDNA locus (rDNA silencing) requires only SIR2. The mechanism of rDNA silencing is therefore distinct from TPE and HM silencing. Few genes other than SIR2 have so far been linked to the rDNA silencing process. To identify additional non-Sir factors that affect rDNA silencing, we performed a genetic screen designed to isolate mutations which alter the expression of reporter genes integrated within the rDNA. We isolated two classes of mutants: those with a loss of rDNA silencing (lrs) phenotype and those with an increased rDNA silencing (irs) phenotype. Using transposon mutagenesis, lrs mutants were found in 11 different genes, and irs mutants were found in 22 different genes. Surprisingly, we did not isolate any genes involved in rRNA transcription. Instead, multiple genes associated with DNA replication and modulation of chromatin structure were isolated. We describe these two gene classes, and two previously uncharacterized genes, LRS4 and IRS4. Further characterization of the lrs and irs mutants revealed that many had alterations in rDNA chromatin structure. Several lrs mutants, including those in the cdc17 and rfc1 genes, caused lengthened telomeres, consistent with the hypothesis that telomere length modulates rDNA silencing. Mutations in the HDB (RPD3) histone deacetylase complex paradoxically increased rDNA silencing by a SIR2-dependent, SIR3-independent mechanism. Mutations in rpd3 also restored mating competence selectively to sir3Delta MATalpha strains, suggesting restoration of silencing at HMR in a sir3 mutant background.
Chromatin↗
Targeting of human retrotransposon integration is directed by the specificity of the L1 endonuclease for regions of unusual DNA structure.
L1 elements are polyA retrotransposons which inhabit the human genome. Recent work has defined an endonuclease (L1 EN) encoded by the L1 element required for retrotransposition. We report the sequence specificity of this nicking endonuclease and the physical basis of its DNA recognition. L1 endonuclease is specific for the unusual DNA structural features found at the TpA junction of 5'(dTn-dAn) x 5'(dTn-dAn) tracts. Within the context of this sequence, substitutions which generate a pyrimidine-purine junction are tolerated, whereas purine-pyrimidine junctions greatly reduce or eliminate nicking activity. The A-tract conformation of the DNA substrate 5' of the nicked site is required for L1 EN nicking. Chemical or physical unwinding of the DNA helix enhances L1 endonuclease activity, while disruption of the adenine mobility associated with TpA junctions reduces it. Akin to the protein-DNA interactions of DNase I, L1 endonuclease DNA recognition is likely mediated by minor groove interactions. Unlike several of its homologues, however, L1 EN exhibits no AP endonuclease activity. Finally, we speculate on the implications of the specificity of the L1 endonuclease for the parasitic relationship between retroelements and the human genome.
Base Sequence↗
Libraries of green fluorescent protein fusions generated by transposition in vitro.
Two artificial transposons have been constructed that carry a gene encoding Green Fluorescent Protein and can be used for generating libraries of GFP fusions in a gene of interest. One such element, AT2GFP, can be used to generate GFP insertions in frame with the amino acid sequence of the protein of interest, with a stop codon at the end of the GFP coding sequence; AT2GFP also contains a selectable marker that confers trimethoprim resistance in bacteria. The second element, GS, can be used to generate tribrid GFP fusions because there is no stop codon in the GFP transposon, and the resulting fusion proteins contain the entire amino acid sequence encoded by the gene. The GS element consists of a gfp open reading frame and a supF amber suppressor tRNA gene; the supF portion of the GS transposon can be utilized as a selectable marker in bacteria. Its sequence contains a fortuitous open reading frame, and thus it can be translated continuously with the gfp amino acid sequence. As a target for GFP insertions, we used a plasmid carrying the native Ty1 retrotransposon of the yeast Sacharomyces cerevisiae. The resulting multiple GFP fusions to Ty1 capsid protein Gag and Ty1 integrase were useful in determining the cellular localization of these proteins. Libraries of GFP fusions generated by transposition in vitro represent a novel and potentially powerful method to study the cell distribution and cellular localization signals of proteins.
Animals↗
A hotspot for the Drosophila gypsy retroelement in the ovo locus.
The Drosophila retroelement gypsy has a number of unusual features including an unusual LTR terminal sequence and an apparent target sequence preference. The ovo locus is a known hotspot for gypsy insertion. We examined the target sequence preference of gypsy within ovo by isolating 26 new insertions and sequencing the gypsy/ovo junctions. Insertions were found at multiple sites within the ovo locus. The insertions clustered within an approximately 150 bp region in the non-translated region of the ovo beta transcript, with most insertions falling within the first intron. There were seven sites of insertion within this region and these mostly conform to the consensus sequence YRYRYR (where Y = pyrimidine and R = purine). However, this target sequence is at best necessary but not sufficient to specify a hotspot, as there were several other sequences conforming to this consensus in the ovo locus that were not hit. The results indicate that gypsy may have a higher degree of target specificity than most infectious LTR retroelements.
Animals↗
Yeast retrotransposons: finding a nice quiet neighborhood.
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Retroelements↗
Skipper, an LTR retrotransposon of Dictyostelium.
The complete sequence of a retrotransposon from Dictyostelium discoideum , named skipper , was obtained from cDNA and genomic clones. The sequence of a nearly full-length skipper cDNA was similar to that of three other partially sequenced cDNAs. The corresponding retrotransposon is represented in approximately 15-20 copies and is abundantly transcribed. Skipper contains three open reading frames (ORFs) with an unusual sequence organization, aspects of which resemble certain mammalian retroviruses. ORFs 1 and 3 correspond to gag and pol genes; the second ORF, pro, corresponding to protease, was separated from gag by a single stop codon followed shortly thereafter by a potential pseudoknot. ORF3 (pol) was separated from pro by a +1 frameshift. ORFs 2 and 3 overlapped by 32 bp. The computed amino acid sequences of the skipper ORFs contain regions resembling retrotransposon polyprotein domains, including a nucleic acid binding protein, aspartyl protease, reverse transcriptase and integrase. Skipper is the first example of a retrotransposon with a separate pro gene. Skipper is also novel in that it appears to use stop codon suppression rather than frameshifting to modulate pro expression. Finally, skipper and its components may provide useful tools for the genetic characterization of Dictyostelium.
Amino Acid Sequence↗
Genetic selection of intragenic suppressor mutations that reverse the effect of common p53 cancer mutations.
Several lines of evidence suggest that the presence of the wild-type tumor suppressor gene p53 in human cancers correlates well with successful anti-cancer therapy. Restoration of wild-type p53 function to cancer cells that have lost it might therefore improve treatment outcomes. Using a systematic yeast genetic approach, we selected second-site suppressor mutations that can overcome the deleterious effects of common p53 cancer mutations in human cells. We identified several suppressor mutations for the V143A, G245S and R249S cancer mutations. The beneficial effects of these suppressor mutations were demonstrated using mammalian reporter gene and apoptosis assays. Further experiments showed that these suppressor mutations could override additional p53 cancer mutations. The mechanisms of such suppressor mutations can be elucidated by structural studies, ultimately leading to a framework for the discovery of small molecules able to stabilize p53 mutants.
Animals↗
Retrotransposon R1Bm endonuclease cleaves the target sequence.
The R1Bm element, found in the silkworm Bombyx mori, is a member of a group of widely distributed retrotransposons that lack long terminal repeats. Some of these elements are highly sequence-specific and others, like the human L1 sequence, are less so. The majority of R1Bm elements are associated with ribosomal DNA (rDNA). R1Bm inserts into 28S rDNA at a specific sequence; after insertion it is flanked by a specific 14-bp target site duplication of the 28S rDNA. The basis for this sequence specificity is unknown. We show that R1Bm encodes an enzyme related to the endonuclease found in the human L1 retrotransposon and also to the apurinic/apyrimidinic endonucleases. We expressed and purified the enzyme from bacteria and showed that it cleaves in vitro precisely at the positions in rDNA corresponding to the boundaries of the 14-bp target site duplication. We conclude that the function of the retrotransposon endonucleases is to define and cleave target site DNA.
Amino Acid Sequence↗
Designer deletion strains derived from Saccharomyces cerevisiae S288C: a useful set of strains and plasmids for PCR-mediated gene disruption and other applications.
A set of yeast strains based on Saccharomyces cerevisiae S288C in which commonly used selectable marker genes are deleted by design based on the yeast genome sequence has been constructed and analysed. These strains minimize or eliminate the homology to the corresponding marker genes in commonly used vectors without significantly affecting adjacent gene expression. Because the homology between commonly used auxotrophic marker gene segments and genomic sequences has been largely or completely abolished, these strains will also reduce plasmid integration events which can interfere with a wide variety of molecular genetic applications. We also report the construction of new members of the pRS400 series of vectors, containing the kanMX, ADE2 and MET15 genes.
Alleles↗
Moloney murine leukemia virus protease expressed in bacteria is enzymatically active.
Replication of Moloney murine leukemia virus requires a readthrough translation mechanism to generate the Gag-Pol polyprotein. One of the final products of this polyprotein is the protease (PR), which is required to generate the mature virion proteins. The assembly of Gag and Gag-Pol polyprotein into a virion followed by activation of the viral protease is necessary to produce a mature, infectious particle. These events are believed to occur near the cell membrane just prior to the budding of the virion. We report here the autoproteolytic activity of the viral PR when a Gag-PR fusion protein is expressed in E. coli. Efficient cleavage at the p12/CA, CA/NC and NC/PR junctions was observed. Thus the Moloney murine leukemia virus PR is capable of cleaving its substrates in the absence of specific host factors.
Endopeptidases↗
Intronic snoRNA biosynthesis in Saccharomyces cerevisiae depends on the lariat-debranching enzyme: intron length effects and activity of a precursor snoRNA.
The eukaryotic small nucleolar RNAs (snoRNAs) are involved in processing of pre-rRNA and modification of rRNA nucleotides. Some snoRNAs are derived from mono- or polycistronic transcription units, whereas others are encoded in introns of protein genes. The present study addresses the role of the RNA lariat-debranching enzyme (Dbr1p) in the synthesis and function of intronic snoRNAs in the yeast Saccharomyces cerevisiae. Intronic snoRNA production was determined to depend on Dbr1p. Accumulation of mature intronic snoRNAs is reduced in a dbr1 mutant; instead, intronic snoRNAs are "trapped" within host intron lariats. Interestingly, the extent of intronic snoRNA accumulation in the form of lariats in dbr1 cells varied among different intronic snoRNAs. Intronic snoRNAs encoded within shorter introns, such as U24 and snR38, accumulate more unprocessed lariat precursors than those encoded within longer introns, e.g., U18 and snR39. This correlation was corroborated by experiments conducted with model intron:U24 snoRNA constructs. These results support a splicing-dependent exonucleolytic pathway for the biosynthesis of intronic snoRNAs. Curiously, U24 in a lariat may be functional in directing methylation of ribosomal RNA.
Animals↗
Distribution of a limited Sir2 protein pool regulates the strength of yeast rDNA silencing and is modulated by Sir4p.
Transcriptional silencing in Saccharomyces cerevisiae occurs at the silent mating-type loci HML and HMR, at telomeres, and at the ribosomal DNA (rDNA) locus RDN1. Silencing in the rDNA occurs by a novel mechanism that depends on a single Silent Information Regulator (SIR) gene, SIR2. SIR4, essential for other silenced loci, paradoxically inhibits rDNA silencing. In this study, we elucidate a regulatory mechanism for rDNA silencing based on the finding that rDNA silencing strength directly correlates with cellular Sir2 protein levels. The endogenous level of Sir2p was shown to be limiting for rDNA silencing. Furthermore, small changes in Sir2p levels altered rDNA silencing strength. In rDNA silencing phenotypes, sir2 mutations were shown to be epistatic to sir4 mutations, indicating that SIR4 inhibition of rDNA silencing is mediated through SIR2. Furthermore, rDNA silencing is insensitive to SIR3 overexpression, but is severely reduced by overexpression of full-length Sir4p or a fragment of Sir4p that interacts with Sir2p. This negative effect of SIR4 overexpression was overridden by co-overexpression of SIR2, suggesting that SIR4 directly inhibits the rDNA silencing function of SIR2. Finally, genetic manipulations of SIR4 previously shown to promote extended life span also resulted in enhanced rDNA silencing. We propose a simple model in which telomeres act as regulators of rDNA silencing by competing for limiting amounts of Sir2 protein.
DNA, Ribosomal↗
Transposable elements and genome organization: a comprehensive survey of retrotransposons revealed by the complete Saccharomyces cerevisiae genome sequence.
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.
Base Sequence↗
Schizosaccharomyces pombe retrotransposon Tf2 mobilizes primarily through homologous cDNA recombination.
The Tf2 retrotransposon, found in the fission yeast Schizosaccharomyces pombe, is nearly identical to its sister element, Tf1, in its reverse transcriptase-RNase H and integrase domains but is very divergent in the gag domain, the protease, the 5' untranslated region, and the U3 domain of the long terminal repeats. It has now been demonstrated that a neo-marked copy of Tf2 overexpressed from a heterologous promoter can mobilize into the S. pombe genome and produce true transposition events. However, the Tf2-neo mobilization frequency is 10- to 20-fold lower than that of Tf1-neo, and 70% of the Tf2-neo events are homologous recombination events generated independently of a functional Tf2 integrase. Thus, the Tf2 element is primarily dependent on homologous recombination with preexisting copies of Tf2 for its propagation. Finally, production of Tf2-neo proteins and cDNA was also analyzed; surprisingly, Tf2 was found to produce its reverse transcriptase as a single species in which it is fused to protease, unlike all other retroviruses and retrotransposons.
Cloning, Molecular↗