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Jef D Boeke

Publications and source records attributed to Jef D Boeke.

At least 55 records · Page 3Linked to original sources
PubMed2003

DNA helicase gene interaction network defined using synthetic lethality analyzed by microarray.

We describe a new synthetic lethality analysis by microarray (SLAM) technique that uses approximately 4,600 Saccharomyces cerevisiae haploid deletion mutants with molecular 'bar codes' (TAGs). We used SGS1 and SRS2, two 3'-->5' DNA helicase genes, as 'queries' to identify their redundant and unique biological functions. We introduced these 'query mutations' into a haploid deletion pool by integrative transformation to disrupt the query gene in every cell, generating a double mutant pool. Optimization of integrative transformation efficiency was essential to the success of SLAM. Synthetic interactions defined a DNA helicase genetic network and predicted a role for SRS2 in processing damaged replication forks but, unlike SGS1, not in rDNA replication, DNA topology or lagging strand synthesis. SGS1 and SRS2 have synthetic defects with MRC1 but not RAD9, suggesting that SGS1 and SRS2 function in a parallel pathway with MRC1 to transduce the DNA replication stress signal to the general DNA damage checkpoint pathway. Both helicase genes have rad51-reversible synthetic defects with 5'-->3' DNA helicase RRM3, suggesting that RRM3 helps prevent formation of toxic recombination intermediates. SLAM detects synthetic lethality efficiently and ranks candidate genetic interactions, making it an especially useful method.

DNA Helicases↗
PubMed2003

Identifying related L1 retrotransposons by analyzing 3' transduced sequences.

BACKGROUND: A large fraction of the human genome is attributable to L1 retrotransposon sequences. Not only do L1s themselves make up a significant portion of the genome, but L1-encoded proteins are thought to be responsible for the transposition of other repetitive elements and processed pseudogenes. In addition, L1s can mobilize non-L1, 3'-flanking DNA in a process called 3' transduction. Using computational methods, we collected DNA sequences from the human genome for which we have high confidence of their mobilization through L1-mediated 3' transduction. RESULTS: The precursors of L1s with transduced sequence can often be identified, allowing us to reconstruct L1 element families in which a single parent L1 element begot many progeny L1s. Of the L1s exhibiting a sequence structure consistent with 3' transduction (L1 with transduction-derived sequence, L1-TD), the vast majority were located in duplicated regions of the genome and thus did not necessarily represent unique insertion events. Of the remaining L1-TDs, some lack a clear polyadenylation signal, but the alignment between the parent-progeny sequences nevertheless ends in an A-rich tract of DNA. CONCLUSIONS: Sequence data suggest that during the integration into the genome of RNA representing an L1-TD, reverse transcription may be primed internally at A-rich sequences that lie downstream of the L1 3' untranslated region. The occurrence of L1-mediated transduction in the human genome may be less frequent than previously thought, and an accurate estimate is confounded by the frequent occurrence of segmental genomic duplications.

Base Sequence↗
PubMed2003

Use of mixture models in a microarray-based screening procedure for detecting differentially represented yeast mutants.

We describe the use of a statistical model in a genome-wide microarray-based yeast genetic screen performed by imposing different genetic selections on thousands of yeast mutants in parallel. A mixture model is fitted to data obtained from oligonucleotide arrays hybridized to 20-mer oligonucleotide "barcodes'' and a procedure based on the fitted model is used to search for mutants differentially represented under experimental and control conditions. The fitted stochastic model provides a way to assess uncertainty. We demonstrate the usefulness of the model by applying it to the problem of screening for components of the nonhomologous end joining (NHEJ) pathway and identified known components of the NHEJ pathway.

Journal Article↗
PubMed2003

Short-chain fatty acid activation by acyl-coenzyme A synthetases requires SIR2 protein function in Salmonella enterica and Saccharomyces cerevisiae.

SIR2 proteins have NAD(+)-dependent histone deacetylase activity, but no metabolic role has been assigned to any of these proteins. In Salmonella enterica, SIR2 function was required for activity of the acetyl-CoA synthetase (Acs) enzyme. A greater than two orders of magnitude increase in the specific activity of Acs enzyme synthesized by a sirtuin-deficient strain was measured after treatment with homogeneous S. enterica SIR2 protein. Human SIR2A and yeast SIR2 proteins restored growth of SIR2-deficient S. enterica on acetate and propionate, suggesting that eukaryotic cells may also use SIR2 proteins to control the synthesis of acetyl-CoA by the level of acetylation of acetyl-CoA synthetases. Consistent with this idea, growth of a quintuple sir2 hst1 hst2 hst3 hst4 mutant strain of the yeast Saccharomyces cerevisiae on acetate or propionate was severely impaired. The data suggest that the Hst3 and Hst4 proteins are the most important for allowing growth on these short-chain fatty acids.

Acetate Kinase↗
PubMed2003

The unusual phylogenetic distribution of retrotransposons: a hypothesis.

Retrotransposons have proliferated extensively in eukaryotic lineages; the genomes of many animals and plants comprise 50% or more retrotransposon sequences by weight. There are several persuasive arguments that the enzymatic lynchpin of retrotransposon replication, reverse transcriptase (RT), is an ancient enzyme. Moreover, the direct progenitors of retrotransposons are thought to be mobile self-splicing introns that actively propagate themselves via reverse transcription, the group II introns, also known as retrointrons. Retrointrons are represented in modern genomes in very modest numbers, and thus far, only in certain eubacterial and organellar genomes. Archaeal genomes are nearly devoid of RT in any form. In this study, I propose a model to explain this unusual distribution, and rationalize it with the proposed ancient origin of the RT gene. A cap and tail hypothesis is proposed. By this hypothesis, the specialized terminal structures of eukaryotic mRNA provide the ideal molecular environment for the lengthening, evolution, and subsequent massive expansion of highly mobile retrotransposons, leading directly to the retrotransposon-cluttered structure that typifies modern metazoan genomes and the eventual emergence of retroviruses.

Animals↗
PubMed2003

Transcriptional interactions between yeast tRNA genes, flanking genes and Ty elements: a genomic point of view.

Retroelement insertion can alter the expression of nearby genes. The Saccharomyces cerevisiae retrotransposons Ty1-Ty4 are transcribed by RNA polymerase II (pol II) and target their integration upstream of genes transcribed by RNA polymerase III (pol III), mainly tRNA genes. Because tRNA genes can repress nearby pol II-transcribed genes, we hypothesized that transcriptional interference may exist between Ty1 insertions and pol III-transcribed genes, the preferred targets for Ty1 integration. Ty1s upstream of two pol III-transcribed genes (SNR6 and SUP2) were recovered and analyzed by RNA blot analysis. Ty1 insertions were found to exert a neutral or modest stimulatory effect on the expression of these genes. Further RNA analysis indicated a modest tRNA position effect on Ty1 transcription. To investigate the possible genomic relevance of these expression effects, we compiled a comprehensive tRNA gene database. This database allowed us to analyze a genome's worth of tRNA genes and Ty elements. It also enabled the prediction and experimental confirmation of tRNA gene position effects at native chromosomal loci. We provide evidence supporting the hypothesis that tRNA genes exert a modest inhibitory effect on adjacent pol II promoters. Direct analysis of PTR3 transcription, promoted by sequences very close to a tRNA gene, shows that this tRNA position effect can operate on a native chromosomal gene.

3' Flanking Region↗
PubMed2002

Human L1 element target-primed reverse transcription in vitro.

L1 elements are ubiquitous human transposons that replicate via an RNA intermediate. We have reconstituted the initial stages of L1 element transposition in vitro. The reaction requires only the L1 ORF2 protein, L1 3' RNA, a target DNA and appropriate buffer components. We detect branched molecules consisting of junctions between transposon 3' end cDNA and the target DNA, resulting from priming at a nick in the target DNA. 5' junctions of transposon cDNA and target DNA are also observed. The nicking and reverse transcription steps in the reaction can be uncoupled, as priming at pre-existing nicks and even double-strand breaks can occur. We find evidence for specific positioning of the L1 RNA with the ORF2 protein, probably mediated in part by the polyadenosine portion of L1 RNA. Polyguanosine, similar to a conserved region of the L1 3' UTR, potently inhibits L1 endonuclease (L1 EN) activity. L1 EN activity is also repressed in the context of the full-length ORF2 protein, but it and a second cryptic nuclease activity are released by ORF2p proteolysis. Additionally, heterologous RNA species such as Alu element RNA and L1 transcripts with 3' extensions are substrates for the reaction.

Base Sequence↗
PubMed2002

SIRT3, a human SIR2 homologue, is an NAD-dependent deacetylase localized to mitochondria.

The SIR2 (silent information regulator 2) gene family has diverse functions in yeast including gene silencing, DNA repair, cell-cycle progression, and chromosome fidelity in meiosis and aging. Human homologues, termed sirtuins, are highly conserved but are of unknown function. We previously identified a large imprinted gene domain on 11p15.5 and investigated the 11p15.5 sirtuin SIRT3. Although this gene was not imprinted, we found that it is localized to mitochondria, with a mitochondrial targeting signal within a unique N-terminal peptide sequence. The encoded protein was found also to possess NAD(+)-dependent histone deacetylase activity. These results suggest a previously unrecognized organelle for sirtuin function and that the role of SIRT3 in mitochondria involves protein deacetylation.

Amino Acid Sequence↗
PubMed2002

Molecular archeology of L1 insertions in the human genome.

BACKGROUND: As the rough draft of the human genome sequence nears a finished product and other genome-sequencing projects accumulate sequence data exponentially, bioinformatics is emerging as an important tool for studies of transposon biology. In particular, L1 elements exhibit a variety of sequence structures after insertion into the human genome that are amenable to computational analysis. We carried out a detailed analysis of the anatomy and distribution of L1 elements in the human genome using a new computer program, TSDfinder, designed to identify transposon boundaries precisely. RESULTS: Structural variants of L1 elements shared similar trends in the length and quality of their target site duplications (TSDs) and poly(A) tails. Furthermore, we found no correlation between the composition and genomic location of the pre-insertion locus and the resulting anatomy of the L1 insertion. We verified that L1 insertions with TSDs have the 5'-TTAAAA-3' cleavage site associated with L1 endonuclease activity. In addition, the second target DNA cut required for L1 insertion weakly matches the consensus pattern TTAAAA. On the other hand, the L1-internal breakpoints of deleted and inverted L1 elements do not resemble L1 endonuclease cleavage sites. Finally, the genome sequence data indicate that whereas singly inverted elements are common, doubly inverted elements are almost never found. CONCLUSIONS: The sequence data give no indication that the creation of L1 structural variants depends on characteristics of the insertion locus. In addition, the formation of 5' truncated and 5' inverted L1s are probably not due to the action of the L1 endonuclease.

Algorithms↗
PubMed2002

A core nucleosome surface crucial for transcriptional silencing.

Transcriptional silencing in yeast provides a genetically tractable system for analyzing the formation and maintenance of heterochromatin, a transcriptionally repressive chromatin structure found in all organisms. The nucleosome constitutes the central structure of chromatin and comprises two chains each of histones H2A, H2B, H3 and H4. The structure of the nucleosome consists of a central globular core surrounded by outwardly protruding amino-terminal histone tails. We show that a specific surface of the assembled nucleosome core is required for silencing in yeast. This surface is located at a H3/H4 histone-fold motif and contains amino-acid side chains located on the nucleosome disk surface and on an adjacent surface that interacts with DNA. The side chains, identified from mutants in which all three forms of silencing (rDNA, telomere and silent mating locus silencing) are eliminated, are centered around Lys79 of histone H3, a residue methylated by the yeast Dot1 protein. Moreover, mutations in the genes encoding H3 (HHT1 and HHT2) and H4 (HHF1 and HHF2) mapping to spatially adjacent amino-acid residues affected the three forms of silencing distinctly, suggesting that specific interactions mediate each form of silencing. Several of the mutations that we identified resemble those in a cluster of previously identified mutations affecting a distinct histone-fold motif elsewhere in the nucleosome core. These two clusters relieve distinct forms of transcriptional repression (silencing versus repression resulting from lack of Swi/Snf chromatin remodeling activity).

Amino Acid Sequence↗
PubMed2002

Human l1 retrotransposition is associated with genetic instability in vivo.

Retrotransposons have shaped eukaryotic genomes for millions of years. To analyze the consequences of human L1 retrotransposition, we developed a genetic system to recover many new L1 insertions in somatic cells. Forty-two de novo integrants were recovered that faithfully mimic many aspects of L1s that accumulated since the primate radiation. Their structures experimentally demonstrate an association between L1 retrotransposition and various forms of genetic instability. Numerous L1 element inversions, extra nucleotide insertions, exon deletions, a chromosomal inversion, and flanking sequence comobilization (called 5' transduction) were identified. In a striking number of integrants, short identical sequences were shared between the donor and the target site's 3' end, suggesting a mechanistic model that helps explain the structure of L1 insertions.

5' Flanking Region↗
PubMed2002

Functional profiling of the Saccharomyces cerevisiae genome.

Determining the effect of gene deletion is a fundamental approach to understanding gene function. Conventional genetic screens exhibit biases, and genes contributing to a phenotype are often missed. We systematically constructed a nearly complete collection of gene-deletion mutants (96% of annotated open reading frames, or ORFs) of the yeast Saccharomyces cerevisiae. DNA sequences dubbed 'molecular bar codes' uniquely identify each strain, enabling their growth to be analysed in parallel and the fitness contribution of each gene to be quantitatively assessed by hybridization to high-density oligonucleotide arrays. We show that previously known and new genes are necessary for optimal growth under six well-studied conditions: high salt, sorbitol, galactose, pH 8, minimal medium and nystatin treatment. Less than 7% of genes that exhibit a significant increase in messenger RNA expression are also required for optimal growth in four of the tested conditions. Our results validate the yeast gene-deletion collection as a valuable resource for functional genomics.

Cell Size↗
PubMed2002

Inhibition of reverse transcription in vivo by elevated manganese ion concentration.

Mutations in PMR1, a yeast gene encoding a calcium/manganese exporter, dramatically decrease Ty1 retrotransposition. Ty1 cDNA is reduced in pmr1 mutant cells, despite normal levels of Ty1 RNA and proteins. The transposition defect results from Mn(2+) accumulation that inhibits reverse transcription. Cytoplasmic accumulation of Mn(2+) in pmr1 cells may directly affect reverse transcriptase (RT) activity. Trace amounts of Mn(2+) potently inhibit Ty1 RT and HIV-1 RT in vitro when the preferred cation, Mg(2+), is present. Both Mn(2+) and Mg(2+) alone activate Ty1 RT cooperatively with Hill coefficients of 2, providing kinetic evidence for a dual divalent cation requirement at the RT active site. We propose that occupancy of the B site is the major determinant of catalytic activity and that Mn(2+) at this site greatly reduces catalytic activity.

Binding Sites↗
PubMed2002

Structure of a Sir2 enzyme bound to an acetylated p53 peptide.

Sir2 proteins are NAD(+)-dependent protein deacetylases that play key roles in transcriptional regulation, DNA repair, and life span regulation. The structure of an archaeal Sir2 enzyme, Sir2-Af2, bound to an acetylated p53 peptide reveals that the substrate binds in a cleft in the enzyme, forming an enzyme-substrate beta sheet with two flanking strands in Sir2-Af2. The acetyl-lysine inserts into a conserved hydrophobic tunnel that contains the active site histidine. Comparison with other structures of Sir2 enzymes suggests that the apoenzyme undergoes a conformational change upon substrate binding. Based on the Sir2-Af2 substrate complex structure, mutations were made in the other A. fulgidus sirtuin, Sir2-Af1, that increased its affinity for the p53 peptide.

Acetylation↗
PubMed2002

Telomeric and rDNA silencing in Saccharomyces cerevisiae are dependent on a nuclear NAD(+) salvage pathway.

The Sir2 protein is an NAD(+)-dependent protein deacetylase that is required for silencing at the silent mating-type loci, telomeres, and the ribosomal DNA (rDNA). Mutations in the NAD(+) salvage gene NPT1 weaken all three forms of silencing and also cause a reduction in the intracellular NAD(+) level. We now show that mutation of a highly conserved histidine residue in Npt1p results in a silencing defect, indicating that Npt1p enzymatic activity is required for silencing. Deletion of another NAD(+) salvage pathway gene called PNC1 caused a less severe silencing defect and did not significantly reduce the intracellular NAD(+) concentration. However, silencing in the absence of PNC1 was completely dependent on the import of nicotinic acid from the growth medium. Deletion of a gene in the de novo NAD(+) synthesis pathway BNA1 resulted in a significant rDNA silencing defect only on medium deficient in nicotinic acid, an NAD(+) precursor. By immunofluorescence microscopy, Myc-tagged Bna1p was localized throughout the whole cell in an asynchronously growing population. In contrast, Myc-tagged Npt1p was highly concentrated in the nucleus in approximately 40% of the cells, indicating that NAD(+) salvage occurs in the nucleus in a significant fraction of cells. We propose a model in which two components of the NAD(+) salvage pathway, Pnc1p and Npt1p, function together in recycling the nuclear nicotinamide generated by Sir2p deacetylase activity back into NAD(+).

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↗