Jef D. Boeke

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.

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.

5-Fluoroorotic acid as a selective agent in yeast molecular genetics.

5-FOA is an extremely useful reagent for the selection of Ura- cells amid a population of Ura+ cells. The selection is effective in transformation and recombination studies where loss of URA3+ is desired. A new plasmid shuffling procedure based on the 5-FOAR selection permits the recovery of conditional lethal mutations in cloned genes that encode vital functions.

In vitro mutagenesis and plasmid shuffling : from cloned gene to mutant yeast

Publisher Summary This chapter describes the In Vitro mutagenesis and plasmid shuffling in yeast genes. method for generating mutant alleles uses replicating yeast episomes as a means of exchanging the wild-type gene for mutant copies. The basic scheme for the exchange, known as plasmid shuffling In the first step, one copy of the gene of interest is inactivated in a diploid, and a wild-type copy is propagated in the cell on an episome. This allows the generation of a haploid strain with a chromosomal null allele. Mutagenized copies of the gene are then introduced into this cell on a second episome and exchanged (or shuffled) with the wild-type version. Removal of the wild-type gene, YFG in our example, is the key step in any plasmid shuflting scheme. This can be accomplished by taking advantage of two factors. First, even relatively stable YCp episomes are lost from a cell by missegregation or misrepfication at a rate of 10 –2 per generation. Second, compounds are available that prevent the growth of cells carrying specific yeast genes, and in the presence of such compounds these genes act as counterselectable markers, allowing one to directly select for cells which have lost this marker. By including one of these counterselectable markers on the same plasmid that contains the wild-type YFG gene, an investigator can select for cells that have lost the entire plasmid.

Transcriptional disruption by the L1 retrotransposon and implications for mammalian transcriptomes

LINE-1 (L1) elements are the most abundant autonomous retrotransposons in the human genome, accounting for about 17% of human DNA. The L1 retrotransposon encodes two proteins, open reading frame (ORF)1 and the ORF2 endonuclease/reverse transcriptase. L1 RNA and ORF2 protein are difficult to detect in mammalian cells, even in the context of overexpression systems. Here we show that inserting L1 sequences on a transcript significantly decreases RNA expression and therefore protein expression. This decreased RNA concentration does not result from major effects on the transcription initiation rate or RNA stability. Rather, the poor L1 expression is primarily due to inadequate transcriptional elongation. Because L1 is an abundant and broadly distributed mobile element, the inhibition of transcriptional elongation by L1 might profoundly affect expression of endogenous human genes. We propose a model in which L1 affects gene expression genome-wide by acting as a ‘molecular rheostat’ of target genes. Bioinformatic data are consistent with the hypothesis that L1 can serve as an evolutionary fine-tuner of the human transcriptome.

Analysis of the human protein interactome and comparison with yeast, worm and fly interaction datasets.

We present the first analysis of the human proteome with regard to interactions between proteins. We also compare the human interactome with the available interaction datasets from yeast (Saccharomyces cerevisiae), worm (Caenorhabditis elegans) and fly (Drosophila melanogaster). Of >70,000 binary interactions, only 42 were common to human, worm and fly, and only 16 were common to all four datasets. An additional 36 interactions were common to fly and worm but were not observed in humans, although a coimmunoprecipitation assay showed that 9 of the interactions do occur in humans. A re-examination of the connectivity of essential genes in yeast and humans indicated that the available data do not support the presumption that the number of interaction partners can accurately predict whether a gene is essential. Finally, we found that proteins encoded by genes mutated in inherited genetic disorders are likely to interact with proteins known to cause similar disorders, suggesting the existence of disease subnetworks. The human interaction map constructed from our analysis should facilitate an integrative systems biology approach to elucidating the cellular networks that contribute to health and disease states.

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.

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.

Regulated nucleosome mobility and the histone code

Post-translational modifications of the histone tails are correlated with distinct chromatin states that regulate access to DNA. Recent proteomic analyses have revealed several new modifications in the globular nucleosome core, many of which lie at the histone-DNA interface. We interpret these modifications in light of previously published data and propose a new and testable model for how cells implement the histone code by modulating nucleosome dynamics.

Total Synthesis of a Functional Designer Eukaryotic Chromosome

Designer Chromosome One of the ultimate aims of synthetic biology is to build designer organisms from the ground up. Rapid advances in DNA synthesis has allowed the assembly of complete bacterial genomes. Eukaryotic organisms, with their generally much larger and more complex genomes, present an additional challenge to synthetic biologists. Annaluru et al. (p. 55, published online 27 March) designed a synthetic eukaryotic chromosome based on yeast chromosome III. The designer chromosome, shorn of destabilizing transfer RNA genes and transposons, is ∼14% smaller than its wild-type template and is fully functional with every gene tagged for easy removal. A synthetic version of yeast chromosome III with every gene tagged can substitute for the original. Rapid advances in DNA synthesis techniques have made it possible to engineer viruses, biochemical pathways and assemble bacterial genomes. Here, we report the synthesis of a functional 272,871–base pair designer eukaryotic chromosome, synIII, which is based on the 316,617–base pair native Saccharomyces cerevisiae chromosome III. Changes to synIII include TAG/TAA stop-codon replacements, deletion of subtelomeric regions, introns, transfer RNAs, transposons, and silent mating loci as well as insertion of loxPsym sites to enable genome scrambling. SynIII is functional in S. cerevisiae. Scrambling of the chromosome in a heterozygous diploid reveals a large increase in a-mater derivatives resulting from loss of the MATα allele on synIII. The complete design and synthesis of synIII establishes S. cerevisiae as the basis for designer eukaryotic genome biology.