Targeted Forward Genetics: Saturating Mutational Analyses of Specific Target Loci Within the Genome.

Precise allele replacement by homologous recombination (also known as "gene targeting" or "genome editing") allows scientists to engineer altered DNA sequences, insertions, or deletions at specific locations in the genome. Such reverse genetics provides powerful tools to elucidate the structure and function of regulatory DNA elements, genes, RNAs, and proteins within their natural, endogenous context. Here, we describe in detail the methodology for Targeted Forward Genetics (TFG), which supports population-scale, saturating screens of allele replacements spanning thousands of base pairs at a specific target locus in the genome.

Agar lot-specific inhibition in the plating efficiency of yeast spores and cells.

The fission yeast Schizosaccharomyces pombe and the budding yeast Saccharomyces cerevisiae are highly diverged (530 mya), single-celled, model eukaryotic organisms. Scientists employ mating, meiosis, and the plating of ascospores and cells to generate strains with novel genotypes and to discover biological processes. Our three laboratories encountered independently sudden-onset, major impediments to such research.

Creating Meiotic Recombination-Regulating DNA Sites by in Fission Yeast Reveals Inefficiencies, Target-Site Duplications, and Ectopic Insertions.

Recombination hotspot-activating DNA sites (e.g., , , ) and their binding proteins (e.

Distance-dependent effects on CRISPR/Cas9-mediated genome editing in compromise efficiency and create unsought alleles.

Discrete DNA sites position meiotic recombination at hotspots. We sought to create four different, 15 bp long, candidate regulatory DNA sites within the reporter gene. Each effort employed a fission yeast-optimized CRISPR system (SpEDIT), optimal guide RNA, and one of four homologous recombination templates with 10 to 15 bp substitutions.

Eukaryotic Pif1 helicase unwinds G-quadruplex and dsDNA using a conserved wedge.

G-quadruplexes (G4s) formed by guanine-rich nucleic acids induce genome instability through impeding DNA replication fork progression. G4s are stable DNA structures, the unfolding of which require the functions of DNA helicases. Pif1 helicase binds preferentially to G4 DNA and plays multiple roles in maintaining genome stability, but the mechanism by which Pif1 unfolds G4s is poorly understood.

Laboratory horror stories: Poison in the agars.

The fission yeast is a single-celled eukaryote that can be cultured as a haploid or as a diploid. Scientists employ mating, meiosis, and the plating of ascospores and cells to generate strains with novel genotypes and to discover biological processes. Our two laboratories encountered independently sudden-onset, major impediments to such research.

Two residues in the DNA binding site of Pif1 helicase are essential for nuclear functions but dispensable for mitochondrial respiratory growth.

Pif1 helicase functions in both the nucleus and mitochondria. Pif1 tightly couples ATP hydrolysis, single-stranded DNA translocation, and duplex DNA unwinding. We investigated two Pif1 variants (F723A and T464A) that have each lost one site of interaction of the protein with the DNA substrate.

DNA sequences and distinct mechanisms for and alleles of .

The gene of the fission yeast supports both positive and negative selection; consequently, this gene is widely employed as a powerful tool to study diverse biological processes. Here we report the DNA sequences of two functionally null alleles, and . The allele has a four bp duplication of bp +63 to +66 (5'-CAAG-3') within the ORF and the allele has a nonsynonymous substitution (G to A) at bp +679.

Adaptive Control of the Meiotic Recombination Landscape by DNA Site-dependent Hotspots With Implications for Evolution.

Meiosis is an essential component of the sexual life cycle in eukaryotes. The independent assortment of chromosomes in meiosis increases genetic diversity at the level of whole chromosomes and meiotic recombination increases genetic diversity within chromosomes. The resulting variability fuels evolution.

Molecular mechanisms for environmentally induced and evolutionarily rapid redistribution (plasticity) of meiotic recombination.

It has long been known (circa 1917) that environmental conditions, as well as speciation, can affect dramatically the frequency distribution of Spo11/Rec12-dependent meiotic recombination. Here, by analyzing DNA sequence-dependent meiotic recombination hotspots in the fission yeast Schizosaccharomyces pombe, we reveal a molecular basis for these phenomena. The impacts of changing environmental conditions (temperature, nutrients, and osmolarity) on local rates of recombination are mediated directly by DNA site-dependent hotspots (M26, CCAAT, and Oligo-C).

Accurate and Sensitive Quantitation of the Dynamic Heat Shock Proteome Using Tandem Mass Tags.

Cells respond to environmental perturbations and insults through modulating protein abundance and function. However, the majority of studies have focused on changes in RNA abundance because quantitative transcriptomics has historically been more facile than quantitative proteomics. Modern Orbitrap mass spectrometers now provide sensitive and deep proteome coverage, allowing direct, global quantification of not only protein abundance but also post-translational modifications (PTMs) that regulate protein activity.

Targeted Forward Genetics: Population-Scale Analyses of Allele Replacements Spanning Thousands of Base Pairs in Fission Yeast.

Precise allele replacement (genome editing), without unwanted changes to the genome, provides a powerful tool to define the functions of DNA elements and encoded factors in their normal biological context. While CRISPR is now used extensively for gene targeting, its utility for precise allele replacement at population scale is limited because: (A) there is a strict requirement for a correctly positioned PAM motif to introduce recombinogenic dsDNA breaks (DSBs); (B) efficient replacements only occur very close to the DSBs; and (C) indels and off-target changes are frequently generated. Here we show, using a saturated mutation library with about 15,000 alleles of the gene of , that pop-in, pop-out allele replacement circumvents these problems.

Diverse DNA Sequence Motifs Activate Meiotic Recombination Hotspots Through a Common Chromatin Remodeling Pathway.

In meiosis, multiple different DNA sequence motifs help to position homologous recombination at hotspots in the genome. How do the seemingly disparate -acting regulatory modules each promote locally the activity of the basal recombination machinery? We defined molecular mechanisms of action for five different hotspot-activating DNA motifs (, , , , ) located independently at the same site within the locus of the fission yeast Each motif promoted meiotic recombination (, is active) within this context, and this activity required the respective binding proteins (transcription factors Atf1, Pcr1, Php2, Php3, Php5, Rst2). High-resolution analyses of chromatin structure by nucleosome scanning assays revealed that each motif triggers the displacement of nucleosomes surrounding the hotspot motif in meiosis.

In Vivo Metabolic Tracing Demonstrates the Site-Specific Contribution of Hepatic Ethanol Metabolism to Histone Acetylation.

Background: Epigenetic dysregulation through ethanol (EtOH)-induced changes in DNA methylation and histone modifications has been implicated in several alcohol-related disorders such as alcoholic liver disease. EtOH metabolism in the liver results in the formation of acetate, a metabolite that can be converted to acetyl-CoA, which can then be used by histone acetyltransferases to acetylate lysine residues. EtOH metabolism in the liver can also indirectly influence lysine acetylation through NAD -dependent sirtuin activity that is altered due to increases in NADH.

The NIH must reduce disparities in funding to maximize its return on investments from taxpayers.

New data from the NIH reveal that the scientific return on its sponsored research reaches a maximum at around $400,000 of annual support per principal investigator. We discuss the implications of this 'sweet spot' for funding policy, and propose that the NIH should limit both the minimum and maximum amount of funding per researcher.

Biases in grant proposal success rates, funding rates and award sizes affect the geographical distribution of funding for biomedical research.

The ability of the United States to most efficiently make breakthroughs on the biology, diagnosis and treatment of human diseases requires that physicians and scientists in each state have equal access to federal research grants and grant dollars. However, despite legislative and administrative efforts to ensure equal access, the majority of funding for biomedical research is concentrated in a minority of states. To gain insight into the causes of such disparity, funding metrics were examined for all NIH research project grants (RPGs) from 2004 to 2013.

Nonsense codon suppression in fission yeast due to mutations of tRNA(Ser.11) and translation release factor Sup35 (eRF3).

In the fission yeast Schizosaccharomyces pombe, sup9 mutations can suppress the termination of translation at nonsense (stop) codons. We localized sup9 physically to the spctrnaser.11 locus and confirmed that one allele (sup9-UGA) alters the anticodon of a serine tRNA.

A CRISPR-based approach for proteomic analysis of a single genomic locus.

Any given chromosomal activity (e.g., transcription) is governed predominantly by the local epiproteome.

Binding of the transcription factor Atf1 to promoters serves as a barrier to phase nucleosome arrays and avoid cryptic transcription.

Schizosaccharomyces pombe displays a large transcriptional response common to several stress conditions, regulated primarily by the transcription factor Atf1. Atf1-dependent promoters contain especially broad nucleosome depleted regions (NDRs) prior to stress imposition. We show here that basal binding of Atf1 to these promoters competes with histones to create wider NDRs at stress genes.

Rapid, efficient and precise allele replacement in the fission yeast Schizosaccharomyces pombe.

Gene targeting provides a powerful tool to modify endogenous loci to contain specific mutations, insertions and deletions. Precise allele replacement, with no other chromosomal changes (e.g.

A stress-activated, p38 mitogen-activated protein kinase-ATF/CREB pathway regulates posttranscriptional, sequence-dependent decay of target RNAs.

Broadly conserved, mitogen-activated/stress-activated protein kinases (MAPK/SAPK) of the p38 family regulate multiple cellular processes. They transduce signals via dimeric, basic leucine zipper (bZIP) transcription factors of the ATF/CREB family (such as Atf2, Fos, and Jun) to regulate the transcription of target genes. We report additional mechanisms for gene regulation by such pathways exerted through RNA stability controls.