RESUMO
A broad variety of biomolecules is industrially produced in bacteria and yeasts. These microbial expression hosts can be optimized through genetic engineering using CRISPR tools. Here, we designed and characterized such a modular genome editing system based on the Cas12a-like RNA-guided nuclease MAD7 in Escherichia coli. This system enables the efficient generation of single nucleotide polymorphisms (SNPs) or gene deletions and can directly be used with donor DNA from benchtop DNA assembly to increase throughput. We combined multiple edits to engineer an E. coli strain with reduced overflow metabolism and increased plasmid yield, highlighting the versatility and industrial applicability of this approach.
Assuntos
Sistemas CRISPR-Cas , Edição de Genes , Escherichia coli/genética , Engenharia Genética , PlasmídeosRESUMO
Although the formation of rod-shaped chromosomes is vital for the correct segregation of eukaryotic genomes during cell divisions, the molecular mechanisms that control the chromosome condensation process have remained largely unknown. Here, we identify the C2H2 zinc-finger transcription factor Zas1 as a key regulator of mitotic condensation dynamics in a quantitative live-cell microscopy screen of the fission yeast Schizosaccharomyces pombe By binding to specific DNA target sequences in their promoter regions, Zas1 controls expression of the Cnd1 subunit of the condensin protein complex and several other target genes, whose combined misregulation in zas1 mutants results in defects in chromosome condensation and segregation. Genetic and biochemical analysis reveals an evolutionarily conserved transactivation domain motif in Zas1 that is pivotal to its function in gene regulation. Our results suggest that this motif, together with the Zas1 C-terminal helical domain to which it binds, creates a cis/trans switch module for transcriptional regulation of genes that control chromosome condensation.
Assuntos
Proteínas de Ciclo Celular/genética , Cromossomos Fúngicos/genética , Mitose/genética , Proteínas de Schizosaccharomyces pombe/genética , Adenosina Trifosfatases/genética , Proteínas de Ligação a DNA/genética , Regulação Fúngica da Expressão Gênica/genética , Complexos Multiproteicos/genética , Mutação , Schizosaccharomyces/genéticaRESUMO
Even though the formation of compact cylindrical chromosomes early during mitosis or meiosis is a prerequisite for the successful segregation of eukaryotic genomes, little is known about the molecular basis of this chromosome condensation process. Here, we describe in detail the protocol for a quantitative chromosome condensation assay in fission yeast cells, which is based on precise time-resolved measurements of the distances between two fluorescently labeled positions on the same chromosome. In combination with an automated computational analysis pipeline, this assay enables the study of various candidate proteins for their roles in regulating genome topology during cell divisions.
Assuntos
Segregação de Cromossomos/genética , Cromossomos Fúngicos/genética , Biologia Molecular/métodos , Schizosaccharomyces/genética , Corantes Fluorescentes/química , Genoma Fúngico , Meiose/genética , Mitose/genéticaRESUMO
Condensins associate with DNA and shape mitotic chromosomes. Condensins are enriched nearby highly expressed genes during mitosis, but how this binding is achieved and what features associated with transcription attract condensins remain unclear. Here, we report that condensin accumulates at or in the immediate vicinity of nucleosome-depleted regions during fission yeast mitosis. Two transcriptional coactivators, the Gcn5 histone acetyltransferase and the RSC chromatin-remodelling complex, bind to promoters adjoining condensin-binding sites and locally evict nucleosomes to facilitate condensin binding and allow efficient mitotic chromosome condensation. The function of Gcn5 is closely linked to condensin positioning, since neither the localization of topoisomerase II nor that of the cohesin loader Mis4 is altered in gcn5 mutant cells. We propose that nucleosomes act as a barrier for the initial binding of condensin and that nucleosome-depleted regions formed at highly expressed genes by transcriptional coactivators constitute access points into chromosomes where condensin binds free genomic DNA.
Assuntos
Adenosina Trifosfatases/metabolismo , Cromossomos Fúngicos/metabolismo , Proteínas de Ligação a DNA/metabolismo , Mitose , Complexos Multiproteicos/metabolismo , Nucleossomos/metabolismo , Schizosaccharomyces/fisiologia , Acetiltransferases/metabolismo , Composição de Bases , Proteínas de Schizosaccharomyces pombe/metabolismo , Fatores de Transcrição/metabolismoRESUMO
BACKGROUND: Mechanoreceptors contain compliant elements, termed "gating springs," that transfer macroscopic stimuli impinging on the cells into microscopic stimuli that open the mechanosensitive channels. Evidence for gating springs comes from mechanical experiments; they have not been identified molecularly or ultrastructurally. RESULTS: We show that the filamentous structures that connect the plasma membrane to the microtubules are compliant structural elements in the mechanoreceptive organelle of fly campaniform receptors. These filaments colocalize with the ankyrin-repeat domain of the transient receptor potential (TRP) channel NOMPC. In addition, they resemble the purified ankyrin-repeat domain in size and shape. Most importantly, these filaments are nearly absent in nompC mutants and can be rescued by the nompC gene. Finally, mechanical modeling suggests that the filaments provide most of the compliance in the distal tip of the cell, thought to be the site of mechanotransduction. CONCLUSIONS: Our results provide strong evidence that the ankyrin-repeat domains of NOMPC structurally contribute to the membrane-microtubule connecting filaments. These filaments, as the most compliant element in the distal tip, are therefore good candidates for the gating springs.
