A Toolkit and Robust Pipeline for the Generation of Fosmid-Based Reporter Genes in C. elegans

Tursun, Baris; Cochella, Luisa; Carrera, Inés; Hobert, Oliver

doi:10.1371/journal.pone.0004625

Cited by 168 publications

(184 citation statements)

References 30 publications

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“…Fosmid clone WRM069bE04 was recombineered using galK selection to replace the nos-2 coding sequence and introns with yfp, as described (Tursun et al, 2009). yfp containing FRT-flanked galK within an intron was amplified Fig.…”

Section: Constructing Nos-2(−) Strainmentioning

confidence: 99%

A novel germ cell determinant reveals parallel pathways for germ line development inCaenorhabditis elegans

2015

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Despite the central importance of germ cells for transmission of genetic material, our understanding of the molecular programs that control primordial germ cell (PGC) specification and differentiation are limited. Here, we present findings that X chromosome NonDisjunction factor-1 (XND-1), known for its role in regulating meiotic crossover formation, is an early determinant of germ cell fates in Caenorhabditis elegans. xnd-1 mutant embryos display a novel 'one PGC' phenotype as a result of G2 cell cycle arrest of the P 4 blastomere. Larvae and adults display smaller germ lines and reduced brood size consistent with a role for XND-1 in germ cell proliferation. Maternal XND-1 proteins are found in the P 4 lineage and are exclusively localized to the nucleus in PGCs, Z2 and Z3. Zygotic XND-1 turns on shortly thereafter, at the ∼300-cell stage, making XND-1 the earliest zygotically expressed gene in worm PGCs. Strikingly, a subset of xnd-1 mutants lack germ cells, a phenotype shared with nos-2, a member of the conserved Nanos family of germline determinants. We generated a nos-2 null allele and show that nos-2; xnd-1 double mutants display synthetic sterility. Further removal of nos-1 leads to almost complete sterility, with the vast majority of animals without germ cells. Sterility in xnd-1 mutants is correlated with an increase in transcriptional activation-associated histone modification and aberrant expression of somatic transgenes. Together, these data strongly suggest that xnd-1 defines a new branch for PGC development that functions redundantly with nos-2 and nos-1 to promote germline fates by maintaining transcriptional quiescence and regulating germ cell proliferation.

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Section: Constructing Nos-2(−) Strainmentioning

confidence: 99%

A novel germ cell determinant reveals parallel pathways for germ line development inCaenorhabditis elegans

2015

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“…Recombineering was used to generate p845, which encodes C-terminal tagged LIN-1::GFP, as described in Tursun et al (2009). Fosmid WRM0629cG08 was used as the template.…”

Section: Lin-1::gfp Fosmid Reporter and Rescuementioning

confidence: 99%

Spatial Regulation of lag-2 Transcription During Vulval Precursor Cell Fate Patterning in Caenorhabditis elegans lag-2

Zhang

Greenwald

2011

Genetics

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lag-2 encodes a ligand for LIN-12/Notch and is a component of the lateral signal that activates LIN-12/Notch during Caenorhabditis elegans vulval precursor cell (VPC) fate patterning. lag-2 is specifically transcribed in one VPC, named P6.p, in response to activation of EGFR/Ras/MAPK by the inductive signal that initiates vulval development. Here, we show that a critical molecular event linking inductive and lateral signaling is the relief of VPC-wide lag-2 repression in P6.p. We find that the lag-2 promoter contains an element, VPCrep, which mediates repression in all VPCs when the inductive signal is absent, and another promoter element, VPCact, which is required for activation when repression is relieved by the inductive signal. We show that repression through VPCrep is mediated by the Elk1 ortholog LIN-1, and that the level and subcellular accumulation of a functional LIN-1::GFP protein is similar in all six VPCs before and after vulval induction, suggesting that relief of LIN-1-mediated repression in P6.p is likely due to the known MAPK-dependent phosphorylation of LIN-1. We also provide evidence that the factor(s) acting through VPCact is present in all VPCs but is not modulated by the inductive signal, and that transcription of lag-2 requires the Hth/Meis ortholog UNC-62 and the Mediator complex component SUR-2. Relief of repression of lag-2 in P6.p offers a plausible mechanistic basis for spatial restriction of lag-2 in generating the precise spatial pattern of VPC fates.

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“…The cog-1 and die-1 fosmid based reporters were generated following the procedure described by Tursun et al (2009).…”

Section: Generation Of Fosmid Based Reportersmentioning

confidence: 99%

Neuron-type specific regulation of a 3′UTR through redundant and combinatorially acting cis-regulatory elements

Didiano

Cochella²,

Tursun³

et al. 2009

RNA

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ABSTRACT39 Untranslated region (UTR)-dependent post-transcriptional regulation has emerged as a critical mechanism of controlling gene expression in various physiological contexts, including cellular differentiation events. Here, we examine the regulation of the 39UTR of the die-1 transcription factor in a single neuron of the nematode C. elegans. This 39UTR shows the intriguing feature of being differentially regulated across the animal's left/right axis. In the left gustatory neuron, ASEL, in which DIE-1 protein is normally expressed in adult animals, the 39UTR confers no regulatory information, while in the right gustatory neuron, ASER, where DIE-1 is normally not expressed, this 39UTR confers negative regulatory information. Here, we systematically analyze the cis-regulatory architecture of the die-1 39UTR using a transgenic, in vivo assay system. Through extensive mutagenesis and sequence insertions into heterologous 39UTR contexts, we describe three 25-base-pair (bp) sequence elements that are both required and sufficient to mediate the ASER-specific down-regulation of the die-1 39UTR. These three 25-bp sequence elements operate in both a redundant and combinatorial manner. Moreover, there are not only redundant elements within the die-1 39UTR regulating its left/right asymmetric activity but asymmetric 39UTR regulation is itself redundant with other regulatory mechanisms to achieve asymmetric DIE-1 protein expression and function in ASEL versus ASER. The features of 39UTR regulation we describe here may apply to some of the vast number of genes in animal genomes whose expression is predicted to be regulated through their 39UTR.

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A Toolkit and Robust Pipeline for the Generation of Fosmid-Based Reporter Genes in C. elegans

Cited by 168 publications

References 30 publications

A novel germ cell determinant reveals parallel pathways for germ line development inCaenorhabditis elegans

A novel germ cell determinant reveals parallel pathways for germ line development inCaenorhabditis elegans

Spatial Regulation of lag-2 Transcription During Vulval Precursor Cell Fate Patterning in Caenorhabditis elegans lag-2

Neuron-type specific regulation of a 3′UTR through redundant and combinatorially acting cis-regulatory elements

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