Double-joint PCR: a PCR-based molecular tool for gene manipulations in filamentous fungi

Yu, Jae Hyuk; Hamari, Zsuzsanna; Han, Kap Hoon; Seo, Jeong Ah; Reyes-Domínguez, Yazmid; Scazzocchio, Claudio

doi:10.1016/j.fgb.2004.08.001

Cited by 1,031 publications

(915 citation statements)

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“…The complementation construct YEC4 was made via single-joint PCR strategy (Sagaram et al, 2006;Yu et al, 2004). Primers CPP1-LC-F and CPP1-LC-R amplified the complete CPP1 gene plus the 850 bp 59 untranslated region (UTR) and the 450 bp 39 UTR.…”

Section: Methodsmentioning

confidence: 99%

“…DRestriction enzyme sites (underlined) were incorporated in the primers to insert amplicons into pBP15. THE M13-R primer sequence for single joint PCR application is shown in bold (Yu et al, 2004).…”

Section: Methodsmentioning

confidence: 99%

“…The complementation construct YEC5 was generated by single-joint PCR strategy (Fig. 1a) (Yu et al, 2004). WT ppe-1 (1.43 kb ppe-1 gene plus 1.6 kb 59 UTR and On: Mon, 08 Apr 2019 04:50:41 500 bp 39 UTR) was amplified from the N. crassa genomic DNA using the primers PPE1-com-F1 and PPE1-com-R1.…”

Section: Methodsmentioning

confidence: 99%

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Functional characterization of Fusarium verticillioides CPP1, a gene encoding a putative protein phosphatase 2A catalytic subunit

Choi

Shim

2008

Microbiology

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Fusarium verticillioides produces the mycotoxin fumonisin B 1 (FB 1 ) on maize kernels. In this study, we identified a putative protein phosphatase gene CPP1 in F. verticillioides, and investigated its role in FB 1 regulation. Previous work has shown that CPP1 expression is elevated in an FB 1 -suppressing genetic background. Thus, we hypothesized that CPP1 is negatively associated with FB 1 production. To test this hypothesis, we generated a CPP1 knockout mutant, PP179, and studied the effects of gene deletion on FB 1 biosynthesis and fungal development. PP179 showed elevated expression of FUM genes, and in turn produced higher levels of FB 1 than the wild-type progenitor. Other significant mutant phenotypes included reduced radial growth on agar plates, reduced conidia germination rates, significantly increased macroconidia formation, and hyphal swelling. To verify that these phenotypes were directly due to CPP1 deletion, we complemented PP179 with the wild-type CPP1 gene. The complemented strain PPC4 showed FUM1 expression and FB 1 production similar to that of the wild-type, providing evidence that CPP1 is negatively associated with FB 1 biosynthesis. Other PP179 phenotypes, such as macroconidiation and hyphal swelling, were also restored to that of wild-type progenitor. Furthermore, we complemented F. verticillioides PP179 strain with Neurospora crassa wild-type ppe-1 gene, demonstrating that Cpp1 and PPE-1 proteins are functionally conserved. Pleiotropic effects of CPP1 deletion led us to hypothesize that CPP1 is associated with multiple downstream signalling pathways in F. verticillioides. Identification and functional characterization of downstream Cpp1-interacting proteins are necessary to better understand the complex regulatory mechanisms associated with Cpp1.

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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Functional characterization of Fusarium verticillioides CPP1, a gene encoding a putative protein phosphatase 2A catalytic subunit

Choi

Shim

2008

Microbiology

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“…A 6.8-kb cryA genomic fragment was subcloned into pGEM5 (SpeI/NsiI), creating pME3182. For construction of the complementation cassette, by using primers OZG189/192, nat R cassette was amplified from pNV1 (Seiler et al, 2006), and 3Ј untranslated region (UTR) of cryA was amplified from genomic (OZG191/ 10) DNA, and these two fragments were fused by fusion PCR (Yu et al, 2004) and nat R ϩ 3Ј UTR fusion fragment was cloned in pCR-Blunt II-TOPO (Invitrogen). VspI site of this plasmid served as recipient for OZG199/200 amplified and VspI digested 5Ј UTR ϩ cryA ORF from pME3182, resulting in the complementation plasmid pME3183 from which 6.5-kb complementation fragment was released with PfIMI digest and used for complementation of cryA⌬ mutant.…”

Section: Plasmids and Oligonucleotidesmentioning

confidence: 99%

More Than a Repair Enzyme:Aspergillus nidulansPhotolyase-like CryA Is a Regulator of Sexual Development

Bayram

Biesemann

Krappmann

et al. 2008

MBoC

129

116

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Cryptochromes are blue-light receptors that have presumably evolved from the DNA photolyase protein family, and the genomes of many organisms contain genes for both types of molecules. Both protein structures resemble each other, which suggests that light control and light protection share a common ancient origin. In the genome of the filamentous fungus Aspergillus nidulans, however, only one cryptochrome/photolyase-encoding gene, termed cryA, was identified. Deletion of the cryA gene triggers sexual differentiation under inappropriate culture conditions and results in upregulation of transcripts encoding regulators of fruiting body formation. CryA is a protein whose N-and C-terminal synthetic green fluorescent protein fusions localize to the nucleus. CryA represses sexual development under UVA 350-370 nm light both on plates and in submerged culture. Strikingly, CryA exhibits photorepair activity as demonstrated by heterologous complementation of a DNA repair-deficient Escherichia coli strain as well as overexpression in an A. nidulans uvsB⌬ genetic background. This is in contrast to the single deletion cryA⌬ strain, which does not show increased sensitivity toward UV-induced damage. In A. nidulans, cryA encodes a novel type of cryptochrome/photolyase that exhibits a regulatory function during light-dependent development and DNA repair activity. This represents a paradigm for the evolutionary transition between photolyases and cryptochromes. INTRODUCTIONCryptochromes (CRYs) are blue-light receptors that regulate growth, development, and the circadian clock in higher eukaryotes and that are believed to have evolved from the DNA photolyase protein family (Daiyasu et al., 2004;Lin and Todo, 2005). Photolyases, in contrast, repair UV-induced DNA damage by using a mechanism referred to as photorepair. They absorb light in the blue spectrum and transfer an excited electron from the cofactor FAD to an enzyme-bound cyclobutane pyrimidine dimer (CPD), which is thereby cleaved (Sancar, 1990(Sancar, , 1994Sancar, 1994). Cryptochromes are characterized by an N-terminal photolyase-related (PHR) region without significant photorepair activity and by a C-terminal domain of varying length, which is absent in members of the CRY-DASH subfamily (Daiyasu et al., 2004). The exact function of the CRY-DASH proteins was elusive; however, recent data imply that they are actually photolyases that specifically repair CPDs in single-stranded DNA (Selby and Sancar, 2006). The PHR domain is the most conserved region of the CRY proteins and contains the cofactor FAD required for electron transfer reactions. In Xenopus and Drosophila, the PHR domain is physiologically active even in the absence of the C-terminal domain. The C-terminal domain is important for either localization or protein stability, and its expression in Arabidopsis results in constitutive growth (Lin and Todo, 2005). Plant CRYs are phosphorylated under illumination, and gene expression control ranges from protein interactions to direct chromatin interactions (Shalitin et al....

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“…This method has been used to create constructs for gene replacement in A. awamori [99] and for targeted in vivo promoter exchange [110]. A variation of the technique, in which overlapping segments were present on both primers at a join (termed double-jointed PCR), was used to create linear constructs for biolistic transformation of Cryptococcus neoformans [111] and for protoplast transformations of A. nidulans, A. fumigatus and Fusarium graminarium [112].…”

Section: High Throughput Gene Targeting Strategiesmentioning

confidence: 99%

Approaches to functional genomics in filamentous fungi

et al. 2006

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The study of gene function in filamentous fungi is a field of research that has made great advances in very recent years. A number of transformation and gene manipulation strategies have been developed and applied to a diverse and rapidly expanding list of economically important filamentous fungi and oomycetes. With the significant number of fungal genomes now sequenced or being sequenced, functional genomics promises to uncover a great deal of new information in coming years. This review discusses recent advances that have been made in examining gene function in filamentous fungi and describes the advantages and limitations of the different approaches.

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Double-joint PCR: a PCR-based molecular tool for gene manipulations in filamentous fungi

Cited by 1,031 publications

References 19 publications

Functional characterization of Fusarium verticillioides CPP1, a gene encoding a putative protein phosphatase 2A catalytic subunit

Functional characterization of Fusarium verticillioides CPP1, a gene encoding a putative protein phosphatase 2A catalytic subunit

More Than a Repair Enzyme:Aspergillus nidulansPhotolyase-like CryA Is a Regulator of Sexual Development

Approaches to functional genomics in filamentous fungi

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