Dataset Information

A high-throughput gene knockout procedure for Neurospora reveals functions for multiple transcription factors.

ABSTRACT: The low rate of homologous recombination exhibited by wild-type strains of filamentous fungi has hindered development of high-throughput gene knockout procedures for this group of organisms. In this study, we describe a method for rapidly creating knockout mutants in which we make use of yeast recombinational cloning, Neurospora mutant strains deficient in nonhomologous end-joining DNA repair, custom-written software tools, and robotics. To illustrate our approach, we have created strains bearing deletions of 103 Neurospora genes encoding transcription factors. Characterization of strains during growth and both asexual and sexual development revealed phenotypes for 43% of the deletion mutants, with more than half of these strains possessing multiple defects. Overall, the methodology, which achieves high-throughput gene disruption at an efficiency >90% in this filamentous fungus, promises to be applicable to other eukaryotic organisms that have a low frequency of homologous recombination.

SUBMITTER: Colot HV

PROVIDER: S-EPMC1482798 | biostudies-literature | 2006 Jul

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

A high-throughput gene knockout procedure for Neurospora reveals functions for multiple transcription factors.

Colot Hildur V HV Park Gyungsoon G Turner Gloria E GE Ringelberg Carol C Crew Christopher M CM Litvinkova Liubov L Weiss Richard L RL Borkovich Katherine A KA Dunlap Jay C JC

Proceedings of the National Academy of Sciences of the United States of America 20060626 27

The low rate of homologous recombination exhibited by wild-type strains of filamentous fungi has hindered development of high-throughput gene knockout procedures for this group of organisms. In this study, we describe a method for rapidly creating knockout mutants in which we make use of yeast recombinational cloning, Neurospora mutant strains deficient in nonhomologous end-joining DNA repair, custom-written software tools, and robotics. To illustrate our approach, we have created strains bearin ...[more]

PMID: 16801547

Similar Datasets

Project description:Liver cancer, including hepatocellular carcinoma (HCC), is a malignant tumor that has high rates of metastasis and mortality worldwide. Upstream transcription factor 1 (USF1) is a canonical transcription factor (TF) and is associated with the pathogenesis of several cancers, but its biological functions and molecular targets in HCC remain unclear. Huh7 cells that overexpress USF1 were used with whole transcriptome profiling through RNA sequencing and chromatin immunoprecipitation (ChIP) sequencing methods to investigate the downstream targets of USF1. Reverse transcription-quantitative PCR was then used to validate the downstream targets. The results showed that USF1 significantly regulates 350 differentially expressed genes (DEGs). The upregulated DEGs were primarily protein-coding genes enriched in immune and inflammation response pathways, while the downregulated DEGs were mainly coding long non-coding (lnc)RNAs, indicating the regulatory function of USF1. It was also demonstrated that USF1 directly binds to the promoter region of 2,492 genes, which may be involved in the viral progression and cell proliferation pathways. By integrating these two datasets, 16 overlapped genes were detected, including downregulated lncRNA-NEAT1 and upregulated TF-ETV5. The downregulated lncRNA-NEAT1 showed reverse expression pattern and prognosis result compared with that of USF1 in patients with liver cancer, while upregulated TF-ETV5 showed consistent results with USF1. Promoter region motif analysis indicated that ETV5 has more binding motifs and genes than USF1 itself for USF1-regulated DEGs, indicating that USF1 may indirectly modulate gene expression by regulating ETV5 expression in Huh7 cells. The study also validated the direct interaction between USF1 and the promoter of ETV5 using ChIP-qPCR. In summary, the results demonstrated that USF1 binds to the promoter region of thousands of genes and affects a large part of DEGs indirectly. Downstream genes, including lncRNA-NEAT1 and TF-ETV5, may also have potential functions in the regulated network by USF1 and have potential functions in the progression of HCC. The present findings suggested that USF1 and its downstream targets could be potential targets for HCC therapy in the future.

Dataset Information

A high-throughput gene knockout procedure for Neurospora reveals functions for multiple transcription factors.

Publications

A high-throughput gene knockout procedure for Neurospora reveals functions for multiple transcription factors.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets