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A Tool Set for the Genome-Wide Analysis of Neurospora crassa by RT-PCR.

ABSTRACT: Neurospora crassa is an important model organism for filamentous fungi as well as for circadian biology and photobiology. Although the community-accumulated tool set for the molecular analysis of Neurospora is extensive, two components are missing: (1) dependable reference genes whose level of expression are relatively constant across light/dark cycles and as a function of time of day and (2) a catalog of primers specifically designed for real-time PCR (RT-PCR). To address the first of these we have identified genes that are optimal for use as reference genes in RT-PCR across a wide range of expression levels; the mRNA/transcripts from these genes have potential for use as reference noncycling transcripts outside of Neurospora. In addition, we have generated a genome-wide set of RT-PCR primers, thereby streamlining the analysis of gene expression. In validation studies these primers successfully identified target mRNAs arising from 70% (34 of 49) of all tested genes and from all (28) of the moderately to highly expressed tested genes.

SUBMITTER: Hurley JH

PROVIDER: S-EPMC4592987 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Transcription factors play a key role in transcription regulation as they recognize and directly bind to defined sites in promoter regions of target genes, and thus modulate differential expression. The overall process is extremely dynamic, as they have to move through the nucleus and transiently bind to chromatin in order to regulate gene transcription. To identify transcription factors that affect glycogen accumulation in Neurospora crassa, we performed a systematic screen of a deletion strains set generated by the Neurospora Knockout Project and available at the Fungal Genetics Stock Center. In a wild-type strain of N. crassa, glycogen content reaches a maximal level at the end of the exponential growth phase, but upon heat stress the glycogen content rapidly drops. The gene encoding glycogen synthase (gsn) is transcriptionally down-regulated when the mycelium is exposed to the same stress condition. We identified 17 deleted strains having glycogen accumulation profiles different from that of the wild-type strain under both normal growth and heat stress conditions. Most of the transcription factors identified were annotated as hypothetical protein, however some of them, such as the PacC, XlnR, and NIT2 proteins, were biochemically well-characterized either in N. crassa or in other fungi. The identification of some of the transcription factors was coincident with the presence of DNA-binding motifs specific for the transcription factors in the gsn 5'-flanking region, and some of these DNA-binding motifs were demonstrated to be functional by Electrophoretic Mobility Shift Assay (EMSA) experiments. Strains knocked-out in these transcription factors presented impairment in the regulation of gsn expression, suggesting that the transcription factors regulate glycogen accumulation by directly regulating gsn gene expression. Five selected mutant strains showed defects in cell cycle progression, and two transcription factors were light-regulated. The results indicate that there are connections linking different cellular processes, such as metabolism control, biological clock, and cell cycle progression.