Exploring the circadian outputs and function of HPT-1 in Neurospora crassa
MetadataShow full item record
Although many genomes have now been sequenced, genes without a known function still comprise approximately 40% of both the human and Neurospora crassa genomes. The ability to control gene expression by both activation and repression is a powerful method to elucidate biological function. This is often accomplished through the use of a gene promoter that can be manipulated to direct expression levels. However, current promoters used in N. crassa respond to multiple environmental cues, or only induce expression in one direction (up or down). To aid in the study of genes, we developed a strategy using PCR and homologous recombination to insert the tcu-1 promoter in front of any gene of interest. The tcu-1 gene in N. crassa encodes a high affinity copper transporter that is tightly controlled by copper availability. Excess copper represses expression, while copper depletion, via the use of a copper chelator, activates expression. This promoter is ideal for gene control studies in that it displays a high level of specificity, and can direct expression in both directions (up and down). The kinetics of induction and repression were found to be rapid, and the effects long lived. To test if copper repression can recapitulate the loss of viability that an essential gene knockout causes, we placed Ptcu-1 upstream of the essential gene, hpt-1. The addition of excess copper drastically reduced the growth rate as expected. Thus, this strategy will be useful to probe the biological function of any N. crassa gene through controlled expression. Importantly, essential genes can be studied without affecting viability, as would occur if mutated or deleted.
SubjectGene fucntion, Protein overexpression, Regulation of essential genes, Copper sulfate, bathocuproinedisulfonic acid
Vickery, Justin Wayde (2014). Exploring the circadian outputs and function of HPT-1 in Neurospora crassa. Honors and Undergraduate Research. Available electronically from