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Scheffersomyces stipitis

ABSTRACT: ChIP-Seq of constitutively expressed Ndt80A-Myc and Ndt80B-Myc in S. stipitis

PROVIDER: PRJNA355299 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	SRR5061372.fastq.gz	Fastqsanger.gz
	SRR5061373.fastq.gz	Fastqsanger.gz
	SRR5061374.fastq.gz	Fastqsanger.gz
	SRR5061375.fastq.gz	Fastqsanger.gz
	SRR5061376.fastq.gz	Fastqsanger.gz

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Expression analysis of L-rhamnose catabolism in the yeast Scheffersomyces stipitis

Project description:In Scheffersomyces stipitis and related fungal species the genes for L-rhamnose catabolism RHA1, LRA2, LRA3 and LRA4 but not LADH are clustered. We find that located next to the cluster is a transcription factor, TRC1, which is conserved among related species.Our transcriptome analysis shows that all the catabolic genes and all genes of the cluster are up-regulated on L-rhamnose. Among the genes that were also up-regulated on L-rhamnose were two transcription factors including the TRC1. In addition, in 16 out of the 32 analysed fungal species only RHA1, LRA2 and LRA3 are in a cluster. The clustering of RHA1, LRA3 and TRC1 is also conserved in species not closely related to S. stipitis. Since the LRA4 is often not part of the cluster and it has several paralogs in L-rhamnose utilising yeasts we analysed the function of one of the paralogs, RHA41 by heterologous expression and biochemical characterization. Rha41p has similar catalytic properties but the transcript was not up-regulated on L-rhamnose. The RHA1, LRA2, LRA4 and LADH genes were previously characterized in Sheffersomyces (Pichia) stipitis. We expressed the L-rhamnonate dehydratase, Rha3p, in S. cerevisiae, estimated the kinetic constants of the protein and showed that it indeed has activity with L-rhamnonate. A six chip study using total RNA recovered from three separate cultures of S. stipitis CBS 6054 grown glucose and respectively three separate cultures grown on rhamnose

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Project description:The purpose of this experiment was to identify the genes bound by the two Ndt80 paralogs in S. stipitis when constitutively expressed. Both paralogs were tagged with c-myc, the native Ndt80 promoter was replaced with the E. gossypii Tef1 promoter and the protein was immunoprecipitated with a c-myc antibody. Cells were grown unti log-phase in YPD. Each experiment was repeated twice and sequenced on an Illumina HiSeq 2500.

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