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Using Zinc-based probes to generate pseudoislets from purified human alpha-cells for functional analyses

ABSTRACT: Using Zinc-based probes to generate pseudoislets from purified human alpha-cells for functional analyses

PROVIDER: PRJNA819965 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	SRR18481800_1.fastq.gz	Fastqsanger.gz
	SRR18481800_2.fastq.gz	Fastqsanger.gz
	SRR18481801_1.fastq.gz	Fastqsanger.gz
	SRR18481801_2.fastq.gz	Fastqsanger.gz
	SRR18481802_1.fastq.gz	Fastqsanger.gz

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Project description:Zinc is an essential nutrient because it is a required cofactor for many enzymes and transcription factors. To discover genes and processes in yeast that are required for growth when zinc is limiting, we used genome-wide functional profiling. As a result, we identified over 400 different genes required for optimal growth under zinc-limiting conditions. Among these were several targets of the Zap1 zinc-responsive transcription factor. Their importance is consistent with their up-regulation by Zap1 in low zinc. We also identified genes that implicate Zap1-independent processes as important. These include endoplasmic reticulum function, oxidative stress resistance, vesicular trafficking, peroxisome biogenesis, and chromatin modification. Our studies also indicated the critical role of macroautophagy in low zinc growth. Finally, as a result of our analysis, we discovered a previously unknown role for the ICE2 gene in maintaining ER zinc homeostasis. Pools of homozygous diploid deletion mutants (n = 4,607 strains) were grown in zinc defined media (LZM) at 2 concentrations for 5 and 15 generations (5g, 15g). Each strain has a deletion in a different gene. In each strain, the gene was replaced by a deletion cassette containing a antibiotic resistance gene and two BARCODES, up and down tags (Please see Giaever et al, 2002, Nature). These tags in the DNA are specific to each strain. We pool all deletion strains and grow them under a selective condition; extract DNA; amplify barcodes using universal primers and hybridize to arrays containing complemetary sequences to the up and down tags. In this way, we can look at growth of each of the strains. Our strategy is to analyze separately the up and down tags. Therefore, for each array (CEL file), we generate two data files, one for all ups and another one for all downs. In these files, the list of genes are the same but the data come from different set of probes, up or down. The values are log2 averages of replicate probes for the same tag. These pre processed files were used to identify strains with differential growth in 1uM zinc by comparing to the 100uM zinc arrays.

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Using Zinc-based probes to generate pseudoislets from purified human alpha-cells for functional analyses

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