AccuOligo® S. pombe Validation Primer Set Ver 3.0 (3,308 primers)

The yeast genome-wide knockout library is a set of mutant yeasts with defects in the functions of specific yeast genes and consists of mutants targeting approximately 98.5% of the genes of the entire fission yeast (S. pombe) genome. Gene function-deficient variants were created by inserting an antibiotic resistance gene and barcode instead of the target gene using the homologous gene recombination method. The process is as follows:

1. Target gene selection: Identify target genes in the yeast genome (Shizosaccharomyces pombe).

2. Preparation of DNA cassette: Prepare a DNA cassette to be recombined with the yeast target gene through homologous recombination. The DNA cassette contains a recombinant target gene ORF (open reading frame) on both flanks, a geneticin (G418) resistance gene (KanMX) cassette for selection of knockout strains, and a unique barcode for identification of each variant.

3. Yeast transformation: Transformation technology is utilized to introduce the DNA cassette into yeast cells. The DNA cassette entered into the yeast cell recognizes the homologous sequence of the flanking region of the target gene on the genome and is then inserted into the corresponding location. The target gene ORF is removed, preventing its function in yeast. Additionally, yeast cells with the DNA cassette inserted at the same time become G418 resistant.

4. Selection of knockout strains: Transformed yeast is grown in medium containing G418 to select only knockout yeast. This process is repeated for all target genes, and finally, all knockout strains are prepared as one set and provided as a library.

S.pombe grows at a temperature of 30℃, and the main medium composition is as follows:

YES for General culture and maintenance medium:

S.pombe knockout library strains have the KanMX4 gene, so they can be selected on medium containing G418 (100 mg/L).

First, the definitions of ploidy and genotype are as follows:

Ploidy:

• Diploid: Diploid cells have two sets of chromosomes (2n). This means that you usually have two copies of each chromosome, one inherited from each parent.
• Haploid: Haploid cells have a single set of chromosomes (n). Haploid yeast cells usually arise through a mating event or specific genetic manipulation.

Genotype:

• Heterozygous: Having different alleles, or in the case of diploid cells, having two different alleles. One allele comes from each parent.
• Homozygous: Having identical alleles, or in the case of diploid cells, having two identical alleles. In haploid cells, there is always only one allele and therefore only homozygous.

Yeast can have different mating types (e.g., MATa and MATα). Heterozygous diploids can be formed by crossing suitable haploid strains (e.g., MATa and MATα). Diploid yeast cells can also undergo meiosis under certain conditions, resulting in the formation of four haploid spores.

In summary:

• Heterozygous diploid strain: A diploid heterozygous strain with chromosome 2n, where one allele is normal, but the other gene is defective. Bioneer heterozygous diploid strains include both essential and non-essential genes (4,845 strains).
• Homozygous haploid strain: A strain on the n chromosome with one allele. Bioneer homozygous haploid strains consist only of non-essential genes (3,497 strains).

Agar type: A method of cultivating and storing yeast strains on a pre-prepared solid medium rich in nutrients. Agar serves to solidify the liquid medium. Agar type strains are cultured at 30℃ and stored at 4℃ to inhibit growth.

Glycerol type: A storage condition that allows liquid cultured strains to be cryopreserved (-70℃) by treating them with 30% (v/v) glycerol. Glycerol binds to water molecules within cells and acts as a cryoprotectant that protects cells by suppressing ice crystal formation even at low temperatures.

When ordering yeast strains from Bioneer, you can choose between agar type and glycerol type as the shipping type below:

• Agar type: Rich medium + supplements solid medium (2.0 microtube)
• Glycerol type: Rich medium + supplements + 30% glycerol liquid medium (2.0 microtube)

* Glycerol type is shipped frozen, so a dry ice fee is added.

In general, a DNA barcode refers to a specific DNA sequence used to identify a biological species. In the yeast genome-wide knockout library, a DNA barcode refers to the unique DNA sequences of the target gene knockout strains that make up the library. DNA barcodes can be used to identify knockout strains using DNA sequencing.

Schizosaccharomyces pombe (S. pombe) and Saccharomyces cerevisiae (S. cerevisiae) are both yeasts widely used as model organisms, but each has different research advantages. The advantages of using S. pombe include:

1. Cell cycle study:

• G2/M transition: S. pombe has a longer G2 phase than S. cerevisiae, making it more advantageous for studying the G2/M transition of the cell cycle. Because human cells also have a relatively long G2 phase, S. pombe is more suitable for human cell cycle studies. Key regulatory proteins such as Cdc25 and Cdk1 were first discovered and characterized in S. pombe.
• Similarity: The cell cycle regulation mechanism of S. pombe is similar to that of human cells, making it ideal for studying the function of genes and proteins involved in cell cycle regulation.

2. Chromosome structure and separation:

• Chromosome structure: The centromere and telomere structures of S. pombe are similar to human cells, making it an important model for studying chromosome segregation and maintenance.
• Pseudochromatin: S. pombe contains both heterochromatin and pseudochromatin, making it useful for studies related to chromatin remodeling.

3. Cell division:

• Microtubule formation: S. pombe forms microtubules using spindle pole bodies rather than centrosomes. This is similar to plant and some animal cells, making it a model for studying cell division.
• Cell plate formation: S. pombe forms a cell plate during cell division and divides into daughter cells, similar to plant cells.

4. Gene expression and regulation:

• Gene expression studies: S. pombe provides a suitable system for studying gene expression and regulatory mechanisms, especially transcriptional regulation and chromatin structural changes. The gene expression RNAi pathway is functional in S. pombe but absent in S. cerevisiae.

The Schizosaccharomyces pombe genome database can be found at PomBase.

PomBase provides a comprehensive suite of data and tools for S. pombe research, including:

• Genome sequence and annotation: Access the complete S. pombe genome sequence, gene models, and functional annotation.
• Genetic information: Explore gene function, phenotypes, and interactions through curated collections of information and mutations.
• Expression data: Analyze gene expression patterns under various conditions using RNA-seq and microarray data.
• Protein information: Provides protein sequence, structure, and function information of S. pombe proteins.
• Tools and Resources: Utilize a variety of tools for sequence analysis, genetic homology searches, and experimental design.

There are two main approaches to finding the human ortholog gene of an S. pombe gene:

1. Using PomBase

PomBase, the primary S. pombe genome database, offers resources for finding human orthologs. Here's how to use it:

• Navigate to the gene page of your S. pombe gene of interest on PomBase. You can search for the gene by name or gene ID using the search bar.
• Look for the "Orthologs" section: This section might be located within the gene summary or under a dedicated "Orthologs" tab depending on the gene page layout.
• PomBase curates human orthologs for many S. pombe genes. If a human ortholog is available, it will be listed in this section, often with a link to the corresponding human gene page on NCBI or other databases.

2. Using Sequence Similarity Search Tools

If a curated human ortholog isn't available on PomBase for your S. pombe gene, you can utilize sequence similarity search tools like BLAST (Basic Local Alignment Search Tool). Here's a general workflow:

• Obtain the protein sequence of your S. pombe gene of interest. You can find this information on the PomBase gene page or by searching for the protein sequence using the gene ID at NCBI protein database
• Use a protein BLAST tool: Options include NCBI BLAST or EBI BLAST
• Set the database to "human protein" and run the search. BLAST will identify human proteins with the most sequence similarity to your S. pombe protein.
• Evaluate the results: The top hits with significant sequence similarity (indicated by E-value) are potential human orthologs. Analyze the protein function and domain architecture of these human genes to determine the most likely ortholog.