Related Experiment Video
Updated: Aug 12, 2026
14:57
Yeast As a Chassis for Developing Functional Assays to Study Human P53
Published on: August 4, 2019
Expression of a cDNA derived from the yeast killer preprotoxin gene: implications for processing and immunity
Summary
Yeast killer toxin and immunity are encoded by M1-dsRNA. Researchers created a fusion plasmid expressing a preprotoxin, demonstrating that both toxin and immunity are within this precursor molecule.
Area of Science:
- Molecular Biology
- Yeast Genetics
- Protein Biochemistry
Background:
- Type I killer strains of Saccharomyces cerevisiae produce a 19-kDa dimeric toxin that disrupts the cytoplasmic membrane of sensitive cells.
- The toxin and immunity are encoded by the M1-dsRNA plasmid.
- A 35-kDa preprotoxin is the presumed in vivo gene product.
Purpose of the Study:
- To investigate the structure and maturation of the yeast killer toxin preprotoxin.
- To demonstrate that both toxin and immunity determinants reside within the preprotoxin molecule.
- To facilitate studies on preprotoxin processing and glycosylation.
Main Methods:
- Constructed a yeast expression vector (p1A1) with a partial M1-dsRNA cDNA copy under the PHO5 promoter.
- Created an in-frame gene fusion replacing the native secretion leader with the PHO5 leader sequence.
- Transformed sensitive yeast strains lacking M1-dsRNA with the fusion plasmid.
- Utilized L-1-Tosylamido-2-phenylethyl chloromethyl ketone to inhibit glycosylation for comparative analysis.
Main Results:
- Transformed yeast strains became phosphate-repressible, immune killers, confirming the presence of both toxin and immunity within the preprotoxin.
- Preprotoxin processing involves three glycosylation events and does not require cotranslational leader peptidase action.
- The PHO5 leader sequence was removed from the fusion preprotoxin, unlike the native secretion leader.
Conclusions:
- The preprotoxin molecule contains all necessary determinants for both killer toxin activity and immunity.
- The PHO5 promoter provides a controllable system for studying preprotoxin expression and maturation in yeast.
- Understanding the glycosylation and processing of preprotoxin offers insights into protein maturation pathways in Saccharomyces cerevisiae.
Related Concept Videos
01:28Yeast Signaling
Yeasts are single-celled organisms, but unlike bacteria, they are eukaryotes (cells with a nucleus). Cell signaling in yeast is similar to signaling in other eukaryotic cells. A ligand, such as a protein or a small molecule released from a yeast cell, attaches to a receptor on the cell surface. The binding stimulates second-messenger kinases to activate or inactivate transcription factors that further regulate gene expression. Many of the yeast intracellular signaling cascades have similar...
02:01pre-mRNA Processing
In eukaryotic cells, transcripts made by RNA polymerase are modified and processed before exiting the nucleus. Unprocessed RNA is called precursor mRNA or pre-mRNA to distinguish it from mature mRNA.
Once about 20-40 ribonucleotides have been joined together by RNA polymerase, a group of enzymes adds a “cap” to the 5’ end of the growing transcript. In this process, a 5’ phosphate is replaced by modified guanosine that has a methyl group attached to it (7-Methyl guanosine). This 5’ cap helps the...
Once about 20-40 ribonucleotides have been joined together by RNA polymerase, a group of enzymes adds a “cap” to the 5’ end of the growing transcript. In this process, a 5’ phosphate is replaced by modified guanosine that has a methyl group attached to it (7-Methyl guanosine). This 5’ cap helps the...
02:32Exon Recombination
The evolution of new genes is critical for speciation. Exon recombination, also known as exon shuffling or domain shuffling, is an important means of new gene formation. It is observed across vertebrates, invertebrates, and in some plants such as potatoes and sunflowers. During exon recombination, exons from the same or different genes recombine and produce new exon-intron combinations, which might evolve into new genes.
Exon shuffling follows “splice frame rules.” Each exon has three reading...
Exon shuffling follows “splice frame rules.” Each exon has three reading...
01:36What is Gene Expression?
A gene is a stretch of DNA that serves as the blueprint for functional RNAs and proteins. Since DNA is comprised of nucleotides and proteins are comprised of amino acids, a mediator is required to convert the information encoded in DNA into proteins. This mediator is the messenger RNA (mRNA). mRNA copies the blueprint from DNA by a process called transcription. In eukaryotes, transcription occurs in the nucleus by complementary base-pairing with the DNA template. The mRNA is then processed and...
01:31Antigen Processing Pathways
MHC molecules are key players in the immune response, enabling T cells to recognize and respond to specific antigens. They are present on the surface of all nucleated cells in the body and are instrumental in presenting antigens to T cells and activating them. T cells recognize the MHC-antigen complex and initiate an immune response. MHC class I and MHC class II are two main types of MHC molecules, each associated with a distinct antigen processing pathway.
MHC Class I: Presenting Endogenous...
MHC Class I: Presenting Endogenous...
01:22T Cell Activation and Clonal Selection
T cells are integral to our adaptive immune system, recognizing and effectively responding to foreign antigens. T cell activation and clonal selection are pivotal in orchestrating this immune response. This article elucidates these mechanisms, detailing the roles of cluster of differentiation (CD) markers, major histocompatibility complex (MHC) molecules, costimulatory signals, and the process of clonal selection.
Naive T cells that have not yet encountered an antigen express two primary CD...
Naive T cells that have not yet encountered an antigen express two primary CD...