|Injection of C. elegans Gonads |
In this video, Amy Fabritius, a post-doctoral fellow in Dr. Andy Golden's lab, demonstrates microinjection of DNA into the gonad of C. elegans hermaphrodites. This is an essential skill one needs to master to edit the genome using the CRISPR/Cas9 tools.
|mel-15 embryos lack paternal DNA|
Wild type and mel-15(it7) hermaphrodites were shifted to 24°C as L4s. After ~24 hours, hermaphrodites were dissected and embryos imaged with a confocal microscope. Both maternal and paternal DNA are present in wild type embryos (paternal DNA, arrow). Embryos fertilized by mel-15(it7) males do not have paternal DNA, but other paternal products (i.e. centrosomes, arrowheads) are transferred to the embryo during fertilization. M=maternal DNA, P=paternal DNA, white circle demarks area of missing paternal DNA. Red: Histone::mCherry. Green: nuclear pore::GFP, tubulin::GFP. Scale bar= 30 μm.
|Figure 3. Model of yeast prion amyloid structure|
The parallel in-register architecture of yeast prion amyloids features the favorable interaction between aligned identical amino acid side-chains. This structure can explain how proteins can template their own conformation and how a protein can act as a gene.
|Figure 2. Prion domains of yeast prion proteins|
For each of the yeast and fungal prions, a restricted portion of the protein is necessary and sufficient for generation and propagation of the prion. These domains are generally rich in glutamine and asparagine and are the part of the protein that comprises the core of the amyloid in an in-register parallel structure.
|Figure 1. Three genetic criteria for a yeast prion|
[URE3] is a non-Mendelian genetic element of S. cerevisiae that makes cells able to take up ureidosuccinate when ammonia is the nitrogen source. [PSI] is a non-Mendelian genetic element that increases the efficiency with which weak suppressor tRNAs allow read-through of translation termination codons. Three properties of these genetic elements indicated to us that they were prions of Ure2p and Sup35p, respectively: (1) reversible curability, (2) overproduction of the prion protein inducing prion formation de novo, and (3) similarity of prion phenotype and phenotype of mutation of the gene encoding the prion protein (with prion propagation depending on that gene). These three properties allowed us to first identify these yeast non-chromosomal genetic elements as prions.
|Spectra of poly-L-lysine.||Circular dichroism, absorbance, and g-factor spectra of poly-L-lysine, in the random coil (H2O), alpha-helical (TFE), and beta-sheet (SDS) forms.||Enlarge|
|Biosynthetic pathway E. coli||This image depicts a diagram of the major polyamine biosynthetic pathway in E. coli. Plants have a nearly similar pathway from ornithine and arginine; humans and other animals mostly have the ornithine decarboxylase pathway.||Enlarge|
|Ure2-GFP aggregates in a [URE3] prion cell.||Ure2-GFP fluorescence in a strain containing [URE3] and in a strain lacking the prion ([ure-o])||Enlarge|
|Model of two amyloid variants.||The model depicts two variant structures of a yeast prion amyloid formed by in-register parallel beta sheets. The amyloid filament direction is indicated by a big gray arrow. Parallel interacting sheets of neighboring proteins are indicated by aligned black and white arrows. The yellow oval and star represent two of the in-register amino acids.||Enlarge|
|Filaments of Ure2 protein in a [URE3] prion cell.||Electron microscopy reveals filaments (red) of Ure2 protein in a [URE3] prion cell.||Enlarge|