Journal: PLoS ONE

Article Title: Deficient Sumoylation of Yeast 2-Micron Plasmid Proteins Rep1 and Rep2 Associated with Their Loss from the Plasmid-Partitioning Locus and Impaired Plasmid Inheritance

doi: 10.1371/journal.pone.0060384

Figure Lengend Snippet: ( A ) Five-fold serial dilutions of a cir + or cir 0 yeast one-hybrid reporter strain encoding STB upstream of a HIS3 reporter gene and transformed with plasmids encoding the indicated Rep1 and Rep2 proteins fused to B42 AD -HA were spotted onto galactose media to induce expression of fusion proteins. Recruitment of the Rep proteins to STB was monitored by growth on medium lacking histidine supplemented with 5 mM 3-aminotriazole. (B) Levels of the B42 AD -HA fusion proteins were monitored by western blot analysis of total protein extracted from the yeast transformants 24 h after galactose induction. (C) ChIP assays were performed with anti-Rep1, anti-Rep2, or anti-FLAG antibodies and the precipitated DNA amplified using primers specific for STB . ChIP efficiency is indicated by the percent of input DNA immunoprecipitated (avg ± sd from triplicate assays) (left) and ethidium-stained agarose gels of PCR products from a representative assay are shown (right). Template DNA amplified in “input” PCR reactions represented 40% of the DNA that was immunoprecipitated and used as template in “ChIP” PCR reactions.

Article Snippet: PCR products were resolved by agarose gel electrophoresis, stained with ethidium bromide, imaged using a VersaDoc MP 4000 imaging system (BioRad) and quantified by densitometry using QuantityOne software (BioRad).

Techniques: Transformation Assay, Expressing, Western Blot, Amplification, Immunoprecipitation, Staining