ABSTRACT
A universal response to elevated temperature and other forms of physiological stress is the induction of heat shock proteins (HSPs). Hsp16 in Schizosaccharomyces pombe encodes a polypeptide of predicted molecular weight 16 kDa that belongs to the HSP20/alpha-crystallin family whose members range in size from 12 to 43 kDa. Heat shock treatment increases expression of the hsp16 gene by 64-fold in wild-type cells and 141-fold in cdc22-M45 (ribonucleotide reductase) mutant cells. Hsp16 expression is mediated by the spc1 MAPK signaling pathway through the transcription factor atf1 and in addition through the HSF pathway. Nucleotide depletion or DNA damage as occurs in cdc22-M45 mutant cells, or during hydroxyurea or camptothecin treatment, is sufficient to activate hsp16 expression through atf1. Our findings suggest a novel role for small HSPs in the stress response following nucleotide depletion and DNA damage. This extends the types of damage that are sensed by the spc1 MAPK pathway via atf1.
Subject(s)
Chaperonin 60/genetics , Mitogen-Activated Protein Kinases/metabolism , Nucleotides/pharmacology , Schizosaccharomyces pombe Proteins , Schizosaccharomyces/drug effects , Cell Cycle Proteins/genetics , Chaperonin 60/metabolism , Gene Expression Regulation, Fungal/drug effects , Glucose/pharmacology , Green Fluorescent Proteins , Luminescent Proteins/genetics , Luminescent Proteins/metabolism , Microscopy, Fluorescence , Mutation , Nitrogen/pharmacology , RNA, Fungal/drug effects , RNA, Fungal/genetics , RNA, Fungal/metabolism , Recombinant Fusion Proteins/drug effects , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Saccharomyces cerevisiae/genetics , Schizosaccharomyces/genetics , Schizosaccharomyces/metabolism , Signal Transduction/drug effects , Two-Hybrid System TechniquesABSTRACT
An efficient insertional mutagenesis system has been developed for Schizosaccharomyces pombe based on linear PCR-generated cassettes containing selectable markers. It depends upon illegitimate recombination for integration into the genome. Various selectable markers of different sizes can be used to obtain sufficiently high transformation and integration frequencies. Based on Southern blotting, a single insertion is found in each strain and integration sites are broadly distributed in the genome. Sequence analysis of the insert junctions frequently reveals small regions of homology (4-10 bp) between the ends of the integrated cassette and the disrupted gene. The system has been used for simple genetic screens of various types and as a promoter trap for in-frame GFP fusions.
