1. Screening for genes important cold acclimatization in Neurospora crassa.
Holly Howell*, Victor Manzanilla*
(*=Undergraduate Researchers)
Valparaiso University
Valparaiso, Indiana
Abstract:
The environmental conditions that life must contend with can vary widely. Organisms have evolved a wide range of mechanisms for contending with these changing conditions. For relatively simple, sessile organisms (such as fungi), they must adapt themselves to the extremes of the environment. When subjected to rapid drops of temperature (cold shock), Neurospora responds with a dramatic, but temporary shift in its branching pattern. The fungal response, seems to parallel the physiological response to similar cold shock which has been observed in E. coli in which a set of genes is activated transiently in order to adjust the cell for growth in the cold. While the Neurospora response has been described morphologically, it has yet to be examined to any serious extent genetically. This project aimed to begin the genetic characterization of the cold shock response. Selected mutants from the Neurospora knockout library were screened for their response to cold shock with those displaying variation from the wild-type identified and categorized according to phenotype. Several knockout mutants were identified with altered responses. The genes impacted in these mutants are listed and discussed below.
Results:
Failure (pex-4)
Dense (acon-3)
“cot-like” (pex-14)
Vision:
Understanding how life adjusts to survive stressful environments (including cold) will help determine how terrestrial organisms could be modified to make them more suited for these extremes as well as predict the characteristics of organisms endogenous to such environments. This will prove valuable in designing experiments aimed at detecting these organisms.
Objectives:
Identify mutants which alter the response to cold shock, and thus identify genes important to cold acclimatization in Neurospora. These results will be used to identify classes of gene functions which will then be the subject of later studies.
Methods:
Selected gene knockout strains were already in-hand in the lab. These strains were tested for their response to cold shock to determine their cold acclimatization capacities. Mutants were grown at 33°C and then subjected to a rapid shift to 4°C and scored and photographed the following day. Wild-type strains go through a predictable multistage morphological response following this shift as they adjust to cold growth conditions. Deviations from this normal response were used to identify strains with mutations in cold acclimatization.
Conclusions:
~200 selected mutant strains from the Neurospora knockout library were screened for alterations to their response to cold shock.
35 strains with variant cold shock responses
The altered responses fell into several phenotypic categories.
The genes impacted in each knockout strain were identified
The genes involved appear to be involved in a number of different cellular functions including lipid/membrane metabolism, protein degradation/turnover, gene regulation, protein regulation, and reactive oxygen control
Wild type cold shock response
Burst (sod-1)
Thin (ACW-10)
Future Work:
Draw connections between genes which play a role in cold shock
Confirm phenotypes
Screen additional knockout strains generally as well as those related to the ones identified here and those related to known Ecoli cold acclimatization genes.
Acknowledgements
This work was supported by a grant from the Indiana Space Grant Consortium (INSGC).