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Heat shock proteins and temperature adaptation by native minnows of Kansas Keith B. Gido and Gerald R. Reeck. Our primary goal is to gain an understanding of the genetic basis of the stress response by native stream fishes in Kansas in response to harsh environmental conditions that are predicted to occur as a result of global warming. General circulation models predict that the duration between rainfall events is likely to increase, thus increasing the intensity of drought conditions such as increased stream temperature and toxic solute concentrations (i.e., ammonia). Although heat-shock proteins (HSPs) are known to play an important role in the stress response and thermal tolerance of fishes, little is known about variation in the expression of these proteins among different fish species; particularly among low molecular weight (30 kDa range) and high molecular weight (90 kDa range) HSPs. We contend that an understanding of the genetic regulation of HSPs will help predict the consequences of global climate change on fish populations in the Great Plains. Our goals are to use quantitative genetic and proteomic approaches to examine changes in the HSP expression in prairie stream minnows that are exposed to elevated stream temperatures and ammonia concentrations. The focal species include red shiner (Cyprinella lutrensis), southern redbelly dace (Phoxinus erythrogaster) and central stoneroller (Campostoma anomalum). Zebrafish (Danio rerio) will also be tested as a control, because the genetics of this species are well known and it is a member of the same family (Cyprinidae) as the other species. After the heat-shock response is quantified by Western blotting, we will use quantitative RT-PCR to examine changes in concentrations of HSP-transcripts in these species when exposed to elevated temperatures and ammonia concentrations. We are using a proteomics approach to search for, and identify stress proteins in these fishes. They have developed a technique to use 2-D gels and specialized software to identify proteins that change in intensity under different experimental conditions. This can be followed with an analysis by a MALDE TOF-TOF mass spectrophotometer to perform partial sequencing of protein spots from those gels. These goals are parallel to those of the Ecological Genomics initiative at Kansas State University, in that we will attempt to link genetic variation in DNA and gene expression to the ecological interactions of fishes to various climate change scenarios. Progress to date includes hiring and training one graduate and three undergraduate students to maintain captive populations of stream fishes and run experiments. Methods have been developed for the capture and housing of prairie stream fishes as well as for manipulating environmental conditions (i.e., temperature and ammonia concentrations). Several pilot studies have allowed us to define the thermal limits of the target species. We have determined temperature limits and time durations that can be used to elicit a heat-shock response in these fishes. Muscle from zebrafish has been used for 2D gel analysis of heat-induced proteins. Numerous heat-induced spots (proteins) were observed in several molecular weight regions, thus suggesting induction of several families of heat-shock proteins. RT-PCR has been used for Hsp30, again indicating a heat induction. Zebrafish Hsp30 has been expressed in E. coli and purified, in small amounts. This will be used to test whether zebrafish Hsp30 can function as a chaperone. Future plans include continuing to refine our techniques (extending them to include proteomic identification of protein spots on 2D gels and real-time RT-PCR) and use them to characterize the response in zebrafish muscle under different heat shock regimens. At the same time we will be examining 2D gel patterns of muscle proteins in Kansas minnow species, particularly red shiner, southern redbelly dace, and central stoneroller, and comparing them to that of zebrafish. Results from these experiments will be used as baseline information to develop a proposal to the National Science Foundation.
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