Identification,characterization And Sequence Analysis Of The Genes Differentially Expressed In Hypothalamus Of Rats With Heat Adaptation

The ability of mankind to adapt to the severe environment is of prime interest for both basic-medical and clinical researcher because it is the key factor determining whether mankind can survive the environment when they interact with each other. The high-temperature and high-humidity climate of South-China is one of the major focuses of study on special environment. The army is a unique group who are required to survive such environment and keep fighting capacity. The crucial point to solve the problem is using some effective approaches to help people achieve and conserve adaptation to the environment. However, the knowledge about heat adaptation is limited and its molecular mechanism is still uncertain, although the fact of acclimatization is widely accepted and its physiological mechanism has been elucidated clearly. Little is known about the function of hypothalamus in heat adaptation on molecular level, and the differential expression of the genes in this tissue of heat-adaptive organisms has not been reported, though it plays an important role in lots of regulatory functions of the whole organism including body temperature regulation. So, there are not more effective methods to improve heat adaptation and evaluate its level than some traditional ones. ?? Aim: Investigating the differential expression of the genes in the hypothalamus of rats with heat adaptation using differential display PCR in order to clarify the heat adaptation molecular mechanism. Method: The differential display PCR technique was improved on the basis of former studies and was used to identify the differentially expressed genes. The improvements included repeating the comparison simultaneously, applying one-base anchored primers instead of two-base anchored ones, elongating the primers to more than 20 bases, using high- stringency PCR condition and increasing the dNTP concentration in PCR. Result: A differentially displayed segment was identified which showed greater density in the heat adaptation group than in the control group after total RNA isolation, RT-PCR, sequencing electrophoresis and autoradiography sequentially, and was confirmed by dot-blot hybridization. It was cloned and sequenced to be a 1 64bp DNA sequence. This sequence was then registered in the genebank with the accession number of BG642391. Similarity analysis revealed that the differentially expressed gene was the homologue of the gene encoding mammalian cytochrome C oxidase subunit Vile. Cytochrome C oxidase(COX) is an enzyme complex at the end of the mitochondrial respiratory chain, catalyzing the redox reaction and energy conversion. This enzyme is conservative in both structure and function, and the mammalian subunit VIIc of COX has great homology with the yeasty subunit VIII which can optimize the function of the holoenzyme. Study on the subunit VIIc from several mammals showed that this subunit is expressed without tissue specificity. Thus, ii can be suggested that the increased expression of this subunit is a generalized reaction of the heat-adaptive mammals and that the improved aerobic respiration contributes a lot to mammals?maintaining heat adaptation. Conclusion: The differential display PCR was improved. An increasingly expressed gene was identified from heat-adaptive rats? ?? hypothalamus...