Cloning And Sequencing Of A CDNA Coding A Putative Ser/thr Protein Kinase From Thermomyces Lanuginosus

All living cells must constantly be interfered by all kinds of factors during their life cycle of proliferation, differentiation, development and death. These factors include not only the intercellular hormones but also extracellular light, temperature and water so on. All these request the cell to recognize and receive these signal factors correctly and respond to them as soon as possible to control the courses of progression and cellular activities. In this way, the receivers lie in the membrane of the cell recognizing the external ligand. Then the external signals are transducted into the nucleolus to regulate the gene expression and enzyme activity leading to the cell response. We can conclude that signal transduction plays an important role in the processes of environment-cell interaction.A lot of studies have resulted that kinases and phosphatases are likely to be important mediators of signal transduction. Through phosphrylation and dephosphorylation, the activity and structure of protein changed. External signal are transducted into the cell nucleolus by the signal cascade. Protein kinase mediated phosphorylation regulates protein function, membrane channels and pumps and transcription factors by which changed the gene expression. Indian scientist Ray found in 1994 that three membrane proteins they isolated from Pseudomonas syringae which were phosphorylated in response to temperature changes and resulted that one of these proteins was possibly a histidine kinase. Thermomyces lanuginosus is a kind of heat-endurable fungus which can live normally at about 50℃.In order to probe the molecular mechanism of the characteristics of heat-enduration we have been trying to clone the gene of a protein kinase and looking for the molecules upstream and downstream of it. Using RT-PCR, one 1243bp cDNA fragment was obtained. Sequencing analysis showed that the cloned cDNA of T. lanuginosus was similar to that of fission yeast protein kinase dsk. Comparison results showed that the homogeneity of the 3'-deduced amino acids was above 60%. Six conservative amino acid subdomains which most ser/thr protein kinase contain can be found in the deduced amino acid sequence. Comparison of the necleotide sequence resulted that the homogeneity is not high(10%). Homogeneity domains is as short as 40nt. However, we can find that GC% of the cDNA is higher (about 56%), which showed an organism specificity. Looking into the preference codons of T. lanuginosus , we found the average value of GC% is about 53%. We surmise that such a high GC% value may be important factor for this kind of fungus tolive normally at about 50℃. We tried to obtain the 5'-end of the cDNA by 5'RACE(rapid amplification of cDNA ends). Unfortunately ,due to the complexity of 5'RACE and use of a single gene-specific primer(with the anchor primer),the length of 5'-end fragment we estimated is too long to be captured. We failed to isolate the 5'-ends of the cDNA. Southern blot analysis showed that T. lanuginosus genome may contain one homology of the cDNA fragment.Bioinformatics is pushed ahead quickly because of the internet development. Using the internet information to explore the distinction of life becomes one major research field. Human genome project was carried out and brought rapid growth to bioinformation. All these make it possible to use bioinformation for gene cloning, sequence analysis and protein function prediction. We used the bioinformation in internet to predict the transmembane domain, secondary structure and 3D structure for the deduced amino acid sequence of the cDNA fragment.