| Although most species grow at normal temperature ranging from 20°C to 50°C, some microorganisms have been found able to thrive far beyond this range. Those the growth temperatures of which are beyond 50°C are thermophiles. The mechanism of the thermophilic adaptation of the thermophiles has been a very interesting topic to which many works have been devoted. Due to the execrable condition the thermophiles live in, it is difficult researching it by means of the experimental methods. Thanks to the development of the genomics and bioinformatics, more and more complete genomes of the thermophiles have been sequenced and the increased data made it easier to research the thermophiles.Research in the thermophilic adaptation of the thermophiles mainly focused on the gene and protein levels. Our former work has revealed that there exists a strong positive correlation between the percentage of predicted highly expressed (PHX) genes and the optimal growth temperatures (OGTs) of several deep-sea microorganisms. This enlightens us about the contribution from gene expression level to thermophilic adaptation. 33 prokaryotes with different OGTs are chosen in present work and they are analyzed in the aspect of gene expression level. However, the gene function may be influential to the results, and annotation of these prokaryotes in public databases is not satisfactory resulting in the mass of the unknown function genes. Consequently, the first step in present work is the reannotation of genomes. To describe easily, two hyperthermophiles, Pyrococcus abyssi and Pyrococcus furiosus are taken for examples. First, non-coding ORFs are recognized and then translation initiation sites (TISs) of all the annotated genes have been re-located, both of which are based on the Z-curve theory. At last, functions of the refined'hypothetical genes'are predicted by using sequence alignment tools such as Blast and COGs, and more than 200 originally annotated'hypothetical genes'in either of the two hyperthermophiles have been assigned with functions.Then, by analyzing the predicted gene expression levels of 33 prokaryotes by E(g) and CAI, a universal positive correlation was found between the percentage of predicted highly expressed genes and the organisms' optimal growth temperature. Attempting to trace the imprint of the phenomena, a comprehensive indicator for the thermostability of protein based on amino acids composition, CIT, is defined. The proteins coded by the highly express genes have higher CIT values, meaning that they are more thermostable. Besides, as AG content and ApG content have been regarded as an indicator of the DNA stability, this indicator of the highly expressed genes are also calculated, with the finding that the AG and ApG content of the highly expressed genes are significant higher than that of lowly expressed genes. So, these results indicate that highly expressed genes are statistically more thermostable than lowly expressed genes on both protein and DNA levels. These findings show the possibility of the significant contribution of gene expression level to the prokaryotic thermal adaptation and provide evidence for the translational selection pressure on the thermostability of natural proteins during evolution.Moreover, contribution of topology of protein tertiary structure in the thermostability is also referred to. Nine parameters collected from literature of 127 proteins of which the PDB structures and melting temperatures have been determined are calculated. However, because of the lack of the protein data, it is hardly to find a definite result. Despite, this work could be regarded as a basis for the advanced research in this topic.
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