Functional Analysis Of A Novel Protein Containing Hmg-box Domain From Tetrahymena Thermophila

High-mobility-group （HMG） proteins are abundant nonhistone chromosomal proteins that are present in a wide range of eukaryotic cells. Three distinct families of HMG proteins have been defined and named based on the structure of their DNA binding domains and their substrate binding specificity, including:HMGA （the HMG-AT-hook family）, HMGN （the HMG-nucleosome binding family）, and HMGB （the HMG-box family）.HMG box is well conserved and has a characteristic domain of about80amino-acid residues consisting of three a-helices arranged in an "L" shape which can fit into the minor groove of duplex DNA and involve in DNA binding. The proteins containing HMG-box are important components of the nucleus that participate in transcription regulation, DNA replication, transcription, repair, recombination, differentiation, apoptosis, and serving as molecular chaperones.The ciliated protozoan Tetrahymena thermophila provides an extreme example for exploring the function of containing HMG-box protein. Tetrahymena contains two nuclei:a germ line micronucleus （MIC） and a somatic macronucleus （MAC） in a single cell. Two small, abundant HMG-like proteins, HMG B and HMG C have been reported. HMG C are abundant only in macronuclei, HMG B is abundant in micronuclei as well as in macronuclei. In vegetative cells, the presence of HMG B in micronuclei correlates well with known periods of micronuclear DNA replication, and in mating cells it correlates with meiotic DNA repair synthesis as well as with scheduled S-phase DNA replications.In this study, we identified a noval HMG-box protein, HmgB3, from the Tetrahymena genome database. Cellular localization and structure of HmgB3were analysed, the results are as follows: 1. Bioinformatics analysis of HMGB3. HMGB3（TTHERM₀0155590） encoded a predicted protein of670amino acids harboring a single HMG-box domain, a SK-rich repeat domain and a neutral carboxy-terminal domain. And S598, S602, S603, S612, S614, S615and S616in the C-terminal domain were phosphoralated and an acidic carboxy-terminal domain was formed. RT-PCR results showed that HmgB3expressed at a low level in vegetative growing stage and upregulated during conjugation stage, especially at the2-4h.2. The immunofluorescence localization of HmgB3. HA-HMGB3was constructed and transformed into CU428and B2086cells, respectively. Transformed cells were selected by paromomycin and identified by PCR. Immunofluorescence localization of HA-HmgB3showed HmgB3is specially localized on the micronucleus during vegetative growth. In starvation stage, HmgB3is also observed around micronuclear memebrane. HmgB3specially localized on the functional micronucleus were related to meiosis and mitotic during conjugation stage. In addition, overexpressed HmgB3abnormally localized at new macronuclei in2MAC-1MIC stage and half of the overexpressed HmgB3progeny did not grow return to nutrient and eventually died.3. The immunofluorescence localization of truncated-HmgB3. To investigate the mechanism of HmgB3micronuclear specific localization, we evaluated the localization of various truncated forms of HmgB3. HMGB3△NC lacking the HMG-box domain and C-terminal strains showed HmgB3△NC diffusion in the cytoplasm during conjugation stage. Removal of the C-terminal tail does not attenuate micronuclear localization. But, once HMG-box was disrupted, HmgB3△N diffuse throughout the cytoplasm, which is the same as the HmgB3△NC cell. This indicated that HMG-box domain is necessary for HmgB3binding to micronuclei. In this study, we first reported a noval HMG-box gene, HMGB3, from the Tetrahymena genome database. Immunofluorescence localization of HA-HmgB3indicated HmgB3was a micronuclear specific protein, and overexpressed HmgB3affected the viability of progeny. These studies provide new ideas for further exploring the function of the HMG box protein in chromosome assembly and transcripition regulation.