Studies on the Triangular-shaped Extremely Halophilic Archaeon

盐生盐藻(Haloarcula Ja Onica)是一株以三角形圆盘状嗜盐古生菌为主的菌株，迄今已对该菌株的分类特征、细胞分裂方式和细胞膜超微结构进行了研究。然而，维持三角形的机制仍然不清楚。我们证明了在其细胞表面存在一种糖蛋白(细胞表面糖蛋白：CSG)，这似乎对维持日本血吸虫特有的形状是重要的。对日本血吸虫CSG进行了纯化和鉴定。经十二烷基硫酸钠-聚丙烯酰胺凝胶电泳法测定，其表观分子量为170 kDa，糖类含量约为12%(w/w)。多肽部分含有较大比例的酸性氨基酸。合成了针对盐生盐杆菌和盐生盐藻CSGs之间保守区的寡核苷酸，并将其作为聚合酶链式反应的引物。从染色体…中扩增出与CSG相关的序列用聚合酶链式反应扩增日本血吸虫csg基因，扩增出约890bp的DNA片段，在大肠杆菌中进行克隆和测序。根据预测的氨基酸序列，日本血吸虫的CSG与Hb、Halobium和Hf.Volcaniia的CSG有明显的同源性。用EcoRI酶切日本血吸虫染色体DNA，将其连接到载体上，转化大肠杆菌，构建基因组文库。以扩增产物为探针，通过菌落杂交对转化子进行筛选。阳性克隆含有相同的3.5kb EcoRI插入片段的重组质粒。部分核苷酸序列表明CSG基因的5‘端和上游区域。因此，通过Walding基因从日本哈密尔顿的另一个基因组文库中克隆了2.5kb的KpnI片段。3.5kb和2.5kb片段重叠1.4kb。将上述3.5kb和2.5kb的KpnI-EcoRI片段在相同的SacI位点进行重组，获得了4.6kb的KpnI-EcoRI片段，并将其插入到pUC119载体中构建了表达载体pJAG16。较少

Haloarcula japonica strain TR-1 is a predominantly triangular disc-shaped halophilic archaeon.Taxonomic characteristics, mode of cell division and ultrastructure of cell envelope have been studied for the strain to date. However, the mechanisms maintaing the triangular shape have been still unclear. We demonstrated occurrence of a glycoprotein on its cell surface (cell surface glycoprotein : CSG), which seemed to be important in maintenance of the characteristic shape of Ha.japonica. The Ha.japonica CSG was purified and characterized. It had an apparent molecular mass of 170 kDa in sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE)and the carbohydrate content was about 12%(w/w). The polypeptide portion contained a large proportion of acidic amino acids. Oligonucleotides, directed against conserved regions between the CSGs from Halobacterium halobium and Haloferax volcanii, were synthesized and used as PCR primers. CSG-related sequences were amplified from the chromosom … More al DNA of Ha.japonica by PCR.The PCR-generated DNA fragment of about 890 bp, presumed to be a part of the CSG gene of Ha.japonica, was cloned in Escherichia coli and sequenced. According to the predicted amino acid sequence, the CSG from Ha.japonica showed apparent homology with those from Hb, halobium and Hf.volcanii.Chromosomal DNA from Ha.japonica was digested with EcoRI,ligated to a vactor plasmid, and then transformed into E.coli to construct a genomic library. Transformants were screened by colony hybridization using the PCR product as a probe. Positive clones were found to contain recombinant plasmids with an identical 3.5-kb EcoRI insert. The partial nucleotide sequence indicated that the 5'end of the CSG gene and upstream region. Therefore, the KpnI fragment of 2.5 kb was cloned from another genomic library of Ha.japonica by gene walding. The 3.5-kb and 2.5-kb fragments overlapped by 1.4 kb. The 4.6-kb KpnI-EcoRI fragment was obtained by recombining the above 3.5-kb and 2.5-kb fragments at a common SacI site, and then inserted in pUC119 vector to construct plasmid pJAG16.SDS-PAGE of cell extract of E.coli harboring pJAG16 revealed that the Ha.japonica CSG gene was expressid in E.coli. Less