Journal of Genetics and Genomics

Drosophila sbo regulates lifespan through its function in the synthesis of coenzyme Q in vivo

Jiyong Liu, Qinghua Wu, Dianlu He, Tengyu Ma, Li Du, Wen Dui, Xiaoyan Guo, Renjie Jiao

2011, 38(6): 225-234. doi: 10.1016/j.jgg.2011.05.002

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Abstract:
CoQ is an essential electron carrier in the mitochondrial respiratory chain of both eukaryotes and prokaryotes. It consists of a benzoquinone head group and a hydrophobic polyisoprenoid tail. The genes (COQ1–9) involved in CoQ biosynthesis have been characterized in yeast. In this study, we generated and molecularly characterized a mutant allele of a novel Drosophila gene, sbo, which encodes a protein that is predicted to catalyze the prenylation of p-hydroxybenzoate with the isoprenoid chain during the process of CoQ synthesis. Expression of sbo in yeast rescues the lethality of ∆COQ2 mutant cells, indicating that sbo is a functional homolog of COQ2. HPLC results show that the levels of CoQ₉ and CoQ₁₀ were significantly reduced in sbo heterozygous adult flies. Furthermore, the mean lifespans of males and females heterozygous for sbo are extended by 12.5% and 30.8%, respectively. Homozygous sbo animals exhibit reduced activities of the insulin/insulin-like growth factor signaling (IIS) pathway. Taken together, we conclude that sbo is an essential gene for Drosophila development, mutation of which leads to an extension of lifespan most likely by altering endogenous CoQ biosynthesis.

c-Jun binding site identification in K562 cells

Minli Li, Qinyu Ge, Wei Wang, Jinke Wang, Zuhong Lu

2011, 38(6): 235-242. doi: 10.1016/j.jgg.2011.05.004

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Abstract:
Determining the binding sites of the transcription factor is important for understanding of transcriptional regulation. Transcription factor c-Jun plays an important role in cell growth, differentiation and development, but the binding sites and the target genes are not clearly defined in the whole human genome. In this study, we performed a ChIP-Seq experiment to identify c-Jun binding site in the human genome. Forty-eight binding sites were selected to process further evaluation by dsDNA microarray assay. We identified 283 c-Jun binding sites in K562 cells. Data analysis showed that 48.8% binding sites located within 100 kb of the upstream of the annotated genes, 28.6% binding sites comprised consensus TRE/CRE motif (5′-TGAC/GTCA-3′, 5′-TGACGTCA-3′) and variant sequences. Forty-two out of the selected 48 binding sites were found to bind the c-Jun homodimer in dsDNA microarray analysis. Data analysis also showed that 1569 genes are located in the neighborhood of the 283 binding sites and 191 genes in the neighborhood of the 42 binding sites validated by dsDNA microarray. We consulted 38 c-Jun target genes in previous studies and 16 among these 38 genes were also detected in this study. The identification of c-Jun binding sites and potential target genes in the genome scale may improve our fundamental understanding in the molecular mechanisms underlying the transcription regulation related to c-Jun.

Unraveling the Acidithiobacillus caldus complete genome and its central metabolisms for carbon assimilation

Xiao-Yan You, Xu Guo, Hua-Jun Zheng, Ming-Jiang Zhang, Li-Jun Liu, Yong-Qiang Zhu, Baoli Zhu, Sheng-Yue Wang, Guo-Ping Zhao, Ansgar Poetsch, Cheng-Ying Jiang, Shuang-Jiang Liu

2011, 38(6): 243-252. doi: 10.1016/j.jgg.2011.04.006

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Abstract:
Acidithiobacillus caldus is one of the dominant sulfur-oxidizing bacteria in bioleaching reactors. It plays the essential role in maintaining the high acidity and oxidation of reduced inorganic sulfur compounds during bioleaching process. In this report, the complete genome sequence of A. caldus SM-1 is presented. The genome is composed of one chromosome (2,932,225 bp) and four plasmids (pLAtc1, pLAtc2, pLAtc3, pLAtcm) and it is rich in repetitive sequences (accounting for 11% of the total genome), which are often associated with transposable genetic elements. In particular, twelve copies of ISAtfe and thirty-seven copies of ISAtc1 have been identified, suggesting that they are active transposons in the genome. A. caldus SM-1 encodes all enzymes for the central metabolism and the assimilation of carbon compounds, among which 29 proteins/enzymes were identifiable with proteomic tools. The SM-1 fixes CO₂ via the classical Calvin–Bassham–Benson (CBB) cycle, and can operate complete Embden-Meyerhof pathway (EMP), pentose phosphate pathway (PPP), and gluconeogenesis. It has an incomplete tricarboxylic acid cycle (TCA). Four putative transporters involved in carbohydrate uptake were identified. Taken together, the results suggested that SM-1 was able to assimilate carbohydrates and this was subsequently confirmed experimentally because addition of 1% glucose or sucrose in basic salt medium significantly increased the growth of SM-1. It was concluded that the complete genome of SM-1 provided fundamental data for further investigation of its physiology and genetics, in addition to the carbon metabolism revealed in this study.

Deletion of the topoisomerase III gene in the hyperthermophilic archaeon Sulfolobus islandicus results in slow growth and defects in cell cycle control

Xiyang Li, Li Guo, Ling Deng, Deqin Feng, Yi Ren, Yindi Chu, Qunxin She, Li Huang

2011, 38(6): 253-259. doi: 10.1016/j.jgg.2011.05.001

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Abstract:
Topoisomerase III (topo III), a type IA topoisomerase, is widespread in hyperthermophilic archaea. In order to interrogate the in vivo role of archaeal topo III, we constructed and characterized a topo III gene deletion mutant of Sulfolobus islandicus. The mutant was viable but grew more slowly than the wild-type strain, especially in a nutrient-poor medium. Flow cytometry analysis revealed changes of the mutant in growth cycle characteristics including an increase in proportion of cells containing either more than two genome equivalents or less than one genome equivalent in exponentially-growing cultures. As shown by fluorescence microscopy, a fraction of mutant cells in the cultures were drastically enlarged, and at least some of the enlarged cells were apparently capable of resuming cell division. The mutant also shows a different transcriptional profile from that of the wild-type strain. Our results suggest that the enzyme may serve roles in chromosomal segregation and control of the level of supercoiling in the cell.

Development of pyrF-based gene knockout systems for genome-wide manipulation of the archaea Haloferax mediterranei and Haloarcula hispanica

Hailong Liu, Jing Han, Xiaoqing Liu, Jian Zhou, Hua Xiang

2011, 38(6): 261-269. doi: 10.1016/j.jgg.2011.05.003

Abstract (66) HTML PDF (0)

Abstract:
The haloarchaea Haloferax mediterranei and Haloarcula hispanica are both polyhydroxyalkanoate producers in the domain Archaea, and they are becoming increasingly attractive for research and biotechnology due to their unique genetic and metabolic features. To accelerate their genome-level genetic and metabolic analyses, we have developed specific and highly efficient gene knockout systems for these two haloarchaea. These gene knockout systems consist of a suicide plasmid vector with the pyrF gene as the selection marker and a uracil auxotrophic haloarchaeon (▵pyrF) as the host. For in-frame deletion of a target gene, the suicide plasmid carrying the flanking region of the target gene was transferred into the corresponding ▵pyrF host. After positive selection of the single-crossover integration recombinants (pop-in) on AS-168SY medium without uracil and counterselection of the double-crossover pyrF-excised recombinants (pop-out) with 5-fluoroorotic acid (5-FOA), the target gene knockout mutants were confirmed by PCR and Southern blot analysis. We have demonstrated the effectiveness of these systems by knocking out thecrtB gene which encodes a phytoene synthase in these haloarchaea. In conclusion, these well-developed knockout systems would greatly accelerate the functional genomic research of these halophilic archaea.

2011 Vol. 38, No. 6