Ling Hong

Generation and initial analysis of more than 15,000 full-length human and mouse cDNA sequences.

The National Institutes of Health Mammalian Gene Collection (MGC) Program is a multiinstitutional effort to identify and sequence a cDNA clone containing a complete ORF for each human and mouse gene. ESTs were generated from libraries enriched for full-length cDNAs and analyzed to identify candidate full-ORF clones, which then were sequenced to high accuracy. The MGC has currently sequenced and verified the full ORF for a nonredundant set of >9,000 human and >6,000 mouse genes. Candidate full-ORF clones for an additional 7,800 human and 3,500 mouse genes also have been identified. All MGC sequences and clones are available without restriction through public databases and clone distribution networks (see http://mgc.nci.nih.gov).

A Novel Golgi Retention Signal RPWS for Tumor Suppressor UBIAD1

UBIAD1 plays critical roles in physiology including vitamin K and CoQ10 biosynthesis as well as pathophysiology including dyslipimedia-induced SCD (Schnyder’s corneal dystrophy), Parkinson’s disease, cardiovascular disease and bladder carcinoma. Since the subcellular localization of UBIAD1 varies in different cell types, characterization of the exact subcellular localization of UBIAD1 in specific human disease is vital for understanding its molecular mechanism. As UBIAD1 suppresses bladder carcinoma, we studied its subcellular localization in human bladder carcinoma cell line T24. Since fluorescent images of UBIAD1-EGFP in T24, human prostate cancer cell line PC-3, human embryonic kidney cell line HEK293 and human hepatocyte cell line L02 are similar, these four cell lines were used for present study. Using a combination of fluorescent microscopy and immunohistochemistry, it was found that UBIAD1 localized on the Golgi and endoplasmic reticulum (ER), but not on the plasma membrane, of T24 and HEK293 cells. Using scanning electron microscopy and western blot analysis, we found that UBIAD1 is enriched in the Golgi fraction extracted from the L02 cells, verifying the Golgi localization of UBAID1. Site-directed mutagenesis showed that the RPWS motif, which forms an Arginine finger on the UBIAD1 N terminus, serves as the Golgi retention signal. With both cycloheximide and brefeldin A inhibition assays, it was shown that UBIAD1 may be transported from the endoplasmic reticulum (ER) to the Golgi by a COPII-mediated mechanism. Based upon flow cytometry analysis, it is shown that mutation of the RPWS motif reduced the UBIAD1-induced apoptosis of T24 cells, indicating that the proper Golgi localization of UBIAD1 influences its tumor suppressant activity. This study paves the way for further understanding the molecular mechanism of UBIAD1 in human diseases.

Vitamin K2 Induces Mitochondria-Related Apoptosis in Human Bladder Cancer Cells via ROS and JNK/p38 MAPK Signal Pathways.

The effects of vitamin K2 on apoptosis in a variety of cancer cells have been well established in previous studies. However, the apoptotic effect of vitamin K2 on bladder cancer cells has not been evaluated. The aim of this study is to examine the apoptotic activity of Vitamin K2 in bladder cancer cells and investigate the underlying mechanism. In this study, Vitamin K2 induced apoptosis in bladder cancer cells through mitochondria pathway including loss of mitochondria membrane potential, cytochrome C release and caspase-3 cascade. Furthermore, the phosphorylation of c-Jun N-terminal kinase (JNK) and p38 MAPK was detected in Vitamin K2-treated cells and both SP600125 (an inhibitor of JNK) and SB203580 (an inhibitor of p38 MAPK) completely abolished the Vitamin K2-induced apoptosis and loss of mitochondria membrane potential. Moreover, the generation of reactive oxygen species (ROS) was detected in bladder cancer cells, upon treatment of vitamin K2 and the anti-oxidant N-acetyl cysteine (NAC) almost blocked the Vitamin K2-triggered apoptosis, loss of mitochondria membrane potential and activation of JNK and p38 MAPK. Taken together, these findings revealed that Vitamin K2 induces apoptosis in bladder cancer cells via ROS-mediated JNK/p38 MAPK and Mitochondrial pathways.

Extensive structural conservation exists among several homologs of two Euglena chloroplast group II introns.

Abstract 82 of the 155 chloroplast introns in Euglena gracilis have been categorized as group II introns. Because they are shorter and more divergent than group II introns from other organisms, the assignment of these Euglena introns to the group II class has been questioned. In the current study, two homologs of E. gracilispetB intron 1 and four homologs of psbC intron 2 have been isolated from related species and characterized. Based on a comparative sequence analysis of intron homologs, the intron core and four of the six helical domains present in the canonical group II intron structural model are conserved in E. gracilispetB intron 1 and psbC intron 2 and all of their homologs. Distal portions of domain I, which are involved in most of the tertiary interactions, are less well conserved than the central core.

Two new group-II twintrons in the Euglena gracilis chloroplast are absent in basally branching Euglena species

Abstract Studies of the phylogeny and chloroplast intron content of selected Euglena species have led to insights in our understanding of the timing of intron acquisition. In the current study, two new twintrons, found in E. gracilis, have been characterized by the analysis of partially spliced pre-mRNAs. Intron 1 of atpE is a 463-nt group-II intron interrupted by a second group-II intron 320 nt long. Intron 1 of psbD is also a group-II twintron with external and internal introns of 635 nt and 463 nt, respectively. The two introns composing the psbD twintron, as well as six additional group-II introns found in the E. gracilispsbD gene, are not present in several basally branching Euglena species, including E. myxocylindracea, E. stellata and E. viridis. The distribution of psbD introns in Euglena is consistent with a late evolutionary acquisition of group-II introns in this lineage.

Heixuedian (heix), a potential melanotic tumor suppressor gene, exhibits specific spatial and temporal expression pattern during Drosophila hematopoiesis

The Drosophila heixuedian (heix) is the ortholog of human UBIAD1 gene (a.k.a TERE1). The protein product of UBIAD1/heix has multiple enzymatic activities, including the vitamin K2 and the non-mitochondrial CoQ10 biosynthesis. However, the expression pattern of UBIAD1/Heix during metazoan development has not been systematically studied. In this paper, we found that loss of function of heix resulted in pathological changes of larval hematopoietic system, including lymph gland hypertrophy, hemocyte overproliferation and aberrant differentiation, and melanin mass formation. Overexpression of heix cDNA under the tubulin Gal4 driver rescued the above hematopoietic defects. Interestingly, Heix was specifically expressed in plasmatocyte/macrophage lineage in srp driven EGFP positive cells on the head mesoderm during embryogenesis, while it was highly expressed in crystal cells in the primary lobes of the third instar larval lymph gland. Using qRT-PCR analysis, loss of function of heix caused aberrant activation of multiple hemocyte proliferation-related as well as immune-related pathways, including JAK/STAT pathway, Ras/MAPK pathway, IMD pathway and Toll pathway. These data suggested that heix is a potential melanotic tumor suppressor gene and plays a pivotal role in both hemocytes proliferation and differentiation in Drosophila.

Transcriptome sequencing and annotation of the halophytic microalga Dunaliella salina

The unicellular green alga Dunaliella salina is well adapted to salt stress and contains compounds (including β-carotene and vitamins) with potential commercial value. A large transcriptome database of D. salina during the adjustment, exponential and stationary growth phases was generated using a high throughput sequencing platform. We characterized the metabolic processes in D. salina with a focus on valuable metabolites, with the aim of manipulating D. salina to achieve greater economic value in large-scale production through a bioengineering strategy. Gene expression profiles under salt stress verified using quantitative polymerase chain reaction (qPCR) implied that salt can regulate the expression of key genes. This study generated a substantial fraction of D. salina transcriptional sequences for the entire growth cycle, providing a basis for the discovery of novel genes. This first full-scale transcriptome study of D. salina establishes a foundation for further comparative genomic studies.摘要目 的解析杜氏盐藻代谢过程, 主要关注盐胁迫下累积的代谢物 (渗透平衡产物、多胺和类胡萝卜素) 的代谢。创新点本研究通过高通量测序产生了大量来自杜氏盐藻整个生长周期的转录组数据, 描述了杜氏盐藻在盐胁迫下累积的渗透平衡产物、 多胺和类胡萝卜素的代谢过程。另外通过该手段也进一步分析了盐胁迫处理下, 抑制精胺合成底物的供应可能会缓解盐藻增殖对胡萝卜素含量的影响。方 法以来自3 个不同生长时期的杜氏盐藻为材料, 进行大规模转录组测序。 在转录组功能注释的基础上, 预测了杜氏盐藻盐胁迫下累积的渗透平衡产物 (图3)、 多胺 (图4) 和类胡萝卜素 (图5) 的代谢路径。 利用相对定量聚合酶链反应 (qPCR) 技术构建了相关代谢路径中关键基因的表达谱 (图6)。结 论通过杜氏盐藻转录组测序共获取了 39820 条单一序列。 在功能注释和聚类分析的基础上预测了杜氏盐藻盐胁迫下累积的渗透平衡产物 (甘油和脯氨酸)、 多胺以及类胡萝卜素的代谢路径。 相关代谢途径的关键酶的表达谱分析, 说明盐能够调节甘油、脯氨酸以及多胺的代谢过程。 抑制精胺合成底物的供应可能会缓解盐藻增殖对胡萝卜素含量的影响。