Sequencing of a 5.5-kb DNA Fragment and Identification of a Gene Coding for a Subunit of the Helicase/Primase Complex of Avian Laryngotracheitis virus (ILTV)

The nucleotide sequence of a 5,520-bp EcoRI restriction fragment of avian infectious laryngotracheitis (ILTV) DNA was reported and submitted to GeneBank with an accession number of AF001078. Computer prediction revealed one large potential open reading frame (ORF) with sequence similar to one subunit of the DNA helicase-primase complex of α-herpesviruses. The DNA helicase/primase complex of HSV-1 consists of three sub-units with molecular weights of 12,000, 97,000 and 70,000, encoded by genes UL52, UL5 and UL8, respectively. This enzyme complex is essential for herpesvirus DNA replication. The UL52 and UL5-equivalent genes of ILTV have been reported previously (Fuchs, W. and Mettenleiter, T.C., J Gen Virol, 1996, 77: 2221–2229; Johnson, M.A. et. al., Arch Virol, 1995, 14: 623–634). Amino acid sequence comparison and homology search revealed that this ORF shares sequence similarity to the UL8-equivalent gene of α-herpesviruses, that is, the ORF 52 of vericura-zoster virus (VZV), the ORF 54 of equine herpesvirus type-1 (EHV-1), as well as the equivalent gene of bovine herpesvirus type 1(BHV-1) and canine herpesvirus (Vlcek, C. et al., Virology, 1995, 210: 100–108; Remond, M. et al., J Gen Virol, 1996, 77: 37–48). This revised version was published online in July 2006 with corrections to the Cover Date.     

Increased nasal mucosal interferon and CCL13 response to a TLR7/8 agonist in asthma and allergic rhinitis

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对一种源于蜗牛的壳聚糖水解酶的不同纯化方法的选择

目的研究源于蜗牛的壳聚糖水解酶的纯化方法，得到壳聚糖水解酶的纯品，从而为氨基酸序列分析、基因克隆及工业菌制备奠定前期基础。方法对比疏水作用层析、离子交换层析、羟基磷灰石柱（HA）层析、凝胶过滤层析等方法纯化蜗牛酶的实际效果，确定纯化的最佳条件，从而设计出最合理的纯化方案。结果分别用苯基琼脂糖柱层析，DEAE—Sepharose离子交换层析，羟基磷灰石柱层析，Sephacryl S-300凝胶过滤层析分离蜗牛酶，最终确定为用弱阴离子交换填料DEAD—Sepharose，疏水作用层析，凝胶过滤层析进行分离。结论本文探讨了蜗牛酶的不同纯化方法，建立了一种从蜗牛酶中分离高效高纯度壳聚糖水解酶的方法，为壳寡糖的酶解工业生产提供了新方法。     

微小染色体维持蛋白与复制

微小染色体维持蛋白家族是复制执照系统的重要组成成分,共有八个亚基.该蛋白家族呈周期性表达,调节DNA复制,使得每个细胞周期的DNA复制一次且仅有一次.微小染色体维持蛋白1和10虽然不归属前者,但其功能却与复制密切相关.本文就微小染色体维持蛋白在复制中的作用简单综述.     

Ocular manifestations in the inherited DNA repair disorders

Deoxyribonucleic acid (DNA) repair is a fundamental process designed to keep the integrity of genomic DNA that is continuously challenged by intrinsic or environmental induced alterations. Numerous genes involved in DNA repair have been cloned and are involved in different DNA repair pathways: base excision repair, nucleotide excision repair, mismatch repair, DNA recombination. Inherited conditions due to mutations in DNA repair genes include mainly: xeroderma pigmentosum, Cockayne syndrome, Trichothiodystrophy, Bloom syndrome, Rothmund-Thomson syndrome, and Werner syndrome. Minor to major ocular manifestations occur in these syndromes. For example, eyelid skin cancers in xeroderma pigmentosum and retinal dystrophy in Cockayne syndrome are major features of these syndromes. This review focuses on the DNA repair pathways, the general and ocular features of the related syndromes, the laboratory tests useful for diagnosis, and the general processes implied with DNA repair (ultraviolet sensitivity, carcinogenesis, apoptosis, oxydative stress, and premature aging).     

丙型肝炎病毒解旋酶在大肠杆菌中的表达研究

目的：在大肠肝菌中表达完整的丙型肝炎病毒的解旋酶。方法：用异硫氰酸胍法从HCV阳性的病人血清中提取RNA,经RT－PCR法扩增出NS3区的基因片段,将其克隆入PQE30表达载体中转化大肠杆菌XLI－Blue进行表达。表达出的蛋白用western blot方法鉴定。结果：扩增出的基因片段大小为1350bp,表达出的蛋白分子量为52KD,经Western blot鉴定为阳性。结论：成功地在大肠杆菌中表达出完整的HCV解旋酶蛋白,为HCV的诊断和疫苗研究奠定了基础。     

A Role for the Cytoplasmic DEAD Box Helicase Dbp21E2 in Rhodopsin Maturation and Photoreceptor Viability

Abstract:

The Dbp21E2 (DEAD box protein 21E2) is a member of a family of DEAD box helicases active in RNA processing and stability. The authors used genetic mosaics to identify mutants in Dbp21E2 that affect rhodopsin biogenesis and the maintenance of photoreceptor structure. Analysis of a green fluorescent protein (GFP)-tagged Rh1 rhodopsin construct placed under control of a heat shock promoter showed that Dbp21E21 fails to efficiently transport Rh1 from the photoreceptor cell body to the rhabdomere. Retinal degeneration is not dependent on the Rh1 transport defects. The authors also showed that GFP- and red fluorescent protein (RFP)-tagged Dbp21E2 proteins are localized to discrete cytoplasmic structures that are not associated with organelles known to be active in rhodopsin transport. The molecular genetic analysis described here reveals an unexpected role for the Dbp21E2 helicase and provides an experimental system to further characterize its function.     

MDA5 assembles into a polar helical filament on dsRNA

Melanoma differentiation-associated protein 5 (MDA5) detects viral dsRNA in the cytoplasm. On binding of RNA, MDA5 forms polymers, which trigger assembly of the signaling adaptor mitochondrial antiviral-signaling protein (MAVS) into its active fibril form. The molecular mechanism of MDA5 signaling is not well understood, however. Here we show that MDA5 forms helical filaments on dsRNA and report the 3D structure of the filaments using electron microscopy (EM) and image reconstruction. MDA5 assembles into a polar, single-start helix around the RNA. Fitting of an MDA5 homology model into the structure suggests a key role for the MDA5 C-terminal domain in cooperative filament assembly. Our study supports a signal transduction mechanism in which the helical array of MDA5 within filaments nucleates the assembly of MAVS fibrils. We conclude that MDA5 is a polymerization-dependent signaling platform that uses the amyloid-like self-propagating properties of MAVS to amplify signaling.     

A unique DNA entry gate serves for regulated loading of the eukaryotic replicative helicase MCM2–7 onto DNA

The regulated loading of the replicative helicase minichromosome maintenance proteins 2–7 (MCM2–7) onto replication origins is a prerequisite for replication fork establishment and genomic stability. Origin recognition complex (ORC), Cdc6, and Cdt1 assemble two MCM2–7 hexamers into one double hexamer around dsDNA. Although the MCM2–7 hexamer can adopt a ring shape with a gap between Mcm2 and Mcm5, it is unknown which Mcm interface functions as the DNA entry gate during regulated helicase loading. Here, we establish that the Saccharomyces cerevisiae MCM2–7 hexamer assumes a closed ring structure, suggesting that helicase loading requires active ring opening. Using a chemical biology approach, we show that ORC–Cdc6–Cdt1-dependent helicase loading occurs through a unique DNA entry gate comprised of the Mcm2 and Mcm5 subunits. Controlled inhibition of DNA insertion triggers ATPase-driven complex disassembly in vitro, while in vivo analysis establishes that Mcm2/Mcm5 gate opening is essential for both helicase loading onto chromatin and cell cycle progression. Importantly, we demonstrate that the MCM2–7 helicase becomes loaded onto DNA as a single hexamer during ORC/Cdc6/Cdt1/MCM2–7 complex formation prior to MCM2–7 double hexamer formation. Our study establishes the existence of a unique DNA entry gate for regulated helicase loading, revealing key mechanisms in helicase loading, which has important implications for helicase activation.