Dissection of the methyl-CpG binding domain from the chromosomal protein MeCP2.

MeCP2 is a chromosomal protein which binds to DNA that is methylated at CpG. In situ immunofluorescence in mouse cells has shown that the protein is most concentrated in pericentromeric heterochromatin, suggesting that MeCP2 may play a role in the formation of inert chromatin. Here we have isolated a minimal methyl-CpG binding domain (MBD) from MeCP2. MBD is 85 amino acids in length, and binds exclusively to DNA that contains one or more symmetrically methylated CpGs. MBD has negligable non-specific affinity for DNA, confirming that non-specific and methyl-CpG specific binding domains of MeCP2 are distinct. In vitro footprinting indicates that MBD binding can protect a 12 nucleotide region surrounding a methyl-CpG pair, with an approximate dissociation constant of 10(-9) M.     

Recognition of methylated DNA through methyl-CpG binding domain proteins

DNA methylation is a key regulatory control route in epigenetics, involving gene silencing and chromosome inactivation. It has been recognized that methyl-CpG binding domain (MBD) proteins play an important role in interpreting the genetic information encoded by methylated DNA (mDNA). Although the function of MBD proteins has attracted considerable attention and is well characterized, the mechanism underlying mDNA recognition by MBD proteins is still poorly understood. In this article, we demonstrate that the methyl-CpG dinucleotides are recognized at the MBD-mDNA interface by two MBD arginines through an interplay of hydrogen bonding and cation-π interaction. Through molecular dynamics and quantum-chemistry calculations we investigate the methyl-cytosine recognition process and demonstrate that methylation enhances MBD-mDNA binding by increasing the hydrophobic interfacial area and by strengthening the interaction between mDNA and MBD proteins. Free-energy perturbation calculations also show that methylation yields favorable contribution to the binding free energy for MBD-mDNA complex.     

赤拟谷盗功能基因组学研究进展

赤拟谷盗Tribolium castaneum是一种重要的模式生物,在遗传、发育、生化与免疫等研究领域均取得了重要的研究进展。同时它也是一种危害极大的鞘翅目类储粮害虫,在世界各地都有分布,每年给储藏物造成了数十亿美元的经济损失。其全基因组测序的完成、遗传操作体系的构建及系统RNAi方法的应用都极大地促进了其功能基因组学的研究。本文综述了近年来赤拟谷盗基因组计划及功能基因组学的研究进展,拟为赤拟谷盗的生物学研究和防治奠定基础。     

Identification and characterization of novel ER-based hsp90 gene in the red flour beetle,Tribolium castaneum

Heat-shock protein 90 (HSP90) is a highly conserved molecular chaperone found in all species except for Archaea, which is required not only for stress tolerance but also for normal development. Recently, it was reported that HSP83, one member of the cytosolic HSP90 family, contributes to oogenesis and responds to heat resistance in Tribolium castaneum. Here, a novel ER-based HSP90 gene, Tchsp90, has been identified in T. castaneum. Phylogenetic analysis showed that hsp90s and hsp83s evolved separately from a common ancestor but that hsp90s originated earlier. Quantitative real-time polymerase chain reaction illustrated that Tchsp90 is expressed in all developmental stages and is highly expressed at early pupa and late adult stages. Tchsp90 was upregulated in response to heat stress but not to cold stress. Laval RNAi revealed that Tchsp90 is important for larval/pupal development. Meanwhile, parental RNAi indicated that it completely inhibited female fecundity and partially inhibited male fertility once Tchsp90 was knocked down and that it will further shorten the lifespan of T. castaneum. These results suggest that Tchsp90 is essential for development, lifespan, and reproduction in T. castaneum in addition to its response to heat stress.

Electronic supplementary material

The online version of this article (doi:10.1007/s12192-013-0487-y) contains supplementary material, which is available to authorized users.     

Molecular cloning and characterization of neutral ceramidase homologue from the red flour beetle, Tribolium castaneum

Ceramidase plays an important role in regulating the metabolism of sphingolipids, such as ceramide, sphingosine (SPH), and sphingosine-1-phosphate (S1P), by controlling the hydrolysis of ceramide. Here we report the cloning and biochemical characterization of a neutral ceramidase from the red flour beetle Tribolium castaneum which is an important storage pest. The Tribolium castaneum neutral ceramidase (Tncer) is a protein of 696 amino acids. It shares a high degree of similarity in protein sequence to neutral ceramidases from various species. Tncer mRNA levels are higher in the adult stage than in pre-adult stages, and they are higher in the reproductive organs than in head, thorax, and midgut. The mature ovary has higher mRNA levels than the immature ovary. Tncer is localized to the plasma membrane. It uses various ceramides (D-erythro-C₆, C₁₂, C₁₆, C_18:1, and C_24:1-ceramide) as substrates and has an abroad pH optimum for its in vitro activity. Tncer has an optimal temperature of 37 °C for its in vitro activity. Its activity is inhibited by Fe²⁺. These results suggest that Tncer has distinct biochemical properties from neutral ceramidases from other species.     

Identification of microsatellite markers in the red flour beetle,Tribolium castaneum

We isolated and characterized microsatellite markers for the red flour beetle Tribolium castaneum, an important model species for studies in various areas of evolutionary biology and ecology. A microsatellite‐enriched genomic library was constructed and screened with single stranded oligonucleotide probes [(CCT)₁₇, (AAT)₁₇ and (CAG)₁₇]. Forty‐five primer pairs were designed of which 19 pairs produced successful amplification. Polymorphism screening involved beetles from five beetle strains and revealed 15 polymorphic and four monomorphic markers. The development of polymorphic microsatellite markers will facilitate future ecological and genetic studies involving T. castaneum beetles.     

Identification and functional characterization of liposome tubulation protein from magnetotactic bacteria

Magnetotactic bacteria synthesize intracellular magnetosomes that are comprised of membrane‐enveloped magnetic crystals. In this study, to identify the early stages of magnetosome formation, we isolated magnetosomes containing small magnetite crystals and those containing regular‐sized magnetite crystals from Magnetospirillum magneticum AMB‐1. This was achieved by using a novel size fractionation technique, resulting in the identification of a characteristic protein (Amb1018/MamY) from the small magnetite crystal fraction. The gene encoding MamY was located in the magnetosome island. Like the previously reported membrane deformation proteins, such as bin/amphiphysin/Rvs (BAR) and the dynamin family proteins, recombinant MamY protein bound directly to the liposomes, causing them to form long tubules. We established a mamY gene deletion mutant (ΔmamY) and analysed MamY protein localization in it for functional characterization of the protein in vivo. The ΔmamY mutant was found to have expanded magnetosome vesicles and a greater number of small magnetite crystals relative to the wild‐type strain, suggesting that the function of the MamY protein is to constrict the magnetosome membrane during magnetosome vesicle formation, following which, the magnetite crystals grow to maturity within them.     

The methyl-CpG binding domain and the evolving role of DNA methylation in animals

DNA methylation occurs in bacteria, fungi, plants and animals, however its role varies widely among different organisms. Even within animal genomes, methylation patterns vary substantially from undetectable in nematodes, to global methylation in vertebrate genomes. The number and variety of proteins containing methyl-CpG binding domains (MBDs) that are encoded in animal genomes also varies, with a general correlation between the extent of genomic methylation and the number of MBD proteins. We describe here the evolution of the MBD proteins and argue that the vertebrate MBD complement evolved to exploit the benefits and protect against the dangers of a globally methylated genome.