Heterochromatin Protein 1 Deleted Chromo Domain Decreases Gene Silencing of Transgene in Mouse

The heterochromatin protein 1 (HP1) regulates epigenetic gene silencing by promoting and maintaining chromatin condensation. To decrease gene silencing, the chromo domain (CD) in the M31 (the main HP1 in mouse) was deleted by site-directed mutagenesis. Vector pcDNA3.1(+)/M31-DeltaCD, in which the M31-DeltaCD is driven by the CMV promoter, and vector pcDNA3.1(+)/P1A3-M31-DeltaCD, in which the M31-DeltaCD is driven by a goat ss-casein promoter were constructed. The former vector was transfected into a murine fibroblast cell line, which can express enhanced green fluorescent protein (EGFP). EGFP expression, which was determined by flow cytometric analysis, increased approximately 80% in the transfected cells. After injection of the latter vector into transgenic mouse mammary glands, which can express human clotting factor IX (hFIX), the hFIX expression level in the mouse milk increased approximately 40-60% and hFIX in one mouse milk was maintained at a high concentration for over 10 days.     

Differential Toxicity of Cis and Trans Isomers of Dichlorodiammineplatinum

Nephrotoxicity is the dose-limiting toxic effect of cis-dichlorodiammineplatinum (cis-platin) in humans. Its stereoisomer transplatin does not have any toxicity at equimolar concentrations, and it also possesses little antitumor activity. In this study, subcellular localization of both the platinum isomers was examined in the liver and kidney of the mouse 24 hours following the drug administration. Levels of the platinum isomers were measured using flame-less atomic absorption. The results showed that higher concentrations of the cis isomer were localized in the liver and kidney, while the concentration of the trans isomer was higher in blood. This indicates that trans isomer is sequestered in the central compartment, whereas cis isomer is distributed in the organs. We also measured metallothionein mRNA and protein levels in both liver and kidney following cisdichlorodiammineplatinum and transdichlorodiammine-platinum treatment to distinguish if the differential toxicity of the two stereo-isomers could be related to metallothionein induction. We report here that cisplatin was capable of inducing metallothionein expression in mice in vivo and that there is an inverse relationship between metallothionein expression and the pattern of tissue toxicity induced by the drug.     

RNA介导的转基因沉默:原理及应用

RNA介导的转基因沉默是有dsRNA参与指导的,以外源和内源mRNA为降解目标的转基因沉默现象。本文对植物中RNA介导的转基因沉默的机理做了详细的阐述,并且将各种生物中在此过程中起关键作用的蛋白及其功能进行了总结,最后介绍dsRNA作为一种基因组学研究手段的优势和它实际的应用前景。 Abstract:RNA-based transgene silencing is a phenomenon that endogenous and exogenous mRNAs are degraded specifically directed by double-stranded RNA.Here we review the recent advances on its mechanism and summarize related proteins and expatiate their functions.Furthermore,we introduce the enormous potential of dsRNA as a tool in functional genomics research and practical biotechnology.     

Heterochromatin, Position Effect, and Genetic Silencing

Genomes of higher eukaryotes consist of two types of chromatin: euchromatin and heterochromatin. Heterochromatin is densely packed material typically localized in telomeric and pericentric chromosome regions. Euchromatin transferred by chromosome rearrangements in the vicinity of heterochromatin is inactivated and acquires morphological properties of heterochromatin in the case of position effect variegation. One of the X chromosomes in mammal females and all paternal chromosome set in coccides become heterochromatic. The heterochromatic elements of the genome exhibit similar structural properties: genetic inactivation, compaction, late DNA replication at the S stage, and underrepresentation in somatic cells. The genetic inactivation and heterochromatin assembly are underlain by a specific genetic mechanism, silencing, which includes DNA methylation and posttranslational histone modification provided by the complex of nonhistone proteins. The state of silencing is inherited in cell generations. The same molecular mechanisms of silencing shared by all types of heterochromatic regions, be it unique or highly repetitive sequences, suggest the similar organization of these regions. No type of heterochromatin is a permanent structure as they all are formed at the strictly definite stages of early embryogenesis. Based on the bulk of evidence accumulated today, heterochromatin can be regarded as a morphological manifestation of genetic silencing.     

Cis and Trans Cooperativity of E-Cadherin Mediates Adhesion in Biomimetic Lipid Droplets

The regulation of cell-cell adhesion is important in cell motility, tissue growth, and for the mechanical integrity of tissues. Although the role of active cytoskeleton dynamics in regulating cadherin interactions is crucial in vivo, here we present a biomimetic emulsion system to characterize the passive E-cadherin-mediated adhesion between droplets. The visualization of a three-dimensional assembly of lipid droplets, functionalized with extracellular E-cadherin domains, reveals a hierarchy of homophilic interactions. First, the high interfacial tension of droplets facilitates trans cadherin-cadherin adhesion, which is strong enough to stabilize looser than random close packing configurations. Second, fluorescence enhancement shows that adding clustering agents, such as calcium or chelating ligands, favor the lateral cis adhesion of the already bound cadherin pairs over the clustering of monomer cadherin on the surface. Finally, above a threshold cadherin and calcium concentration, the cis and trans protein interactions become strong enough to trigger and promote droplet fusion. While E-cadherin is not known to participate in cellular fusion, this mechanism is general because replacing calcium with cholesterol to cluster the cadherin-carrying lipids also promotes fusion. These results suggest that passive clustering, via calcium-induced dimerization or membrane ordering, may contribute to the reinforcement of cell-cell contacts. Alternatively, a molecular switch for fusion offers a route to mixing droplet contents and controlling their size in situ.     

植物转基因沉默研究进展、对策及应用

转基因沉默 (ｔｒａｎｓｇｅｎｅｓｉｌｅｎｃｉｎｇ)是导入并整合进受体基因组中的外源基因在当代转化体或其后代中表达受到抑制的现象。自Ｐｅｅｒｂｏｌｔｅ在 1986年首次报道发现转基因沉默现象以来 ,相关报道不断发表。如今 ,转基因沉默现象已成为遗传转化技术实用化 ,商品化过程中的巨大障碍。从九十年代开始 ,人们就致力于转基因沉默方面的研究。随着研究的不断深入 ,转基因沉默的各种机理不断被揭示 ,并研究出相应的对策。1.植物转基因沉默研究进展转基因沉默的各种机理都涉及到各种核酸之间的相互作用 ,包括转录水平的基因沉默 (ｔ…     

Modulation of Opioid Receptor Binding by Cis and Trans Fatty Acids

In synaptosomal brain membranes, the addition of oleic acid (cis), elaidic acid (trans), and the cis and trans isomers of vaccenic acid, at a concentration of 0.87 mumol of lipid/mg of protein, strongly reduced the Bmax and, to a lesser degree, the binding affinity of the mu-selective opioid [3H]Tyr-D-Ala-Gly-(Me)Phe-Gly-ol ([3H]DAMGO). At comparable membrane content, the cis isomers of the fatty acids were more potent than their trans counterparts in inhibiting ligand binding and in decreasing membrane microviscosity, both at the membrane surface and in the core. However, trans-vacenic acid affected opioid receptor binding in spite of just marginally altering membrane microviscosity. If the receptors were uncoupled from guanine nucleotide regulatory protein, an altered inhibition profile was obtained: the impairment of KD by the fatty acids was enhanced and that of Bmax reduced. Receptor interaction of the delta-opioid [3H](D-Pen2,D-Pen5)enkephalin was modulated by lipids to a greater extent than that of [3H]DAMGO: saturable binding was abolished by both oleic and elaidic acids. The binding of [3H]naltrexone was less susceptible to inhibition by the fatty acids, particularly in the presence of sodium. In the absence of this cation, however, cis-vaccenic acid abolished the low-affinity binding component of [3H]naltrexone. These findings support the membrane model of opioid receptor sequestration depicting different ionic environments for the mu- and delta-binding sites. The results of this work show distinct modulation of different types and molecular states of opioid receptor by fatty acids through mechanisms involving membrane fluidity and specific interactions with membrane constituents.     

正、反杂交二西蜂及其父、母本DNA基因组的多态性研究

使用RAPD-PCR(random amplified polymorphic DNA-polymerase chain reaction）技术,分析了正杂交松丹一号西蜂,反杂交松丹二号西蜂及其父,母本的DNA多态性,结果正杂交松丹一号西峰蜂,反杂交松丹二号西蜂及其父,母本的DNA多态性均有明显的差别,这说明通过正杂交松丹一号与反杂交松丹二号的DNA分子发生了变异。     

Intragenic Cis and Trans modification of genetic susceptibility in DYT1 torsion dystonia

A GAG deletion in the DYT1 gene is a major cause of early-onset dystonia, but clinical disease expression occurs in only 30% of mutation carriers. To gain insight into genetic factors that may influence penetrance, we evaluated three DYT1 single-nucleotide polymorphisms, including D216H, a coding-sequence variation that moderates the effects of the DYT1 GAG deletion in cellular models. We tested DYT1 GAG-deletion carriers with (n=119) and without (n=113) clinical signs of dystonia and control individuals (n=197) and found the frequency of the 216H allele to be increased in GAG-deletion carriers without dystonia and to be decreased in carriers with dystonia, compared with the control individuals. Analysis of haplotypes demonstrated a highly protective effect of the H allele in trans with the GAG deletion; there was also suggestive evidence that the D216 allele in cis is required for the disease to be penetrant. Our findings establish, for the first time, a clinically relevant gene modifier of DYT1.     

Cis and Trans Actions of the Cholinesterase-like Domain within the Thyroglobulin Dimer

Thyroglobulin (Tg, precursor for thyroid hormone synthesis) is a large secreted glycoprotein composed of upstream regions I-II-III, followed by the ∼570 residue cholinesterase-like (ChEL) domain. ChEL has two identified functions: 1) homodimerization, and 2) binding to I-II-III that facilitates I-II-III oxidative maturation required for intracellular protein transport. Like its homologs in the acetylcholinesterase (AChE) family, ChEL possesses two carboxyl-terminal α-helices. We find that a Tg-AChE chimera (swapping AChE in place of ChEL) allows for dimerization with monomeric AChE, proving exposure of the carboxyl-terminal helices within the larger context of Tg. Further, we establish that perturbing trans-helical interaction blocks homodimerization of the Tg ChEL domain. Additionally, ChEL can associate with neuroligins (a related family of cholinesterase-like proteins), demonstrating potential for Tg cross-dimerization between non-identical partners. Indeed, when mutant rdw-Tg (Tg-G2298R, defective for protein secretion) is co-expressed with wild-type Tg, the two proteins cross-dimerize and secretion of rdw-Tg is partially restored. Moreover, we find that AChE and soluble neuroligins also can bind to the upstream Tg regions I-II-III; however, they cannot rescue secretion, because they cannot facilitate oxidative maturation of I-II-III. These data suggest that specific properties of distinct Tg ChEL mutants may result in distinct patterns of Tg monomer folding, cross-dimerization with wild-type Tg, and variable secretion behavior in heterozygous patients.     

Evidence for a Set of Closely Linked Autosomal Genes That Interact with Sex-Chromosome Heterochromatin in DROSOPHILA MELANOGASTER

It is proposed that there exists a special region in the euchromatin of the left arm of chromosome 2 (contained within sections 31-32 of the standard salivary gland chromosome map) that is defined by a set of genes, each one of which interacts with a specific sex-chromosome heterochromatic segment. The evidence for the existence of this region is, first, the exhibition, mapping, and analysis of five different maternal-effect, embryonic semi-lethals located in region 31-32. Secondly, in each case the consequence of the maternal effect is markedly influenced by the amount of X- or Y-chromosome heterochromatin carried by the progeny of mutant mothers. The nature of this interaction and possible reasons for the existence of the cluster of autosomal genes are discussed.     

Organization of the 378-bp Satellite Repeat in Terminal Heterochromatin of Allium fistulosum

Telomeres, DNA–protein structures, are important elements of the eukaryotic chromosome. Telomeric regions of the majority of higher plants contain heptanucleotides TTTAGGG arranged into a tandem repeat. However, some taxa have no such repeats. These are some species of Liliaceae and Alliaceae. For example, terminal regions of chromosomes of bunching onion (Allium fistulosum) contain satellite DNA whose unit repeats are 380 bp in length, and the short arm of its chromosome 8 contains rDNA repeats. This study deals with the terminal heterochromatin and organization of the satellite repeat in A. fistulosum. Fluorescent in situ hybridization (FISH) was used to locate the satellite DNA on chromosomes and on extended DNA of A. fistulosum.Nonsatellite DNA was found in the structure of telomeric repeat. Polymerase chain reaction (PCR) and Southern hybridization were used for analysis of terminal heterochromatin. Various rearrangements were found in the satellite repeat. The roles of retrotransposons and microsatellites in the formation of terminal heterochromatin are discussed.