转录后水平沉默与基因表达

基因沉默是1个非常复杂和普遍的现象。转录后水平的基因沉默是指转基因在细胞核里能稳定转录,细胞质里却无相应的稳定态mRNA存在的现象。它往往被称为共抑制、静息作用或RNA干预等。本文介绍了转录后水平的基因沉默现象的发现、分子机理和应用等方面的进展。提出了克服转录后水平基因沉默的一些对策。     

Horizontal gene transfers as metagenomic gene duplications

While it is well accepted that horizontal gene transfer plays an important role in the evolution and the diversification of prokaryotic genomes, many questions remain open regarding its functional mechanisms of action and its interplay with the extant genome. This study addresses the relationship between proteome innovation by horizontal gene transfer and genome content in Proteobacteria. We characterize the transferred genes, focusing on the protein domain compositions and their relationships with the existing protein domain superfamilies in the genome. In agreement with previous observations, we find that the protein domain architectures of horizontally transferred genes are significantly shorter than the genomic average. Furthermore, protein domains that are more common in the total pool of genomes appear to have a proportionally higher chance to be transferred. This suggests that transfer events behave as if they were drawn randomly from a cross-genomic community gene pool, much like gene duplicates are drawn from a genomic gene pool. Finally, horizontally transferred genes carry domains of exogenous families less frequently for larger genomes, although they might do it more than expected by chance.     

基因治疗中外源基因的导入

基因治疗是将遗传物质导入靶细胞以达到治疗疾病的目的,目前基因治疗研究中的主要障碍是如何格外源基因导入靶细胞。本介绍基因治疗的原理和外源基因导入靶细胞时的常用方法,包括显微注射法、电穿孔法、基因枪粒子轰击法等。对基因治疗的现状、存在的问题及未来发展前景作了简要探讨。     

Human globin gene expression after gene transfer

Human globin genes can be transferred into mouse and human erythroid cells in culture, and can be appropriately expressed at the mRNA level in these cells. A plasmid containing a human beta globin gene is expressed in mouse erythroleukemia cells (MELC), and another containing a human epsilon or gamma gene is expressed in human erythroleukemia (K562) cells. A neomycin resistance (neoR) gene on the plasmids has been used to select for those cells containing the transferred globin genes; this selection may favor the expression of the globin genes by providing chromosomal positions requiring neoR expression. Analyzing clones resistant to G418, a neomycin analogue, demonstrated globin mRNA expression and induction. Retroviral vectors have also been used to transfer and appropriately express human beta genes in MELC. In addition, a plasmid containing a dihydrofolate reductase (DHFR) gene as well as neoR and beta globin genes has been used to amplify and express beta globin mRNA in MELC. These experiments suggest that high level appropriate expression of human beta globin genes is feasible and provides potentially useful approaches to the long-range goal of gene therapy for sickle cell anemia and beta thalassemia.     

SNAPper: gene order predicts gene function

SNAPper is a network service for predicting gene function based on the conservation of gene order. AVAILABILITY: The SNAPper server is available at http://pedant.gsf.de/snapper. SNAPper-based functional predictions will soon be offered as part of the PEDANT genome analysis server http://pedant.gsf.de.     

Heterochromatin,gene position effect and gene 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.     

Tumor-specific gene expression patterns with gene expression profiles

Gene expression profiles of 14 common tumors and their counterpart normal tissues were analyzed with machine learning methods to address the problem of selection of tumor-specific genes and analysis of their differential expressions in tumor tissues. First, a variation of the Relief algorithm, “RFE_Relief algorithm” was proposed to learn the relations between genes and tissue types. Then, a support vector machine was employed to find the gene subset with the best classification performance for distinguishing cancerous tissues and their counterparts. After tissue-specific genes were removed, cross validation experiments were employed to demonstrate the common deregulated expressions of the selected gene in tumor tissues. The results indicate the existence of a specific expression fingerprint of these genes that is shared in different tumor tissues, and the hallmarks of the expression patterns of these genes in cancerous tissues are summarized at the end of this paper.     

Identifying time-lagged gene clusters using gene expression data

MOTIVATION: Analysis of gene expression data can provide insights into the time-lagged co-regulation of genes/gene clusters. However, existing methods such as the Event Method and the Edge Detection Method are inefficient as they compare only two genes at a time. More importantly, they neglect some important information due to their scoring criterian. In this paper, we propose an efficient algorithm to identify time-lagged co-regulated gene clusters. The algorithm facilitates localized comparison and processes several genes simultaneously to generate detailed and complete time-lagged information for genes/gene clusters. RESULTS: We experimented with the time-series Yeast gene dataset and compared our algorithm with the Event Method. Our results show that our algorithm is not only efficient, but also delivers more reliable and detailed information on time-lagged co-regulation between genes/gene clusters. AVAILABILITY: The software is available upon request. CONTACT: jiliping@comp.nus.edu.sg SUPPLEMENTARY INFORMATION: Supplementary tables and figures for this paper can be found at http://www.comp.nus.edu.sg/~jiliping/p2.htm.     

Site-specific gene targeting for gene expression in eukaryotes

Major advances in the use of site-specific recombinases to facilitate sustained gene expression via chromosomal targeting have been made during the past year. New tools for genomic manipulations using this technology include the discovery of epitopes in recombinases that confer nuclear localization, crystal structures that show the precise topology of recombinase-DNA-substrate synaptic complexes, manipulations of the DNA recognition sequences that select for integration over excision of DNA, and manipulations that make changes in gene expression inducible by drug administration. In addition, endogenous eukaryotic and mammalian DNA sequences have been discovered that can support site-specific recombinase-mediated manipulations.     

Multifunctional nanocomplexes for gene transfer and gene therapy

DNA formulated into aggregates with polycationic reagents are referred to by a variety of terms including non-viral vectors, synthetic vectors, lipoplexes, polyplexes and more recently nanoparticles. The capacity for delivery of multiple genes, genomic-sized constructs and siRNA delivery, with a diversity of possible formulations, as well as the possibilities of improved efficiency of in vivo gene deliveries, means that nanoparticles, or nanocomplexes to reflect self-assembling systems, will be investigated with increasing vigour in the coming years. This review briefly outlines the applications and challenges for nanoparticle technologies in the field of gene therapy then focuses on the development of a specific kind of formulation, receptor-targeted nanocomplex (RTN), that we have found to be particularly useful in our gene therapy research. An overriding guiding concept that has emerged in the development of synthetic nanodelivery systems is the idea to develop formulations and structures that mimic viruses, whilst retaining the safety elements of synthetic, non-viral systems. RTNs have been optimised and developed for airway epithelial transfection, leading towards gene therapy for cystic fibrosis and for vascular transfection in vein grafts used in bypass surgery. The modular design of the RTN platform further allows for the testing of specific hypotheses relating to the structure and functional role of components in the formation of stable particles and in the transfection pathway, leading to their ultimate disassembly in the nucleus.     

Tumor-specific gene expression patterns with gene expression profiles

Gene expression profiles of 14 common tumors and their counterpart normal tissues were analyzed with machine learning methods to address the problem of selection of tumor-specific genes and analysis of their differential expressions in tumor tissues. First, a variation of the Relief algorithm, "RFE_Relief algorithm" was proposed to learn the relations between genes and tissue types. Then, a support vector machine was employed to find the gene subset with the best classification performance for distinguishing cancerous tissues and their counterparts. After tissue-specific genes were removed, cross validation experiments were employed to demonstrate the common deregulated expressions of the selected gene in tumor tissues. The results indicate the existence of a specific expression fingerprint of these genes that is shared in different tumor tissues, and the hallmarks of the expression patterns of these genes in cancerous tissues are summarized at the end of this paper.     

Designing gene delivery vectors for cardiovascular gene therapy

Genetic therapy in the cardiovascular system has been proposed for a variety of diseases ranging from prevention of vein graft failure to hypertension. Such diversity in pathogenesis requires the delivery of therapeutic genes to diverse cell types in vivo for varying lengths of time if efficient clinical therapies are to be developed. Data from extensive preclinical studies have been compiled and a certain areas have seen translation into large-scale clinical trials, with some encouraging reports. It is clear that progress within a number of disease areas is limited by a lack of suitable gene delivery vector systems through which to deliver therapeutic genes to the target site in an efficient, non-toxic manner. In general, currently available systems, including non-viral systems and viral vectors such as adenovirus (Ad) or adeno-associated virus (AAV), have a propensity to transduce non-vascular tissue with greater ease than vascular cells thereby limiting their application in cardiovascular disease. This problem has led to the development and testing of improved vector systems for cardiovascular gene delivery. Traditional viral and non-viral systems are being engineered to increase their efficiency of vascular cell transduction and diminish their affinity for other cell types through manipulation of vector:cell binding and the use of cell-selective promoters. It is envisaged that future use of such technology will substantially increase the efficacy of cardiovascular gene therapy.     

Intracellular signal-responsive gene carrier for cell-specific gene expression

We designed a peptide-polymer conjugate (CPCCtat) as a novel gene carrier that could control gene expression responding to the intracellular caspase-3 signal. This carrier consists of an uncharged main polymer chain and a cationic peptide side chain, which includes the substrate sequence of caspase-3 and the protein transduction domain sequence of HIV-1 Tat. In the present study, CPCCtat formed a tight complex with DNA through an electrostatic interaction, and in this state the gene expression was totally suppressed. In contrast, the complex disintegrated in the presence of caspase-3 due to cleavage of the cationic portion from CPCCtat. This event led to an activation of gene expression. Our results also indicate that the complex can be delivered into living cells due to the cell-permeable peptide side chain of CPCCtat. This intracellular signal-responsive system with CPCCtat will be useful for the cell-specific gene expression system.