High-throughput RNAi in Caenorhabditis elegans: genome-wide screens and functional genomics

The phenomenon of RNA-mediated interference (RNAi) was first discovered in the nematode Caenorhabditis elegans, in which introduction of double-stranded RNA causes specific inactivation of genes with corresponding sequences. Technical advances in RNAi methodology and the availability of the complete genome sequence have enabled the high-throughput, genome-wide RNAi analysis of this organism. Several groups have used large-scale RNAi to systematically examine every C. elegans gene for knock-down phenotypes, providing basal information to be mined in more detailed studies. Now, in addition to functional genomic RNAi analyses, high-throughput RNAi is also routinely used for rapid, genome-wide screens for genes involved in specific biological processes. The integration of high-throughput RNAi experiments with other large-scale data, such as DNA microarrays and protein-protein interaction maps, enhances the speed and reliability of such screens. The accumulation of RNAi phenotype data dramatically accelerates our understanding of this organism at the genetic level.     

Origins and evolution of eukaryotic RNA interference

Small interfering RNAs (siRNAs) and genome-encoded microRNAs (miRNAs) silence genes via complementary interactions with mRNAs. With thousands of miRNA genes identified and genome sequences of diverse eukaryotes available for comparison, the opportunity emerges for insights into the origin and evolution of RNA interference (RNAi). The miRNA repertoires of plants and animals appear to have evolved independently. However, conservation of the key proteins involved in RNAi suggests that the last common ancestor of modern eukaryotes possessed siRNA-based mechanisms. Prokaryotes have an RNAi-like defense system that is functionally analogous but not homologous to eukaryotic RNAi. The protein machinery of eukaryotic RNAi seems to have been pieced together from ancestral archaeal, bacterial and phage proteins that are involved in DNA repair and RNA processing.     

病毒诱导的基因沉默及其在植物基因功能研究中的应用

RNA介导的基因沉默是近年来在生物体中发现的一种基于核酸水平高度保守的特异性降解机制.病毒诱导的基因沉默（virus induced gene silencing, VIGS）是指携带植物功能基因cDNA的病毒在侵染植物体后,可诱导植物发生基因沉默而出现表型突变,进而可以研究该目的基因功能.至今,已经建立了以RNA病毒、DNA病毒、卫星病毒和DNA卫星分子为载体的VIGS体系,这些病毒载体能在多种寄主植物（包括拟南芥、番茄和大麦）上有效抑制功能基因的表达.VIGS已开始应用于N基因和Pto基因介导的抗性信号途径中关键基因的功能研究、抗病毒相关的寄主因子研究以及植物代谢和发育调控研究.在当前植物基因组或EST序列大量测定的情况下,VIGS为植物基因功能鉴定提供了有效的技术平台.     

The utility of transposable elements as tools for the isolation of plant genes

Transposable elements are segments of DNA which have the unique capability of being able to excise from one site in the genome and reintegrate into new, different sites elsewhere in the genome. When transposition takes place and integration occurs within a gene locus, mutations are frequently generated producing variegated or recessive phenotypes. This ability of transposable elements to act as mutagenic agents through their association with particular gene sequences has lead to the development of the procedure of transposon tagging or gene tagging in higher plants. Through this technique, transposable elements can be used to clone and isolate genes of interest for which little or nothing is known about the final product (i.e., polypeptide). This offers tremendous potential for the isolation of a variety of agronomically important genes, which are virtually impossible to recover by other currently available gene cloning methodologies. To date, the technique has been used successfully to isolate genes from corn and snapdragon. Using gene transfer technologies, the potential now exists to extend this approach to clone genes from other plant species. Advantages and limitations of transposon tagging for isolating plant genes will be discussed.     

Sex is for sisters: intragenomic recombination and homology-dependent mutation as sources of evolutionary variation

Sex and recombination generate variation via processes that depend on an underlying complementarity between participants. Sex between DNA segments depends on their sequences having enough in common. Viewed in this way, sex does not depend on genes that originate in separate cells. Sex in the single genome uses many of the same mechanisms as intergenomic sex but has not been properly appreciated as a source of variation or as a selectable process. Mutation is the generation of new sequences rather than the novel grouping of pre-existing alleles. Mechanisms of mutation that depend on pre-existing sequence similarities in the haploid genome are a source of variation with significant and special characteristics.     

丝状真菌目标基因替换过程中的策略与方法

目标基因替换是基因功能分析的重要手段。随着基因组测序和转化技术的进展, 该技术在丝状真菌功能基因组学中的应用越来越广泛, 新的体系与方法不断建立和完善。文章在转化体系、打靶载体构建、突变体筛选等方面综述了丝状真菌目标基因替换过程中的策略与方法, 并对各方法的优缺点及发展趋势进行了评述。     

cDNA-AFLP analysis of seed germination in Arabidopsis thaliana identifies transposons and new genomic sequences

A cDNA-AFLP experiment was designed to identify and clone nucleotide sequences induced during seed germination in Arabidopsis thaliana. Sequences corresponding to known genes involved in processes important for germination, such as mitochondrial biogenesis, protein synthesis and cell cycle progression, were isolated. Other sequences correspond to Arabidopsis BAC clones in regions where genes have not been annotated. Notably, a number of the sequences cloned did not correspond to available sequences in the databases from the Arabidopsis genome, but instead present significant similarity with DNA from other organisms, for example fish species; among them, some may encode transposons. A number of the sequences isolated showed no significant similarity with any sequences in the public databases. Oligonucleotides derived from these new sequences were used to amplify genomic DNA of Arabidopsis. Expression analysis of representative sequences is presented. This work suggests that, during germination, there may be a massive transposon mobilization that may be useful in the annotation of new genome sequences and identification of regulatory mechanisms.     

植物瞬间表达系统与功能基因组学研究

结构基因组学和功能基因组学的发展使特定植物基因组和转录组序列的获取更为方便和快捷。随之而来的是对各种基因和调控序列的功能注释,探索植物生长和发育的遗传机理。表达和调控表达是遗传物质的自身语言和动态属性,因此通过植物细胞内表达来分析目标基因和序列的表达和调控行为是功能分析的主要立足点。除创造转基因植株外,近几年来植物细胞瞬间表达系统得到了广泛的使用,与基因重排、病毒诱导基因沉默和RNA干扰等新兴技术的结合使其在植物功能基因组研究中扮演了越来越重要的角色。     

Analysis of developmentally interesting genes cloned from higher plants by insertional mutagenesis

The ability of transposable elements to generate gene mutations by excising from one site in the genome and reintegrating into new, different sites elsewhere in the genome has led to the development of procedures whereby the elements can be used to tag specific gene sequences for eventual isolation and analysis through gene cloning. This transposon tagging strategy is particularly useful in those situations where limited knowledge of the biochemistry of the target gene precludes gene cloning by conventional strategies. This approach, in conjunction with the more general insertional mutagenesis approach using T-DNA, has led to the cloning and subsequent analysis of several genes from higher plants involved in particular developmental processes. Studies of this nature should eventually shed light on the precise molecular mechanisms utilized to regulate and control cellular differentiation in plants.     

Programs of Gene Expression During the Laying Down of Memory Formation as Revealed by DNA Microarrays

Many experiments in the past have demonstrated the requirement of de novo gene expression during memory formation. In contrast to the initial reductionistic view that genes relevant to learning and memory would be easily found and would provide a simple key to understand this brain function, it is becoming apparent that the genetic contribution to memory is complex. Previous approaches have been focused on individual genes or genetic pathways and failed to address the massively parallel nature of genome activities and collective behavior of the genes that ultimately control the molecular mechanisms underlying brain function. In view of the broad variety of genes and the cross talk of genetic pathways involved in this regulation, only gene expression profiles may reflect the complete behavior of regulatory pathways. In this review we illustrate how DNA microarray-based gene expression profiling may help to dissect and analyze the complex mechanisms involved in gene regulation during the acquisition and storage of memory in the mammalian brain.     

Insertional mutagenesis in Arabidopsis thaliana

Recently, a number of mutant gene loci in the Arabidopsis thaliana plant genome have been identified through insertional mutagenesis. In this review, we evaluate different methods used for Agrobacterium tumefaciens-mediated T-DNA insertional mutagenesis with regard to their mutation frequencies and conclude that a major breakthrough in the isolation of genes involved in plant development has been acheived. To provide a specific example, we summarize recent progress made in the understanding of flower morphogenesis at the molecular level through the study of homeotic genes obtained via gene tagging. T-DNA gene fusion vectors are being discussed that will allow the isolation of plant regulatory sequences with particular cell or tissue specificity, or that are controlled by specific external stimuli. Finally, we report on the approaches followed to convert the maize transposons Ac/Ds into valuable gene tags for use in a heterologous host such as Arabidopsis.     

Applications of DNA genetic markers to the study of plant growth and development

DNA genetic markers, such as restriction fragment length polymorphisms (RFLPs) and random amplified polymorphic DNA markers (RAPDs), are powerful tools for studying the genetics of plant growth and development. DNA markers are defined sequences of DNA that can be used in traditional linkage mapping. Using DNA marker technology, scientists can uncover relationships between cloned cDNA sequences and classically characterized genes. DNA markers make it possible to dissect the contributions of multiple genetic loci underlying complex developmental processes. Moreover, changes in genome organization that occur during development or in response to environmental signals can be monitored using RFLP technology. In the future, it may be possible to clone any gene based solely on its map position. This will involve the use of tightly linked DNA markers as entry points for chromosome walking, in which a series of overlapping genomic clones reaching from the tightly linked DNA marker to the gene of interest are identified.     

Technological advances in the molecular biology of Leptospira

Pathogenic members of the genus Leptospira have been refractory to genetic study due to lack of known mechanisms of genetic exchange. To bypass this limitation, several techniques have been useful for Leptospira gene discovery, including heterologous complementation of Escherichia coli mutants, screening of DNA libraries with probes, and random sequence analysis. Construction of combined physical and genetic maps revealed the presence of two circular chromosomal replicons. The organization of the L. interrogans genome is quite variable, with genetically similar strains differentiated by many rearrangements. These rearrangements likely occur through recombination between repetitive DNA elements found scattered throughout the genome. Analysis of intervening sequences and genes encoding LPS biosynthetic enzymes provide evidence of lateral transfer of DNA between Leptospira spp. We have also gained insight into the biology of these bacteria by analyzing genes encoding LPS and outer membrane proteins (OMPs). Some of these OMPs are differentially expressed. Characterization of mechanisms governing the expression of the OMP genes should provide insight into host-parasite interactions. Furthermore, recent advances in heterologous expression of leptospiral OMP genes are opening new avenues of vaccine development.     

DNA Repair Systems in Archaea: Mementos from the Last Universal Common Ancestor?

DNA repair in the Archaea is relevant to the consideration of genome maintenance and replication fidelity in the last universal common ancestor (LUCA) from two perspectives. First, these prokaryotes embody a mix of bacterial and eukaryal molecular features. Second, DNA repair proteins would have been essential in LUCA to maintain genome integrity, regardless of the environmental temperature. Yet we know very little of the basic molecular mechanisms of DNA damage and repair in the Archaea in general. Many studies on DNA repair in archaea have been conducted with hyperthermophiles because of the additional stress imposed on their macromolecules by high temperatures. In addition, of the six complete archaeal genome sequences published so far, five are thermophilic archaea. We have recently shown that the hyperthermophile Pyrococcus furiosus has an extraordinarily high capacity for repair of radiation-induced double-strand breaks and we have identified and sequenced several genes involved in DNA repair in P. furiosus. At the sequence level, only a few genes share homology with known bacterial repair genes. For instance, our phylogenetic analysis indicates that archaeal recombinases occur in two paralogous gene families, one of which is very deeply branched, and both recombinases are more closely related to the eukaryotic RAD51 and Dmc1 gene families than to the Escherichia coli recA gene. We have also identified a gene encoding a repair endo/exonuclease in the genomes of several Archaea. The archaeal sequences are highly homologous to those of the eukaryotic Rad2 family and they cluster with genes of the FEN-1 subfamily, which are known to be involved in DNA replication and repair in eukaryotes. We argue that there is a commonality of mechanisms and protein sequences, shared between prokaryotes and eukaryotes for several modes of DNA repair, reflecting diversification from a minimal set of genes thought to represent the genome of the LUCA.