MicroRNAs are important negative regulators of protein-coding gene expression and have been studied intensively over the past years. Several measurement platforms have been developed to determine relative miRNA abundance in biological samples using different technologies such as small RNA sequencing, reverse transcription-quantitative PCR (RT-qPCR) and (microarray) hybridization. In this study, we systematically compared 12 commercially available platforms for analysis of microRNA expression. We measured an identical set of 20 standardized positive and negative control samples, including human universal reference RNA, human brain RNA and titrations thereof, human serum samples and synthetic spikes from microRNA family members with varying homology. We developed robust quality metrics to objectively assess platform performance in terms of reproducibility, sensitivity, accuracy, specificity and concordance of differential expression. The results indicate that each method has its strengths and weaknesses, which help to guide informed selection of a quantitative microRNA gene expression platform for particular study goals.

The advent of systems biology necessitates the cloning of nearly entire sets of protein-encoding open reading frames (ORFs), or ORFeomes, to allow functional studies of the corresponding proteomes. Here, we describe the generation of a first version of the human ORFeome using a newly improved Gateway recombinational cloning approach. Using the Mammalian Gene Collection (MGC) resource as a starting point, we report the successful cloning of 8076 human ORFs, representing at least 7263 human genes, as mini-pools of PCR-amplified products. These were assembled into the human ORFeome version 1.1 (hORFeome v1.1) collection. After assessing the overall quality of this version, we describe the use of hORFeome v1.1 for heterologous protein expression in two different expression systems at proteome scale. The hORFeome v1.1 represents a central resource for the cloning of large sets of human ORFs in various settings for functional proteomics of many types, and will serve as the foundation for subsequent improved versions of the human ORFeome.

DNA damage-inducible (din) (16,41). Regulation of the SOS system in E. coli is controlled by the products of the recA and lexA genes. The RecA protein has many functions in E. coli and is involved in the processes of recombination, DNA repair, and mutagenesis (31,41,45). The LexA protein is a repressor of as many as 20 unlinked, coordinately regulated loci which include the recA and lexA genes themselves (19,41). Following exposure of E. coli to agents that alter DNA structure or interfere with DNA replication (such as UV radiation, mitomycin, nalidixic acid, etc.), an inducing signal is generated. (4,7,17,34,35), and the UmuD protein (2, 39). As levels of LexA repressor decline, damage-inducible loci are derepressed, resulting in expression of'the physiological phenomena that compose the SOS response (16, 41).The SOS system of E. coli has served as a model for the study of similar inducible DNA repair systems in other gram-negative bacteria (14,36,37,43,44 and essentially error free (7a). This contrasts with W reactivation in E. coli, which is capable of repairing a variety of DNA lesions by an error-prone mechanism (32). Furthermore, while induction of all SOS phenomena in E. coli is dependent upon a functional RecA protein, filamentation in B. subtilis is a RecA-independent response (21). Finally, the SOB system in B. subtilis is developmentally regulated. As B. subtilis differentiates into the physiological state of natural competence (6), SOB phenomena are spontaneously induced in the absence of externally generated DNA damage (20,23,47,49,50).DNA damage-inducible loci in B. subtilis were first identified by using transposon-mediated gene fusions (20). Tn917-lacZ transposon insertions within din loci were isolated from a library of insertions by selecting those fusions that induced expression of the lacZ reporter gene after exposure to DNA-damaging agents (20). Fifteen independently isolated din gene transposon insertions were genetically mapped and localized to three loci (dinA, dinB, and dinC) on the B. subtilis chromosome (11). As mentioned above, induction of all three din loci was demonstrated to be dependent upon a functional RecA protein (20). In order to elucidate the mechanisms that regulate damage-inducible gene expression in B. subtilis, we have cloned and sequenced DNA fragments that contain the dinA, dinB, and dinC promoter regions. Described here is our initial characterization of these cloned din promoter regions and the identification of a putative SOB operator sequence.

The ability to clone and manipulate DNA segments is central to molecular methods that enable expression, screening, and functional characterization of genes, proteins, and regulatory elements. We previously described the development of a novel technology that utilizes in vitro site-specific recombination to provide a robust and flexible platform for high-throughput cloning and transfer of DNA segments. By using an expanded repertoire of recombination sites with unique specificities, we have extended the technology to enable the high-efficiency in vitro assembly and concerted cloning of multiple DNA segments into a vector backbone in a predefined order, orientation, and reading frame. The efficiency and flexibility of this approach enables collections of functional elements to be generated and mixed in a combinatorial fashion for the parallel assembly of numerous multi-segment constructs. The assembled constructs can be further manipulated by directing exchange of defined segments with alternate DNA segments. In this report, we demonstrate feasibility of the technology and application to the generation of fusion proteins, the linkage of promoters to genes, and the assembly of multiple protein domains. The technology has broad implications for cell and protein engineering, the expression of multidomain proteins, and gene function analysis.[Supplemental material is available online at www.genome.org.]The cloning and manipulation of DNA segments, typically encoding functional elements such as promoters, genes, protein domains, or fusion tags, are central to methods of cell engineering, protein production, and gene-function analysis. The large number of available genome sequences now makes it possible to create and apply repositories of defined functional elements to conduct high-throughput, genome-wide analyses. The Gateway Cloning Technology (Hartley et al. 2000) uses in vitro sitespecific recombination to clone and subsequently transfer DNA segments between vector backbones. This approach has been used to generate several large clone collections (Entry Clones), in some cases comprising the entire or nearly entire coding capacity of model genomes as open reading frames (ORFs). These ORFeomes include Caenorhabditis elegans (Walhout et al. 2000b;Reboul et al. 2001Reboul et al. , 2003, Pseudomonas aeruginosa (LaBaer et al. 2004), and Saccharomyces cerevisiae (G. Marsischky, pers. comm.), Arabidopsis (Yamada et al. 2003; also see Atome project http:// genoplante-info.infobiogen.fr/Databases/CT_Nouveaux_Outils/ NO2001054/), human (clones available from several commercial sources), and an incipient collection of Drosophila ORFs (http:// www.fruitfly.org/EST/gateway.shtml). A collection of sequenced, full-length Arabidopsis cDNAs in the Gateway Vector pCMV-SPORT6 will shortly be made available through INRA-Genoscope (Castelli et al. 2004). Repositories of full-length clones, some of which are in the Gateway format, are available for Xenopus (http://xgc.nci.nih.gov/), zebrafish (http://zgc.nci.nih.gov/), as well as many hu...

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The SOS response of Escherichia coli has become a paradigm for the study of inducible DNA repair and recombination processes in many different organisms. While these studies have demonstrated that the components of the SOS response appear to be highly conserved among bacterial species, as with most models, there are some significant variations. Perhaps the best example of this comes from an analysis of the SOS-like system of the developmental organism, Bacillus subtilis. Accordingly, the most striking difference is the complex developmental regulation of the SOS system as this organism differentiates into its competent state. In this review we have given an overview of the elements that comprise the SOS system of B. subtilis. Additionally, we have summarized our most recent findings regarding the regulation of this regulon. Using these results along with new findings from other laboratories we have provided provocative molecular models for the regulation of the B. subtilis SOS system in response to DNA damage and during competent cell formation.

A novel consensus sequence (GAAC-N4-GTTC) has been identified within the promoter regions of DNA damage-inducible (din) genes from Bacillus subtilis. This sequence has been proposed to function as an operator site that is required for regulation of the SOS system of B. subtilis. To test this hypothesis, a deletion analysis of the dinA and recA promoter regions was utilized. A single consensus sequence is sufficient and necessary for damage-inducible regulation of the dinA and recA promoters. Deletion of the consensus sequences upstream of these promoters derepressed their expression under uninduced conditions. In addition, this deletion analysis has further defined sequences upstream of the recA promoter that are required for expression of the recA gene in cells that have differentiated to the state of natural competence. Northern (RNA) hybridization and S1 nuclease protection experiments have demonstrated that the damage-inducible and competence-inducible recA-specific transcripts initiate from a single promoter. Mutations within the comA, srfA, and degU loci each completely abolish the competence-inducible expression of the recA gene.The inducible response to DNA damage (the SOS response) has been extensively studied in Escherichia coli and other closely related gram-negative bacteria (13, 28). These organisms share a high degree of similarity in the regulation and function of their SOS systems (12,26,30,31). In E. coli, the presence of DNA damage generates a signal which activates the RecA protein, which then facilitates the inactivation of the LexA repressor. These events result in the coordinate derepression of a global regulatory network of loci that are responsible for expression of the phenomena that are associated with the SOS response. Examination of the SOS systems of other organisms has led to the realization that different organisms have adapted their SOS systems to suit their own individual requirements. A striking example of this adaptation can be found in the gram-positive bacterium Bacillus subtilis. Characterization of the SOS system of B. subtilis began following the initial observation that certain prophages were induced in cells that had differentiated to the state of natural competence (35). From this observation it was hypothesized that the SOS system of B. subtilis was induced, not only as a consequence of the presence of DNA damage, but also spontaneously within cells that had differentiated to the state of natural competence. This hypothesis was subsequently confirmed following an extensive characterization of the SOS system of B. subtilis (14,15,32,34).Natural competence in B. subtilis is a physiological state that enables the cell to bind and internalize exogenous DNA (6). The development of natural competence is influenced by many regulatory genes, which include spoOA, comP, comA, srfA, and degU (2,19,20,29). These genes as well as other regulatory genes are required for the proper expression of the late-competence genes, which are subject to nutritional, growth stage-specific, and c...