Organization Of Transcription Units Research Articles

Integrative characterization of the near-minimal bacterium Mesoplasma florum.

The near‐minimal bacterium Mesoplasma florum is an interesting model for synthetic genomics and systems biology due to its small genome (~ 800 kb), fast growth rate, and lack of pathogenic potential. However, fundamental aspects of its biology remain largely unexplored. Here, we report a broad yet remarkably detailed characterization of M. florum by combining a wide variety of experimental approaches. We investigated several physical and physiological parameters of this bacterium, including cell size, growth kinetics, and biomass composition of the cell. We also performed the first genome‐wide analysis of its transcriptome and proteome, notably revealing a conserved promoter motif, the organization of transcription units, and the transcription and protein expression levels of all protein‐coding sequences. We converted gene transcription and expression levels into absolute molecular abundances using biomass quantification results, generating an unprecedented view of the M. florum cellular composition and functions. These characterization efforts provide a strong experimental foundation for the development of a genome‐scale model for M. florum and will guide future genome engineering endeavors in this simple organism.

Studying Gene Expression: Database Searches and Promoter Fusions to Investigate Transcriptional Regulation in Bacteria

A laboratory project was designed to illustrate how to search biological databases and utilize the information provided by these resources to investigate transcriptional regulation in Escherichia coli. The students searched several databases (NCBI Genomes, RegulonDB and EcoCyc) to learn about gene function, regulation, and the organization of transcriptional units. A fluorometer and GFP promoter fusions were used to obtain fluorescence data and measure changes in transcriptional activity. The class designed and performed experiments to investigate the regulation of genes necessary for biosynthesis of amino acids and how expression is affected by environmental signals and transcriptional regulators. Assessment data showed that this activity enhanced students' knowledge of databases, reporter genes and transcriptional regulation.

The entire organization of transcription units on the Bacillus subtilis genome

BackgroundIn the post-genomic era, comprehension of cellular processes and systems requires global and non-targeted approaches to handle vast amounts of biological information.ResultsThe present study predicts transcription units (TUs) in Bacillus subtilis, based on an integrated approach involving DNA sequence and transcriptome analyses. First, co-expressed gene clusters are predicted by calculating the Pearson correlation coefficients of adjacent genes for all the genes in a series that are transcribed in the same direction with no intervening gene transcribed in the opposite direction. Transcription factor (TF) binding sites are then predicted by detecting statistically significant TF binding sequences on the genome using a position weight matrix. This matrix is a convenient way to identify sites that are more highly conserved than others in the entire genome because any sequence that differs from a consensus sequence has a lower score. We identify genes regulated by each of the TFs by comparing gene expression between wild-type and TF mutants using a one-sided test. By applying the integrated approach to 11 σ factors and 17 TFs of B. subtilis, we are able to identify fewer candidates for genes regulated by the TFs than were identified using any single approach, and also detect the known TUs efficiently.ConclusionThis integrated approach is, therefore, an efficient tool for narrowing searches for candidate genes regulated by TFs, identifying TUs, and estimating roles of the σ factors and TFs in cellular processes and functions of genes composing the TUs.

The mammalian transcriptome and the cellular complexity of the brain

The mammalian transcriptome and the cellular complexity of the brain

The complexity of the mammalian transcriptome

A comprehensive understanding of protein and regulatory networks is strictly dependent on the complete description of the transcriptome of cells. After the determination of the genome sequence of several mammalian species, gene identification is based on in silico predictions followed by evidence of transcription. Conservative estimates suggest that there are about 20,000 protein-encoding genes in the mammalian genome. In the last few years the combination of full-length cDNA cloning, cap-analysis gene expression (CAGE) tag sequencing and tiling arrays experiments have unveiled unexpected additional complexities in the transcriptome. Here we describe the current view of the mammalian transcriptome focusing on transcripts diversity, the growing non-coding RNA world, the organization of transcriptional units in the genome and promoter structures. In-depth analysis of the brain transcriptome has been challenging due to the cellular complexity of this organ. Here we present a computational analysis of CAGE data from different regions of the central nervous system, suggesting distinctive mechanisms of brain-specific transcription.

Chromosomal organization is shaped by the transcription regulatory network

Transcription regulation, a key step in the control of gene expression, has been the focus of several large-scale studies; however, little attention has been given to its relationship with the chromosomal arrangement of transcription units. We studied this relationship systematically in Escherichia coli and Saccharomyces cerevisiae using network analysis methods. Our analysis reveals links between transcription regulation and chromosomal organization, suggesting that in both organisms transcription regulation has shaped the organization of transcription units on the chromosome. Differences found between the organisms reflect the inherent differences in transcription regulation between prokaryotes and eukaryotes.

The eukaryotic genome: a system regulated at different hierarchical levels.

Eukaryotic gene expression can be viewed within a conceptual framework in which regulatory mechanisms are integrated at three hierarchical levels. The first is the sequence level, i.e. the linear organization of transcription units and regulatory sequences. Here, developmentally co-regulated genes seem to be organized in clusters in the genome, which constitute individual functional units. The second is the chromatin level, which allows switching between different functional states. Switching between a state that suppresses transcription and one that is permissive for gene activity probably occurs at the level of the gene cluster, involving changes in chromatin structure that are controlled by the interplay between histone modification, DNA methylation, and a variety of repressive and activating mechanisms. This regulatory level is combined with control mechanisms that switch individual genes in the cluster on and off, depending on the properties of the promoter. The third level is the nuclear level, which includes the dynamic 3D spatial organization of the genome inside the cell nucleus. The nucleus is structurally and functionally compartmentalized and epigenetic regulation of gene expression may involve repositioning of loci in the nucleus through changes in large-scale chromatin structure.

A probable mixed-function supraoperon in Pseudomonas exhibits gene organization features of both intergenomic conservation and gene shuffling.

Sequencing of an 8182-bp chromosomal region in Pseudomonas stutzeri revealed the major portion of an apparent mixed-function supraoperon (defined as a nested organization of transcriptional units encoding gene products which function in more than one biochemical pathway). A nearly identical supraoperon organization was apparent in the unpublished Pseudomonas aeruginosa genome database, where the complete Pseudomonas supraoperon was deduced. The serC(pdxF)-aroQp. pheA-hisHb-tyrAc-aroF-cmk-rpsA supraoperon encodes 3-phosphoserine aminotransferase, a bidomain chorismate mutase/prephenate dehydratase, imidazole acetol-phosphate aminotransferase, cyclohexadienyl dehydrogenase, 5-enolpyruvylshikimate 3-phosphate synthase, cytidylate kinase, and ribosomal protein S1. The member genes were identified by homology analysis, enzyme assay, and/or functional complementation. Although SerC(PdxF) and HisHb exercise their primary functions in serine, pyridoxine, and histidine biosynthesis, they also have critical catalytic roles in provision of the sidechain amino groups of tryptophan, phenylalanine, and tyrosine. The likelihood of supraoperon-wide translational coupling is suggested by the highly compressed intergenic spacing (including overlapping stop and start codons), as well as by possible hairpin structures in mRNA which may sequester some of the ribosome-binding sites and thus provide a mechanism for translational coupling. A comparison of the organization of the supraoperon genes in other organisms represented in the database revealed unmistakable conservation of the linkage of these genes across wide phylogenetic boundaries, albeit with considerable gene shuffling. At least remnants and shuffled portions of the entire supraoperon are distributed throughout the Gram-negative bacteria with the hisHb-tyrA-aroF gene block being conserved as distantly as the gram-positive bacteria. Such conservation of mixed-function genes may reflect the selective value of still-unknown global relationships of protein-protein interaction or regulation.

The Legionella pneumophila icmGCDJBF genes are required for killing of human macrophages.

Previously, a collection of mutants of Legionella pneumophila that had lost the ability to multiply within and kill human macrophages was generated by Tn903dIIlacZ transposon mutagenesis and classified into DNA hybridization groups. A subset of these mutants was complemented by a plasmid, pMW100, containing a 13.5-kb genomic DNA insert. This plasmid restored the ability to multiply within and produce cytopathic effects on human macrophages to members of DNA hybridization groups II, IV, VI, and XVII. A region of the genomic insert of pMW100 was sequenced, and eight potential genes were identified and named icmE, icmG, icmC, icmD, icmJ, icmB, icmF, and tphA. None of the genes encode potential protein products with significant homology to previously characterized proteins, except for tphA, whose product has significant homology to a family of metabolite/H+ symport proteins from gram-negative bacteria. The positions of the Tn903dIIlacZ insertions within the genes were determined by nucleotide sequencing. No Tn903dIIlacZ insertions mapped to icmG, icmJ, or tphA; therefore, these loci were mutated to test whether they were required for macrophage killing. Complementation analysis was used to evaluate the roles of the potential gene products and provide information on the organization of transcriptional units within the region. The results indicate that all identified open reading frames except tphA are required for killing of human macrophages.

In vitro and in vivo analysis of transcription within the replication region of plasmid pIP501.

Derivatives of the conjugative streptococcal plasmid pIP501 replicate stably in Bacillus subtilis. The region essential for replication of pIP501 has been narrowed down to a 2.2 kb DNA segment, the sequence of which has been determined. This region comprises two genes, copR and repR, proposed to be involved in copy control and replication. By in vitro and in vivo transcriptional analysis we characterized three active promoters, pI, pII and pIII within this region. A putative fourth promoter (pIV) was neither active in vitro nor in vivo. We showed that copR is transcribed from promoter pI while the repR gene is transcribed from promoter pII located just downstream of copR. The pII transcript encompasses a 329 nucleotide (nt) long leader sequence. A counter transcript that was complementary to a major part of this leader was found to originate from a third promoter pIII. The secondary structure of the counter transcript revealed several stem-loop regions. A regulatory function for this antisense RNA in the control of repR expression is proposed. Comparative analysis of the replication regions of pAM beta 1 and pSM19035 suggested a similar organization of transcriptional units, suggesting that an antisense RNA is produced by these plasmids also.

Characterization of the Caulobacter crescentus flbF promoter and identification of the inferred FlbF product as a homolog of the LcrD protein from a Yersinia enterocolitica virulence plasmid

We have investigated the organization and expression of the Caulobacter crescentus flbF gene because it occupies a high level in the flagellar gene regulatory hierarchy. The nucleotide sequence comprising the 3' end of the flaO operon and the adjacent flbF promoter and structural gene was determined, and the organization of transcription units within this sequence was investigated. We located the 3' ends of the flaO operon transcript by using a nuclease S1 protection assay, and the 5' end of the flbF transcript was precisely mapped by primer extension analysis. The nucleotide sequence upstream from the 5' end of the flbF transcript contains -10 and -35 elements similar to those found in promoters transcribed by sigma 28 RNA polymerase in other organisms. Mutations that changed nucleotides in the -10 or -35 elements or altered their relative spacing resulted in undetectable levels of flbF transcript, demonstrating that these sequences contain nucleotides essential for promoter function. We identified a 700-codon open reading frame, downstream from the flbF promoter region, that was predicted to be the flbF structural gene. The amino-terminal half of the FlbF amino acid sequence contains eight hydrophobic regions predicted to be membrane-spanning segments, suggesting that the FlbF protein may be an integral membrane protein. The FlbF amino acid sequence is very similar to that of a transcriptional regulatory protein called LcrD that is encoded in the highly conserved low-calcium-response region of virulence plasmid pYVO3 in Yersinia enterocolitica (A.-M. Viitanen, P. Toivanen, and M. Skurnik, J. Bacteriol. 172:3152-3162, 1990).

Organization of transcription units around the Drosophila melanogaster rudimentary locus and temporal pattern of expression.

The molecular organization of 90 kb of DNA derived from a region of the X chromosome that encompasses the rudimentary locus of D. melanogaster is presented. This segment spans the cytogenetic region 14F2-3 to 15A1-2, and there are, in addition to the rudimentary gene several transcription units present, whose functions are still unknown. We have determined the pattern of expression of all these genes at several stages of development, and found that they all show a different temporal modulation of their activity. The accumulation of the r product correlates well with the enzymatic activity determined for the protein product of the gene, being highest in very early embryos and adult females.

Physical mapping and complementation analysis of transposon Tn5 mutations in Caulobacter crescentus: organization of transcriptional units in the hook gene cluster

Using the cloned DNA from the hook protein gene region of Caulobacter crescentus ( Ohta et al., Proc. Natl. Acad. Sci. U.S.A. 79:4863-4867, 1982), we have identified and physically mapped 19 Tn5-induced and 2 spontaneous insertion mutations to this region of the chromosome. These nonmotile mutants define a major cluster of fla genes that covers approximately 17 kilobases on the chromosome (hook gene cluster). Complementation analysis of the mutants using DNA fragments from the region subcloned in the broad host range plasmid pRK290 has shown that these fla genes are organized into at least five transcriptional units (I to V). Transcriptional unit II contains at least one gene in addition to the hook protein gene, which makes this the first operon described in C. crescentus. Expression of the hook protein gene and the genetically unlinked flagellin A and B genes by this set of mutants also furnishes additional insights into the hierarchial regulation of flagellar genes. We have found that the spontaneous insertion mutant ( SC511 ) of the hook protein gene ( flaK ) makes no flagellin A or B and that genes downstream from the hook protein gene are required in trans for expression of the hook protein operon and the flagellin A and B genes. Recombination and complementation results thus place flaK , flaJ , flaN , and flaO (R. C. Johnson and B. Ely , J. Bacteriol . 137:627-634, 1979) in the hook gene cluster, identify at least three new genes ( flbD , flbG , and flbF ), and suggest that this cluster may contain several additional, as yet unidentified, fla genes.

Expression of the vaccinia virus genome: analysis and mapping of mRNAs encoded within the inverted terminal repetition.

We have investigated the organization of transcriptional units within a 9000 bp segment of the terminally repeated region of the DNA genome of vaccinia virus, which uses its own enzyme system to synthesize mRNA within the cytoplasm of infected cells. RNA splicing, which has been demonstrated for DNA viruses that replicate within the nucleus of infected cells, does not appear to be involved in the formation of these first vaccinia virus mRNAs to be examined. Three immediate early mRNAs, approximately 1050, 600 and 1100 nucleotides long, were mapped between 3.21 and 4.24, 6.54 and 7.16, and 7.20 and 8.23 kb from the end of the genome, respectively. The direction of transcription was toward the end of the genome for the two larger mRNAs and in the opposite direction for the smallest one. Additional minor RNAs, which were larger in size, were mapped between and to the same DNA strand as the mRNAs of 1050 and 1100 nucleotides. No evidence for interrupted genes was obtained by nuclease S1 analysis after hybridization of RNA to labeled DNA. In addition, the 5' ends of the mRNAs, which were specifically labeled by in vitro capping, hybridized to DNA adjacent to the body of the message.