This study describes the meta-analysis and kinetic modelling of gene expression control by sigma factor SigA of Bacillus subtilis during germination and outgrowth based on microarray data from 14 time points. The analysis computationally models the direct interaction among SigA, SigA-controlled sigma factor genes (sigM, sigH, sigD, sigX), and their target genes. Of the >800 known genes in the SigA regulon, as extracted from databases, 311 genes were analysed, and 190 were confirmed by the kinetic model as being controlled by SigA. For the remaining genes, alternative regulators satisfying kinetic constraints were suggested. The kinetic analysis suggested another 214 genes as potential SigA targets. The modelling was able to (i) create a particular SigA-controlled gene expression network that is active under the conditions for which the expression time series was obtained, and where SigA is the dominant regulator, (ii) suggest new potential SigA target genes, and (iii) find other possible regulators of a given gene or suggest a new mechanism of its control by identifying a matching profile of unknown regulator(s). Selected predicted regulatory interactions were experimentally tested, thus validating the model.
- Klíčová slova
- Bacillus subtilis, Gene expression, Kinetic modelling, Regulatory network, Sigma A,
- MeSH
- Bacillus subtilis genetika MeSH
- bakteriální proteiny genetika MeSH
- genetická transkripce genetika MeSH
- genové regulační sítě genetika MeSH
- kinetika MeSH
- regulace genové exprese u bakterií genetika MeSH
- sigma faktor genetika MeSH
- spory bakteriální genetika MeSH
- transkripční faktory genetika MeSH
- Publikační typ
- časopisecké články MeSH
- metaanalýza MeSH
- práce podpořená grantem MeSH
- Názvy látek
- bakteriální proteiny MeSH
- sigma faktor MeSH
- transkripční faktory MeSH
Changes in synthesis and abundance of proteins associated with chlortetracycline (CTC) production in Streptomyces aureofaciens were investigated by two-dimensional polyacrylamide gel electrophoresis of proteins pulse-labelled in vivo with L-[35S]methionine. Eleven individual protein spots were selected as being related to formation of the antibiotic. Expression of these prominent proteins was not observed in the non-producing mutant; moreover, they were overexpressed in cultures grown in the presence of benzyl thiocyanate, a specific stimulator of CTC biosynthesis used in industrial fermentations. The expression kinetics of the selected proteins was assessed using the technique of computer-assisted image analysis with the EQIAS software and the elongation factor Tu as an internal standard. Interestingly, the kinetic profiles were generally not identical. including those of anhydrotetracycline monooxygenase and the 13-kDa subunit of tetracycline dehydrogenase, two enzymes involved, in the terminal sequential steps of the CTC biosynthetic pathway. The presence of more forms of these enzymes with different charge characteristics was observed. The data presented demonstrated how dramatically the industrial microorganism can change its protein repertoire during the production phase; at least five proteins were nearly comparable in level to the most prominent proteins, exemplified by elongation factor Tu.
- MeSH
- 2D gelová elektroforéza MeSH
- antibakteriální látky biosyntéza MeSH
- bakteriální proteiny genetika izolace a purifikace metabolismus MeSH
- chlortetracyklin biosyntéza MeSH
- exprese genu účinky léků MeSH
- kinetika MeSH
- oxidoreduktasy genetika izolace a purifikace metabolismus MeSH
- oxygenasy genetika izolace a purifikace metabolismus MeSH
- proteom MeSH
- Streptomyces aureofaciens účinky léků genetika metabolismus MeSH
- thiokyanatany farmakologie MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- anhydrotetracycline oxygenase MeSH Prohlížeč
- antibakteriální látky MeSH
- bakteriální proteiny MeSH
- benzyl thiocyanate MeSH Prohlížeč
- chlortetracyklin MeSH
- oxidoreduktasy MeSH
- oxygenasy MeSH
- proteom MeSH
- tetracycline 5a(11a)dehydrogenase MeSH Prohlížeč
- thiokyanatany MeSH
Many cell control processes consist of networks of interacting elements that affect the state of each other over time. Such an arrangement resembles the principles of artificial neural networks, in which the state of a particular node depends on the combination of the states of other neurons. The lambda bacteriophage lysis/lysogeny decision circuit can be represented by such a network. It is used here as a model for testing the validity of a neural approach to the analysis of genetic networks. The model considers multigenic regulation including positive and negative feedback. It is used to simulate the dynamics of the lambda phage regulatory system; the results are compared with experimental observation. The comparison proves that the neural network model describes behavior of the system in full agreement with experiments; moreover, it predicts its function in experimentally inaccessible situations and explains the experimental observations. The application of the principles of neural networks to the cell control system leads to conclusions about the stability and redundancy of genetic networks and the cell functionality. Reverse engineering of the biochemical pathways from proteomics and DNA micro array data using the suggested neural network model is discussed.
Many natural processes consist of networks of interacting elements that, over time, affect each other's state. Their dynamics depend on the pattern of connections and the updating rules for each element. Genomic regulatory networks are networks of this sort. In this paper we use artificial neural networks as a model of the dynamics of gene expression. The significance of the regulatory effect of one gene product on the expression of other genes of the system is defined by a weight matrix. The model considers multigenic regulation including positive and/or negative feedback. The process of gene expression is described by a single network and by two linked networks where transcription and translation are modeled independently. Each of these processes is described by different network controlled by different weight matrices. Methods for computing the parameters of the model from experimental data are discussed. Results computed by means of the model are compared with experimental observations. Generalization to a 'black box' concept, where the molecular processes occurring in the cell are considered as signal processing units forming a global regulatory network, is discussed.-Vohradský, J. Neural network model of gene expression.
- MeSH
- genetická transkripce genetika MeSH
- kinetika MeSH
- modely genetické * MeSH
- neuronové sítě (počítačové) * MeSH
- proteiny analýza MeSH
- proteosyntéza genetika MeSH
- regulace genové exprese * MeSH
- RNA analýza genetika MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- proteiny MeSH
- RNA MeSH
In the automation of proteome analysis, matching of two-dimensional (2-D) electropherograms represents a bottleneck in the process. Here we present a point pattern recognition approach to the matching of spots in 2-D electropherograms. The algorithm is based on a comparison of spot neighborhoods, converted to point patterns between reference and compared gels. The neighborhood was characterized by a syntactic descriptor which minimized the influence of spot displacements. A combined criterion utilizing the similarity of point patterns and a metric definition of position similarity was derived. The efficiency and accuracy of the algorithm was tested on a set of 69 gels with different levels of similarity. For a typical gel the accuracy of matching was higher than 98% and the number of correctly identified spots was higher than 95%.
- MeSH
- 2D gelová elektroforéza metody MeSH
- algoritmy MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
The interpretation of two-dimensional gel electrophoresis spot profiles can be facilitated by statistical and machine learning programs. Two different approaches to classification of spot profiles - cluster analysis and neural networks - are discussed. Neural networks for two different model patterns were designed and an algorithm for training of the net for the classification was developed. It was shown that the performance of neural networks is higher compared to cluster and principal component analysis. The possibility of combining both approaches into one process can increase reliability and speed of classification. Artificially created training sets with added random noise can be used for network training. The analysis was applied on the Streptomyces coelicolor developmental two-dimensional (2-D) gel database.
Multivariate statistical comparisons of two-dimensional protein (2-D) gel patterns were used for the first time to define stages of a biological developmental system. The differentiating procaryote, Streptomyces coelicolor, was radiolabeled in liquid cultures at 16 intervals during development, and radioactive proteins were separated and quantified on 2-D gels. Cluster, principal component, and correlation analyses classified these gel patterns into four distinct groups, each reflecting a pattern of gene expression specific for a stage of development. These studies focused our attention on a phase of arrested growth as a key regulatory transition leading to secondary metabolism and a phase of renewed growth. Proteins whose synthesis was switched on or off during the "transitional" phase (some 21 and 18, respectively) were identified and will be the focus of future studies designed to identify their physiological or regulatory function.
- MeSH
- 2D gelová elektroforéza statistika a číselné údaje MeSH
- algoritmy MeSH
- bakteriální proteiny genetika izolace a purifikace MeSH
- databáze faktografické MeSH
- genom bakteriální MeSH
- interpretace statistických dat MeSH
- multivariační analýza MeSH
- peptidové mapování statistika a číselné údaje MeSH
- prokaryotické buňky MeSH
- shluková analýza MeSH
- Streptomyces chemie genetika růst a vývoj MeSH
- vývojová regulace genové exprese MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- bakteriální proteiny MeSH
MOTIVATION: The principal motivation was to design an environment for the development of image-analysis applications which would allow the integration of independent modules into one frame and make available tools for their build-up, running, management and mutual communication. RESULTS: The system was designed as modular, consisting of the core and work modules. The system core focuses on overall management and provides a library of classes for build-up of the work modules, their user interface and data communication. The work modules carry practical implementation of algorithms and data structures for the solution of a particular problem, and were implemented as dynamic-link libraries. They are mutually independent and run as individual threads, communicating with each other via a unified mechanism. The environment was designed to simplify the development and testing of new algorithms or applications. An example of implementation for the particular problem of the analysis of two-dimensional (2D) gel electrophoretograms is presented. The environment was designed for the Windows NT operating system with the use of Microsoft Foundation Class Library employing the possibilities of C++ programming language. AVAILABILITY: Available on request from the authors.
- MeSH
- 2D gelová elektroforéza statistika a číselné údaje MeSH
- navrhování softwaru MeSH
- počítačová grafika MeSH
- počítačové zpracování obrazu metody MeSH
- proteiny chemie izolace a purifikace MeSH
- software * MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- proteiny MeSH
The complete nucleotide sequence (4,826 bp) of the cryptic plasmid pGA1 from Corynebacterium glutamicum was determined. DNA sequence analysis revealed four putative coding regions (open reading frame A [ORFA], ORFA2, ORFB, and ORFC). ORFC was identified as a rep gene coding for an initiator of plasmid replication (Rep) according to the high level of homology of its deduced amino acid sequence with the Rep proteins of plasmids pSR1 (from C. glutamicum) and pNG2 (from Corynebacterium diphtheriae). This function was confirmed by deletion mapping of the minimal replicon of pGA1 (1.7 kb) which contains only ORFC. Deletion derivatives of pGA1 devoid of ORFA exhibited significant decreases in the copy number in C. glutamicum cells and displayed segregational instability. Introduction of ORFA in trans into the cells harboring these deletion plasmids dramatically increased their copy number and segregational stability. The ORFA gene product thus positively influences plasmid copy number. This is the first report on such activity associated with a nonintegrating bacterial plasmid. The related plasmids pGA1, pSR1, and pNG2 lacking significant homology with any other plasmid seem to be representatives of a new group of plasmids replicating in the rolling-circle mode.
- MeSH
- bakteriální proteiny chemie genetika fyziologie MeSH
- Corynebacterium genetika MeSH
- DNA vazebné proteiny * MeSH
- DNA-helikasy chemie genetika MeSH
- molekulární sekvence - údaje MeSH
- otevřené čtecí rámce MeSH
- plazmidy genetika MeSH
- promotorové oblasti (genetika) MeSH
- regulace genové exprese u bakterií MeSH
- replikon genetika MeSH
- sekvence aminokyselin MeSH
- sekvence nukleotidů MeSH
- sekvenční seřazení MeSH
- trans-aktivátory chemie genetika MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- bakteriální proteiny MeSH
- DNA vazebné proteiny * MeSH
- DNA-helikasy MeSH
- ORFA protein, Corynebacterium MeSH Prohlížeč
- replication initiator protein MeSH Prohlížeč
- trans-aktivátory MeSH
Cell protein profiles of submerged cultures of Streptomyces aureofaciens cultivated in the absence or presence of 12 microM benzyl thiocyanate (BT) were analyzed by one-dimensional SDS polyacrylamide gel electrophoresis. Substantial increase in the intensity of the 13, 35, 37, 60, and 100 kDa protein bands was observed in cultures treated with BT. Similar increase in the 35, 37, and 60 kDa bands was found in a mutant blocked in the last chlortetracycline biosynthesis step. Effect of BT on the solid medium-grown cultures was also observed, with a more intensive substrate mycelium pigmentation and alteration in the spore size and shape as the most characteristic features. Earlier studies of BT effect involving those on the stimulation of chlortetracycline biosynthesis are summarized and a possible signal-transducing mechanism is discussed from the point of view of adaptation of S. aureofaciens to the uncoupling of oxidative phosphorylation.
- MeSH
- bakteriální proteiny metabolismus MeSH
- elektronová mikroskopie MeSH
- Streptomyces aureofaciens účinky léků metabolismus ultrastruktura MeSH
- tetracykliny biosyntéza MeSH
- thiokyanatany farmakologie MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- bakteriální proteiny MeSH
- benzyl thiocyanate MeSH Prohlížeč
- tetracykliny MeSH
- thiokyanatany MeSH
