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Many programs have been developed to identify transcription factor binding sites. However, most of them are not able to infer two-word motifs with variable spacer lengths. This case is encountered for RNA polymerase Sigma (σ) Factor Binding Sites (SFBSs) usually composed of two boxes, called -35 and -10 in reference to the transcription initiation point. Our goal is to design an algorithm detecting SFBS by using combinational and statistical constraints deduced from biological observations.
We describe a new approach to identify SFBSs by comparing two related bacterial genomes. The method, named SIGffRid (SIGma Factor binding sites Finder using R'MES to select Input Data), performs a simultaneous analysis of pairs of promoter regions of orthologous genes. SIGffRid uses a prior identification of over-represented patterns in whole genomes as selection criteria for potential -35 and -10 boxes. These patterns are then grouped using pairs of short seeds (of which one is possibly gapped), allowing a variable-length spacer between them. Next, the motifs are extended guided by statistical considerations, a feature that ensures a selection of motifs with statistically relevant properties. We applied our method to the pair of related bacterial genomes of Streptomyces coelicolor and Streptomyces avermitilis. Cross-check with the well-defined SFBSs of the SigR regulon in S. coelicolor is detailed, validating the algorithm. SFBSs for HrdB and BldN were also found; and the results suggested some new targets for these σ factors. In addition, consensus motifs for BldD and new SFBSs binding sites were defined, overlapping previously proposed consensuses. Relevant tests were carried out also on bacteria with moderate GC content (i.e. Escherichia coli/Salmonella typhimurium and Bacillus subtilis/Bacillus licheniformis pairs). Motifs of house-keeping σ factors were found as well as other SFBSs such as that of SigW in Bacillus strains.
We demonstrate that our approach combining statistical and biological criteria was successful to predict SFBSs. The method versatility autorizes the recognition of other kinds of two-box regulatory sites.
The identification of Transcription Factor Binding Sites (TFBSs) is a fundamental problem in the understanding of regulatory networks. A large number of software programs have been designed for the identification of TFBSs. Some of them have been compared in a recent survey [1] that shows the diversity of proposed solutions. Many algorithms are devoted to single motifs prediction [2-11]. They include genetic algorithm [10], expectation maximization or Gibb sampling methods [2,5,7], with incorporated phylogeny data [11], or other methods often based on multiple alignments [4,6] or statistical over-representation [12] and can identify some kinds of TFBSs, but these approaches are not adapted to regulatory binding sites composed of two boxes (a box refers to a conserved part of a signal modelled by a word).
Indeed, in bacterial RNA polymerase, an interchangeable subunit, the sigma (σ) factor, recognizes motifs usually composed of two boxes called -35 and -10 in reference to their location with respect to the transcription initiation point. For close σ factors in related bacterial species, the spacer length between the two boxes of the sigma factor binding sites (SFBSs) can vary slightly [13]. This characteristic, however, is not tackled by most of the existing methods, such as the popular MEME program [2].
Consider the methods dedicated to finding two-box motifs. Most of them can not take into account the variability of spacer length between the two boxes [14-21]. At least four approaches deal with this property. Smile [22], and the more efficient and recent RISO [23], can search for two-box motifs and allows for variable spacer lengths, but they require defining precisely structural constraints applied to the motif in order to avoid a high number of output motifs. In addition, they require the user to define the minimal proportion of input sequences owning the motif looked for. Using a quorum as small as 0.8% to obtain motifs concerned by at least 8 sequences in a set of 1000 sequences gives in a very high number of results. A quorum as high as 10% needs the input set of sequences to be previously selected by another way to ensure that at least 10% of the sequences share the motif we search for. A motif recognized by a σ factor but corresponding to a small number of SFBSs could not be found. In practice, such algorithm can only be applied to sets of promoter regions of known possibly co-regulated genes. Nevertheless, they infer the more general problem of defining TFBS in eukaryotic organisms.
Closer to prokaryotic considerations, Jacques et al. algorithm [24] does not need transcriptional data and uses the supposed enrichment of transcription factor binding sites in intergenic regions. But it requires a matrix that represents the genomic distribution of hexanucleotide pairs, deduced from a training set composed of experimentally verified promoters, often from other bacteria when little is known in the bacterium we are interested in. The advantage of this algorithm is the variability of the spacer between boxes authorized for a same candidate as SFBS consensus. Unfortunately, this approach can not determine which nucleotides are important within each box and can not define the contribution of a position in a given SFBS. This contribution is variable depending on the bacterium for a same SFBS (illustrated by the Figure 6 of a recent article related to structural basis for -35 element recognition [25]). Given motifs are quite long compared to the number of conserved letters in the known promoters of S. coelicolor for example. This last remark is also applicable to MITRA application [18], and the algorithms implemented by Vanet et al. (tested on Helicobacter pylori [26]) which define 12-letter motifs. Another method based on Gibbs Sampling algorithm (Bioprospector [17]) requires specification of the width of the motif for the entire run, whereas motif length of SFBSs seems to be quite variable (see the review article [27]).
An appropriate way to improve results is the footprinting method, and more generally phylogenetic approaches because of the relative conservation of regulatory elements across evolution process. Current comparative approaches need either distantly related species or more than two species [10]. In the first case, the number of shared regulatory motifs will tend to decrease (in parallel to the decrease of motif conservation). In the last one, the need of a high enough number of known closely related bacteria will limit the approach to well-studied families of bacteria.
We present an algorithm, named SIGffRid, for identifying SFBSs, taking into account the limitations reported above. SIGffRid uses a comparative approach to guide word comparisons and defines two-box motifs, whose spacer length can vary sligthly. This possible variation is an important characteristic of SFBS motifs [13,28-32] that we have to take into account in the detection process. By restricting the set of searched conserved boxes to over-represented words at its footprinting stage, SIGffRid allows a comparison of closely related species that are more likely to share common regulatory elements and does not need a great number of bacteria. This phylogenetic footprinting limits false positive rate. The following stages treat each bacterium separately in order to obtain their peculiar motifs.
Our algorithm extends short pairs of patterns shared by conserved pairs of selected words, adapting box widths, until the global motif obtained reach a significant over-representation in upstream regions. It does not fix a strongly constrained structure for final SFBS candidates.
Within the treated set of orthologue pairs, SIGffRid looks for two-word motifs conserved in upstream sequences. If at least eight of those motifs in the same bacterium share the same seven-letter pattern (called motif in the following explanations), it can be considered to be a putative SFBS. The program does not need additional transcriptional data, but can use them with improved performances, if provided. Moreover, SIGffRid's final motifs can be composed with only seven bases. Therefore, subtle motifs can be found by our algorithm.
Most of the characteristics of SFBS motifs (spacer length and variability, box length) exploited by SIGffRid are already described by Hertz et al. [33] but had been combined only once, in an algorithm [24] that defines a SFBS with 12 nucleotides while some of known would need only seven, as is used in SIGffRid.
Phylogenetic relationships, motif properties, and statistical characteristics of SFBSs are the only selection criteria currently retained by our algorithm.
Properties of SFBSs: parameters for the program
The parameters of SIGffRid are correlated to the biological characteristics of the SFBSs:
• the related -35 and -10 boxes, 3 to 7 letters wide (default values in SIGffRid), are sufficiently conserved for a same σ factor to be detected (6 fixed letters in the two boxes, at least 2 fixed letters per box). This motivates our interest to group putative SFBSs by homology of pairs of words.
In practice, in many cases, only one of the two boxes is well defined (the aptly-named extended -10 element for instance [34]), a fact taken into account by the capability of our algorithm to obtain motifs with various structures,
• minimal and maximal spacer lengths between -35 and -10 boxes, taking into account the binding sites of all σ factors can vary in a wide range of values (from 14 to 20 nucleotides by default for σ70 family in SIGffRid),
• spacer length between the two boxes can vary slightly for the same σ factor in the same bacterium and for two orthologous σ factors in two related bacteria, characteristics taken into account by using variable spacer (± 1 by default in the same bacteria, reinforced by Agarwal et al. experiments [35] in an actinomycete; ± 1 by default in two related bacteria),
• SFBSs are located upstream of CDSs, property used for defining our a posteriori statistical test,
• each of the -35 and -10 boxes is over-represented in the whole genome if we consider frequencies of their sub-words. At its footprinting stage, SIGffRid restricts its set of conserved words to those significantly over-represented.
General description
The main program needs following input data:
• the GenBank files of bacterial species of interest (from NCBI database),
• the file giving orthologous relationships (from MBGD database [36] possibly with a user file defining a list of interesting genes in one of the bacteria, or a user file defining orthologous gene IDs).
For the sake of clarity, we describe step by step, globally, the broad lines of the algorithm before refining their description.
We know that SFBSs occurrences are rare in a genome, because useless occurrences of SFBSs can represent a handicap for the bacterium which has to overcome the pressure of selection. The higher number of SFBS-like sequences the bacterium has (in non regulatory regions), the higher is the risk that it is counter selected as suggested by a recent study on density of promoter-like sequences for σ70 [37]. When a transcription factor diffuses in the cell volume (or along with the DNA helix), it has to recognize its binding sites. It can only detect something which is exceptional compared to every possible motifs present in the genome. Selection pressure contributes both to the motif rareness and its conservation. Accordingly, we hypothesized -35 and -10 motifs of SFBSs as exceptional motifs in the genome. This was verified in S. coelicolor where all known sites owned either over-represented boxes or over-represented sub-words of boxes (minimal width of 3 letters)(data not shown). The algorithm is summarized as below:
Restrict dictionary of searched boxes
The searched boxes are the words detected by R'MES [38] as significantly over-represented in the whole genome of the bacterium of our interest. We chose the whole genome model because it is expected to be further from SFBS model than upstream sequence model. Therefore, SFBS boxes are more unexpected in the whole genome model.
Support for SFBS search
Using another closely related bacterium, intergenic sequences of probable orthologous genes are extracted and grouped by pairs. We chose to extract sequences from position -349 (largest value) to 0 in reference to translation start site because most of SFBSs are found in this range of positions (as shown by studies of Escherichia coli [39] or Streptomyces [40] promoters). We fixed their minimal length to 30 nucleotides.
Though some SFBSs can occur in coding sequences, we use only intergenic sequences, otherwise we would have word conservation related to coding sequences, and consequently a high number of false positives. Nevertheless, for a putative SFBS motif, every occurrences located in the -349 to 0 regions are given in final result.
Defining pairs of orthologues
We use orthologous relationships based on MBGD database [36], and group pairs of promoter regions of orthologous according to families given in MBGD, to decrease the number of sequences treated simultaneously.
Although, these "families" are used to split the set of promoter regions in functionally consistent subgroups, we cannot systematically infer co-regulation relationships.
SIGffRid gives the possibility for the user to define a subset of genes of one bacterium, thus, pairs of promoter regions obtained from orthologous relationship, if existing.
Defining conserved pairs of words
Then pairs of conserved over-represented words with a compatible spacer for a SFBS are searched: for each pair of orthologous promoter regions, a list of SFBS candidates shared by the two bacteria is obtained.
Here, we consider the over-representation of each box on the whole genome even if we search only those conserved in promoter regions. Final statistical test will consider over-representation of the complete motif in upstream sequences of coding sequences.
Grouping conserved pairs of words
Further, these pairs of words are grouped according to pairs of sub-words they share satisfying a spacer constraint. For this purpose, we fix sub-word profiles, called seeds.
Motifs extensions
From this stage, we treat the sequences of each bacterium separately, in order to find close motifs which could have diverged.
Finally, an extension of the shared sub-words is carried out according to a probabilistic model. Each one-letter extension concerns only one position and is followed by the design of a regular expression describing the conserved extended area.
Final statistical tests on candidate motifs
A statistical test is led on this regular expression to find out if it is specific to upstream regions of CDSs.
Our statistical test is based on counting in two sets of sequences, and requires the using of:
○ whole genome sequences,
○ lists of upstream sequences of CDS, merged if they overlap each other on a same strand, for each bacterium (see Figure 1). We count occurrences of possible SFBSs in these sets of sequences. Some SFBSs of a particular gene are known to be located in the upstream CDS. Therefore we use upstream sequences instead of intergenic upstream sequences to take every occurrence of SFBSs into account in the final statistical test.
Figure 1. Merging of upstream overlapping sequences on a same strand. The final statistical test of motifs needs to count the number of occurrences in the upstream sequences. If genes overlap each other, their upstream sequences could overlap each other. We avoid to count twice the same motif occurrence by merging upstream overlapped sequences which are on a same strand.
If the motif is considered as an interesting one, we then obtain annotations of genes located downstream the motif occurrences, and stop the process. Otherwise, it goes on recursively until an interesting motif is found.
We give a more detailed description of these techniques in the following paragraphs.
Definition of searched words
R'MES [38] is a statistical software dedicated to finding words with exceptional frequencies in a sequence or a set of sequences. The exceptionality is evaluated by a statistical comparison between the observed counts and the ones expected under Markov models taking the sequence composition into account. A score of exceptionality is then calculated for each word. The study of -35 and -10 known boxes in Streptomyces coelicolor has shown that corresponding words, or sub-words they are composed of, get a high positive score, i.e. are significantly over-represented (data not shown). We have used this general property to restrict the number of searched words.
Here, we consider maximal order Markov models meaning that one takes the (h-1)-letter word composition of the sequences into account to find exceptional h-letter words. Since we consider words shorter than 8 bases in genomes longer than 8 Mb, i.e. very frequent words on average, scores are calculated using a Gaussian approximation of the count distribution [41]. Moreover, we analyze each word simultaneously with its reverse complement (considered like a word family in R'MES) because we run R'MES on the whole genome ; this is important as a mutation into a word on one strand leads to a mutation into its reverse complement on the other strand. Therefore, the frequency of a word is closely related to that of its reverse complement.
The scores of exceptionality produced by R'MES can be converted into approximate p-values. The p-value of an over-represented word is its probability to occur so many times in random sequences having the same short oligonucleotide composition than the observed sequence. More precisely, if X ~ (0, 1) then the approximate p-value is the probability for X to be greater than the observed score. Because of multiple testing, only words of length h with a p-value smaller than α/4h, with α = 5 × 10-3, will be considered as exceptionally frequent; e.g. it corresponds to scores greater than 4.11 for h = 4 or than 4.71 for h = 6.
We applied this procedure to all words of length 3 ≤ h ≤ 7 which gave us a set W of exceptionally frequent words on the alphabet = {a, c, g, t}. These words were then searched in each pair of promoter regions of orthologues.
Properties of candidate motifs as possible SFBSs
Let spmin and spmax be the minimal and maximal authorized spacers between -35 and -10 boxes (deduced from known SFBSs), and let δ be the spacer variation accepted in the SFBSs of two promoter regions.
Consider a triplet C = {w1, w2, {s1i, s2i}} corresponding to words w1 and w2 ∈ W in promoter regions of orthologues s1i and s2i. C is said to be interesting if w1 and w2 occur in s1i and s2i with spacers sp1 and sp2 in [spmin, ...,spmax] respectively, such that -δ ≤ sp2 - sp1 ≤ + δ (see Figure 2). For each pair of orthologous sequences, we keep only interesting triplets C. These are candidates as SFBS.
Figure 2. Conservation of interesting words in promoter regions of orthologues. We search for pairs of conserved significantly over-represented words with approximately the same spacer in the two promoter regions: sp2 - sp1 = δ, δ ∈ {-1, 0, 1}.
Motif extensions
Next, we group interesting triplets according to pairs of seeds. We define a seed as a pattern g on the alphabet {*, #} where '*' can match with any character and '#' corresponds to an exact match.
For instance, from the seed g = ##*#, we get 34 searching motifs, or keys, on the alphabet ∪ {*}:
Let t1 and t2 be two keys obtained from seeds g1 = ### and g2 = ### respectively, and let dt1-t2 be a spacer that separates t1 and t2 in a given C. By considering SS1 = ∪ s1i and SS2 = ∪ s2i (see Figure 3), a set = {t1, t2, [e, ..., e + δ], SS1, SS2} is deduced from all interesting C = {w1, w2, {s1i, s2i}} which verify, for a given integer e:
Figure 3. Grouping of pairs of interesting words found in promoter regions according to pairs of hits. From the conservation of pairs of words in the two bacteria (on the left of the Figure), we deduce the sets of sequences SS1 and SS2 – one for each bacterium – sharing a given pair of patterns.
For instance, using the key pair {gaa, gtt} obtained from seed pair {g1, g2} with g1 = g2 = ### and a spacer of e = 19 ± δ, the following pairs of words given by R'MES (in uppercase) for one bacterium will be grouped together (seeds are underlined):
We consider λ as the minimal number of distinct sequences (by default 8) involved in a candidate SFBS motif. We keep each set SS1 or SS2 only if it presents at least λ distinct sequences. Note that, for a given pair t1 and t2, we merge the sets whose [e, ..., e + d] intervals overlap each other.
A set G of possible seeds of length 3 ≤ L ≤ 5 is fixed before the run. For grouping we use all non-redundant pairs of keys deduced from pairs of seeds {g1, g2} that verify
where ℓmin and ℓmax are respectively the minimal and maximal number of fixed authorized letters in the two seeds, and #(g) is the number of # in seed g (by default ℓmin = ℓmax = 6). To correspond with the usual form of SFBS motifs, we chose the set of seeds G = {###, ####, ##**#, #**##, #*#*#, #***#, #**#, #*#}. Motif extensions only concern seeds without '*' and composed of at least 3 letters (if one of the two seeds has gaps or is to short, only the other will be used for motif extension).
For the sake of clarity, we will illustrate it for the case of pairs of trinucleotides (two seeds ###). Let t1 be the trinucleotide on the left which will be included in the -35 box of a potential SFBS and t2 be the trinucleotide on the right which will be included in the -10 box of the same potential SFBS. For each set SS1 and SS2, sequences are sorted according to the letters adjoining t1 and t2 (see Figure 4). We define the positions of letters as follows:
Figure 4. Extension of shared trinucleotides, classifying of related promoter regions. The set SS1 corresponds to n promoter regions of a given bacterium sharing a pair of given trinucleotides t1 and t2. We compute the probabilities to obtain the encountered letters at the positions neighbouring t1 and t2, considering our n sequences. We retain the position associated with the letter which has the lowest probability to be obtained as soon as observed in this set of n sequences. We group sequences according to the letters at this position which have a low probability to be obtained (with at least eight related sequences). They constitute new sets of sequences to be evaluated with LRT statistical test (see Section "Computing a consensus motif and its statistical evaluation"). "INTERESTING SETS" means sets of promoter regions whose shared motif is over-represented in merged usptream sequences.
• position 1: immediately on the left of t1,
• position 2: immediately on the right of t1,
• position 3: immediately on the left of t2,
• position 4: immediately on the right of t2,
Note that if t1 (respectively t2) corresponds to a gapped seed, positions 1 and 2 (respectively 3 and 4) are not used for extension and probability computations.
Our statistical criteria uses the transition probabilities of a third-order Markov model adjusted on the whole genome. It means that probabilities are computed according to the three letters which come before/after the considered letter (depending on its position according to seed).
Let n be the number of sequences concerned, t be the trinucleotide to extend, and j ∈ {1, 2} be the fixed subscript determining the treated sequences set. For a one letter extension on the right, we define:
The number Nr(a) of sequences having the nucleotide a at position r, i.e. , follows the Binomial law (n, N(ta)/N(t)), where N(·) is the counting function and ta the tetranucleotide formed with t followed by a. We can also compute the significance pr(a) of the observed number x of sequences with an a at position r :
For a one letter extension on the left, we apply the same principle: the number Nℓ(a) of sequences having a at position ℓ ∈ {1, 3} is distributed according to the Binomial (n, N(at)/N(t)) and a p-value pℓ(a) will be calculated.
We search for the position k ∈ {1, 2, 3, 4} containing the minimal probability pk(a) over all a ∈ satisfying Nk(a) ≥ λ . Let be the set of every letters a at position k which verify (Nk(a) ≥ λ) ∦ (pk(a) ≤ 0.15). We group sequences according to each letter a ∈ for the next steps (see Figure 4). A motif corresponding to this set of sequences is generated and evaluated (Section "Computing a consensus motif and its statistical evaluation").
• If it is considered to be an interesting one, we record the corresponding set of sequences as results,
• If the number of involved sequences becomes too low (<λ), the process is stopped,
• If the motif is not interesting, a new evaluation is done on each subset of sequences defined by letters from .
○ if the evaluation is conclusive, we record the corresponding set of sequences as results,
○ otherwise the extension goes on every set of sequences defined by letters a from , by replacing:
- t1 by t1' := a.t1[1].t1[2], if k = 1,
- t1 by t1' := t1[2].t1[3].a, if k = 2,
- t2 by t2' := a.t2[1].t2[2], if k = 3,
- t2 by t2' := t2[2].t2[3].a, if k = 4,
where . is the concatenation operator and t[u] stands for the u-th letter of trinucleotide t.
Therefore, the extended area includes both t1 and t1' (respectively t2 and t2') if k = 1 or k = 2 (respectively k = 3 or k = 4). In the first case, the extension process concerns the letters on the left of t1 and the right of t1'. In the second case, these are the letters on the left of t2 and on the right of t2' which are concerned.
Other sequences are grouped and evaluated with the same criteria of probability for motif extension. Here, we take into account the fact that σ subunits of RNA polymerase are closely related if we consider regions of these proteins involved in -35 and -10 DNA binding (these regions are called 2.4 and 4.2 regions). Therefore, SFBSs might be so closely related that they differ only by one letter.
Note: We verify that the sequence set cannot be split into several distinct subsets, each one corresponding to a spacer length with a narrower range of variation. If it is the case, we record each one of the results corresponding to subset, otherwise we record the global result.
Computing a consensus motif and its statistical evaluation
At each grouping step, a generic motif m is deduced corresponding to two words with a variable spacer. It is built by adding to (extended) trinucleotide pairs, bordering letters a ∈ satisfying:
where pk(a) is obtained from Equation (2) and λ is the minimal number of distinct sequences (by default 8) involved in a candidate SFBS motif.
The method evaluates the specificity of m for upstream sequences. The motif is then searched in the set U of upstream sequences of CDSs (we will call them merged sequences in this paper) by considering each strand separately (we merge sequences if they overlap each other on the same strand, see Figure 1). It means that we take into account the motif orientation when we search it in merged sequences. The number of occurrences is also computed on direct and reverse strands of the whole genome G (composed of |G| elements: genome, plasmids). We took into account plasmids because they usually contain genes with one particular interest like antibiotic resistance genes. We chose not to neglect regulatory elements located in plasmids.
Let ℓU (respectively ℓG) the length of U (resp. G) and NU (resp. NG) the number of occurrences of the motif m into U (resp. G). We then define the following ratio
that measures the specificity of the motif for merged sequences. To test the significance of R, we use the likelihood ratio test (LRT) [42]: the LRT statistics given below follows the chi-square distribution χ2(1) with one degree of freedom.
where N+ = NU + NG and is the expected proportion of m occurrences in the merged sequences. LU and LG are the corrected lengths of sequences U and G (LU = ℓU - (|m| × |U|), LG = 2 [ℓG - (|m| × |G|)]) and μU (resp. μG) is the probability for the motif to occur in sequence U (resp. G) at a given position. μU and μG are calculated under the Bernoulli model (obtained from the sequence sets U and G) to take U and G nucleotide compositions into account. This is a crucial point because intergenic sequences are known to be richer in AT than other sequences in known bacteria whatever their GC letters proportion is [43].
LRT measures the difference of concentration of a given motif in two sets of sequences. The continuation or stop of the consensus motif extension -by sorting sequences- depends on LRT. A selection of the more interesting results is made according to the ratios R and LRT.
The relationship (R ≥ Rmin) ∦ (LRT ≥ LRTmin) must be verified, with LRTmin the quantile at 5% of the χ2(1) law and Rmin = the minimal threshold for specificity, where M corresponds to the minimal ratio between number of occurrences of SFBS supposed to be in merged sequences, and the number of occurrences in the whole genome in terms of number of occurrences (three by default). Considering that most SFBSs are in the upstream regions of CDSs, we suppose that sites which are located upstream are two times more represented in this set than in the whole genome (measurement of the density of the motif). This evidence makes SIGffRid to continue motif extension while motifs are not sufficiently specific to merged sequences (see Figure 4). Therefore, general elements quite frequent in upstream sequences but largely distributed on the whole genome are not in SIGffRid results.
Visualization of the results
Each motif is displayed with all related gene identifiers, scores R and LRT. Two related files complete these results corresponding to all the occurrences found in the complete set of upstream sequences of the related bacterium (including plasmids), their positions according to the translation start point and the annotations of the involved genes. For validation, only cross checking with known biological pathway is necessary to ensure the coherence of related gene functions linked by the same regulation motif.
We ran SIGffRid on phylogenetically related bacterial species belonging to the same genus, Streptomyces coelicolor A3(2) and Streptomyces avermitilis MA-4680 [44,45]. These mycelial Gram-positive bacteria have large genomes (8,667,507 bp and 9,025,608 bp, respectively) and a high GC content (72.1% and 70.7%, respectively). Sixty nine percents of S. avermitilis genes have orthologues in S. coelicolor [45]. These bacteria present a complex regulatory network, as suggested by the high number of predicted σ factors (65 and 60, respectively), whose very few consensus regulatory binding sites are known. And approximately 12.3% of their genes are supposed to be regulators [44]. As proposed by Konstantinidis et al. [46], many regulation systems are expected to cross talk, because their genes share high sequence similarity (paralogous genes of expanded gene families), which suggests increased complexity in regulation as well.
In this context, defining SFBSs, and more generally TFBSs is a true challenge.
Genes of S. coelicolor and S. avermitilis were grouped according to functions defined in MBGD database [36] to reduce the memory and processor usage for large genomes. A total of 3,148 promoter pairs of orthologues were extracted, distributed in 15 functional categories (1,476 orthologous pairs), and the rest that could not be assigned to a function (1,672 pairs) were put in one single category. Spacer range was chosen to correspond to σ70 family spacers (from 14 to 20). We used seeds {###, ####, ##**#, #**##, #*#*#, #***#, #**#, #*#} and the dictionary of exceptional words from S. coelicolor for the two bacteria (using S. avermitilis dictionary gave similar results, data not shown).
From our data set, 113 motifs (two words with a variable spacer) were obtained for S. coelicolor and 65 for S. avermitilis.
Additional file 1 summarizes most interesting results from SIGffRid (Table2_summary.pdf). The complete lists of putative binding sites, positions and sequences, and related gene functions for S. coelicolor, are also available on SIGffRid web page dedicated to results [47]. The SIGffRid web server can be found at [48]. To assign biological function to genes, we used the protein classification scheme available on Sanger Institute website [49] based on that originally created for E. coli in the EcoCyc database [50].
Additional file 1. Summary of results for Streptomyces. Summary of results. Interesting motifs given by SIGffRid when applied on S. coelicolor and S. avermitilis and comparison with known σ factor binding sites. Are given motifs whose occurrences overlap known SFBS (known motif is given in front of the name of the concerned Sigma factor).
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Motifs and genes related to SigR binding site
To validate our approach, we looked for the presence of the SigR binding site among SIGffRid results. The regulon of SigR, a σ factor involved in the response to oxidative stress, is the largest described so far in S. coelicolor. Paget et al. show that SigR activates directly the response of at least 30 genes, and recognizes the motif GGAATN18GTT [51].
Two different motifs obtained with S. coelicolor overlapped the SFBS of SigR regulon (see Table 1). Among the 79 occurrences of the first motif GGAATN16,19GTT, 29 occured in the promoters previously described by Paget et al. [51]. The 30th, SCO3162 motif, was not found because it overlapped CDS. Rest of the 50 potential binding sites were cross referenced, with microarray data showing variation of genes transcription under thiol specific oxidative stress condition triggered by diamide containing medium (Paget MSB, personal communication). Four among them were differently expressed in the microarray data (SCO4956, SCO0569-0570, SCO4297, and SCO6061). Two of these motifs had a promoter with a 18 nt spacer (SCO4956, SCO0569-0570) and the other two had a 19 nt spacer (SCO4297, SCO6061). The unaltered expression of the genes related to the 46 other occurrences can be explained by either particular stress conditions inducing their transcription (not used in this microarray experiment) or by the fact that they are not real promoters.
Table 1. Summary of found motifs similar to known SigR SFBSs
The second motif GGGAAN18,20CGTT corresponds to previously reported promoters likely regulated by the orthologue of SigR, named SigH, in another actinomycete Mycobacterium tuberculosis [51]. Twelve out of the 58 occurrences of this motif were related to differently expressed genes under oxidative conditions (SCO4039, SCO5805, SCO0888, and SCO6061 also reported above). Among these, eight were similar to the motif observed by Paget et al. [51]. Further, two occurrences (of the 12) shared the motif GGGAAGAN16CGTT (SCO0888, SCO4039), very close to the one previously deduced from SigH-dependent promoters in M. tuberculosis (GGGAACAN16CGTT [52]). One occurrence (SCO6061) also overlapped that of the first motif.
The Additional file 2 describes gene functions and proposed binding sites according to SIGffRid motifs similar to SigR binding site (Table3_SigR_motifs.pdf).
Additional file 2. SIGffRid motifs related to SigR. SIGffRid predictions related to the SigR target sequence in S. coelicolor. Gene functions and putative binding sites for SigR σ factor or its homologue(s). It shows overlaps of binding sites of the various motif declinations for SigR binding site.
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Other putative binding sites of known sigma factors
Some motifs detected by SIGffRid correspond to proposed sigma factor binding sites. The motif CGTAAN18,19GTT matched the promoter of bldM (SCO4768 [53]), which is the sole known binding site for BldN. BldN is involved in morphological differenciation and recognizes the motif CGTAACN16CGTTGA.
The SIGffRid motif was found in 24 other regions upstream of coding sequences (see Additional file 3: Table4_BldN_motifs.pdf) suggesting new targets for the σ factor BldN.
Additional file 3. SIGffRid motifs related to BldN. BldN related motif. Gene functions and putative binding sites for BldN σ factor in S. coelicolor.
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HrdB, the major σ factor in S. coelicolor [54], has at least 12 known binding sites [54-62] of which six overlapped four SIGffRid motifs (TGACAN17,20AN3T, TTGAN18,19CTA, TTGACN19,20ANCNT, CNGN18,21TAGGCT). Five among the six remaining motifs, and the motif determined as HrdD binding site [59] (a close homologue of HrdB), were also close to the above four SIGffRid motifs. Approximately 390 genes would be concerned by those motifs.
Other putative SFBSs
The SIGffRid motif, CNGN14,16AGTAA, could correspond to a SFBS consensus. Indeed, the motif CNGN14,16AGTAA is present in the promoter region of the S. coelicolor bldB gene and AGTAA has been proposed to be the -10 box of bldB [63]. The bldB gene encodes a 98 amino acids polypeptide involved in morphogenesis, antibiotic production, and catabolite control in S.coelicolor [63]. Interestingly, this motif is found in the DNA region preceding bldKC, belonging to a five gene cluster encoding an oligopeptide permease responsible for the import of an extracellular signal governing aerial mycelium formation in S. coelicolor.
Two SIGffRid results, TGTCAGTN14,15TnG and TGTCAGTN14TnG, found in both S. coelicolor and S. avermitilis, could correspond to DNA damage-inducible promoters. They are declinations of the Streptomyces rimosus UV-inducible recA promoter, given by Ahel et al. (TTGTCAGTGGCN6TAGggT [64]) and whose variation was proposed by Studholme et al. through a bioinformatic method [21]. Two additional motifs, TGTCAGTGN9,12ANG and TGTCAGTN12,14TNG, could be retrieved when the spacer length parameter range was made from 8 to 14. In S. coelicolor, 67 genes were featured by these motifs, and 39 of them were assigned to a function (see Additional file 4: Table5_recA_motifs.pdf). Several functional groups could be distinguished, the most significant being related to DNA repair (13–20 genes) and includes homologues of the E. coli genes dinP, priA, radA, dinG, recQ. This group also included DNA glycosylases (e.g. ung), excinuclease (e.g. uvrB SC), and polymerase I genes. Another set of genes was related to DNA replication (e.g. dnaE, dnaN encoding respectively α and β subunits of PolIII, and recF).
Additional file 4. SIGffRid motifs possibly related to recA promoter motif. Interesting motif related to recA promoter motif. Gene functions and putative regulatory binding sites for DNA-damage related motifs (recA promoter) in S. coelicolor.
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TFBS motifs other than SFBSs
A SIGffRid motif, [TA]GTGAN18,20TN2C overlaps the BldD binding site whose consensus was proposed by Elliot et al. (AGTgANmTCACc [65]). BldD is a key transcriptional regulator involved in morphological differentiation and antibiotic production in S. coelicolor [65]. This motif was found upstream of bldG (anti-sigma factor antagonist) and five σ factor encoding genes (including HrdB, and whiG which encodes an alternative sigma factor essential for sporulation [66]).
Another SIGffRid motif [TA]GTGAN16,18CNT overlapping the above motif was found upstream of seven σ factors, including HrdD and those found downstream of the first motif. We speculate that these motifs may be declinations of BldD binding site.
Application to other bacterial genomes
The efficiency of SIGffRid was further tested onto pairs of related bacterial species with lower G+C genome contents (i.e. Escherichia coli K12, 50% and Salmonella typhimurium LT2, 52% on one hand, and Bacillus subtilis 168, 43% and Bacillus licheniformis ATCC 14580 (DSM13), 46% on the other hand, [67-71]). Approximately 80% of the predicted B. licheniformis coding sequences have B. subtilis orthologues [70]. The phylogenetic relationships inferred from the 16S rDNA identities, 97.0% and 97.4% between the species of each pairs, was similar to those between the Streptomyces species (97.3%) previously used to develop the algorithm. In contrast to Streptomyces where functional gene categories were used to limit computational times and result quantities, the whole orthologue gene sets were used on E. coli/S. typhimurium and B. subtilis/B. licheniformis analyses.
Several motifs were proposed by SIGffRid for each pair. Among these motifs, some could describe the binding sites of the house keeping σ factors, σ70 of E. coli and SigA of B. subtilis. Thus, for B. subtilis, the motifs TTGAN18,19TATAAT and TTGACN18,20ATAAT for instance perfectly match the known consensus. Some other motifs describe SFBSs for alternative σ factors such as SigW (TGAAACN16,17CGTA [72]) which is implied in stress response in B. subtilis. SIGffRID extends the -10 motif by one nucleotide to give TGAAACN16,17CGTAT. For B. licheniformis, the motif proposed match exactly the SigW consensus of B. subtilis described in the litterature. The data and additional motifs are detailed in Additional file 5 (Table6_eco_stm_bsu_bld.pdf).
Additional file 5. SIGffRid motifs similar to known E. coli, S. typhimurium, B. subtilis, or B. licheniformis SFBSs. SIGffRid results compared with known SFBS motifs in E. coli/S. typhymurium on one hand, and B. subtilis/B. licheniformis on the other hand. Interesting results obtained from E. coli K12/S. typhimurium LT2 and B. subtilis 168/B. licheniformis ATCC 14580 pairs of bacterial genomes by using all pairs of orthologues.
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Our algorithm proved to be relevant in finding different SFBSs and TFBSs, and can be applied to any bacterial species because it only uses general properties. SIGffRid is particularly suited to the detection of SFBSs with a high number of occurrences (those of house-keeping σ factors, e.g. SigA in B. subtilis) or with a small number of well-conserved occurrences (those of some alternative σ factors, e.g. BldN or SigR binding sites in S. coelicolor).
We combine the knowledge of footprinting, constraints of motif structures, phylogeny and statistical models to ameliorate motif characteristics in TFBSs prediction.
Beyond phylogenetic footprinting, some features specific to our method take better into account the variations of the same SFBS in two closely related bacteria. The first being the extension of shared pairs of seeds applied separately in each bacterium. We eventually obtain different variations of the same SFBS in two related bacteria, where the differences concern boxes and/or spacer lengths. Another is its capabilities to group putative sites of the same transcription factor using probabilities. By analysing possible regulons found by SIGffRid, we have shown that regulatory networks could be deduced from annotations, in addition to consensus motifs. Finally, it features an independant statistical test to evaluate the pertinence of the motif. Based on a biological hypothesis, it has the advantage of allowing SIGffRid to be applicable on any subset of sequences (e.g. list of genes obtained from microarray data). Though SIGffRid can be improved by refining probabilistic models used for motif extension and statistical evaluation, it clearly infers motifs close to known consensuses of TFBS.
The nucleotidic motif is probably only one aspect of the SFBS recognition, but is a necessary first bioinformatic step for its prediction. It would be undoubtedly complicated to account for the large number of parameters implicated in DNA recognition by σ factors in all potential promoter regions.
Authors' contributions
FT designed the combinatorial algorithm, with assistance from GK and IDR, and developed the SIGffRid application. SS developed the statistical methodology and wrote the statistical parts of the paper. FT wrote the biological parts of the paper, with assistance from PL and BA, and the computational parts, with assistance from GK and IDR. Results were interpreted by FT, with assistance from PL and BA. All authors read and approved the final manuscript.
We are grateful to Dimitris Kallifidas and Mark Paget (University of Sussex, UK) for providing us with microarray data showing variation of genes transcription under oxidative stress condition, to Wayne Sleeth for comments, and to Charu Asthana for friendly help. FT is supported by the Région Lorraine and by the ACI IMPBio from the Ministère de l'Education Nationale, de l'Enseignement supérieur et de la Recherche.
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Obey Balk Flannel Jacket
Item # 229786
Buy1 Get1 Free
Stash Points: 4,498
What is this?This is the number of points you get in The Zumiez Stash for purchasing this item. Stash points are redeemable for exclusive rewards only available to Zumiez Stash members. To redeem your points check out the rewards catalog on thestash.zumiez.com.
This boyfriend fit jacket is crafted with a heavyweight brushed flannel in a bold plaid pattern, and features adjustable back pleats for a more fitted option.
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Jacque Weidner turned her life around in middle school thanks to influential teachers, and she now wants to be a mentor for girls at Roosevelt Middle School in Blaine....Read More
Roosevelt Middle School
Five of 13 local Destination Imagination (DI) teams competing in last weekend's state tournament have advanced to the Global Finals tournament set for May 22-25 in Knoxville, Tenn....Read More
Five of 13 local Destination Imagination (DI) teams competing in last weekend's state tournament have advanced to the Global Finals tournament, set for May 22-25 in Knoxville, Tenn....Read More
Leapin' lizards! Roosevelt Middle School presents the largest theater production ever to be presented by the school....Read More
Competing for bookworm bragging rights, Anoka-Hennepin middle school students compete in a Battle of the Books, devouring hundreds of pages of the written word, memorizing character traits, story lines, plot twists and subtle innuendos. ...Read More
At Roosevelt Middle School, random acts of kindness are becoming a regular part of everyday life....Read More
Last year when Brooke Larson, then in seventh grade at Roosevelt Middle School (RMS), was bullied by a classmate, her friends gave her support....Read More
When Carol Gutt was invited to present curriculum she developed to the Minnesota Council on Economic Education (MCEE) at the "Conference on Teaching Economics and Personal Finance" in August, little did she know she was doing so as an award winner....Read More
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Roosevelt Middle School in Blaine presents a fast-paced, fun play - Anthony Drewe's "Honk! Jr."...Read More
Middle schoolers in Anoka-Hennepin School District 11 showed off their reading knowledge at the districtwide Battle of the Books....Read More


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As part of my running route, every time I passed the wading pool the sounds of children and families having a great day could be heard wafting through the trees.
The wading pool is a wonderful place to take younger children. Younger kids can play in the shallow water and enjoy the water bubbling up on the edge (where they can crawl or walk in). A bonus is that it is free!
We really like to go to this pool. It's fairly small and only a couple feet deep. And the best part about it is it's free! It's usually very clean, too. And it's right next to the playground. A great choice for an outing with the kids!
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Select Local Merchants
Fat Buddah Tattoo & PiercingRead MoreSince first putting ink to skin in 2000, Fat Buddah Tattoo & Piercing outgrew two locations before finally taking residence in a 3,900-square-foot space. There, the experienced staff adorns clients with piercings and permanent artwork ranging from custom lettering to portraits. The shop's portfolio boasts a large selection of designs, though they welcome clients to bring in their own ideas via sketches, photos, or vivid recollections of dreams.180 NW 183rd St.Miami Gardens, FL US
Allstar Tattoo & Body PiercingsRead MoreThe epidermal embellishers at AllStar Tattoos & Body Piercing adorn bodies with eye-catching jewelry and precisely drawn tattoos in a safe, clean environment. Licensed tattoo artists emblazon customer exteriors with vivid images and text arranged into shapes such as upper-arm half sleeves, chest pieces, and lower-back trapezoids. Piercing specialist William Roldan practices his skin-puncturing craft with a portfolio of gently inserted piercings and dermals that glitter from earlobes, eyebrows, and backs. Purchasers of the first option can start a tattoo collection with a subtle star, rose, or cursive name, while those choosing the second option can finish up a full sleeve or ascend to the upper crust with a set of diamond freckles.1562 West 68th StreetHialeah, FL US
Goa Exotic TattooRead MoreGOA is a company that has been in the tattoo and piercing industry for 15 years. It started in Colombia and is now also located in Miami, Florida, next to the Florida International University main campus.10740 SW 24th StMiami, FL US
Tattoo and Co.Read MoreUnder the direction of owner Javier Acero, who has been a tattoo artist for over a decade, Tattoo and Co. provides tattooing, piercing and body art applications to a discerning crowd of hardcore Miami locals. The bright, clean shop on SW 40th Street is filled with a wild array of art pieces, paintings and design elements, from tiki-style carvings to colorful bits of art that make the white-walled space pop. Each of Tattoo and Co.'s artists operate in much the same way, with differing and unique styles offered by each individual staff member. Luckily, photo books and design samples are readily available, meaning guests can find the look that matches their personality before making an appointment with the appropriate artist. Piercings are also widely available, as are permanent makeup tattoos, with procedures being done in private rooms that run along a wide, narrow corridor.9550 SW 40th StMiami, FL US
Salvation Tattoo LoungeRead MoreSalvation Tattoo Lounge has been making its mark since 2005. At a pair of Miami Beach locations, professional ink artists transform their clients into living, breathing pieces of art. They can even recreate recognizable faces with striking precision, such as those of movie characters, pop culture stars, and beloved mailmen. When they're not peering through the crosshairs of a tattoo gun, the artists administer piercings and apply permanent makeup that is safe to wear by the pool and outdoors.1413 Washington AvenueMiami Beach, FL US
South Seas Tattoo CompanyRead MoreSouth Seas Tattoo Company's artist and founder Rob Soli's expertise extends from a lifetime of professionally painting, sketching, and airbrushing in his hometown of Honolulu. His tattooing philosophy reflects the Oceanic and Pacific Islander tradition, which views tattoos as a rite of passage rather than a mere accessory. With his custom pieces, he aims to commemorate significant events or honor one's heritage. A team of artists carry out piercings and body images such as portraits, fantasy designs, and Polynesian tribal work in stainless-steel stations equipped with privacy screens. They match their pride in sterilization techniques with that of their designs by exceeding all local, state, and industry standards.833 Southeast 8th AvenueDeerfield Beach, FL US


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Latest Car News & Features
Tuesday, June 30, 2015
Mazda Eunos: PH Fleet
Skids temporarily off the agenda as the LSD bites the dust - what to do?
Tuesday, June 30, 2015
Dodge launches new Challenger Drag Pak
Turn key drag racer is claimed to be able to turn in eight second quarters all day long
Tuesday, June 30, 2015
Ford Focus RS: Spotted
An unmolested fast Ford icon for £10K? Best get in before the new one bumps them up!
Tuesday, June 30, 2015
Bentley builds a Harlequin
Well, almost - the Breitling Jet Team Continental will certainly stand out in a crowd.
Tuesday, June 30, 2015
£100K Garage: Lee Bracken
An Alpina, an Atom, a Mini and a Disco cover all the bases for Lee's fantasy garage
Tuesday, June 30, 2015
Button drives Senna's McLaren: Time for Coffee?
One of the most beautiful Formula 1 cars of the modern era on the hill at Goodwood
Monday, June 29, 2015
Mercedes-AMG GLE 63 S: Driven
Is it a truck? Is it a supercar? Nobody knows, least of all us. But we drove it anyway...
Monday, June 29, 2015
Noble fastest in Goodwood supercar shootout
Anthony Reid brings British supercar maker bragging rights at the Festival of Speed. Again.
Monday, June 29, 2015
Motorsport on Monday: 29/06/2015
Formula E: Were you electrified?
Monday, June 29, 2015
Mercedes AMG A45 gets faster and louder
Facelift time already for the A Class, including even more power for the A45
Monday, June 29, 2015
New Audi A4 revealed
Coming soon to a fast lane near you...
Monday, June 29, 2015
MotoGP Assen: PH2
Dutch delight for MotoGP fans after another thrilling race
Monday, June 29, 2015
Porsche 928 S4: PH Carpool
A lovely example of 'the forgotten Porsche' very much enjoyed by one enthusiastic PHer
Monday, June 29, 2015
Euro Hillclimbing: Beaujolais Villages
After last outing's dramas Charlie was hoping for a solid outing at Beaujolais [Updated with video]
Saturday, June 27, 2015
Lotus 3-Eleven - official!
No more silly hashtags or speculation - first details of the latest Lotus here [Updated pics]


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Forum Menu
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Post Response
Re: Strong JRTPosted by: Marie Evans
Posted on: April 22, 2003 at 14:59:15
In Reply to: Strong JRT
: Hello.
The best way to strengthen your JRT is to give him good exercise daily. This will help to build lean muscle mass and keep him toned.
It's not hard to do, it just takes time on your part to make sure he's getting enough hard exercise.
Take him out with a tennis ball and have him chase and fetch it. Remember though, if he's been a couch potato to gradually get him into a routine. Just like us humans, any type of physical exercise should be increased gradually so muscles don't get sore or your dog gets too exhausted. These dogs will go until they drop, so it's up to you to pace him.


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This is something that we learned of today that has my family basically speechless..
Background: My "nephew" Brendon has a rare metabolic condition that was destroying his kidney and liver. He had many hospitalizations throughout his life.Ā He was fed only via g-tube. He is very behind developmentally and was extremely cautious and easily frightened. He also suffers from Sensory Integration Disorder and hates all new things. Two years ago he received a kidney/liver transplant at Stanford at the age of 11. After the transplant he became obsessed with the movie Surfs Up and watched it 50+ times in the hospital.He loved all things surfing.Ā He joined the softball team, which we had tried talking him into many times before. He began eating normally. His new love was roller coasters where before he hated them and any rides for that matter.
Today we got a letter from the donor's family. His donor was a 15 year old girl who was an avid softball player, She loved trying new things to eat and lived for roller coasters. She died in a surfing accident.
Is this just strange coincidence? Does somehow part of her soul reside in him now? Thoughts?


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Lying With Pixels
Seeing is no longer believing. The image you see on the evening news could well be a fake - a fabrication of fast new video-manipulation technology.
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Allows a central login page for password protected child pages. Enter a password and you are taken to the newest child page with a matching password.
Extract the zip file and just drop the contents in the wp-content/plugins/ directory of your WordPress installation and then activate the Plugin from Plugins page.
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prevent tobacco-use initiation and promote cessation would need to be implemented at the multiple outlined levels (IOM, 2001). Individually oriented interventions would be most effective when the environment in which people live and make choices is in synchrony with the knowledge and behaviors addressed in the programs. Environmental and policy changes will be most effective when they are combined with programs that motivate and educate people to respond to the changes (Kumanyika, 2007).
Progress made in tobacco control in the general population has been based on a socioecologic understanding of health and human behavior (Hovell et al., 2009; Martinez-Donate et al., 2008). The greatest changes in smoking prevalence have resulted from populationwide interventions: economic measures to reduce access to tobacco; laws and regulations restricting tobacco use, advertising, promotion, and sales of tobacco products; and multicomponent public-education campaigns (Fisher et al., 2004; Task Force on Community Preventive Services, 2005). Lessons from tobacco control illustrate a compounding effect due to the interaction of interventions at different levels; tobacco-control interventions at the population level have proved most effective when conducted in combination with individual-level interventions. For example, smoking restrictions in workplaces and other public places can increase smokers' motivation to seek cessation services and to restrict smoking in their homes (Borland et al., 2006), which in turn may


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The Magazine
Clinging to Her Religion
The faith journey of Sarah Palin, 'Bible-believing Christian.'
Sep 29, 2008, Vol. 14, No. 03 • By TERRY EASTLAND
A few weeks before the Republican convention, Time magazine asked Sarah Palin what her religion was. "Christian," she said. Asked whether she was any particular kind of Christian, she replied, "No. Bible-believing Christian." Ever since John McCain asked Palin to be his running mate, her religion has been high on the list of subjects journalists have pursued. Although Palin herself hasn't brought it up and has mostly declined to be interviewed on the matter, it is already clear that her religious background contains material unfamiliar to media and political elites. Few politicians at Palin's level describe themselves as Bible-believing Christians.
Palin was baptized a Roman Catholic as an infant. When she was a teenager, she and her mother began attending the Wasilla Assembly of God. There she was "saved," as she has said, and also rebaptized, by full immersion, in Beaver Lake.
At Wasilla High School, Palin was known for her Christian faith. In an interview, John Bitney, who went to high school with her and later worked for her in the governor's office, recalls that she was "just a Christian girl" who was well regarded for her character. He adds that she "didn't preach" at anyone. A basketball star, she led a chapter of the Fellowship of Christian Athletes.
Palin, her husband Todd, and their growing family attended Wasilla Assembly of God until 2002, when they moved to Wasilla Bible Church. Palin also has worshipped at other churches, including the Church on the Rock in Wasilla. In Juneau, the state capital, she has gone to Juneau Christian Center.
Of these four churches, two--Wasilla Assembly of God and Juneau Christian Center--are members of the Assemblies of God. Founded in 1914, the Assemblies of God is the largest Pentecostal denomination in the country. Pentecostalism--which takes its name from the day of Pentecost when, according to the Book of Acts, the Holy Spirit descended upon the Apostles--is a movement that began in 1901 and is best known for its emphasis on the gifts of the Spirit, including speaking in tongues. The other two churches are freestanding congregations. The Church on the Rock is "charismatic," a term usually applied to more recent forms of Pentecostalism, while Wasilla Bible, the Palins' present church, is neither Pentecostal nor charismatic.
Reporters ask whether Palin has ever spoken in tongues. Her spokeswoman has said that Palin doesn't consider herself a Pentecostal. A friend of Palin's told the New York Times that her family left Wasilla Assembly of God for Wasilla Bible in part because the latter's ministry was "less extreme." Exactly what Palin may have found "extreme" at Wasilla Assembly of God is unclear. In any case, Palin retains an evident affection for Wasilla Assembly of God, as does the church for her.
The explicitly nondenominational Wasilla Bible Church was started more than 30 years ago by a small group of families. The word "Bible" was included in the church's name to reflect the Scripture's centrality in the lives of Christians, says pastor Larry Kroon. In interpreting the Bible, he says, "we try to get at the author's intent" by considering the text as well as its history and structure.
Kroon says that his church has had programs for children with special needs, that it supports the pro-life Heart Reach Pregnancy Center, which helps women in crisis pregnancies, and that it participates in house-building efforts undertaken by Habitat for Humanity. Rarely does Wasilla Bible have outside speakers, the most recent one a leader of Jews for Jesus. The church sometimes promotes events sponsored by outside groups, such as a recent Focus on the Family conference on overcoming unwanted same-sex attraction held in Anchorage.
Those who attend Wasilla Bible tend to be social conservatives. Kroon describes himself as "pro-life." But the church, he says, doesn't get involved in politics. "We're extreme the other way. We put everything else down when we worship, whether it's politics or anything else. The church is the church. Worship is worship." In contrast, there has been some political preaching at Wasilla Assembly of God, where the senior pastor asked in a sermon whether people who voted for John Kerry in 2004 would make it into heaven.


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Results 1 to 8 of 8
July 12th, 2011 #1
Triceratops skull + Ceratosaurus + Deinonychus
My first finnished and painted model.
Around 11xm lonng. I used an air drying clay for this, (because it was cheap ^^) and started from the middle. I had a side photo of a skull as a start and went into one direction - because I didnt fix it on a wire, so it had to lay on the table. XD
I used wire in the horns and in tha jaw, which is a single piece. You can detach it, but it actually fits that well into the skull that you can move the jaw, lift the model and it still doesnt fall off.
Painted with acrylics, burnt umber, raw/burnt sienna, ultramarine blue.
I can say I am really proud of this.
Last edited by Vaejoun; July 20th, 2011 at 11:38 AM.
Hide this ad by registering as a memberJuly 12th, 2011 #2
Looks great! Could you post a photo with a ruler or something as a scale reference? You have every reason to be proud.
July 13th, 2011 #3
Thanks, coming from you, it means a lot to me. . Always looking up to your works.
Here are two shots for the size:
(The ruler is upsite down, so dont be irritated by the strange numbers XD)
The Following User Says Thank You to Vaejoun For This Useful Post:
July 16th, 2011 #4
@Thagomizer: I tried out your paper method for skulls.
Tricky to get the 3d shape right, but it worked out nicely for the first shot.
Didnt have a good front view of the skull though, I just had to make a drawing myself and guess. ^^
Should be 1:10, if the data is correct that the skull is ~75 cm long.
July 16th, 2011 #5
Very cool! I've not really done this sort of thing as a finished piece but used it as a study aide or stepping stone to a more "finished" skull or flesh restoration. Well done.
July 20th, 2011 #6
@Thagomizer: Thanks again.
I made another step and finnished my first complete model of a dinosaur, using your papertechique again. That was a challenge.
Though I am not confident enough to go down to a 1m dinosaur in a 1:10 scale (anything below 1m should barely be possible to do)
I picked a Deinonychus antirrhopus. Simmilar to a Velociraptor but bigger. (~3m)
Here the WIP. I still have to make a podest or something for him to stand on, than I will post pictures of the finnished model.
July 20th, 2011 #7
Looking good! I hope you can post some photos of the finished skeleton.
My only criticism is the orientation of the hands. I think the palms should face inwards rather than downwards.
Keep up the good work!
July 21st, 2011 #8
Here you go ^^


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Originally written on Fox Sports Carolinas | Last updated 2/1/12
Clemson and Milton Jennings seemed like a perfect match three years ago when the Summerville, S.C. product chose to stay in-state and play basketball for the Tigers. But Monday for the second time this season Jennings has been suspended, this time for academic reasons. Jennings, a junior, was suspended earlier this season for the final game of Hawaiis Diamond Head Classic after an on-court confrontation with Brad Brownell late in a game the day before. Jennings later said he learned from the incident and was a better man for it. Its an unfortunate development for an athlete-program relationship that began with so much promise. Jennings was the first McDonalds All-American to sign with Clemson since Sharone Wright in 1991 and he was the Palmetto States first burger boy since Raymond Felton in 2002. Jennings was a hot commodity, offered by Connecticut, UCLA and Georgetown among others, but he saw Clemsons run of NCAA Tournaments and that it was close to home made it attending s...
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eco: 1/25 of 1272
next »
Health and wellbeing are at the heart of our Green future
Natalie Bennett
3rd July 2015The NHS is one of our greatest institutions and we must defend it to the hilt, writes Natalie Bennett. But to build the healthy society we all want and deserve, we need joined up policies across the policy spectrum, valuing human wellbeing above crude economic growth. more...
Secret report: fracking could hurt house prices, health and environment
Adam Vaughan & Rowena Mason / Guardian Environment
2nd July 2015House prices could fall 7% near fracking rigs, according to the 'secret' Defra report on fracking in rural areas, only published in full after a legal battle, while leakage of waste water could damage human health and contaminate food. more...
To live within planetary boundaries, we must contract the global economy
Samuel Alexander
5th July 2015We have a problem, writes Samuel Alexander. Even the most eco-friendly rich world lifestyles are overconsuming resources and over-dumping wastes. To put us on track to 'single planet living' will mean far deeper changes than any yet envisaged, including deliberate 'degrowth'' and the abandonment of consumer culture. more...
Exposing technocracy - the mindset of industrial capitalism
David King
27th June 2015Technology is crucial to all the big issues, but criticism is hampered by mythologies and structures of power, writes David King. Designed by and for corporate interests, modern industrial technologies embody a 400-year old technocratic philosophy of control of nature and people which must be confronted. more...
Labour's choice: neoliberalism, more neoliberalism - or Jeremy Corbyn?
Ben Whitham
4th July 2015Within minutes of Labour's election defeat its MPs were denouncing Miliband for failing to 'embrace aspiration' and alienating 'wealth creators', writes Ben Whitham. But the real problem was that he never expressed a coherent alternative to neoliberalism and austerity, presenting at best a 'Tory lite' agenda that failed to inspire. And who are the real wealth creators anyway? more...
Anti-austerity movement revives radical urban squatting
Almudena Serpis
24th June 2015Long a feature of British urban life, domestic squatting has now been criminalised, writes Almudena Serpis. But suddenly two social centres have come to life in a squatted bank in Brighton, and a long abandoned pub in London, reviving the rebellious spirit of the squatting movement, and promulgating a radical anti-austerity message that evokes the struggles in Greece, Spain and beyond. more...
Social and environmental justice: the struggle is one!
Pope Francis
28th June 2015We must beware an 'environmental' agenda that excludes the human dimension, Pope Francis writes in his recent Encyclical: the poor are least to blame for the ecological and climate crises, yet they are its primary victims. Humanity must make enduring decisions about the world we and our children will share. more...
Pope Francis's climate letter is a radical attack on the logic of the market
Steffen Böhm, University of Essex
18th June 2015The Encyclical published today by Pope Francis represents a profound religious and philosophical challenge to the mainstream narratives of our times, writes Steffen Böhm, and a major confrontation with the great corporate, economic and political powers, as it spells out the potential of a new world order rooted in love, compassion, and care for the natural world. more...
To support 'green' farming, officials must learn that small is beautiful
Alicia Miller / Sustainable Food Trust.
16th June 2015It's a hard life being an organic farmer, writes Alicia Miller - and specially when it comes to engaging with a bureaucracy that's trying to 'green' our agriculture. Should small scale farmers change their farming practices to fit in with it? Or the other way round?
Is Canada's government trying to kill off the wild salmon?
Jeff Matthews
8th June 2015Does the Canadian Government actually want to wipe out its wild salmon? To ordinary, sane people, the idea is completely mad, writes Jeff Matthews. But for resource extraction industries, salmon farmers and right wing neoliberal politicians, it could make perfect sense. more...
Sustainable fashion is slow fashion - because fast comes at a price
Ruth Styles
8th June 2015The mainstream fashion industry is trapped in a competitive spiral of ever shorter and faster sales and production cycles, writes Ruth Styles - and that inevitably stresses both textile workers and natural resources. But there is another way: the way of 'slow fashion' in which clothes are timeless, beautiful and made to last. more...
Endangered species don't need an Ark - they need a Living Planet!
Derrick Jensen
11th June 2015While we face 'hard choices' about which species and ecosystems to conserve, it's odd how we face no such quandaries over which of our frivolous luxuries to refrain from, or what murderous weapons system not to build, writes Derrick Jensen. And of course, there's no question at all of tackling the root causes of global ecocide. more...
eco: 1/25 of 1272
next »
Giving aid money to big business doesn't solve poverty. Who knew?
Kevin Smith
23d May 2015The UK government has showered £500 million of its aid budget on 'partnerships' with global corporations that are meant to help the poor, writes Kevin Smith. Surprise - an independent assessment has found that the only ones to benefit were the companies themselves. This ideologically-driven farce must stop now! more...
Over-grazing and desertification in the Syrian steppe are the root causes of war
Gianluca Serra
5th June 2015Civil war in Syria is the result of the desertification of the ecologically fragile Syrian steppe, writes Gianluca Serra - a process that began in 1958 when the former Bedouin commons were opened up to unrestricted grazing. That led to a wider ecological, hydrological and agricultural collapse, and then to a 'rural intifada' of farmers and nomads no longer able to support themselves. more...
Sustainable abundance - rebuilding fisheries to support coastal communities in Madagascar
Alasdair Harris
12th May 2015Marine conservation is usually expressed in austere and negative terms, writes Alasdair Harris, with strict quotas and exclusion zones. But the truth is the exact opposite: it's about working with natural ecosystems to unlock their productive potential, creating sustainable wealth and abundance for fishing communities while enhancing marine biodiversity. more...
Renewable energy alone cannot reverse global warming or make a sustainable world
Pete Dolack
25th May 2015The renewable power boom is excellent news for people and planet, writes Pete Dolack. But let's not get carried away: much energy that claims to be 'renewable; like biomass and big hydro, is no such thing. And greening our energy is just one of many steps to a sustainable world. The greatest challenges - like tackling the monster of infinite 'growth' - all lie ahead. more...
Cullers beware - killing 'pest' animals can increase their abundance
Christopher Johnson
8th May 2015A study of feral cats in Tasmania shows that culling them to reduce their impact on native wildlife had a paradoxical effect - their population went up! If you can't take 'pest' animals out faster than they can reproduce and move in from nearby areas, writes Christopher Johnson, you're better off not bothering at all. more...
If modern farming can't sustain bees, how much longer can it sustain us?
Dave Goulson
11th May 2015Our bees and wider farmland ecosystems have been seriously harmed by neonicotinoids, writes Dave Goulson. But that's just the start of the damage that modern farming is doing to wildlife in a countryside stripped of wild flowers and drenched by cocktails of pesticides. The problem is not just neonics, but the entire model of industrial agriculture. more...
For a Green future of tremendous possibility
Natalie Bennett
6th May 2015Whatever government we end up with after tomorrow's election, there's just one way to make sure it takes climate change and wider environmental issues seriously, Natalie Bennett told a Cambridge audience yesterday - for us to deliver the strongest Green vote ever! more...
The oceans are not worth $24 trillion
Charles Eisenstein
15th May 2015Some things are beyond measure and beyond price, writes Charles Eisenstein. No amount of money is enough to compensate for the loss of the sacred or the essential - and to pretend that it is, is to capitulate to the very mentality that is driving the destruction of the biosphere. more...
To save the world's large herbivores, we too must make them welcome
Matt Hayward & William Ripple
5th May 2015Most of the world's large herbivores - from elephants to gorillas, tapirs and sloths - are at risk of extinction, a new study shows. With most of these species in poorer countries, rich nations should dedicate substantial resources to their survival. But we must also lead by example - by reintroducing them ourselves. more...
Can agroecology save us from 'scorched-earth' agriculture?
Professor Henrietta Moore
6th May 2015Industrial agriculture has become a prime driver of many of the world's most serious problems, writes Henrietta Moore: the loss of wild and farmed biodiversity, huge climate-changing emissions, and the entrapment of small farmers in ever-deepening cycles of poverty. But there is a solution: the widespread adoption of agroecological farming. more...
'Beyond petroleum' - fracking's collapse heralds the arrival of peak oil
Paul Mobbs
5th May 2015The 'death of peak oil' has been much exaggerated, writes Paul Mobbs. Take out high-cost 'unconventional' oil and production peaked ten years ago, and even North America's fracking and tar sands boom has failed to open up new resources both big enough to make good the shortfall, and cheap enough to reward investors. We really do need to be thinking 'beyond petroleum'. more...
In Nepal's next big quake, hydropower dams threaten catastrophe
Michael Buckley
4th May 2015A spate of hydroelectric dam building in Nepal means that future earthquakes could send inland tsunamis flooding down the steep mountain valleys, writes Michael Buckley. Disaster was averted in last month's quake - a badly damaged dam was not yet filled. But despite the risks and the damage to river ecology, tourism and rural livelihoods, there's no sign of any policy shift. more...
Future dustbowl? Fracking ravages Great Plains land and water
Tim Radford
4th May 2015The fracking boom has caused massive vegetation loss over North America's rangelands, writes Tim Radford, as 3 million hectares have been occupied by oil and gas infrastructure and 34 billion cubic metres of water have been pumped from semi-arid ecosystems. more...
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Lather and use as required on body and hair.
Aloe barbadensis leaf juice extract, Aqua, Coco-glucoside, Sodium coco-sulfate, Citric acid, Butylene glycol, Cocoyl proline, Glyceryl oleate, Punica granatum extract, Sodium chloride, Sodium benzoate, Potassium sorbate, Parfum*, Citrus aurantium dulcis (Orange) oil, Citronellol, Geraniol, Hexyl cinnamal, Limonene. Suitable for vegetarians and vegans
Advisory information:
Avoid contact with eyes, if this should occur, immediately rinse with warm water. Due to the ingredients natural origin, colour and consistency may vary from batch to batch. This does not affect the quality of the product.