text:
- © 2008 American Society of Plant Biologists
In addition to the major serine/threonine-specific phosphoprotein phosphatase, Mg2+-dependent phosphoprotein phosphatase, and protein tyrosine phosphatase families, there are novel protein phosphatases, including enzymes with aspartic acid-based catalysis and subfamilies of protein tyrosine phosphatases, whose evolutionary history and representation in plants is poorly characterized. We have searched the protein data sets encoded by the well-finished nuclear genomes of the higher plants Arabidopsis (Arabidopsis thaliana) and Oryza sativa, and the latest draft data sets from the tree Populus trichocarpa and the green algae Chlamydomonas reinhardtii and Ostreococcus tauri, for homologs to several classes of novel protein phosphatases. The Arabidopsis proteins, in combination with previously published data, provide a complete inventory of known types of protein phosphatases in this organism. Phylogenetic analysis of these proteins reveals a pattern of evolution where a diverse set of protein phosphatases was present early in the history of eukaryotes, and the division of plant and animal evolution resulted in two distinct sets of protein phosphatases. The green algae occupy an intermediate position, and show similarity to both plants and animals, depending on the protein. Of specific interest are the lack of cell division cycle (CDC) phosphatases CDC25 and CDC14, and the seeming adaptation of CDC14 as a protein interaction domain in higher plants. In addition, there is a dramatic increase in proteins containing RNA polymerase C-terminal domain phosphatase-like catalytic domains in the higher plants. Expression analysis of Arabidopsis phosphatase genes differentially amplified in plants (specifically the C-terminal domain phosphatase-like phosphatases) shows patterns of tissue-specific expression with a statistically significant number of correlated genes encoding putative signal transduction proteins.
The phosphorylation and dephosphorylation of proteins has been found to modify protein function in a multitude of ways (Cohen, 2002). The protein kinase content (kinome) of many eukaryotes and their evolutionary relationships have been studied in depth, revealing both the importance and diversity of these proteins (Manning et al., 2002a, 2002b; Caenepeel et al., 2004; Champion et al., 2004). With the exception of the few phosphatidyl inositol 3-kinase-like kinases, the protein kinases share a highly conserved catalytic domain. In contrast, the protein phosphatases are more diverse, displaying three different catalytic signatures, and thus can be divided into three broad groups (Moorhead et al., 2007). Although many of the phosphatases have been cataloged in the genomes of several organisms (Koh et al., 1997; Kerk et al., 2002; Alonso et al., 2004), this list continues to grow and, in organisms like higher plants, classification schemes are often quite incomplete.
Protein phosphatases were originally identified as enzymes responsible for dephosphorylating Ser and Thr residues on enzymes involved in mammalian glycogen metabolism. Purification of these enzymes, cloning, and genomics have revealed that this group is composed of two families (Ser/Thr-specific phosphoprotein phosphatase [PPP] and Mg2+-dependent phosphoprotein phosphatase [PPM]) that represent the major group of Ser and Thr phosphatases in eukaryotes (Table I ; Rayapureddi et al., 2003; Alonso et al., 2004; Gohla et al., 2005; Moorhead et al., 2007). Ten years after the cloning of the first Tyr kinase, the first Tyr phosphatase was purified and then cloned. Its catalytic signature (C[X]5R) defined the large protein Tyr phosphatase (PTP) superfamily (Table I), which now, in addition to the Tyr specific enzymes, includes enzymes that specifically dephosphorylate Ser or Thr as well as Tyr (the dual specificity phosphatases [DSPs]), mRNA, and phosphoinositides. Based on this catalytic signature, the group has expanded to 107 transcribed genes in humans (Homo sapiens). Several of these are catalytically inactive but their gene products function in the cell, and several have been linked to human diseases (Robinson and Dixon, 2006). The third major group of phosphatases was identified most recently and is characterized by a catalytic signature DXDXT/V and is referred to here as the Asp-based enzymes (Table I). The phosphatase responsible for dephosphorylation of the C-terminal domain (CTD) of RNA polymerase II (Pol II; FCP1) was the first enzyme of this group to be identified as a protein phosphatase and the related small CTD phosphatases (SCPs) have been recognized as part of the group. Several haloacid dehalogenase (HAD) superfamily members, such as EYES ABSENT (EYA), which acts as a transcription factor, and chronophin, which controls cofilin phosphorylation, have now been demonstrated to function as protein phosphatases. Like FCP1 and SCP enzymes, HAD superfamily members have a DXDXT/V catalytic signature, utilizing a unique Asp-based catalytic mechanism. The HAD superfamily is potentially very large, but to date only a few members have been demonstrated to display Ser or Tyr phosphatase activity (Moorhead et al., 2007).
A catalog of enzymes that comprises the PPP, PPM, and some of the PTP family members of Arabidopsis (Arabidopsis thaliana) was presented several years ago (Kerk et al., 2002). Since then both the number of known protein phosphatases and the set of completely sequenced reference genomes have expanded considerably. In this situation a systematic revisitation of the protein phosphatase repertoire is warranted. We have focused on these new or novel phosphatases by using the 11 phosphatase classes from humans to identify homologs from the genomes of Arabidopsis, green algae (Chlamydomonas reinhardtii and Ostreococcus tauri), Oryza sativa, and Populus trichocarpa. These proteins, along with their counterparts in humans and other animals, were analyzed to determine their interrelationships. When combined with previous studies from our laboratory, this work defines a complete set of all known varieties of protein phosphatases in Arabidopsis.
Homologs were identified using BLAST searches as well as hidden Markov models (HMMs) of the catalytic domains. The overall results of this study are summarized in Table II . This lists the number of homologs for each protein phosphatase type that was found in each of the subject organism-specific databases. The structural classes are derived from table I in the recently published study of Moorhead et al. (2007). Results will be discussed in the order of their appearance in this table, followed by an analysis of expression and promoters of a subset of the identified genes. Evidence of expression of all proteins is summarized in Supplemental Table S1 (see "Materials and Methods" for details).
Using the sequence of human SSU72 (gi:7661832) to search the target protein databases, we found one candidate homolog each in C. reinhardtii (Cre130182), O. tauri (Ot16g02480), Populus (Pop775960), Arabidopsis (At1g73820.1), and O. sativa (Os12g07050.1 and .2). We constructed a multiple sequence alignment, which is presented as Supplemental Figure S1. Phylogenetic trees show that both of the algal sequences cluster with the higher plant sequences in two of the three inference methods with high bootstrap support (82.2% maximum parsimony [Pars]; 80.0% maximum likelihood [ML]).
Slingshots, along with chronophin (included in "Gene Expression"), dephosphorylate cofilin and, as a result, stimulate the depolymerization of F-actin (Huang et al., 2006). We used the human sequences SSH1 (gi:40254884), SSH2 (gi:37674210), and SSH3 (gi:24586675) to search the target protein databases. We found no candidate slingshot homologs in the algae, nor in any of the plant species, indicating that this method of regulation is animal specific.
CDC14 belongs to the family of PTPs and is responsible for control of mitotic exit in organisms of the fungi/metazoan group (Trinkle-Mulcahy and Lamond, 2006). We used the human proteins CDC14A (gi:55976620) and CDC14B (gi:55976216) to search the target protein databases. A single candidate CDC14 homolog was found in C. reinhardtii (Cre112184). Our work shows clearly through several different methods that this sequence has all the structural features expected of a true CDC14. First, analysis with the FFAS03 technique (Rychlewski et al., 2000) shows that there is sequence similarity between Cre112184 and the solved structure of human CDC14B (Protein Data Bank entry: 1ohc; Gray et al., 2003) extending for over 300 amino acids, encompassing both the upstream A (unique to CDC14) and the downstream B (PTP/DSP catalytic) domains. The score for this comparison is very high (Z ∼ 90 versus Z ∼ 110 for HuCDC14B versus itself; for this technique a Z score of 9.5 or greater is considered significant). Second, the FFAS03 alignment shows high conservation (10/11) of a set of critical residues described in the solved structure of human CDC14B [these include the canonical PTP/DSP catalytic residues (HC[X]5R) in the B domain, plus a number of others unique to CDC14s]. Third, a multiple sequence alignment was constructed encompassing the B domain of animal CDC14s and, as an outgroup, the protein phosphatase domains of a set of DSPs previously characterized from Arabidopsis (Kerk et al., 2002, 2006; presented as Supplemental Fig. S2). The corresponding phylogenetic tree is presented as Figure 1 . It is clear that in this tree Cre112184 is part of a clade with human and Xenopus laevis CDC14s, sharing a common node in all three phylogenetic tree inference methods, with high bootstrap support (100% neighbor joining [NJ]; 98.8% Pars; 78.2% ML).
In the green alga O. tauri there is a domain that is related to CDC14s. It was initially detected with BLAST searches utilizing human CDC14 sequences (E ∼ e-5). It is found as an N-terminal domain in a sequence (Ot15g00870) that is annotated as containing a nicotinamide adenine dinucleotide kinase (NADK) domain (PF01513). In a reciprocal BLAST search, the best hit to this O. tauri domain is human CDC14A (E = 0.001), indicating a specific relationship. Using this O. tauri domain sequence as a query, we found similar sequences (BLAST hits E ∼ e-15) in P. trichocarpa (two sequences: Pop347211 and Pop714589), Arabidopsis (A1g21640.1), and O. sativa (Os11g08670). Upon further analysis, it is very clear that these sequences are all related to CDC14. First, when the FFAS03 technique is applied to each of them, there is similarity to the solved structure of human CDC14B (Protein Data Bank entry: 1ohc) along a 300-amino-acid region that encompasses both the A and B domains. The scores for these comparisons are strong, between Z = 20 and Z = 50. (Note, however, that these scores are much weaker than those obtained with the C. reinhardtii sequence Cre112184, presented above.) The O. tauri sequence (Z ∼ 50) retains the canonical PTP/DSP catalytic residues (HC[X]5R), and therefore could be enzymatically active. It retains some of the hydrophobic pocket residues described in the solved structure of human CDC14B, but not all of them, and has only two of six acidic residues in the acidic groove region. In the solved structure these acidic residues are thought to be critical to binding basic residues in the target cyclin-dependent kinase, whereas the hydrophobic pocket residues are responsible for maintaining substrate specificity of CDC14 for Pro in the pSer + 1 position (Gray et al., 2003). Therefore, although structural resemblance is readily apparent, it is doubtful that this domain could function specifically as a CDC14. The higher plant sequences obtain FFAS03 scores of between Z = 22 and Z = 36. They lack nearly all of the set of specific CDC14 residues, and furthermore have a C to S substitution in the PTP/DSP catalytic loop sequence. They therefore could not function as catalytic domains. A multiple sequence alignment encompassing the length of the full A and B domains of the CDC14-like sequences is presented as Supplemental Figure S3.
When considered in the context of the shorter multiple sequence alignment (Supplemental Fig. S2) and the subsequent phylogenetic tree (Fig. 1), it is apparent that sequence Ot15g00870 and the higher plant sequences form a second, distinct clade, sharing a common node with high bootstrap support in all tree inference methods (99.1% NJ; 98.4% Pars; 82.7% ML). Finally, the C. reinhardtii/animal cluster and the O. tauri/higher plant cluster are clearly related, compared with the generic Arabidopsis DSPs, sharing a common node in all three tree inference methods, with high bootstrap support in two of them (88.6% NJ; 95% Pars; 40.2% ML). It is thus very clear that all these sequences are CDC14 like and evolved from a common ancestor, if not from CDC14 itself. All other clusters and nodes in this DSP tree are as previously published (Kerk et al., 2006; data not shown).
As an additional note of interest, the higher plant CDC14-like sequences are found as a domain on NADKs. NADK2, the Arabidopsis protein containing the domain, is a chloroplast-localized protein that has been shown to be a calcium-dependent calmodulin-regulated protein (Turner et al., 2004; Chai et al., 2005). Calmodulin binding takes place on the N terminus of the protein, which is the location of the CDC14-like domain. The calmodulin-binding site was mapped to a 45-amino-acid region of the domain, presented in figure 7 of Turner et al. (2004). When comparing with our multiple sequence alignment, the heart of this binding motif is the mutated PTP consensus site (H[C→S]X5R). This is reminiscent of substrate trapping mutants observed at the altered consensus catalytic motifs of other PTPs (Bliska et al., 1992; Milarski et al., 1993; Sun et al., 1993).
The CDC25 proteins also have a role in control of progression through the cell cycle in fungi and metazoans. Although CDC14 controls mitotic exit, CDC25 is involved in the transition from G2 to M phase (Trinkle-Mulcahy and Lamond, 2006). Humans have three CDC25s (CDC25A [gi:50403734]; CDC25B [gi:21264471]; CDC25C [gi:125625350]). Using these sequences to search the target databases, we found a number of similar sequences, whose protein phosphatase catalytic domains are collected in the multiple sequence alignment presented as Supplemental Figure S4. The resulting phylogenetic tree is presented as Figure 2 . O. tauri has a candidate CDC25 homolog (Ot02g05470) that is a member of a CDC25 clade encompassing human, animal, and yeast (Saccharomyces cerevisiae and Schizosaccharomyces pombe) sequences, sharing a common node with high bootstrap support in all three tree inference methods (100% NJ; 97.8% Pars; 75.9% ML). Previous work supports this, as this gene has previously been cloned, and the expressed protein acted as a CDC25 in both yeast complementation and starfish oocyte cell division assays (Khadaroo et al., 2004). They noted the divergence of the N-terminal domain of this sequence (see Supplemental Fig. S5 for an alignment of the O. tauri and human sequences), but asserted that there was a conserved 14-3-3-binding site and several potential phosphorylation sites. Presumably the 14-3-3-binding site reported was 252-RPLASPP-258, the closest match to either of the consensus binding sites (Rxxx[S/T]xP in this case), which, while matching the consensus, has a Pro in both the S − 3 and S + 1 positions. This has been shown to be unfavorable (Yaffe et al., 1997), making it unlikely that the O. tauri protein has any 14-3-3-binding capability. Thus, although this protein clearly can act like a true CDC25 in functional assays and we support its classification as a CDC25 based on our sequence analysis, we anticipate that its regulation in vivo might well differ from that previously described for the fungal/animal proteins. Our searches also revealed sequences sharing some similarity in C. reinhardtii (Cre153947, Cre171654, Cre183511, Cre167673), P. trichocarpa (Pop282198), Arabidopsis (A5g03455.1), and O. sativa (O10g39860, O03g01770). The Arabidopsis sequence was originally published as a CDC25 (Landrieu et al., 2004), but more recent work indicates that it may functionally be an arsenate reductase, indicated by a lack of arsenate reductase activity in a T-DNA plant line, and in vitro arsenate (V) reductase activity (Bleeker et al., 2006; Dhankher et al., 2006). The sequences of several known arsenate reductases from fern (Pteris vittata; PvACR2), yeast (ScACR2, SpACR2), and the protist Leishmania major (LmACR2) form a clade with the above candidate algal and higher plant sequences, with strong bootstrap support in two phylogenetic tree inference methods (98.8% Pars; 82.7% ML), supporting the categorization of these proteins as arsenate reductases. Our findings confirm and extend, with larger sequence sets, the phylogenetic analyses previously reported (Dhankher et al., 2006; Ellis et al., 2006). The higher plant and C. reinhardtii sequences also lack any significant N terminus, which is known to contain regulatory sites in animal CDC25s, such as phosphorylation and 14-3-3 protein-binding sites. It is apparent that the algal/plant proteins lack the conserved regulatory sites, as well as the catalytic activity, of the fungal/animal CDC25s.
This viewpoint is supported by a recent article questioning the existence of CDC25 in higher plants, and suggesting that cell cycle control has been reorganized along lines distinctly different than the fungal/metazoan (presumably ancestral) model (Boudolf et al., 2006). It seems that with our data, an expansion of this concept is in order. It appears as if this reorganization of cell cycle control has occurred in higher plants, and that this process began during the radiation of the green algae. When combined with the above information on CDC14 in plants, it appears that different algal species are frozen at different points in this reorganization, some retaining one mitotic phosphatase or the other, but both lost by the transition to higher plants. Study of these algal species, as well as the higher plants, may give a unique insight into the evolution of these processes.
Low-Molecular-Weight PTPs
We used the sequence for the low-molecular-weight PTPs (LMWPTPs) from humans (ACP1 [gi:1709543]) to search the target protein databases. We found one candidate homolog in C. reinhardtii (Cre117512), none in O. tauri, two in P. trichocarpa (Pop821042, Pop594818), one in Arabidopsis (At3g44620.1), and one in O. sativa (Os08g44320.1). The multiple sequence alignment constructed from these sequences is presented as Supplemental Figure S6. As seen in the alignment, the proteins are remarkably conserved, indicating an essential, conserved function for the protein in eukaryotes. The lack of a homolog in O. tauri is puzzling, however, and is possibly the result of secondary loss of the protein because the O. tauri genome is remarkably streamlined (Derelle et al., 2006).
Asp-Based Catalysis: FCP Like
The TFII-interacting RNA Pol II CTD protein phosphatase FCP1 is an essential yeast protein that acts to dephosphorylate the CTD of the largest subunit of RNA Pol II (Archambault et al., 1997). This subunit contains an array of repeats of a heptad unit containing Ser residues at positions 2 and 5. The transcription initiation and elongation process consists of a variety of mRNA modifying proteins being recruited to the Pol II complex. This appears to be modified by the state of Pol II phosphorylation—it is recruited to the complex in a hypophosphorylated state, phosphorylated during the transcription process, then dephosphorylated to allow termination and recruitment to a new complex (Meinhart et al., 2005; Moorhead et al., 2007). Complex modifications of the phospho-array are thus possible and with it modulation of the transcription process. FCP1 is a metal-binding protein, possessing a DXDXT/V motif that is essential to catalytic activity (Kobor et al., 1999). The isolated phosphatase domain is sufficient for catalytic activity. In plants and algae, we found a large set of proteins sharing a degree of similarity to this prototype sequence. A large multiple sequence alignment of the protein phosphatase catalytic domain of 99 sequences was constructed (presented as Supplemental Fig. S7) and the corresponding phylogenetic trees inferred (Fig. 3 ). The Arabidopsis proteins CPL1 (CTD phosphatase-like protein phosphatase) and CPL2 contain an FCP-like catalytic domain; however, they and their homologs in other plants are further characterized by the presence of one or two double-stranded RNA (dsRNA)-binding domains, and are discussed separately from the other members of this family. The trees were composed of several distinct subclusters, which are each presented in turn, based upon the topology of the NJ tree. The amino acid residues required for protein phosphatase catalytic activity have been well studied in FCP1 (Hausmann and Shuman, 2003; Hausmann et al., 2004). A set of 11 critical sequence positions have been identified through biochemical analysis and are indicated in Supplemental Figure S7. The majority of sequences in this FCP1-like data set retain conservation at all these residue positions. However, 44 sequences deviate from the yeast residue pattern in at least one position (see Supplemental Fig. S7 legend).
SCP (Subcluster A)
SCP proteins are small RNA Pol II CTD protein phosphatases. In humans there are three proteins: SCP1 (gi:15278033), SCP2 (gi:31074179), and SCP3 (gi:34392247). We found one sequence in Chlamydomonas (Cre149388) and one in Ostreococcus (Ot12g02910) that share similarity with the animal proteins in the phosphatase catalytic domain, and none in the higher plants. Upon multiple sequence alignment and phylogenetic tree inference, these algal sequences form part of a clade with the human and other animal proteins, sharing a common node with varying bootstrap support in the three tree inference methods (99.7% NJ; 44.8% Pars; 34.7% ML). This support is not to our minimum threshold of majority support in two of three methods. To clarify the situation, sequences were added (duplicates with different accession numbers) and removed (more divergent sequences) from the alignment used to generate the trees. In both these situations, the modified alignments, with more or less sequences, met our requirements of support of two of three methods. However, N-terminal (noncatalytic domain) motif analysis shows that the O. tauri sequence does not share motifs found in the animal sequences, and the C. reinhardtii sequence lacks this N-terminal region. Thus, while not unequivocal, these data support the assignment of these two algal sequences to the SCP cluster.
Subclusters B to F
Subcluster B is a mixed group of 19 animal/algal/plant sequences that achieve high to moderate bootstrap support in two tree inference methods (96.7% NJ; 70.1% ML). The sequences in subcluster B are: Hu6841480 (also known as HSPC129), Xt56605878, Ot14g03430, Cre118402, Pop415530, Pop179057, Pop586980, Pop568466, A5g11860.1, A5g46410.1, O7g10690.3, O07g10690, O05g49120, O1g43870.2, O01g43870, O1g47540.3, O01g47540, O1g47540.4, and O1g47540.2. N-terminal motif analysis of the sequences in subcluster B shows that the Os01g43870 isoforms lack motifs shared by the other sequences. Characterization of the proteins in this group is limited to a very recent study indicating CTD phosphatase activity of the human protein HSPC129 (Qian et al., 2007). There has been moderate radiation of proteins belonging in this subcluster in higher plants, where, discounting splice variants, higher plants have between two and four homologs.
Subcluster C comprises human Dullard and its animal homologs (100% NJ; 100% Pars; 98.4% ML). Dullard is a fairly recent discovery in this gene family, and has been implicated in neural tube development, in the BMP (bone morphogenetic protein) pathway, and in nuclear membrane biogenesis (Satow et al., 2002, 2006; Kim et al., 2007). N-terminal sequence analysis shows that these sequences share common motifs. The presence of a Dullard homolog in yeast, involved in a conserved pathway (Kim et al., 2007), and the lack of homologs in algae/plants suggest it arose after the plant/animal evolutionary split.
Subcluster D is a group of six higher plant sequences (Pop545127, A1g29780.1, A1g29770.1, A5g45700.1, O05g11570, and Pop235176). This group has high bootstrap support in all three tree inference methods (99.4% NJ; 91.8% Pars; 80.6% ML). N-terminal sequence analysis shows that these sequences share common motifs. Subcluster E contains both algal and higher plant sequences (Cre111940, Pop560916, Pop659512, A3g55960.1, Os01g61640, and O05g39070). This group receives high to moderate bootstrap support in two tree inference methods (99.3% NJ; 60.46% ML) and these sequences share common N-terminal motifs. Subcluster F contains two C. reinhardtii sequences that are not splice variants (Cre187551 and Cre142839) and have high to moderate bootstrap support in two of three inference methods (99.7% NJ; 79.4% ML). The proteins of subclusters D to F have yet to be characterized.
Subcluster G
This is a set of eight sequences from animals, algae, and higher plants (TIM50Hu, Cre169672, Ot03g04550, Pop642296, Pop568582, A1g55900.1, O05g43770, and O1g55700.1). This group receives moderate bootstrap support in all three tree inference methods (82.0% NJ; 88.7% Pars; 74.7% ML). N-terminal motif analysis shows that most sequences in this cluster share a common signature. TIM50 only weakly shares some elements of this signature, and is clearly the most distantly related sequence in this group. TIM50 (sometimes referred to as TIMM50) is the homolog of the yeast protein of the same name, and is named as translocase of inner mitochondrial membrane 50 kD. As indicated by its name, TIM50 is involved with the translocation of proteins through the inner membrane into the matrix, as part of the TIM23 complex (mitochondrial protein import; for review, see Neupert and Herrmann, 2007), although a nuclear-localized isoform has also been identified in humans (Xu et al., 2005). The Arabidopsis protein was identified in a previous proteomic characterization of mitochondrial import proteins, demonstrating the conservation of the localization of this protein, at the very least (Lister et al., 2004).The human isoform has also been shown to possess phosphatase activity, intriguingly active on phosphorylated Ser, Thr, and Tyr (Guo et al., 2004). This subcluster is the only one containing proteins that we can be all but assured are not involved in the dephosphorylation of the CTD (due to their mitochondrial localization), raising questions not only about the substrate specificity of other FCP-like proteins but also about the specific target of dephosphorylation by these TIM50 homologs.
"FCP Assemblage" (Subcluster H)
This is a large group of sequences (24) from animals, algae, and higher plants. The assemblage as a whole receives moderate to low bootstrap support from all three tree inference methods (64.1% NJ; 67.6% Pars; 43.7% ML; note that several sequences are excluded from the subcluster in the Pars tree; see the Fig. 3 legend for details). Within it are distinct subclusters formed by the yeast/animal FCP1 group (FCP1_Spomb, FCP1_Hu, Dr49618915, Dr94734487, FCP1_Xle, and Xt62858037), the higher plant CPL3 (Pop708815, CPL3_Ath, and OsCPL3), and CPL4 (Pop262722, CPL4_Ath, and OsCPL4) groups, and associated algal sequences (Cre187332, Cre141879, Ot04g02710, and Ot03g04040). Of particular note is a subcluster made up exclusively of Arabidopsis sequences (A5g23470.1, A2g02290.1, A1g20320.1, A1g43600.1, and A1g43610.1). There is also a subcluster of closely related sequences from the algae as well as P. trichocarpa (Ot03g00770, Cre149314, and Pop560900). Each of these subclusters receives high bootstrap support, but their relative topological interrelationships within the assemblage varies slightly among the different tree inference methods.
N-terminal motif analysis of these sequences indicates that some of these sequences share more than simply the same catalytic domain. The higher plant CPL3s have a distinct motif signature (data not shown). Elements of this signature are weakly shared by the two algal sequences: Ot03g00770 and Cre187332. This indicates that these sequences are most closely related to the CPL3s. The higher plant CPL4s also have a distinct motif signature (data not shown). The algal sequence Ot03g04040 shares this motif signature, as does the algal sequence Cre141879 (though with reduced similarity). Finally, the CPL4 upstream motif signature is shared by the Arabidopsis sequences A1g20320, A2g02290, and A5g23470. It is likely that all these CPL4-like sequences are related. The animal FCP1s share a common motif signature, which is shared to some extent by yeast FCP1. The CPL4-like sequences share elements of this motif signature with the FCP1s, whereas the CPL3s do not, suggesting a closer relationship of CPL4-like and FCP1 groups. The algal sequence Ot04g02710 shares elements of the FCP1 motif signature, suggesting it is more closely related to the animal and yeast FCP1s. Sequences A1g43600 and A1g43610 have nonexistent or short N termini, respectively, and are likely to be regulated differently from other proteins in this cluster.
Fungal and animal FCP1 proteins have, in addition to the protein phosphatase catalytic domain, a downstream phosphoprotein-binding BRCA-related C-terminal (BRCT) domain. The only sequences in our data set containing this domain are in the "FCP Assemblage", confirming the relationship of these algal and plant sequences to the FCP1s. However, although most sequences contain the BRCT domain, some do not. Fifteen of the 24 sequences in the cluster contain it, with the exception being the small Arabidopsis-only sequence cluster (five sequences), the three algal sequences Ot03g00770, Cre141879, Cre149314, and the P. trichocarpa sequence Pop560900, which is likely a CPL1/2 relative but was included here because of its ambiguity. Multiple sequence alignment of the BRCT domain sequences show them to be well conserved, and we would therefore expect them to be functional (data not shown). Although the Arabidopsis sequences without BRCT domains are likely a result of secondary loss of the domain, the algal sequences could also be an indicator of the original state of the FCP-like proteins, before gaining the BRCT domain.
Limited study of the Arabidopsis CPL3 and CPL4 proteins sheds some light on the comparative function of these proteins in plants. Both of these proteins contain a functional BRCT domain, which binds to AtRAP74, a homolog of animal/yeast TFIIF (Bang et al., 2006). Knockout plants for CPL3 display hyperactivation of abscisic acid (ABA)-mediated transcription, as well as a general alteration of plant growth and maturation, which can be duplicated with mutations to either the BRCT or catalytic domains (Koiwa et al., 2002). RNAi knockdown of CPL4 also leads to plant growth and maturation defects (Bang et al., 2006).
Subcluster I
This cluster is composed of 10 plant and algal sequences (O07g01850, A4g261190.1, Pop290290, A3g29760.1, Pop296466, Pop195150, O3g54870.1, O3g54850.1, Ot05g00010, and Ot05g00160; Cre166215 is also included depending on tree). This group receives a range of support from the three tree inference methods (99.5% NJ; 60.4% Pars; 68.1% ML; Cre166215 is included in this subcluster in the Pars and ML trees). The sequences do not seem to have any significant relation outside of the catalytic domain.
Subcluster J
This cluster is composed of eight plant, animal, and algal sequences (Cre115803, Ot01g02920, and MGC10067Hu [also known as UBLCP1, ubiquitin-like domain-containing C-terminal phosphatase 1; Zheng et al., 2005], O01g6450, O1g65450.2, A4g06599.1, Pop274109, and Pop830623). This group receives high to moderate support from the three tree inference methods (100% NJ; 99.0% Pars; 58.2% ML). This group is defined by the presence of a ubiquitin-like domain on the N terminus of the proteins. This domain is listed in the National Center for Biotechnology Information conserved domain database, as cd01813, as shared with ubiquitin-specific proteases; however, all entries appear to be CTD phosphatase homologs. Despite this, these proteins do have what appears to be a proteasome interacting motif based on the work of Upadhya and Hegde (2003). The human protein in this group, UBLCP1 (listed on the tree and alignment as MGC10067), has been studied and determined to be a functional CTD phosphatase with a possible preference for Ser-5 (Zheng et al., 2005). The combination of the ubiquitin-directed proteolysis and RNA Pol II phosphatase activity, in addition to the apparent conservation, make this group of proteins intriguing, and further study of their role in the cell is awaited.
CPL1 and CPL2
CPL1 and CPL2 are CTD phosphatase-like protein phosphatases initially described in Arabidopsis (At4g21670.1 and At5g01270.1, respectively; Koiwa et al., 2002, 2004). As mentioned above, these FCP-like phosphatases are characterized by the presence of dsRNA-binding domain(s) on the C terminus (two in CPL1 and one in CPL2). We used the Arabidopsis sequences to search the target protein databases. We found four candidate homologs in P. trichocarpa (Pop555554, Pop743771, Pop90064, and Pop560900), and six candidate homologs in O. sativa (O02g42600, O01g63820, O1g63820.2, O4g44710.1, O4g44710.2, and Os38346621; the last being a possible isoform of the previous two). The multiple sequence alignment encompassing these full-length sequences and that of the Arabidopsis proteins is presented in Supplemental Figure S8. From an inspection of the C-terminal region of this alignment, it is evident that six of these newly identified sequences have two full predicted RNA-binding domains, with a high degree of similarity to CPL1_Ath, and are therefore CPL1s (O02g42600, Os38346621, O4g44710.1, O4g44710.2, Pop555554, and Pop743771). In contrast, two of the new sequences (Pop90064 and Pop560900) have a greatly truncated second RNA-binding region (very similar to CPL2_Ath) and are therefore CPL2 proteins. The situation with the remaining new sequence is more complex.
The sequence O01g63820 (both isoforms) occupies an intermediate position between the well-defined CPL1 and CPL2 clusters in the phylogenetic trees. There is disagreement between tree inference methods as to whether it is included within the CPL1 cluster (NJ) or the CPL2 cluster (Pars). Although the protein contains a well-conserved second RNA-binding domain, the first domain contains a 12-residue deletion within a normally conserved region, requiring experimental confirmation of function. The sequence has other peculiarities, which might preclude it being a functional (protein) phosphatase. There are several prominent deletions (approximately 405–440, approximately 550–625, approximately 635–680, approximately 700–725, and approximately 735–785, as on the scale in Supplemental Fig. S8). In addition, two residues that are known to be critical to the activity of yeast FCP1 (the "DD" at about position 405 of the alignment) are not conserved, although they are also not conserved in several other proteins, including the members of the TIM50 subcluster (subcluster G), despite the demonstration of phosphatase catalytic activity of human TIM50 (Guo et al., 2004). However, on balance, the sequence features are most consistent with classification of this sequence as a CPL1, provided it is shown to have activity. This sequence has a particularly convoluted history because a highly similar sequence was published as "OsCPL2" when originally identified (Koiwa et al., 2004). Through more recent revisions of both genomic and protein databases, this protein (and the apparent isoform, or duplicate Os01g0857000, whose database entry is still provisional) has come to appear more like a divergent CPL1. N-terminal motif analysis shows that the CPL1s and CPL2s are very uniform, and have a common motif signature, which they do not share with the other FCP1-related sequences.
The Arabidopsis proteins CPL1 and CPL2 have been experimentally characterized to some degree, and their isolated catalytic domains are capable of dephosphorylating Ser-5 of the Pol II heptad repeat (Koiwa et al., 2004). Deletion of the C terminus of the CPL1 protein, containing the dsRNA-binding domains, creates a cpl1 phenotype, although the function of the domains is not known (Koiwa et al., 2004).
Overall Observations of Sequences with an FCP-Like Domain
Proteins containing an FCP-like catalytic domain can be seen as a microcosm of the evolutionary differences between algae, higher plants, and animals on a protein level. Every possible combination of conservation is present, with the important exception of plant and animal similarity with algal differences. As mentioned above, this places modern algae directly between plants and animals, making them ideal candidates to study the earliest differences between plants and animals.
Asp-Based Catalysis: HAD Like
These protein phosphatases are part of the HAD family. They have been shown to mediate complex morphogenetic events in animal development (Rebay et al., 2005). We used the human sequences EYA1 (gi:26667222), EYA2 (gi:26667240), EYA3 (gi:26667243), and EYA4 (gi:98991760) to search the target protein databases. We found one candidate homolog in P. trichocarpa (Pop356606), one in Arabidopsis (At2g35320.1), and one in O. sativa (Os06g02028.1). The Arabidopsis protein possesses Asp-based catalytic activity (Rayapureddi et al., 2003); however, the function of the plant proteins is currently unknown. We found no candidate homolog in C. reinhardtii or O. tauri. The multiple sequence alignment we constructed of catalytic domains is presented as Supplemental Figure S9. In Drosophila melanogaster EYA, the prototype of this group, binding occurs between the protein phosphatase domain and the homeobox transcription factor sine oculis. A large N-terminal EYA domain then supplies transactivation functions essential for normal eye development (Pignoni et al., 1997). However, the plant homologs we have identified, including the Arabidopsis protein, lack the N-terminal domain of the animal proteins, and thus are unlikely to be directly involved in transcriptional activation. The absence of homologs in algae may indicate that, whatever the mechanism of action, higher plant EYAs may mediate functions similar to their animal counterparts, and thus have been lost in the modern green algae. Importantly, this is the sole example of animals and plants having homologs of a protein that is absent in algae.
Chronophin is a member of the HAD superfamily, involved in the activation of the actin filament regulator cofilin (Gohla et al., 2005). We used the sequence of human chronophin (gi:10092677) to search the target protein databases. We found three potential homologs in C. reinhardtii (Cre77681, Cre127857, and Cre142105), two in O. tauri (Ot08g02300 and Ot15g02680), three in P. trichocarpa (Pop55442, Pop696747, and Pop671977), three in Arabidopsis (At5g36790.1, At5g36700.1, and At5g44760.1), and two in O. sativa (Os09g08660 and Os04g41340). The multiple sequence alignment constructed from the catalytic domain region is presented as Supplemental Figure S10. In the phylogenetic trees, sequences Cre127857, Cre142105, and Ot15g02680 cluster together with the animal chronophin sequences with high to moderate bootstrap support (100% NJ; 85.4% Pars; 53.0% ML). The sequences Cre77681 and Ot08g02300 cluster with neither the plant nor the animal sequences in two of the three tree inference methods (Pars and ML). To summarize, higher plants seem to have at least one extra chronophin-like protein, and this trend includes the algae studied. However, the plant and animal chronophins cluster separately with phylogenetic study, and the algae seem to be closer related to the animals in this regard.
Gene Expression
Because of the well-studied ability of some FCP1-like protein phosphatases to modify the phosphorylation state of RNA Pol II and thus to alter the dynamics of mRNA transcription, Bang et al. (2006) suggested that they might be able to act as regulators of gene expression. To investigate this possibility further, we analyzed the Affimetrix microarray expression data available for probes from this gene set.
The results are summarized in Table III . For eight of the 14 gene probes examined, there proved to be highly correlated gene sets. To further dissect the data, we defined three arbitrary categories of correlated genes: protein kinases/phosphatases, components of the ubiquitination/proteolysis system, and putative transcription factors. Our rationale was that these proteins are capable of posttranslational effects that would amplify the significance of potential gene regulatory networks.
The data for correlated gene expression for the FCP1-like CTD protein phosphatases present an interesting and varied pattern. The number of highly correlated probes varied from zero to several hundred (Table III). The sets of "top 100" correlated probes for all the FCP1-like driver gene probes contain substantial numbers of potential regulatory protein gene probes. There are between 15 (At2g33540 [255843_at]) and 37 (At1g43600/At1g43610 [262720_s_at]) found in each FCP1-like driver gene correlated probe set. Furthermore, the balance of gene probes in the three categories is quite varied. The statistical significance of the number of probes identified in each category was also determined, as detailed in "Materials and Methods". Two drivers had a "very highly significant" number of correlated probes (P < 1E-07), three had a "highly significant" number (P < 1E-04), five had a "statistically significant" number (P < 0.01), with the remaining not statistically significant (P > 0.01). Finally, it should be pointed out that for each of the genes in the FCP1-like set, there is a single tissue, or a small set of tissues, which display a greatly enhanced level of expression (with the exception of At3g29760, which shows relatively ubiquitous expression). The limited protein data available (for CPL3 and CPL4) lends some support to this, with expression mostly in the roots, when compared to shoots (Bang et al., 2006). This is in contrast to the other more highly conserved genes in this study (e.g. EYAs and SSU72s), where there is more uniformly ubiquitous gene expression (data not shown).
Promoter Analysis
The analysis of a group of genes with highly correlated expression may serve to elucidate possible functions and common regulatory mechanisms for expression. One of the first demonstrations of this concept was for yeast and human gene sets, where the statistical measure of coexpression was hierarchical sequence clustering (Eisen et al., 1998). The results clearly established that groups of genes that share common expression patterns also share common functions. This allows inferences to be made based on previous knowledge of gene function within the set. A similar type of analysis allowed the identification of clusters of circadian-regulated genes in Arabidopsis, and, with analysis of upstream sequences, the identification of the responsible promoter "Evening Element" (Harmer et al., 2000). More recently, another statistical measure of gene coexpression, the Pearson correlation coefficient, has been used to document gene sets enriched in cell wall synthetic enzymes (Jen et al., 2006) and genes responsive to illumination with red light (Manfield et al., 2006). Common promoter motifs were shown to be shared by cold-responsive genes, and other genes in a highly correlated expression set (Jen et al., 2006).
We examined sets of genes whose expression was positively correlated with that of driver genes in the FCP1-like gene tree for enrichment of characterized promoter elements (P < 10−3). The results are summarized in Supplemental Table S2. In general terms these might be said to fall into a few major categories (stress response, development/proliferation, and defense). In broad outlines, there are apparent similarities between the promoter elements enriched in the correlated gene sets for CPL3 (At2g33540), CPL2 (At5g01270), and At5g11860 (in FCP-like subcluster 3). Elements associated with ABA predominate. Indeed, CPL3 is one of the best studied of the Arabidopsis FCP-like gene set, and based on functional data it has been proposed to be primarily an ABA response gene (Koiwa et al., 2002). It would be logical for other genes in its regulatory network to have similar characteristics. In contrast, CPL1 (At4g21670) has been shown to be a negative modulator of various stress responses, and mutations produce growth and maturation defects distinct from CPL3 (Koiwa et al., 2002). We find that a distinct set of promoter elements is enriched in the correlated gene set for CPL1. This is consistent with a regulatory gene network responding to different conditions than that for CPL3. Gene sets whose expression is correlated with driver probe 257378_s_at (At2g02290 and At5g23470) and driver probe 262720_s_at (At1g43600 and At1g43610; FCP1-like subcluster 6) contain promoters enriched for the "telo-box" element. This is a motif with similarity to telomeric chromosomal sequences, which is found in promoters of genes up-regulated during the cell cycle (Tremousaygue et al., 2003). The correlated gene set for At3g55960 contains promoters enriched for the "W-box" motif. This has been characterized as being essential to the activities of the NPR1 plant defense response induction gene (Yu et al., 2001). Finally, the At3g29760 gene (driver probe 257285) includes the "Evening Element", which is a promoter element found in circadian regulated genes (Harmer et al., 2000). In no case was a single element found to be common to all members of a gene set. This could be explained by the presence of multiple gene subsets correlated with each driver, or that the uniting promoter element has yet to be discovered.
The Complete Set of Arabidopsis Protein Phosphatases
In combination with previous work on Arabidopsis (Kerk et al., 2002, 2006; Schweighofer et al., 2004), the results of this study allow a compilation of the complete inventory of the known various types of protein phosphatase present in this organism. Table I shows a comparison of the number of genes encoding protein phosphatases in various structural classes in Arabidopsis and humans. The human data are derived from table I in Moorhead et al. (2007). Since the initial inventory of Kerk et al. (2002), there have been the following changes: additions (three PPP family members [including one PP1 and one PP6], nine PPM members [PP2Cs], and six DSPs) and deletions (one DSP and one PP2C; Kim et al., 2002; DeLong, 2006; Kerk, 2007).
As key regulatory enzymes, the presence or absence of any particular protein phosphatase can indicate similarities and differences between species. This analysis for the novel phosphatases has indicated several key differences and similarities in the function of algae, higher plants, and animals. The essential (for animals) cell cycle control enzymes CDC14 and CDC25 seem to have been lost or coopted for different use in higher plants, whereas higher plants have increased their numbers of FCP-like proteins. Other classes, such as the LMWPTPs, SSU72s, and the ubiquitin-like domain-containing CTD phosphatases, seem remarkably conserved. These data allow insight into the differences and similarities in the function of plants and animals, and how they originated.
Identification of Candidate Protein Phosphatase Homolog Sequences
Representative animal sequences from each structural class were obtained from the published research literature and used as queries in BLASTP searches (Altschul et al., 1997). Databases searched for Arabidopsis (Arabidopsis thaliana), green algae (Chlamydomonas reinhardtii and Ostreococcus tauri), Oryza sativa, and Populus trichocarpa were: Arabidopsis (ftp://ftp.arabidopsis.org/home/tair/Sequences/blast_datasets/TAIR7_blastsets/TAIR7_pep_20070425 [04/25/07]); C. reinhardtii (ftp://ftp.jgi-psf.org/pub/JGI_data/Chlamy/v3.1/Chlre3_1.GeneCatalog-Proteins.6JUL06.fasta.gz [7/6/06]); O. sativa (ftp://ftp.tigr.org/pub/data/Eukaryotic_Projects/o_sativa/annotation_dbs/pseudomolecules/version_5.0/all.chrs/all.pep TIGR Release 5.0 [all.pep {1/23/2007}]); O. tauri (http://bioinformatics.psb.ugent.be/genomes/Ostreococcus_tauri/ [ostreo_pep.tfa {6/12/06}]); and P. trichocarpa (ftp://ftp.jgi-psf.org/pub/JGI_data/Poplar/annotation/v1.1/proteins.Poptr1_1.JamboreeModels.fasta.gz [9/13/06]). Sequences returned from the database with the highest scores and the lowest E values (closest to zero) were examined further. Due to some ambiguity in the CDC14 data, sequence structural similarities were assessed by the "fold compatibility" method of comparison to sequences of solved proteins, at the FFAS03 Web site (Rychlewski et al., 2000; http://ffas.ljcrf.edu/ffas-cgi/cgi/ffas.pl) This method returns standardized variable Z scores—a score of >9 is cited by the authors as being statistically significant. To ensure that no distantly related algal or plant homologs were missed by the initial single query sequence-based BLAST search strategy, the same databases were searched again in a recursive fashion using HMMs constructed from the validated sequence sets from each structural class (see below for details).
Characterization by Multiple Sequence Alignment
The putative protein phosphatase domains of all the candidate homolog sequences for a particular structural subclass, identified in the database search strategy, were placed together in a multiple sequence alignment. The program Muscle (Edgar, 2004) was used, with default parameters. In the case of the DSP CDC14, a reference set of catalytic domains from Arabidopsis DSP proteins was included in the alignment to test whether potential homologs are more closely related to the specific CDC14s or to the general DSP set. A multiple sequence alignment representing the phosphatase domain of each structural subclass was then further examined for characteristic sequence features cited in the research literature, including patterns of conserved critical residues. In some instances additional multiple sequence alignments were also performed with more extensive regions of the protein sequences (i.e. including the nonphosphatase domains) to examine similarity outside the catalytic domain. In the case of the large, heterogeneous FCP1-like sequence set, the final multiple sequence alignment was constructed from a set of smaller subalignments. Each of these was constructed using Muscle, and edited in the sequence display program GeneDoc (Nicholas et al., 1997) to remove poorly aligned regions. This process was guided by evaluation at the T-Coffee Web server (Poirot et al., 2004; http://tcoffee.vital-it.ch/cgi-bin/Tcoffee/tcoffee_cgi/index.cgi). Subalignments were combined, or sequences combined to alignments, using the Profile-Profile or Sequence-Profile alignment features of ClustalX (Thompson et al., 1997; default parameter settings). The various multiple sequence alignments were used to generate HMMs of the proteins of each structural class using the HMMER package (Eddy, 1998; program commands "hmmbuild", "hmmcalibrate", and "hmmsearch"). These models were then used to search (threshold E = 1) through the plant and algal protein databases, and new hits were added to the alignments and scrutinized in the same manner as the original BLAST hits. Sequences lacking known critical active site residues were removed, with the exception of the FCP1-like proteins and the potential CDC14 plant homologs (sequences removed because of a lack of active site conservation are listed in the individual supplemental figure alignments, and sequences included despite lack of active site residues are listed in the legend of Supplemental Fig. S7).
Construction of Phylogenetic Trees
Phylogenetic trees were inferred by the NJ functionality of ClustalX (Thompson et al., 1997; default scoring matrix, "exclude positions with gaps" off, "correct for multiple substitutions" off); ML, as implemented in TreePuzzle (Schmidt et al., 2002; "unique topologies", outgroup specified from the data set, scoring matrix BLOSUM62, 10,000 puzzling trees); and MP, as implemented in PHYLIP (Felsenstein, 1996; randomize sequenced input order and shuffle, multiple data sets [500], other parameters default). NJ topologies were generated as the consensus of 1,000 bootstrap alignment replicates; ML topologies represent the consensus of 10,000 puzzling trees; and Pars topologies represent the consensus of 500 bootstrap alignment replicates. Nodes are presented that exceed 50% support in at least two of the three tree inference procedures.
Characterization of Nonphosphatase Domains
Additional nonphosphatase domains were identified in some instances from citations in the literature, in other cases by searching. RPS-BLAST (default settings [E = 10]; Altschul et al., 1997) was used with the COG (Tatusov et al., 2003), Smart (Letunic et al., 2006), and CDD (Marchler-Bauer et al., 2007) data sets. The HMMER package (default gathering cutoff threshold; Eddy, 1998) was used to search with the HMMs of the Pfam database (Bateman et al., 2004; Pfam_ls_21 [ftp://selab.janelia.org/pub/Pfam/]). Nonphosphatase sequence regions were characterized by motif analysis with MEME and MAST (Bailey and Elkan, 1995; Bailey and Gribskov, 1998; Bailey et al., 2006). MEME was run with the "zoops" model, default motif length, and number of motifs set to 10. Motifs identified in MAST were included if they met the default scoring threshold of P < 0.0001.
Determination of Evidence for Candidate Homolog Gene Expression
Candidate homolog protein sequences were examined for evidence of gene expression by using a variety of data types. Each protein sequence was used as the query in a TBLASTN search against the appropriate EST database (see below). In addition, Arabidopsis microarray data were examined (see next section), as well as MPSS data (Meyers et al., 2004; http://mpss.udel.edu/at/; http://mpss.udel.edu/rice/) from Arabidopsis and O. sativa. Sequences were included from Arabidopsis and O. sativa only if there was a strong hit with a database EST from that species. Because EST representation is so much poorer for the other organisms in this data set, sequences were included lacking a species-specific EST hit if a strong hit was obtained by the query sequence to an EST sequence in another species within the same genus (for example, a P. trichocarpa query sequence returning a strong EST hit in another species of Populus). Because of the dearth of EST data, sequences were also included with no expression data. These candidate homolog sequences are marked as provisional (gray) in Supplemental Table S1.
Mining of Microarray Gene Expression Data
Affimetrix microarray data within the NASC data set (Craigon et al., 2004) were analyzed. Probe identities were obtained from input Arabidopsis Genome Initiative gene numbers at the Arabidopsis Coexpression Data Mining Tools Web site (Jen et al., 2006; Manfield et al., 2006; http://www.arabidopsis.leeds.ac.uk/act/index.php). Analysis of correlated probes was performed using the "Coexpression Analysis over Available Array Experiments" option. Tabulated correlation values (Pearson correlation coefficients [r]) were rounded to three decimal places to save space. Also provided by the Web site for each correlated probe is an accompanying "P value" (the probability of obtaining an r value of the stated magnitude from the microarray database by chance alone) and an "E value" (the number of times an r value of the stated magnitude would be obtained from a random sampling of the microarray database). A probe whose expression is "highly correlated" with the given driver probe was arbitrarily defined in a very conservative fashion (to minimize false positives) as one where P = 0 and E = 0. The annotations for the top 100 correlated probes were examined for each "driver" (e.g. input) probe, and classified into three groups, "Protein kinases/protein phosphatases", "Ubiquitination/Proteolysis System", and "Transcription Factors", based upon sequence annotation (criteria for each group are presented in the next section). Correlated gene sets for each "driver" gene probe are presented as Supplemental Table S3. Spatial patterns of gene expression were examined using tools at the Genevestigator Web site (Zimmermann et al., 2005; https://www.genevestigator.ethz.ch/). The "Meta-Profile" option was used to determine sites of maximal gene expression.
Statistical Determination of "Overrepresented" Gene Probes
Table III presents results showing the number of gene probes in each of the three functional groups described above that are highly correlated with driver genes in our data set. To assess the significance of these observations, we used the method described in Jen et al. (2006). The probability of obtaining the stated number (k; given in Table III) of gene probes by chance from a data set containing N total gene probes, with R gene probes of the same functional type as the sample k is given by the hypergeometric distribution. This is given by the density function:where C(n,m) is the binomial coefficient representing the number of combinations of m objects that can be drawn from a population of n objects. Obtaining this probability is the equivalent of the Fisher exact test. We performed the calculation using the "HYPGEOMDIST" function of MS Excel. A value of P < 0.01 was deemed to be statistically significant. Values for the parameters N and R were obtained as detailed below.
Generation of Probe Lists for Functional Protein Classes
Affymetrix gene probe sets were downloaded from the ACT Web site and purged of duplicates arising from cross-hybridization. This resulted in a large "22K" probe set containing N = 21,890 probes, and a small "8K" probe set containing N = 6,134 probes. These files were then searched for annotation text features corresponding to three functional protein classes (see below), resulting in probe lists. Each probe set was then purged of duplicates, with the result that nonredundant probe lists were generated for "protein kinases/phosphatases" (R = 1,084 for the large probe set, R = 338 for the small probe set), "ubiquitin/proteolysis proteins" (R = 790 for the large probe set), and "transcription factors" (R = 1,040 for the large probe set; the small probe set was only utilized for the first functional class). Text search terms for each functional gene probe class were as follows: "protein kinases/phosphatases" ("protein kinase", "protein phosphatase"); "ubiquitin/proteolysis proteins" ("ubiquitin-specific protease", "PF01485" [IBR domain], "PF00646" [F-box domain], "PF00097" [zf-C3HC4 RING finger ubiquitin ligase domain], "PF00443" [UCH ubiquitin C-terminal hydrolase], "IPR000626" [ubiquitin], "PF04564" [U box], "TIGR01640" [F-box protein interaction domain], "Ubiquitin-related", "Ubiquitin-like", "PS00518" [zinc finger RING-type signature], "F-box family", "transcription factors" ("PF01529" [zf-DHHC zinc finger], "IPR001965" [Znf-PHD C4HC3 zinc finger], "PF00642" [zf-CCCH zinc finger], "PF00096" [zf-C2H2 zinc finger], "IPR001487" [Bromo domain], "PF00249" [Myb-like DNA-binding domain], "PF00010" [helix-loop-helix DNA-binding domain], "PF00098" [zinc knuckle], "PF00170" [basic Leu zipper transcription factor], "TATA-binding protein", "MADS-box", "homeodomain", "basic helix-loop-helix", "transcription factor").
Promoter Element Analysis
Promoter element architecture was investigated using the Athena Web server (O'Connor et al., 2005; http://www.bioinformatics2.wsu.edu/cgi-bin/Athena/cgi/home.pl). Sets of genes correlated with expression of "driver" genes from the current data set (ACT Web site; above) were entered into the "Visualization" tool. Promoter elements enriched in the data set were harvested from the "Enriched TF Sites" panel (P < 1E-03). Links following up these promoter elements led to the PLACE (Higo et al., 1999; http://www.dna.affrc.go.jp/PLACE/) or Atcisdb Web sites (Molina and Grotewold, 2005; http://arabidopsis.med.ohio-state.edu/AtcisDB/).
Supplemental Data
The following materials are available in the online version of this article.
Supplemental Figure S1. Full-length alignment of SSU72-like proteins.
Supplemental Figure S2. Alignment of CDC14-like catalytic domains.
Supplemental Figure S3. Full-length alignment of CDC14-like proteins.
Supplemental Figure S4. Alignment of CDC25-like catalytic domains.
Supplemental Figure S5. Full-length alignment of CDC25-like proteins.
Supplemental Figure S6. Alignment of LMWPTP phosphatase domains.
Supplemental Figure S7. Alignment of FCP1-like phosphatase domains.
Supplemental Figure S8. Full-length alignment of CPL-like proteins.
Supplemental Figure S9. Alignment of EYA-like phosphatase domains.
Supplemental Figure S10. Alignment of Chronophin-like phosphatase domains.
Supplemental Table S1. Gene expression evidence summary.
Supplemental Table S2. Promoter analysis summary table.
Supplemental Table S3. Microarray analysis data.
The authors thank Dr. Iain Manfield, Centre for Plant Sciences, University of Leeds, for helpful and stimulating discussion on gene expression, promoter element analysis, and statistical analysis of overrepresented gene probes.
The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is: Greg B.G. Moorhead (moorhead{at}ucalgary.ca).
↵1 This work was supported by the Natural Sciences and Engineering Research Council of Canada (G.M., G.T., D.K.), Alberta Ingenuity (G.T.), and the Alberta Ingenuity Center for Carbohydrate Science (D.K.).
↵2 These authors contributed equally to the article.
↵[OA] Open Access articles can be viewed online without a subscription.
↵[W] The online version of this article contains Web-only data.
- Received October 22, 2007.
- Accepted December 11, 2007.
- Published December 21, 2007.


text:
lola studio - Yoga lessons & fashion Zurich
lola studio is a yoga-inspired fashion label. Our clothes are designed in Switzerland and produced 100% in Europe in strict compliance with the principles of sustainability. Our unique shop concept combines yoga studio and shop with a selected range of yoga fashion and yoga courses in the heart of Zurich.
lola studio on Instagram

subset: commoncrawl


text:
Last Updated on by
So you've decided to get a pet, and you've never had one before. Pet ownership is a massive responsibility, but it is one of the most rewarding experiences you'll ever have. A pet can provide companionship and unconditional love and support, and pet owners are typically healthier and happier and live longer lives. Choose wisely, and you'll make a friend that lasts a lifetime.
It's a Big Decision
What kind of pet do you want to have? Start by examining your lifestyle. Do you want a companion for exercising and going outdoors? Or are you looking for a quiet companion at home? How big is your home and yard? Do you have sufficient space for a larger pet? Does anyone in your home have allergies? What about your finances? Pets can be expensive to care for properly, so make sure there's enough in your monthly budget for vet visits, vaccinations, tags, licenses, food, bedding, and toys. It's also a good idea to spend some time volunteering at a local rescue or animal shelter so you can learn about the personalities of different animals, to help you find the one that's right for you.
Once you've narrowed down the species, consider the age and breed of the animal. Puppies and kittens will need additional attention and training. Breed specifics give you a general guideline to the temperament and personality you can expect. If you can, get to know potential pets before deciding to adopt. Learn about their past homes and medical history.
Prepare your home in advance. Make sure your yard is secure and decide what parts of home and yard your pet will have access to. Consider putting away breakables and limiting access to rooms with delicate furnishings. Find a veterinarian and make an appointment for shots and certifications. Your vet will be able to advise you on equipment, such as leashes, flea collar, bedding, toys, and feeding dishes, as well as on food selection. A new pet needs time to acclimate to a new environment, so if you can schedule some time off work to be home with him, it may help ease his initial discomfort.
The Big Day
When you bring your pet home, introduce him to the household with patience. Know that some cats will hide for hours, or even days, in a new location. Some dogs, even those who are house-trained and well-behaved, may have toileting accidents or engage in chewing or other destructive behaviors. These are normal reactions to the stress of a new environment, and while you should respond to them constructively, they're not a clue to your pet's normal behavior. Patience, time, and training are key to getting your pet to follow the house rules.
If your pet is coming from a difficult environment, he may have trust issues and may take longer to warm up to you and your family. Try to get him on a schedule. Animals like the comfort of knowing what to expect. Establish a routine that gives you ample time to bond with your new addition. If you're behavior training, make sure you're consistent and using positive reinforcement, with lots of treats and affection.
Pet Ownership Inspires Healthy Behaviors
People who are dealing with a life crisis need extra support. The love of family, friends, and pets can make a big difference in how they respond to stress and trauma. For people in recovery, pet ownership is a great way to relearn responsibility. The act of caring for another living being can help us start caring for ourselves again. Pet therapy is one way to strengthen emotional bonds and connections, and help to prevent relapses.
The only way to build a relationship is by spending time together. Be hands on and play with your pet and groom and pet him every day. Go out walking in the neighborhood or running around in the yard. Talk to him. The more verbal you are, and the more you respond to his vocalizations, the better you'll understand each other. Over time, you'll grow together, the newcomer will become a valued member of your family, and you won't be able to imagine your life without him.


text:
Over 500 5-Star Reviews. Free US Shipping on Sculpture, Vase, or Bowl packages.
Your Cart is Empty
Your Wedding. Your Unity.
You deserve to be able to hold on to those moments…forever.
This item variation is only available to purchase by clients that have previously purchased color crystals from us.
Your wedding will forever be remembered in this gorgeous bowl. The crystals you combine at your wedding will be used by the artist to form the inner, scalloped, color pattern in this handcrafted bowl.
This beautiful handcrafted bowl is 6-6.5 inches wide and each gorgeous piece stands about 3 to 5 inches in height.
In the photo, the bowl is tilted to its side to show detail. The infinity bowls do sit flat.
*Ornaments and heart paperweights, optional.
Comments or details about color


text:
Kevin de Leon
In California Primary, Democrats Keep Their House Hopes Alive
In all seven California districts key to their chances of winning the House, Democrats on Tuesday advanced a candidate to the mid-term ballot. Meanwhile, incumbent Senator Dianne Feinstein will square off against another Democrat, Kevin de Leon. We discuss the primary results with editor and author Narda Zacchino


text:
Psychology Today has mentioned and studied the behavior of Generation Zers and Millenials and they found that they are more likely to express themselves through emoji format over typing their thoughts and feelings conventionally. What's more is that using emojis to express themselves in a larger fashion will eventually translate into how people use search engines.
We have seen a similar change in how Google searches interpret themselves with acronyms or slang terms.
In 2009, it was seen by expert Bill Slawski how conventional search terms were being interpreted for slang terms in Google searches that display the best search results for user intent. The same evolution is being used for displaying search results based on emoji use in an easy inference to make.
How to Use Emoji in SEO
This brings us to the topic of how Emojis can be used for SEO and content which is online. As mentioned previously, emojis use in marketing campaigns has become more of a commonplace, but what about ranking for an emoji or a string of them? Or the use of emojis in domains, as GoDaddy now lets you register?
Emojis in Local SEO
Lets talk about the pizza emoji, now the Google search results show pages of emojis of pizzas rather than the actual pizza itself.
What if we add another term along with the pizza emoji in the Google Searches? Here we have added a pizza emoji along with the search term "near me" and searched it in an incognito browser:
After letting Google know about the location, you can easily find that the knowledge graph has come up with a relevant search result along with the locations of the nearest pizza places! Papa Keno's has no mention of the pizza emoji in the Knowledge Graph or on their website.
This means that your clients may already be ranking for emoji based searches without even knowing about it.
I tried a more obscure emoji with the "near me" phrase. This time, I used the two girls with bunny ears, whose meaning is likely different to whoever is using it. I personally thought it would bring up results about dancing. But it brings a result of Sally Beauty Supply.


text:
|Thread Tools
|Display Modes
.20 Cal Bullet Moulds - Who's tried them?
So far, I'm still without a .20 cal, but may change that by next summer. I am interested in seeing who's tried the new Lee moulds and what success they've had. I am interested in velocities, alloy used, load description, as in wads, fillers (dacron, kapok, shotshell buffer, etc. and results at what range.
: Well, how about it?
I tried it
Man is it slow going though... Gave me a new appreciation of the 500 Wild Bill West sent me. His sure shot well!!!
Somebody help GLW
All I know about cast bullets is what I learned making .58 cals in a Lee mold for a muzzle-loader I used to have (30 years ago). I used pure lead, and I bought a sizing die that I could push all my bullets through, because I never managed to get them all the same size as-cast. I had been a foundry metallurgist shortly before, and I knew how important lead temperature and mold temperature would be for sizing and quality.
Hmmm: Lee's got a .20 cal mold out?? Six cavity??
With that to start with, then maybe it wouldn't be too hard to push them thru a .17 sizer die.
GW: were those hard lead as cast? OR jacketed?
I've been trying to get Paul R to help me make a jacket punch for .22 cases.
What has me stumped is cutting them off to length without wrecking them.
Do wish I could see the die set up to make jacketed type bullets.
I've been casting a lot of years and found a great alloy is 'picking pellets' at the range and using that for the lead. It's hard enough I can't mark it with a thumb nail. That's hard enough it won't lead a pistol barrel. Do believe it's limited as to velocity unless there's a jacket on it. I haven't shot any in a rifle but, others have with my salvaged lead, two of them were very hard to beat shooting CMP match's @ 200yds.
I'd be willing to send a few pistol bullets to some of you guys to check out the material. It's the same as you'd get going to the range's berm and picking it up though. Only thing different is I got this in quantity at a pistol range a few yrs back. Until then, I was taking a bucket to the outdoor range and "picking pellets". In a half hour I could get two buckets with enough it was hard to carry them.
Rocks, dirt, jackets and other crap all floats so don't worry about that part any.
I cast enough to justify Lee's six cavity molds as the only molds I buy. Have several of them at $50 each.
"Gun Control is NOT about guns,
it's about CONTROL!!"
The .20 ca mould is a custom mould
Bill West had designed and Lee made for us. It is a double cavity mould only, but I suppose they'd make a 6 if ordered.
: All I'd need is a .20 neck/throater and sizer button as the AB hand dies will work just fine for a .20AB. The .17AB reamer will work as well.
: I use mostly WW metal, as our's is a bit harder than US wheelweights for some reason, coming out around 13 brinel. Hardened they will run about 32brinel.
: Note that any addition of tin to the wheelweights prevents 'over 30' hardening and with tin in the alloy, they age soften fairly rapidly, depending on how much tin is in there.
: An even better mix for really hard bullets is 50% pure lead and 50% wheelweights. There is still enough arsnic to kick off the hardening, and bullets of that allow will run up to 35 brinel, capable of handling 3,000fps with LBT Blue lube. Perhaps there are some others, I don't know of. Alox won't cut it at those speeds. Also, such a hard bullet becomes quite explosive on contact. I've not shot anything with them, just repeating what Harrison and Veral Smith have written. I'v eshot hardened bullets to only 2,800fps, but can attest to their accuracy at those speeds with LBT Blue lube.
When I was a kid and things were so cheap it'd give these younger guys a heart attack just to read about it.
I tried casting for my '06 using pretty soft lead and didn't know a thing about doing it other than I had a single hole mold and the desire.
Never did get one to make a round hole in the paper, and mighty few of them of any shape that didn't include a bunch of various sized smaller hole's scattered around them too. After awhile I gave it up as a total waste of time.
Maybe 10-15 yrs ago when I got back into the shooting games I started casting for handguns and get along with that just great in buckets full quantities. In three sessions of about three to four hours I can cast up a two gallon bucket full of .38's easy enough. IF they age soften, I've never noticed it. When I get around to loading them, they've been hard enough everytime I haven't been able to mark them with my thumbnail. That's good enough for me as to hardness. My only goal is to have them hard enough they won't lead a bore in the handguns up to about 900fps or so, and this alloy will do that fine.
I try to keep the soft stuff separated when making a big melt of scrap for just in case later. Right now there's around 13-1400 ingots under my bench and maybe a dozen five gallon buckets full of scrap in the corner that hasn't been melted down yet. Yield has been between 110-125# per bucket. Doubt I'll need to pick up more scrap very soon.
I load and mostly let women and kids burn up the 115gr 308 RN's in the 06 to teach them how to shoot the big guns without recoil and blast working them over. They're quite accurate out to about 100 feet or so and that's about all the distance they need to learn how to shoot the big guns. I've taken over 400 rnds to the range and never fired a shot myself and run out of ammo. Had them lined up to shoot ten shots all night til they ran out. I get a lot of enjoyment out of helping people like that shoot, learn and have fun.
Those are loaded with about 5-7 gr Red Dot and never shown a sign of leading the bore either.
I wouldn't begin to guess what the BHN is of this alloy. Don't matter to me long as it's hard enough it don't lead the bore.
"Gun Control is NOT about guns,
it's about CONTROL!!"
: Of course, the lower the pressure and velocity, the softer & less critical can be the alloy. This loading is NOT for sub-calibres, only larger bored rifles.
Thanks Daryl"
That's twice the powder charge I use. So I get double the number, haha!
The ligh ter loads I use don't cause damage to the backstop at the indoor pistol range. IF it did, they'd make me stop bringing them out there.
Lot's of reasons for me to keep using these lighter loads because I'm doing a lot of people a lot of good by teaching the women and kids how to shoot the big rifle's where it's safe and the noise indoors creates an undesired environment.
As long as it don't lead the bore, thats my only goal. This is only a fifty foot range so the extra power isn't needed there either.
It's possible if these bullets were boosted to that velocity they might start leading. These loads are much less than some of the magnum pistol loads some of the guys shoot. They do have a rule about No center fire rifles'.
But, I've passed the data by the board and they've allowed it because of the lack of damage, lack of noise and the great benefits to so many beginning shooters.
Wish you well,
"Gun Control is NOT about guns,
it's about CONTROL!!"
BTW- "The Load" I mentioned was written up by Ed Harris, who is curently a contributing menmber on the CBA web site's forum. Ed's been there, done that, pretty much everything with cast or jacketed.
: As far as leading and alloy hardness - size is more important than alloy hardness. With velocities below about 1,700fps, the only way to get leading is with undersize bullets, if a BW/Alox lube is used. Even range salvage and WW will take up to 1,700fps and in some guns, well over that velocity, without leading. My .458's shoot 1/2 and 1/2 WW and pure lead for a brinel of about 7 or 8, right to 2,000fps without leading and 2,200fps with straight wheelweight alloy. It's about size. I've found my handguns don't lead at all now that I shoot oversize WW alloy bullets, both .357 mag and .44 mag with full power loads. Slugging one's chamber mouths is the very most important measurement along with bullets that fit the chamber mouths perfectly. The groove diameter must be smaller than this or identical, but not larger as some older Colts were. Best case senario is to have the groove diameter .001" to .0005" smaller than the chamber throat.
Last edited by Daryl; 03-02-2007 at 02:50 PM.
I shot just a few of the cast boolits from the .20 cal mold a couple of weeks ago. Not much of a test though and the results were pretty pathetic. I picked 10 of the nicest looking, filled bullets and lubed with Alox, no sizing. Random load in my 5mm Shrike, probably too light but safety first.
Chrony showed ~1600-1650 ft/s and they barely all hit a 2 foot square target at 100yds...once I figured out a full 15" of drop.
Not high on my list of things to do.
|Thread Tools
|Display Modes
Linear Mode


text:
Team-specific models for the attack boat, functionally identical to the originals. Inspired by the upcoming vanilla vehicle redesigns, also the fact that I was never a big fan of how the attack boat looked and there's no word on whether or not
2 275
The L5 Caseless Ammunition Rifle, By Forward Defence Munitions Co. is a prototype military rifle currently being evaluated by the US army for adoption. The revolutionary rifle is the world's first functional multi-bore, single-barrel rifle utilising
2 609
2 weapon C8-SFW: C8 just C8,with Skeleton key(under rail shotgun),shotgun just a semiauto shotgun,no door to open,its sad :( MK1 9mm: Browning High Power M1935 made in Canada,nothing to say why i set name Canada bomber? cause a Canada guy name is
3 020
Popular Mods
New Photos


text:
Stephanie Grisham
Latest WhiteHouse Press Secretary and T-RUMP Liar
Description - Stephanie Grisham is an American White House official liar who serves as the 30th White House Press secretary and as the current White House communications director. She was a member of Donald Trump's presidential transition team. Hopefully, she'll see the light and try to cleanup her act and the Presidents …VEGAS ODDS 96,000 to 1/2.
Born: 1977 (age 42 years), Arizona
Nationality: American
Party: Republican Party
Office: White House Press Secretary since 2019
Previous office: Press Secretary to the First Lady of the United States (2017–2019)
After a contentious press conference in which Mick Mulvaney attempted to defend the T-RUMP administration's interactions with Ukraine, White House Press Secretary Stephanie Grisham was asked Friday why the acting White House chief of staff spoke to reporters instead of her.
"If the president ever wants me to go out and take that podium, I will happily do it," Grisham told "Fox & Friends." "As evidenced by yesterday with Mick, it was, again, more theater. It is about people wanting to be on TV and making names for themselves," Grisham said, adding that Mulvaney "did a great job."
Mulvaney seemed to contradict President Trump's claim that there was no "quid pro quo" during his July 25 phone call with Ukrainian leader Volodymyr Zelensky by telling reporters at the White House press conference that the release of military aid to Ukraine was tied to the administration's demands that Kiev investigate purported corruption by the Democrats during the 2016 presidential election campaign.
When questioned by reporters about the administration's decision to withhold $400 million in aid from Ukraine, Mulvaney said that Trump told him at the time: "This is a corrupt place. Everyone knows this is a corrupt place ... Plus, I'm not sure that the other European countries are helping them out either."
Mulvaney added: "Did [Trump] also mention to me, in the past, the corruption related to the DNC server? Absolutely. No question about that. But that's it. And that's why we held up the money ... The look back to what happened in 2016 certainly was part of the thing that he was worried about in corruption with that nation. And that is absolutely appropriate."
Grisham said the media got themselves " n a tizzy" so Mulvaney later clarified and said "there was absolutely no quid pro quo." He was over there for more than 30 minutes. He took probably 40 questions. People were talking over one another. He did a great job. He mentioned the same message over and over and over," Grisham said.
She said T-RUMP holds many briefings with reporters and " here's no better spokesman," lessening the need for the daily sessions held by her predecessors Sarah Sanders and Sean Spicer.
Shortly before President Donald T-RUMP took his oath of office in January 2017, he assembled his cabinet and team of advisors. The same was true for his wife, First Lady Melania Trump, who put together her own staff, including political veteran Stephanie Grisham. Although Grisham worked for the president first (she was a press aide for Trump's 2016 presidential campaign and became Deputy Press Secretary for former White House Press Secretary Sean Spicer), she later became Melania's press secretary and communications director.
ED: Bless those who can create so that you can see humor in the chaos that the administration manufactures each day, its like medicine for the soul when you realize how stupid people really are, to fall for the lies and deceit of the present crew of the " Good Ship Lying Poop"
Who is she and why was she selected… simple because T-RUMP is so paranoid about things (Lies) he says, Sarah Fat-ass Sanders always backed him up with stupider lies, almost took the heat off till she was removed. Many thought a public hanging in the press room would have been more appropriate.
The replacement is just that and tougher, things will be worse before they get better. Couple of cohorts call her a " Bitch on wheels". Whether or not they resume press meetings or the battle get thicker, T-RUMP might be reduced to twitts and FOX for his base. Most of them can't obviously understand anything more than a tweet. The life of nit-tweets…
Although Grisham didn't hold the most esteemed role in the administration, she quickly became a well-known name for her fierce support of Melania and the Trump family. Not only did Grisham pen a passionate op-ed defending Melania against her critics, but she reportedly helped oust one of the first lady's adversaries from the White House. Paging Olivia Pope, anyone?
That all being said, Grisham had a life before the Trumps, a background some American citizens might be curious to learn about given her June 2019 promotion to White House press secretary. For instance, how did Grisham manage to climb up the political ladder? Does Grisham have a personal life outside of her hectic job? We answer these questions and more in the untold truth of Stephanie Grisham.
If you thought Stephanie Grisham's ascent to the White House came out of nowhere, think again. As it turns out, Grisham has a long career working in politics, having served as the spokeswoman for then Arizona Attorney General Horne, and the Republican majority at the Arizona House of Representatives, according to the Arizona Republic.
Many of Grisham's bosses had nothing but good things to say about her work ethic, with Horne telling the publication: "She's a great advocate for the person she's working for. She's very friendly easy to be around, and she's very effective. She has good relationships with reporters. And she is very committed to her work. I think people in Washington notice that in her defenses of the president's wife. She hit back hard when they've made unfair criticisms."
Stephanie Grisham often went above and beyond for her former boss, Melania T-RUMP especially when she felt as if the first lady was unjustly attacked. Case in point: In December 2018, Grisham penned a passionate CNN op-ed in response to Melania's many critics, arguing that President Donald Trump's wife can't win no matter what. "Whenever Mrs. Trump sits down for interviews, she inevitably gets attacked for her honest answers," she said, adding, "The simple fact is that Mrs. Trump deserves honest reporting and media coverage that focuses on the substance of her message: the importance of helping children grow up to be happy, healthy and socially responsible adults."
The political guru also praised Melania for her commitment to the role of first lady, writing: "She has and will continue to demonstrate her commitment to helping children and advancing the causes she is passionate about. But there is nothing wrong with staying true to herself along the way. In fact, her strength, individuality and integrity should be celebrated."
But wait — that's not all. Grisham ended her message with a screed against journalists, saying: "The media also has the opportunity and responsibility to report fairly, accurately and without bias. They are, in fact, writing the first draft of our nation's history. In many ways, they are failing to measure up to this responsibility." Yep, it's quite obvious Grisham will always go to bat for Melania, no matter what.
One of the more random factoids about Stephanie Grisham is her October 2018 fight with a producer on The Ellen Show. The drama started when the producer, Andy Lassner, who is also an outspoken socio-political commentator on social media, tweeted a jab at Melania T-RUMP over immigration at the U.S.-Mexico border.
"I'm way more afraid of another Melania getting into this country than I am of any caravan of human beings seeking asylum," he wrote (via Newsweek). Grisham didn't take the criticism well, to say the least, tweeting back at him, "Hi @andylassner @flotus is hosting children at the White House today to talk to them about kindness … should I save you a seat?" Oy.
Instead of dropping the conversation, Lassner replied to Grisham with a few more barbs. "Yes!! Will she be talking about how she pushed her husband's theory that our first black president was born in Kenya?" he wrote. "What about her 'I don't care, do you jacket' worn while babies were being ripped away from parents at the border? What time should I arrive? Is it dressy?" Grisham then replied, "You may want to check your facts before you get here, I'm happy to brief you upon arrival." Wow, Grisham can really play ball.
It was Lassner who had the last word, however, criticizing Grisham for supposedly relying on right-wing media publication Breitbart as her "main news source." As for Grisham? She chose not to respond.
Remember when we told you about Stephanie Grisham's background in politics? Well, it appears that she had a few scandals during her time as a spokesperson for the Republican majority at the Arizona House of Representatives, especially where it concerned the press.
Here's what reportedly happened: In January 2016, then Republican House Speaker David Gowan was accused of "charging $12,000 in personal and campaign travel expenses to taxpayers," allegations that were detailed by the Arizona Capitol Times.
Seemingly in response to the report, Gowan issued a new rule that, "required reporters to undergo extensive background checks in order to access the floor of the chamber," according to the Arizona Republic, therefore revoking many reporters' credentials.
Arizona Capitol Times editor Jim Small said the move was "an attack on the press and an affront to the concept of accountable government" as well as "direct retaliation" against reporters "who exposed how House Speaker David Gowan was using state vehicles to crisscross the state and advance his congressional bid."
Small also accused Grisham of playing a role in Gowan's decision, stating: "When I called Grisham to get an explanation, she made no bones about the fact that the paper's access had been pulled because of the story. She accused the paper of working for Chandler attorney Tom Ryan, who was quoted in the original story saying he intended to file a complaint against Gowan and the others for misusing the state vehicles."
Stephanie Grisham can be forceful when she wants to, a personality trait she put on display amid a scandal involving former Deputy National Security Advisor Mira Ricardel.
Per reports, Melania Trump didn't get along with Ricardel due a laundry list of personal issues, a frustration that came to an explosive head in November 2018. Melania was supposedly upset over Ricardel's decisions involving a trip to Africa, including "seating on the plane and the use of National Security Council resources," according to CNN. In response to the disagreements, Grisham released a pointed statement (via The New York Times) about Ricardel, saying: "It is the position of the Office of the First Lady that she no longer deserves the honor of serving in this White House." Yikes — tell us how you really feel, ladies.
Shortly after Grisham released the statement, then White House press secretary Sarah Huckabee Sanders announced Ricardel's dismissal. "Mira Ricardel will continue to support the president as she departs the White House to transition to a new role within the administration," she said (via The New York Times). "The president is grateful for Ms. Ricardel's continued service to the American people and her steadfast pursuit of his national security priorities."
So Grisham fits right into this sick f*ckin administration. Her cavalier disregard for the truth is matched by her equally cavalier disregard for the law. In September 2018, Grisham was reprimanded by the Office of Special Counsel for violating the Hatch Act, which prohibits White House employees from engaging in party-political activities.
Rather than fire her, Trump decided to promote her. And why wouldn't he? There don't seem to be any consequences for T-RUMP advisers who tell lies or break the law.
I cannot print my reply after I had heard of the dismissel of Mira Ricardel, an excellent Security Analyst assigned to Melania T-RUMP because of the personal frustrations of Melania. This was a trifecta of uselessness, a clusterf*ck of stupidity and another good qualified person who just happened to be in the wrong WhiteHouse.
What happened? She somehow didn't kiss the drooping ass of the Zombie first lady who follows T-RUMP around as on a leash like a puppy dog and does nothing but pose and pose and she would wear stilettos to a sand-pit volleyball tournament and break a leg.
She's tall enough to play and looks great in a thong bikini or even nude as she did for all those epic pictures that T-RUMP dumped all over the media. Just another useless White House fake, who has been tainted by T-Rumpism and fits in well with fake images. I thought she might be more of an influence over the misogamist panderer she's married to.
CURRENT WhiteHouse JOKE: We joked about whistleblowers, Donald loves whistle blowers, especially when it's his whistle… Then she didn't have the courage to dismiss her herself so she brought in STEPHANIE: "THE XXXX" GRISHAM, who did what was told and is a great supporter of Melania T-RUMP because that where her paycheck is and possibly some Melania Hand Me Downs….
In the last part of this disgusting way to treat people, the arrogance brought the number one human slob Sarah Not-A-Wara AKA Brunhilda the Mforphodite to spread the news to the press.


text:
You don't have any saved vehicles!
Look for this link on your favorites:
Once you've saved some vehicles, you can view them here at any time.
Delivers 25 Highway MPG and 17 City MPG! This Kia Stinger boasts a Twin Turbo Premium Unleaded V-6 3.3 L/204 engine powering this Automatic transmission. SEAT BACK CARGO TRAY, REMOTE START PUSH, PUDDLE LIGHTS.
This Kia Stinger Comes Equipped with These Options
HICHROMA RED, DOOR SILL APPLIQUE, CARPETED FLOOR MAT, BLACK, LEATHER SEAT TRIM, Window Grid Antenna, Wheels: 19 x 8.0 Front & 19 x 8.5 Rear Alloy, Wheels w/Machined w/Painted Accents Accents, Valet Function, Trip Computer, Transmission: Electronically Controlled 8-Spd Auto -inc: drive mode select and paddle shifters.
Stop By Today
You've earned this- stop by Smart Kia/Hyundai of Davenport located at 3700 N Harrison St, Davenport, IA 52806 to make this car yours today!
We invite you to join our family here at Smart Kia of Davenport when considering your next purchase. We will provide you with a world class experience that dares to challenge dealerships from Moline to Bettendorf. Our experienced team of Sales Professionals, Finance Managers, Service Advisors, Certified Technicians and Parts Specialists are here to make your car buying experience as stress-free as possible.
New vehicle pricing includes all offers and incentives. Tax, Title and Tags not included in vehicle prices shown and must be paid by the purchaser. While great effort is made to ensure the accuracy of the information on this site, errors do occur so please verify information with a customer service rep. This is easily done by calling us at (563) 447-9547 or by visiting us at the dealership.
**With approved credit. Terms may vary. Monthly payments are only estimates derived from the vehicle price with a 72 month term, 4.9% interest and 20% downpayment.
*Number of views in last 30 days
† Based on 2020 EPA mileage ratings. Use for comparison purposes only. Your actual mileage will vary depending on how you drive and maintain your vehicle.
You don't have any saved vehicles!
Look for this link on your favorites:
Once you've saved some vehicles, you can view them here at any time.
(563) 447-9547Call Now
3707 N. Harrison St • Davenport, IA 52806Get Directions
Open Today! Sales: 9am-8pm
Open Today! Service: 7am-6pm
Open Today! Parts: 7am-6pm
Phone: (888) 719-3482http://maps.google.com/?q=3707%20N.%20Harrison%20St%2C%20%2C%20Davenport%2C%20IA%2052806


text:
"This was the ONLY outing of our DIY laser/smoke light show, before we flogged it all, as venues were refusing to put us on due to health and safety. Spot the DIY stompbox by Lewy….240V going through that sucker."
From Broken Links Live @ Hamptons 03/11. Posted by Broken Links on 3/08/2011 (25 items)
Generated by Facebook Photo Fetcher 2


text:
Tips On Locating A Reputable Exterminator Near Me
Sometimes you have to deal with cockroaches and ants that have come into your home. You are going to need to find a way to get rid of them. You can try to use store-bought products, but many of these are not going to provide you with the results that you need. For those that are searching for exterminators, you can find a reputable one in your area. There will be several to choose from, all of which will have different levels of experience. These tips will lead you to the best and most reputable exterminator in your area that will provide you with affordable services.
Where To Find These Companies
The phone book can often provide you with all of the information that you will need. It is better, however, to look on the Internet. You should have no problem at all finding a local exterminator that will have the time to come out. By the end of the day, you will have an appointment set. This will be with one of these businesses that can deal with your problem. Whether you have ants, cockroaches, termites, or even bats that are in your attic, they will know exactly what to do to get rid of them.
Best Ways To Save Money With These Exterminators
Exterminators can save you a lot of money but you have to compare them. Some of them are going to be running specials. Others are going to be spending a lot of time advertising on the web. This is where you can save the most money in most cases. They will have advertisements that will be on Google and Facebook. You can click on those to see how much the savings will be. After talking with the representative, and clarifying the price of their visit, you can hire that one to come out to help you.
There are always going to be exterminators in both rural and urban areas. They will take care of your pest infestation that you have. Whether this is wild animals coming onto your property, or cockroaches that won't go away, they will soon be gone by using these professionals. It may take a few treatments to complete the process, or they may have to bring several cages, but they will soon be gone. It's better than trying to do this on your own, especially if you have an infestation that is substantial.
For more queries, visit http://discoverziehler.com/pest-control-mason/


text:
In spite of the geographic isolation of the Ozark area, national events did have effects on the residents of the Buffalo River area.
The Civil War along the Buffalo was not a matter of large scale military maneuvers of opposing armies. Instead regiments or companies skirmished with guerilla bands or one side bushwhacked the other to gain resources and supplies.
The Great Depression came to Arkansas long before the stock market crash of 1929. Floods, failing crop prices, and drought crippled the mostly agricultural state. By 1933 President Franklin D. Roosevelt, initiated federal programs that were developed to assist in the country's financial recovery. At least 2 historic homesteads along the Buffalo have concrete outhouses provided by the Works Progress Administration (WPA). The development at Buffalo Point was begun by the Civilian Conservation Corps in 1938.
Last updated: December 22, 2017