Introduction
============

Homologous DNA recombination is a fundamental process in the biochemistry of DNA repair and replication, which contributes to the generation of the genetic diversity critical for natural selection. An important step in the recombination process is the pairing of homologous double-stranded DNAs followed by the exchange of DNA strands between the paired molecules. Experimental studies have shown that members of the archetypal RecA family of recombinases are central to this reaction in all extant forms of life \[[@B1],[@B2]\].

Studies in *Escherichia coli* have shown that, although RecA is the principle protein involved in pairing and strand exchange, unrelated proteins, that have a much more restrictive phyletic distribution, can also promote similar reactions in a RecA dependent or RecA-independent manner \[[@B3]\]. These alternative or additional mediators of homologous recombination include the well-characterized prophage RecT, phage λ Redβ and phage P22 ERF proteins \[[@B4],[@B5]\]. Similarly, in yeast and vertebrates, the RAD52 protein is involved in the pairing and strand exchange reaction and can promote recombination in a RAD51 (the eukaryotic RecA homolog)-dependent or independent manner \[[@B6]\]. The RecT protein works in conjunction with the RecE-nuclease \[[@B7]\] and was initially described in genetic studies on the complemention of mutations in the RecBCD pathway of DNA repair \[[@B8]-[@B10]\]. Biochemically, RecT has been shown to bind single-stranded (ss) DNA 3\' overhang regions generated by the RecE nuclease, and promote strand exchange between homologous DNA partners by assisting the pairing of complementary single-stranded regions \[[@B4],[@B10]\]. The reaction catalyzed by the RecT/RecE system is similar to that described for the phage λ exonuclease (exo/Redβ) and the single-strand annealing protein Redβ. The similarity between these two systems is further extended by the observation that the RecT/E system can complement mutations in the λ exo/Redβ system \[[@B10],[@B12]\]. In eukaryotes, RAD52 protein has been shown to exhibit properties similar to those of RecT and Redβ proteins: it binds ssDNA and promotes strand exchange via the pairing of complementary single strands \[[@B6],[@B13]\]. *In vitro* studies on quaternary structures have shown that the single strand annealing proteins (SSAPs), RecT, Redβ, ERF and RAD52, form similar helical super-structures \[[@B14]-[@B17]\]. This has led to the proposal that RecT, Redβ, ERF and the eukaryotic RAD52 function in an analogous fashion, and even are \"structural homologs\" \[[@B14]\].

However, no sequence or secondary structural similarities have been noticed between different SSAPs and current understanding of their evolutionary history and phyletic range remains poor. Here, we describe the results of an in-depth sequence analysis of these proteins and delineate their evolutionary relationships and phyletic horizon in available genomes. We show that, in spite of the functional similarities, and the similar quaternary structures, there are three distinct superfamilies of SSAPs, namely the RecT/Redβ, RAD52 and ERF, that appear to be evolutionarily unrelated to each other. These superfamilies show a wide distribution in viral and cellular genomes, but appear to have originally evolved in large DNA bacteriophages. Through an analysis of the contextual information provided by the predicted operons, in which the SSAPs occur, we predict several previously undetected functional connections of these proteins, which might shed new light on the corresponding DNA repair/recombination pathways.

Results
=======

RecT and Redβ are evolutionarily related and define a widespread family of DNA recombination proteins
-----------------------------------------------------------------------------------------------------

Several lines of evidence, including genetic analyses, and similarities in biochemistry and quaternary structures, suggest that the *E. coli* RecT and phage λ Redβ proteins are functionally equivalent as mediators of single-strand exchange in DNA recombination \[[@B10],[@B12]\]. However, no sequence similarity has been detected between these proteins leaving their actual evolutionary relationships unresolved. In order to gain a better understanding of their functions and origins, we undertook a detailed sequence analysis of these two proteins using iterative sequence profile searches with the PSI-BLAST program with a inclusion threshold of .01 iterated until convergence. Such searches, with Redβ proteins from different lambdoid bacteriophages as queries, retrieved not only other obvious Redβ homologs, but also the RecT protein family. For example, searches initiated with the Redβ homolog, PF161 protein (Genbank gi: 9836834 amino acids 1 to 188) from *Borrelia hermsii*\[[@B18]\], detected the *E. coli* RecT protein in the 5^th^ iteration with significant expectation (e) values (3 × 10^-3^). Subsequent iterations retrieved several more RecT-related proteins from diverse sources. Further, transitive searches with the proteins detected in the above searches resulted in the identification of more divergent homologs, such as a protein termed the \'enterohemolysin associated protein\' (EHAP1) from *E. coli*\[[@B19]\] and its orthologs in *Salmonella* (Fig. [1](#F1){ref-type="fig"}). An examination of the pairwise alignments generated by these searches showed that all these proteins shared a characteristic set of residues, including two highly conserved aromatic residues at the N- and C-termini, respectively, and two consecutive acidic residues near the C-terminus. These observations strongly suggested that RecT and Redβ, along with several other proteins, could be unified into a single protein superfamily with a core conserved domain of approximately 200 amino acid residues.

![**Multiple sequence alignment of the RecT/Redβ superfamily of proteins.** Proteins are denoted with their gene names, species abbreviation and gi numbers. The coloring reflects the amino acid conservation at 85% consensus. The consensus abbreviations and coloring scheme are as follows: h: hydrophobic residues (L,I,Y,F,M,W,A,C,V), l: aliphatic (L,I,A,V) and a: aromatic (F,Y,W,H) residues shaded yellow; o: alcohol (S,T), colored blue, c: charged (K,E,R,D,H) residues, +: basic (K/R/H) residues, -: acidic (D,E) residues, and p: polar (S,T,E,C,D,R,K,H,N,Q) residues colored purple; s: small (S,A,C,G,D,N,P,V,T) and u:tiny (G,A,S) residues, colored green; b: big (L,I,F,M,W,Y,E,R,K,Q) residues shaded gray. Secondary structure assignments are as follows: H: Helix, E: Extended (Strand). Species abbreviations are as follows: Bh: *Bacillus halodurans,* Borhercp: *Borrelia hermsii* circular plasmid, BPA118: Bacteriophage A118, BPbIL309: Bacteriophage bIL309, BPphi31_1: Bacteriophage phi31.1, BPPVL: Bacteriophage PVL, BPR1T: Bacteriophage R1T, Bs: *Bacillus subtilis,* ec: *Escherichia coli,* lambda: Bacteriophage λ, Li: Listeria innocua, Lp: *Legionella pneumophila,* Ml: *Mesorhizobium loti,* Salent: *Salmonella enterica* subsp. enterica serovar Typhi, SaN315: *Staphylococcus aureus* N315 subsp. aureus N315,Sd: *Shigella dysenteriae,* SPP1: Bacteriophage SPP1, StLT2: *Salmonella typhimurium* LT2, Strpy: *Streptococcus pyogenes,* Uu: *Ureaplasma urealyticum,* Xf; *Xylella fastidiosa*](1471-2164-3-8-1){#F1}

A multiple alignment of all members of the RecT/Redβ superfamily was generated using the T_coffee program followed by adjustments based on the PSI-BLAST search results. This alignment was used to predict their secondary structure using the JPRED and PHD methods; these predictions pointed to an α + β domain with a core of five β-strands and five α-helices (Fig. [1](#F1){ref-type="fig"}). Some of the strongest conservation is concentrated in the long helices, and the pattern includes some charged or polar residues, suggesting that they are probably exposed and participate in the protein-protein and protein-DNA interactions that are typical of this superfamily (helices 2,3, 4 in Fig. [1](#F1){ref-type="fig"}). The conserved, regularly spaced hydrophobic residues in the RecT/Redβ superfamily are predicted to be buried, allowing these domains to assume a globular structure. Experimental studies have shown that the strand transfer reaction mediated by RecT and its binding to dsDNA are sensitive to Mg^+2^ concentrations and it was proposed that the levels of free Mg^+2^ could regulate RecT activity \[[@B4]\]. Similarly, Redβ has been shown to promote single strand annealing in a Mg^+2^-dependent manner \[[@B20]\]. In this context, the conservation of the two C-terminal acidic residues in the majority of members of this superfamily suggests that these might be involved in the coordination of Mg^+2^ and implies that the metal ion-dependent conformational switching is likely to be a generic feature of this family.

Phylogenetic analyses of the RecT/Redβ superfamily using the least squares and maximum likelihood methods distinguished three distinct groups, namely the RecT-like, the Redβ-like and the EHAP1-like families (Fig. [2](#F2){ref-type="fig"}). Previously, the RecT proteins have been known from very few bacteria and Redβ has only been detected in λ and closely related phages. However, we showed that the Redβ family is widespread in bacteria, such as *Borrelia hermsii, Xylella, Ureaplasma, Listeria, Streptococcus pyogenes, Mesorhizobium loti.* The RecT family is predominantly seen in the low-GC Gram-positive bacteria, such as *Bacillus, Streptococcus, Lactococcus and Listeria,* and their phages (Fig. [2](#F2){ref-type="fig"}). *E. coli* and *Legionella pneumophila* are the only two γ-proteobacteria that possess this protein, suggesting that they might have acquired RecT via a relatively recent horizontal transfer from Gram-positive bacteria. The sporadic distribution of the RecT and Redβ family proteins in bacterial genomes and their presence in phages suggest that these proteins ultimately are of phage origin and have been co-opted by the bacterial DNA recombination/repair systems. Consistent with this, practically all the bacterial members of these families appear to belong to prophages or their remnants, as they are mostly in the neighborhood of what appear to be clearly phage-derived genes. The EHAP1-like family is extremely divergent and represented thus far only in *E. coli* and the closely related *Salmonella.* Practically all members of the RecT/Redβ superfamily are single-domain proteins showing extended similarity to each other throughout their globular regions. The only exceptions are the *E. coli* EHAP1 and the PF161 protein encoded in the *Borrelia hermsii* circular plasmid, which are fused to C-terminal fragments of the ERF protein (see below).

![**Maximum likelihood tree for the RecT/Redβ superfamily of proteins.** The internal branches with RELL bootstrap support \>70% are indicated by blue circles. Proteins are designated by their gene names and species abbreviations as in Fig. [1](#F1){ref-type="fig"}. The gene neighborhoods of the RecT/Redβ superfamily genes are shown in association with the corresponding branches whenever they they contained genes for proteins with plausible functional connections with SSAPs. The hatched boxes represent fragments of ERF (Indicated by E) and SSB (indicated by S) genes encoding C-terminal regions as described in the text. Gene abbreviations are as follows: GP46: GP46 of bacteriophage PSA,  of bacteriophage PSA,  of *Streptococcus thermophilus* bacteriophage 7201, ORF40: ORF40 of bacteriophage PVL  of *Staphylococcus aureus* temperate phage φSLT,  of *Staphylococcus aureus* temperate phage φSLT, ORF364: ORF364 of bacteriophage φ31.1, Ec2360: b2360 of *E. coli,* AcylTr: N-Acyltransferase, Bro: Bro-N domain fused to XF0704, Met: DNA Methyltransferase](1471-2164-3-8-2){#F2}

ERF defines a superfamily of SSAPs that are evolutionarily distinct from the RecT/Redβ super family
---------------------------------------------------------------------------------------------------

The ERF protein of phage P22 is involved in the circularization of the linear dsDNA phage genome upon entry into the host cell \[[@B21]-[@B23]\]. Experimental studies have shown that, mutations in ERF are complemented by Redβ and that *in vitro* ERF adopts quaternary structures analogous to those of Redβ and RecT \[[@B14],[@B17],[@B24],[@B25]\]. However, in the comprehensive analysis of the RecT/Redβ superfamily no statistically significant similarity could be detected between these proteins and the ERF proteins. To explore the evolutionary affinities of the ERF domains, we carried out a sequence profile analysis as described above for the RecT case using transitive PSI-BLAST analysis. As a result of these searches, homologs of ERF encoded in several bacterial and phage genomes from diverse taxa were identified. The alignments generated in these searches consistently pointed to a region of approximately 150 amino acids that is conserved in all these proteins, with a characteristic motif of the form: GuXXoYhp + YXhXXhh (where G is glycine, Y-tyrosine, u is a tiny residue, h-hydrophobic, p is a polar residue, o is an alcohol residue, + is a basic residue, and X is any residue; Fig. [3](#F3){ref-type="fig"}). This suggested that ERF was the prototype of a family of conserved bacterial domains.

![**Multiple sequence alignment of the ERF protein superfamily.** The coloring reflects the amino acid conservation at 85% consensus. The coloring scheme and secondary structure assignment abbreviations are as in Fig. [1](#F1){ref-type="fig"}. Species abbreviations are as follows: Bbcp32-4: *Borrelia burgdorferi* cicular plasmid (cp) 32-4, Bbcp32-7: *B. burgdorferi* cp 32-7, Bbcp32-8: *B. burgdorferi* cp 32-8, Bbcp8.3: *B. burgdorferi* cp 8.3, Bbcp9: *B burgdorferi* cp 9, bIL67: bacteriophage IL67, c2: *Lactococcus* phage c2, D3: *Pseudomonas* phage D3, HK97: bacteriophage HK97, Lj771: *Lactobacillus johnsonii* prophage Lj771, Lm: *Listeria monocytogenes,* MM1: *Streptococcus pneumoniae* bacteriophage MM1, BPmv4: Bacteriophage mv4, P22: Bacteriophage P22, phiPV83: Bacteriophage φPV83, phiSLT: *Staphylococcus aureus* temperate phage φSLT, ST7201: *Streptococcus thermophilus* bacteriophage 7201, Unk: Unknown.](1471-2164-3-8-3){#F3}

Secondary structure prediction based on the multiple alignment of the ERF domain suggests a globular α + β fold with five helices and three or four strands (Fig. [3](#F3){ref-type="fig"}). The above-mentioned motif that is typical of this family is associated with helix 4 of this domain; given the presence of conserved basic residues, it may be critical for DNA-binding and strand-transfer activity of the ERF-like proteins. Additionally, in the loop between helices 4 and 5 of the ERF domain there is a universally conserved acidic motif of the form DXD. Analogous to the RecT superfamily, this acidic dyad might coordinate a divalent cation and undergo a conformational change dependent on metal-binding. However, the average size of the core domains, the patterns of conserved residues, and the predicted secondary structures of the RecT/Redβ and ERF domains show no correspondence to each other, implying that there is no direct evolutionary link between these protein groups.

ERF homologs are encoded by the genomes of several temperate phages of Gram-positive bacteria and γ-proteobacteria; additionally, we detected members of this superfamily in *Listeria* and in all the circular plasmids and one linear plasmid of *Borrelia burgdorferi* (Fig. [4](#F4){ref-type="fig"}). Thus, like the RecT/Redβ superfamily, the ERF family is likely to have emerged in the temperate phages, and was disseminated to the *Borrelia* circular plasmids and some bacterial genomes via prophages.

![**Maximum likelihood tree for the ERF superfamily of proteins.** The designations, gene names and species abbreviations are as in Fig. [2A](#F2){ref-type="fig"}. The internal branches with RELL bootstrap support \>70% are indicated by blue circles. The gene neighborhoods of the ERF proteins are shown whenever they contained gene coding for proteins with potential functional relevance. Gene abbreviations are as in Fig. [1B](#F1){ref-type="fig"}](1471-2164-3-8-4){#F4}

Detection of bacterial homologs of RAD52 and identification of an aberrant HhH domain in these proteins
-------------------------------------------------------------------------------------------------------

The baker\'s yeast protein RAD52 and its paralog RAD59 define a small family of proteins thus far represented in fungi, vertebrates and the early-branching ameboid eukaryote, *Entamoeba histolytica.* Rad52 functions in conjunction with the RecA ortholog, the RAD51 recombinase in double-strand break repair and meiotic recombination \[[@B6]\]. RAD52 binds ssDNA during recombination and also shows a quaternary organization similar to those of RecT/Redβ and ERF \[[@B16],[@B26]\]. However, RAD52-like proteins showed no detectable sequence similarity with either the ERF or the RecT/Redβ-like proteins. Sequence searches initiated with the conserved globular region of the eukaryotic RAD52 proteins readily detected their homologs from other eukaryotes and, at convergence, also retrieved from the database certain bacterial proteins, such as DR0423 from *Deinococcus* and CAC1936 from *Clostridium* respectively, with border-like statistical significance (e \~ .05). These bacterial proteins form a small family that is additionally represented in *Salmonella paratyphi* A, the temperate bacteriophage u136 of *Lactococcus lactis* (ORF252-encoded protein) and a Shiga toxin-converting phage from *E. coli*. Iterative profile searches initiated with CAC1936 from *Clostridium acetobutylicum* and its *S. paratyphi* A ortholog correspondingly retrieved *S. cerevisiae* RAD52 and its eukaryotic homologs, with borderline e-values at convergence (\~0.043). The alignment between these bacterial proteins and the eukaryotic Rad52 homologs was co-linear throughout the entire length of their shared globular region and the Gibbs sampling procedure detected two motifs of greater than 20 residues, with a probability of chance occurrence in these proteins less than 10^-18^ (Fig. [5](#F5){ref-type="fig"}). In addition to the similar conservation pattern, separate secondary structure predictions for both the eukaryotic RAD52 family and their potential bacterial homologs showed a complete concordance of the predicted structural elements between RAD52 and the bacterial proteins, strongly suggesting that they all belong to a single homologous superfamily (hereinafter the RAD52 superfamily).

![**Multiple sequence alignment of the RAD52 protein superfamily.** The coloring reflects the consensus at 90% conservation. The coloring scheme and secondary structure assignment abbreviations are as in Fig. [1](#F1){ref-type="fig"}. Species abbreviations are as follows: BPul36: Bacteriophage ul36. Cab: *Clostridium acetobutylicum,* Dr: *Deinococcus radiodurans,* Hs: Homo sapiens, Kla: *Kluyveromyces lactis,* NC: *Neurospora crassa,* Sc: *Saccharomyces cerevisiae,* Sp: *Schizosaccharomyces pombe,* Sparatyphi: *Salmonella paratyphi* A, Ralbus: *Ruminococcus albus.* The Shiga toxin-converting phage RAD52-like protein (gi: 17977996) is nearly identical to the *Salmonella paratyphi* A RAD52 like protein. The RAD52-like proteins from Bacteriophage ul36 (gi: 8248159) and *Ruminococcus albus* are respectively adjacent to genes encoding the single-strand binding protein and the λ-type exonuclease.](1471-2164-3-8-5){#F5}

The secondary structure predictions showed that the Rad52 superfamily proteins adopt a structure with interspersed α-helices and β-strands (Fig. [5](#F5){ref-type="fig"}). Additionally, fold predictions using 3DPSSM (E-value=.0085, corresponding to a 90% confidence in the prediction) and the hybrid fold method (Z-score = 19.5) predicted the presence of a potential Helix-hairpin-Helix (HhH) fold in members of the RAD52 superfamily. The HhH domain is a small nucleic acid-binding module comprised of two helices joined by a central loop (hairpin), which functions as the DNA-binding moiety of numerous repair and recombination proteins\[[@B27],[@B28]\]. Two HhH modules are predicted in the core conserved domain of the RAD52 family, the first one bounded by the predicted helices 2 and 3, and the second one bounded by helices 5 and 6 (Fig. [5](#F5){ref-type="fig"}). Although these predicted HhH modules are very divergent in sequence from the typical versions, the hairpin in both HhH modules of the RAD52 family proteins is bounded by small residues, typically glycine; this conforms to the signature motif characteristic of the classical HhH modules \[[@B28],[@B29]\]. However, in the case of the RAD52 superfamily the predicted HhH modules appear to have been welded into a large globular superstructure that maintained its evolutionary distinctness over time. The conservation pattern and predicted structural elements of the RAD52 superfamily are distinct from those predicted for the ERF and RecT/Redβ superfamilies (Fig [1](#F1){ref-type="fig"}, [3](#F3){ref-type="fig"}, [5](#F5){ref-type="fig"}), supporting the lack of a direct evolutionary relationship between these proteins.

The RAD52 superfamily shows a sporadic phyletic distribution, and even in the crown-group eukaryotes, might have been secondarily lost in certain lineages, such as plants, nematodes and insects. The sporadic distribution of this family among phylogenetically distant bacteria, along with its presence in several prophages, suggests that, like the RecT/Redβ and ERF superfamilies, at least the bacterial RAD52-proteins might be of predominantly phage origin. The core of the eukaryotic recombination system appears to have been inherited from the system present in the common ancestor shared with the archaea \[[@B29]\]. However, RAD52 is thus far absent in all archaeal genomes and is restricted to a single orthologous group in the eukaryotes \[[@B29]\]. Thus, it appears plausible that eukaryotic RAD52 was ultimately derived through lateral transfer either from a bacterial genome or directly from a viral source, at a point at least predating the divergence of the crown group eukaryotes and *Entamoeba.*

Contextual information from gene neighborhoods provides details regarding functional interactions of the SSAPs with DNA recombination pathways
----------------------------------------------------------------------------------------------------------------------------------------------

The clustering of functionally related genes in prokaryotic genomes into co-transcribed and co-regulated units, operons, often allows functional assignments through the principle of \'guilt by association\' \[[@B30]-[@B32]\]. Generally, genes whose products physically interact to form a complex or are involved in successive steps in a biochemical pathway form operons that are conserved over large evolutionary distances \[[@B30]\]. On previous occasions, we have used gene neighborhoods or operons to predict novel DNA repair complexes and their components \[[@B33]\]. Accordingly, a similar approach was applied to the three families of SSAPs (RecT/Redβ, ERF, Rad52), to shed light on their functional links.

Notably, the genes encoding the three evolutionarily distinct SSAPs co-occurred with similar sets of DNA repair/recombination-related proteins (Figs. [2](#F2){ref-type="fig"},[4](#F4){ref-type="fig"}). In at least one case, each of them was found adjacent to the gene for the single-strand-binding protein (SSB), an OB-fold protein that binds ssDNA (Figs. [2](#F2){ref-type="fig"},[4](#F4){ref-type="fig"}). This association ties in with the function of the SSAPs in single-strand annealing, suggesting that they closely interact with SSB. It has been suggested in the case of RecT that it may compete with SSB for binding single strand overhangs and thereby make them available for the annealing process \[[@B3]\]. Similar interactions between other SSAPs and SSB, that probably coats the ssDNA generated by nucleases, appear likely. Genes for SSAPs from all the 3 distinct superfamilies may also occur adjacent to or in the vicinity of genes encoding nucleases or Holliday junction resolvases (HJRs). Genes for RecT/Redβ superfamily proteins are associated with genes encoding a λ-type exonuclease (LE) of the type II restriction enzyme fold, RecE, which also might be a divergent member of this fold, and a nuclease of the Endonuclease VII (EndoVII) fold \[[@B7],[@B34]\] (Fig. [2](#F2){ref-type="fig"}). The ERF superfamily genes are associated with a RusA superfamily nuclease/HJR and EndoVII fold nucleases (Fig. [4](#F4){ref-type="fig"}) \[[@B34]\]. Furthermore, the *Borrelia* plasmids that encode ERF, also almost always additionally encode a λ-type exonuclease, even if it is not the adjacent gene. In a single instance, in the Gram-positive bacterium *Ruminococcus albus,* the gene encoding a RAD52 superfamily protein occurs adjacent to a gene for a λ-type exonuclease. These nucleases probably contribute to the repair process, in which SSAPs are involved, by providing the initial break in the dsDNA and/or in digesting the nicked target to generate ssDNA.

The RecT and Redβ family proteins often co-occur with the SbcC gene that encodes an ABC ATPase with a large coiled-coil segment. These proteins are known to cooperate with SbcD, nuclease of the calcineurin-like phosphoesterase superfamily and to degrade dsDNA in the 3\' → 5\' direction generating ssDNA \[[@B35],[@B36]\]. It seems likely that RecT/Redβ proteins, at least in certain cases, function in conjunction with the SbcCD-pathway, by utilizing the single-stranded regions generated by the SbcCD nuclease. Additionally, several genes, whose functions are less clear, tend to co-occur with the genes coding for the SSAPs. These include DNA methyltransferases and the primosomal protein DnaD from low-GC Gram-positive bacteria \[[@B37]\] that co-occur with both ERF and RecT superfamily members (Figs. [2](#F2){ref-type="fig"},[4](#F4){ref-type="fig"}). The poorly characterized phage-or prophage-specific genes that are frequently observed in these neighborhoods include ORF15 (*Streptococcus thermophilus* bacteriophage 7201), ORF86, ORF100a (*Staphylococcus aureus* temperate phage φSLT) and ORF364 (bacteriophage φ31.1) (Figs. [2](#F2){ref-type="fig"},[4](#F4){ref-type="fig"}). Secondary structure predictions indicate a high α-helical content for these proteins. It is likely that these α-helical proteins are phage innovations that could function as adaptors in the recombination pathway either as accessory protein-protein interacting domains or as DNA-binding domains.

Evidence for convergent operon evolution and in situ non-orthologous displacement of genes in operons encoding SSAPs
--------------------------------------------------------------------------------------------------------------------

A superposition of the gene neighborhood information upon the phylogenetic trees for the SSAP superfamilies provides insights into the evolutionary processes that led to the emergence of the operons that include the SSAP genes. As discussed above, the RecT/Redβ superfamily clearly splits into three distinct families (Fig. [2](#F2){ref-type="fig"}). The phylogenetic tree shows that SbcC co-occurs with the SSAP once within Redβ-family and once within the RecT-family. An examination of the tree and the respective gene neighborhoods suggests that independent juxtaposition of SbcC with Redβ-like and RecT-like genes on two separate occasions is the most parsimonious explanation. The alternative explanation, namely that the gene coding for the common ancestor of the Redβ and RecT already co-occurred with the *sbcC* gene is far less likely because it would require over 10 independent losses of this, apparently, functionally advantageous organization in different bacterial and bacteriophage lineages. Likewise, the observation that, in one or more cases, genes encoding each of the SSAPs co-occur in the same predicted operon with SSB or a λ-type exonuclease, suggests that similar operon structures may also emerge independently in evolution. Thus, the same or analogous operon organizations may emerge convergently on multiple occasions, probably due to the selective pressure arising from the strong interactions between the SSAPs and their functional partners such as SbcC, SSB and LE.

The distribution of the gene neighborhoods, in which a member of the RecT superfamily occurs next to the RecE on the phylogenetic tree of the RecT/Redβ superfamily, indicates that the RecE-RecT combination was probably the ancestral state for at least the RecT and EHAP1 families (Fig. [2](#F2){ref-type="fig"}). This implies that, on at least two occasions, the gene for λ endonuclease displaced the functionally analogous *recE* gene and became the adjacent gene to RecT (Fig. [2](#F2){ref-type="fig"}). That this displacement might have occurred by *in situ* insertion of a non-orthologous gene is suggested by the detection, on three separate occasions, of unusual remnants of pre-existing genes. The RecT/Redβ superfamily members, namely EHAP1 from the enterobacteria and PF161 from *Borrelia hermsii,* contain a small, C-terminal fragment of the core conserved domain of the ERF superfamily, which is located C-terminal of their *bona fide* RecT/Redβ domains. These fragments of the ERF protein are closely related to other ERF domains from related organisms and are unlikely to fold into the native conformation characteristic of the full-length ERF domain. For example the ERF fragment fused to the EHAP1 RecT/Redβ domain is closely related to the P22 phage ERF domain. This suggests that in each of these cases a RecT superfamily gene was inserted in frame into a pre-existing ERF gene leaving behind only a non-functional fragment of it (Fig. [2](#F2){ref-type="fig"}). In a very similar case, the bacterial RAD52-like protein from a Shiga toxin encoding temperate phage is fused to an extreme C-terminal fragment that is nearly identical to the C-terminal most portion of the P22 ERF protein. In this case, it appears that the pre-existing ERF gene was displaced through the insertion of a bacterial RAD52-like gene. Interestingly, and in the same vein, the RecT proteins from *Bacillus* species contain a short C-terminal acidic module that is missing in other RecT proteins, but is highly similar to the C-terminal region of SSBs, particularly those from Gram-positive bacteria (data not shown). This suggests that, at some stage in their evolution, the *Bacillus recT* gene protein has recombined with the gene coding for SSB, which might even have resulted in a functional replacement of an SSB with an SSAP.

Thus it appears likely that functionally equivalent genes may displace their analogs in operons via insertion into the same position.

Conclusions
===========

We show that functionally similar SSAPs belong to at least three evolutionarily distinct superfamilies. We unify the Redβ and RecT proteins and their homologs, which have not been reported as being related at the sequence level, into a single superfamily, supporting the notion that these proteins share a similar mechanism of action. The second superfamily typified by the ERF proteins is predominantly found in bacteriophages and is also present on all circular plasmids from *Borrelia,* suggesting a role in the recombination of these plasmids. The third superfamily, typified by the yeast RAD52 protein and previously detected only in eukaryotes, was shown to include bacterial and phage homologs and to contain a modified HhH domain. By comparing the gene neighborhoods of the SSAPs, we show that the predicted operons that include the SSAP genes evolve according to the \"LEGO\" principle. In these operons, the SSAP genes are linked to the genes for various DNAses and DNA repair related proteins, such as SSB and SbcC, which implies functional connections between the encoded proteins. Evidence is presented of convergent emergence of similar SSAP-encoding operons in different lineages and of *in situ* non-orthologous displacement of functionally similar genes in these operons.

Materials and Methods
=====================

Sequence searches of the non-redundant (NR) and the unfinished genomes databases, were done using the gapped BLAST and PSI-BLAST programs \[[@B38]\]. Iterative PSI-BLAST searches used for in-depth sequence analysis were done with the profile inclusion cutoff expectation value (E value) set at 0.1. Multiple sequence alignments were generated using the T_Coffee program \[[@B39]\] and the output was adjusted using PSI-BLAST search results and secondary structure predictions, which were conducted using the PHD \[[@B40],[@B41]\] and Jpred \[[@B42]\] programs. Fold predictions were done using the 3-D position specific score matrix (3DPSSM) \[[@B43]\] and the Hybrid fold method \[[@B44]\]. Phylogenetic analysis was carried out using the neighbor-joining algorithm, with subsequent local rearrangements using the maximum likelihood algorithm \[[@B45]\]. The robustness of tree topology was assessed with 10000 Resampling of Estimated Log Likelihoods (RELL) bootstrap replicates. The MOLPHY and Phylip software packages were used for the analyses \[[@B46],[@B47]\].

Authors\' contributions
=======================

Author 1 (LMI) contributed to the discovery process, preparation of the manuscript and multiple sequence alignments. Author 2 (EVK) contributed to the analysis of the predicted operons encoding RecT and SSB proteins from Gram positive bacteria, Author 3 (LA) contributed to the discovery process, preparation of the manuscript and conceived the study.


Background
==========

*Crocus sativus* L. (Iridaceae), commonly known as saffron is used in folk medicine for various purposes such as an aphrodisiac, antispasmodic and expectorant \[[@B1]\]. Modern pharmacological studies have demonstrated that saffron extracts have antitumour \[[@B2]-[@B4]\], radical scavenger, hypolipaemic \[[@B5]\], anticonvulsant effects \[[@B6]\] and improve activity on learning and memory \[[@B5],[@B7]\].

Chemical studies on *C. sativus* have shown the presence of constituents such as crocin, crocetin, safranal and picrocrocin \[[@B8]-[@B10]\]. Among the constituents of saffron extract, crocetin is mainly responsible for these pharmacological activities \[[@B5]\].

In traditional medicine, the stigma of this plant is used as an antiedematogenic remedy \[[@B1]\]. The aim of this study was to validate the anti-inflammatory and antinociceptive effect of the saffron stigma and petal extracts.

Results
=======

The maximum non-fatal doses of stigma aqueous and ethanolic extracts were 0.8 g/kg and 2 g/kg (i.p.), respectively.

The maximum non-fatal doses of petal aqueous and ethanolic extracts were 3.6 g/kg and 8 g/kg (i.p.), respectively. LD~50~ values of the petal aqueous and ethanolic extracts were 6.67 g/kg, i.p. (4.95, 8.99) and 9.99 g/kg, i.p. (8.13,12.28), respectively.

Phytochemical screening of the extracts indicated the presence of flavonoids, tannins and anthocyanins in the petal aqueous and ethanolic extracts. Alkaloids and saponins were found in the stigma aqueous and ethanolic extracts (Table [1](#T1){ref-type="table"})

::: {#T1 .table-wrap}
::: {.caption}
######

Phytochemical screening of *Crocus sativus* petal and stigma aqueous and ethanolic extracts.
:::

  **Extract**    **Alkaloids**   **Flavonoids**   **Tannins**   **Saponins**   **Anthocyanins**
  -------------- --------------- ---------------- ------------- -------------- ------------------
  **Petal**
   *Aqueous*     \-              \+               \+            \-             \+
   *Ethanolic*   \-              \+               \+            \-             \+
  **Stigma**
   *Aqueous*     \+              \-               \-            \+             \-
   *Ethanolic*   \+              \-               \-            \+             \-
:::

The aqueous and ethanolic extracts of *C. sativus* petal and stigma significantly reduce the number of mouse abdominal constrictions induced by a 0.7% acetic acid solution in a dose dependent manner (P\<0.001, except for the dose of 0.36 g/kg of aqueous stigma which was P\<0.01). Morphine and diclofenac induced a protection against abdominal constriction (P\<0.001). Naloxone, (2 mg/kg, s.c.) pretreatment after i.p. injection of the extracts practically did not inhibit the antinociceptive activity of both extracts (figures [1](#F1){ref-type="fig"}, [2](#F2){ref-type="fig"}, [3](#F3){ref-type="fig"}, and [4](#F4){ref-type="fig"}). Only, the antinociceptive effect of aqueous stigma extract, 0.32 g/kg, was partially blocked by naloxone (figure [3](#F3){ref-type="fig"}). Naloxone completely antagonized the antinociceptive activity of morphine.

![Effect of a subcutaneous injection of naloxone on the antinociceptive effect of intraperitoneally administered *Crocus sativus* petal aqueous extract, morphine and diclofenac on acetic acid-induced writhing test in mice. Values are the mean ± S.E.M. of writhes number for 8 mice, ^\*\*^P\<0.01, ^\*\*\*^P\<0.001, compared to control (normal saline); +++P\<0.001, compared to morphine plus naloxone, Tukey-Kramer test.](1471-2210-2-7-1){#F1}

![Effect of a subcutaneous injection of naloxone on the antinociceptive effect of intraperitoneally administered *Crocus sativus* petal ethanolic extract, morphine and diclofenac on acetic acid-induced writhing test in mice. Values are the mean ± S.E.M. of writhes number for 8 mice, ^\*\*\*^P\<0.001, compared to control (normal saline); +++P\<0.001, compared to morphine plus naloxone, Tukey-Kramer test.](1471-2210-2-7-2){#F2}

![Effect of a subcutaneous injection of naloxone on the antinociceptive effect of intraperitoneally administered *Crocus sativus* stigma aqueous extract, morphine and diclofenac on acetic acid-induced writhing test in mice. Values are the mean ± S.E.M. of writhes number for 8 mice, ^\*\*\*^P\<0.001, compared to control (normal saline); +++P\<0.001, compared to morphine (or extract) plus naloxone, Tukey-Kramer test.](1471-2210-2-7-3){#F3}

![Effect of a subcutaneous injection of naloxone on the antinociceptive effect of intraperitoneally administered *Crocus sativus* stigma ethanolic extract, morphine and diclofenac on acetic acid-induced writhing test in mice. Values are the mean ± S.E.M. of writhes number for 8 mice, ^\*\*\*^P\<0.001, compared to control (normal saline); +++P\<0.001, compared to morphine plus naloxone, Tukey-Kramer test.](1471-2210-2-7-4){#F4}

In hot plate test, aqueous (1.4, 2.5 and 3.6 g/kg, i.p.) and ethanolic (0.8, 3.2 and 5.6 g/kg, i.p.) petal extracts showed no significant antinociceptive activity (P\>0.05)(figures [5](#F5){ref-type="fig"} and [6](#F6){ref-type="fig"}). The aqueous (0.32, 0.56 and 0.8 g/kg, i.p.) and ethanolic (0.8, 1.4 and 2 g/kg, i.p.) stigma extracts also exerted no significant analgesic activity (P\>0.05)(figures [7](#F7){ref-type="fig"} and [8](#F8){ref-type="fig"}). Morphine, a positive reference, (10 mg/kg, i.p.) showed significant analgesic effect in the hot-plate test beginning 30 min after treatment (P\<0.001). In the xylene induced ear edema, the aqueous and ethanolic extracts of petal showed no significant anti-inflammatory activity but diclofenac and dexamethasone reduced the edema about 50% (Tables [2](#T2){ref-type="table"} and [3](#T3){ref-type="table"}). In higher doses, the aqueous (0.56 g/kg, P\<0.05; 0.8 g/kg, P\<0.01) and ethanolic (1.4 g/kg, P\<0.05; 2 g/kg, P\<0.05) extracts of stigma showed significant activity against the acute inflammation (Tables [4](#T4){ref-type="table"} and [5](#T5){ref-type="table"}).

![Effect of the aqueous extract of *Crocus sativus* petal and morphine (i.p.) on pain threshold of mice in the hot-plate test. Each point represents the mean ± S.E.M. of reaction time for n = 8 experiments on mice. ^\*\*\*^P\<0.001, compared to control (normal saline), Tukey-Kramer test.](1471-2210-2-7-5){#F5}

![Effect of the ethanolic extract of *Crocus sativus* petal and morphine (i.p.) on pain threshold of mice in the hot-plate test. Each point represents the mean ± S.E.M. of reaction time for n = 8 experiments on mice. ^\*\*\*^P\<0.001, compared to control (normal saline), Tukey-Kramer test.](1471-2210-2-7-6){#F6}

![Effect of the aqueous extract of *Crocus sativus* stigma and morphine (i.p.) on pain threshold of mice in the hot-plate test. Each point represents the mean ± S.E.M. of reaction time for n = 8 experiments on mice. ^\*\*\*^P\<0.001, compared to control (normal saline), Tukey-Kramer test.](1471-2210-2-7-7){#F7}

![Effect of the ethanolic extract of *Crocus sativus* stigma and morphine (i.p.) on pain threshold of mice in the hot-plate test. Each point represents the mean ± S.E.M. of reaction time for n = 8 experiments on mice. ^\*\*\*^P\<0.001, compared to control (normal saline), Tukey-Kramer test.](1471-2210-2-7-8){#F8}

::: {#T2 .table-wrap}
::: {.caption}
######

Effect of the intraperitoneal doses of *Crocus sativus* petal aqueous extract, diclofenac and dexamethasone on xylene-induced ear swelling in mice.
:::

  Treatment       Dose       Ear swelling (mg)   Inhibition (%)
  --------------- ---------- ------------------- ----------------
  Control         10 ml/kg   7.6 ± 0.6           --
  Diclofenac      15 mg/kg   3.9 ± 0.4^\*\*\*^   49.1
  Dexamethasone   15 mg/kg   3.2 ± 0.3^\*\*\*^   58.2
  Extract         1.4 g/kg   6.8 ± 0.3           11.7
  Extract         2.5 g/kg   6.4 ± 0.4           15.9
  Extract         3.6 g/kg   6.3 ± 0.3           17.2

The increase in weight caused by the irritant (xylene) was measured by subtracting the weight of the untreated left ear section from that of the treated right ear sections. Values are the mean ± S.E.M. for 7 mice, ^\*\*\*^P\<0.001, compared to control (normal saline), Tukey-Kramer.
:::

::: {#T3 .table-wrap}
::: {.caption}
######

Effect of the intraperitoneal doses of *Crocus sativus* petal ethanolic extract, diclofenac and dexamethasone on xylene-induced ear swelling in mice.
:::

  Treatment       Dose       Ear swelling (mg)   Inhibition (%)
  --------------- ---------- ------------------- ----------------
  Control         10 ml/kg   6.8+0.5             --
  Diclofenac      15 mg/kg   3.7 ± 0.6^\*\*^     45.3
  Dexamethasone   15 mg/kg   3.3 ± 0.5^\*\*^     51.4
  Extract         0.8 g/kg   5.5 ± 0.6           19.2
  Extract         1.6 g/kg   5.5 ± 0.5           19.4
  Extract         2.5 g/kg   5.1 ± 0.6           25.8
  Extract         3.2 g/kg   4.8 ± 0.6           29.0

The increase in weight caused by the irritant (xylene) was measured by subtracting the weight of the untreated left ear section from that of the treated right ear sections. Values are the mean ± S.E.M. for 7 mice, ^\*\*^P\<0.01, compared to control (normal saline), Tukey-Kramer.
:::

::: {#T4 .table-wrap}
::: {.caption}
######

Effect of the intraperitoneal doses *of Crocus sativus* stigma aqueous extract, diclofenac and dexamethasone on xylene-induced ear swelling in mice.
:::

  Treatment       Dose        Ear swelling (mg)   Inhibition (%)
  --------------- ----------- ------------------- ----------------
  Control         10 ml/kg    7.6 ± 0.6           --
  Diclofenac      15 mg/kg    3.9 ± 0.4^\*\*\*^   53.6
  Dexamethasone   15 mg/kg    32+0 3^\*\*\*^      63.1
  Extract         0.32 g/kg   5.0 ± 0.4           20.9
  Extract         0.56 g/kg   4.3 ± 0.4^\*^       32.6
  Extract         0.8 g/kg    3.5 ± 0.5^\*\*^     44.1

The increase in weight caused by the irritant (xylene) was measured by subtracting the weight of the untreated left ear section from that of the treated right ear sections. Values are the mean ± S.E.M. for 7 mice, ^\*^P\<0.05, ^\*\*^P\<0.01, ^\*\*\*^P\<0.001, compared to control (normal saline), Tukey-Kramer.
:::

::: {#T5 .table-wrap}
::: {.caption}
######

Effect of the intraperitoneal doses of Crocus sativus stigma ethanolic extract on xylene-induced ear swelling in mice.
:::

  Treatment       Dose       Ear swelling (mg)   Inhibition (%)
  --------------- ---------- ------------------- ----------------
  Control         10 ml/kg   6.2 ± 0.3           --
  Diclofenac      15 mg/kg   2.6 ± 0.4^\*\*\*^   58.3
  Dexamethasone   15 mg/kg   3.1 ± 0.4^\*\*\*^   49.5
  Extract         0.2 g/kg   5.1 ± 0.3           18.2
  Extract         0.8 g/kg   4.8 ± 0.3           22.4
  Extract         1.4 g/kg   4.4 ± 0.4^\*^       29.3
  Extract         2.0 g/kg   4.5 ± 0.6^\*^       27.8

The increase in weight caused by the irritant (xylene) was measured by subtracting the weight of the untreated left ear section from that of the treated right ear sections. Values are the mean ± S.E.M. for 7 mice, ^\*^P\<0.05, ^\*\*\*^P\<0.001, compared to control (normal saline), Tukey-Kramer.
:::

In the chronic inflammation (formalin test), the aqueous petal extracts did not exhibit significant anti-inflammatory activity (P\>0.05) but the ethanolic petal extract showed significant activity (figure [9](#F9){ref-type="fig"}). Both extracts of the stigma exerted significant anti-inflammatory activity (figure [10](#F10){ref-type="fig"}). In diclofenac and the ethanolic stigma extract (1.4 g/kg) groups; the hind paw edema of rat disappeared after 6 days (P\<0.001) (figure [10](#F10){ref-type="fig"}). Diclofenac as well as the aqueous (0.8 g/kg) and ethanolic stigma extract (1.4 g/kg) showed anti-inflammatory effect on day one (P\<0.05) (figure [10](#F10){ref-type="fig"}). On days 4 and 5, both diclofenac and the extracts did not demonstrate anti-inflammatory activity (P\>0.05).

![Effect of petal *Crocus sativus* aqueous and ethanolic extracts and diclofenac on formaldehyde induced arthritis in hind paw of rats. The inflammation was produced by subaponeurotic injection of 0.1 ml of 2% formaldehyde in the right hind paw of the rats on the first and third day. The animals were treated daily with the extracts or diclofenac intraperitoneally for 10 days. All agents were administered intraperitoneally. Each point represents the mean ± S.E.M. of change of hind paw size for 6 rats. Only the ethanolic extracts and diclofenac were effective compared to control (normal saline), Tukey-Kramer.](1471-2210-2-7-9){#F9}

![Effect of stigma *Crocus sativus* aqueous and ethanolic extracts and diclofenac on formaldehyde induced arthritis in hind paw of rats. The inflammation was produced by subaponeurotic injection of 0.1 ml of 2% formaldehyde in the right hind paw of the rats on the first and third day. The animals were treated daily with the extracts or diclofenac intraperitoneally for 10 days. All agents were administered intraperitoneally. Each point represents the mean ± S.E.M. of change of paw size for 6 rats. Both extracts and diclofenac were effective compared to control (normal saline), Tukey-Kramer.](1471-2210-2-7-10){#F10}

Discussion
==========

The present results indicate that aqueous and ethanolic extracts of *C. sativus* petal and stigma have antinociceptive activity in chemical pain tests. The stigma extracts showed activity against acute and chronic inflammation. The petal ethanolic extract showed anti-inflammatory activity in the chronic inflammatory test.

In respect to LD~50~ values and maximum non-fatal doses, the stigma extracts were more toxic than the petal extracts. LD~50~ values of aqueous and ethanolic stigma extracts have been reported as 1.6 g/kg, i.p. (1.16, 2.22) and 3.38 g/kg, i.p. (2.55, 4.52) in mice \[[@B6]\]. According to a toxicity classification \[[@B11]\], stigma and petal extracts are \"relatively toxic\" and \"low toxic\", respectively. Treatment with the *C. sativus* extract also significantly prolonged the life span of cisplatin-treated mice almost three-fold \[[@B12]\]. Toxicity studies showed that the hematological and biochemical parameters were within normal range with saffron extract \[[@B2]\].

As preliminary phytochemical results indicated, it could be suggested that the antinociceptive and anti-inflammatory effects of the petal extracts may be due to their content of flavonoids, tannins and anthocyanins. Other studies have demonstrated that various flavonoids such as rutin, quercetin, luteolin, hesperidin and biflavonoids produced significant antinociceptive and/or anti-inflammatory activities \[[@B13]-[@B16]\]. There are few reports on the role of tannins in antinociceptive and anti-inflammatory activities \[[@B17]\]. Recently, it has been shown that crocins, *Crocus* glycosides, exhibited an anti-inflammatory effect in some models of inflammation \[[@B18]\].

In the present study, morphine, a centrally acting analgesic drug, produced an inhibitory effect on the nociceptive response in the hot plate test, a central antinociceptive test \[[@B19]\], while the aqueous and ethanolic extracts showed no antinociceptive activity in this test. Thus, the extracts may not act via central mechanisms, although agents that alter the motor performance of animals may increase the latency time on the hot plate test without acting on the central nervous system \[[@B19]\]. Antinociceptive activity of opioid agonists, opioid partial agonists, and non-steroidal anti-inflammatory agents can be determined by the writhing test \[[@B20]\]. It has also been shown that some plants such as *Hunteria zeylanica*\[[@B21]\] and *Ocotea suaveolens*\[[@B22]\] decrease stretching induced by acid acetic acid but have no effect on heat-induced pain. The association of both antinociceptive activity and moderate anti-inflammatory effect observed with the extracts has also been shown in non-steroidal anti-inflammatory drugs (NSAIDs). It is a well-established fact that NSAIDs exert their analgesic and anti-inflammatory activity by the inhibition of cyclo-oxygenase activity \[[@B23]\]. As the antinociceptive activity of most extracts in the writhing test was not inhibited by naloxone, the extracts may not act via opioid receptors and may exert their activity via a peripheral mechanism. The stigma extracts and the ethanolic petal extracts significantly diminished in a dose dependent way the induced paw edema in rats. The extracts were able to reduce the inflammation in acute and chronic phases.

It is concluded that saffron stigma and petal aqueous and ethanolic maceration extracts have antinociceptive effects in chemical pain tests and have acute and/or chronic anti-inflammatory activity. The antinociceptive and anti-inflammatory effects of the extracts may be due to their content of flavonoids, tannins, anthocyanins, alkaloids and saponins. However, the chemical constituents and mechanism(s) responsible for the pharmacologoical activities remain to be investigated.

Materials and Methods
=====================

Animals
-------

Male and female albino mice 25--30 g and Wistar rats weighing 150--210 g were obtained from a random bred colony in the animal house of Mashhad University of Medical Sciences. Animals were housed in colony room 12/12 hr light/dark cycle at 21 ± 2°C and had free access to water and food.

Plant material
--------------

Plants were collected from Torabat Hydarieh (in south of Khorassan province, I.R. of Iran) in October 1998 and dried in shadow and ground. The *C. sativus* L. was identified by Ferdowsi University (Ms. Molaei) and voucher samples were preserved for reference in the herbarium of School of Pharmacy, Mashhad, IR. Iran (143-0319-1).

Preparation of extracts
-----------------------

The powder of stigma or petal was extracted using maceration with ethanol or water. The powdered plant was macerated in water or ethanol (80 %, v/v) for 3 days and, subsequently, the mixture was filtered and concentrated under reduced pressure at 40°C. The yield (w/w) of aqueous and ethanolic extracts of stigma was 50.8% and 56.6%, respectively. The yield (w/w) of aqueous and ethanolic extracts of petal was 15.5% and 19%, respectively. The extracts were dissolved in normal saline.

Phytochemical screening
-----------------------

Phytochemical screening of the extract was performed using the following reagents and chemicals: Alkaloids with Dragendorffs reagent, flavonoids with the use of Mg and HCl; tannins with 1% gelatin and 10% NaCl solutions and saponins with ability to produce suds \[[@B24]\].

The maximum non-fatal dose (MNFD) and acute toxicity
----------------------------------------------------

Different doses of the extracts were injected to separate groups of five animals. After 48 h, the highest dose that did not induce any mortality was considered as the maximum non-fatal dose. The number of deaths was counted at 48 h after treatment. LD~50~ values and the corresponding confidence limits (CL, 95%) were determined by the Litchfield and Wilcoxon method (PHARM/PCS Version 4). Doses of 10, 40 (50), 70 and 100 % MNFD were chosen for most tests. Lower doses were found to be effective in the writhing test.

Antinociceptive study
---------------------

### Hot-plate test

The hot-plate test was assessed on groups of 8 mice. The temperature of a metal surface was maintained at 55 *±* 0.2°C. Latency to a discomfort reaction (licking paws or jumping) was determined before and after drug administration. The cut-off time was 20 s. The latency was recorded before and 30, 60, 120, 150 and 240 min following intraperitoneal administration of the agents. The prolongation of the latency times compared with the values of the control was used for statistical comparison. Control received normal saline (10 ml/kg, i.p.) and morphine (10 mg/kg, i.p.) was used as reference drug \[[@B25]\].

### Writhing test

Groups of 8 mice were used for controls and test mice. One hour after the administration of the extract, the mice were given an intraperitoneal injection of 0.7% v/v acetic acid solution (volume of injection 0.1 ml/10 g). The mice were placed individually into glass beakers and five min were allowed to elapse. The number of writhes produced in these animals was counted for 30 min. For scoring purposes, a writhe is indicated by stretching of the abdomen with simultaneous stretching of at least one hind limb. Control received normal saline (10 ml/kg, i.p.), diclofenac (10 mg/kg, i.p.) and morphine (10 mg/kg, i.p.) were used as reference drugs. Naloxone (2 mg/kg, s.c.) was administered 15 min prior to the extracts or morphine injections \[[@B25]\].

Anti-inflammatory study
-----------------------

### Xylene-induced ear edema

Mice were divided into groups of seven. Thirty minutes after i.p. injection of the extract, diclofenac and dexamethasone, 0.03 ml of xylene was applied to the anterior and posterior surfaces of the right ear. The left ear was considered as control. Two hours after xylene application, mice were killed and both ears were removed. Circular sections were taken, using a cork borer with a diameter of 7 mm, and weighed. The increase in weight caused by the irritant was measured by subtracting the weight of the untreated left ear section from that of the treated right ear sections. The formula for computing percent inhibition was: average writhes in the control group (normal saline) minus writhes in the drug group divided by writhes in the control group times 100%. Control received normal saline (10 ml/kg, i.p.), diclofenac (10 mg/kg, i.p.) and dexamethasone (15 mg/kg, i.p.) were used as reference drugs \[[@B25]\].

Formalin induced inflammation
-----------------------------

Rats were divided into groups of six. The inflammation was produced by subaponeurotic injection of 0.1 ml of 2% formaldehyde in the right hind paw of the rats on the first and third day. The animals were treated daily with the extracts or diclofenac intraperitoneally for 10 days. The daily changes in paw size were measured by wrapping a piece of cotton thread round the paw and measuring the circumference with a meter rule \[[@B26]\].

Materials
=========

The following reagents were used: morphine and dexamethasone (Dam Pakhsh, I.R. Iran), naloxone hydrochloride (Tolid Daru, I.R. Iran), diclofenac (Zahrawi, I.R. Iran), acetic acid, xylene and formaldehyde (Merck).

Statistical analysis
--------------------

The data were expressed as mean values ± S.E.M. and tested with analysis of variance followed by the multiple comparison test of Tukey-Kramer.

Acknowledgments
===============

The authors are thankful to T.W. Stone .Professor of Pharmacology, Division of Neuroscience and Biomedical Systems, University of Glasgow, Scotland, for his linguistic help.


Background
==========

Differentiation is commonly associated with intermingled changes in gene expression and cellular proliferation. In some differentiating cell types, changes in both gene transcription and proliferation are regulated by the same transcription factor \[[@B1]-[@B9]\]. CCAAT/Enhancer Binding Protein alpha (C/EBPα) is a transcription factor that is required for the differentiation of a number of tissues \[[@B10]-[@B18]\]. Mice homozygous for C/EBPα null alleles have severe defects in tissues involved in metabolic homeostasis \[[@B19]-[@B21]\]. Cellular proliferation is elevated in the liver of these knockout mice \[[@B22]\] suggesting that C/EBPα blocks proliferation *in vivo.* In cultured cells, C/EBPα expression leads to decreased colony formation upon antibiotic selection \[[@B23]-[@B25]\], decreased DNA synthesis \[[@B22],[@B24]-[@B27]\] and an enhanced proportion of cells in the G1 phase of the cell cycle \[[@B26]\]. Thus, C/EBPα regulates cellular proliferation, as well as gene transcription.

C/EBPα contains a bZIP domain conserved at the carboxy terminus of a number of transcription factors \[[@B12],[@B28]\]. The bZIP domain consists of a basic region that binds directly to DNA, followed immediately by a leucine zipper. C/EBPα dimerizes via the leucine zipper. This dimerization is required for DNA binding \[[@B28],[@B29]\]. At least three transcription activation functions have been described in the more amino terminal regions of C/EBPα \[[@B30]-[@B32]\]. C/EBPα domains and activities associated with proliferation arrest also have been identified, but vary considerably between studies \[[@B23],[@B25],[@B33]-[@B36]\].

C/EBPα binds to and activates transcription of the gene promoter for the p21 inhibitor of cyclin-dependent kinase (CDK) \[[@B37]\]. This led to speculation that p21 gene activation may contribute to cell cycle arrest by C/EBPα \[[@B37]\]. Similarly, suppression of mitotic growth during adipocyte differentiation was associated with C/EBPα activation of the promoters of gadd45 (growth arrest and DNA damage-inducible gene 45), gas2 and gas3 (growth arrest-associated genes 2 and 3) \[[@B5],[@B6],[@B38],[@B39]\]. However, C/EBPα mutants defective in DNA binding still blocked proliferation \[[@B25],[@B33]\]. This suggested that direct promoter activation was unnecessary, or redundant, for C/EBPα proliferation arrest \[[@B18]\].

Possible mechanisms of transcription-independent proliferation arrest by C/EBPα have been suggested by a number of studies. Decreased proliferation was associated with C/EBPα stabilization of the p21 protein \[[@B22],[@B24]\]. p21 interacted directly with a large internal segment of C/EBPα that included transcription activation domain 3 \[[@B25]\] (see Fig. [1A](#F1){ref-type="fig"}). CDK2 and CDK4 also interacted with segments of C/EBPα close to, and within, transcription activation domain 3 \[[@B36]\]. CDK2 also interacted with the basic region of the C/EBPα \[[@B25]\]. p21 also has a second interaction site, within the leucine zipper of C/EBPα \[[@B25]\]. *in vitro,* C/EBPα enhanced p21 inhibition of CDK2 activity. C/EBPα inhibition of CDK2 activity correlated with p21 binding to C/EBPα transcription activation domain 3 \[[@B25]\]. However, proliferation arrest by C/EBPα still occurred in cell lines not containing p21 genes \[[@B40]\]. This indicated that proliferation arrest by C/EBPα did not rely solely upon C/EBPα enhancement of CDK inhibition by p21.

![**A,** Positions of the transcription activation (TA), DNA binding (basic) and dimerization (ZIP) domains along the linear sequence of C/EBPα. The numbers below the C/EBPα diagram indicate the amino acid positions at the boundaries of the domains. Also shown are C/EBPα sequences involved in known actions with the indicated cell-cycle proteins: p107 \[[@B34]\], p21 \[[@B25]\], CDK2 \[[@B25],[@B36]\], CDK4 \[[@B36]\] and E2F \[[@B33]\]. **B,** Schematic of the GFP fusions with full-length and mutant C/EBPα used in the studies reported here.](1471-2121-3-6-1){#F1}

Another mechanism reported for proliferation blockage by C/EBPα involves the E2F-DP1 transcription complexes. E2F complexes activate genes required for entry into S phase. C/EBPα binds to and inhibits transcriptional activation by E2F \[[@B26],[@B33]\]. The complex of E2F with the retinoblastoma-related p107 protein is prevalent in cycling cells. Transcription activation domain 2 of C/EBPα was observed to interact specifically with p107 to disrupt p107/E2F complex formation \[[@B34],[@B35]\]. Disruption of E2F activity also was associated the basic domain of C/EBPα \[[@B33],[@B34]\].

Thus, a variety of interactions and mechanisms are potentially involved in proliferation arrest by C/EBPα. This variability may indicate divergent mechanisms for the block of proliferation by C/EBPα in different study conditions and/or cell types. In pituitary cells, C/EBPα is part of a multi-subunit complex \[[@B41],[@B42]\] that activates the growth hormone \[[@B42]\] and prolactin \[[@B43],[@B44]\] promoters. Full-length C/EBPα is absent from the pituitary progenitor GHFT1-5 cell line \[[@B42],[@B45]\] in which ectopically expressed C/EBPα activates co-transfected growth hormone and prolactin promoters \[[@B42],[@B44],[@B45]\]. We show here that ectopically expressed C/EBPα prolongs GHFT1-5 cells in the G1 and S phases of the cell cycle in a transcription-independent fashion. Different C/EBPα domains were required for G1- and S-phase arrest. C/EBPα domains previously described for the interaction with p21 were not required for arrest in GHFT1-5 cells. Rather, domains previously implicated in the regulation of the E2F and CDK2 pathways by C/EBPα were required for arrest of these progenitor cells.

Results
=======

Transcriptionally active and inactive GFP fusions with C/EBPα
-------------------------------------------------------------

The domains of C/EBPα and their known interactions with cell-cycle proteins are summarized in figure [1A](#F1){ref-type="fig"}. We conducted studies to determine which, if any, of those activities affect the proliferation of GHFT1-5 pituitary progenitor cells. In prior studies, we noticed that fewer cells were present in GHFT1-5 cell cultures transfected with the C/EBPα expression vector than in cultures transfected with control vectors (X. Wang, F. S., unpublished data). The decreased cell number might be due to impaired proliferation of GHFT1-5 cells expressing C/EBPα. To examine if C/EBPα affected the progression of GHFT1-5 cells through the cell cycle, we needed to distinguish transfected cells that expressed C/EBPα from those that did not. We fused the cDNA for the green fluorescence protein (GFP) to either the 5\' (GFP-C/EBPα) or the 3\' (C/EBPα-GFP) end of the coding sequence of the C/EBPα cDNA. The cDNAs for the GFP-C/EBPα and C/EBPα-GFP fusion proteins were inserted into an expression vector and transfected into GHFT1-5 cells. Flow cytometry was used to specifically identify green fluorescent, C/EBPα-expressing cells.

We initially characterized the abilities of the C/EBPα-GFP and GFP-C/EBPα fusion proteins to bind DNA and activate transcription in GHFT1-5 cells (Fig. [2](#F2){ref-type="fig"}). The C/EBPα-GFP or GFP-C/EBPα expression vectors were transfected into GHFT1-5 cells with a promoter consisting of a single C/EBPα binding site upstream of the growth hormone TATA box. This minimal promoter was specifically responsive to C/EBPα expression in GHFT1-5 cells \[[@B45],[@B46]\]. Cells transfected with the C/EBPα-GFP expression vector showed a statistically significant (p\<0.05, n = 5) 9.66 +/- 6.08-fold higher promoter activity than did cells sham-transfected with the same expression vector deleted of the C/EBPα cDNA (Fig. [2A](#F2){ref-type="fig"}). In contrast, activation by GFP-C/EBPα was a statistically insignificant 1.88 +/- 1.48-fold (Fig. [2A](#F2){ref-type="fig"}). Promoter activation by C/EBPα-GFP was reproducibly less than promoter activation by unfused C/EBPα. Western blots of nuclear extracts from the transfected cells showed that unfused C/EBPα was expressed at marginally higher levels (Fig. [2B](#F2){ref-type="fig"}).

![**A,** Chloramphenicol acetyltransferase activity expressed from a promoter transiently transfected into GHFT1-5 cells together with vectors expressing C/EBPα, fused or not, with GFP. The promoter contained a single C/EBPα binding site upstream of a TATA box \[[@B45]\]. CAT activities were normalized to the activity present in cells transfected with the expression vector not containing the C/EBPα cDNA (\"Sham\") and plotted as the mean +/- standard deviation from five independent experiments. **B,** Nuclear extracts from sham-transfected cells and cells expressing C/EBPα, C/EBPα-GFP and GFP-C/EBPα were separated by SDS-polyacrylamide gel electrophoresis. The separated proteins were transferred to a membrane and stained with an antibody directed against the FLAG epitope, which was appended to the amino terminus of C/EBPα in all the constructs. Arrow, expressed C/EBPα-GFP and GFP-C/EBPα. **C,** Whole cell extracts, from cells transfected with C/EBPα-GFP and GFP-C/EBPα or GFP-C/EBPαΔLZ expression vectors, were incubated with a radiolabeled oligonucleotide containing a high affinity consensus C/EBPα binding site. The observed complexes were competed with a 1, 10 and 100 fold molar excess of unlabeled oligonucleotide or were supershifted with an antibody directed against C/EBPα.](1471-2121-3-6-2){#F2}

C/EBPα-GFP and GFP-C/EBPα both were expressed as intact, full-length proteins of the appropriate molecular weight \[[@B45]\](Fig. [2B](#F2){ref-type="fig"}, arrow). In addition, both C/EBPα-GFP and GFP-C/EBPα bound similarly to DNA (Fig. [2C](#F2){ref-type="fig"}). Extracts of cells expressing either C/EBPα-GFP or GFP-C/EBPα shifted the electrophoretic mobility of a radiolabeled oligonucleotide containing a C/EBPα binding site (Fig. [2C](#F2){ref-type="fig"}). No shift was observed with sham-transfected cells (not shown). The shifted bands were specifically competed by a 100-fold molar excess of the cold oligonucleotide and were supershifted with an antibody directed against C/EBPα. GFP-C/EBPα truncated of its leucine zipper (Fig. [1B](#F1){ref-type="fig"}) was ineffective at shifting the binding site (Fig. [2C](#F2){ref-type="fig"}, ΔLZ). Flow cytometry (discussed later) showed that comparable levels of green fluorescence were emitted from cells expressing C/EBPα-GFP, GFP-C/EBPα and ΔLZ. Thus, C/EBPα-GFP and GFP-C/EBPα were expressed and could bind to C/EBPα binding sites in DNA, but differed in their ability to activate gene transcription.

Cell cycle distribution of GHFT1-5 cells
----------------------------------------

Transcriptionally active C/EBPα-GFP and transcriptionally inactive GFP-C/EBPα were compared for their effects on proliferation of GHFT1-5 cells. We first determined the distribution of untransfected GHFT1-5 cells in each phase of the cell cycle. GHFT1-5 cells were grown to subconfluence, collected and the DNA within the cells was stained with propidium iodide. The DNA content within each cell was quantified by flow cytometry as the amount of orange fluorescence from the propidium iodide-stained DNA (see Materials and Methods for details). The fluorescence intensity measured from each cell falls into one of three populations: cells centered around the lowest level of orange fluorescence, cells centered around the highest level of orange fluorescence which is twice that of the lowest, and cells with intermediate levels of orange fluorescence. This corresponds, respectively, to cells containing a 2n DNA complement prior to duplication of the genome (in G1 phase), cells containing a 4n DNA complement following genome duplication (in growth phase 2 or in mitosis, G2/M), and cells containing a partially replicated genome intermediate between 2n and 4n (in S phase). As averaged from nine independent experiments, the proportion of growing GHFT1-5 cells in G1, S and G2/M corresponded to 42%, 38% and 20%, respectively (Fig [3](#F3){ref-type="fig"}, white bars).

![GHFT1-5 cells were grown to subconfluence and treated for 20 hours with DMSO (white bars), 100 ng/ml nocodazole (gray bars) or 0.5 mM mimosine (speckled gray bars), both in DMSO. The amount of DNA within each cell was measured by flow cytometric quantification of the orange fluorescence emitted from propidium iodide-stained DNA. The proportion of GHFT1-5 cells containing a 2n complement of DNA (G1), a 4n complement of DNA (G2/M), and an intermediate amount of DNA (S) was determined for each treatment and presented as the mean +/- standard deviation from nine independent experiments.](1471-2121-3-6-3){#F3}

We next synchronized GHFT1-5 cells in particular stages of the cell cycle. GHFT1-5 cells were treated for 20 hours with nocodazole, which prevents microtubule polymerization/depolymerization and exit from M phase \[[@B47]\]. The delivery vehicle, DMSO, was added to parallel cell cultures. Cells also were treated with mimosine (in DMSO), which blocks cells in late G1 immediately before entry into S phase \[[@B48]\]. Mimosine also prolongs some cell types in the S phase itself \[[@B48]\]. GHFT1-5 cells grow with an average doubling time of approximately 18 to 24 hours. 20 hours of treatment allowed most cells to pass through one cell cycle and to accumulate as 4n cells upon nocodazole treatment (Fig. [3](#F3){ref-type="fig"}, light gray bars) or as 2n cells at the G1/S boundary upon mimosine treatment (Fig. [3](#F3){ref-type="fig"}, stippled gray bars). Thus, GHFT1-5 cells can be synchronized at particular stages of the cell cycle. Synchronization facilitated characterization of the cell cycle stages blocked or prolonged by C/EBPα expression.

Transcriptionally active and inactive C/EBPα both retain GHFT1-5 cells in the G1 and S phases of the cell cycle
---------------------------------------------------------------------------------------------------------------

We determined if either C/EBPα-GFP or GFP-C/EBPα altered the distribution of GHFT1-5 cells in the G1, S or G2/M phases of the cell cycle. GHFT1-5 cells were transfected with the expression vectors for C/EBPα-GFP or GFP-C/EBPα, or with the sham expression vector not containing the C/EBPα cDNA. The cells were incubated for one day to allow time for C/EBPα expression. Cells were then synchronized in M-phase or in G1/S by 20-hour incubations with nocodazole (Fig. [4A](#F4){ref-type="fig"}) or mimosine (Fig. [4B](#F4){ref-type="fig"}). Cells were collected and stained with propidium iodide. Flow cytometry was used to measure DNA content in 1) cells transfected with the sham expression vector (Fig. [4](#F4){ref-type="fig"}, Sham), 2) green fluorescent cells expressing GFP-tagged C/EBPα (Fig. [4](#F4){ref-type="fig"}, C/EBPα-GFP or GFP-C/EBPα) or 3) the subpopulation of cells from the C/EBPα-GFP or GFP-C/EBPα transfections that did not express measurable amounts of green fluorescent C/EBPα (Fig. [4](#F4){ref-type="fig"}, No C/EBP). The \"No C/EBP\" cells represent an internal control for cells not expressing C/EBPα collected simultaneously with cells expressing C/EBPα. The No C/EBP controls also indicated the extent to which green fluorescent, C/EBPα-expressing cells were distinguished from non-expressing cells by flow cytometry. For all our experiments, the distribution of cells in the G1, S and G2/M phases was similar for the \"No C/EBPα \" and sham-transfected cells.

![Cells were transfected with the control expression vector (Sham), the transcriptionally active C/EBPα-GFP expression vector or the transcriptionally inactive GFP-C/EBPα expression vector and treated one day later with **A,** nocodazole or **B,** mimosine. Cells from the transfections with the C/EBP-GFP and GFP-C/EBP expression vectors were separated into cells with green fluorescence above background levels (C/EBP-GFP and GFP-C/EBP) or at background (No C/EBP). The proportion of cells in the G1, S and G2/M phases were plotted as the mean +/- standard deviation from A, six or B, three independent experiments. Statistically significant differences in the proportion of cells in G1, S or G2/M, relative to the proportions determined for the sham-transfected cells, are indicated (one-way ANOVA: ^\*\*^, p \< 0.01; ^\*^p \< 0.05; -, no difference).](1471-2121-3-6-4){#F4}

The expression of C/EBPα-GFP or GFP-C/EBPα resulted in a statistically significant decrease in the amount of cells blocked in G2/M upon nocodazole treatment compared to sham-transfected cells (Fig. [4A](#F4){ref-type="fig"}) (p \<\< 0.01, n = 6). This indicated that C/EBPα expression prevented a significant proportion of GHFT1-5 cells from reaching G2/M to be blocked by nocodazole. When cells were blocked in G1 by mimosine treatment, C/EBPα expression caused no decrease in the proportion of cells in G2/M (Fig. [4B](#F4){ref-type="fig"}). This demonstrated that C/EBPα expression did not actively decrease the proportion of cells in G2/M.

The C/EBPα-induced reduction in cells blocked in G2/M was associated with a highly statistically significant increase in the proportion of GHFT1-5 pituitary progenitor cells in G1 (p \< 0.001) and a statistically significant increase in the proportion of cells in S phase (p \< 0.03) (Fig. [4A](#F4){ref-type="fig"}). This retention of GHFT1-5 cells in the G1 and S phases of the cell cycle occurred for both the transcriptionally active C/EBPα-GFP and inactive GFP-C/EBPα fusions. The independence of C/EBPα-induced proliferation arrest from C/EBPα-regulated transcriptional activation also has been observed in other studies \[[@B25],[@B26]\]. Thus, simple transcription activation mechanisms do not account for the effect of C/EBPα on the cell cycle.

Targeting of C/EBPα to the presumed centromeric G1/S checkpoint is insufficient for prolongation of G1
------------------------------------------------------------------------------------------------------

Our prior fluorescence microscopy studies showed that C/EBPα-GFP, GFP-C/EBPα, and antibody-stained C/EBPα \[[@B45],[@B46]\], concentrated at specific intranuclear domains in GHFT1-5 cells (see Fig. [5A](#F5){ref-type="fig"}, GFP-C/EBPα). These domains coincide with regions that stain with the blue fluorescent DNA-binding dye Hoechst 33342 (Fig. [5A](#F5){ref-type="fig"}, Hoechst 33342). Hoechst 33342 stains AT-rich DNA that concentrates around the centromeres \[[@B45],[@B49]\]. Given the role of the centromere as a checkpoint for regulation of the mitotic phase of the cell cycle \[[@B50],[@B51]\], it was hypothesized that C/EBPα localization around the centromere could play a role in C/EBPα regulation of the cell cycle \[[@B49]\].

![**A,** GFP-C/EBP or, **B,** GFP-C/EBP deleted of amino acids 310 to 358 (ΔLZ) were expressed in GHFT1-5 cells. The ΔLZ construct is depicted in figure [1B](#F1){ref-type="fig"}. The transfected cells were counter-stained with the blue fluorescent, DNA-binding dye Hoechst 33342 immediately before analysis by fluorescence microscopy. Green fluorescent (left panels) and blue fluorescent (right panels) images were collected from a single cell using filter sets that selectively discriminate GFP and Hoechst 33342 fluorescence (see Materials and Methods). The subnuclear position of GFP-C/EBPα and ΔLZ were compared to the subnuclear position of the peri-centromeric chromatin stained by Hoechst 33342. Representative images are shown.](1471-2121-3-6-5){#F5}

To investigate if targeting of C/EBPα to the peri-centromeric chromatin is necessary or sufficient to cause G1 and S phase prolongation in GHFT1-5 cells, we first determined the domain of C/EBPα required to target C/EBPα to the peri-centromeric chromatin. It was previously suggested that the peri-centromeric targeting of C/EBPα was a function of DNA binding: C/EBPα binding sites are concentrated within the repeated DNA sequences that comprise the bulk of peri-centromeric chromatin \[[@B49]\]. Indeed, C/EBPα truncated to contain little more than the DNA binding, bZIP domain still concentrated at peri-centromeric chromatin \[[@B45]\]. By contrast, C/EBPα deleted of amino acids 310 to 358 (Fig. [1B](#F1){ref-type="fig"}, ΔLZ) no longer targeted to the peri-centromeric DNA (Fig. [5B](#F5){ref-type="fig"}). This deletion disrupts DNA binding (Fig. [2C](#F2){ref-type="fig"}) by eliminating the leucine zipper required for DNA binding by all members of the bZIP family of transcription factors \[[@B52]\]. Thus, as predicted \[[@B49]\], the bZIP domain is necessary and sufficient for C/EBPα targeting to peri-centromeric chromatin.

When expressed in GHFT1-5 cells, ΔLZ was as effective as full-length GFP-C/EBPα in prolonging both G1 and S (Fig. [6A](#F6){ref-type="fig"}). Thus, leucine zipper dimerization, site-specific DNA binding and targeting of C/EBPα to peri-centromeric were not required for C/EBPα prolongation in G1 and S. In contrast, the isolated C/EBPα bZIP DNA binding domain (Fig. [1B](#F1){ref-type="fig"}, DBD), which still targeted to peri-centromeric chromatin, did not prolong G1 (Fig. [6B](#F6){ref-type="fig"}). S phase remained prolonged upon DBD expression. The different effects of the isolated DBD on G1 and S blockage indicated that C/EBPα regulation of G1 and S phase arrest was mechanistically distinct. Thus, C/EBPα regulates proliferation by two distinct pathways. Both pathways do not depend upon site-specific DNA binding, which is commonly considered a prerequisite for gene-specific transcription.

![GHFT1-5 cells were transfected with the **A,** Sham, GFP-C/EBP and ΔLZ expression vectors or **B,** Sham, C/EBP-GFP and DBD expression vectors and treated with nocodazole. The proportions of cells in G1, S and M phase were determined and plotted as the mean +/- standard deviation from six independent experiments for both A and B. No C/EBP, the subpopulation of cells with background levels of green fluorescence (i.e. did not express GFP-linked C/EBPα). Data from No C/EBP cells are shown adjacent to the green fluorescent cells for each expression construct. Statistically significant differences in the proportion of green fluorescent cells in G1, S or G2/M, relative to the proportions determined for the sham-transfected cells, are indicated (One-way ANOVA: ^\*\*^, p \< 0.01; ^\*^p \< 0.05; -, no difference).](1471-2121-3-6-6){#F6}

Prolongation in G1 requires the amino terminal domain of C/EBPα
---------------------------------------------------------------

The C/EBPα DBD therefore was insufficient for prolongation of G1 (Fig. [6B](#F6){ref-type="fig"}). We next determined which additional domains of C/EBPα were required for G1 prolongation. C/EBPα amino acids 1--154 or 154--257 were appended to the DBD (see Fig. [1B](#F1){ref-type="fig"}). The addition of amino acids 1--154 (Fig. [7](#F7){ref-type="fig"}, DBD + 1--154) caused a statistically significant increase, relative to sham-transfected cells, in the proportion of cells in G1 (p \<\< 0.01, n = 6). The proportion of cells in G1 was not statistically different in cells expressing DBD + 1--154 from that in cells expressing full-length C/EBPα-GFP. Adding amino acids 1--125, instead of amino acids 1--154, caused a similar prolongation in G1 (Fig. [7](#F7){ref-type="fig"}, DBD + 1--125). Thus, an element in the amino terminal domain of C/EBPα was required for G1 prolongation.

![The indicated amino acids of C/EBPα were appended to DBD-GFP (see Fig. [1B](#F1){ref-type="fig"}) and expressed in GHFT1-5 cells. The proportions of green fluorescent cells in G1, S and M phase in nocodazole-treated cells were determined and plotted as the mean +/- standard deviation from three independent experiments. The DBD + 1--154 mutant was repeated a total six times (not shown), for which the statistical significance remained p \< 0.01. Statistically significant increases in the proportion of G1 cells, compared to Sham cells, are indicated (One-way ANOVA: ^\*\*^, p \< 0.01; ^\*^p \< 0.05; -, no difference).](1471-2121-3-6-7){#F7}

In contrast, addition of amino acids 154--257 to C/EBPα DBD did not rescue G1 prolongation (Fig. [7](#F7){ref-type="fig"}, DBD + 154--257). Again, appending amino acids 1--125 to this mutant restored G1 prolongation (A126--153), as did amino acids 1--97 (A 98--153). All of the mutants contained the DBD and prolonged the cell cycle in S phase (Fig. [7](#F7){ref-type="fig"}). Thus, the DNA binding, bZIP domain was sufficient to direct S phase regulation by C/EBPα whereas amino acids 1--97 contained a domain required for G1 regulation.

Discussion
==========

C/EBPα prolongs GHFT1-5 cells in G1 and S
-----------------------------------------

The differentiative and anti-mitotic actions of C/EBPα have been observed in a variety of cell culture models \[[@B23],[@B24],[@B26],[@B40],[@B53]\]. To date, studies of the C/EBPα block of proliferation have relied on comparing the growth \[[@B23]-[@B25],[@B40]\] or DNA synthetic \[[@B22],[@B24]-[@B27]\] rates of C/EBPα-expressing and non-expressing cells. These studies suggest that multiple, possibly independent, mechanisms may contribute to C/EBPα arrest in a variety of cells \[[@B18],[@B23]-[@B26],[@B33]-[@B37],[@B40],[@B54]-[@B56]\]. Studies that distinguish the anti-proliferative actions of C/EBPα at different phases of the cell cycle, and in specific cell types, may clarify these discrepancies.

Our studies showed that C/EBPα expression prolonged the proliferation of GHFT1-5 pituitary progenitor cells at two different phases of the cell cycle, G1 and S (Fig. [4](#F4){ref-type="fig"}). A G1 block has been reported previously in C/EBPα-expressing mouse L cells \[[@B26]\]. However, those studies did not define the activities within C/EBPα required for that blockage. Here, we observed that prolongation in G1 required the amino terminal 97 amino acids of C/EBPα (Fig. [7](#F7){ref-type="fig"}.). In contrast, amino acids 1 to 97 were not required for S phase arrest (Figs. [6](#F6){ref-type="fig"}, [7](#F7){ref-type="fig"}). This showed that C/EBPα prolongation of GHFT1-5 cells in the G1 and S phases occurred through molecularly distinct pathways. Such distinct effects on G1 and S may contribute to the wide variations in mechanisms reported, to date, to participate in proliferation arrest by C/EBPα.

Cell Cycle Proteins and C/EBPα Inhibition of Proliferation
----------------------------------------------------------

Candidate mechanisms for the block of proliferation by C/EBPα include 1) activation of the gene promoters of the cyclin-dependent kinase inhibitor p21 \[[@B37]\], gadd45 \[[@B5],[@B6]\], gas2 and gas3 \[[@B38],[@B39]\], 2) post-transcriptional stabilization of p21 \[[@B22]\], or 3) interactions of C/EBPα with cell cycle proteins including p21 \[[@B22],[@B24],[@B25],[@B55]\], CDK2 \[[@B25],[@B36]\], CDK4 \[[@B36]\], retinoblastoma \[[@B54]\], the retinoblastoma-related protein p107 \[[@B34]\] and the G1/S-regulating E2F transcription complex \[[@B26],[@B33]\] (Fig. [1A](#F1){ref-type="fig"}). We found that transcriptional activation domain 3 and the leucine zipper were not required for prolongation of either G1 or S (Fig [7](#F7){ref-type="fig"}). These domains interact with p21, CDK2 and CDK4 \[[@B22],[@B24],[@B25],[@B36],[@B55]\]. Thus, C/EBPα interactions with at least p21 and CDK4 were not required for the anti-proliferative actions of C/EBPα in GHFT1-5 cells. CDK2, and other proteins, also interact with another domain of C/EBPα \[[@B25]\] required for proliferation arrest (discussed below).

Arrest in S phase was associated with amino acids 259 to 310 (Fig. [6](#F6){ref-type="fig"}), which include the basic amino acid-rich, DNA binding region of C/EBPα. C/EBPα deleted of the leucine zipper dimerization domain, also required for DNA binding \[[@B25]\] (Fig. [2C](#F2){ref-type="fig"}), was as effective as full-length GFP-C/EBPα in prolonging both S and G1 (Fig. [6A](#F6){ref-type="fig"}). Thus, DNA binding *per se* was not required for proliferation arrest. The basic region of C/EBPα blocks the transcriptional activity of E2F by unknown mechanisms \[[@B33]\] and contains another known CDK2-interaction site \[[@B25]\]. Either of these activities may contribute to basic region-dependent, S phase arrest of GHFT1-5 cell proliferation.

G1 block in GHFT1-5 cells required C/EBPα amino acids 1--97 (Fig. [7](#F7){ref-type="fig"}), comprising activation domains 1 and 2 \[[@B31]\]. Amino acids 1 to 97 have been implicated in C/EBPα growth arrest via the E2F/pl07 complexes \[[@B34],[@B35]\]. C/EBPα interaction with p107 is believed to prevent the formation of an S-phase promoting complex containing p107 and E2F \[[@B26],[@B34],[@B35]\]. Thus, G1 block in GHFT1-5 cells may be related to the ability of C/EBPα to interact with p107. However, the amino terminus of C/EBPα also functionally interacts with a number of other factors \[[@B31],[@B45],[@B46]\], any of which might contribute to proliferation arrest.

Subnuclear Architecture and C/EBPα Inhibition of Proliferation
--------------------------------------------------------------

Tang and Lane \[[@B49]\] showed that C/EBPα becomes associated with peri-centromeric chromatin upon expression during adipocyte differentiation. We observed that C/EBPα, expressed in mouse pituitary progenitor GHFT1-5 cells, also localized to peri-centromeric chromatin \[[@B45]\]. Here we show that targeting of C/EBPα to peri-centromeric chromatin in GHFT1-5 cells required the DNA binding domain of C/EBPα (Fig. [5](#F5){ref-type="fig"}). However, C/EBPα deleted of the DNA binding domain was as effective as full-length C/EBPα in prolonging GHFT1-5 cells in the G1 and S phases of the cell cycle (Fig. [6A](#F6){ref-type="fig"}). Thus, the specific association of C/EBPα with peri-centromeric chromatin, speculated to contribute to C/EBPα growth arrest \[[@B49]\], was not necessary for disruption of the cell cycle by C/EBPα. The independence of C/EBPα regulation of proliferation from its intranuclear localization may even be necessary since we have recently determined that pituitary cell differentiation is associated with the dispersal of C/EBPα from peri-centromeric heterochromatin by the pituitary-specific transcription factor Pit-1 (J. F. E., M. A. Kawecki, F. S., R. N. D., submitted).

Transcription-Independent Inhibition of Proliferation
-----------------------------------------------------

Transcription factors, like C/EBPα, may regulate cell proliferation by directly controlling the expression of proteins required for cell cycle progression. Indeed, C/EBPα activates transcription of the p21 \[[@B37]\], gadd45 \[[@B5],[@B6]\], gas2 and gas3 \[[@B38],[@B39]\] genes. However, similar levels of p21 mRNA in the livers of wild-type and homozygous C/EBPα knock-out mice \[[@B22]\] argue against a physiologically significant contribution of C/EBPα to p21 gene transcription in the liver. We also observed that transcriptionally inactive and DNA-binding-defective forms of C/EBPα still blocked GHFT1-5 cell proliferation in both the G1 and S phases of the cell cycle (Figs. [2](#F2){ref-type="fig"}, [4](#F4){ref-type="fig"},[6](#F6){ref-type="fig"}). DNA-binding-defective mutants of C/EBPα have also been observed to block proliferation in other cell lines \[[@B25],[@B33]\]. Together, this suggests that C/EBPα regulation of transcription, in general, might not contribute to the regulation of cell proliferation. It remains possible that C/EBPα activation of the transcription of some cell cycle regulatory genes does control proliferation in some cell types or under certain conditions \[[@B8],[@B37]-[@B39],[@B56]\].

The absence of a dependence of GHFT1-5 cell proliferation on transcription factor activities that are more classically associated with transcription regulation strongly suggests that C/EBPα, and perhaps other transcription factors, control proliferation and transcription through completely separable pathways. The effects of human papilloma virus E7 proteins on C/EBPα-induced differentiation and growth arrest also suggest that the effects of C/EBPα on proliferation are mechanistically divergent \[[@B40]\]. Having distinct mechanisms for regulation of gene transcription and proliferation allows a transcription factor to regulate these two critical processes completely independently of one another. This may be particularly important in differentiated, non-proliferating cells in which events that regulate a transcription factor\'s contribution to gene expression must be disconnected from that transcription factor\'s continuing blockage of proliferation.

Conclusions
===========

C/EBPα is a transcription factor that controls both proliferation and gene expression. C/EBPα uses molecularly distinct mechanisms to prolong the G1 and S phases of pituitary progenitor GHFT1-5 cells. The anti-proliferative effects of C/EBPα do not require activities in C/EBPα commonly required for gene-specific transcription. Proliferation arrest instead corresponds with C/EBPα domains previously implicated in post-transcriptional effects on E2F and/or CDK2 activity.

Materials and Methods
=====================

Plasmid construction
--------------------

The GFP-C/EBPα vector was constructed by inserting the cDNA encoding the S65T derivative of GFP \[[@B57]\] at the start codon of the cDNA encoding rat C/EBPα. The fused cDNA was cloned into a previously described expression vector \[[@B58]\]. The GFP and C/EBPα portions of the fusion were separated by a 16 amino acid long linker including the epitope for the FLAG antibody. The C/EBPα-GFP vector was constructed by inserting the S65T GFP into the NcoI site present near the carboxy terminus of rat C/EBPα (also tagged at the amino terminus with the FLAG epitope) in our previously described C/EBPα expression vector \[[@B42]\]. This resulted in a deletion of the last four amino acids of C/EBPα. The DBD and DBD + 154--257 derivatives of C/EBPα were constructed by replacing, respectively, the amino terminal 257 amino acids of C/EBPα-GFP (to the SgrAI site of C/EBPα) and the amino terminal 153 amino acids of C/EBPα-GFP (to the NotI site of C/EBPα) with oligonucleotides containing a strong Kozak sequence. DBD + 1--154 was constructed by replacing amino acids 155 to 257 (from the NotI to SgrAI sites) with an oligonucleotide. The remaining constructs (Fig. [7](#F7){ref-type="fig"}) were generated by appending C/EBPα fragments to the NotI site of DBD + 1--154, or to the SgrAI site of DBD. ΔLZ was constructed by deleting amino acids from amino acid 310 (Tth111II site) at the junction of the basic region and leucine zipper of C/EBPα to the carboxy terminus of the GFP-C/EBPα fusion. The C/EBP-TATA reporter used to determine the transcriptional activity of the two GFP fusion proteins was previously described \[[@B45]\].

Transfection
------------

GHFT1-5 cells were propagated at 37°C and 5% CO~2~ in Dulbecco\'s modified Eagle (DME-H21) medium supplemented with 10% fetal calf serum. Approximately 1 × 10^7^ cells were transfected by electroporation, as previously described \[[@B42]\], with 5 μg of either the C/EBPα-GFP, GFP-C/EBPα, or control expression vectors. Following transfection, cells were propagated at 33°C. Incubation at 33°C results in significantly higher levels of green fluorescence (unpublished data), presumably because of better folding of the jellyfish GFP. Transfected cells were maintained in the dark throughout the experiment to minimize fluorescence activation of GFP prior to flow cytometry.

DNA Binding and Promoter Activation
-----------------------------------

Electrophoretic mobility shifts assays were done by mixing a radiolabeled oligonucleotide containing a consensus, high affinity C/EBP binding site (GATCGAGCCCCATTGCGCAATCATAGATC) together with extracts prepared from transfected cells as previously described \[[@B58],[@B59]\]. Competition with the same, unlabeled oligonucleotide in 1, 10 or 100 molar excess of the radiolabeled probed indicated specific binding. The protein DNA complex was supershifted with an antibody directed against C/EBPα (Santa Cruz Biotechnology, sc-61). For studies of the transcriptional activation properties of the fusion proteins, 1 μg of the C/EBP-TATA vector \[[@B45]\] was co-transfected and transcriptional activity was assessed by measuring the amount of chloramphenicol acetyltransferase reporter expressed with and without C/EBPα expression \[[@B45]\].

Fluorescence Microscopy
-----------------------

Following transfection, most cells were plated into 14 cm dishes and allowed to grow for 24 hours. This ensured that the cells were not confluent by the time of collection for cell cycle analysis. A small portion of the transfected cells were cultivated on a 22 × 22 mm No. 1 borosilicate glass cover slip in a separate 6-well dish and treated with nocodazole, mimosine or vehicle alone as described below. The coverslips were removed for fluorescence microscopic observation immediately before collecting cells for flow cytometric analysis of DNA content and GFP expression. Cells were stained 10--20 minutes with 5 μg/ml of the cell-permeable DNA binding dye Hoechst 33342. The green fluorescence emitted from C/EBPα tagged with GFP and the blue fluorescence emitted from Hoechst 33342-stained DNA were readily distinguished by selectively exciting each fluorophore and capturing fluorescence emissions specific for each fluorophore using appropriate excitation and emission filter sets (Chroma Technology Corporation, Brattelboro, VT)\[[@B45]\]. The images shown in Fig. [5](#F5){ref-type="fig"} were acquired using an Olympus 40X PlanApochromat (0.95 numerical aperture) objective on an Olympus IX-70 microscope. Metamorph acquisition software (Universal Imaging Corporation, Downingtown, PA) on an Orca cooled interline camera (Hamamatsu, Bridgewater, NJ) were used to collect the images.

Post-transfection Cell Synchronization
--------------------------------------

The C/EBPα fusion proteins were expressed for one day following transfection, after which we added to the cell culture media 100 ng/ml nocodazole (Sigma) in dimethylsulphoxide (DMSO), 0.5 mM mimosine (Sigma) in DMSO, or DMSO vehicle. In some experiments, cells were also grown for 48 and 72 hours post-transfection before applying nocodazole (data not shown). The distributions of cells in G1, S and G2/M, in the presence or absence of C/EBPα expression, were similar for cells grown for 24 (reported here), 48 or 72 hours (data not shown) prior to nocodazole addition. Following nocodazole, mimosine or DMSO addition, the transfected GHFT1-5 cells continued to be cultivated at 33°C.

Flow Cytometry
--------------

24 hours after drug or vehicle treatment, transfected cells were trypsinized, collected in DME-H21 media containing 10% fetal calf serum, and washed twice with PBS. The harvested cells (typically 5 × 10^6^ to 1 × 10^7^ cells) were resuspended in 300 μl of 8 μg/ml propidium iodide (Molecular Probes) in PBS containing 0.1% NP-40 and 10 μg/ml RNAse A, then incubated at room temperature in the dark for 30 minutes. The cell suspension was then analyzed on a FACScan flow cytometer (Becton Dickinson) at a flow rate of 12 μl/min. Fluorescence of the GFP and propidium iodide-bound DNA was excited with a 488 nm argon-ion laser. Green GFP fluorescence was collected using a 530/30-nm band pass filter, and orange emission from propidium iodide-bound DNA was detected using a 585/42-nm band pass filter. Photomultiplier tube voltage and spectral compensation were initially set using single-stained cells (cells expressing GFP fusions with C/EBPα but not stained with propidium iodide and cells transfected with the control vector containing no GFP cDNA but stained with propidium iodide). Electronic compensation was adjusted among the fluorescence channels to remove residual spectral overlap. The area and width of each event were measured to discriminate intact single cells from debris and from doublet or multiple cells stuck together. GFP fluorescence and propidium iodide-stained DNA fluorescence was thus collected from single cells. A minimum of 10,000 cellular events was collected for each sample. Data was analyzed using CELLQuest software (Becton Dickenson), and cell cycle subset analyses of DNA histograms were performed using ModFitLT™ software (Verity Software House, Topsham, ME).

Statistical Analysis
--------------------

One-way analysis of variance was used to compare data from multiple independent experiments. Statistically significant differences (\*\*, p \< 0.01; \*, p \< 0.05 compared to the Sham-transfected cells) and experiment number are indicated in the figures and legends. All significant differences were confirmed by paired, one-tailed t-tests.

List of abbreviations
=====================

C/EBPα CCAAT/Enhancer Binding Protein alpha

G1 Growth phase 1 of the cell cycle

S DNA Synthesis phase of the cell cycle

G2 Growth phase 2 of the cell cycle

M Mitsosis phase of the cell cycle

CDK Cyclin-Dependent Kinase

GFP Green Fluorescent Protein

GFP-C/EBPα Fusion protein with GFP attached to the amino terminus of C/EBPα

C/EBPα-GFP Fusion protein with GFP attached to the carboxy terminus of C/EBPα

Δ Symbol signifying \"deletion\" when naming constructs

LZ Leucine Zipper

DBD DNA Binding Domain

DMSO Dimethyl Sulfoxide

Acknowledgements
================

We thank Dr. Paul Webb (U.C.S.F.) for critical reading of the manuscript. This work was supported by Public Health Service grant DK-54345 from the National Institutes of Health.