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193606

Biological Clock Depends on Many Parts Working Together

How do people subjected to the endless dark days of winter in the far northern latitudes maintain normal daily rhythms? Though many might feel like hibernating, a highly regulated internal system keeps such impractical yearnings in check. From fruit flies to humans, nearly every living organism depends on an internal clock to regulate basic biological cycles such as sleep patterns, metabolism, and body temperature. And that clock runs on similar molecular mechanisms. Specific clusters of neurons in the brain are known to control the biological clock. Scientists believed these brain “clock cells” function as independent units. But new research described in this issue shows that the neurons do not act in isolation; rather, they collaborate with other neurons in a cell-communication network to sustain the repeating circadian rhythm cycles. Clock cells within the brain maintain an organism's circadian rhythms, even in the absence of cyclical environmental signals like light, in a state scientists call “free running.” Though it has long been clear that the circadian rhythms of an organism persist under such free-running conditions (for example, constant darkness), it was thought that the gene-expression patterns within the cells governing these biorhythms did not require any external, or extracellular, signals to continue ticking. In experiments described here, Michael Rosbash and his colleagues show that the key brain clock cells in fruit flies ( Drosophila ), called ventral lateral neurons , do indeed support the fly's circadian rhythms during periods of constant darkness and that the molecular expression patterns associated with these rhythms continue to cycle as well within other clock cells. These sustained expression patterns, however, require intercellular communication between different groups of brain clock cells. In other words, the ventral lateral neurons do not act alone. When the molecular clock machinery was manipulated so that only the ventral lateral neurons were active, the fly's circadian rhythms were not sustained, suggesting the rhythms depend on other neuronal groups as well. The researchers also demonstrate that the persistence of normal cycling during constant darkness depends on a protein (called PDF) secreted by the ventral lateral cells. The PDF neuropeptide protein was thought to connect the molecular expression pattern of the ventral lateral neurons with the manifestation of circadian rhythms, but the researchers found evidence of a larger influence. When mutant flies lacking a functional PDF gene were exposed to constant darkness, the molecular expression patterns gradually stopped. The scientists say this suggests that the ventral lateral neurons and the PDF protein it produces help coordinate the entire neural network that underlies circadian rhythms. Drosophila lateral neuron (green)

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193607

New Genomic Approach Predicts True Evolutionary History of Bacterial Genomes

Bacteria are an indiscriminate lot. While most organisms tend to pass their genes on to the next generation of their species, bacteria often exchange genetic material with totally unrelated species. That is why skeptics doubted that bacteria researchers could ever hope to map a reliable history of cell lineages in bacteria over time. But now, thanks to the availability of sequenced genomes for groups of related bacteria, researchers at the University of Arizona demonstrate that constructing a bacterial family tree is indeed possible. Previous efforts to trace the ancestry of bacteria were constrained by a dearth of related bacterial genomes, which, among other things, prevented scientists from successfully accounting for bacteria's tendency to exchange genes with unrelated organisms. In this process, called lateral gene transfer , organisms acquire genetic material not from their ancestors, the most prevalent route, but from unrelated organisms. Lateral gene transfer greatly complicates the issue of who descended from whom, because two organisms could appear closely related based on the similarity of some genes but distantly related based on other genes. The problem is to determine which genes have been faithfully vertically transmitted—from parent cell to offspring—and thus reflect the history of the bacterial cell lineages. In this issue, Nancy Moran, Emmanuelle Lerat, and Vincent Daubin propose an approach that solves this problem by identifying a set of genes that serve as reliable indicators of the vertical transfer of bacterial cell lineages. This method has important implications for biologists studying the evolutionary history of organisms by establishing a foundation for charting the evolutionary events, such as lateral gene transfer, that shape the structure and substance of genomes. With this method, scientists can begin to understand how bacteria have evolved and how their genomes have changed. Bacteria promise to reveal the most information about genomic evolution, because so many clusters of related bacterial genomes have been sequenced—allowing for broad comparative analysis among species—and their genomes are small and relatively compact. In this study, the researchers chose the ancient bacteria group Proteobacteria, an ecologically diverse group (including Escherichia coli and Salmonella species) with the most documented cases of lateral gene transfer and the highest number of species with sequenced genomes. The researchers identified a set of likely single-copy orthologs (homologous genes that diverged due to the speciation of ancestral lineages) with widespread distribution in the different species of Proteobacteria that could be used to trace the history of the cell lineages. Surprisingly, they found that almost all of the 205 ortholog gene families they selected supported the same evolutionary branching pattern. Only two did not, which the researchers then investigated and found to be the result of lateral gene transfer. These results, the researchers say, support the ability of their method to reconstruct the important evolutionary events affecting genomes. By mapping out the evolutionary path of genetic information on a genomic level, their approach promises to elucidate not only the evolution of bacterial genomes but also the diversification of species. Electron micrograph of Proteobacteria in eukaryotic cell

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212687

A Functional Analysis of the Spacer of V(D)J Recombination Signal Sequences

During lymphocyte development, V(D)J recombination assembles antigen receptor genes from component V, D, and J gene segments. These gene segments are flanked by a recombination signal sequence (RSS), which serves as the binding site for the recombination machinery. The murine Jβ2.6 gene segment is a recombinationally inactive pseudogene, but examination of its RSS reveals no obvious reason for its failure to recombine. Mutagenesis of the Jβ2.6 RSS demonstrates that the sequences of the heptamer, nonamer, and spacer are all important. Strikingly, changes solely in the spacer sequence can result in dramatic differences in the level of recombination. The subsequent analysis of a library of more than 4,000 spacer variants revealed that spacer residues of particular functional importance are correlated with their degree of conservation. Biochemical assays indicate distinct cooperation between the spacer and heptamer/nonamer along each step of the reaction pathway. The results suggest that the spacer serves not only to ensure the appropriate distance between the heptamer and nonamer but also regulates RSS activity by providing additional RAG:RSS interaction surfaces. We conclude that while RSSs are defined by a “digital” requirement for absolutely conserved nucleotides, the quality of RSS function is determined in an “analog” manner by numerous complex interactions between the RAG proteins and the less-well conserved nucleotides in the heptamer, the nonamer, and, importantly, the spacer. Those modulatory effects are accurately predicted by a new computational algorithm for “RSS information content.” The interplay between such binary and multiplicative modes of interactions provides a general model for analyzing protein–DNA interactions in various biological systems.

Introduction During B- and T-lymphocyte development, the immunoglobulin (Ig) and T-cell receptor (TCR) genes are assembled from discrete V, D, and J gene elements via a process of genomic rearrangements known as V(D)J recombination ( Fugmann et al. 2000a ; Hesslein and Schatz 2001 ). V(D)J recombination occurs in two steps: a cleavage phase, in which DNA double-strand breaks are created, followed by a joining phase ( Fugmann et al. 2000a ). During cleavage, the lymphoid-specific recombinase proteins, RAG1 and RAG2, presumably together with the accessory DNA-binding factor HMG-1/2, bind recombination signal sequences (RSSs) located adjacent to each rearranging gene element. A complex consisting of RAG and HMG proteins bound to a single RSS is then thought to capture a second RSS ( Jones and Gellert 2002 ; Mundy et al. 2002 ); within this synaptic complex, the RAG proteins introduce double-strand breaks at the junctions between each RSS and its associated gene element ( Hiom and Gellert 1998 ). In the joining phase, ubiquitous DNA repair factors involved in nonhomologous end joining, in the presence of the RAG proteins, ligate the cleaved ends, generating two types of recombinant junctions: precise signal joints (SJs) and imprecise coding joints (CJs) ( Bassing et al. 2002 ). RSSs are an essential part of V(D)J recombination, as their presence is both necessary and sufficient to direct RAG-mediated recombination on artificial substrates. Sequence alignments of RSSs suggested that each signal can be dissected into three components: a conserved heptamer (consensus: 5′-CACAGTG) and a conserved nonamer (consensus: 5′-ACAAAAACC), separated by a poorly conserved spacer of either 12 ± 1 or 23 ± 1 bp ( Tonegawa 1983 ; Akira et al. 1987 ; Ramsden et al. 1994 ). The heptamer is the site of DNA cleavage ( Roth et al. 1992 ), while the nonamer provides a major binding surface for RAG1 ( Difilippantonio et al. 1996 ; Spanopoulou et al. 1996 ; Nagawa et al. 1998 ; Swanson and Desiderio 1998 ). Spacer length restricts recombination according to the “12/23 rule”; efficient recombination occurs between two gene elements only when one element is flanked by an RSS with a 12 bp spacer (12-RSS) and the other by an RSS with a 23 bp spacer (23-RSS) ( Tonegawa 1983 ). Despite the enormous specificity that RSSs confer on the recombination process, the recombination signals themselves demonstrate a remarkable degree of sequence heterogeneity. Only the first three nucleotides of the heptamer and the fifth and sixth positions of the nonamer show almost perfect conservation ( Ramsden et al. 1994 ) and are therefore thought to be the major determinants of RSS specificity and function. Mutations in any of these five “critical” nucleotides, alone or in combination, essentially abolish recombination ( Tonegawa 1983 ; Akira et al. 1987 ; Hesse et al. 1989 ). The roles of the remaining “noncritical” heptamer and nonamer nucleotides are less understood. Some studies observed that mutations in these lesser-conserved residues have comparatively milder phenotypes unless present in combination ( Tonegawa 1983 ; Hesse et al. 1989 ). Others, however, reported that nonconsensus deviations of noncritical residues lead to vastly different recombination efficiencies, resulting in significant differences in gene element usage in the unselected antigen receptor repertoire ( Ramsden and Wu 1991 ; Suzuki and Shiku 1992 ; Connor et al. 1995 ; Larijani et al. 1999 ). Our current knowledge about the functional role of the spacer is that its length is crucial in directing V(D)J recombination ( Tonegawa 1983 ; Hesse et al. 1989 ). Comprehensive sequence alignments show that the spacer possesses some degree of sequence conservation, albeit at a level much lower than that of the heptamer or nonamer ( Ramsden et al. 1994 ). This suggests that there is little or no selective pressure for spacers to adopt a given sequence. Studies examining the effects of different spacer sequences on recombination activity have yielded seemingly conflicting results. An early report found up to a 15-fold effect of different spacer sequences ( Akira et al. 1987 ), while follow-up studies observed either no effect ( Wei and Lieber 1993 ; Akamatsu and Oettinger 1998 ) or up to 6-fold effects ( Fanning et al. 1996 ; Nadel et al. 1998 ; Larijani et al. 1999 ). This suggests that spacer sequence may affect recombination activity, but a comprehensive picture of the rules that govern how it does so is lacking. One limitation inherent in many prior RSS studies is that they have often been performed in the context of RSSs with a preponderance of consensus nucleotides. While such analyses have been useful in characterizing the most conserved or critical determinants of RSS function, the contributions of other nucleotides are potentially masked in RSSs with high consensus nucleotide representation. That most endogenous RSSs do not contain consensus heptamer and/or nonamer motifs further suggests the need for a careful study of individual RSS nucleotides in the context of physiologically relevant RSSs. We have performed an extensive analysis of the functional properties of RSS elements in the context of endogenous recombination signals. To explore the nature of the complex relationships that might exist among different elements and positions in the RSS, we started with the nonfunctional RSS associated with the murine Jβ2.6 pseudogene element of the TCRβ locus (Jβ2.6 RSS). While most such pseudogene elements are flanked by RSSs with crippling mutations ( Akira et al. 1987 ), Jβ2.6 is unique in that the sequence of its flanking RSS suggests no obvious explanation for its complete lack of activity ( Figure 1 ). All of the critical residues are conserved, and each nonconsensus nucleotide in the heptamer and nonamer is represented in at least one other functional RSS in the TCRβ locus ( Figure 1 ). A systematic analysis of Jβ2.6/consensus hybrid RSSs revealed that the nonamer, by itself, is the biggest determinant of Jβ2.6 RSS activity and that the lack of Jβ2.6 RSS function is due to the concerted action of nonconsensus nucleotides throughout the entire RSS, including the spacer. Surprisingly, we found that in combination with other consensus elements, an artificial consensus spacer can markedly boost recombination activity, while an anticonsensus spacer strongly impairs activity. Furthermore, in a genetic screen for functional spacer sequences, we observe a selective pressure for substrates with an increased representation of consensus nucleotides. Our results provide strong support for the model that RSS activity is a summation of numerous complex interactions between the RAG proteins and the RSS, involving not only the heptamer and nonamer but also most (if not all) basepairs of the spacer. Figure 1 Recombination Signal Sequences Heptamer, spacer, and nonamer elements of 12-RSSs referred to in this study are shown. “Cons.” and “Anti-Cons.” denote the consensus and anticonsensus 12-RSSs, respectively. VκL8, Jβ2.6, and Jβ2.2 are murine 12-RSSs. “Jβ Cons.” denotes the consensus RSS compiled for all functional 12-RSSs in the murine Jβ1 and Jβ2 clusters. Where more than one nucleotide is listed at any given position, this indicates a shared preponderance of those nucleotides. For consensus RSSs, nucleotides in bold indicate almost absolute conservation; for the anticonsensus RSS, bold nucleotides are almost completely absent. Nucleotides in lowercase italics appear at slightly reduced frequencies compared to the other nucleotides listed. “Jβ-G/-A/-T/-C” and the corresponding numbers indicate the number of functional RSSs in the murine Jβ1/Jβ2 clusters at which the respective nucleotide appears at the designated position. At the top of the figure, the position of each nucleotide is labeled with respect to the first position of the respective element. Results In Vivo Assay for Recombination We generated a series of recombination substrates to measure the ability of various hybrid Jβ2.6/consensus 12-RSSs to rearrange to a “standard” 23-RSS (consisting of consensus heptamer and nonamer elements flanking a spacer from the functional Ig Jκ1 RSS). This standard 23-RSS was used instead of the natural Jβ2.6 RSS partner (the 23-RSS flanking Dβ2), since the substrates containing the Dβ2 23-RSS showed much lower levels of recombination in our hands (data not shown). The 12-RSS coding flank was the same for all constructs, namely that of Jβ2.6. For our study, a polymerase chain reaction (PCR)-based assay ( Figure 2 , top) was employed, which allowed us to visualize recombination efficiencies across a >1,000-fold range. The recombination substrates were transfected into the human embryonic kidney cell line 293T along with constructs expressing full-length RAG1 and RAG2 proteins, and recombination frequencies were measured by PCR using primers that amplify SJs. To confirm that the amplified products in our PCR assay were bona fide SJs, we demonstrated that they could be cleaved efficiently with Apa LI restriction endonuclease, which cuts precise RSS–RSS junctions (data not shown). The amount of recombination substrate recovered from each transfection was measured by PCR and used to normalize the recombination activity. Although we assayed primarily for SJ formation, analyses of CJ formation yielded parallel results (data not shown). As a reference, we used a substrate containing the 12-RSS from the TCR Jβ2.2 gene element (see Figure 1 ), which recombines at low but detectable levels, as measured both in our system and during T-lymphocyte development ( Figure 2 , lanes 1–4) ( Livàk et al. 2000 ). Figure 2 Recombination Activities on Hybrid Jβ2.6/Consensus RSSs A diagram of the recombination assay (SJ formation) is shown (top). Activities were measured on substrates containing the indicated hybrid 12-RSS and a standard 23-RSS. H, Sk, Sc, or N denotes the consensus heptamer, VκL8 spacer, consensus spacer, or consensus nonamer, respectively; each 12-RSS bears the indicated combination of consensus/VκL8 elements, with the remaining elements belonging to Jβ2.6 RSS. To determine relative recombination efficiencies, the amount of SJs was first corrected for DNA recovery, then normalized to the values obtained for the substrate containing the Jβ2.2 RSS. Relative recombination efficiencies for each of three experiments are shown as bar graphs; the average value is shown below each sample. The gels shown here correspond to Experiment 3 and represent products of PCRs on 10-fold dilutions of recovered plasmid DNA. Consensus Heptamer, Spacer, and Nonamer Replacements Recombination of Jβ2.6 RSS is below the level of detection of our assay ( Figure 2 ). Substitution of a consensus heptamer (H) into the Jβ2.6 RSS elevates the recombination frequency to levels just above background ( Figure 2 , lanes 13–16). Similarly, substitution of a spacer from a standard, functional 12-RSS (recombination signal sequence spacer [Sk], from Ig VκL8; see Figure 1 ) or of an artificial consensus spacer (Sc) only marginally restores recombination ( Figure 2 , lanes 17–24). By contrast, substitution of a consensus nonamer (N) boosts recombination activity to the level of Jβ2.2 RSS ( Figure 2 ; compare lanes 1–4 to 25–28), approximately 20-fold higher than substitution of H, Sk, or Sc alone and at least two orders of magnitude above Jβ2.6 RSS. Therefore, the nonamer, by itself, is the biggest single determinant of Jβ2.6 RSS activity. The combination of a consensus heptamer and nonamer (H–N) further increases activity approximately 10-fold above N alone ( Figure 2 , lanes 45–48). Hence, the cumulative effects of nonconsensus mutations in the heptamer and nonamer elements of Jβ2.6 RSS are quite large. In combination with a consensus heptamer and/or a consensus nonamer, the presence of either the VκL8 or the consensus spacer markedly enhances recombination activities above those observed with the Jβ2.6 RSS spacer ( Figure 2 , lanes 29–44). Although there is some fluctuation between experiments, in each replicate the greatest enhancement by the Sk or Sc spacer is seen in combination with a consensus heptamer: on average, H–Sk and H–Sc are 30- to 50-fold higher than H alone. By comparison, Sk–N and Sc–N are 3- to 8-fold higher than N, while H–Sk–N and H–Sc–N are 3- to 9-fold higher than H–N. Thus, a functional spacer can, in most cases, “rescue” the effects of a nonconsensus nonamer more fully than the effects of a nonconsensus heptamer, suggesting that the spacer has greater functional overlap with the nonamer than with the heptamer. Single-Nucleotide Consensus Replacements The heptamer and nonamer of Jβ2.6 RSS differ from the consensus in only five positions (see Figure 1 ): the last three nucleotides of the heptamer and the second and fourth nucleotides of the nonamer. To determine which of these nucleotides make the greatest contributions to Jβ2.6 RSS activity, we introduced the respective consensus nucleotides individually at each of these positions. Since substitution of a consensus heptamer alone yields very low recombination levels ( Figure 2 ), we assayed single-nucleotide heptamer replacements (H[5], H[6], and H[7]) in combination with a consensus spacer. We also assayed substrates containing H(5) combined with a consensus nonamer or with both consensus spacer and nonamer elements. All single-nucleotide heptamer replacements result in significant partial restoration of activity, to levels at least 50% of those obtained with the full consensus heptamer (data not shown). This suggests that the low activity of the Jβ2.6 RSS heptamer is due to contributions of all three nonconsensus nucleotides. Substitution of a consensus nucleotide at either the second or fourth position of the nonamer (N[2] or N[4], respectively), alone or in combination with a consensus heptamer and/or spacer, partially reproduces the effects of the full consensus nonamer ( Figure 3 A). Interestingly, in each set of constructs, N(2) confers a greater restoration of activity than N(4): on average, constructs containing N(2) recombine at 50% the level of N, while constructs containing N(4) recombine at roughly 10% of N. This suggests that the recombination process has a greater preference for preserving a consensus C at the second position of the nonamer than a consensus A at the fourth position. Figure 3 In Vivo Recombination Activities on Hybrid 12-RSSs with Nonamer Point Mutations or with the Anticonsensus Spacer The plots, error bars, and values listed below each sample represent the averages of three experiments. Note that all recombination efficiencies presented in this figure were obtained from transfections/PCRs that were completely independent from those shown in Figure 2 . Abbreviations are identical to those used in Figure 2 . (A) N(2) or N(4) denotes point substitution of the consensus nucleotide at the second or fourth position of the nonamer, respectively. (B) Sac indicates substrates that contain an anticonsensus 12-RSS spacer. Anticonsensus Spacer Replacements In the presence of a consensus heptamer and/or nonamer, a consensus spacer markedly enhances recombination levels over the Jβ2.6 RSS spacer. We therefore wondered whether the presence of an artificial anticonsensus spacer (Sac) (see Figure 1 ), containing the least-conserved nucleotide at each position ( Ramsden et al. 1994 ), would impair recombination. In all cases, Sac reduced recombination levels 10- to 20-fold compared to the already inefficient Jβ2.6 RSS spacer ( Figure 3 B; compare N to Sac–N, and H–N to H–Sac–N). In our experimental system, the consensus and anticonsensus spacer sequences are therefore capable of specifying a surprisingly large range of recombination efficiencies of up to two orders of magnitude. Coupled Cleavage In Vitro Two important questions arise from the results of these in vivo assays. First, do the differences in the RSS nucleotide sequences affect the cleavage or the joining phase of the reaction? Second, are the RAG proteins by themselves the only proteins that mediate the discrimination between various RSSs? To address these questions, we performed standard 12–23 coupled cleavage reactions using purified, truncated (core) RAG proteins ( Figure 4 A). The linear substrates for these reactions were amplified by PCR from the plasmids used in the transient recombination assay. The amount of coupled cleavage products from three independent sets of reactions was quantified ( Figure 4 C). While the consensus RSS (H–Sc–N) promotes efficient cleavage of up to 23% of the input substrate, the Jβ2.6 RSS is cleaved at extremely low levels, at or below the limit of detection ( Figure 4 A, lane 2). As expected from the in vivo experiments, Jβ2.2 is sufficient for low but clearly detectable cleavage ( Figure 4 A, lane 26). In agreement with the SJ formation data, the consensus nonamer substitution (N) boosts the level of cleavage significantly ( Figure 4 A, lane 6), while the introduction of Sk or Sc has less effect ( Figure 4 A, lanes 8 and 10). In contrast to our findings on SJ formation, the substrate containing a consensus heptamer (H) is as efficiently cleaved as that containing N ( Figure 4 A; compare lanes 4 and 6). Interestingly, all substrates containing a consensus nonamer (and to a lesser extent those harboring a consensus spacer) show a high level of single-site cleavage at the 12-RSS ( Figure 4 A, lanes 6, 10, 12, 18, and 20); such products, which are only rarely generated on extrachromosomal substrates in vivo ( Steen et al. 1997 ), could account for a reduced level of coupled cleavage compared to the recombination efficiencies obtained for the respective constructs in our SJ assays. The underlying mechanism of this phenomenon is the topic of ongoing studies. Figure 4 In Vitro Cleavage Reaction (A and B) Coupled cleavage was performed using body-labeled DNA substrates containing a standard 23-RSS (filled triangle) and different 12-RSSs (open triangle) as indicated above the lanes. Reaction products were separated on 4% polyacrylamide gels. The identity of the bands is indicated by symbols located between the gels; an arrow indicates the double cleavage product, while an asterisk marks single-site cleavage products. The gels shown here correspond to Experiment 2. (C and D) The intensity of the bands from three individual experiments (see legend) was quantified and the average cleavage efficiency calculated for each individual substrate (indicated below the chart). The efficiencies are displayed as relative to those obtained for Jβ2.2, which were arbitrarily set to 1. Interestingly, a favorable spacer sequence (Sk or Sc), when paired with H or N, boosts cleavage over the Jβ2.6 RSS spacer ( Figure 4 A, lanes 12, 14, 16, and 18). The levels of cleavage for H–Sk or H–Sc are reproducibly higher than those for Sk–N or Sc–N; although the effect is less striking than for SJ formation, the limits of detection in the coupled cleavage assay dictate that this assay spans a much narrower range of activities than the SJ formation assay. To further address the role of spacer sequences in our coupled cleavage system, we performed another set of experiments using the substrates containing the anticonsensus spacer (Sac) ( Figure 4 B and 4D). In conjunction with either consensus heptamer (H–Sac) or consensus nonamer (Sac–N), the anticonsensus spacer reduces cleavage 5- to 10-fold compared to the consensus spacer (H–Sc or Sc–N) ( Figure 4 C and 4D) and 3-fold compared to the Jβ2.6 RSS spacer (H or N) ( Figure 4 B; compare lanes 4 and 8 to lanes 6 and 10, respectively). This suggests that the Jβ2.6 RSS spacer, although “poor” compared to Sk or Sc, is still more proficient for cleavage than Sac. RSS Binding It is likely that differences in the nucleotide sequences of the RSS lead to variations in the stability of RAG–RSS complexes ( Hiom and Gellert 1997 ; Akamatsu and Oettinger 1998 ; Swanson and Desiderio 1998 ). This idea provides one obvious explanation for the observed differences in SJ formation and cleavage efficiency among the various analyzed 12-RSSs. To address this possibility, we analyzed binding of the RAG proteins to individual isolated 12-RSSs, since the 23-RSS remained identical in all experiments described above. Binding was assessed in standard gel-shift assays using oligonucleotide substrates containing the respective 12-RSSs ( Figure 5 A). All binding assays were performed three times; the quantitation of binding for each RSS relative to Jβ2.2 is displayed in Figure 5 B. (Note that the amount of shifted complex has been normalized for the amount of free probe, which contributes to the fact that, between some samples, visual assessment of relative binding activities are less striking than quantitative measurements.) As expected, the consensus 12-RSS (H–Sc–N) shows the highest binding efficiency, while binding to the endogenous Jβ2.6 RSS is weak, about 2-fold reduced compared to our standard, the functional Jβ2.2 12-RSS. Given that, as with the coupled cleavage assay, the range of activities in the binding assay is much narrower than in the SJ formation assay, these results correlate well with those obtained in the other assays. Substitution of the individual consensus elements H, Sc, and N, however, led to surprising results. While the consensus nonamer (N) sequence, as expected, increases the level of binding (up to that of Jβ2.2), the consensus spacer (Sc) alone has no effect on binding at all, and the consensus heptamer (H) consistently reduces the level of binding. The consensus spacer boosts binding only in the context of a consensus nonamer (the ratios of Sc–N:N and H–Sc–N:H–N are greater than H–Sc:H), and the consensus heptamer contributes significantly to RAG–RSS interactions in this assay only when both spacer and nonamer are consensus sequences (H–Sc–N:Sc–N > H–N:N or H:Jβ2.6 RSS). This indicates that the nonamer is the predominant element determining the stability of the initial RAG–HMG–RSS complex while the heptamer makes additional important contributions to cleavage and recombination not reflected in this binding assay. Figure 5 In Vitro Binding (A) Binding assays were performed using the 5′-end-labeled 12-RSS substrates indicated above the lanes. Each reaction contained identical amounts of DNA substrate. Owing to differences in the end-labeling efficiencies, the quantitation (shown in [B]) is required to make quantitative comparisons. The gels shown here correspond to Experiment 3. (B) The relative amount of substrate in the shifted complex was determined. The binding efficiencies from three independent experiments were calculated relative to the binding seen for Jβ2.2 oligonucleotides (which were arbitrarily set to 1). The average value is displayed below the chart. In the context of a consensus nonamer, the consensus spacer reproducibly enhances binding more than a consensus heptamer (Sc–N > H–N). In contrast, the anticonsensus spacer (H–Sac–N) reduces binding about 3-fold compared to H–Sc–N ( Figure 5 A and 5B). The effects of Sc–N compared to Sac–N are also clearly visible. Interestingly, the levels of binding in the presence of Sac are very similar to those obtained for the respective RSSs containing the original Jβ2.6 RSS spacer, in contrast to the comparative effects of the two spacers on cleavage (see Figure 4 ). Taken together, the results of our binding studies underline clearly that the reduced ability of the Jβ2.6 RSS to participate in the initial interaction with the RAG complex, and hence the subsequent steps of V(D)J recombination, is caused not solely by the Jβ2.6 RSS nonamer but also by the “inefficient” spacer sequence. This indicates that the spacer helps the nonamer to efficiently lock the RAG proteins onto the RSS. The heptamer can contribute to this only when interactions with the other two elements are favorable. Genetic Screen for Functional Spacer Sequences Although the RSS spacer is poorly conserved and no naturally occurring RSS has yet been identified that bears the published consensus spacer sequence, our results show that the presence of the most- or least-conserved nucleotides at all positions of the spacer dramatically alters recombination activities of RSSs that contain a consensus heptamer and/or nonamer. This suggests that a functional preference exists for certain spacer sequences over others. We therefore established a genetic screen for functional spacer sequences in which each position of the spacer was randomized to contain either a consensus or an anticonsensus nucleotide (Sc/Sac). Because the greatest effect of the consensus spacer in our experiments is seen in combination with a consensus heptamer (H–Sc), the randomized spacer was analyzed in the context of 12-RSSs containing a consensus heptamer and the Jβ2.6 RSS nonamer (H–Sc/Sac). The H–Sc/Sac library contained roughly 80,000 clones, sufficient to represent each of the 4,096 possible spacer sequences multiple times (data not shown). We transfected the H–Sc/Sac library into 293T cells together with vectors expressing full-length RAG1 and RAG2 , and we cloned and sequenced PCR-amplified SJs. As a control, we analyzed PCR products corresponding to unrearranged substrates from library pools transfected in the absence of RAG1 and RAG2 ( Figure 6 ). This control pool shows a bias toward the presence of C nucleotides (the consensus nucleotide at positions 4 and 7–9 of the spacer, and the anticonsensus nucleotide at positions 1 and 6), such that the overall bias of the unselected library is slightly toward the consensus spacer (total consensus/total anticonsensus nucleotides = 1.19), consistent with sequence analysis of untransfected library clones (data not shown). Sequence analysis of amplified SJs reveals an overall enrichment for consensus spacer nucleotides over the unrearranged control (total consensus/total anticonsensus nucleotides = 1.73 for SJs, versus 1.19 for control). Spacer positions 1–5 (adjacent to the heptamer) and 8–11 all show a preference for the consensus nucleotide; the remaining positions show little or no preference for the consensus or in one case (position 7) even an enrichment for the anticonsensus nucleotide ( Figure 6 , white bars). The strongest preference for consensus is seen at position 5, which shows almost a 3-fold enrichment over the unrearranged control; interestingly, previous mutation analyses have implicated this spacer position as having a role in affecting recombination levels ( Fanning et al. 1996 ; Larijani et al. 1999 ). In general, the degree of enrichment at any given position reflects the degree to which the consensus nucleotide is represented among the endogenous RSS repertoire ( Figure 6 ) ( Ramsden et al. 1994 ). Figure 6 Genetic Screen for Preferred Spacer Sequences A plasmid library containing 12-RSSs with a consensus heptamer and either consensus or anticonsensus nucleotides at each position of the spacer was screened for spacers with higher activity using either in vivo recombination or in vitro coupled cleavage assays (see text for details). The number of library clones screened was >10 5 . In total, 240 sequences from two independent in vivo experiments and 205 sequences from two in vitro screens were analyzed. The relative enrichment for a consensus over an anticonsensus nucleotide at each position was calculated (taking the bias in the starting library into account). The average from two experiments is displayed in the bar graph and the values are displayed above or below the bars. The log 2 of the ratio of the frequency of consensus and anticonsensus nucleotides at each position is displayed; hence, a value of one indicates that the respective nucleotide occurs two times more frequently in the selected population than in the starting library. In addition, the degree of conservation of each nucleotide is indicated ( Ramsden et al. 1994 ). To determine whether the preferred spacer sequences for SJ formation and cleavage differ, the library screen was also performed in vitro. To obtain artificial SJs from our biochemical cleavage assays, T4 ligase was added to the deproteinized cleavage products, which circularized the cleavage product containing two signal ends. The sequence analysis of such artificial SJs from two independent cleavage reactions showed that positions 2–5 as well as positions 8–11 of the spacer are enriched for consensus over anticonsensus sequences ( Figure 6 , black bars). While these observations mirror the SJ formation data, the nucleotide located at position 1 (and to some extent position 3) seems less important for coupled cleavage than for recombination in vivo. Similar to the in vivo experiment, position 5 shows the highest magnitude of enrichment for the consensus (about 4-fold). The differences between the results of the two experimental systems (SJ formation in vivo and cleavage in vitro) could be a reflection of the number of sequences obtained in each type of analysis (200–250) or could represent differences in the nucleotide requirements of spacer participation in cleavage versus SJ formation. Overall, our experiments indicate that spacer effects are largely mediated by the RAG proteins and occur, at least in part, in the first phase of V(D)J recombination: the recognition of the RSSs, their synapsis, and the cleavage step. Correlation with a Computational Model for RSS Function The observation that an RSS spacer can act in concert with the noncritical residues of the heptamer and nonamer to drastically modulate RSS activity suggests the need for models of RSS function that take into account complex functional relationships among the different nucleotides. A predictive algorithm for quantitatively assessing the potential of a given DNA sequence to undergo V(D)J recombination has recently been developed ( Cowell et al. 2002 , 2003 ). This algorithm calculates the theoretical recombination potential, or RSS information content (RIC) score, by examining internucleotide relationships within a given DNA sequence. We calculated RIC scores for the hybrid Jβ2.6/consensus RSSs used in this study, and we compared them to the experimental binding, cleavage, and recombination values ( Figure 7 A and 7B; data not shown). The correlation between RIC scores and our experimental data is striking. The RIC score for Jβ2.6 RSS is below the threshold (−40) for sequences that would be expected to recombine. The addition of consensus heptamer and/or nonamer elements boosts RIC scores, mirroring the increases in binding, cleavage, and SJ formation. Of particular interest is the fact that effects of consensus and anticonsensus spacers on binding/cleavage/recombination are prominently reflected in the RIC scores as well. Intriguingly, RIC scores appear to be more strongly correlated with cleavage ( r S = 0.90) than with binding ( r S = 0.86) and most correlated with SJ formation ( r S = 0.96). The correlations between our experimental data and RIC scores suggest that the failure of Jβ2.6 RSS to recombine and the ability of consensus heptamer, spacer, and nonamer elements to rescue Jβ2.6 RSS activity are functions of how well RSS structure corresponds to that of a preferred sequence. In this case, the selective advantage of the consensus RSS is not limited to a few critical nucleotides in the heptamer or nonamer but, rather, extends throughout the length of the RSS, even in regions (e.g., the spacer) that were previously thought to be unimportant. Figure 7 Theoretical Predictions of RSS Qualities The average recombination/cleavage efficiencies obtained in the in vivo experiments (A) and in vitro assays (B) are plotted against the RIC scores for the 12-RSS in the respective recombination substrates. Note that the values obtained from the in vitro cleavage assays were normalized to account for differences in the detection range of individual experiments. Further support for the potential of the RIC score as a theoretical measure for RSS activity arises from our genetic screen. For both the in vivo and the in vitro screens, the mean RIC score of the 12-RSSs in the enriched population is higher than that of the starting pool (data not shown), and those differences are statistically significant (Student's t test and the Mann–Whitney test, p<0.0002 for all tests). This indicates that the RIC score is able to predict the quality of RSSs and that this ability is not limited to the well-conserved heptamer and nonamer but also applies to the far more diverse spacer. Discussion RSSs are the DNA elements that direct and control the V(D)J recombination reaction. In the TCR loci, differences in the abilities of individual RSSs to recombine with each other are a significant determinant of variations in the frequencies with which gene elements appear in the mature TCR population ( Livàk and Petrie 2002 and references therein). The molecular basis of such differences in intrinsic recombination activities lies in the remarkable sequence diversity of endogenous RSSs. Previous studies using consensus or nearly consensus RSSs suggested that only a handful of absolutely conserved nucleotides in the heptamer and nonamer serve as the major determinants of RSS specificity and function. These studies, however, did not take into account the fact that the vast majority of endogenous RSSs do not contain fully consensus elements; hence, the physiologic roles of lesser-conserved RSS nucleotides are likely of much greater significance than previously estimated. Contributions of Individual Elements Starting from the nonfunctional Jβ2.6 RSS, we asked the following question: what effects do a perfect heptamer, nonamer, or spacer and combinations thereof have in an inactive or poorly active RSS? We show that a number of mutations in noncritical RSS positions are required to convert Jβ2.6 RSS into a highly active 12-RSS or to convert a highly active RSS (H–Sk–N or H–Sc–N) into a completely nonfunctional, pseudogene-type RSS. Our experiments demonstrate that all RSS nucleotides, including the spacer element and the noncritical positions of the heptamer and nonamer, have some sequence-directive roles. In general, we observe that the magnitude of the effects of unfavorable nucleotides in noncritical RSS positions is dependent on the presence of other unfavorable nucleotides. This explains why, in previous studies using largely consensus RSSs, the effects of nonconsensus nucleotides at the noncritical positions were concluded to be less significant ( Tonegawa 1983 ; Hesse et al. 1989 ). Contributions of Individual Nucleotides in Jβ2.6 RSS The Jβ2.6 RSS heptamer differs from the consensus in the fifth, sixth, and seventh positions; none of these is drastically more important than any other in specifying overall heptamer function (data not shown). The Jβ2.6 RSS nonamer differs from the consensus in the second and fourth positions (see Figure 1 ), and the G at the fourth position disrupts the poly(A) tract present in the consensus nonamer. Previous footprint analyses and studies on the homologous DNA-binding domain of the bacterial Hin recombinase ( Feng et al. 1994 ) suggest that RAG1 may bind the nonamer in the minor groove of this poly(A) tract ( Spanopoulou et al. 1996 ; Akamatsu and Oettinger 1998 ; Nagawa et al. 1998 ). Hence, we expected that restoration of the poly(A) tract of the nonamer would have a greater boosting effect on recombination levels than a consensus substitution at the second position. Instead, the opposite is true, regardless of the sequences in the remainder of the RSS (see Figure 3 ). Having the consensus cytidine at position 2 creates a CA step within the nonamer. Such CA steps have been implicated in alternative DNA structures ( Gorin et al. 1995 ); while previous discussion has focused on the CA steps present at the site of cleavage in the heptamer, it is possible that a single CA step in the nonamer is important for the RAG complex to identify the subsequent downstream poly(A) tract. Defects in RAG Binding to Jβ2.6 RSS Previous binding studies have shown that the nonamer is the key element for initial RAG–RSS interactions and that mutations within the nonamer can strongly reduce or even completely abolish formation of the 12-SC (signal complex) ( Hiom and Gellert 1997 ; Akamatsu and Oettinger 1998 ). In contrast, mutating the entire heptamer leads only to a partial decrease in 12-SC formation, and, importantly, the absolutely conserved “CAC” triplet contributes only as much to binding as the last four nucleotides of the heptamer ( Akamatsu and Oettinger 1998 ). Our gel-shift studies recapitulate these observations with the Jβ2.6 RSS heptamer and nonamer (see Figure 5 ). Moreover, a hybrid Jβ2.6/consensus RSS containing a consensus nonamer can promote 12-SC formation as efficiently as the functional Jβ2.2 RSS (see Figure 5 ). This explains why replacement of the Jβ2.6 RSS nonamer with a consensus nonamer can restore recombination to low but physiologically relevant levels (see Figure 2 ). The effect of a consensus spacer on 12-SC formation exhibits striking plasticity (see Figures 2–5 ). Additionally, in our in vitro screen, the areas of the 12-RSS spacer most highly enriched for consensus nucleotides (see Figure 6 ) correlate with sites of spacer contacts identified in previous footprinting studies (spacer positions 2–5 and 9–11) ( Akamatsu and Oettinger 1998 ; Nagawa et al. 1998 ; Swanson and Desiderio 1998 ; Swanson 2002 ). Given that the nonamer provides the most important contact surfaces, if strong interactions with the nonamer can form, then the presence of a consensus spacer may allow additional favorable contacts to be established, not only in the spacer itself, but even farther away, in the heptamer. By contrast, an unfavorable spacer (e.g., the Jβ2.6 RSS spacer or Sac) may structurally “insulate” protein–DNA contacts seen in the nonamer, such that potential heptamer contact surfaces that could otherwise contribute to overall 12-SC stability remain hidden. This may explain why a consensus heptamer, in the absence of a good nonamer, is unable to promote formation of a stable 12-SC complex. Our in vitro cleavage assay integrates the effects of RSS binding, pairing, and actual DNA cleavage. Hence, the differences between the results of binding and cleavage assays suggest that the steps following initial binding (paired complex [PC] formation and DNA cleavage) are also regulated by spacer sequences. PC formation requires the recognition of the partner RSS with respect to its spacer length, and thus it is plausible that the sequence of spacers influences the protein–DNA contacts required for this compatibility test. Since it is within the PC that coordinated, synchronous DNA cleavage takes place ( Hiom and Gellert 1998 ; West and Lieber 1998 ), it is conceivable that RSSs “communicate” with each other and that their spacer sequences therefore may affect the alignment of the cleavage site with respect to the recombinase active site. Such structural changes may underlie the phenomenon of the “beyond 12/23 rule” that restricts V(D)J recombination of the TCRβ locus, preventing recombination of certain 12–23 RSS pairs and favoring recombination of others ( Jung et al. 2003 ). The 23 bp spacer of the Vβ RSSs is the critical element in dictating the strong preference of Vβ RSSs for the 12-RSS flanking the D segments as compared to the 12-RSS flanking the J segments, and this preference is regulated before or at the cleavage step ( Jung et al. 2003 ). These intriguing findings, however, did not provide experimental insight into how a DNA motif whose sequence had previously been deemed unimportant could paradoxically play such an important role. Our findings provide a framework with which to understand how such an unexpected phenomenon might occur. Finally, the differences between the in vitro cleavage and in vivo recombination assays indicate an additional role of the spacer sequence in the joining phase of the reaction. This seems plausible, since joining is thought to start with the controlled disassembly of the postcleavage complex in which the four DNA ends, including the RSSs, are held in intimate contact with each other, presumably by the RAG proteins ( Hiom and Gellert 1998 ; Tsai et al. 2002 ). Spacer sequences might thus be involved in controlling the structure and stability of such complexes. Relationship between Spacer Sequence Conservation and Recombination Activity Based on comprehensive sequence alignments showing a small but significant degree of spacer sequence conservation ( Ramsden et al. 1994 ), a few studies demonstrated reproducible effects of up to 6-fold of naturally occurring spacers on recombination levels ( Fanning et al. 1996 ; Nadel et al. 1998 ). In transient transfection assays, we infer a much wider range of recombination efficiencies solely due to differences in spacer sequence. Strikingly, we observe that spacer sequence variably affects RSS activity depending on the extent to which each nucleotide of the spacer matches either the most- or the least-conserved nucleotide. This observation resolves some of the apparent discrepancies observed among previously published studies. For example, a poly(G) spacer, which reduces recombination 15-fold compared to a highly active control ( Akira et al. 1987 ), contains one consensus and five anticonsensus residues; by contrast, a spacer containing intermixed G and C residues, which has no effect on recombination activity ( Wei and Lieber 1993 ), contains five consensus and four anticonsensus residues. A Structural Basis for the Ability of RAG Proteins to Recombine Highly Diverse RSSs We find that progressive accumulation of nonconsensus nucleotides within an RSS progressively impairs recombination activity and that, at the less-conserved positions of an RSS, a multitude of nonconsensus nucleotides acting in concert can render the RSS completely inactive. This suggests that the RAG–RSS complex can tolerate or correct for a considerable amount of sequence and/or structural diversity. UV–cross-linking studies previously demonstrated RAG1 and RAG2 cross-linking to the heptamer, particularly near the site of cleavage ( Eastman et al. 1999 ; Mo et al. 1999 ; Swanson and Desiderio 1999 ). Footprint analyses of the 12-SC show that complex formation is at least partly blocked by base or phosphate group modification on the spacer side of the heptamer, on both the heptamer- and nonamer-proximal sides of the spacer, and throughout the nonamer ( Akamatsu and Oettinger 1998 ; Nagawa et al. 1998 ; Swanson and Desiderio 1998 ; Swanson 2002 ). The identified contact sites in the spacer coincide with the areas of the spacer that were preferentially found to be consensus type in our genetic screen (see Figure 6 ). Moreover, the observed recombination efficiencies of our hybrid substrates correlate well with the predicted recombination efficiencies from RIC analyses (see Figure 7 A and 7B). Together, these findings support a unifying model in which the RAG proteins establish multiple contacts throughout the length of an RSS (including the spacer) that allow for fine-tuning of activity. Such an extensive network of RAG–RSS contacts within the recombinase complex would create a “structural buffer,” in which unfavorable nucleotides at only a few noncritical positions might be compensated for by favorable protein–DNA interactions at other positions. Conceptually similar models exist for the I- Ppo I and I- Cre I homing endonucleases, which cleave at recognition sites approximately 20 bp in length ( Argast et al. 1998 ; Jurica et al. 1998 ), and which can tolerate sequence heterogeneity in cleavage sites. Both I- Ppo I and I- Cre I form direct sidechain interactions with most of the nucleotides in their recognition sites, and it is believed that the extensive protein–DNA contacts contribute to tolerance of sequence diversity. Based on our in vivo, in vitro, and in silico analyses, we propose that the RAG–RSS complex contains two distinct types of protein–DNA interactions: “digital” (or binary) interactions of a strictly sequence-specific nature, and “analog” (or multiplicative) contacts that fine-tune the strength of the digital contacts ( Travers 1993 ). Digital interactions are established with those nucleotides for which proper sequence is absolutely critical for activity (e.g., the first three nucleotides of the heptamer and positions 5 and 6 of the nonamer). Analog interactions describe local structural variations brought about by different sequences along the rest of the RSS. Disruption of digital interactions completely precludes complex formation (e.g., a single mutation of a critical residue in the consensus RSS can render it entirely inactive), yet digital interactions alone are not sufficient to establish complex formation (e.g., the critical residues by themselves cannot confer activity to the Jβ2.6 RSS). This duality in the nature of protein–DNA contacts present within the RAG–RSS recombinase may be applicable to other biological systems, including other transposases, transcription factors, and DNA-binding proteins. In most protein–DNA interaction systems, the target sequence to which a protein binds contains some nucleotides that are absolutely critical, and others that are noncritical. Digital interactions are established with the absolutely conserved nucleotides in the form of sequence-specific binding, conferring a binary specificity; the digital contacts therefore determine whether a protein will bind (+1) or not (0). Analog contacts are then established with the lesser-conserved nucleotides; the analog interactions act as functional multipliers that determine the efficiency of complex stability, yielding a spectrum of binding efficiencies ranging from full activity (1 × A max , where A = effect on binding efficiency due to analog interactions) to no activity (0 × A min ). Hence, the noncritical residues are crucial for determining how well a protein complex can exert its biological function. By including so many nucleotides as requirements for RSS function, the V(D)J recombination system may have evolved to avoid random cleavage of DNA and translocation errors. If only the critical heptamer and nonamer nucleotides were required for activity, the frequency of cleavage at inappropriate or “cryptic” sites in the genome would be expected to be quite high. By contrast, the required participation of noncritical nucleotides in complex stability safeguards the reaction against uncontrolled cleavage. Hence, from the standpoint of controlled diversification of reaction specificity, it is beneficial for the recombinase to have evolved a spacer with a high degree of sequence heterogeneity, while maintaining intimate contact with the spacer nucleotides via analog interactions. The complex multiplier effect of analog contacts throughout the length of the RSS, superimposed onto specific digital contacts in the heptamer and nonamer, therefore confers upon the recombinase the critical ability to distinguish between inappropriate sites that happen to contain the requisite absolutely conserved nucleotides (e.g., the Jβ2.6 RSS) versus true binding sites whose sequences diverge markedly from the consensus (e.g., most endogenous RSSs). Theoretical Predictions of RSS Quality RIC scores provide a powerful tool for the prediction of RSS quality based on nucleotide sequence. This method generates statistical predictions of RSS function based on the physiologic 12- and 23-RSSs in the mouse antigen receptor gene loci. In our study, RIC scores accurately predicted the relative efficiencies with which RSSs were bound, cleaved, and rearranged (see Figure 7 ; data not shown). Interestingly, the capacity of RIC models to predict RSS quality is not restricted to sequence variability in the conserved RSS heptamer and nonamer; RIC scores also predict the effects of the RSS spacer sequence on RSS function with considerable accuracy. It is striking that RIC scores correlate so well with SJ formation, less well with cleavage, and less well still with RSS binding. This supports the idea that individual nucleotides (and groups thereof) make distinct contributions to the different steps of the V(D)J recombination reaction. This concept is consistent with previous findings showing that the nonamer is a major determinant of binding while the influence of the heptamer becomes most apparent at the level of cleavage. Hence, the efficiency with which an RSS recombines represents an integration of its protein–DNA interactions throughout all steps of the reaction, and RIC scores provide a remarkably accurate prediction of this. RIC models should be useful not only in guiding RSS mutation studies, but also in identifying potential cryptic RSSs in the genome, whose usage could lead to genomic alterations as an initial event leading to chromosomal translocations and cancer ( Cowell et al. 2002 , 2003 ). Furthermore, an identical mathematical approach could be useful for predicting binding sites for DNA-binding complexes (e.g., transcription factors) in general, since the algorithm incorporates the combination of both the digital and the analog DNA–protein interactions that determine the biological function of a given protein complex on a potential DNA target. Materials and Methods Oligonucleotides and plasmids. The sequence of oligonucleotides used for cloning of recombination substrates and libraries are presented in Table S1 . The oligonucleotides used in the gel-shift experiments are listed in Table S2 , and the sequences of oligonucleotides used for PCR ( INNE1 , CIT4A , TL1 , TL2 , TL3 , TL4 , TL5 , and TL6 ) have been described previously ( Eastman et al. 1996 ; Leu et al. 1997 ). The pSJΔ series of substrates for the in vivo recombination and in vitro cleavage assays was created as follows: pSF299 ( Fugmann and Schatz 2001 ) was modified to create p299-Jβ2.6 by replacing the original 12-RSS with a Jβ2.6 12-RSS such that the 12/23-RSS pair is in deletional orientation; for all other substrates, the 12-RSS of p299-Jβ2.6, flanked by Hind III and Sal I sites, was replaced with the respective annealed oligonucleotides (see Table S1 ). To generate the library for the genetic screen, the oligonucleotide HSCSAC1 was synthesized that contained a 1:1 molar ratio of consensus:anticonsensus nucleotides at each position of the spacer and an additional randomized trinucleotide sequence downstream of the nonamer. The oligonucleotide SJLIBREV was annealed, the overhang was filled in using Klenow fragment (New England Biolabs, Beverly, Massachusetts), and the double-stranded fragment was digested with Hind III and Sal I and ligated into the linearized p299-Jβ2.6 vector. Ligation reactions were transformed into DH5α, colonies were harvested into 120 ml of Luria broth (containing 100 μg/ml ampicillin), and plasmid DNA was prepared after an additional incubation at 37°C at 250 rpm for 15 min. pEBB, pEBB-RAG1, and pEBB-RAG2 expression constructs have been described elsewhere ( Roman et al. 1997 ). Recombination assays. Human embryonic kidney 293T cells were transfected with 6 μg of recombination substrate and 3 μg each of pEBB-RAG1 and pEBB-RAG2 using calcium phosphate as described previously ( Fugmann and Schatz 2001 ); for control samples without RAG expression constructs, 6 μg of pEBB was substituted. After 48 h, DNA was recovered by rapid alkaline lysis preparation (RAP) ( Hesse et al. 1987 ). PCR was performed on 10-fold serial dilutions in 20 μl reaction volumes containing 1× Taq buffer (Invitrogen, Carlsbad, California), 2 mM MgCl 2 , 0.1 mM each dNTP, 0.5 μM each oligo, and 0.2 U Taq (Invitrogen). To quantify DNA recovery, the oligonucleotide pair TL5/TL6 was used for the PCR (94°C for 15 s, 60°C for 15 s, 72°C for 30 s, for 18 cycles). To detect SJs, DNA samples were treated with Dpn I, Mlu I, and Xho I to remove unreplicated and unrecombined plasmids. Oligonucleotides INNE1 and CIT4A were used to amplify SJs (94°C for 15 s, 60°C for 15 s, 72°C for 30 s, for 28 cycles). To detect CJs, RAP samples were treated with Dpn I and CJs were amplified using primers TL2 and TL3 . All PCR products were electrophoresed on native 4.5% polyacrylamide gels, stained with SYBR green, visualized using a Fluoroimager 595 (Molecular Dynamics, Sunnyvale, California), and quantified using ImageQuant software (Molecular Dynamics). Genetic screen for functional spacer sequences. 293T cells were transfected with the plasmid library and RAG or pEBB constructs as described in the Results. Extrachromosomal DNA was extracted and samples were digested with either Dpn I/ Mlu I/ Xho I (for cloning of SJs) or Dpn I only (for cloning of unrearranged bands in no-RAG controls). PCR was performed using INNE1 and CIT4A primers, and samples were electrophoresed and stained as indicated above. The products corresponding to the appropriate SJ or unrearranged bands were excised, purified, and cloned into pCR2.1 using a TOPO-T/A cloning kit (Invitrogen). DNA was prepared from individual transformed colonies and sequenced. The in vitro screen was performed using the plasmid library as the substrate in a standard coupled cleavage reaction. After proteinase K digestion, the products were precipitated and dissolved in 100 μl of 1× ligase buffer. T4 DNA ligase (1 μl) (New England Biolabs) was added and the mixture incubated at 16 °C for 4 h to create artificial SJs. The resulting plasmids were treated identically to the plasmids recovered after transfection in the in vivo screen. Protein expression. Recombinant GST-RAG2, MBP-RAG1, and HMG2 were expressed and purified as described previously ( Spanopoulou et al. 1996 ; Eastman et al. 1999 ; Rodgers et al. 1999 ). DNA-binding and cleavage assays. The body-labeled DNA substrates for the cleavage assay were generated by PCR using the oligonucleotides TL1 , TL4 , and the respective recombination substrate as a template. The 12-RSS oligonucleotide substrates used in EMSA were generated by annealing the 5′-end-labeled top strand with an equimolar amount of the unlabeled respective bottom strand (see Table S2 ). Binding and cleavage reactions were performed as reported previously ( Fugmann et al. 2000b ), and gels were quantified using a Storm 820 PhosphorImager and ImageQuant software (Molecular Dynamics). RIC score calculation and other computational analysis. Statistical models of RSS correlation structure have been previously reported ( Cowell et al. 2002 ) ( Data S1 ). Supporting Information Data S1 RIC Score Calculation and Other Computational Analysis (23 KB DOC). Click here for additional data file. Table S1 Oligonucleotides for Cloning of Recombination Substrates (31 KB DOC). Click here for additional data file. Table S2 Oligonucleotides for Gel Shift Experiments (23 KB DOC). Click here for additional data file.

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The Guanine Nucleotide Exchange Factor ARNO mediates the activation of ARF and phospholipase D by insulin

Background Phospholipase D (PLD) is involved in many signaling pathways. In most systems, the activity of PLD is primarily regulated by the members of the ADP-Ribosylation Factor (ARF) family of GTPases, but the mechanism of activation of PLD and ARF by extracellular signals has not been fully established. Here we tested the hypothesis that ARF-guanine nucleotide exchange factors (ARF-GEFs) of the cytohesin/ARNO family mediate the activation of ARF and PLD by insulin. Results Wild type ARNO transiently transfected in HIRcB cells was translocated to the plasma membrane in an insulin-dependent manner and promoted the translocation of ARF to the membranes. ARNO mutants: ΔCC-ARNO and CC-ARNO were partially translocated to the membranes while ΔPH-ARNO and PH-ARNO could not be translocated to the membranes. Sec7 domain mutants of ARNO did not facilitate the ARF translocation. Overexpression of wild type ARNO significantly increased insulin-stimulated PLD activity, and mutations in the Sec7 and PH domains, or deletion of the PH or CC domains inhibited the effects of insulin. Conclusions Small ARF-GEFs of the cytohesin/ARNO family mediate the activation of ARF and PLD by the insulin receptor. Electronic supplementary material The online version of this article (doi:10.1186/1471-2121-4-13) contains supplementary material, which is available to authorized users.

Background Small GTPases of the ADP-ribosylation factor (ARF) family play a major role in membrane trafficking in eukaryotic cells [ 1 ]. ARF activation is facilitated by specific guanine nucleotide exchange factors (ARF-GEFs). Several ARF-GEFs have been identified, varying in size, structure and subcellular distribution [ 2 – 6 ]. Of particular interest in signaling events are the members of the cytohesin/ARNO family of ARF-GEFs. These proteins have been found to associate with the plasma membrane under certain conditions, and consist of three well-defined motifs: an N-terminal coiled-coil domain (CC domain), a central domain with homology to the yeast protein Sec7 (Sec7 domain), and a C-terminal pleckstrin homology domain (PH domain) (Fig. 1 ). The catalytic activity of ARNO for guanine nucleotide exchange is localized in the Sec7 domain and appears to be regulated through the interaction of the PH domain with phosphatidylinositol (PtdIns) (3,4,5)-P3 [ 7 , 8 ], an intermediate in signaling cascades regulated by insulin and other agonists [ 3 ]. Figure 1 Schematic structure of ARNO constructs. Full length of wild type ARNO and ΔPH-ARNO were subcloned either in pCMV-myc or pEGFP-C1. PH-ARNO and ΔCC-ARNO were subcloned in pEGFP-C1. CC-ARNO was subcloned in pEGFP-N1. Phospholipase D (PLD) catalyzes the hydrolysis of phosphatidylcholine (PC) to produce phosphatidic acid (PA). It is involved in a variety of signaling pathways and membrane traffic processes [ 9 , 10 ]. Many hormones, neurotransmitters, and growth factors, including insulin, have been shown to induce the activation of PLD [ 11 , 12 ]. Several factors are involved in the regulation of cellular PLD activity, such as Ca 2+ , protein kinase C, tyrosine kinases, and G proteins [ 13 – 17 ]. Among these, the members of the ARF and Rho families of GTPases appear to be the most potent physiological activators [ 18 – 24 ]. However, the mechanism of the activation of PLD by ARF and Rho has not yet been fully established. This study was designed to investigate the role of ARNO in the regulation of PLD activity by insulin in HIRcB cells, a Rat-1 fibroblast cell line that overexpresses human insulin receptors. The objectives were: 1) to test if insulin induces the translocation of wild type ARNO to the plasma membrane in transiently transfected HIRcB cells; 2) to determine whether ARNO translocation is accompanied by activation and subcellular translocation of ARF; 3) to explore if overexpression of wild type ARNO in HIRcB cells alters insulin-dependent PLD activity; and 4) to investigate the function of individual domains of ARNO in insulin-dependent PLD and ARF activation. Results Insulin–dependent binding of ARNO to cell membranes The translocation of ARNO and ARNO mutants to the membranes was studied in HIRcB cells using a digitonin permeabilization assay. For these experiments, HIRcB cells were transiently transfected with myc-tagged wild type ARNO and the following mutants: ΔPH-ARNO, PH-ARNO, ΔCC-ARNO, CC-ARNO, E156K-ARNO and R280D-ARNO. This assay is based on the formation of pores in the plasma membrane induced by digitonin to allow cytosolic proteins to leak out of treated cells upon centrifugation. Fig. 2 shows that, after digitonin permeablization, a significant fraction of ARNO proteins leaked out of serum-starved HIRcB cells that transiently overexpressed the wild type ARNO and its mutants. Since these proteins were mostly recovered from the supernatant fractions, suggesting that wild type ARNO and the mutants tested are predominantly cytosolic in non-stimulated cells. In contrast, when digitonin permeablization was performed in the presence of insulin (100 nM), most of wt-ARNO, E156K-ARNO, and ΔCC-ARNO as well as a part of CC-ARNO were recovered from the particulate membrane fraction, suggesting that these ARNO proteins can be recruited to the membrane by insulin to various degrees. However, neither R280D-ARNO nor ΔPH-ARNO was recovered from the particulate fraction after insulin stimulation, suggesting that the translocation of ARNO to the membrane requires an intact PH-domain. It should be noted that, although the CC domain alone binds to the membranes under stimulation conditions, the degree of the binding is much less than that of wild type ARNO (Fig. 2 ). Surprisingly, a construct containing only the PH domain of ARNO could not be recruited to the membranes by insulin, indicating that the PH domain is essential but not sufficient for the translocation of ARNO. Figure 2 Insulin promotes the translocation of ARNO to cell membranes. HIRcB cells were transfected with myc-wt-ARNO, myc-E156K-ARNO, myc-R280D-ARNO, myc-ΔPH-ARNO, EGFP-PH-ARNO, EGFP-ΔCC-ARNO, and CC-ARNO-EGFP. The cells were treated with/without (Control) 10 μM digitonin (Dig). Where indicated, 100 nM insulin, 1 mM ATP, and 100 μM GTPγS were present during permeablization reaction. Pellets and supernatants were separated by centrifugation and the presence of myc-ARNO and its mutants or ARNO-EGFP in each fraction was determined by immunoblotting. ARNO recruits ARF1 to the plasma membrane in an insulin-dependent manner Since ARNO is an activation factor of ARF, we tested the hypothesis that agonist-dependent ARNO translocation facilitates the local binding of ARF proteins to the membrane. An initial set of real-time studies was done using HeLa cells that had been stably transfected with an ARF1-GFP construct [ 25 ]. These cells were transfected with myc-ARNO, serum-starved overnight, and imaged with a confocal microscope equipped with a constant-temperature microperfusion incubator to maintain the temperature at 37°C. Time-lapse images were collected at 30-second intervals. A representative experiment was shown in Fig. 3A . Prior to insulin stimulation, ARF1-GFP protein was mostly cytosolic or bound to the Golgi apparatus, although a small amount of ARF-GFP was localized on the surface of the cells. Ten minutes after the insulin stimulation, most of the ARF1-GFP was found on the plasma membrane. Similar results were obtained with HIRcB cells co-transfected with ARNO-myc and ARF1-GFP (Fig. 3B ). It should be noted that a significant accumulation of ARF1-GFP on the plasma membrane was not observed in the cells that had not been transfected with ARNO (not shown), or that had been transfected with the inactive mutant E156K-ARNO (Fig. 3B ). Since the endogenous levels of ARNO in HeLa cells were so low that the protein could not be detected in Western blots, it is reasonable to assume that under physiological conditions only a very small fraction of ARF1 translocates to the plasma membrane in response to extracellular agonists. Figure 3 A. Real time image of the translocation of ARF1-GFP to the plasma membrane. HeLa cells that had been stably transfected with ARF1-GFP were transiently transfected with myc-ARNO, serum starved overnight, and treated with 100 nM insulin. Images were collected every 30 seconds using a Molecular Dynamics 2001 confocal microscope. The time intervals that were indicated on the upper right hand corner of each panel represent the time after the addition of insulin. B. The translocation of ARF1-GFP to the plasma membrane by the effects of insulin requires ARNO. ARF1-GFP/HeLa cells were transfected with myc-ARNO, treated, fixed, and stained for myc-epitope as described in the Materials and Methods section. Images displaying ARF1-GFP (green) and myc-ARNO (red) were merged us ing Adobe Photoshop software. ARNO interacts directly with the insulin receptor Our previous work has shown that the insulin receptor co-immunoprecipitates with ARF in an agonist-dependent manner [ 23 ]. Furthermore, we have also shown that an ARF-GEF activity is associated with the insulin receptor and that this activity is not a function of the receptor itself [ 23 ]. Given that many receptor tyrosine kinases form complexes with their target proteins, we tested the hypothesis that ARNO binds the insulin receptor. Figure 4 shows that insulin receptors that were immunoprecipitated in the presence of insulin were associated with an ARF-GEF activity (Fig 4 ●), and that the ARF-GEF activity that was co-immunoprecipitated with the insulin receptor was significantly increased in the cells that had been transiently transfected with myc-ARNO (Fig. 4 ■). Insulin receptors that were immunoprecipitated in the absence of insulin did not accelerate the binding of GTPγS to the recombinant ARF1 as much as those obtained in the presence of insulin (Fig. 4 ○), indicating that the association of ARF-GEF activity with the insulin receptor was dependent on the presence of insulin. Figure 4 The ARF-GDP exchange activity of the coimmunoprecipitates with the insulin receptor. The exchange activity was determined as described in Materials and Methods. (○,□) Receptors were immunoprecipitated in the absence of insulin from cells transfected with empty vector (○) or with myc-ARNO (□). (●,■) Receptors were immunoprecipitated in the presence of insulin from cells transfected with empty vector (●) or with myc-ARNO (■). We then transfected HIRcB cells with myc-tagged ARNO constructs. Fig. 5 shows that the wild type ARNO co-immunoprecipitated with the insulin receptor in an insulin-dependent manner. E156K-ARNO was also co-immunoprecipitated with the insulin receptor upon insulin stimulation. However, none of the deletion mutants, including ΔPH-ARNO, PH-ARNO, ΔCC-ARNO, and CC-ARNO, as well as a site-directed mutant R280D-ARNO, was found co-immunoprecipitated with the insulin receptor. These data suggest that ARNO directly interacts with the insulin receptor and that the interaction requires intact PH and CC domains, but the catalytic activity of the Sec7 domain does not alter the interaction. Figure 5 Immunoprecipitation of the insulin receptor with ARNO and its mutants. Immunoprecipitated proteins were resolved by SDS-PAGE and myc-ARNO, myc-E156K-ARNO, myc-R280D-ARNO and myc-ΔPH-ARNO were detected by immunoblotting with a monoclonal anti-myc epitope antibody. PH-ARNO-EGFP, ΔCC-ARNO-EGFP, and CC-ARNO-EGFP were detected by immunoblotting with a polyclonal antibody against EGFP. Effects of the overexpression of ARNO or its mutants on insulin-dependent PLD activity We have shown so far that ARNO mediates the translocation of ARF proteins to the plasma membrane with insulin stimulation. Since ARF proteins mediate the activation of PLD by insulin [ 23 ], we tested the hypothesis that ARNO may play a role in the regulation of PLD activitiy upon insulin stimulation. To prove this point, the PLD activity of HIRcB cells that had been transiently transfected with the wild type ARNO, and mutant ARNO constructs. Fig. 6 shows that the overexpression of the wild type ARNO significantly increased insulin-induced PLD activity when compared with that of non-transfected cells. In contrast, the overexpression of the indicated ARNO mutants significantly decreased the ability of insulin to stimulate PLD. We conclude, therefore, that members of the cytohesin/ARNO family of ARF GEFs play an important role in the regulation of PLD activity by insulin. Figure 6 Effects of overexpression of the wild type and mutant ARNO constructs on the activation of phospholipase D by insulin. HIRcB cells were trans fected with empty vector, myc-wt-ARNO, myc-E156K-ARNO, myc-R280D-ARNO, and myc-ΔPH ARNO, PH-ARNO-EGFP, ΔCC-ARNO-EGFP, and CC-ARNO-EGFP. PLD activity was determined by a transphosphatidylation assay as described in Materials and Methods. Discussion Several studies have demonstrated that ARF proteins may mediate receptor-dependent activation of PLD. Stimulation of cell surface receptors with agonists, such as insulin, promotes the translocation of ARF proteins to the cell membranes and the activation of ARF proteins and the subsequent activation of PLD [ 16 , 18 , 21 , 23 ]. However, the mechanisms by which ARF proteins are activated by cell surface receptors remain obscure. ARF GEFs of the cytohesin/ARNO family have been shown to be recruited to cell membranes by mechanisms that are influenced by extracellular agonists [ 7 , 26 ]. These GEFs have been implicated in the regulation of many cellular processes, ranging from the regulation of cell motility [ 27 ] to cell adhesion [ 28 ] and, more recently, oncogenesis [ 29 ]. It has been speculated that PLD activation may mediate several of the cellular events regulated by cytohesin/ARNO GEFs [ 30 ]. However, a direct proof of a role for these factors in the regulation of the receptor-mediated PLD activation is still lacking. To address these and other related issues, we have studied in detail some of the mechanistic aspects of this pathway using a fibroblast cell line that overexpresses human insulin receptors as a model. This model and other similar ones have been used in our laboratory and others to examine specific aspects of insulin receptor function, such as receptor phosphorylation and traffic [ 23 , 31 – 33 ] and the regulation of the MAPK pathway [ 34 ]. Our studies showed that insulin promoted the translocation of myc-tagged ARNO constructs to the plasma membrane. This result is in agreement with data previously published by Venkateswarlu et al [ 7 ] and Langille et al [ 35 ] who demonstrated the insulin-dependent translocation of ARNO and the related protein GRP-1 to the plasma membrane, respectively. A detailed analysis of ARNO deletion and point mutants demonstrated that: 1) the translocation of ARNO to the membrane is independent of its ARF-GEF activity; 2) ARNO translocation to the plasma membrane requires an intact PH domain; 3) the CC domain of ARNO plays a role in targeting ARNO to the plasma membrane; 4) neither the PH domain of ARNO nor its CC domain alone sufice to target the protein to the plasma membrane; and 5) the plasma membrane translocation of ARNO is strongly regulated by insulin and, perhaps, other extracellular agonists. The linkage between ARNO translocation to specific subcellular fractions and ARF activation was studied using myc-tagged ARNO and ARF-GFP constructs in two different cell types. Our data showed conclusively that insulin promoted the co-localization of wild type myc-ARNO and ARF1-GFP on the surface of HIRcB and HeLa cells. Interestingly, insulin, acting through ARNO, promoted the translocation of ARF1-GFP to the plasma membrane. ARF1, like most members of the ARF family, is primarily a cytosolic protein that exerts its function on specific membranes to which it is recruited by specific activators that promote the binding of GTP. However, ARF1 seems to act primarily at the Golgi, promoting the binding of coatomer proteins to the Golgi membrane [ 36 , 37 ]. Nevertheless, the fact remains that ARF1 is primarily cytosolic, and that only a small fraction of it is bound to the Golgi membrane at any time [ 36 ]. It is not surprising, therefore, that some ARF1 may bind to the plasma membrane after being locally activated by ARNO, which is in turn recruited to the cell surface by the action of insulin. It should be remembered that our cells overexpress ARF1-GFP. Whether ARF1 does in fact work at the plasma membrane under physiological conditions or not remains to be established. Our data simply establish the fact that a receptor-dependent mechanism to recruit ARF1 to the plasma membrane does exist. On the other hand, ARF6 is normally found associated with the plasma membrane [ 36 , 38 ], and there is evidence that ARF6 might be the primary target for ARF-GEFs of the cytohesin/ARNO family [ 27 ]. However, when ARF dominant negative mutants were tested for their ability to inhibit agonist-dependent PLD activation, the data showed that ARF1 dominant negative mutants (T31N-ARF1) were as efficient as ARF6 mutants (T27N-ARF6) [ 23 ]. These observations strongly support the idea that ARF-GEFs of the cytohesin/ARNO family have full access to the cytosolic ARF proteins. Therefore, although ARF6 might be the primary intermediate for ARNO-regulated PLD activation, other ARF proteins may as well play an important role in the pathway. The ability of insulin to promote the translocation of ARNO and ARF to the plasma membrane correlated well with the ability of insulin to promote the activation of PLD. Therefore, our data support the hypothesis that the activation of PLD by insulin is mediated by ARF-GEFs of the cytohesin/ARNO family by a mechanism that involves the interaction of the PH and CC domains of these GEFs with some specific cellular targets. This conclusion is based on the demonstration that ARNO constructs with catalytically inactive domain or the mutants with defective PH and CC domains acted as dominant inhibitors of insulin-dependent PLD activation. The dominant negative effects of E156K-ARNO were not unexpected, since this mutant contains the intact PH and the CC domains and is therefore likely to compete with endogenous ARNO. The dominant negative effect of the PH and the CC domain deletion mutants on PLD activation was of particular interest. These mutants were at best partially translocated to the membrane but blocked the ability of insulin to promote ARF and PLD activation. This result was somewhat surprising since these deletion mutants contain an intact Sec7 domain and, therefore, would have been expected to support ARF and PLD activity. However, this was not the case, suggesting that all regions of ARNO play an important role in the regulation of this protein. Moreover, the failure of the ΔCC mutant to activate ARF and PLD indicates that other cellular targets that bind to the CC doma in of ARNO and regulate the subcellular location or the function of the signaling protein complex may exist. In fact, some proteins that interact strongly with the CC domain of members of the ARNO family, such as CASP and GRASP, have already been identified [ 39 , 40 ]. Consistent with these ideas was the observation that the overexpression of either the PH or the CC domain alone was sufficient to block insulin-dependent PLD activation. Therefore, we propose that cellular targets that recognize both the PH and CC domains of ARNO are important for the regulation of the function of this protein by cell surface receptors. On the other hand, our data also strongly support the hypothesis that the regulation of ARNO activity by insulin involves, at least transiently, a direct interaction of the insulin receptor with ARNO. Consistently, the presence of an ARNO-like activity and ARNO in the immunoprecipitated materials was confirmed by biochemical experiments. Finally, ARNO constructs lacking either the CC or the PH domain, or with a defective PH domain, failed to co-immunoprecipitate with the insulin receptor. These findings suggest a mechanism of the activation in which the binding of ARNO to the membrane is regulated by the insulin receptor at two different levels: 1) ARNO must interact with the receptor; and 2) ARNO must interact with the membrane, either via binding to polyphosphoinositides or through the interaction with specific protein targets. Our data strongly support the idea that both CC and PH domains play a crucial role in this phenomenon. Conclusions This study suggests a general model for the activation of PLD with insulin stimulation. Insulin, upon binding to its receptor, promotes the phosphorylation of IRS-1 and the activation of PI3 kinase. This results in the accumulation of polyphosphoinositides on the plasma membrane. In parallel, the insulin-bound receptor promotes the recruitment of ARNO (and/or other members of the ARNO family, such as GRP-1) to the plasma membrane, either by direct interaction with their CC and PH domains or by promoting the interaction of ARNO with other as yet unidentified targets. The binding of ARF-GEFs to the plasma membrane is stabilized by the interactions of their PH domain with polyphosphoinositides generated by the action of PI3 kinase. Once on the membrane, the ARF-GEFs catalyze the activation of membrane-bound ARF6 or cytosolic ARF proteins that are then recruited to the membrane where they may activate PLD. Cell culture Rat-1 fibroblasts overexpressing the human insulin receptors (HIRcB cells) were cultured in Dulbecco's modified Eagle's medium (DMEM)/Ham's F-12, supplemented with 10% fetal bovine serum, antibiotics, and 100 nM methotrexate, as previously described [ 20 ]. Cells were subcultured, transfected as indicated in the figure legends, and serum starved for overnight (approximately 20 hrs) prior to insulin stimulation. HeLa cells were cultured in DMEM supplemented with 10% fetal bovine serum and antibiotics. HeLa-ARF1-GFP stable transfectants were obtained by using G418 as a selection agent as described elsewhere [ 25 ]. Clonal populations were obtained and used in the assays described here. Transient Transfection Subconfluent (70–90%) HIRcB cells were transfected with LipofectAMINE (Invitrogen) for biochemical analyses or Superfect (QIAGEN) for imaging analyses. Transfection was performed according to the manufacturer's instructions. Transfection efficiencies were 70–90% for LipofectAMINE and 40–50% for Superfect transfection reagent as previously described [ 41 ]. Generation of fusion proteins It has been reported that the members of the cytohesin/ARNO family of ARF-GEFs each exist in two isoforms in terms of existence of extra G (glycine) in PH domain [ 42 ]. In this study, we used the isoform of ARNO with GGG (tri-glycine), which has similar binding affinities for both PI-(3,4,5)-P 3 and PI-(4,5)-P 3 . The following myc-tagged ARNO constructs were generated: wt-ARNO, ΔPH-ARNO, PH-ARNO, ΔCC-ARNO, E156K-ARNO, and R250D-ARNO. wt-ARNO, ΔPH-ARNO (amino acids 1 to 269), PH-ARNO (amino acids 262–399), and ΔCC-ARNO (amino acids 51–399) (Fig. 1 ) were amplified by PCR and subcloned in the multiple cloning site of the vector pEGFP-C1 (CLONTECH) and fused to green fluorescent protein (GFP) as described by Venkateswarlu and coworkers [ 7 ]. The CC domain of ARNO (amino acids 1 to 55) (Fig. 1 ) was PCR out of wt-ARNO and subcloned into pEGFP-N1 using BglII and EcoRI restriction sites. E156K-ARNO (inactive Sec7 domain) was generated by site-directed mutagenesis as described by Frank and coworkers [ 43 ]. R280D-ARNO was designed on the basis of that a mutation on an analogous arginine impairs the binding of cytohesin-1 to polyphosphoinositides [ 26 ]. The sequences of the constructs were verified by direct sequencing and the expression of appropriate fusion proteins was examined by Western blotting. The level of expression of all constructs was found to be comparable. Immunoprecipitation assay Transfected and serum-starved HIRcB cells were washed with ice-cold PBS, scraped, and collected by centrifugation. The cell pellets were solubilized on ice for 1 hr in a solution of 50 mM Hepes, pH 7.45, containing 100 mM NaCl, 1.5% sodium cholate, 1 mM EDTA, 1 mM EGTA, 5 ug/ml leupeptin, 1 mM PMSF, and 1 mg/ml soybean trypsin inhibitor. Insoluble materials were removed by centrifugation. The cell lysate was immunoprecipitated with anti-mouse IgG agarose that had been equilibrated with a monoclonal antibody 83.7 (which recognizes the α subunit of the human insulin receptor). Immunoprecipitation was carried out overnight (approximately 20 hrs) at 4°C. The immunoprecipitates were washed with lysis buffer, resuspended in SDS-PAGE sample buffer, and subjected to Western blotting analysis. Immunoblotting Proteins were separated by SDS-PAGE, transferred to a nitrocellulose membrane, and blocked with 5% non-fat milk in PBS containing 0.1% Tween at room temperature for 2 hrs. The membrane was then cut in half horizontally. The upper part was used to detect the β subunit of the insulin receptor with a monoclonal antibody, CT-1, that recognizes the carboxyl terminus of the β subunit of the human insulin receptor. The lower part was used to detect ARNO proteins with a monoclonal antibody anti-myc or a polyclonal antibody anti-GFP. PLD activity assay Serum-starved HIRcB cells were labeled overnight with 3 H-palmitate (5 μCi/ml) in serum-free medium. The cells were stimulated with insulin (100 nM) in the presence of 0.5–1% ethanol for 20 min. The reaction was stopped by addition of chloroform: methanol (1:1). The lipid phase was extracted and developed by thin layer chromatography (TLC) on silica gel 60 plates using ethyl acetate: trimethylpentane: acetic acid (9: 5: 2) as a solvent. The position of major phospholipids was determined using true standards (Avanti Biochemicals) and autoradiography. The TLC plates were scraped and the total amount of radioactivity associated with each lipid species was determined by liquid scintillation counting. The data were expressed as the number of counts associated with the phosphatidylethanol (PtdEtOH) spot normalized by the total number of counts of lipid. Digitonin treatment Serum-starved HIRcB cells were collected, resuspended in PBS, and treated with 10 μM digitonin in the presence or absence of insulin (100 nM), ATP (1 mM), and GTPγS (100 μM) at 37°C for 15 min. To release intracellular proteins, the digitonin-treated cells were centrifuged in a microcentrifuge for 20 min. The supernatants and the cell pellets were collected separately, and subjected to SDS-PAGE. ARNO proteins were detected by immunoblotting as described above. In vitro ARF activation assay ARF activation was determined by the binding of GTPγS to the purified, myristoylated recombinant human ARF1 (mhARF1), as described by Shome and coworkers [ 23 ]. The insulin receptor was immunoprecipitated in the presence or absence of 100 nM insulin as described above. Four to 8 μg mhARF1 and the immunoprecipitated insulin receptors were incubated with 100 nM GTPγ[ 35 S] (1 μCi) in 20 mM Hepes buffer containing 2 mM MgCl 2 / 0.1% Na-cholate / 1 mM ATP. At the indicated time points, the reaction was quenched by addition of 100 μM ice-cold, unlabeled GTPγS and the protein-bound nucleotide was determined by filtration through nitrocellulose filters as described [ 23 ]. Confocal microscopy HIRcB cells were plated on poly-L-lysine coated glass coverslips and transfected with the constructs as indicated above. Cells were serum starved overnight and stimulated with 100 nM insulin. Live cells were imaged in a LSM5 Zeiss laser scanning confocal microscope equipped with a 63X oil immersion objective. For ARF and ARNO colocalization experiments, HIRcB cells were plated on poly-L-lysine coated coverslips as described above and co-transfected with myc-ARNO and ARF-GFP constructs using Superfect transfection reagent according to the manufacturer's instructions. Following insulin stimulation, the cells were fixed with 4% fresh paraformaldehyde in PBS at 4°C for 30 min, and permeabilized in 0.1% Triton X-100 at room temperature for 2 min. After permeabilization, the cells were blocked with 3% bovine serum albumin in PBS at room temperature for 30 min, and immunostained with a monoclonal antibody 9E10 (Upstate Biotechnology) that recognizes the myc epitope. After extensively washing, the cells were incubated with a Cy5-conjugated donkey anti-mouse secondary antibody (Jackson Immunoresearch) and imaged using a Zeiss laser scanning confocal microscope with filters appropriate for the detection of GFP and Cy5.

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